i k i ^9 n^ '-' ^ ', ■ ^^ THE BIOLOGY OF THE CELL SURFACE ci 7 6 J 9^ THE BIOLOGY OF THE CELL SURFACE B y ERNEST EVERETT JUST Natur hat weder Kern Noch Schale, Alles ist sie mit einemmale — goethe R BLAKISTON'S SON & CO., INC. 1 nilaaelpnia Copyright, 1939, by P. Blakiston's Son & Co., Inc. PRINTED IN U. S. A. BY THE MAPLE PRESS COMPANY, YORK, PA. To the memory of ^My ^Mother \\(:Af X ^ TreJ. ace 'ZS^HEX I BEGAN THE WRITING OF THIS BOOK, I INTENDED merely to set forth my views on some biological problems which I had studied during a period of more than twenty- five years of research on the eggs of marine animals. I had thought my task easy; that it would be a simple matter to present my ideas and findings against the clearly delineated back-drop of general biological theories and on the basis of well-established fact. Merely to hint at the theories and to indicate briefly the factual evidence would suffice, I thought, to make myself clear to my colleagues. But in thinking thus, I had deluded myself; soon I realized that the back-drop had to be cleared in order to show its con- tours; that instead of a substantial platform I had only pieces of material that had first to be ordered and put together before they could serve me. This work had to be done, I found, before I had a stage on which I could present my own ideas and let them speak their parts in my inter- pretation of the drama of life unfolding before our eyes. It thus became imperative for me to examine and to appraise hypothesis and factual evidence and to define first every problem the discussion of which I had projected for my book. Thus my book developed from a mere statement of my views for biologists engaged in a narrowly restricted field of investigation into a thoroughgoing presentation and dis- cussion of biological problems for a wider audience. To all those who look with interest upon the manifestations of life in animals and in man, who desire to know more, and more vii PREFACE exactly what answers to their questions concerning life biology can give, this book would speak. Biologists in other fields, students in biology and related sciences and all who have general interest, I have endeavored to address in the following pages. I have the strong feeling that many thoughtful and serious men in engineering, practical physics and chemistry and laymen outside of these professions do not know what modern biology is; even medical men often wonder what this biology of to-day offers. They have genuine interest in animal life and give much reflection to biological ques- tions; yet present day biology leaves them untouched. For such readers text-books offer little. What the professional biologist denominates as biology — taxonomy, palaeontol- ogy, ecology, physiology, morphology, embryology, phyto- and zoo-geography, as well as genetics, biometry and the like, with their various subdivisions, each of which is again subdivided, as, for instance, cytology with its separate domains ruled by karyologists, " protoplasmatikers," "Golgiologists" and " mitochondriacs" etc., etc., — needs to be brought into relation with the world outside biological institutes and recording offices. To the uninitiated this biological regimentation inspires awe because it connotes the abstruse too far removed from everyday life. Even the most abstract truth needs to be expressed with simplicity and clearness and thus relate itself to everyday human experience. Complexity of expression is often a sign of incomplete knowledge and certainly it is not a sijie qua non of learning, though there be those who consider profound and erudite that which they can never understand. However cloistered biology may be as a scientific research, as the science of life and having appeal to all men it should make itself articulate beyond its cloistered walls. I have endeavored in writing this book to express myself with such clearness that even the uninitiated via PREFACE can follow my argument at least in the main. At the same time I bring the reader at once into the arena of conflicting biological thought, for only thus, I think, can he realize the status of the science to-day. I trust that what to biologists are well-known facts I have presented in a manner that elicits their interest. Together with a definition of the general problems, my own work as well as my ideas are presented. The out- come of these is my theory which I set forth in a more explicit manner than heretofore. The conception upon which the book is built, though latent in my earlier researches, did not come fully awake until 1930 while I was enjoying the hospitality of the Kaiser-Wilhelm-Institut fiir Biologic at Berlin-Dahlem. There I fell under the inspiration of Adolf von Harnack's personality. I like to feel that my work was influenced by the rich experience of personal contact with him. The studies which gave rise to my conception were made during some twenty years, largely at the Marine Biological Laboratory, Woods Hole, Mass.; some few were made at the Zoological Station at Naples. For the support of many of these researches I am indebted to the late Mr. Julius Rosenwald, whose friendship I esteemed. However, this book could not have been finished but for the spontaneous and sympathetic understanding of my work shown by Dr. F. W. Keppel, President of the Carnegie Corporation. A grant from this corporation made possible a year's study necessary to complete the work. In the task of writing a book understandable also to non-biologists, I have further been sustained and encouraged by many friends, biologists, medical men, and others outside of these fields. The book may be divided Into three parts. Part I, com- prising Introduction, Life and Experiment, Protoplasmic System, Ectoplasm, General Properties of the Ectoplasm and Water, though dealing primarily with the animal egg, ix PREFACE embodies principles which concern the fundamental organi- zation of any living thing. Part II, including the Fertiliza- tion-process, the Fertilization-reaction, Parthenogenesis, Cell-division and Cleavage and Differentiation, discusses in particular the problems that refer directly to animal eggs in their earliest stages of development. Part III, embrac- ing chapters on the Chromosomes and Ectoplasm, Ecto- plasm and Evolution, and Conclusion, has to do with more or less theoretical discussions. Throughout the whole treatment, the principle that the cell is the biological umt is kept in mind. In particular, structure and function of the ectoplasm are emphasized; upon these my theory of the state of being alive is in large measure grounded. Concerning the literature cited, I should point out that I have made no attempt to refer to all of the many original papers which I have studied and abstracted. Instead, with each revision of the manuscript I have reduced the number of titles in the bibliography. Nevertheless, the list of titles retained is ample for the argument set forth. E. E. J. Paris, France, Novrynher , 1938. X Contents XI Page Chapter 1 . Introduction ^ 2. Life and Experiment ^^ 3. The Protoplasmic System 3^ 4. The Ectoplasm 75 5. General Properties of the Ectoplasm lO^ 6. Water ^^4 7. The Fertilization-process ^47 8. The Fertilization-reaction J^^4 9. Parthenogenesis ^^^ 10. Cell-division ^47 I I . Cleavage and Differentiation 280 12. Chromosomes and Ectoplasm 34^ 13. Ectoplasm and Evolution 354 14. Conclusion 3^- 15. Bibliography 37^ Index of Authors 3^^ Index of Subjects 3^4 50120 Introduction inY, REALM OF LIVING THINGS BEING A PART OF NATURE is contiguous to the non-living world. Living things have material composition, are made up finally of units, mole- cules, atoms, and electrons, as surely as any non-living matter. Like all forms in nature they have chemical struc- ture and physical properties, are physico-chemical systems. As such they obey the laws of physics and chemistry. Would one deny this fact, one would thereby deny the possibility of any scientific investigation of living things. No matter what beliefs we entertain, the noblest and purest, concerning life as something apart from physical and chemi- cal phenomena, we can not with the mental equipment which we now possess reach any estimate of living things as apart from the remainder of the physico-chemical world. But although any living thing, being matter, is a physico- chemical system, it differs from matter which constitutes the non-living. This difference exists and would continue to exist were some chemist at this moment to succeed to synthesize out of non-living matter a living thing. The analysis of living things reveals that they are composed of no peculiar chemical elements — instead, they are made up of those most commonly occurring. The difference can not then be attributed to the elements. To be sure, certain complex compounds, as proteins, carbohydrates and lipins (fats and fat-like substances) — themselves compounded mainly of the commonly occurring elements, carbon, hydro- gen and oxygen, and never of rare elements — are peculiar I THE BIOLOGY OF THE CELL SURFACE to living matter; but the synthesis of protein-like bodies, of sugar and of fat as well as the synthesis of thyroxin (a compound in the internal secretion of the thyroid gland), of products of other internal secretions and of vitamins must dissipate whatever belief may have lingered on since Wohler's classic synthesis of urea (more than a hundred years ago) that some unknown vital principle sets apart the chemistry of living things from that of non-living. And yet there is a difference which expresses itself in the chemical make-up of the living thing. It is its organiza- tion.^ The difference with respect to chemistry thus lies in the peculiar combination of compounds which together make a heterogeneous system. This acts as a unit-struc- ture, whose behavior or manifestations are those of a single thing and not the sum-total of the multitudinous chemical components in an agglomerate mass. Living matter has an organization peculiar to itself. Nowhere except in the living world does matter exhibit this organization. Life, even in the simplest animal or plant, so far as we know, never exists apart from it. Resting above and conditioned by non-living matter, life perhaps arose through the chance combination of the compounds which compose it. But who knows 1 A living thing is not only structure but structure in motion. As static, it reveals the superlative combination of compounds of matter; as a moving event, it presents the most intricate time-pattern in nature. Life is exquisitely a time-thing, like music. And beyond the plane of life, out of infinite time may have come that harmony of motion which endowed the combination of compounds with life. Clearly then, the state of being alive reposing in com- binations, in the order in which the constituents are 1 Dujardin, iSjj; Brucke, iS6i; Jllman, iSyg; Verzvorn, iSgg; Moore, ig2i. 2 INTRODUCTION assembled both in space and in time, its investigation is limited. The direct analysis of the state of being alive must never go below the order of organization which charac- terizes life; it must confine itself to the combination of compounds in the life-unit, never descending to single com- pounds and, therefore, certainly never below these. Whereas physical science has to do finally with ultimate units of matter, the scope of biological research embraces the behavior of more heterogeneous combinations of these units. The physicist aims at the least, the indivisible, particle of matter. The study of the state of being alive is confined to that organization which is peculiar to it. It may be that life can never be written as a formula because it may be a physics and chemistry in a new dimension which, though superimposed upon the now known physics and chemistry, lies in an infinity which the human mind can not ever embrace — as a tone which theoretically we know exists but which the human ear can never hear. But be this as it may, life as an event lies in a combination of chemical stuffs exhibiting physical properties; and it is in this combination, i.e., its behavior and activities, and in it alone that we can seek life. A living thing represents in its unit of structure and behavior the highest order of com- plexity in nature. All this implies that the method employed in the investigation of a living thing can not be identical with that used in physical sciences. With right we glorify refined and precise measurements — ■ great accomplishments in the physical sciences rest upon them. The desire to extend their use to embrace the science of living things is understandable; nevertheless, quantita- tive measurements can never be used in biology to the extent that they are used in physics. To be sure biology also employs measurements. He who studies the living animal or plant often has recourse, most of the time entirely unconsciously, to the estimation of 3 THE BIOLOGY OF THE CELL SURFACE weight and size. The classification of living things, the earliest scientific treatment of life, relies upon measure- ments. The anatomist, be he interested in bones or in chromosomes, reckons size and number. These kinds of simple and direct measuring have a definite place in biology. Similarly have those relating to the functions of the living thing. No one questions the value of measurement for the analysis of the gases in human blood. He who would be foolhardy enough to deny the dependence of biology upon physics and chemistry would do well to ponder the history of the research on animal respiration, beginning with Lavoisier, whose work stands as a new starting point for both chemistry and biology. The final proof that the blood carries most of its contained oxygen in chemical rather than in physical union constitutes one of the most brilliant chap- ters in biology to read which quickens the pulse. The measurements on temperature, on pressure, of electrical conditions, of light, of the chemical breaking down of com- plex foods and the building up of these, the photosynthetic process in green plants without which human life would become immediately impossible — these and many others show that biology comprises a welter of physico-chemical measurements, a fact to occasion no astonishment since living things are physico-chemical entities. The measurements employed in physical science which do not apply to living things concern states of matter below that level of organization which characterizes a living thing. True, matter in the living state as all matter is compounded of electrons. The high and enviable excellence of modern physics rests upon the beautiful and enthralling analysis of the ultimate unit-structure of the least particle of which all nature is composed. However, the fact that life as far as we know exists as a composite, and only as such, renders pure physical analysis, i.e., into electrons, inappli- cable to the state of being alive. I'he fact that life dissolves 4 INTRODUCTION even when it is resolved only into compounds means, in the present state of our knowledge of electrons, an insuperable obstacle to an analysis of life into electrons. This would still be true were we suddenly to discover a peculiar com- pound which we could define as the compound of the state — or event — of being alive. As matter, a living thing may be analyzed with the means utilized in the resolution of non-living matter; but the moment we end the living state we render impossible any direct analysis of life. Clearly: if life exists only in a super-compound-state, contingent upon aggregation, analysis of its compounds being useless, analysis of molecular and atomic structure becomes equally futile. A quantitative biology that does not recognize this fact is doomed to failure. Without needlessly elaborating, I do nevertheless wish to make myself understood. It would be shamefully unfair and ungrateful of any biologist even to appear to discount the researches in chemistry upon which modern biology so largely rests. The greatest biological investigator of our time, Pasteur, w^as a chemist. His work indicates the extent to which chemistry may carry biology. The work of Emil Fischer, of Albrecht Kossel and of a host of other chemists has fructified and fortified biology. Still the fact remains: the exact analysis of the compounds which com- prise a living thing is only analysis of the compounds and by destroying life, such analysis fails to reveal the secret, the goal of all biology, the answer to the question, what is life ? Here one may interpose a question, an inquiry concerning the postulated life-molecule. '^ In earlier times one thought of life as reposing in a peculiar molecule, as the biogen- molecule. Chemical researches have however so far failed to reveal the life-molecule. Instead they show that living ^ See Hopkins, ig/2. See also, Minot, i8g6. 5 THE BIOLOGY OF THE CELL SURFACE matter Is composed of proteins, carbohydrates and lipins together with water, electrolytes and gases. It would be necessary, unless life resides only in protein, and this has not yet been proved, to obtain evidence to indicate that all these constituents named as components of living matter in some way combine as a single molecule. Further, with respect to proteins, carbohydrates and fats, we have no sufficient ground to warrant the conclusion that they exist in living matter in the same form which they have when they are Isolated. This is especially true of the proteins; all of them known to us outside the living substance are probably changed in their nature chemically and physically by the methods of isolation. In the chemist's test-tube the organic constituents of the living substance may repre- sent merely end-products of something In the living. In this, however, lies no reason for supporting the theory of a blogen-molecule; rather should we try to refine our methods of isolation, so that the results allow a more direct con- clusion. If one says that the blogen-molecule depends upon a certain milieu — if, for example, being protein-like it depends upon fat and fat-like substances, sugars, various electrolytes and water — we need the postulate that the life- molecule is totally unlike molecules with which physical science has to do. I have no wish to quarrel with words; either the term molecule used in the expression, life-mole- cule, carries the connotation ordinarily assigned It by physical scientists or it embodies a non-physical conception. As a concept consonant with physical science it stands open to serious doubt; as a non-physical postulate it defeats Its own purpose. What is said of the blogen-molecule may be said of the gene-molecule, the hypothetical unit of which the chromo- somes are assumed to be built. Like the former it too appears incapable of self-maintenance — It grows by what it feeds upon, the cellular matrix in which it lies; and more, never stands apart from other genes in the same chromo- 6 INTRODUCTION some. Since, according to the gene-theory, it represents in the body of the organism in which it exists a character which another gene (or genes) in another chromosome (or other chromosomes) may represent, it appears to act not singly but as part of the chromosome-structure and of the whole chromosome-garniture. We know that life exists in size below that recognizable by the microscope; but we possess no proof that life consists of a single gene. Much has been said recently of the size of the gene; but we should recall that size does not condition molecularity. Molecules vary in size. In addition, many investigators have become cautious in speaking of the gene as a molecule, preferring the idea that it may be several molecules. The most potent objection to the gene-molecule- (or molecules) concept as the unit of life lies in the fact that the gene-theory fails to explain how a single cell, the egg, becomes a complex animal. A detailed discussion of this point is given beyond. Here it suffices to say that a particle (or all the particles together) which is only in part engaged in the unfolding of life during its course from Qgg to adult can not constitute life itself.^ The conception of a hypothetical life-molecule is barren and indicates again the limitation of the quantitative method of analyzing life below the level where life is. The investi- gator of the living state can and must use physics and chemistry since the living state is a zone in nature and his method of investigation can parallel that of the physical scientist inasmuch as he finally comes to the unit-organi- zation of life. Below this he can not go, for life is the harmonious organization of events, the resultant of a communion of structures and reactions. In general, the organization of living matter, that is, of protoplasm, appears as consisting of two components, a nuclear and a cytoplasmic. Although most often these ^ Bui cf. Jennings, ^93^' 7 THE BIOLOGY OF THE CELL SURFACE are set off as two distinct regions, as a sphere (nucleus) within a sphere (cytoplasm), this sharp differentiation is not invariable. For several reasons, as will be shown beyond, much of modern biological investigation has centered upon the nuclear component as though it were indeed the kernel of life. Not only has the cytoplasmic component been relatively neglected but also have those protoplasmic systems which lack sharply defined and set- off nuclei received scant attention, although a bacterium whose protoplasmic organization fails to show a discrete nucleus is a living system. Because of the rapid rise of genetics, hegemony in the protoplasmic organization has been ascribed to the chromosomal structure of the nucleus and the cytoplasm has been subordinated as though it be a mere protective and nutritive shell. It is no part of the purpose of this book to minimize the achievements of genetics and the investigations on chromosome-structure, all outgrowths of descriptive studies on protoplasmic organization. Instead, inasmuch as life, as we know it so far, resides in the whole system, the pages which follow aim to show how far life-processes are related to the dual and reciprocal components, nuclear and cytoplasmic structure. Investigations on cytoplasm have had largely to do with the formed bodies, the so-called cytoplasmic inclusions; only to a very small degree have they dealt with the cyto- plasm itself and its differentiation into an inner and an outer region, endoplasm and ectoplasm. Only in a general way has the role of the ectoplasm, the surface cytoplasm, been hinted at; thoroughgoing descriptions of it, compar- able to those of the nucleus, have not been made, doubtless because of the greater difficulties of observing it. None the less should the ectoplasm, as part of the protoplasmic system, be assigned a role in vital manifestations. An examination of its properties and behavior in life-processes 8 INTRODUCTION is all the more imperative because it has been so grossly neglected. The play of environmental forces demands the continuous adjustment, balance and discrimination of the living thing. How this self-regulation comes about constitutes a great problem in biology. Not less capable is the power of self- differentiation exhibited by the living system. Against and with the outside world, repelling and responding, it makes itself anew, exhibits a series of events in sequence and order ever the same. Self-regulation and self-differentia- tion are fundamental expressions of the organization of living matter. As an integral part of the protoplasmic structure the ectoplasm can not be divorced from this. The proto- plasmic organization however much a composite of many parts — nucleus with its structures and cytoplasm with its regions — acts as a unit. To conceive it as such is to approach an understanding of those series of moving events that we call life. The ectoplasm, standing between the protoplasmic system's inner substance and the outside world, reacts first to environmental stimuli and thus condi- tions the responses of the whole system. Its rapidly occur- ring, highly active structural changes portray self-regula- tion and self-differentiation. Thus by its location and by its peculiar attributes, the ectoplasm becomes the most tangible expression of life-processes. Z>//^ a?id Experiment The method employed in the investigation of the living thing, the foregoing statements clearly indicate, must be different from that in physical sciences: the nature of the thing investigated determines the method of inquiry. Description is the method of most use in biology. It plays a larger role here than in physics not because experimental biology is a younger science than physics, as one often avers, but because of the heterogeneity and complexity of the composite life-unit. The more heterogeneous and complex an object, its parts undergoing multitudinous kaleidoscopic changes, the more important description becomes. And when this object is minute, its changes evanescent, accurate description is imperative. By description the objects may be defined and the changes recorded in more and more closely set stages. This statement, however, by no means implies that experiment has no place in the study of the state of being alive. Both description and experiment are utilized by all the natural sciences — the extent to which each is used being determined by the state of the matter investi- gated. Just as the complex organization of living matter demands the larger employment of description, so it prescribes experimental method. The present chapter aims to evaluate the place of experiment in the study of a living thing in order to clear the ground for the dis- cussion of the problems with which this book deals. Thus I present my conception of the role of experiment in lo LIFE AND EXPERIMENT biology, a conception which resulted from years of expe- rience and which by determining more and more my mode of experimenting had a considerable influence on the formation of my theory of the state of being alive. In order to set my conception off from that maintained by a sub- stantial number of biologists, I begin by defining their point of view. I refer to the so-called "physico-chemical" school of biology. Specific example is often the best of definitions. I could give many examples from the writings of the "physico-chemical" biologists to define their position; one will suffice. Says Loeb^: The physical researches of the last ten years have put the atomistic theory of matter and electricity on a definite and in all probability permanent basis. We know the exact number of molecules in a given mass of any substance whose molecular weight is known to us, and we know the exact charge of a single electron. This permits us to state as the ultimate aim of the physical sciences the visualization of all phenomena in terms of groupings and displacements of ulti- mate particles, and since there is no discontinuity between the matter constituting the living and non-living world the goal of biology can be expressed in the same way. It is often said that in their thinking biologists are the most mechanistic of scientists, holding fast to concepts that the most mechanistic astronomers even have aban- doned. Indeed, many biologists besides Loeb believed — and many even now believe — that biology as a science must finally be concerned with the ultimate particles, that life as well as all physical phenomena can be interpreted on the mechanistic basis of the motion of particles in a system that is rigidly ordered as to time and space. But so far any attempt at "the visualization of all phenomena in terms of groupings and displacements of ultimate particles" has 1 Loeb, igi6. II THE BIOLOGY OF THE CELL SURFACE failed for biology. And it is to be doubted that any such "visualization" can ever succeed. We do not know that there is no discontinuity between the non-living and the living world and we certainly possess no evidence for the postulate that living phenomena can be expressed In "terms of groupings and displacements of ultimate particles." Nowadays, even for the physicist, Loeb's statement is too extreme. The evidence of physics does not yet permit such a view as to the finality of its concepts. In addition, before physical principles can be utilized profitably In biology, they must be sharply defined and accepted by physicists themselves.^ Moreover, even before the advent of the relativity- and the quantum-theory, physicists were not agreed that mechanics constitutes all of physics. Here a statement written forty years ago by the biologist Whitman- is apt: While biology is certainly indebted to physics for some of its metaphysics, it is to the credit of physics to have made it clear that mechanism, indispensable as are its methods, affords no fundamental explanation of anything. As Karl Pearson has so well said, the mystery of life is "no less or no greater because a dance of organic corpuscles is at bottom a dance of inorganic atoms." What dances and why it dances is not explained by reducing size to the lowest limit of divisibility, and just as little by the assumption of ultra- physical causes. This is no criticism — no disparagement; it is only a confession of ignorance. The ultimate mystery is beyond reach of both mechanism and vitalism; let preten- sion be dropped, and approximation to truth be closer on both sides. Further, as we have seen in the preceding chapter, our Investigations of the living thing as such end with its dis- integration. The moment that any such far-going physlco- 1 Watson, 1 93 1. - M'' hitman, 189^. 12 LIFE AND EXPERIMENT chemical analysis, as that postulated by Loeb, is applied, the living thing disappears and only a mere agglomerate of parts remains. The better this analysis proceeds and the greater its yield, the more completely does life vanish from the investigated living matter. The state of being alive is like a snowflake on a window-pane which disappears under the warm touch of an inquisitive child. This objection to the so-called physico-chemical point of view — i.e., that the goal of biology is the reduction of living matter to ultimate particles — is far more potent than those mentioned before. Certainly, it would still remain were the physicists able to state their concepts of the ultimate particles of matter in final terms. Though com- pounded of the same elements found elsewhere in nature and showing physical properties because of this composition, though amenable to physico-chemical laws, the living thing is refractory to analysis into ultimate particles. Here Heisenberg may be quoted to show how we can conceive the particular realm of living things as part of the natural world and the natural sciences: If for instance one thinks of the problems which are con- nected with the existence of living organisms, one will suppose, from the point of view of modern physics, that the powers which act in the organisms limit themselves in a like manner that is rationally exactly perceptible, from the purely physical laws, as, for example, thermodynamics limits itself from classic mechanics. . . . The building of natural science therefore most probably can not become a continuous unit, so that simply by following the prescribed way one can come from one point in it to all the other rooms of the building. Rather, the building is made up of single parts, each of which though standing in manifold relations to the others, nevertheless is a unit that is in itself complete. The step from already completed parts of the building to a newly discovered one or to one to be constructed demands always a mental action which can not be performed simply by developing farther that which already exists. ^ ^ Heisenberg, 1934. 13 THE BIOLOGY OF THE CELL SURFACE The physico-chemical biologists, however, do not picture natural science as Heisenberg does. Rather, they visualize the problems of the living organism as occupying a room in a single apartment of the building of physics, that of mechanics. Their view of physics, far more restricted than that of physicists themselves, has therefore obfuscated both the methodology and the philosophy of biology. This is shown by their loose usage of the terms, mechanism, mechanical, mechanistic and even machine, as though these be interchangeable. In biology the term, mechanistic, is used as the antipode of vitalistic. Since practise has legitimatized this usage, there may be little reason to quarrel with it. Nevertheless I wish to point out that the physico-chemical school of biologists has erred in elevating the term, mechanistic, beyond the meaning assigned it by physicists. By their own doctrine the term should have the connotation assigned it by physics. The true antipode of mechanistic is non- mechanistic. The term, non-mechanistic, by no means implies vitalism. Not every physicist who opposes the mechanistic conception deems it necessary to support a non-physical, super-natural concept. Rather, he holds that the behavior of the ultimate particles of matter is not rigidly determined, perfectly predictable. Logically, those biologists who conceive vital processes as phenomena to be interpreted by physics, should adhere to concepts of physics. The physico-chemical biology should take cognizance of the fact that physics has grown beyond "classical physics." Physico-chemical analysis into ultimate particles and the hypotheses derived from such work establish the fact of the existence of similarities between living and non-living. By virtue of its peculiar organization In space as well as in time, however, the living thing occupies a level in the natural world above that of chemical compounds. From this organization spring those characteristics by which we 14 LIFE AND EXPERIMENT commonly distinguish a living thing from a non-living; both the organization and these characteristics should claim more attention from those — be they biologists, mathematicians, physicists or chemists — who study the living state, than they have up to now received. Having agreed that there exists no chemistry peculiar to living things and that physical properties are possessed by the living and by the non-living as well, we have remaining the task of evaluating the differences. It is not implied that only similarities have been studied and never the differences of the two regions. Nevertheless, those differences which set apart a living thing from a non- living should be studied more extensively as such. I can not see how they can be investigated by physico-chemical methods in the sense of Loeb, that is, by resolution into ultimate particles, by methods suitable for pure compounds used in the chemist's laboratory, or by any other that does not maintain the integrity of the living state. Biology should develop its theories by a method of work adapted to the peculiarities of the living thing and therefore quite distinct from those used in pure physics and pure chemistry. This statement does not imply that we should discard entirely for biology the use of physical and chemical means. Surely, no one would set himself against the use of the microscope or any other most refined apparatus, and of reagents, drugs, dyes, etc., the common equipment in the study of the living thing. Biologists count, measure and weigh and seek to detect cause and effect. But whatever means we employ should be adapted to that particular level which the living thing occupies in the natural world. In an investigation which aims to explain the state of being alive, the first prerequisite is the appreciation of the limits which circumscribe this state. In the utilization of physico-chemical means, then, we need to recognize the extent to which we change the living state; and, if we go 15 THE BIOLOGY OF THE CELL SURFACE beyond it, we must realize this fact. For this reason, the most definite knowledge of the manifestations of the normal living state becomes necessary. Experiments in biology, then, fall into three categories: 1. Experiments on non-living systems. 2. Experiments on killed living systems. 3. Experiments on living systems. Number i includes experiments that furnish comparisons between living and non-living systems and that elucidate the result of cellular activity which expresses itself in secre- tion and excretion. Here belong physical models and artificial systems, as for example, semi-permeable mem- branes, suspensions of soap in water — used to imitate cleavage-patterns of the egg — colloidal solutions, passive iron wire — employed to illustrate nerve-conduction — etc. Also, here belong chemical analyses of substances that are produced by the living cell, as for example, hormones and digestive ferments and excretions, as urine, etc. Number 2 includes experiments on the chemical composi- tion of living matter, killed in the process of analysis. Though not experimental in the strict sense of the word, all histological and cytological studies on fixed tissues and cells are included in this category. When properly used, fixa- tion gives a faithful picture of the living and as such has great value for the study of it. Number 3 embraces experiments on the living system. These are of two kinds: those in which the normality remains unchanged and those in which it is altered. By means of the former, valuable data are collected by which indicia and criteria are established for supplementing and extending as well as interpreting observations and descrip- tions of vital processes such as respiration, etc. Such experiments are also of incalculable value for medicine. By the second type of experiments in this category we often succeed in making evident the roles of various factors 16 LIFE AND EXPERIMENT by comparing their behavior in the altered living with that in the normal. This review makes it at once clear that experimenting in biology requires not only knowledge of physics and chemistry but also, and in no less degree, that of the normal living organism, the fundamental object of biological investigation. The following discussion centers around experiments on eggs. However, it applies also to work on other types of cells. The investigator must possess familiarity with the bio- logical system whose investigation he undertakes. The chemist demands pure standardized materials for his work and the physicist is at pains to be sure that his apparatus always performs perfectly; neither is satisfied with experi- ments contaminated by sources of error that can be eliminated. Just because of the reason that biological systems are not to be compared to "chemically pure" sub- stances and that their performance is not always one hundred per cent, perfect, the condition of the living thing studied should be as fully known as possible. However, a chemist who will use only purest chemicals will often in biological work use cells or organisms in poor or even mori- bund condition; the physicist who in the physical labora- tory demands perfection in apparatus, frequently is content with whatever cell or organism given him for biological research, no matter how it behaves. Unfor- tunately, it is all too true of many biologists, even of many old-fashioned ones, that they investigate living systems which are not in optimum condition. In the haste to make experiments, many find no time to learn the optimum condition of the system under study. Some do not care and are content to report results that vary from day to day, while others would not be able to distinguish a really normal egg or other living structure from an abnormal one. Indeed, it would seem by the manner in which many 17 THE BIOLOGY OF THE CELL SURFACE treat the organisms on which they work, "material" as they call them, that they prefer their living systems in a debased and degraded condition. Surely, of all biologists the investigator who experiments on eggs needs to exercise most scrupulous care in order to be sure that the cells under investigation are in optimum condition. In the investigation of a system of organs, an organ or even a tissue, like nerve or muscle, although one can never be absolutely certain that all cells present are normal, the high number of cells that compose such a structure guarantees a high number of normally reacting cells and thus increases the probability of a normal response. In the case of eggs, one deals with structures which are single units, individuals that show each its own state of normality or abnormality. Furthermore, whilst it is true that out of each egg arise cell-complexes — tissues, organs, and organ-systems — these differ so much in degree of activity, in time of origin and in relation to each other in space, that the determination of the normality of the developing ^^^^ made up at a given stage of a number of cells equivalent to that in a given tissue with which com- parison is made, is more difficult than in this tissue. The developing egg is normal only if all these different com- plexes present are normal. But this necessity of careful investigation of the grade of normality in eggs has one important compensation which makes the study of eggs so fruitful for biology. The behavior in laboratory experi- ments of tissues, e.g., nerve, muscle, or tissues in culture, removed from an organism may not at all be the same as that in the intact organism. That countless investiga- tors have obtained the same laboratory results speaks on the one side for the excellence of laboratory procedures, on the other for the wonderful capacity of tissues removed from an animal's body to withstand experimental treat- i8 LIFE AND EXPERIMENT ment. Whilst the doubt as to the identity in behavior of excised and of intact tissues does not deny the value of studies on the former for laboratory exercises, it should be borne in mind that only rarely and with great difficulty can these tissues be experimented upon in situ (in their normal surroundings). There is doubt that results from experi- ments made on tissues in vitro (excised) could be obtained from such experiments made on tissues in situ. With the egg the situation is different. We can speak with more certainty concerning its normality for in many cases nature gives us the opportunity easily to study it 'S'n situ^ This advantage of a study in situ is also offered by many proto- zoan cells. The control of normality in them is even less difficult than in eggs, because they lack, even those with most complex life-history, the potential diversity of the egg-cell realized as development unfolds; the protozoan cell reduplicates itself only. This difference, however, though showing the greater difficulty in the study of eggs, indicates the greater possibilities of such study. Valuable as studies on Protozoa doubtless are for both biology and medicine, they lack direct bearing on the grand problem of biology, how out of a single egg arises the complex multi- cellular organism. Only in the egg and its development can we hope to trace to its source the pattern of structure, and to resolve into its motif the harmonious behavior which characterizes the many-celled animals. If the condition of the eggs is not taken into account, the results obtained by the use of sub-normal eggs in experi- ments may be due wholly or in part to the poor physiologi- cal condition of the eggs. Thus, the failure of sea-urchin's eggs that are freed of their jelly, fully to separate their vitelline membrane after fertilization, as they normally do, does not mean that the experimental removal of jelly renders membrane-separation impossible but only that the te/* *-"*- *\c3\ THE BIOLOGY OF THE CELL SURFACE eggs are in a bad condition brought about by the injurious action of the agent employed to remove the jelly. ^ The physiological condition of all eggs known to me can be impaired by exposure to low temperatures. Indeed, since low temperature (like high) is an experimental means, to experiment on eggs from animals which have been kept in the ice-chest in order to delay shedding, is equivalent to compounding experimental procedures whose effects may be complementary or antagonistic. Thus, I would never think of exposing eggs of Platyniereis, for example, which had been kept over night in the cold room (at 5°C.) to ultraviolet light because the low temperature alone gives the eflFect of polyploidy to obtain which I use the ultra- violet radiation.^ From a series of experiments made on the capacity of a sea-urchin's eggs to develop without spermatozoa, it was concluded that development can be caused by immersing the eggs in sea-water which had been charged with a sub- stance liberated by others of the same species. This con- clusion was unwarranted because, as I found, the results obtained are solely due to evaporation of the sea-water and not to the presence of substances originating from the eggs.^ Another series of experiments was used to prove that this subtance liberated by the eggs and held to be the cause of their development, had been isolated. But I found that sea-water treated by one or by all of the reagents employed for precipitating the alleged substance was if anything more effective for causing development than sea-water containing the egg-substance. Thus the develop- ment was due not to the isolated (precipitated) sub- ^ For discussion of this subject, see Just, ig2Sc. Derbes, 184"/ knew not only that jelly surrounds the sea-urchin's egg but also that its absence does not impair the egg's development. ~ Just, ig2()e. 2 Just, igzSa. 20 LIFE AND EXPERIMENT stance liberated hy the eggs, but to increased salinity of the sea-water.^ Few authors who quote Wilson's famous experiments on the eggs of the marine mollusc, Patella, I think, realize that Wilson never succeeded in obtaining normal fertilization. - It is not improbable that the results he obtained, upon which he based far-reaching conclusions which have been generally accepted, were in part due to the abnormality invoked by the artificial aid with which he induced fertiliza- tion. To induce cross-fertilization (fertilization of eggs with non-specific spermatozoa) often one has recourse to artificial aid, as changes in alkalinity or temperature of the sea-water. Such changes however bring about variations in the development of the egg (for instance of the sea- urchin) also if these develop from straight fertilization (fertilization of eggs with specific spermatozoa). This fact makes it clear that results obtained after cross-fertiliza- tion can not be ascribed to the influence of the non-specific spermatozoon or its chromosomes unless it is shown that the efTect differs from that obtained in straight fertilized eggs that were subjected to the same changes in the medium by which the cross-fertilization was induced. Since the experiments on cross-fertilization that have been made were not controlled in this way, they allow no conclusion as to the action of the foreign sperm-nucleus.^ Even under constant external conditions the sea-urchin's larval skeleton will vary, as most exact stud}' of normal straight fertilized eggs has shown."* Thus the extent to 1 Just, ig2gd. ~ Wilson, igo4. ^ It must therefore astonish us that Morgan (/gjj) not only accepts these experi?nents.on cross-fertilization but also in a zvide- sweeping statement categorically declares them to be proof for action of the genes on the cytoplasm. ■^ Tennent, igio. 21 THE BIOLOGY OF THE CELL SURFACE which eggs in optimum physiological condition vary must also be known in order that differences discovered can be truly attributed to the experimental means. These few examples show how careful we must be in drawing conclusions from experiments in biology. The highly irritable systems that we investigate demand a most exact control of the experimental factors. If only one of these is left aside in our evaluation of the obtained results, our conclusion may be wholly erroneous, especially if the factor induced a change at the beginning of the treatment. We must know whether we have before us normal or already changed, abnormal, eggs. We must control the effect of our means, even of those that are only aids, by following their effect singly before we use them together in one experi- mental setting. And above all must we be wholly familiar with the normal condition and normal development of the eggs we use, since only on this basis are we able to recognize abnormalities and thus the effect or effects of our experi- mental treatments. The simplest and most generally adopted criterion for the eggs' normality lies in estimating the number of them that develop; one hundred per cent, development means optimum condition. Eggs that are laid already fertilized, and especially those in brood pouches or in capsules almost always develop normally. Also where the eggs and spermatozoa are deposited and fertilization ensues in the laboratory as in nature, the chances for normal develop- ment are enhanced. It is with those eggs whose deposition is induced by the experimenter and which are artificially inseminated and especially with those that are removed from the ovaries that most difficulties are encountered. In many cases such eggs develop, if at all, only in small numbers. They are only useful for studies on development if the cause for the low numbers of development can be discovered and the percentage increased. There are cases, 22 LIFE AND EXPERIMENT however, where eggs removed from the animal yield one hundred per cent, development. Here it is necessary to know that the quality of development parallels that of eggs normally laid under natural conditions. By compar- ing the development of eggs in their normal environment, of those naturally shed in the laboratory and of those removed from the animal, one can decide concerning the normality of the last named. In rare cases, where always some immature eggs incap- able of development are present, one may never obtain large numbers of developing eggs, yet those that develop are normal. Similarly, at the end of a breeding season animals frequently give, among those eggs that develop perfectly, some that fail to develop because they have passed the time of optimum condition for fertilization. For such eggs we can not use the criterion of the percentage of development to detect their condition; they can only then be considered to be wholly normal, if their develop- ment is normal at every stage. Now the necessity of following the egg throughout its complete development can be obviated. I was able to establish definite criteria and simple physiological indicia of the optimum condition of eggs. These signs tell us within the first minutes after fertilization whether the eggs are in optimum condition and even indicate whether the eggs will be normal throughout their development. The signs inhere in the reactions following the mixing of eggs and spermatozoa and are in a measure as specific as the given gametes themselves. For any given egg, they appear differently when the egg is abnormal. For the eggs of a common sea-urchin, Arhacia, I found that their optimum condition, whether they are normally shed, induced to be shed by artificial means, or removed from the ovaries, can be determined within three minutes after insemination by the rate and quality of membrane-separation; by the 23 THE BIOLOGY OF THE CELL SURFACE rapidity with which unfertilized eggs separate fully their membranes while in distilled water; by the rate at which fertilized eggs form extra-ovates when in hypotonic sea- water; and by the failure of heavily inseminated eggs to show polyspermy. Whenever these initial reactions are qualitatively poor, the development of the eggs is below normal.^ These criteria I find hold for three other species of sea-urchins at Naples.- Also, for the egg of Nereis the quality of the striking ectoplasmic changes subsequent to fertilization constitutes an index of the condition of the egg and the quality of its later development.^ Further I found criteria which in later stages of develop- ment indicate whether this will be normal in its whole course. For example, in eggs of Nereis and Platynereis, the behavior of the oil drops is a reliable index of normality; one may be sure that swimming forms which possess one oil-drop and only one in each of the four gut cells can develop through metamorphosis to the adult stage and that no other will.^ Doubtless other criteria can be found for every stage of the development. Every one such will prove valuable in extending our knowledge as to what is a good egg. In all cases however, where such criteria are not known, the whole course of development must be always followed before it can be said that development is normal. It is clear that the condition of eggs largely depends upon that of the animals from which they come. Eggs normally shed from animals in a good state are preferable to those shed by weak animals and by^ animals in abnor- mal conditions. In every experiment comparison with and control by the normal, untreated cell or organism is obviously absolutely necessary. The quality of the ^ Just, ip28c. - Just, ig2gc. •' Just, 1915a. 4 Just, I922g. 24 LIFE AND EXPERIMENT investigated system determines the experimental results to such a high degree that the value of the investigation depends upon recognizing and appreciating this quality of the object. The most exact knowledge of the normal form and form- changes of the living thing to be investigated is thus the prerequisite for present-day attack of biological problems. On the strengthening of our knowledge of these rests all progress of modern biological research, no matter how grandly physical, chemical, or mathematical it is. For a long time to come biology will need accurate description and exact observation. The necessity for confirming the classic and exact studies still remains; it is imperative that these be extended. The demand for filling in gaps persists. Where minute details are wanting, they must be supplied. Wherever uncertainty or doubt obtrudes concerning a descriptive datum, this should as far as possible be removed. However much we desire quantitative instead of qualitative studies in biology, however much more we estimate elaborate experimental studies involving knowledge and skill in the use of physics and chemistry and mathematics, however much we yearn to place biology in the same category with the more exact sciences, we can not abandon purely descriptive work. I Ado not mean, I repeat, that biology stands irreconcilably apart from the other natural sciences. But I see no omen to indicate that all biological phenomena are capable of quantitative treatment.^ By chance to-morrow or it may be in the very instant of this writing by some great discovery made in total ignorance of the morphological substratum of biology, someone might be able to appreciate the secret of life in its entirety. But it is just as likely that this biological millcnium may never come. And I for my ^ Cf. Mellor, ^922^ on the use of mathematics in science. 25 THE BIOLOGY OF THE CELL SURFACE part believe that fully to embrace this faith in a chance discovery would anaesthetize activity and the will to seek, while life lasts, the mystery of life. That we are far from having completely exhausted the possibilities of description in biology is clearly shown by the fact that no one of the grand phenomena treated in this book has been adequately defined by description that fulfils our demands for exactness. My own observations having early taught me how rapidly changes ensue in the living &g^, I have studied stages in development not of minutes' but of seconds' duration. Microscopy by extending the range of our vision increases our powers of observation. But even if there should be revealed to us the ultimate space-pattern, there would still remain the problem of the changes of this pattern in time; a gap between the beginning and the end of an event would persist. Just as with the aid of the various kinds of microscopes^ we uncover the minutest space, so must we register most minute changes in time: we must clock the fleeting changes whose sum total is the living thing. Studies of forms and form-changes must continue as long as there are organisms and their processes, eggs and their development, still unexplored. Since not all work ever done in this field is wholly dependable and perfect, there is all the more reason why the modern student experimenting on eggs must acquaint himself directly with the pattern which he aims to explain. Naturally, In that earlier period in the history of the study of eggs the new findings, as fertilization, chromosome behavior, etc., excited and stimu- lated the discoverers to exuberant expressions that were often In error because they came from work which though honestly well done was not always sufficient to justify the conclusions drawn from it. Work like that of Boverl which ^ Note the recently inverited electron-super microscope. 26 LIFE AND EXPERIMENT has given a great impetus to biology, but which nevertheless does not satisfy our demand for exactness and completeness, should most certainly be repeated on a more extensive scale. Even to-day exactness is often not carried as far as we should demand. Above I gave examples of incompletely controlled experimental work. Where living cells are treated statistically, the larger the number used, the more valuable are the conclusions drawn. Few workers would care to publish results like some of Morgan's who in eleven experiments had i, lo, 4, 8, 8, 3, 3, i, 2, 2, 4 eggs, and in other experiments had no eggs, yet spoke of percentage of development.^ Certainly, modern technique offers its advantageous apparatus also to biology for increasing exactness in observation and experiment. These modern achievements on the other hand have served in devaluating morphological knowledge, and impressed by technical progress many an investigator regards experimentation as a virtue in itself, a reward of its own. Experiment for experiment's sake, ever a dangerous philosophy, becomes exceedingly baneful for biology. True, through It valuable knowledge may be gained for mapping out unknown terrain. On the other hand, by it the accumulation of data may become bewildering because it relates to topographical details impossible of reduction on a scale of value for other explorers. Moreover, such refined plotting of points often has no relation to the whole field — indeed, often the field as such is lost sight of and only unrelated minutiae remain. The main purpose of an experiment in biology should be the explanation of the naturally occurring phenomena. Here we encounter a difficulty not met with in physical science. ^ Morgan, 190^. 27 THE BIOLOGY OF THE CELL SURFACE Animal cells are ready-made. We do not devise them; nor can we have them according to specifications which we ourselves set up. If, as some do, we regard living cells as machines, we appreciate that they are ready-made; we do not make them as physicists make their apparatus to prove a theory or test an hypothesis. Still less do we know them and their variables. We do not use them to prove theories; rather, either we elaborate theories from our observations on them, or, having set up a theory, attempt to establish it by experiment. In either case we seek to know the "machine" and not by machine-making to devise hypothe- sis or establish theory. A cell is never a tool. Nor is it an instrument on which to whet one's physics and chemistry. Living matter Is never an excuse and living phenomenon never an oppor- tunity for the display of the investigator's physico-chemical knowledge. If we use an apparatus in order to determine oxidation in an inanimate system or devise a sensitive instrument to measure light, the apparatus or the instru- ment is a tool; but if one determines oxidation in a cell, the oxidation-determination is the tool; if one measures light produced by a cell, the measurement is the tool. In neither case Is the cell a tool unless one frankly wants to compare its sensitivity for oxidation or light production with that of a man-made machine which of course is an entirely different reason from that of investigating the properties named as peculiarities of a living thing. The physicist devises a tool with which to measure a given change or to test an hypothesis; the biologist tries to mea- sure changes in the cell — and the measurements are then the tool by which he evaluates the properties of the cell or tests his postulates of what these properties are. I think that we can agree that in the experimental study of the egg's development the aim is the analysis of the stages found in nature through which the egg passes to 28 LIFE AND EXPERIMENT attain the normal adult condition. By experiment we attempt to ascertain the meaning of normally occurring stages which are co-existing and successive, co-ordinate and subordinate. We seek to unravel the tangle of the pro- cesses or, from another point of view, to stem the rush of developmental events in order to determine what in them is cause and what effect. The picture that the normal developmental process gives us must we trace In its every line and shadow and by experiment aim to elucidate their meaning. Hence, the method for the study of living systems is this : to extend to the utmost a purely biological attack — that is, to know qualitatively every process; to mark its beginning and its end and in Its duration to map out most closely set phases; to chart its course. This appreciation of the normal life-processes will set the goal which we strive to achieve by means of experimental attack. No matter how far the experimental methods change normal condition and behavior, always should results obtained with them have bearing on the normal. The greater the extent of the change, the more surely must we realize that it Is a change. Conclusions drawn from greatly altered states concerning the normal need most careful scrutiny. Death-changes induced by experiment should be appreciated as such and be considered as revealing the living condition, which they extinguish. In no other way except by extinguishing it. Those experiments which alter a normal process least have to-day especially great value in the study of the egg and its development. Few investigators nowadays, I think, subscribe to the naive but seriously meant compari- son once made by an eminent authority in biology, namely, that the experimenter on an egg seeks to know its develop- ment by wrecking it, as one wrecks a train for understand- ing its mechanism; he might also have said, as a young child breaks up a watch to see the wheels go around. The days 29 THE BIOLOGY OF THE CELL SURFACE of experimental embryology as a punitive expedition against the egg, let us hope, have passed. Instead comes the time of nice, exact, carefully graded treatment, wherever possible reversible in its action, for the purpose of analyzing develop- ment in ever more closely set stages, after this has been mapped out in the perfectly normal egg with meticulous care and scrupulous cleanliness. The most minute space- time organization of the living system makes the instrument on whose strings play the processes by action and inter- action. By experiment we here slightly exaggerate, there lightly fret the tones out of which the harmony of the living state arises. 30 3 The Protoplas/fiic Systef?i ey^S ALREADY STATED, THE PROTOPLASMIC SYSTEM DOES not always reveal itself as consisting of one nucleus and surrounding cytoplasmic mass, though this type is very widespread in occurrence. There are living things in which a discrete nucleus can not be discerned and on the other side we know formations which show many nuclei embedded in one continuous cytoplasmic mass. Thus, we may distin- guish three types of the protoplasmic system according to the extent to which the nuclear substance is differentiated. A classification of the types of the protoplasmic system could also be made on the basis of the degree of the differ- entiation of the ectoplasm. But inasmuch as nuclear differentiation has up to now been more thoroughly studied, it is chosen here as the basis for a classification. Some bacteria and blue-green algae have been reported as non-nuclear organisms. In them nuclear substance may, however, be deposited as granules so finely subdivided that they escape detection.^ Indeed, in some cases whilst no formed nuclei or nuclear areas could be located, the presence ^ The presence of nucleic acid in bacteria has been reported. Schaffer, Folkoff and S. Bayne-Jones {1Q22) extracted from ^00 grams of dehydrated bacteria grown on a syyithetic medium free from purine and pyrimidine compounds {constituents of nucleic acid) a non-hygroscopic^ protein-free^ powder which they consider a nucleic acid, though it contained no pentose, the sugar of plant nucleic acid. In the opinion of W. Jones and Folkoff {ig22) the substance is a nucleic acid. 31 THE BIOLOGY OF THE CELL SURFACE of granules has been demonstrated, which respond to reagents as does chromatin (a substance found in nuclei). In others, by means of tests with dyes, chemical reagents, etc., investigators have demonstrated areas which respond as do nuclei in larger cells. Such systems are said to possess diffuse nuclei. So much is clear: there are living forms which show no discrete nuclei. This fact indicates that in the organiza- tion of protoplasm the presence of a discrete nucleus is not indispensable. The blue-green algae — bacteria are very closely related to them — evoke special interest because they probably represent the first forms of plant-life that arose on the earth. If it be established that there exist non-nucleated blue-green algae, this might mean that in the emergence of life, nucleus and cytoplasm were undifferentiated and only subsequently in the course of evolution became separated. Doubtless there exists living matter of such minute size that it can not be seen with the highest powers of the microscope. Whether or not such protoplasmic systems possess the organization exhibited by cells seen under the microscope remains to be learned. In a discussion of the visible form of organisms it would be futile to include invisible forms. Nor can ultra-filterable viruses be included. In addition to the fact that they are of submicro- scopic size is the question whether they be living things or only the product of living things. If they be living, they may despite their minute size possess thenucleo-cytoplasmic organization of visible cells; or, lacking such they may prove to be the most elementary protoplasmic system — ■ protoplasmic ground-substance without clearly differen- tiated regions. Many organisms consist of a mass of cytoplasm contain- ing two or more nuclei. These may be low forms of life. Protozoa, like the slime-mould or the multi-nucleated 32 THE PROTOPLASMIC SYSTEM Opalina. Also among the higher animals certain sheets of protoplasm as the binding or sustaining (connective) tissue or the heart muscles of vertebrates show Innumerable nuclei throughout their extent without Interposed cell- boundaries. Such multi-nuclear protoplasmic systems are called syncytia. The existence of syncytial protoplasmic systems has given rise to theories opposed to the so-called cell-theory which postulates that the cell Is the unit of structure and of function. Rohde's vlews^ concerning the preeminence of syncytia may be dismissed because they have little founda- tion In fact. Similarly, Studnicka's arguments- as to the living nature of cellular formations, fibres, etc., which come to be extra-cellular, do not warrant discussion here. The argument set up by Whitman^ and by Sedgwick,'^ that the cell-theory of development is inadequate, out of which have grown the so-called organlsmal and organlsm- as-a-whole conceptions merits more attention although It too has slight basis In fact and Is Indeed almost wholly academic. These conceptions originated as protests against the extremist's point of view that the individual cell Is the end-all and be-all of life even In complex organisms; that through exact knowledge of single cells one could win an explanation of the organism as a unit. To-day we appre- ciate the fact that a mere agglomerate of cells equal In number to those that constitute an organism is not an organism. That organisms are units and act as such, be they composed of single cells, as Protozoa, or of myriads of cells, as the higher animals, no one will deny. The organlsmal or organlsm-as-a-whole conceptions have ren- ^ Rohde, 192J. '' Studnicka, ^934- ^ Whitman, iSgj. ^ Sedgzvick, i8q2. 33 THE BIOLOGY OF THE CELL SURFACE dered some service therefore in emphasizing this fact so universally accepted. Enough is known concerning the diiTerence in behavior between cells when en rapport with others that make up the intact organism and those having been isolated thereform, concerning the development of eggs and the significance of the orderly sequence in time and in space when specific embryonic cells are set off and concerning the pathological growths, as tumors, to warrant the clear conclusion that cells alone and as members of a unit-system exhibit different behaviors. However, no suffi- cient reason exists for assigning to cellular differentiation, the integration of cell with cell, or the specificity of organ- isms which resides only in cells and their products — all well accepted biological truisms — the new terms of organismal or organism-as-a-whole.^ Moreover, the principle of unity — the expression of integration, differentiation and of specificity both in form and behavior — which distinguishes an organism from a mere mass of like cells, ought not be elevated to the position of an abstract principle divorced from the concrete physical basis on which living things are organized. - An organism possessing no formed nucleus represents one, and a mass of cytoplasm containing many nuclei represents the other extreme type of protoplasmic systems. If we assume that nuclear structure (and therefore from it the discrete nucleus) is a derivative of the ground- substance, the various types of protoplasmic systems can be reconciled: the single nucleus of a simple cell is then the compounding of nuclear bodies evident in non-nuclear ^ I71 view of the claims of priority pressed by several writers it may he worth-while to note that Descartes {1662) conceived the organism as a whole. See Delage^s {i8g^) discussion of " organicisme.'' 2 See Woodger, i<p2g, 1930 and igii^for recent discussion of the organism-as-a-whole point of view. 34 THE PROTOPLASMIC SYSTEM forms like bacteria; the compounding of nuclei themselves (a result easily obtained experimentally in normally mono- nuclear cells) leads to the multi-nucleate, syncytial or polyenergid condition. To a consideration of that proto- plasmic system which is most widespread in occurrence and which reveals itself as a simple membrane-enclosed cell with a single nucleus, I now turn. For most organisms in both the animal and the plant kingdom this cell is the basic structural unit, no matter how complex or how large such an animal or plant may be. A whale and a giant red-wood tree as well as minute forms like a vinegar eel and a thread of green alga are patterns of cells. Animals and plants also exist as single cells, which within their boundary complete their life-cycles, exhibiting the vital activities shown by the more complexly organized individuals. These activities are perfect; diges- tion, respiration, conduction, contraction, reproduction, in a unicellular organism are as complete, though not so complex, as in man. These individuals In their organiza- tion and behavior reveal the single cell as an Independent unit. At first one Is amazed that microscopic creatures like the slipper-animalcule and the diatom, a plant of micro- scopic size, can carry on the functions of more highly organized forms of life; they seem at first to be imitations of the latter. But observation teaches that the millions of cells which make up the body of a multicellular animal or plant have lost in capacity: no one cell In them can perform all the functions to the same degree that a single-celled Individual can carry out. This loss of capacity is compensated for by an emphasis on some one function, as shown, for example, In cells In the human body. In It some parts, which together make up the nervous system, take over the business of conduction which is developed to a high degree. Other parts, the muscles, emphasize contraction; still others, respiration, 3S THE BIOLOGY OF THE CELL SURFACE digestion, excretion, reproduction. Thus we" speak of the various systems — nervous, muscular, respiratory, etc. Each system is made up of organs; each organ is a combi- nation of different tissues; each tissue is a collection of cells of the same origin and usually of the same form. Out of a single cell, the egg, emerges all this complexity of organization found in the adult human being or in most other multicellular organisms, animal or plant. The human eg^ during its development forms tissues, these aggregate to form organs, and the organs are united into systems. In an animal's origin, as well as in its definite adult struc- ture, therefore, the cell is the unit of organization. This fact, together with that of the existence of unicellular animals and plants, shows that a large part of the world of living things is organized on the basis of cell-structure. The history of the multicellular organism as it develops from the egg, a single cell, to the adult is ver}' much like a synopsis of the history of the whole world of multicellular organisms; this has most probably evolved from a single- cell ancestor. Thus biologists classify animals and plants beginning with the single-cell individuals and ascend- ing through grades of increasing complexity. Among animals, for example, the Protozoa stand first; next are the sponges, animals possessed of tissues only; above the sponges stand animals whose tissues are organized into organs and finally come animals with systems of organs. From the point of view of the history, both of the individual multicellular organism and of the world of multicellular organisms, the cell is the unit of the state of being alive. That this cell must be regarded as a unit becomes further evident if we consider the fact that nucleus without cyto- plasm or cytoplasm without nucleus is incapable of living. It is true that pieces of cytoplasm devoid of nuclei, as the red blood corpuscles of mammals, occur normally and live for a time; but these are specialized structures incapable of 36 THE PROTOPLASMIC SYSTEM the full complement of living functions. One can cut off pieces of a single-cell animal; if these pieces be without nuclei they die, whereas a piece containing the nucleus or pieces containing portions of the nucleus are capable of growth to the full size of the normal creature. In turn, nuclei deprived of their cytoplasm do not live. The unit of living matter in these cases is thus shown to reside in the nucleo-cytoplasmic organization. We therefore adopt the point of view that the proto- plasmic system is the unit of life. For the majority of animals and for all their eggs the protoplasmic system reveals itself as comprised of a single nucleus enclosed by cytoplasm — ground-substance containing suspended mat- ter, the cytoplasmic inclusions — and the whole enclosed by a membrane. Such cells exhibit variations in several directions. Cells may show extreme size-variations. They range in size from one micron in length, discernible only under the highest microscopical magnification, to the sporozoon parasite found in the digestive tract of Crustacea which measures i6 mm. in length, or to the unfertilized egg of the ostrich, 105 mm. in diameter, or that of a shark, 220 mm. in diameter, or to the nerve-cells in the human spinal cord, which may be one meter in length. Organization of the cell thus does not depend upon size. We may imagine that the skein of life is greatly contracted in some while it is more diffuse in others. The maintenance of the cell-size that is characteristic of a species still remains an unsettled problem. Maintenance of a definite and specific size is a revelation of the self-regulative capacity of the proto- plasmic system. Cells also show most varied shapes and forms; some are almost perfect geometrical figures — spheres, cylinders, cones; others are irregular or of forms not easily reducible to conventional geometrical figures. Often when in groups 37 THE BIOLOGY OF THE CELL SURFACE they show the effect of compression; thus cells which, were they free, would be spheres, become polyhedral. The varied shapes of cells depend upon many diverse factors in addition to compression, such as presence of a supporting frame-work, as in Foraminifera, the greater or lesser fluidity of the cytoplasm, the structure of the limiting surface, and most of all the little understood intrinsic structure of the cell itself. Cells, like all matter, are chemical in composition and display physical properties. Their form, structure, and morphology, however varied, are molecular in make-up. Hence, as we have seen, there is, in this respect, no dif- ference between living and non-living matter. All the protoplasms known to us have in common a certain basic chemical structure. Since living things are part of the natural world it is not astonishing that they are com- posed chiefly of those chemical elements which are most frequently found in the world of non-living things: Carbon (C), nitrogen (N), oxygen (O), hydrogen (H), sulphur (S), phosphorus (P), iron (Fe), sodium (Na), potassium (K), calcium (Ca), magnesium (Mg) and chlorine (CI). In some cells are found other elements as lithium, barium and strontium; copper and zinc; fluorine, arsenic, bromine and iodine. Thus the difference between non-living and living systems does not reside in the basic elements of which they are composed. A difference obtains in the compounds into which these elements enter. The elements, C, H, N, O, combined build up the protein molecules; out of C, O, H carbohydrates are formed; and C, O, H makes up most lipins; other lipins in addition contain N or P. These are the "organic" compounds of the living substance and exist in nature only in living sub- stance; non-living things do not contain proteins or the oils found in animals and plants; sugars and starches (carbohvdrates) occur naturally only in animal and plant 3S THE PROTOPLASMIC SYSTEM tissues. Thus, not the elements as such characterize the structure of living matter but the peculiar combination of elements which constitute the protein, the lipin and the carbohydrate molecules of living matter. These compounds are suspended and dissolved in an aqueous medium containing electrolytes as Na, K, Ca,Mg, CI, etc., with oxygen and carbon-dioxide. The whole mass of protoplasm — organic compounds, electrolytes, water and gases — it seems, maintains a fairly constant slightly alkaline reaction. Each of the three organic compounds is built up of simpler molecules strung together: the proteins are strings of amino acids formed in some such manner as the following: C4H9.CH(NH2).CO.NH.CH2.CO.NH.CHo.COOH which is a poly-amino-acid or tripeptide of leucin: CH3CH.CH2.CH(NH2)COOH and two molecules of glycin: CH3CH2.NH2.COOH The more complex proteins show comparable linkage.^ The lipins, as shown by the chemical structure of a simple fat, are combinations of three molecules of fatty acid and one of glycerol, as : CH3.CH2.CH2.COOH + CH2.OH CH3.CH2.CH2.COOH + CHOH CH3.CH2.CH2.COOH + CH2.OH with the loss of three molecules of water. Monosaccharides (simple sugars), disaccharides (second- ary sugars) and polysaccharides (starches and the like) ^ With the aid of the ultra-centrifuge and by means of x-ray- spectroscopy valuable information is to-day being gathered con- cerning the protein-molecule . 39 THE BIOLOGY OF THE CELL SURFACE comprise the carbohydrates. Simple sugars Hke dextrose have the formula, C6.H12.O6, secondary sugars 2(C6Hi206) — HoO, or CioHooO]], whilst more complex carbohydrates are only more molecules of simple sugars piled up. Whereas plants because of their photosynthetic power can in sunlight or in artificial light produce sugars from COo and HoO in the presence of the green substance, chlorophyl, and from sugar thus formed synthesize lipins and proteins when N is present, most animals ingest food as protein, carbohydrate and lipin. Before utilization by the animal organism, these compounds must be broken down, i.e., digested, into their component parts: proteins to amino acids, carbohydrates to dextrose and laevulose and lipins to fatty acids and glycerol. These fractions are built up again by the organism into the more complex molecules. Special significance is attached to the synthesis of amino acids, for the proteins thus formed by the organism are peculiar to it and not to the organism which furnished them; thus herbivorous animals, cattle for example, convert the plant proteins of their food into proteins peculiar to beef. The break-down of these compounds is accomplished by enzymes, chemical bodies which act much as catalysts — i.e., by accelerating chemical changes. Enzymes show a high degree of specificity. For proteins, there exist the enzymes like pepsin found in the human stomach and trypsinogen, produced by the pancreas; for the starches is the enzyme, amylase; for the sugars, sucrose, maltose, lactose, the enzymes are sucrase, maltase, and lactase respectively; for the lipins, lipase. The break-down is one in which water is used, a so-called hydrolytic cleavage. Enzymes also synthesize the end-products of digestion into proteins, carbohydrates and lipins in which case water is lost. Some enzyme-reactions are reversible: thus, the reaction lipin plus lipase = fatty acid plus glycerol, or 40 THE PROTOPLASMIC SYSTEM fatty acid plus lipase = lipin — depending upon the amount of water present.^ The chemical make-up of the three organic constituents of protoplasm renders easy their conversion into different compounds; the chains are broken up and when the links are reformed they show a different relation to each other. The proteins have the greatest lability in this respect. From the 21 amino acids which compose them it is theoreti- cally possible to derive millions of different kinds of proteins . This is one reason why the proteins are regarded as responsible for specificity; all species now in the world can doubtless be distinguished from each other on the basis of the chemical constitution of their proteins. But it should be borne in mind that protoplasm never exists entirely free of carbohydrates and lipins; that from sugars plants make protein; that sugar as a pentose or as hexose is an essential constituent of nucleic acid, that lipins are the greatest source of energy and are the great binding sub- stance; that lipins if not present as such in the nucleus probably give rise to the phosphatized portion of the chemical substance, nucleo-protein, found in the nucleus; and finally, that much of the protein in cells does not exist as simple protein but as a conjugant with lipins or lipin- derived phosphate. Protoplasm is not a compound but a complex of compounds of these three organic constituents together with other constituents, water, gases, salts. Water is the most abundant compound in protoplasm. Roughly, two-thirds of an animal's body is water; in some cases it may amount to more than 90 per cent, of the body weight. Even water-poor cells, as the enamel of the teeth and spermatozoa, still possess a great deal of water. Active protoplasm both liberates and utilizes COo and Oo. Dis- ^ See Bradley and others. 41 THE BIOLOGY OF THE CELL SURFACE solved in the water are several inorganic salts, chiefly chlorides, carbonates, phosphates of sodium, potassium, calcium, etc. The researches on the analysis of carbohydrates and on the synthesis of sugar, on the analysis of proteins (espe- cially nucleo-proteins) stand in the fore-front of bio- chemical achievements. And yet, there remains much to be learned of the chemical make-up of the cell. Farther and more refined analysis may furnish a clue to a better understanding of living matter as a complex of compounds. Up to now chemical analysis tells us only what is found in the living thing after it is killed. The very nature of chemical analysis demands isolation of the protoplasmic components as separate chemical entities and thereby sets up an obstacle in the search for the chemical constitution of the living state. To know even with absolute exactness the chemistry of each compound in the once living cell does not guarantee that we know how these are organized in life. Moreover, living organization is dynamic whereas the application of chemical analysis by necessity demands destruction of the very space-time structure which Is the changing organization characteristic of life. No single datum now at hand warrants the assumption that the chemistry of the cell In the chemist's test tube Is that of the living cell. Indeed, the evidence points to the contrary. These shortcomings of chemical analysis of the living thing have served to focus attention upon its physical attributes. Many biologists, therefore, seek to determine the physical diagnostics of non-living and living on the one hand, and of the living before and after It Is killed, on the other. The presence of water In large amount confers upon protoplasm certain physical properties; similarly, the remaining substances entering Into the composition of the cell are responsible for other such properties. Recently, 42 THE PROTOPLASMIC SYSTEM a great deal of attention has been given to the physical properties of the protoplasmic system. A new and flourish- ing branch of biology, bio-physics, stresses this aspect of the study of life. Some of the more general of these physical properties may be mentioned. Protoplasm is a liquid capable of flow. This fluidity can be demonstrated in several ways. First, direct observation shows that in many cells the cytoplasm streams.^ In Amoebae, in many egg-cells, etc., currents can be discerned. Cells which fail to reveal the presence of such currents often show the cytoplasmic granules in intense Brownian movement. Again the fact that in many cells it is possible by very slight centrifugal force to shift and to separate the cytoplasmic inclusions according to their specific weights, demonstrates the liquid nature of proto- plasm. Also, one may compress eggs, draw them out into long strands or otherwise treat them as if they were elastic bags of water. ^ The cell-liquid and the various formed bodies suspended in it reveal that protoplasm is both a solution and a suspen- sion, somewhat like milk, for example, which is a solution of water, electrolytes, proteins, sugar, and lipins in which globules are suspended. Protoplasm being largely made up of protein and complex carbohydrates in the colloidal state, is itself in this state. A colloid (literally glue- like^) is matter in the state which can not or can only with difficulty pass through an animal membrane; whereas crystalloids, like cane sugar and common salt, can very readily pass through such membranes. Protoplasm exhib- ^ Cf. Goette, iS/^, who not only very clearly observed proto- plasmic streaming in an egg but also proffered an interesting hypothesis as to its significance. 2 Just, igzSa. ^ Armstrong, 192^, p. 6^6, points out the bad usage of the term, colloid. 43 THE BIOLOGY OF THE CELL SURFACE its many properties characteristic of this state of matter. This is not to say that the colloid state is peculiar to organic compounds, such as proteins and carbohydrates; the most thoroughly investigated colloids are those of inorganic substances. It is to be emphasized that the term, colloid, applies to a state or condition only and not to a kind of matter different from crystalloid. Since we know that protoplasm is in the colloid state, it should not astonish us that it behaves as other matter in this state. ^ The specific gravity of the total of the cell-contents is usually greater than that of water. But where much fat is present, as in some fish-eggs, the specific gravity of the cell is lower than that of sea-water, as shown by the fact that such eggs are found floating on the surface of the sea. In some eggs the specific gravity increases after fertiliza- tion, a change undoubtedly due to loss of water. The refractive index of a cell is a physical property deserving attention. It depends upon the ground-sub- stance and the cytoplasmic inclusions. With the state of aggregation of these latter, the shape and the water-content of the cell, the refractive index of the cell varies. The influence of these factors is shown during the process of cell-division when the cell's refractive index changes with the dispersion of the inclusions, loss of water and change of form of the cell. Another physical property of the protoplasmic system much studied in recent years is its viscosity. The signifi- cance attached to such studies has, in my judgment, been grossly exaggerated. Nothing up to now indicates that viscosity has the importance for the state of being alive assigned it by the various investigators who, with different methods, and even with the same method, obtain widely diverging results and announce conflicting opinions. The ^ See Svedberg, /p2j, and Heilbrunn, 1^28. 44 THE PROTOPLASMIC SYSTEM problem of protoplasmic viscosity, with special reference to animal eggs, can be dismissed in a few words. The statement made above that the cell contains a high percentage of water, does not imply that the cell-contents flow as freely as water or that they always maintain the same degree of flow. Before the present vogue of measur- ing the viscosity of protoplasm it was shown that the cell- contents have a higher viscosity than water and that this viscosity varies from time to time in a particular cell and differs in different cells. One thinks at once of correlating viscosity with the water-content of a cell: the more water, the less viscosity and vice-versa. To an extent this is true, but the situation is not so simple. Take the case of eggs actively going through the process of division. During each cleavage-cycle, substances move back and forth between nucleus and cytoplasm, between cytoplasm and cytoplasmic inclusions and between egg and external medium. Only in the case of the last named exchange, obviously, does the cell lose water; in the first and second, water moves from place to place within the protoplasmic system. Now since, as the older observations have abundantly shown, changes in viscosity parallel the cell's rhythmical activity in division, it becomes necessary in measuring viscosity-changes to appreciate the fact that water does not shift between nucleus (and structures associated with it) and cytoplasm only; one must also recognize the shift between cytoplasmic inclusions and cytoplasm, especially if one estimates the change in vis- cosity on the basis of the movement of these inclusions, for example, by means of centrifugal force. Since yolk- spheres undergo physical changes during cell-division, one can not in measurements of protoplasmic viscosity assume them as unchanging in deriving conclusions concerning the viscosity of the cytoplasm. Also, in experimentally treated eggs, one can only draw conclusions as to the effect of the 45 THE BIOLOGY OF THE CELL SURFACE means if one knows definitely that this is on the cytoplasm alone, or on one or another of the inclusions or on the whole of the cell-contents. Finally, the effect of experimental treatment should be known to be reversible, and coagula- tive death-changes should not be confused with what is denominated as the normal viscosity-changes in normal and viable cells. Until the viscosity studies have been refined, changes in this physical property cannot be regarded as an infallible sign of the state of being alive. A leading characteristic of biological researches of the last twenty-five years lies in the emphasis which came to be placed upon the investigation of the physical properties of protoplasm — an emphasis so great that it has virtually created another department in an already heavily depart- mentalized science. Everywhere nowadays bio-physics, the physical chemistry of the cell, and the colloid chemistry of protoplasm are in evidence. Many an interesting new datum concerning electrical conductivity, hydrogen-ion- concentration, temperature-coefficients, etc., has been accumulated, whilst the older conceptions of osmotic pres- sure, surface tension and the like have been refined by more exact mathematical treatment. If from all these studies far less has come than had been hoped, one benefit of them deserves emphasis. No matter how refined a study on a physical property of the living substance may be, its value for the understanding of vital activities is determined by its relation to these vital activities and by its correlation with known form-changes and normal processes in viable cells. Modern studies in bio-physics and the like have served to indicate anew how necessary is adequate knowl- edge of the visible structure and the form-changes of cells, and how much we still need to extend this knowledge. What is called the morphology of a cell — its visible form and its visible form-changes — still remains the basis of biological investigation both for its own sake and as the 46 THE PROTOPLASMIC SYSTEM basis of an attack by bio-physicists, bio-colloid-chemists, bio-physical chemists, by which may possibly come an elucidation of the problem of vital manifestations. I therefore turn to a description of the cell. Under low power of the microscope, a living unfertilized egg of the sea-worm, Platynereis, mounted in a few drops of sea-water, at first glance appears as a greenish-yellow sphere everywhere crowded with smaller spheres except near the centre and at the periphery beneath the well marked egg-membrane; the whole presents a pebbled or shagreen effect. A second glance, especially if the egg is brought into focus so that one obtains an optical section of it, gives clearly the regions first noted. The large clear area near the center is the nucleus, sharply set off from the remainder of the cell by its transparency and apparent homogeneity. Outside of it, closely crowded against the nuclear boundary are innumerable spherules with some twenty or more refringent globules (oil drops) interspersed. Among these spherules and globules minute bright granules are scattered. Under higher magnification still smaller granules may be discerned. Spherules, globules and granules lie in the endoplasm or inner cytoplasmic region. Beyond it is a clear band, the ectoplasm, or outer region of the cytoplasm. In this egg the ectoplasm is crossed by fine radial lines; these appear to reach the egg-membrane, called the vitelline membrane, though actually they end in the plasma-membrane which is difficult to discern and which lies underneath the vitelline membrane. If we fix this egg quickly with some chemical reagent which preserves it in a state closely resembling the living, and cut it up into thin slices, we easily discern structures seen with more difficulty in the living cell. Figure i is of a stained slice from such a fixed egg. In the nucleus, granules of various sizes and two chromosomes are shown. These bodies lie In a faintly granular grayish-blue field 47 THE BIOLOGY OF THE CELL SURFACE enclosed by the nuclear membrane. The cytoplasm Is sharply differentiated Into two regions: the endoplasm, crowded with yolk spheres, oil drops and granules, the smaller of which are mitochondria; and the ectoplasm with Its radially disposed lines which extend to the plasma- / Fig. I. — Section of an unfertilized egg of Platynereis megalops. Surrounding the large nucleus, germinal vesicle, is the cytoplasm, which is clearly marked off into two zones, the endoplasm and the ectoplasm. membrane beneath the vitelline membrane. The q^^ of Arbacia, a sea urchin, Is similar to that of Platynereis except that the whole egg Including the nucleus and the endoplasmic bodies Is smaller, the ectoplasm a mere line, and the vitelline membrane an extremelv fine film. In 48 THE PROTOPLASMIC SYSTEM all cells nucleo-cytoplasmic organization is visible and ecto-endoplasmic differentiation is expressed. Nuclei may vary in size, but their size is generally constant for a given species of cell, though this varies during the process by which the cell reduplicates itself. In eggs it also varies during the end-stages of ripening, as will be shown later. The nucleus also shows diversity in form. Nuclei though most frequently spherical or oval or tending toward these two forms, may be quite irregular as those found in white cells of human blood, for example. The amoeboid, irreg- ularly form-changing nuclei in the spinning gland cells of butterflies have often been described. Again, during the process of division an otherwise spherical or oval nucleus may be very irregular due to incomplete or slow fusion of the separate chromosomes into one nucleus. This is clearly shown in nuclei of some eggs, as those of the thread worm, Ascaris, the water-flea, Cyclops. In these the nucleus may be regarded as a composite of many smaller nuclei, each containing one chromosome. Although this condition is not met with in all animal cells, it may be regarded as a fundamental condition of nuclear structure. That the chromosomes are the best known structures in the cell is due in part to the fact that they can be so easily studied because of their affinity for colors, which gave them their name. In the living cell^ they often appear as refringent drops of the same shape and approximately the same size as when fixed and artificially stained with one or another dye. A constant number of chromosomes is characteristic for the cells of a given species of animal. This number is best ascertained in fixed and stained cells, though it can be made out in some living cells, while they ^ Flemming, iSjg^ Peremeschko, 1879. 49 THE BIOLOGY OF THE CELL SURFACE are In stages of nuclear division for then the chromosomes are most easily visible. The chromosomes also appear as strands of condensed structures lying in the more fluid substance of the nucleus, the so-called karyolymph. When the nucleus is at rest the chromosome-material appears as granules and is designated chromatin. How far this appearance coincides with the real state of the chromosome-material has yet to be deter- mined. Since the chromosomes display regularity in struc- ture and behavior during the process of nuclear division when they are visible and, after fading from view, appear again as bodies of the same number, form and character, it lies close at hand to conceive that they maintain their organization also when they are seen as granules. Theo- retically, for the chromosome-theory of heredity, which will be discussed later, it is also simpler to conceive that the chromosomes always maintain their form and never become disorganized by breaking up Into granules. The rapidity with which the nucleus carries out its changes might be considered sufficient for the maintenance of integrity during both the resting and the dividing stage. But the fact that the nucleus remains separated from the cytoplasm may be due to an interposed membrane. Though such a membrane can be demonstrated in many cells, its presence generally in cells has been denied. It is further not at all clear how this membrane arises. As with the presence or absence of the nuclear membrane, so with the linin, a net-work of material In the fixed nucleus: some hold that it is a real structure, others that it is merely an artefact. There is present In the nucleus a nucleolus (in some cases there are several nucleoli) which may be a double structure. Its function is not known. Indeed, one can not be sure of its origin or fully follow its history from one cell generation to the next. 50 THE PROTOPLASMIC SYSTEM The chemistry of the nucleus is known from studies made especially on blood cells and spermatozoa in which the nuclear material is rich in comparison with the cytoplasmic. Since in the spermatozoa the nuclei (sperm heads) are chromatin in the most condensed state known, their • chemisty is practically the chemistry of chromatin. The following is the chemical constitution of an animal's cell- nucleus (spermhead of the white fish) : C96.H,84.N54.O22(C43H57Ni5P4O30)4 " 24H2O The nucleus essentially is made up of a protein conjugated with a sugar-containing compound which through the presence of phosphoric acid becomes an acid, nucleic acid. The presence of phosphorus suggests that although lipin may not be present as such in the nucleus, it is necessary for the formation of nucleic acid. Nucleo-protein contains protein and carbohydrates which probably come together through the mediation of phosphorus-containing lipin. In the light of this suggestion the synthesis of nucleo- protein represents the highest chemical activity of the living substance since we see here synthesized the three important substances that distinguish living from non- living matter chemically: protein, carbohydrates and fats. For this reason I consider it an approach to the understand- ing of chemical activities in living matter to study nucleo- protein (and especially nuclein) with respect to its rate and duration of formation and its amount. In dividing cells the intact or so-called resting nucleus becomes active and exhibits changes after which it divides. However, the nucleus is not to be thought of as a body implanted in the cytoplasm with no relation to it except during synchronous division. Since in the so-called resting stage the nucleus continually increases in size to the moment in which the activities leading to its division set in we assume that it takes up material from the cytoplasm — • 51 THE BIOLOGY OF THE CELL SURFACE substances in solution. Indeed, with respect to the nucleo- cytoplasmic relations, the so-called resting stage of the nucleus Is as important as the stage of division. During the stages of nuclear division there also occur changes in the cytoplasm, as changes in refraction, in dispersion of granules, changes within the granules them- selves. The fact that these changes can be correlated with the stages of nuclear behavior suggests that chemical syntheses and analyses in the living cell are in part at least controlled by the movement of substances in solu- tion to and from the cytoplasm out of and into the nucleus. To this point I return later. As has been said above, the cytoplasm is composed of an inner core immediately surrounding the nucleus and, external to this, an ectoplasmic region extending to the cell-membrane. In this thus differentiated area of extra- nuclear protoplasm formed bodies of different size are suspended. By determining what in this extra-nuclear mixture of fluid and suspended bodies is indispensable to the life of the cell, we may derive a definition of cytoplasm. The suspended bodies are chiefly: droplets of oil, yolk, small formations known as Golgi-bodies, granules called mitochondria and chromidia; further, starch, crystals of various kinds, secretion-granules, excretory products. But these cytoplasmic inclusions are not to be taken for the cytoplasm itself, as is strongly indicated by the following facts. First, animal cells vary with respect to the content of cytoplasmic inclusions: not all cells contain them all and some even under high power of the microscope appear to contain none. Second, in many cases the inclusions occupy no constant position in cells: thus yolk generally present in eggs is variously distributed. Third, and most important, portions of eggs devoid of all cytoplasmic inclusions having been fertilized develop as do whole eggs with all inclusions present. Consider the egg of the marine worm, Chaetop- 52 THE PROTOPLASMIC SYSTEM terus, after it has been centrifuged with force sufficient to stratify its inclusions according to the specific gravity of each: between the zone of oil-drops, the lightest constituents of the cytoplasm, and the yolk-spheres massed at the opposite pole, lies a clear zone which under high power of the microscope and with bright-field illumination appears optically empty; with the dark-field or after proper fixation this zone appears to be made up of extremely fine granules. This clear area if cut away, even though it be but a small fraction of the egg, will develop if fertilized with almost the same pattern and tempo of development as the whole egg. This is likewise true of eggs of sea-urchins. The conclusion is that of the extra-nuclear substances the only part abso- lutely necessary for life is the clear inclusion-free men- struum. I therefore define as cytoplasm this menstruum only. In the following pages I shall adhere stricth' to this definition. Despite the fact that the cytoplasmic inclusions are not essential to cytoplasm, they have undoubted functions and merit here brief notice. In the following treatment, I speak mostly of egg-cells. The lipins (fats and fatty substances), discussed above, are present in eggs largely as oil and in yolk (lecithin). The oil (fluid fat) in eggs exists as drops in various degrees of emulsification. Whilst it is easy to observe oil drops in some eggs in which they are relatively large — as in the egg of the sea-bass, — it is often difficult to observe them in some other eggs where they are in a state of fine division. In the unfertilized egg of Nereis one can count the twenty to twenty-two oil drops present and can as development ensues after fertilization follow the gradual reduction by coalescence of this number to four. In the living larval worm one can further watch the emulsification of these into finer and finer drops which eventually disappear — as further shown by micro-chemical tests on both living and killed 53 THE BIOLOGY OF THE CELL SURFACE larvae. This observation teaches us one function of the oil In this egg-cell: it is a reserve food-material utilized only in the period before the young worm actively begins to ingest food. A gram of fat, as is well known, is richer in energy than a gram of carbohydrate or of protein. Lipin plays a role both in the water-holding power of cells and in the cells' immiscibility with the surrounding medium. It exists in the cell in other forms than oil drops. So, cholesterol is said to be found in the membranes of some eggs; the human red blood corpuscle likewise contains it. A closely related compound, ergosterol, is nowadays much discussed because of its significance in vitamin-chemistry. Fat Is also associated with pigments and thus may play a part in oxidation-processes. Yolk is another form in which lipin occurs in egg-cells. Yolk in eggs appears in manifold chemical and physical forms. The identification of yolk-bodies in eggs rests largely upon staining reactions. Spherules In the cyto- plasm which are stained dark blue or black with iron haematoxylin are usually assumed to be yolk. If finally they come to lie in the endoderm cells one concludes that they are food material and presumably yolk. Even so, these spherules show striking differences. Take, for example, the yolk spheres in the eggs of two closely related marine worms. Nereis and Platynereis. In the former ^gg the yolk spheres are homogeneously blackened by haema- toxylin; in the latter every yolk sphere is a delicately reticular structure staining faintly with the haematoxylin. In each case these bodies come to lie in the four cells which compose the gut. At the beginning of the process of incubation, yolk constitutes the greatest bulk of the chick's egg, whereas at the time of hatching the yolk has largely disappeared. This fact means, as we shall see In a later chapter, that yolk has been converted into cytoplasmic substance. In the 54 THE PROTOPLASMIC SYSTEM same way, of course, the growing animal after having been hatched from the egg manufactures its peculiar kind of protoplasm from the food which it takes in. So also yolk is used up in the development of other large eggs, as those of fishes and amphibians; in smaller eggs, however, develop- ment proceeds to the stage of hatching without the utiliza- tion of yolk. For example: in the Nereis egg, yolk is not used during cleavage by the blastomeres; it goes into the four macromeres which form the gut. A sea-urchin egg, in which the yolk has been displaced by centrifugal force, develops perfectly. And yolk-free fragments of marine eggs develop normally as stated above. In the egg of Nereis the yolk spheres are polyphasic, i.e., they contain a protein-skein and lipin. If these eggs are placed in hypotonic sea-water, oil in minute drops escapes from the yolk-spheres, leaving a reticular and more watery medium, the whole enclosed by a strong membrane. The yolk spheres now resemble those present normally in the egg of Platy?iereis. On transfer to normal sea-water the oil re-enters the yolk-spheres and their original optical proper- ties are restored.^ The process is thus reversible. An observation like this shows how yolk may vary and that we can not satisfy ourselves with only a rough identification of it as that by staining. Certainly, what thus we identify as yolk is not a pure lipin. The Golgi-bodies or Golgi-apparatus, commonly a net of fibres or a cluster of granules of varying form and size, have been described for almost every type of animal and plant cell. Their meaning and function are yet to be made clear. Undoubtedly in some cases what have been described as Golgi-bodies are drops of fat;'^ in other cases even vacuoles have been described as Golgi-apparatus. ^ Just, 1926. - J'ist, 7927a. THE BIOLOGY OF THE CELL SURFACE Mitochondria are minute spheres, rods or filaments. In the egg of Platy nereis pictured they are rods; while in that of the closely related Nereis they are spheres. In some cells they assume first one then the other form. It has been held that the mitochondria are phospholipins. How- ever, because with the most careful cytological technique yet devised (that invented by Altmann), later workers^ were unable to prove that mitochondria contain fats but, instead, could demonstrate their protein nature, doubt is thrown on the older view. Certainly in those marine eggs which I have studied on this point, the mitochodria do not reveal the specific gravity of fat; for, when the eggs are centrifuged, instead of lying in the zone next to the fat they take the place above the yolk.- Chromldia are generally believed to be cytoplasmic granules derived from the nucleus. I may refer again to the egg of Nereis: in it one can observe granules which seem to come out of the nucleus, wander into the cytoplasm and come to lie at the cell-periphery in which position they can not be distinguished from mitochondria. Under various experimental conditions, as after ultra-violet radi- ation or by treatment with hypotonic sea-water, the number of such granules in the vicinity of the nucleus is increased. Often also with such experimental means chro- mosomes are eliminated which degenerate into granules not easily, if at all, distinguishable from chromidia and mitochondria. This degeneration of chromosomes is quite different from the degeneration of chromosomes in abnormal eggs which fail to develop; these latter can never be mis- taken for chromidia or mitochondria because structurally they offer an entirely different aspect. On the basis of ^ Bensley and Gersh, /QJJ- ~ Bensley and Gersh are extremely cautious in their conclusions and it may be that even under the conditions of their experiments the lipin-fraction of the mitochondria iras destroved. 56 THE PROTOPLASMIC SYSTEM my observations I venture the opinion that chromidia and mitochondria represent stages in the transformation of granules of nuclear origin. Of granules other than mitochondria and chromidia found in eggs, pigment is most striking. Many eggs seen en masse have beautiful colors — various shades of red, orange, yellow or green. There are some species which have eggs of two distinct hues; thus individuals of the little marine worm, Autolytus varians, have either red or green eggs. The pigment of sea-urchin eggs has been shown to be a lipochrome. Occasionally crystals can be demonstrated in eggs. Thus Aleves has described fine slender bodies found in fertilized eggs of Psanvmechuius and I have found them also in ferti- lized eggs of Echinarachnius. That such formations are increased in cross-fertilized eggs^ I do not doubt, but that they are solely due to the effect of the foreign sperma- tozoon is to be questioned in view of Meves' and my own observations. Some of these cytoplasmic inclusions, as well as others, that are not here discussed as, for example, secretion- granules which leave the cell, are certainly temporary structures. Study of the history of the egg from its earliest formation, when Its cytoplasm always appears homogeneous, through the stages during which it becomes laden with Inclusions, teaches that Inclusions appear in the cytoplasm during development. The building up of cyto- plasmic Inclusions Is an indication that the egg takes in food-material from Its surrounding medium. Indeed, in some eggs, as those of the flatworms, the yolk, elaborated by special gland-cells, Is merely deposited around the egg. In limiting the application of the term, cytoplasm, to the clear and almost homogeneous menstruum or ground- ^ Tennent, IQ20; Hibbard, ig22. 57 THE BIOLOGY OF THE CELL SURFACE substance and thereby dismissing the various inclusions suspended in it as necessary constitutents of cytoplasmic structure, we dismiss much of the work done on the chem- istry of the cell as relevant to the chemistry of the cyto- plasm inasmuch as chemical studies have been made so largely en gros — encompassing everything including food- material and effete matter which might be present in each of the thousands of cells which are often necessary for a single chemical determination. The chemistry of the cyto- plasm, from my point of view, would embrace only the chemistry of the inclusion-free ground-substance. Since we do not yet possess such, we can only approximate it by considering that which remains after we have subtracted from what is known of the chemistry of the whole cell that which is known of the chemistry of the nucleus, of the various inclusions and of the cell-membrane. This so little light does not throw its beams far and we remain still much in the dark. Moreover, there is always the obstacle that what we call the chemistry of the whole cell is not that of living protoplasm; the isolation necessary for chemical study may change the cell-constituents. Micro-chemical studies on cell-inclusions are certainly valuable as are those on the inclusion-laden protoplasm. But there is the great- est need for such studies on the ground-substance itself. The definition of cytoplasm here given makes mandatory a revaluation also of studies on the physical properties of protoplasm. Here, in order to know what properties inhere in the ground-substance, unless this be studied as such, the known properties of nucleus and cytoplasmic inclusions should be subtracted from those known for the whole cell. It is a curious fact that in that domain of physical chemis- try which by its definition we should expect to encompass the study of the ground-substance workers have laid more stress on the whole cytoplasmic area and neglected the possibility of investigation of the ground-substance which 58 THE PROTOPLASMIC SYSTEM when isolated from, and free of, cytoplasmic inclusions is wholly viable. I refer to that branch of chemistry, colloid chemistry, so-called, where fact and fancy are dispersed in the medium of a naivete which so often characterizes new adventures along narrow confines. An e^^ like that of Nereis in which the formed bodies, oil-drops and yolk spheres, are visible when magnified only twenty times can not with respect to these bodies be con- sidered by a colloid chemist as other than an extremely coarse suspension. It can not be regarded as a colloidal solution for a colloidal solution is usually defined as one in which the particles range in size from i, 2 or 6 to 250 {xix. The smallest are at the lowest limits of visibility with the ultra-microscope and the largest come just within range of microscopic observation. Even such eggs as sea-urchins', whose oil-drops, yolk-spheres and pigment granules are smaller than the oil-drops and yolk-spheres of the Nereis Qgg, can not be looked upon as colloidal solution for their inclusions are visible under relatively low power of the microscope and thus do not fall within the range of sizes of particles in a colloidal solution. But the ground-substance of these eggs is a colloidal solution since its particles, barely visible under the highest powers of the microscope, are more clearly revealed by dark-field illumination. The definition of cytoplasm here given also means dis- carding theories of the morphological structure of cytoplasm since these, the alveolar, filar and granular theories, refer to the cytoplasm plus inclusions. Even if we would define cytoplasm to embrace the cyto- plasmic inclusions, the alveolar theory of cytoplasmic structure would be untenable. In the first place, although it is true that there are cells which show a vacuolated or foam structure, this is by no means all-pervading but is limited to certain cellular regions. Moreover, such struc- ture is far from being demonstrable in all cells and the 59 THE BIOLOGY OF THE CELL SURFACE experimental evidence indicates that it is non-essential to the living cell-substance. Secondly, the alveolar structure Fig. 2. — Two sections of the unfertilized egg of Nereis limbata (after Lillie). a is badly fixed as shown by the partially dissolved yolk spheres and the wholly dissolved oil drops which in h are intact. Compare b with the figure of the li\-ing egg as shown in Fig. 22 (p. 158). v.m., vitelline membrane; c.l., ectoplasm. described for fixed cells, as it often has been, is the result of improper fixation. In such cells the cytoplasm appears a mesh, empty holes occupying the greatest space, holes 60 THE PROTOPLASMIC SYSTEM which have been brought about through the action of the fixing solution in dissolving out fat, yolk or other cyto- plasmic inclusions. Compare the pictures given, (Fig. 22) of a living, (Fig. 2a) of a poorly fixed, and (Fig. 2b) of a properly fixed egg. In Figs. 22 and 2b oil-drop and yolk- sphere are intact. When one bears in mind that Fig. 2b is from a very thin section, about one-thirtieth of the entire egg, and that the larger inclusions present can very easily be displaced in sectioning, the picture is all the more con- vincing: the resemblance to Fig. 22 is striking. But compare Fig. 2a — a mesh-work like a fish-net. From pictures inferior to this (Fig. 2a), wholly disregarding the structure seen in the living egg, Wilson derived his support of the alveolar theory of protoplasmic structure.^ When one speaks of the physical basis of life and refers only to the morphology of the cell, one is misleading- — doubly so if one has in mind a dead cell showing mostly holes. True, protoplasm is the physical, i.e., the corporeal basis of life; also in the fixed state this basis is of interest. But we shall not understand it, if first we vacuolate it and then ascribe to the holes essential meaning without knowledge even as to the significance of the substances which we replaced by holes. On the other hand, to say that the cytoplasm of the living cell is alveolar in structure because the holes of the dead cell were occupied by formed bodies is to give new meaning to the term, alveolus; I can not see how a droplet of fat or a spherical yolk-mass can be designated an alveolus. I have often pressed them out of an egg into sea-water where they remain intact. If they are moved by centrifugal force from one to another region of the cell, they do not leave empty spaces behind them. Hence, neither they nor the places they occupy in the fluid cyto- plasm are alveoli. 1 Wilson, 1^26; but cf. Mathews, igo6. ~ Wilsoyi, I.e. 61 THE BIOLOGY OF THE CELL SURFACE But let us for the moment dismiss badly fixed eggs, pictures of fenestrated or lacunar substance, also the empty spaces previously occupied by oil and yolk, and extend the meaning of the word alveoli to embrace such Inclusions as oil-drops and yolk-bodies seen in the living e^g as discrete spheres. How far does this assumption strengthen the alveolar theory of protoplasmic structure? Can we con- clude that Inasmuch as each "alveolus" (oil-drop or yolk- sphere) Is laid down by the egg during its history, an egg In that stage before oil or yolk is deposited as easily visible discrete spheres has no structure? Shall we say that In those eggs which distribute their "alveoli" in such manner that some cells possess all the "alveoli" and others none, those cells which do not contain them are structureless ? The alveolar theory has found support from the fact that certain artificial mixtures resemble some kinds of proto- plasm. Witness emulsified oil. If we examine under the microscope a thin layer of mayonnaise — olive oil and vinegar plus egg-yolk, the emulsifying agent — we find that the oil is in drops and among them are scattered yolk- spheres. By carrying the process of emulslfication further and further, we obtain smaller and smaller oil-drops and in this way make structures that resemble those of eggs with larger or smaller oil-drops. Such a film of mayonnaise when fixed by a proper agent for cytoplasmic fixation retains faithfully Its appearance before fixation and simulates the picture of properly fixed egg-cytoplasm. The emulslfication obtained by beating olive oil with vinegar or lemon-juice, without egg-yolk, though less permanent, bears also a close resemblance to the living cytoplasm of a living egg, say that of a star-fish. It has even been held that protoplasm Is a foam-structure because of the resem- blance of some cellular structures to such foams prepared in the laboratory. As I see It, all that these and similar models tell is that the living cell can hold Its oil In the form 62 THE PROrOPLASMIC SYSTEM of discrete drops of larger or smaller dimensions. They give us no information concerning the mode in which the living cytoplasm moulds the oil-drops — now coalescing them, now emulsifying them. They fail to reveal the value of such structure for life phenomena. Finally, for the alveolar theory, a yolk-sphere is as much an "alveolus" as an oil-drop; and yet although we do not know as much about yolk as we would wish, nevertheless, what knowledge we have is sufficient to warrant the conclusion that both physically and chemically a yolk-sphere is something quite different from an oil-drop; a yolk-sphere, unlike an oil-drop, is a combination of fat and protein.^ If one adheres to the alveolar theory by denominating oil-drops and yolk-spheres "alveoli," one excludes from consideration the differences between these "alveoli," thus stressing their shape rather than their physico-chemical make-up. The filar theory of cytoplasmic structure never has attracted many adherents, doubtless because of the limited occurrence of fibrils in cytoplasm. For the most part, when found, they are in fixed cells, in which, before fixation, they can not be seen.^ The evidence for their presence in some other living cells is by no means convincing. Thus, the cytoplasm of outward flowing cell-processes appears to be filar — but this may be due to granules disposed in rows by the streaming of the medium that is forced through the orifice.^ Mitochondria-granules may by close alignment appear as fibrils. Altmann^ suggested that granules in the cytoplasm are the elementary living substance. Both before and since his time others have put forth similar theories, often includ- ing more kinds of cellular granules than Altmann did. ^ See also Konopacki^ 1929. - Lewis and Lewis ^ ig24. 3 See Biitschli, iSgo; also Just, igzSe. 4 Altmann., iSgo. 63 THE BIOLOGY OF THE CELL SURFACE Undoubtedly, Altmann's granules are mitochondria; as such they are only cell-inclusions. As to other granules described in this connection, one must remember that many granules in cells are purely secretory — that is, precursors of the cell's secretion, as the granules found in a salivary gland or in the pancreas — that some others are storage-stuffs, others, excretory products. In other words, granules are derivatives of cytoplasmic substances or expressions of cytoplasmic activity. That to which they owe their origin and not they themselves would seem more likely to be basic. For the reasons given, I think that we may discard the alveolar, filar and granular theories of cytoplasmic struc- ture. Oil-drops, yolk-spheres, threads and granules in the cytoplasm make the contents of a cell a gross suspension. Such a suspension as that of an egg of Nereis or of a sea- urchin does not represent the basic structure of life and its peculiar physical state does not offer any clue to the solution of the problem of vital activity. Turning to the menstruum containing the formed bodies, the ground-substance, we find, as stated above, that it appears to be almost optically empty. If, however, one examines the hyaline region of a centrifuged egg of Arbacia, Nereis or Chaetopterus, with extreme care without pressure on the egg — for pressure induces abnormal formations — one can observe very fine granules as barely visible points. Thus, as also said above, the ground-substance is to be considered as a colloidal solution, for it has particles below the size seen under the ordinary microscope, but which can be seen under dark-field illumination. Until we know more of the physics and chemistry of the ground-substance, we must derive a conception of its structure by observing it in the living state and when properly fixed. The fact that well-fixed ground-substance so closely resembles the living makes fixation a supplementary aid in its investigation and permits us to draw conclusions concerning the living state 64 THE PROrOPLASMIC SYSTEM from the fixed. Processes too fleeting to be followed exactly in the living can be followed in their single steps in the fixed. By thus supplementing the study of the living by that of the fixed ground-substance, we may approach the understanding of the basic structure of cytoplasm. My confidence in the value of the study of properly fixed cells rests upon the fact that such study has confirmed my observations of the extremely delicate momentary changes in the ectoplasm of living cells. Without ever discounting the postulate that the living cell is the basis, control and aim of biological investigation, I have come to appreciate the value of proper fixation. But since the use of fixation has been and is questioned by many workers, I must for a moment dilate on this point. When at the end of the last century Fischer^ and Hardy^ independently inveighed against fixation of protoplasm, neither made much impression. But the impression grew; to-day we discern a reaction of no mean proportion against the study of fixed cells. Some histologists adopt an apologetic air, offering their findings on fixed tissues in all but a furtive manner; others have given themselves over to the cinematograph or to the culture of living tissue removed from the animal. Now without doubt conclusions as to living protoplasmic structure drawn from study of dead cells should ever be subjected to severest criticism. He who works with dead cells should never be under delusions that he has to do with other than a dead thing. I do not believe that any com- petent histologist takes any other view. And yet, there is often inference by those who hold lightly all work on fixed cells, whatever its purpose or conclusions, that the investigator of fixed, sectioned and stained cells forgets 1 Fischer, l8g(). - Hardy, iSgg. 65 THE BIOLOGY OF THE CELL SURFACE not onlv that he deals with dead matter but also that he subjects the corpse to some thirty separate treatments before he places sections of it under his microscope for investigation. Hardy's paper nowadays hailed by so many as having infallible authority falls short in three directions^: First, Hardy studied the action of too few fixing solutions; second, he failed to make comparisons of the fixed with the living cells; and third, he used cells in bad condition. Every student of histology knows that he can not assume that because he has successfully fixed cells of one tissue he can employ this same fixation for every other kind of cell. The technique of fixation has not passed very far beyond the trial and error stage and, therefore, every new type of cell encountered offers a new problem of fixation. Hardy should have used more agents and of a much more diversi- fied constitution Instead of holding so strictly to those of closely related composition. Hardy made comparisons between the fixed cell (for example, gut cells of Oniscus, the so-called pill-bug)^ and protein solutions and came to the conclusion that his fixing agents act upon the protoplasm as they do upon protein. This conclusion revealed nothing new. It would have been far more valuable had he made comparisons between the living and the fixed cells In order to ascertain how far was the deviation of the fixed from the normal. Further, Hardy investigated always cells of tissues and never free-living cells as Protozoa or eggs in their normal environment. This ^ In his more thoroughgoing work Fischer contributed to our knowledge of microscopic technique with respect to the use of dyes. - / may point to the fact that the choice of this object, gut cells of Oniscus, was most unfortunate, for several reasons. These cells, for instance, co7itain much chitin whose presence prevents uniformity of fixation. See further: Scheikewilsch, i8g§; McMurrich, i8g§; Conklin, iSgy. 66 THE PROTOPLASMIC SYSTEM fact warrants my third objection to his conclusions for two reasons: First, his cells were already in some state of post mortem changes of greater or lesser degree depending upon the time elapsing between their removal from the animal and their fixation — these changes being more rapid, and, therefore, a more serious source of error, for tissue-cells removed from the warm-blooded animals which he used. In the case of warm-blooded animals it would also make a great difference whether or not the animal from which the tissues were removed, was anaesthetized. Second, he made no allowance for the bulk of tissue used; that is, his con- clusions would have been far more sound had he used a thin sheet of tissue made up of a layer of one or two cells instead of compact masses from the glands investigated. Not only were his cells In some degree of post mortem change, but also were they fixed unevenly and cut into sections of different thickness. True, there is bad and unreliable fixation of eggs. Never- theless, it is erroneous to condemn all fixation. If I find that after fixation an egg very closely resembles the living I can draw conclusions on the basis of this resemblance espe- cially since knowing that the cell is dead I take into con- sideration that its proteins are changed — coagulated, gelated, precipitated, and that still other changes have taken place. Instead of abandoning the study of fixed cells we should use proper fixation in order to check and extend observation on the living. Neither the cinemato- graph nor tissue culture will quite replace the good fixation of the cell for comparison with the living. Although our ignorance of the ground-substance is pro- found and will remain so until we study it frankly as such,^ isolated from all the coarse particles suspended in it, never- theless we have at our disposal certain evidence which may 1 Jvst, 1 93 6b. 67 THE BIOLOGY OF THE CELL SURFACE form the basis for the extension of our knowledge. That the clear, nearly homogeneous, almost structureless ground- substance of an egg can (with a nucleus) develop, renders it at once amenable to all treatment given an entire egg. As it develops it exhibits the same form-changes. Here I restrict myself to the discussion of one well-known phe- FiG. 3. — Boveri's classic figure of a mitotic spindle in the egg of Echinus microtuberculatus. But cf. the aster in Fig. 29b (p. 174) and the mitotic complex in Fig. 33d (p. 259). nomenon, namely, the appearance of the radiating struc- tures seen in so many cells during cell-division. In many cells division of nucleus and of cytoplasm occur simultaneously. Division of the nucleus may be a very simple direct process, a constriction and separation of the constricted parts or a more complex and indirect process with the chromosomes going through a very orderly sequence of manoeuvres on a bundle that appears to be made up of threads. In this latter type of nuclear behavior there often appear in the cytoplasm (occasionally in the 68 rilE PROTOPLASMIC SYSTEM nucleus) radiations from each polar end of a group of fibre- like structures, the so-called mitotic spindle. (Fig. 3.) The radiations center at a minute granule or at a larger vacuolar sphere-structure; in some cases both granule and sphere are present. The minute body is the centriole or cen- trosome and the sphere is the centrosphere; the radiations because of their disposition are called astral rays. Best seen in cells that possess granules, the rays are also demon- strable in cells which are granule-free. Further, cells which show no rays are often filled with granules. Therefore the rays do not depend upon the cell-inclusions. Seen in the living egg of Platynwreis, for example, every astral radiation is a granule-free track whose greater width is about 3 /i. In the living Arbacia or Echinarachims egg the rays have the same configuration. Nothing in the living or properly fixed egg appears as the stiff coarse "astral fibres" of poorly fixed eggs. The radiations are without doubt no fibres at all. It is often held that they are paths of flow and in living granule-rich cells they do certainly appear to be such — the flow causing a displacement and close packing of granules and thereby accentuating the radiate appearance. There is no unanimity of opinion concerning the nature either of the radiations or of the spindle-" threads" on which the chromosomes move. I share with others the idea that spindle-" threads" and astral radiations are both extremely fine delicately thin sheaths enclosing fluid material, that they are not in any sense fibres with contractile power. (See Fig. zgb and Fig. 33^.) As an almost perfectly homogeneous and fluid system, cytoplasm could scarcely show differentiated pathways of flow even in extremely thin-walled tubes; less could it show contractile fibres. Either then cytoplasm is not structure- less or it has power to elaborate structure sufficient to account for the ray-configuration of the astral system. In either case the cytoplasm is diflferent before and after the 69 THE BIOLOGY OF THE CELL SURFACE appearance of the rays. These rays thus indicate structure in the cytoplasm or at least the capacity of the cytoplasm to form structures. So far I have dealt with the cytoplasm, the ground- substance outside of the nucleus. I should remind the reader that I consider the nucleus likewise as differentiated ground-substance. The formation of intra-nuclear spindles just referred to, the origin of centrosomes from nuclei, sometimes described, can be interpreted on the same basis as the formations in the cytoplasm. Nucleus and nuclear constituents are themselves conceived as expressions of the capacity of the ground-substance to form structures. In its other derivative, the ectoplasm, the ground-sub- stance likewise exhibits capacity for structural formations. Indeed, ectoplasmic structure in many cells, e.g., Protozoa, is a diagnostic characteristic. I wish to consider this peripheral ground-substance as it appears in animal cells. But first I must discuss the question of the cell-membrane, because it has obfuscated a proper appreciation of that part of the cell beneath the membrane, the ectoplasm. The cell-membrane, like many another cell-structure, is a perennial subject for debate. The majority opinion is that cells are invested with membranes but the minority is as insistent that a membrane is neither present nor necessary. Of those in the camp of the majority, some hold that the cell-membrane is an endogenous structure, a living thing built by the cell; whilst others deny its life, considering it a sort of excretion; and still others assert that it is a formation induced by the cell's external medium. Opponents of the membrane's existence declare that since the cell-substance is in the colloid state, it is unnecessary to postulate the presence of a membrane: the mere boundary of the colloidal system is sufficient to preserve the cell's integrity. Accord- ing to them the cell is somewhat like a drop of oil in water; just as the surface of the oil keeps the oil intact, so does the 70 THE PROTOPLASMIC SYSTEM surface of the more heterogeneous cell-substances prevent cellular disintegration. Also, are there those who unwit- tingly hold a foot in each camp. Now, whenever there are so greatly divergent opinions, one suspects either insufficient factual basis, or confusion arising from too loose definition. I strongly suspect that the difficulty here arises out of the fact that investigators in their discussions are often not dealing with the same thing; much of the argument then is beside the point. The cellulose wall of a plant cell is something quite different from the surface of a Paramoecium as both observation and experiment show. Similarly, is it hazardous to compare the surface of the red blood corpuscle in human blood with that of human cells or even with the red blood cells of vertebrates other than mammals since the mammalian red blood corpuscle is not a true cell because it is devoid of a nucleus. Therefore, if the mammalian red blood corpuscle lacks a membrane this is no proof that true cells also lack such. The first proposition is thus clear: in defining a cell- membrane we must be careful to distinguish between the various coverings enclosing cells, excluding, for example, discussion of such formations as cellulose walls around plant cells. The second proposition Is, likewise, obvious: if we speak of cell-membranes, we must speak of cells; the presence or absence of a membrane on a piece of a cell like the mammalian red blood corpuscle does not come within the discussion. The Issue concerns cells and cells only. Indeed, much of the work on mammalian red blood cor- puscles should be used only most judiciously In making comparisons with true cells. Thus restricted, the discussion on the cell-membrane centres around two questions: Is there a membrane around animal cells ? And if present, is it a living part of the living system or an exogenous formation .? If exogenous, I 71 THE BIOLOGY OF THE CELL SURFACE presume that It is non-living. I think that the question of necessity or purpose is not involved. To say that one does not see how it is possible for a cell to remain intact without a membrane most certainly does not mean that it can not thus remain. Here I speak only of those cells that I know best, egg-cells, though I hold that the discussion can apply to some extent also to other animal cells which, like the eggs, are often held to be without membranes. In the description given above I pointed out that on eggs, as, for example, that of Platynereis, two membranes are present; the outer or vitelline, and the inner or plasma- membrane. Whenever egg-membranes are discussed, the two membranes should be clearly distinguished. Further- more, when the vitelline membrane is spoken of, it should be clearly stated whether the unfertilized or the fertilized egg is considered. Much of the disagreement concerning the nature and properties of egg-membranes is due to the confusing of the vitelline and the plasma membrane and to failure to appreciate the difference of the former before and after fertilization. Generally, the essential difference between a vitelline and a plasma-membrane lies in the fact that the latter is con- tinuous with the egg-cytoplasm whilst the former is dis- continuous with it, set apart from it and separated by a space, the so-called perivitelline space. Using this simple distinction we meet, however, with the difficulty that in many eggs that membrane which after fertilization is thus so clearly set off as a vitelline membrane, before fertilization appears as a plasma-membrane, continuous with the cyto- plasm. When after fertilization such a vitelline membrane separates from the egg, on the cytoplasmic surface of the egg appears a new plasma-membrane. In observing one living egg I may be able to say in a moment that a membrane is present, it being so sharply differentiated; observing another species of egg, I may not 72 THE PROTOPLASMIC SYSTEM be so sure but doubt vanishes if I know the history of the egg. Take the unfertilized egg of the sea-urchin concerning the presence of whose vitelline membrane there has been much dispute. Knowing the history of this egg as it develops in the ovary I know that it arises by successive divisions, each egg everywhere bounded by a membrane except at its point of attachment to the ovaria-n wall. I know also that the egg is part of the parent whose cells arose by a long series of cell-divisions, of separations by cell- surfaces, from a fertilized egg. This history strongly indicates the presence of a membrane and the simple experi- ments made previously by others which I repeat and con- firm strengthen the indications. What is true of this egg is true of all other eggs that I have studied: they possess membranes built by the egg, living structures and not adventitious depositions from the outside. In other eggs, those of worms, molluscs, ascidians as well as of other echinoderms, the presence of a membrane before fertilization can not be doubted. The fact that membranes become separated at a distance from the eggs after fertilization does not mean that the eggs are now without plasma-membranes for they at once form them anew. Careful observation can establish this as true. It follows from these considerations that the membrane is neither a deposition nor a precipitation induced by the egg's surrounding medium. This is not to say that the egg-plasma never forms precipitation-membranes. On the contrary, the contents of an egg caused to burst by applica- tion of pressure may often show a congealed surface. But this death-change by no means constitutes evidence that the membrane on the living intact egg is a precipitation- membrane. A pared apple in time develops by dessication an outer tough coat but this is not the same structure as the removed skin. Nor does it follow on the definition of protoplasm "as a film-pervaded s^'stem," which is often set 73 THE BIOLOGY OF THE CELL SURFACE forth, that the external covering of a cell is similar to or identical with such intracellular partitions. The understanding of the cell-surface and of the capaci- ties residing in the ground-substance is forwarded by appreciation of that component of the protoplasmic system, which I now discuss in detail: The ectoplasm. 74 4 The Ectoplasm jt/aeckel,^ describing the cell-structure of sponges, first used the terms, exoplasm and endoplasm. He clearly distinguished two regions of the cytoplasm as follows: In the hyaline contractile ground-substance of the proto- plasm a varying mass of small dark granules is constantly embedded; these usually surround the nucleus. On the living flagellar cell, as long as it is active in situ, is a thin granule-free cortical sheath. Thus one can more or less clearly distinguish between a structureless outer cortical substance and a granular inner medullary substance. The outer cortical substance (exoplasma) is fully hyaline, some- what firmer, less watery, more strongly refractive and contains no granules; the inner medullary substance (endo- plasma) is granular, somewhat softer, more watery, less refractive (and contains the granules) and also now and then vacuolar. No matter how distinctly the regions some- times are set off from each other, they are never sharply separated but pass insensibly the one into the other without fixed limiting layer, much as is the case of hyaline cortex and granular medullary substance in the body of an infusorian. Later Haeckel says^ that through its variously changing surface formation, the exoplasm gives to the entire flagellar cell its characteristic form. This clear description given by Haeckel may serve as the basis of our definition of the ectoplasm (exoplasm of Haeckel) : the ectoplasm is the superficial region of the protoplasmic ground-substance set off from the remainder 1 Haeckel, i8'/2. ' Haeckel, I.e. 75 THE BIOLOGY OF THE CELL SURFACE (both nuclear and cytoplasmic ground-substance) by differ- ences in physical properties, in structure and in behavior; as part of the cytoplasm, as differentiated ground-substance, it is definite!}' a living part of the cell. The inner region of the cytoplasmic ground-substance plus its cytoplasmic inclusions — granules and the like — is defined as the endo- plasm. It is not to be supposed that ecto-endoplasmic differ- entiation in animal cells was unknown before Haeckel's time. Although not named as such, those regions were known to the older students of the cell.^ Almost any original paper and text-book of theirs points out differences between the inner and the outer region of the extranuclear cell-contents. Thus, Henle in reports of his researches and Koelliker in his text-book on human histology gave clear descriptions of what subsequently were known as ectoplasm and endoplasm. Kidney, liver and intestinal cells were both described and figured to show these regions. The cells of the skin were favourite objects for such descriptions. If one observes a section of the kidney taken from man or other vertebrate one notes that certain cells are striated and possessed of radial projections. Thus, these cells reveal an ectoplasmic differentiation. The ectoplasmic area is less clearly marked off in the human liver cell. The manifold functions of the liver — the breaking down of red blood corpuscles, the formation of urea, the storage of sugar as glycogen, the mobilization of fat — are reflected in the many pictures of itself that the small polyhedral liver-cell can exhibit. It also appears differently in conditions of hunger and after rich nourish- ment. Nevertheless, in all its different appearances, the liver-cell shows a differentiation between inner and outer regions of the cytoplasm, as Koellicker observed. ^ For example: Leydig, /S^y; Kiihne, /864. 7(^ THE ECTOPLASM The cylindrical cells of the human intestine, measuring 22-26 by 6 At, show on the side presented to the intestinal lumen a brush border or bordure en hrosse. Thus, in these cells as in those of the kidney and liver one notes an ecto- endoplasmic differentiation. In the skin are cells which interlock by means of fine threads, the so-called intercellular bridges.^ Cells of other tissues are connected similarly, as for example those in the retina of the eye. Though many claim that these connec- tions are artefacts, the abundance of evidence from studies on various tissues from many different animals supports the view that these connections are normal cell-processes. As such they are evidence of ecto-endoplasmic differentiation. - In view of the substantial body of convincing data showing the presence of intercellular connections, it becomes difficult to understand why many authors still deny their existence and assert that fixation creates them. But quite apart from the demonstration of these intercellular connections in well-fixed tissues exist clear cases, some of them among the earliest described, showing living cells in rich connections with each other. As will be shown later connections between cells of the developing egg have been long known and can be easily demonstrated. I think that almost no one to-day doubts that in the nerve-system the neurones are in contact with each other by processes which make the synaptic membranes. The body-structure of higher animals, especially of vertebrates including man, embraces certain groups of cells notable for the presence of extra-cellular substances asso- ciated with and produced by them. These groups of cells comprise the binding or connective tissues of the body. Their products are fibres of various kinds, cartilage and ^ See Schuberg, IQOJ^ and later -writers. - Flemniing, /t?79, on living cells. ' // THE BIOLOGY OF THE CELL SURFACE bone. Studies on the development of fibres in connective tissue, on that of cartilage and of bone indicate that these structures which finally come to lie outside of the cell are products of the ectoplasm. Indeed, it has been postulated that connective tissue fibres even after deposition outside of the cell are living ectoplasm. Whether living or not, the intercellular substance of connective tissue and its deriva- tives (cartilage and bone) are to be regarded as extreme formations of ectoplasm, which have lost their organic connections with their cells of origin. Intercellular fibres cast off hy cells are to be distinguished from such fibres which are true cells, having nuclei. These, muscle and some nerve cells, are characterised by their great length and hence by a preponderance of surface to mass. They thus possess relatively more living ectoplasm as part of the cells than any other cells so far discussed. Muscle-cells are notable for their high degree of contractility and students of physiology regard this as a surface-phe- nomenon. Fixed preparations of smooth or plain muscle- cells (individual fibres) show longitudinal threads or fibrils whilst striated muscle-fibres (both those of the heart and of the skeletal or voluntary muscle) show in addition striations running at right angles to the length of the fibres. Much difference of opinion is expressed concerning the significance of these fibrils because studies both on the developing and the definitive muscle-fibres have yielded conflicting results and led to various interpretations. In addition, many investigators doubt the actual presence of fibrils in muscle-fibres inasmuch as they have been unable to see such in living cells. Although it would seem hazardous to venture an opinion in view of this uncertainty with respect both to the longitudinal threads described in fixed smooth muscle and to the additional cross-striations reported in striated (cardiac and skeletal) muscle as seen in fixed preparations, nevertheless, I may offer suggestions concern- 78 THE ECTOPLASM ing them. These suggestions are consistent with the well- estabUshed findings of studies made on both types of muscle-cells by the method of tissue-culture devised by Harrison and with the fact that in the muscle-cell the ectoplasm is preponderant. Also, they stand in accord with our knowledge of ectoplasmic structure in cells generally. The smooth muscle-fibre, an elongated slender spindle- shaped cell, reveals itself in fixed preparations as possessed of fine longitudinal threads, fibrils or myofibrils. Lewis and Lewis were unable with the use of either bright- or dark- field illumination to observe any fibrils in living smooth muscle-cells grown in tissue-culture. According to them, large flat cells seldom contract whereas the elongated and band-like ones sometimes exhibit rhythmic contraction. These latter show ectoplasmic processes. Very much elongated overlapping processes may simulate fibrils when parallel to the long axis of the cell. In many places the cells seem to spread out under considerable ten- sion, and it appears to be along the lines of tension that the contractile substance coagulates into fibrils of various sizes. ^ From what is known of the contractile power of ectoplasmic processes of cells generally in tissue-culture, it is safe to conclude that the contractility of the smooth muscle-cell is likewise inherent. I suggest that the pictures of fibrils obtained in fixed preparations represent ectoplasmic pro- cesses somewhat altered by the action of the reagent employed to fix the cell. Striated muscle differs from smooth by showing alter- nate light and dark bands traversing its long axis. The exact structure of striated muscle remains in question. Since it begins its development as smooth muscle, it would seem most profitable to attempt an elucidation of its structure through study of its developmental stages. ^ Lewis and Lezvis^ 1924. 79 THE BIOLOGY OF THE CELL SURFACE But in these studies is lack of agreement. According to one view the fibrils arise from cytoplasm which is itself of fibrillar structure; another view maintains that the fibrils arise from cytoplasmic inclusions. The living muscle-cell in tissue-culture often fails to show the structure seen in fixed preparations; according to many investigators cross striations do not appear until after the cell is fixed. I venture the following suggestion as to the cross-striations in the muscle-cell. The young striated muscle-cell, like that of smooth muscle, possesses ectoplasmic prolongations. The pro- longations arising first fuse at their outer tips and thus form a membrane so that this region of the ectoplasm has a palisade-like structure. Repetition of this process through- out the length of the cells forms fibrils (the membranes) crossed by ectoplasmic prolongations. Beyond I shall show that the surface of the fertilized egg of the sea-urchin is made up of radial filaments covered by a thin membrane. My conception of the striated muscle- fibril has its origin in this fact. Since other cells than eggs show such projections, it is not too extreme to suggest that the embryonic muscle-cell also possesses such. The slender muscle-cells have the inherent capacity to build up succes- sively striated fibrils by reconstitution of their surface. The sea-urchin's egg builds one such surface layer which remains inseparably bound to the egg-cell; the muscle-cell builds many such surfaces. Also on other grounds my conception of the origin of the cross-striated muscle-fibril is not so far-fetched as it may seem. In the first place, the generally accepted doctrine of muscle-activity emphasizes the role of surface in the phenomenon of its contraction. The fact that in the contraction process, in one stage, oxygen is utilized, is evidence indicating a surface-reaction. Moreover, the shape of the muscle-fibre strongly supports the theory that 80 THE ECTOPLASM its activity is limited to the surface. On more general grounds, it may be pointed out that it is held that muscle- contraction, ciliary action and amoeboid movement are similar and have a common basis. Ectoplasmic threads may be considered as fine amoeboid processes, delicate pseudopodia; a cilium may be regarded as a modified pseudopodium. On the basis of my suggestion the striae of a cross-striated muscle can be regarded as threads of ectoplasmic substance, and muscle as a strand compounded of pseudopodial processes. This suggestion of mine concerning the origin of the muscle-fibre from repeated building of sheets of ectoplasm is interesting in the light of a passage from Lewis and Lewis in their "Behavior of Cells in Tissues"': There are three conditions resembling fibrillac, which are often observed, not only in heart-muscle cells, but in smooth muscle, endothelium, and mcsothelium as well; namely, a linear arrangement of long mitochondria, an overlapping of long, slender processes, and tension striations. The latter deserve most consideration, because they resemble closely the appearance of iibrillae in fixed cells. Migrating cells become more or less flattened out on the solid supports under considerable tension. The direction of this tension appears to be in line with the cell processes, as though the latter produced a pull on the ectoplasmic layer. The visible striae of various widths and lengths thus produced in the living cell are not permanent but may disappear and new ones may appear in line with new processes. The exact significance of these striations is uncertain. They may extend across the nucleus, indenting it or almost cutting it in two. They seem to be a phenomenon produced by ten- sion and reversible when the tension is altered or relaxed. On fixation they may retain their identity and stain more deeply than the rest of cytoplasm, resembling myo-fibrillae. The latter occur however in fixed cells which do not show tension striae. Certain nerve-cells are also notable for their fibre-like nature. Among these are the longest cells known. One ^ Lewis and Lewis, Lc. 8i t THE BIOLOGY OF THE CELL SURFACE ■■"0^ i^m /^t>>j^^ Fig. 4. — (after Harrison), a, row of ectoderm cells showing amoeboid ectoplasm; b, c and d are protoplasmic processes in de\-eloping nerve cells. 82 THE ECTOPLASM cell and its process, for example, of the sciatic nerve in man which is made up of processes from nerve-cells in the spinal cord, may be easily a meter in length. What we speak of as a nerve is really a bundle of processes from nerve-cells. When, for instance, one makes experiments in the labora- tory on excised nerves — the sciatic nerve of the frog is a favorite object for such — one uses only the ectoplasmic portions of cells. Therefore, conduction by nerves means conduction by ectoplasm. That the nerve-fibre is ecto- plasm was fully demonstrated by Harrison's classic and epoch-making work on the origin of the nerve-fibre. Harrison's researches while establishing the mode of origin of the nerve-fibre also furnish a beautiful demonstra- tion of ecto-endoplasmic differentiation. Harrison, the originator of the now so widely used method of growing tissues outside of the body of an animal, had as his object the settlement of the much debated question of the mode of origin of the vertebrate nerve-fibre from the central nervous system. By growing young embryonic cells taken from the spinal cord of a frog embryo at a stage in which he knew that no nerve fibres had yet developed, he was able to prove that these cells extend pseudopodial processes which by farther growth become the nerve-fibre. Says Harrison^: (Fig. 4-) From the time when the tissue is implanted in the lymph it shows a tendency to spread out, and often broad laminae made up of a single layer of cells are found at the peri- phery of the mass, while individual cells may move off entirely by themselves. This is the case with both nervous and axial mesodermic tissue, as well as with pieces of ecto- derm, though the latter more often roll themselves into com- plete spheres. One notable peculiarity that has frequently been observed is the formation of large round or oval open- ings in the flattened tissue, which may be surrounded by very narrow bands or rings of tissue with cells sometimes in single file. This phenomenon may possibly be due ^ Harrison, igio. S3 THE BIOLOGY OF THE CELL SURFACE to the mechanical action of the fibrin upon the implanted tissue, but the spreading out of the cells into thin sheets seems to result largely from the activities of the cells them- selves. These activities, which are common to several tissues, in fact to all except the very inert yolk-laden endo- derm and, perhaps, the notochord, may be referred to a form of protoplasmic movement having its seat in the hyaline ectoplasm found at the angles and sometimes at the borders of the cells. The movement cannot be observed clearly in the larger masses of cells on account of their opacity, but it may be seen very clearly in those cells which leave the main masses and wander off by themselves. These cells are irregular in shape, varying from unipolar form and having a varying amount of ectoplasm at their angles. The movement is amoeboid in character and results either in a change in shape of the cells or in their movement as a whole. Such cells are found usually in greatest numbers in preparations of the medullary cord, and from the cranial ganglia (branchial ectoderm), that gives rise by its movement to long fibres. Cells of the epidermis show their power of movement in somewhat different form. As has frequently been observed, the general tendency of isolated bits of epidermis is to round off into small vesicles, which, when left in water, may move about for days by means of their cilia. Within the lymph the same thing frequently takes place, although there is apparently greater resistance to the process of rolling up, and the cells may often remain together in the form of extensive sheets. Along the free border of these sheets of cells there often appears a fringe hyaline of protoplasm, which under- goes continuous amoeboid changes. In one case of this kind it was observed that the sheet of cells gradually spread out toward the side on which this fringe was placed. Since the work of Peters (1885— 1889) it has been generally admitted that wound healing in the epidermis is primarily due to the movement, in part amoeboid, of the epithelial cells, so that it seems quite possible that in this fringe of hyaline protoplasm above described, we have one part of the mechanism by which the movement of cells in wound- healing is brought about. The most inert of all the tissues is the endoderm, which will remain for days in the lymph, practically unchanged, gorged with yolk and devoid of hya- line ectoplasm. The notochord is also very inactive, although large pieces of this structure may show after a S4 THE ECTOPLASM time the early stages of normal differentiation, unaccom- panied, however, by growth, i.e., increase in length. . . . The movement of the embryonic cells in the lymph clot is very distinct, and is due beyond doubt to the activi- ties of the hyaline ectoplasm, which is accumulated espe- cially at the angles of the cells. It there forms extremely fine filamentous pseudopodia, through the activity of which the cells may change their shape or move from place to place. The exact character of the movement is not the same in all kinds of cells and it varies greatly in inten- sity. Axial mesoderm and medullary cord yield cells that frequently wander for considerable distances by them- selves; epidermis, when it does not roll up into bands or spheres, may form a hyaline fringe, and spread out con- siderably; pieces of the central nervous system and the primordia of the cranial ganglia give rise to the fibre-like structures described in the last section; the endoderm and notochord remain almost inert. In passing, I should like to refer to two points which baffle students of tissue-culture. I offer an interpretation of them on the basis of my knowledge of ectoplasmic behavior in general. The first concerns the observation so often noted that living cells in tissue-culture tend to assume a spindle- shape, especially when they migrate from the bit of tissue implanted in the culture-medium. There is no correlation between the original form of the cell and the spindle-form subsequently attained as we can conclude from the studies of many observers, who on various types of cells have con- firmed Harrison's original observations. Two suggestions have been offered : that the spindle-form is a reversion to a less differentiated (i.e., embryonic) cellular condition and that it is the effect of the cell's new, and abnormal, environ- ment. My suggestion is that the spindle-shape expresses gain in ectoplasm. I base this view on the fact that cells when isolated usually show relatively more active ectoplasm than when they are in contact and further on the observa- tion made by Harrison that spindle-shaped cells become S5 THE BIOLOGY OF THE CELL SURFACE extremely tenuous as they move along a delicate track, such as the thread of a spider's web, which behavior I interpret as a pseudopod extending in one direction. Concerning the cause of migration, two views are main- tained. According to one, the cells react positively to solids; according to the other, the cells move away from regions of too high acidity. My own interpretation is that cells in tissue-culture are no longer under the restraint imposed upon them when they stand in coordination with and subordination to their normal environment, i.e., other cells and tissue fluids. Thus their ectoplasmic activity is no longer ordered and controlled by the contact but is exagger- ated especially in their free and exposed surfaces — that is, in the regions where they are not in contact with the cells of the bit of tissue implanted on the medium. Hence, they migrate away from the mass. Suspended in the blood-plasma of vertebrates are red blood corpuscles. In mammals these are devoid of nuclei and therefore not true cells. All other vertebrates possess nucleated red blood cells. The structure of these latter has been worked out by Meves, who found that they have a well-marked ectoplasm. The blood of vertebrates contains in addition to the red blood corpuscles the leucocytes or white blood cells. In human blood these cells are sub-divided into four kinds, cell-size, staining reaction of cytoplasmic granules, and nuclear form being the characters on which the sub-divisions are made. In a healthy adult individual there is a fairly constant number of each type of white blood-cell. This number is used as an index for pathological states — many diseases have each a characteristic "blood-picture" which is an important diagnostic aid. On these highly interesting cells ectoplasm can be easily demonstrated. It has largely been assumed that for phagocytic activity and movement of white blood cells the same theories hold that have been 86 THE ECTOPLASM postulated for these activities in free-living Amoebae. But even were there only one accepted theory of the cause of amoeboid movement in free-living Amoebae, there would still remain the necessity to prove that such a theory could apply to white blood cells in the blood-stream of warm-blooded animals. It would be more advantageous further to study the white blood-cells themselves. Since amoeboid movement and capacity for engulfing solid par- ticles reside in the ectoplasm,^ the study of primary im- portance for the elucidation of these phenomena exhibited by human white blood-cells relates to the ectoplasm of the various types of these cells and to each of them in their different physiological states in health and in disease. This cursory review dealing mostly with vertebrate tissue-cells by no means pretends at exhaustion. Tho- roughly to treat the subject of ecto-endoplasmic differentia- tion as found in cells of multi-cellular animals would demand a volume in itself. But the review shows that from sponges to man tissue-cells exhibit ecto-endoplasmic differentiation; that the ectoplasm gives rise to processes which interlock with other cells; that the ectoplasm may be cast off to form inter-cellular substance; that it enters largely into the formation of contractile cells (muscles) ; that it is the conductive material par excellence of nerve-cells and that cells, as amoeboid blood cells, in their locomotor capacity indicate ectoplasmic action. Ecto-endoplasmic differentiation is by no means confined to the tissue-cells of multicellular animals. Very early in the history of the study of Protozoa, it was recognized that in these unicellular organisms the superficial cytoplasm is marked off structurally from the interior. Indeed, nowhere else in the animal kingdom is the ectoplasm so sharply defined by so rich and various sculpture. ^ Metschnikoff on amoeboid cells of sponges, iSjg. 87 THE BIOLOGY OF THE CELL SURFACE To Haeckel we owe the application of the term, exoplasm, to the superficial cytoplasm of the Protozoa. Writing in 1873 on the morphology of the Infusoria, he emphasized the differentiation of the protoplasmic body into a bright, firmer cortical substance and a granular, softer medullary substance, a differentiation found also in Amoeba as in many parenchyma cells of higher animals. This outer layer is further distinguished from the medullary portion of the cytoplasm through a lower water content and an independent contractility. Later authors, as Biitschli, Doflein and others, have merely reiterated Haeckel's original description, confirming it through their own observations. The protozoan ectoplasm may be highly differentiated: for support and protection; for motility; for food-capture; and for oral and anal modifications. In it are located the contractile vacuoles. On the basis of their means of locomotion Protozoa are classified into four groups: those like the Amoeba, that move by means of pseudopodia; those that move by means of flagella; those like Para- moecium that move by means of cilia, and, in a negative way, those like the Sporozoa, to which tl,ie organism that causes malaria belongs, which are characterized at least during one period of their life-history by lack of locomotor- apparatus. In other words, the classification of the groups is built upon ectoplasmic differentiation.^ ^ Though this book concerns itself with the animal cell, I may note in passing that in many plant cells, especially in the higher, multi- cellular, organisms, the living cytoplasm is entirely superficial in location, the interior of the cell being a vacuole of non-living materials. Also, I may call attention to the Bacteria which possess a strongly marked ectoplasm, investigated especially by Gutstein {ic}26 a7id earlier). The ectoplasm of Bacteria has been much discussed in connection with their structure in relation to the valuable diagnostic aid for identifying these organisms furnished by the Gram stain. (See Schumacher igjS and earlier.) 88 THE ECTOPLASM While the ectoplasm of the Protozoa has been well known for years, that of eggs has not been generally recognized, albeit eggs from every phylum in the animal kingdom either have been described as showing ectoplasm or can be shown to possess such. Since here we are concerned primarily with eggs, it is necessary to review in detail the evidence with respect to their ectoplasmic differentiation. The terms, exoplasm and endoplasm were first used by Haeckel to describe the outer and inner cytoplasmic regions of the sponge egg. In his monograph on the sponges referred to at the beginning of this chapter, he speaks of the hyaline exoderm (exoplasma) and a granular endoplasm sharply set off from it. Metschnikoff^ in describing eggs of a sponge emphasized its changes as due to amoe- boid processes of the surface-cytoplasm. Gatenby^ has described the ectoplasm of the sponge egg as follows: (Fig. 5). Even in the youngest oocytes one may notice that at an early stage a clear ectoplasmic zone becomes differentiated from an inner or endoplasmic zone. The ectoplasmic zone contains few or no vacuoles, is smooth, and is often drawn out in the form of blunt pseudopodia or filamentous dendri- form threads. The inner or endoplasmic zone is vacuolated completely and has a fine, frothy appearance; it is in this region that the cytoplasmic inclusions lie, granules in the ectoplasm of the oocyte being rare or never found. Occa- sionally, in preparations fixed in mixtures containing alcohol or acetic acid, the vacuoles collapse, and the egg comes to have a curious radiation of fibres around the nucleus (see Joergensen's figures 8a). Eggs treated with silver nitrate solution show the endoplasm browner than the ectoplasm. ... In favourable cases not only young oocytes, but amoebocytes, may be seen to possess ectoplasmic and endoplasmic zones. As in the case of the older oocyte, the cytoplasmic inclusions lie in the endoplasm, while the pseudopodia consist mainly of ectoplasmic material. ^ Metschnikoff, I.e. -Gatenbv, 1919. 8g THE BIOLOGY OF THE CELL SURFACE During development of the egg, all the blastomeres in most cases come to have an equal portion of the ectoplasm. The ectoplasm can be traced through cleavage up to the formation of a blastula, but it soon either becomes absorbed .^ •••** .»■♦-". Fig. 5. — Egg of a sponge, Grantia (after Gatenby) showing amoeboid ectoplasm. or is invaded by endoplasmic substance. ... In a rare number of cases, it was found that in young blastulae the cells of one side had less ectoplasm than those elsewhere but in no example could I show that this inequality had any relationship to the formation of the flagellated and non-flagellated parts of the amphiblastula. It., i - .-^i.A-^ -. ^-^ >-i >■ v-T ■ •-'■ - ■ ■.■-■■...■■■" ,' -y K-i.--.'X< ■ .■ r-r.r,.>.v'.-^,-~.-.i:-- I ■ ■ %^^ V■/Vr?A:^..^■••i••■•^<^s^i^■ \ :■:■':: ■■■::'.'.' -aft;5;'i^-K-tr-4-;i>:- r^#^ ■••■■. '■. ■■■■. ■■: ; .>-^ ■ .•/- *"^«^^^*«^*^ '■^■■•^.;:.:,.^'''' a i f Fig. 6. — Eggs of medusae (after Metschnikoff). a, Liriope mucronata; b, and c, Rathkea. In the cytoplasm of eggs of all classes of Coelenterates, including the Ctenophores, that is, the jelly fishes and their allies, ecto-endoplasmic differentiation has been frequently 90 THE ECTOPLASM noted. (Fig. 6 and Fig. 7.) Kowalewsky^ without naming the two regions gave a very clear description of them as found in several ctenophore ("comb-jelly") eggs. Sub- sequently, authors have confirmed Kowalewsky,^ often only repeating the words of this great observer. Says Agassiz: The yolk mass of the eggs of Idyia (and all Ctenophorae) consists (after they are laid) of two layers, an inner yolk mass more or less fatty, made up of large, irregular spheres. This inner yolk mass is surrounded by a second thin, outer layer, finely granular. This outer layer and its enclosed central mass perform very different parts in the development of the embryo, and it is of the utmost importance to keep the changes these two layers undergo, clearly distinct, while following the development of the young Ctenophore. The outer layer as has been shown by Kowalewsky, is eminently the embryonic layer, while the inner mass acts as a mere nutritive yolk mass. FoF also described ecto- endoplasmic differentiation in Coelenterate eggs, using the term, ectoplasm, instead of Haeckel's exoplasma. Perhaps the most striking ectoplasmic structure described in eggs is that found in the Beroe-egg noted first by Chun and later Fig. 7. — Egg of a ctenophore, by YatSU.^ The ectoplasm in ^-^^/''^^'^''^ (^fter Kowalewsky). this egg is of a bright green colour, a fact that one ^ Kozvalezvsky, 1866; iSy^. See also Kowalewsky and Marion, 1883. ^ Agassiz, iSj^. ' Fol, 1873. ^ Chun, 1880; Yatsu, 190J. For other observers on the ectoplasm of Coelenterate eggs see: Metschnikoff, 1886; Ziegler, i8g8; Appelof, igoo; Hargitt, 1904; Spek, 1^26; Conklin, Cam. Publ., 103; Ciamician, i8'/g; Korotneff, i88q; Harm, 1903; Maas, igoS; Torrey, igoy; Komai, ig22. 91 THE BIOLOGY OF THE CELL SURFACE can easily confirm under low power of an ordinary microscope. Indeed, I have with the naked eye seen the green cytoplasm on this rather large egg; also have I found that under pressure the endoplasm, which Kowalew- sky did not consider living substance, will stream out as the large q^^ ruptures leaving the green surface-cytoplasm intact. The diflFerence between the peripheral and the inner cyto- plasm of eggs of flat-worms has been often observed,^ though some authors fail to do so, and still others have expressed the opinion that the structural differentiation observed might be due to fixation.^ Many flat- worm eggs present serious difficulties for study in the living state because they are laid in capsules each containing several eggs; removal of the eggs from the capsule may mean their injury. In addition, because of opacity, they do not always lend themselves to observation of their finer structure. Nevertheless, one may assert that eggs of turbellarians and trematodes and even of those of cestodes (tape-worms) exhibit a resolution of the cytoplasm into an endoplasmic and an ectoplasmic region, as Metschnikoff, Selenka, Lang, Pereyaslowzewa and Janicki, among others, have shown. The thread-worms, nematodes, include Ascaris, a para- sitic worm which has been the object of many researches and which will come up for reference frequently in this book. The unfertilized egg of Ascaris shows, accord- ing to Van Beneden and Meves, ectoplasmic differen- tiation; irregular in shape, after fertilization it becomes spherical having in the meantime secreted a substance which forms an enclosing shell. The eggs of free-living nematodes, closely allied to Ascaris, show a similar change; ^ Metschnikoff, /SSj; Selenka, i88j; Lang, iSS^; Pereyaslowzewa, iSg2; Warren, igoj; Janicki, igoj. - Halkin, /go2. 92 THE ECTOPLASM on fertilization, bubbles in the cytoplasm break through the surface which undergoes amoeboid changes, then the eggs attain a regular contour.^ Cerebratuhis, a long ribbon-like worm found in the sea, lays eggs in which the cytoplasmic granules in the endoplasm differ from those at the periph- ery.- This egg also displays remarkable amoeboid changes^ which always speak for the presence of an ectoplasmic structure. Eggs of the wheel animal- cules or Rotifera according to several investigators show a distinct ectoplasm.^ In Yatsu's interesting ac- count of the development of the egg of the mollusk-like animal, Lingula,'' is a de- Fig. 8. — Egg of Lingula anatina (after tailed description together latsu). with pictures showing the surface-located cytoplasm set off from the endoplasm. (Fig. 8.) According to Prouho,*^ the egg of Membranipora possesses delicate projections which he figures. In the unfertilized eggs of the echinoderms (starfishes, serpent starfishes, sea-urchins, sea-cucumbers, sea-lilies) the ectoplasm is well defined. Selenka" has described the changes which a sea-urchin egg undergoes before it reaches the stage when it is ready for fertilization. These ^ Biitschli^ iSjj; also v. Erlanger, iSgiJ and later authors. ~ Coe, i8gg. •^ Andrews., E. A.., iSgj; Wilson^ C. B., iSgg and others. ■* Zelinka, l8g2; Storch, /g2^ and others. '" Yatsu, igo2. ^ Prouho., i8g2. " Selenka, 187S. 93 THE BIOLOGY OF THE CELL SURFACE Fig. 9. — For descriptive legend see page 95. 94 THE ECTOPLASM changes largely concern the ectoplasm, as the appended figures (9) show. Though I have sought to obtain these stages in other species of sea-urchins, I have so far failed. In mature echinoderm eggs the ectoplasm, though difficult to observe, can be made out in the living egg as a homo- geneous extremely narrow rim beneath the vitelline mem- brane. In fixed preparations it is only very faintly marked oflF from the endoplasm. After fertilization the ectoplasm stands out clearly because of the now present hyaline Fig. 9. — Development of the ectoplasm in the egg of Toxopneustes variegatus (after Selenka). 95 THE BIOLOGY OF THE CELL SURFACE plasma-layer which though it has often been described^ (Fig. lo) has never, as we shall see in the chapter on cell- division, been properly interpreted by those who have observed it, and generally been misunderstood by those who have on the basis of experiments on it made theories concerning its role. For eggs of the brittle starfishes and of the sea-cucumbers, Selenka has given good descriptions, pointing out that the layer is most pronounced in eggs of the brittle starfishes, less in those of sea-urchins and least in sea- cucumbers'. I find that in these as in the eggs of the starfish the ectoplasm can Fig. io. Fig. ii. Fig. io. — Egg of Echinocyamus pusillus (after Theel) to show structure of ectoplasm after full separation of vitelline membrane following penetration of a single spermatozoon. Fig. II. — The ectoplasm of the egg of Rhynchelmis limosella (after Vejdovsky and Mrazek). always be distinguished in sectioned eggs fixed with certain reagents for then minute granules appear; also the delicately radial structure is evident. 1 turn now to the eggs of the segmented worms. Very strikingly does the ectoplasm differ from the endoplasm in the egg of Rhynchelmis'^ as pictured here (Fig. ii). But no less clearly or remarkably do the inner and outer regions in the egg of Phascolosoma^ reveal themselves. The ecto- ^ Hertwig, i8j6; Selenka, i8y8; Fol, iS/p; Ludwig, 1880; Selenka, i88j; Berthold, 1886; Theel, i8g2; Hamrnar, 1896; Andrews, /c?p7; Ziegler, 1904; Meves, 1914. 2 Vejdovsky, 1892; Vejdozvsky and Mrazek, 1903. ^ Gerould, 190"/; Bergmann, 190^, on annelids. 96 THE ECTOPLASM plasmic structure of the Rhynchelmis egg is similar to that recorded for the Nereis-egg by several workers beginning with Goette^ and this in turn recalls Spengel's older descrip- tion for the egg of Bonellia.^ Also the Chaetopterus-egg has an interesting ectoplasm,^ so too that of Thalassema^ (Fig. 12). Among the great group of soft- bodied animals, the mollusks, including the clams, snails and ink or cuttle fishes, the following produce eggs in which the ecto- riG. 12. — Ectoplasm on the plasm has been observed to be living egg of Thdlassema mellita marked off from the endoplasm: (after Lefevre). Mactra" and Mytilus^ (clams), various species of Chiton^' Patella,^ (a snail) (Fig. 13), Dentalimn^ (a scaphopod) and the cuttle-fishes (cephalopods).^" In the eggs of Mactra ^ Goette, 1SS2; V. Wistinghausen, iSgi; Wilson^ iSg2; Hempel- mann, igii; Lillie^ F. R., igi2. 2 Spengel, iSyg. '^ Lillie, F. R., igo6. ■* Torrey, igoo; Lefevre^ igoj. ^ Kostanecki, igo^. ^ Meves, igiS- ^ Kowalewski^ iSjS. ^ Patten, iS8^; Jorgensen, igij. ^ Wilson^ igo4. '" Bergmann, I.e. 97 THE BIOLOGY OF THE CELL SURFACE the outer sheath of cytoplasm contains large granules, whilst in that of Mytilus according to Meves' figures the ectoplasm in the fixed egg is a clear margin bounded by a homogeneous and apparently firmer sheath of cytoplasm beneath the very thin vitelline mem- brane. In figures of the eggs of Chiton also one distinguishes ectoplasm from endoplasm. In addition, my observa- tions convince me that in the eggs of the razor clam, Ensis, and of the small clam, Cumingia, the outer region of the cyto- plasm differs from the inner. The large ellipsoid eggs of cephalopods — ink-fishes and allies — show a disc of clear cytoplasm containing the nucleus at the upper more pointed end which is continuous with a very thin layer enclosing the inert yolk. Disc and layer constitute the active sub- stance of the egg; in them development ensues; it is as though the cytoplasm in these eggs were all ectoplasm. Among eggs of arthropods are those of insects,^ whose ectoplasm is even more strongly marked than that in eggs of Fig. 14.— Egg of //y- coelenterates. Beyond we shall note drophilus pisceus (after , , ^ , , j^gjjgx that the ectoplasm 01 these eggs has most interesting behavior. Appended is a picture from Heider showing the ecto-endoplasmic differ- entiation in the egg of a beetle (Fig. 14). Eggs of other arthropods clearly show ectoplasm. ^ Among the lowest forms of Chordates, the Ascidians, we find that the beautifully transparent egg of Phal- 1 Heider, i88g. - Groom, 1894. 98 THE ECTOPLASM lusia'^ reveals ecto-endoplasmic differentiation. The egg of Amphioxus^ likewise has an outer sheath of cytoplasm marked off from the inner (Fig, 15). The ectoplasm of the latter e.gg as pictured by Sobotta recalls that already above described for the e^g of Nereis or of the sea-urchin. That all other authors do not note cytoplasmic differ- entiation in the egg of Amphioxus one may attribute Fig. 15. — Ecto-endoplasmic differentiation in the egg of Amphioxus (after Cerfontaine). either to oversight or to the quality of fixation. The separation of the vitelline membrane exemplified by this egg constitutes additional evidence for similarity of its ectoplasmic structure to that of the other eggs named. However, this membrane separation has seldom been observed because of the difficulty of securing the early fertilization-stages for observation. ^ Meves, igi3. - Sobotta, iSg'j; Cerfontaine, igo6. 99 THE BIOLOGY OF THE CELL SURFACE Ascidians and Amphioxus belong to the chordates with- out back-bone or vertebral column. Fishes, amphibians, reptiles, birds and mammals are chordates with a vertebral column. The lamprey is an example of the low vertebrate, having no true jaws to the mouth as other vertebrates have. Fig. i6. — Egg of Pelrotnyzon fluviatilis (after Herfort). Its egg has been described by several investigators, among these Calberla, whose beautiful work on fertilization of the lamprey egg is classic. According to him this egg shows regional cytoplasmic differentiation.' See also Herfort. (Fig. i6.) ' For eggs of selachians (sharks and their allies), His has written as follows: 1 Calberla, iSjj; Bhhrn, iSSS: Herfort, rgoo. 100 THE ECTOPLASM It thus seems practical to speak instead of kinds of plasma, of zones of the cell-body and we can distinguish: A central condensed zone. ... A zone of network. . . . A hyaline cortical zone. . . . ^ In the egg of teleosts (bony fishes) the protoplasm, at first a thin sheath enclosing the yolk, moves at fertilization to one pole to form a disc leaving only a delicate layer to surround the remainder of the yolk mass.- Here the rela- tions are as in the eggs of cephalopods. Living eggs of amphibians (frogs, toads, salamanders) have been long recognized as showing a differentiated surface due to the presence of pigment extending over approximately one-half the egg. In sections amphi- bian eggs show further differentiation of the superficial cytoplasm.'^ In the eggs of both reptiles and birds the protoplasm is sharply set off from the large mass of inert yolk. The description given by His for the selachian egg and quoted above applies to the eggs of reptiles and birds. Recently, Lewis and Hartmann have described the organization of the living egg of the monkey."* Other eggs of mammals, as that of the mouse, bat, rabbit, etc., also show some difference between the peripheral and the central cytoplasm. In addition to these accounts which describe eggs of every group of animals as having ectoplasm, we should note those studies which show that ectoplasmic structure is revealed by experimental treatment. Hammar's descrip- tion of the ectoplasm on the sea-urchin's tgg as composed of radial striations was based on observations on eggs in 1 His, 1897. " List, iSSj. This process was very beautifully described as early as 18^4 by the English physician, Ransom. ^ King, igoi. Earlier: Goette, 187^. •* Lewis and Hartmann, IQJJ. lOI THE BIOLOGY OF THE CELL SURFACE sea-water made more concentrated by evaporation. In calcium-free sea-water, the ectoplasm of sea-urchins' eggs fairly bristles with delicate filaments. In other eggs also the presence of these filaments can be demonstrated by experimental treatment — especially by placing the eggs In hypertonic sea-water. It Is to be emphasized that the experimental means does not cause the formation of these filaments but only makes them more easily visible. Thus, In eggs of Nereis and of Platynereis the ectoplasmic fila- ments, seen In the unfertilized eggs as ectoplasmic strlatlons and for a short time after fertilization as strands, can always be more strongly revealed In later stages, when they are seen with greater difficulty, by submersing the eggs In sea-water made hypertonic by the addition of NaCl or KCl. The same observation has been made on eggs of other worms, for example, on those of Sabellaria (Fig. 17). The often ex- pressed opinion that the ectoplasmic prolongations thus made visible are Fig. 17. — Egg of Sabel- r , • i i i r laria alveolata to show artefacts leaves Unconsidered the fact effect of hypertonic sea- that the cggs show normally an ecto- water on the ectoplasm pi^smlc Structure, as has been abun- (after Faure-Fremiet). dantly proved for eggs from those of sponges to those of vertebrates. The descriptions In the literature admit of no doubt on this point. When we place alongside this fact the observations detailed by students of tissues In culture on the strongly expressed ectoplasm, the generally accepted fact of ecto- plasmic structures In Protozoa together with the evidence of Intercellular connections between cells In tissues and the knowledge of the mode of union of nerve-cells by processes, we have very good reason for asserting that the filaments and prolongations of the ectoplasm are Its characteristic, and perhaps its most Important, structure. They have 102 THE ECTOPLASM often been compared to protozoan pseudopodia, especially to those that are extremely tenuous and fine. Years ago in a work that never has received adequate attention, Mrs. Andrews^ emphasized the capacity of these threads for ceaseless changes which she spoke of as a spinning activity of the protoplasm. The tremendously impressive fact of the existence of ecto- plasmic structure finds here for the first time its proper appreciation. A structure which can rightfully be denom- inated a sine qua non of living matter can be presumed to have significance for the grand problem of biology, the revelation of vital phenomena. In the following pages I aim to establish the thesis that in ectoplasmic behavior we witness the expression of activities that set apart the living thing from the non-living, that mark how life main- tains itself ever in harmonious tempo Avith the ceaseless changes in its surroundings. ^ Andrews, I.e. 103 5 General Properties oj the Ectopias/// CL/2 LTHOUGH WE CAN NOT TO-DAY ANSWER THE QUESTION, what is life? we can say what are the manifestations of the state of being alive. Respiration, conduction and contrac- tion are fundamental properties of living matter, exhibited by all living cells. Life never appears without them. Whilst inanimate things as well as organisms after death may display properties like these, only the living thing main- tains them in self-regulation through continuous adjust- ment. Now the question arises: to what extent are these biological indices, these manifestations of life, properties of the ectoplasm.^ Study of the egg's behavior in the first moments after sperm-contact will throw light on this problem. The attachment of a single spermatozoon to an egg and the effect produced thereby can best be observed micro- scopicalh' b}' adding a drop of sea-water containing very few spermatozoa to sea-water that contains eggs. To eggs of Arhacia spermatozoa attach themselves within two seconds after the)^ are added to the sea-water containing the eggs. Under the impact of a spermatozoon the egg- surface first gives way and then rebounds;^ the egg-mem- brane moves in and out beneath the actively moving spermatozoon for a second or two. Then suddenly the spermatozoon becomes motionless with its tip buried in a slight indentation of the egg-surface, at which point the ectoplasm develops a cloudy appearance. This turbidity 1 Cf. Dcrhes. I.e. 104 GENERAL PROPERTIES OF THE ECTOPLASM spreads from here so that at twenty seconds after insemina- tion— the mixing of eggs and spermatozoa — the whole ectoplasm is cloudy. Now like a flash, beginning at the point of sperm-attachment, a wave sweeps over the surface of the egg, clearing up the ectoplasm as it passes; careful observation reveals that the ectoplasm now shows a brush- work of threads beneath the membrane. Twenty-five seconds after insemination, a cone of ectoplasm protrudes from the egg and encloses the sperm-head. This is sud- denly pulled into the cone; the ectoplasmic threads break at the site of sperm-attachment and release the membrane. Progressi^'ely from this point the membrane separates in a wave from the surface of the egg, leaving in its wake collaps- ing ectoplasmic strands. Thirty seconds after insemination the membrane is separated from the egg by a narrow peri- vitelline space. During the ensuing twenty-five seconds this space increases in width; the ectoplasmic strands become more sharply defined giving the ectoplasm the appearance of a striated layer. The vitelline membrane becomes equidistant from the Q.gg at all points and the perivitelline space is at its greatest width one hundred twenty seconds after insemination. This progressive lifting of the membrane, first noted by Derbes,' I have studied in several species of eggs in great detail.- On the basis of experiments with physico-chemical means which induce membrane-separation, certain investi- gators ha\'e simply assumed the de novo origin of the mem- brane which thev called the "fertilization-membrane." However, careful observation shows, as described above, that the membrane is already present on the egg and separates at fertilization by a progressive wave beginning at the point of sperm-entry. It thus is not a "fertilization-membrane," ^ Derbes, 1847 . See also Fol, iSyg. -Just, JQiQa, ig2i, i()22e, ig2Sc and f, ig2ga and b. 105 THE BIOLOGY OF THE CELL SURFACE but properly the vitelline membrane. I have repeatedly followed the process in the egg of Arbacia, though it is not the most favorable form for this study. In the eggs of the genus Strongylocentrottis, of Echinus and of Echinarachnius, for example, the separation of the membrane is more easily followed. If eggs of Echinarachnius be inseminated with thin sperm- suspension, they throw off their membranes that are d e Fig. 1 8. — Successive stages of membrane-separation in the living egg of Echi- narachnius parma, site of sperm-entry. fully lifted and equidistant from the egg-surface in about two minutes. Membrane-separation follows sperm-pene- tration. After the sperm-head has entered the ectoplasm, a blister appears on the egg-surface at the point of sperm- entry. This blister contains drops that move toward the membrane and go into solution. The process is gradually continued throughout the whole ectoplasm, and thus the membrane is lifted in a wave that sweeps over the surface (Fig. 1 8). By break-down of material in the ectoplasm, beginning at the site of sperm-entry, the membrane is separated from the surface. After this membrane is fully io6 GENERAL PROPERTIES OF THE ECTOPLASM off the &gg, the ectoplasm again builds a new surface-struc- ture, the hyaline plasma-layer. Before the actual separation of the vitelline membrane, an ectoplasmic change beginning at the point of sperm- entry sweeps over the egg which immunizes it to other spermatozoa, the pole opposite the site of sperm-entry being the last point affected. The site of sperm-entry becomes a "point of injury" and is "negative" to the entry of spermatozoa arriving at this point, all other points around the egg for a brief moment being "positive." Especially in slowly reacting eggs it can be noted that this "wave of negativity" moves over the egg, at a rate which varies with that at which the sperm-head disappears within the ectoplasm. When only the tip of the sperm-head has entered the ectoplasm, the immediate vicinity of the site of penetration can not engulf another spermatozoon. As more of the sperm-head disappears within the ectoplasm, the "negativity" to sperm-entry progresses still farther around the egg; when the head has disappeared, the egg can engulf sperm only at one point — the pole opposite that at which the spermatozoon entered the egg. The "wave of negativity" thus precedes the actual beginning of membrane-separation. Before the membrane begins to separate at the site of sperm-entry, other spermatozoa can no longer enter at any point on the egg. From the point of sperm-entry a definite gradient of membrane-separation is established. This gradient, therefore, follows that of loss of susceptibility to sperm-penetration. When a spermatozoon becomes attached not only the penetration of other spermatozoa but also their fixation to the egg depends upon the degree of penetration of the effec- tive spermatozoon, that is, on the rate at which the "wave of negativity" is propagated around the egg. Thus, at the beginning of penetration, other spermatozoa can not become attached to the egg in the immediate neighborhood of the 107 THE BIOLOGY OF THE CELL SURFACE point of sperm-entry. Those farther removed may become attached, lashing back and forth very vigorously, until with farther penetration of the one successful spermatozoon the "wave of negativity" reaches them and their movement comes to a stand-still.^ The ectoplasmic changes in the egg of Nereis that take place after sperm-attachment are also worthy of note. Before insemination the ectoplasm of this egg is a broad chambered structure. When eggs and spermatozoa are mixed, -sperm-attachment rapidly ensues; then follows the escape of material from the ectoplasm, which rapidly sets in the sea-water as a transparent jelly. The ectoplasmic chambers break down, leaving only strands that cross the space between the inner part of the egg and the membrane. Now comes a period of amoeboid changes in the egg:- it shrinks, becomes markedly irregular in outline, its contents become darker and the perivitelline space much reduced. When this period is over, the t^^ assumes a clearer and more rounded appearance; the perivitelline space widens again. The delicate plasma-membrane beneath the vitelline mem- brane is now easily visible. In this egg, thus, the ectoplasmic changes differ from those observed in the eggs of Arbacia and of Echinarachnius . In all three however the essential response to insemination is the same, namely, an alteration in the ectoplasm. These changes have significance for fertilization and experimental parthenogenesis as we shall see. At present, another aspect of them elicits our interest. By experiments with dilute sea-water, I was able to cor- relate the structural changes at the surface of the eggs named above with an alteration in the physical state of the egg. I found, for example, that the egg of Echinarachnius 1 Just, 1919. - Cf. Torrey, igoj on amoeboid changes in eggs of Corymorpha. loS GENERAL PROPERTIES OF THE ECTOPLASM is many times more susceptible to dilute sea-water during the period of membrane separation than the fertilized e^g before or after this separation, or than the unfertilized &gg. The egg is never again so susceptible, although during the cleavage-cycles it exhibits rise and fall in susceptibility which may be correlated with the rhythm of nuclear division. We may consider in detail this sus- ceptibility to hypotonic sea-water first as seen in the egg of Echinarachnius. If to a drop of sea-water containing unfertilized eggs of Echinarachnius mounted under low power of the micro- scope, tap or distilled water be added, one can observe that the eggs take up water, swell, and finally break down in about two minutes. For example: the time of disintegra- tion in tap water for lots of eggs from ten females was as follows : Number of females Time in seconds to disintegration of the eggs while in tap water 270243 3 60 4 113 5 150 148 7 256, 247 9 155 ID 240 The rate of disintegration in eggs from these same females exposed to the action of tap water, five to ten seconds after insemination, was about the same. Eggs from the same females exposed to the action of tap water two minutes after fertilization, i.e., after the membranes are completely off the eggs, withstood the exposure even better than the unfertilized eggs. But with the beginning of membrane separation, twenty to thirty seconds after insemination, the picture is quite otherwise, for then the eggs are highly susceptible. The following table gives the results of such an experiment made on eggs of the same females as were used in the above described experiment. Once it was well established by experiment with tap water that the period of high susceptibility falls in exactly with the period of membrane separation, attention was log THE BIOLOGY OF THE CELL SURFACE Number of females Time in seconds to disinte- gration of eggs exposed to tap water during mem- brane-separation I 2 3 4 S 6 7 8 9 H 17 19 20 9 II 18 16 7 10 directed to the susceptibility of the egg to less dilute sea- water at different stages of the process of membrane separa- tion. In the experiment with the tap water it appeared that when the egg cytolyzed, the break always came in that part of the ectoplasm from which the membrane was lifting at the time of exposure. However, the cytolysis induced by tap water was far too rapid to allow exact observations Fig. 19. — Diagrams showing disruption of egg-contents at site of membrane- separation in egg of Echinarachnius parma. a is a fertilized egg in which mem- brane-separation has not begun. of this phenomenon. The experiments with less dilute sea-water proved that the observation made during expos- ure to tap water was correct: when eggs are exposed to dilute sea-water during the period of membrane-separation they cytolyze by an outflow of cytoplasm at the points from which the membrane is lifting at the moment of exposure (Fig. 19). Thus the susceptibility travels at the same rate as the wave of membrane-separation. As stated above, membrane-separation in the normal process results from the solution of substances in the ectoplasm. Under the microscope one can easily follow droplets of these as they move across the perivitelline space before they completely disappear. Any point on the egg-surface where this dis- solution is taking place becomes the point of susceptibility at the instant of exposure to dilute sea-water or tap water. The most remarkable characteristic of this susceptibility during membrane-separation is its sharp localization. I no GENERAL PROPERTIES OF THE ECTOPLASM have made observations on thousands of eggs and have yet to see an egg exposed during membrane-separation break in those regions from which the membrane is not lifting. This is especially well brought out by exposing eggs during the early stages of membrane-separation; for then only that part of the egg from which the membrane is just lifting, at or near sperm-entry, is susceptible to dilute sea-water. Similarly, any part of the egg from which the membrane has fully lifted is resistant, as is strikingly shown by exposing eggs just at the moment when the membrane is being lifted from the last point on the egg-surface. Then the break-down is only at this point; the zones from which the membrane is already off are resistant. When the membrane is fully off, the egg leaves the period of susceptibility. We may say, therefore, that a wave of resistance to dilute sea-water follows in the wake of the wave of susceptibility. There is an exceedingly rapid restitution-process in the ectoplasm following a momentary loss of resistance through the normal break-down of material in the ectoplasm which pushes off the vitelline membrane. If we attempt to come to some conclusion as to the mean- ing of this susceptibility, we must take into account the following facts: First, the resistance of the ectoplasm in those regions from which the membrane has not yet sepa- rated; second, the very rapid recovery of the ectoplasm after membrane separation; third, the apparent failure of ^ the ectoplasm at the site of sperm-entry to show any greater susceptibility than the remainder of the egg-surface. In brief, we must keep in mind that this susceptibility is, clearly localized; or, what is more correct, that the break in the t^g which is the expression of decreased resistance to the hypotonic sea-water is only at the site, and in the moment, of membrane-separation. Now this does not mean that only at this point water enters the egg. Rather, it indicates that the point at which the membrane is sepa- III THE BIOLOGY OF THE CELL SURFACE rating is the point at which the ectoplasm is weakest in that moment. In the zone of membrane-separation it is dis- continuous and shows a break. The normal process of membrane-separation being due to a secretory process is bound up with water movement. As the ectoplasmic material goes into solution, it is washed away. In the normal process just enough water is present for this behav- ior. When the egg is placed in dilute sea-water, the picture is different; the normally occurring recovery in the zone of membrane-separation can not take place, and the ectoplasm breaks down further. Moreover, with the ectoplasm now gone, the endoplasm is without protection and complete cytolysis results. We are thus here dealing with a sensi- tivity due to actual progressive dissolution of colloids in the ectoplasm of the egg. The case of the egg of Arbacia is very interesting. Here differences in resistance to dilute sea-water shown by the unfertilized and by the fertilized egg at the time of mem- brane-separation are not so clear-cut. I was, however, able to prove that the period of membrane-separation also in these eggs is a critical one, by studying the swimming larvae reared from eggs treated with dilute sea-water before, during and after membrane-separation.^ Fertilized Arbacia eggs exposed to tap or distilled water before or after mem- brane-separation and then returned to normal sea-water give rise to normal larvae. But eggs exposed to the tap or distilled water during the period of membrane-separation on return to normal sea-water show marked abnormalities in the late stages of gastrulation. There is here, therefore, definite evidence of a differential susceptibility which appears in clear-cut fashion; dilute sea-water exerts a decidedly deleterious action during the process of membrane- 1 Just, /(jjSc. 112 GENERAL PROPERTIES OF THE ECTOPLASM separation and the egg is thereby so profoundly altered that the normal processes of gastrulation are disturbed. The unfertilized eggs of Nereis limbata withstand treat- ment with tap or distilled water for three or four minutes before disintegration takes place. Beginning twenty-five minutes after fertilization, the eggs are even more resistant than the unfertilized. But during the twenty-five minute period immediately following the mixing of eggs and sperma- tozoa, the eggs show a very low resistance for they dis- integrate within ten to sixty seconds after exposure to tap or distilled water. The surface-changes described above for this Q^^ run over this period of twenty-five minutes. Thus, the period of susceptibility to hypotonicity exactly coincides with the period of break-down of material in the ectoplasm — with release of the jelly-forming material — the amoeboid changes and the darkening of the egg. When the &%% rounds up and clears, the period of lowered resistance to dilute sea-water passes oflf. These experimental findings on three forms— and I have used eggs of other forms as well — though they show varia- tions, nevertheless point to one conclusion: the period of ectoplasmic changes following fertilization is one of pro- found physical alteration. The changes that I have dis- cussed here which follow the attachment of the sperm-head to the egg-surface sweep over the egg as a wave of explosions that break down material in the ectoplasm. During this period of visible alteration of the egg-surface occur other measurable physico-chemical changes, as oxygen-consump- tion and heat-production. These we may consider briefly. The oxygen-consumption by eggs during various stages of development following insemination has been investi- gated by several workers.^ Shearer'- has reported his find- ^ Warburg, Loeb and Wasteneys, Meyerhof, Shearer. ^ Shearer, ig22. 113 THE BIOLOGY OF THE CELL SURFACE ings on oxygen-consumption by the eggs of a sea-urchin during the first minute after their insemination. Says Shearer: "On addition of the sperm to the eggs there is an immediate consumption of oxygen. In the course of the first minute the uptake of oxygen is many times that of the same eggs one minute before the addition of the sperm, and more is usually taken up in the first minute than is taken up in the second and third minutes after the addition of the sperm taken together." Shearer thinks that this "great initial inrush of oxygen into the egg and a corre- sponding output of CO2 within the first minute after the addition of the sperm" make it clear "that the sperma- tozoon sets up an instantaneous oxidation-process in the egg, which is unparalleled in the reactions of the animal-cell for its sudden character." This period of a great initial inrush of oxygen established by Shearer's findings coincides with the period of surface changes described above during which time also the egg is so susceptible to the action of dilute sea-water. During this period the colloids in the ectoplasm go into solution, material is destroyed. It is this solution at the sur- face of the egg that is accompanied by the great rise in oxygen-consumption. Shearer has also investigated heat-production in fer- tilized eggs of sea-urchins. Rogers and Cole,^ using meth- ods of higher precision, have reported findings on the heat-production by the eggs of the sea-urchin, Arhacia. Their results on the heat-production immediately following insemination are of most interest to us. These workers find that the rate of heat-production at the instant of insemination is ten to twelve times that of the unfertilized egg. Thereafter the rate of heat-production falls con- stantly for twenty minutes to 65 per cent, of the value at ^ Rogers and Cole, 192^. 114 GENERAL PROPERTIES OF THE ECTOPLASM fertilization, remaining constant for the next thirty minutes to drop again by more than lo per cent., remaining constant as far as the eight-cell stage. Farther than this the obser- vations were not carried. Rogers and Cole's figure gives the approximate rate of heat-production. In their words, "the greatest period of heat-production occurs immediateh' upon fertiliza- tion."^ This period falls in with that of the surface changes described above. Say Rogers and Cole: "The fact that the greatest heat-production by the egg comes immediately after fertilization seems to us to make it plau- sible to say that the entrance of the spermatozoon induces a cortical oxidation-process, and that this process results in the elevation of the fertilization-membrane." On the basis of my observations and experiments, described above, these findings of Rogers and Cole can be more precisely explained. What they have measured is the heat liberated during the disintegration of the ectoplasmic colloids. In addition to increased oxygen consumption and heat- liberation other cellular processes can be related to these surface changes in eggs. The wave-like process of break-down in the ectoplasm by which the membrane is separated from the egg of Echhia- rachnius described above, strikingly resembles the trans- mission of change in various tissues. The nerve fibre may be taken as an example because among animal cells it is the most highly excitable and the most rapidly conducting. As is well known, nerve when stimulated at one point trans- mits this local effect throughout its course. As the propa- gated effect, the nerve impulse travels along the stretch of nerve, each point successively, beginning at that where the stimulus was applied, becomes electro-negative to all other regions of the nerve both behind and in advance of the ^ Rogers and Cole, I.e. THE BIOLOGY OF THE CELL SURFACE point. Thus an electro-negative wave sweeps along the nerve fibre as the impulse traverses it. During the transmission of the nerve-impulse there are no visible structural changes in the nerve. The propaga- tion of the effect of sperm-attachment over the surface of the egg, however, is clearly visible. Thus, in the egg of Arbacia a wave of turbidity followed by one of lightening marks its course. In the egg of Echinarachniiis it reveals itself: by the behavior of supernumerary spermatozoa that are in contact with an egg and are brought to a standstill progressively, the ones nearest the site of sperm-entry being first quieted; by progressive dissolution of ectoplasmic colloids; and by the break-down of pigment granules in the jelly that encloses the egg. Also the response of the ectoplasm during the period of membrane-separation to exposure to hypotonic sea-water reminds us of the action-current in a stimulated nerve. The high degree of susceptibility of the ectoplasm in the zone of membrane-separation and the resistance in the zones from which the membrane has not yet separated or from which it is completely lifted again suggest the electro- negative condition in the nerve fibre. ^ It may well be that this is more than a superficial resemblance; and for this reason these ectoplasmic responses would warrant further investigation. Whilst the egg passes through the period of surface changes under discussion, another property of the ectoplasm rises to sharp visibility, its contractility. Of the eggs described above, that of Nereis is the best in which to observe the shrinking and expansion following insemination. But this phenomenon can be followed in other eggs. Met- schnikoff years ago called attention to the contractile power 1 Lillie, R. S., 1922. 116 GENERAL PROPERTIES OF THE ECTOPLASM of eggs of sponges;^ Berthold^ clearly described it for the &gg of the sea-urchin as an unmistakable consequence of sperm-contact with egg. A year later O. and R. Hertwig suggested that eggs are endowed with contractility.^ The earliest description known to me of contraction in the ecto- plasm is that given by Ransom in 1854 who very clearly described it as a consequence of insemination, a work which has not received the attention it merits.'' That contractility is inherent in the ectoplasm of eggs can be readily demonstrated. Unfertilized Arbacia-eggs, for example, may be drawn out into long tenuous strands by putting them among fibres of lens paper. Since in this egg when it is unfertilized the endoplasm is highly fluid, this elasticity is due to the ectoplasm (including the vitelline membrane). The normal contour is readily regained without loss of fertilization-capacity. Paramoecia likewise can squeeze themselves through fine tubes and assume most bizarre shapes, returning again to the normal appearance when the pressure is released.^ Lillie,^ speaking of the egg of Chaetopterus, says: "The protoplasm of this egg is much more fluid than that of any other egg I have tested; in consequence, the egg elongates even with rela- tively low centrifugal force." And later "The egg is an elastic sphere; it therefore elongates in the direction of centrifugal force."'' I conclude from these statements that the elasticity of this egg is due to its surface. Mrs. Andrews^ attributes elasticity of cells to the ectoplasm. 1 Metschnikoff, iSjg. - Berthold, I.e. ■■' Hertwig, 0. and R., /SS/. ■* Ransom, I.e. 5 Just, igzSc. ^ Lillie, F. R., igoS. ^ Ibid., p. 72. ^ Mrs. Andrews, iSgy. 117 THE BIOLOGY OF THE CELL SURFACE These manifestations definitely coincide with the break- down of material in the ectoplasm and with the rapid recon- stitution whereby the egg-surface builds itself anew. They are fundamental physiological expressions of vital activity and have their cause in the visible structural changes at the egg-surface which have been observed both on the normal e^g and on that exposed to the action of dilute sea-water. Sperm-attachment is for the unfertilized egg the normal and best means of stimulation; as a stimulus the sperma- tozoon calls forth the same responses in the egg — oxygen- consumption, conduction and contraction — elicited by stimuli acting on other cells. This similarity I hold to be of fundamental significance for cell-physiology for I con- ceive these modes of response to be properties of the ecto- plasm of cells generally. The grounds for this conception follow. Few investigators to-day support the old theory of the nucleus as the seat of cellular oxidation. The human red blood corpuscle is pre-eminantly an oxygen-carrier; it has no nucleus. If there be evidence of increased accumulation of oxygen in the region of the nucleus of a cell, this more likely is caused by the presence of the nuclear membrane rather than by nuclear substance. Positive evidence indi- cates that oxygen-consumption depends upon cell-struc- tures, that is, upon minute surfaces in the protoplasm; even triturated cells consume oxygen.^ Then the cell surface constitutes, par excellence, an oxygen-uptaking structure. And since this surface is subdivided into processes and its area therefore increased many times, its effectiveness is enhanced. In higher organisms, as the vertebrates, the cells which take up oxygen are rich in surface — as cells of the gills in aquatic and of the lungs in terrestrial animals. ^ 1 Warburg, 1914. - The entire inner surface of a 7nan's lungs aviounts to about go square meters, more than 100 times the area of the skin. 118 GENERAL PROPERTIES OF THE ECTOPLASM Red blood corpuscles are discs, measuring seven to eight by two microns; they thus possess a large surface area, the sum of which amounts in an adult man of 78 kilograms, according to one estimate, to 3840 square meters. Hart- ridge^ has suggested that the shape of the red blood corpus- cle is the best possible one for insuring easy oxygen-intake. No one can object to the proposition that oxygen to enter a cell must cross the cell-boundary. Even on the theory that in oxygen-consumption only the most deeply located structures of the cell, nucleus or otherwise, are concerned, the oxygen enters the cell by way of the cell-surface. Rea- soning by analogy we may say that just as there exist special structures as gills and lungs to obtain oxygen, so in cells generally the modified surface permits easy access of oxygen. These surface-modifications are delicate prolongations. It is also interesting to note that pigment granules, said to be carriers of oxygen, line up at the cell surface. Oxidation means liberation of heat. The heat is lost by the cell-surface. Here again the cell-surface is admir- ably constructed for the rapid conduction and radiation of heat. Since heat is produced when colloids take up water and also when they liquefy, it may be that cell-surfaces in breakdown through swelling and liquefaction liberate heat. Another fundamental property of living substance is conduction. Any living cell when stimulated has the capa- city to transfer the effect of this stimulus. The degree of conduction constitutes one of the chief differences that dis- tinguish animals from plants. In animals conduction is the function of the nervous system, which possesses this property, general to all cells, in the highest degree. Now among other characteristics of nerve cells we note an extreme differentiation of the ectoplasm, as has been stated. Nerve cells show one or more processes, some of Hartridge, ig20. See also Ponder^ ^934- iig THE BIOLOGY OF THE CELL SURFACE which attain great length. Even the simplest unipolar nerve- cell, that is, a cell with one prolongation, is a cell in which the ectoplasm is drawn out into an extensive filament. The more richly branched cells and those with the longest prolongations show no cytoplasmic inclusions in the branches, i.e., the fibres; instead, the cytoplasmic inclusions are located in what is called the nerve cell body. Struc- turally, therefore, the nerve cell is not only rich in surface, but also in a special kind of ectoplasm. It is more than an accident that this structure appears in highly conducting tissues. The minute attenuated threads of nerve fibres are admirably adapted for transmitting impulses. I have already mentioned the work of Harrison who demonstrated that the growing ner^'e fibre shows definite surface changes at the growing tip.^ This tip eventually becomes the means by which one nerve cell establishes con- tact with another, the junction being known as the synaptic membrane. The fibre from one nerve cell does not pierce the cell body of another, but simply comes into delicate contact with it; and so the impulse passes from one nerve fibre to another. Conduction thus is carried on primarily by the fibre. Conduction in nerve cells, in other words, being a peri- pheral phenomenon, is not unlike that in an egg cell, since the interior of the egg cell is not involved in the conduction of the fertilization-stimulus. In free living unicellular ani- mals one can not always clearly identify conduction as a surface phenomenon because often conduction is followed so closely by contraction that it is difficult to separate the two processes. Nevertheless, there are forms in which it can be shown that the conduction is purely a surface phenomenon. It is to be borne in mind that conduction by nerve cells in higher organisms is not restricted to one cell. In the ^ Harrjso7i, I.e. 120 GENERAL PROPERTIES OF THE ECTOPLASM propagation of the nerve-impulse, for example, from a sensory ending which receives the stimulus to end-organ (e.g., muscle) which is aiTected, the impulse in traveling over afferent nerves to central nervous system and from the central nervous system to the efferent nerve, involves at least two and, more frequently, several nerve cells. The transfer of the impulse from nerve cell to nerve cell as well as the connection between the efferent nerve and the end- organ has been clearly demonstrated to be by means of fine fibrils. These, the prolongations of the nerve-fibre, are as clearly ectoplasmic as the fibres themselves. Thus, the integrative action of the nervous system is established by intercellular connections. I conceive all modes of inte- gration in the body of a complex organism, as the verte- brates and man, to be at basis dependent on intercellular — ectoplasmic — connections. The central nervous system integrates all systems of the body; these are at some distance from it and unlike it; the glands of internal secretion in animals which possess them, exercise even more remote control over one or another system. A more passive integration — and of a lower order, since it binds tissues only — is that by connective tissue. Finally, by means of intercellular connections, like cells, i.e., cells in the same sheet of tissue, are held together. These different modes of integration one can not think of as mutually exclusive; nor can one regard them as categori- cally distinct and separate from the point of view of func- tion. Only the order or level of integration — of all systems of the organism, of some systems, of organs, of a tissue — is a special, distinct one in each case. So much has been said by authors and in so great detail concerning nerve- and hormone-integrations, that no time here need to be taken to discuss these types of integration. Suffice it to point out that nerve action influences the chemical output of the glands of internal secretion and that the secretions in turn ma}' affect nerve-tissues; to an extent, therefore, they 121 THE BIOLOGY OF THE CELL SURFACE depend upon each other. Apparently, connective tissue exercises merely a passive restraint upon the subjoined tissues; it isolates and insulates. It is otherwise with the interdigitated cells in a given tissue. Their interdigita- tions constitute a means for bringing the cells directly into connection. On the side of theoretical biology intercellular prolonga- tions deserve our interest. Hammar suggested that by them the unity of the multicellular organism may be main- tained, seeing in them a basis for some support of Whitman's idea of the inadequacy of the cell theory for development, on which I have commented above; here I wish to point out the following: A multicellular organism, as a human being, is a unit and acts as such through integration. The cellular con- nections of the first grade in importance are those of nerve. To secure unified action of the organism is a function of the nerve cells primarily. Alongside with nerve integration, we place that by the hormones; for they come from glands upon which life depends, without which the organism dies; these, except the pancreas, are derivatives of or include derivatives of the nerve tissue or that from which the nerve tissue is derived, namely, the ectoderm; these glands are the pituitary body, the thyroid apparatus and the adrenal body.^ This nerve integration, as that of the three important hormones named above, is remarkable because of the fact of the ectodermal origin of its carriers. As we shall see in a later chapter, ectoderm cells arise from those cells in the developing egg which are richest in ectoplasm. Hence the highest form of integration in the complex multicellular ^ The internal secretion of the pancreas is derived from cells which are neither nervous nor ectodermal in origin and therefore the pan- creas cells arc an exception here. 122 GENERAL PROPERTIES OF THE ECTOPLASM organism is by means of ectoplasmic processes formed by originally ectoplasm-rich cells and by chemicals furnished by originally ectoplasm-rich cells. Now the integration of cell with cell — the simplest mode of integration — is by means of the ectoplasm. Thus it is evident that all forms of integration in the complex organ- ism are in the last analysis ectoplasmic. Contraction is like conduction a fundamental property of the living cell. The lowest forms of animal and plant life have capacity for contractility either permanently or at some time or other in their life-history. All the cells of a highly organized multicellular organism possess it in some degree. But this endowment may be reduced to such a low point that it is almost extinguished. With evolu- tion animals developed cells which were set apart as con- tractile tissues. These are the muscle cells in which this fundamental property of living substance is highly emphasized. Muscle cells are also characterized by having great length. The smooth muscle fibre is thicker than either type of the striated muscle. It is also the least responsive and shows the longest reaction-time. Theories of muscle-contraction that have the widest acceptance explain it as a surface-phenomenon. It is certainly true that contractile tissues have relatively large surface area. Their elasticity is undoubtedly a function of the surface — as in the case of egg-cells. The life-process of a cell is the sum and interaction of its manifold activities. The phenomena which together inhere in the state of being alive reveal themselves as activities of the protoplasm. No protoplasm exists without them. For respiration, conduction and contraction, the foregoing presentation offers proof that they are in particular mani- festations of ectoplasmic activity. That nutrition is like- wise bound up with the ectoplasm will become evident in the next chapter. 123 6 Water lI^^ater, quantitatively the most important com- pound in the inanimate world, and the most ubiquitous, holds the same place in the animate. Roughly, living cells can be said to be made up of two-thirds of water. Water is a structural part of every living protoplasmic system; life does not exist without it. Even when in a highly dessicated state, animals, such as rotifers and tardigrades, are not completely anhydrous. Living protoplasm is an aqueous colloidal solution. In view of the prevailing emphasis nowadays placed upon the colloid state of protoplasm, it is astonishing how little we know of the manner in which water forms a part of the living state. Is protoplasm an emulsoid colloid t To what extent is It a suspensoid colloid 1 Is it a structure in which water encloses other substances or one In which the other substances enclose water, or is It of both types t With these questions I refer to the ultra-microscopic structure of protoplasm — that is, of the ground-substance — for to this, as already pointed out, the colloid-chemist by his own definition of colloid-chemistry should address himself if he wants to study living substance, and not to the micro- scopically visible inclusions. The solution of many funda- mental problems in biology is postponed because of our Ignorance of protoplasmic structure. The colloid-chemist could render great service to medicine and biology by dis- sipating this ignorance. As I see It, the main significance attached to the study of matter in that physical state called the colloidal is that it deals with solutions; and where it 124 WATER concerns watery solutions it may give not only information concerning the physical properties of particles which lie within a certain range of size but additional knowledge of the chemical action of water in a system of colloid particles. The study of watery solutions is especially valuable inas- much as protoplasm is such a solution. Water also is a functional component in the vital reac- tions which take place in the cell. It plays a leading role in the utilization of food materials: when these are broken up into simpler compounds by the process of hydrolytic cleavage water is taken up; when in turn these hydrolyzed cleavage products are synthesized, water is lost. Whether in a cell this cleavage or synthesis will occur, depends upon the presence of water. Thus the movement of water from place to place in a cell hangs together with the direction in which the reactions run. Cellular oxidations demand the presence of water. Water is an end-product of all energy changes. Both conduction and contraction in cells depend upon water. In the removal of secretory products and in the elimination of excretory, water is necessary. Water thus is both part of the protoplasmic structure and a functional component in vital manifestations. In other words, it is bound up with the two fundamental problems of cell biology, protoplasmic structure and func- tion. The rhythm of water loss and water gain which every cell exhibits during its history, serves by these fluctuations, by its transitory nature, to emphasize the significance of water — to offer us, as it were, a key to the puzzle of the oscillatory changes which characterize life. Although dur- ing its development the animal egg progressively loses water — a dehydration imposed upon the rhythmical water-loss and gain — it by no means ever reaches the stage of complete dehydration. Its changing water-content therefore indi- cates to us a possible approach to the problems, how water 125 THE BIOLOGY OF THE CELL SURFACE forms part of protoplasmic structure and how it plays a role in protoplasmic activities. Since the structure of the whole protoplasmic system Is composed so largely of water, and its manifestations as a reacting system depend so greatly upon water, this structure must in some way dictate the distribution of water within the cell and hence determine the entrance and exit of water. When by chance I saw drops of water leaving an egg-cell, I was alert to seize the opportunity to follow by means of simple observations the fate of water within the egg.^ These observations will now be detailed and conclusions derived from them. Also suggestions will be made con- cerning the entrance of substances in solution into cells and concerning the question, why cells take In certain substances and not others. First, some mention should be made of method. When unfertilized eggs of marine animals are placed in dilute sea-water, they take up water and swell at a rate which depends, for specific eggs, upon the degree of dilu- tion. Thus, for the eggs of the sea-urchin, Arbacia, a favorite object for this type of work, the rate of swelling in 40 per cent, sea-water (60 parts tap water plus 40 parts sea-water) has been measured by LlUie- and confirmed by others. After thirty minutes In this dilution the eggs break down. If the sea-water is still more dilute, the water- intake is more rapid and the eggs' break-down occurs earlier. If the sea-water Is less dilute, the rate at which water enters is slower and break-down comes on later. Thus I found that in 60 per cent, sea-water Arbacia-eggs remain intact for twenty-four hours. Unfertilized eggs of Echinarachnius are more sensitive, being injured by dilutions which are innocuous to Arbacia-eggs. ^ Just, /g26b, ig2Sb, 1930a and h. 2 Lillie, R. S., 1916. 126 WATER According to Lillie, among any lot of unfertilized eggs of Arhacia which have been placed in 40 per cent, sea-water are some which disintegrate after a very short time whilst 50 per cent, disintegrate after thirty minutes. From this observation it is clear that, since more than half of the eggs are killed by this dilution, the action of the 40 per cent, sea-water on them is not reversible. Also, it is questionable that this dilution is innocuous to those eggs that do not disintegrate during these thirty minutes; they would scarcely return fully to normal condition when brought back to normal sea-water. Lillie points out that the eggs after eighteen minutes exposure continue to swell, i.e., fail to reach equilibrium with the diluted sea-water; instead they break down. Thus this dilution gives information concerning the rate of swelling only in injured eggs about to die. The very obvious question arises, namely, how much of the swelling is due to injury. It is thus difficult, if not impossible, to draw conclusions from these experiments concerning the behavior of normal eggs. Lucke and McCutcheon in their many calculations of the swelling of the Arhacia egg^ have likewise used 40 per cent, sea-water as well as other solutions including non-electrolytes (sugars). But inasmuch as to their work the same objections hold as to that of Lillie — it is almost certain that the sugar solu- tions used injured the cells further — we can not here con- sider it, although, for their purpose, the demonstration of the osmometer-properties of the egg, its moribund condi- tion is of no consequence. In all my work I was at great pains to fix very definitely the lowest limit of hypotonicity that a given marine egg can withstand with recovery to the perfectly normal condi- tion. The study of dead protoplasm as it exudes from an egg membrane because of pressure put upon it either by 1 Lucke and McCutcheon, iQ2g and earlier. 127 THE BIOLOGY OF THE CELL SURFACE mechanical means or by hypotonicity may be interesting and even fascinating and may allow the drawing of some conclusions concerning the normal processes in the living state. However, in the elucidation of the normal processes in the living state, observations and experiments on living eggs whose conditions are only slightly altered, are, as was said in a previous chapter, of higher value for drawing con- clusions than the study of dying or dead eggs. My studies, first made for another purpose, on several species of marine eggs of the rate at which the deleterious effects of hypo- tonic sea-water set in, led me to establish those hypotonic solutions in which the eggs did not break down though they remained in the solutions for hours; indeed, in these solutions these eggs remained intact as long as, or longer than, normal eggs in normal sea-water. We may for our purpose here at once dismiss those grades of hypotonicity which destroy the eggs.^ There are grades of hypotonicity which though allowing unfertilized eggs to survive for hours, do in time destroy them, as does normal sea-water which also finally is destruc- tive. Such solutions one must use with caution because what is essential in these studies is the most satisfactory demonstration that the action of the solution can be fully removed — i.e., that eggs after residence in it on return to normal sea-water are as normal as those which never have been in the solution. Usually one measures the size of the unfertilized eggs in sea-water after they are removed from the hypotonic medium; the return to normal size is taken as the criterion that the egg has recovered completely. This, I find, is not enough, I have pointed out that the great ' It -was deemed necessary clearly to establish in this elaborate fashion that the phenomenon occurred in viable eggs because after the first communication had been given and whenever demonstrations were made, the question, are the eggs ahve? was raised. 12S WATER advantage of using eggs instead of other cells, especially tissue-cells from a many-celled animal, lies in the fact that one has more and sharper criteria for determining that the cells are alive and normal. One very excellent criterion is the fertilizability of the egg. Eggs that after return from a hypotonic solution regain normal size, may sometimes not be capable any more of fertilization or of develop- ment. Or, as has happened in my studies, the hypotonic sea-water induces parthenogenetic development. Fertili- zation-capacity is then the best expression of normality. If on return to normal sea-water the eggs not only regain normal size but fertilize and develop as normally as eggs that never had been in hypotonic sea-water, we may con- clude that they have fully returned to normal condition. In the table appended figures are given to show how far eggs of Nereis recover after exposure to various degrees of hypotonicity as measured by the per cent, of cleavage and of swimming larvae. Table I. — Per Cent, of Cleavage and of Swimming Forms of Eggs Fertilized in Sea-water One Hour after Having Been Returned from Dilution of Sea-water, in Which They Had Been for One Hour* Per cent, of sea-water Per cent, of normal cleavage Per cent, of normal trochophore 60 94 96 SS 90 94 SO 100 86 45 96 80 40 91 85 33M 92 82 * This table represents one experiment of 10 made for each dilution. In most of the 60 experiments the controls showed 100 per cent, cleavage and more than 90 per cent, normal trochophores. If one bars accidents and uses proper care to guard the eggs against overcrowding and high temperature, they develop as if never having been out of sea-water of normal concentration. I2g THE BIOLOGY OF THE CELL SURFACE But I went farther in insuring the viability of the eggs. I used dilutions of a degree which allowed the eggs to fer- tilize and develop whilst in the dilution. Naturally, this development in the more dilute sea-water is far from normal. Nevertheless, if fertilization and development proceed in a solution, that solution is not as destructive as one in which both egg and spermatozoon are killed. The table appended gives figures to show the per cent, of development of eggs fertilized in hypotonic sea-water of various grades in which they develop. Table II. — Per Cent, of Cleavage and of Swimming Forms of Eggs Fertilized in Various Dilutions of Sea-water, in Which They Remain During Development* Per cent, of sea-water Per cent, of cleavage Per cent, of swimming forms 60 6S 61 5S 100 64 SO 92 79 45 98 82 40 88 60 333^ 96 66 * This table represents one experiment of 10 made for each dilution. In most of the 60 experiments the controls showed 100 per cent, cleavage and more than 90 per cent, normal trochophores. I spent a great deal of time in taking these precautions because I wished very clearly to establish that the eggs in which I observed drops of water were living cells capable of normal processes. Many times indeed I fertilized eggs immediately upon their return to normal sea-water — that is, during the process of their return to normal condition it was possible to initiate development in them; hence the drop-formation did not interfere with fertilization. Thus, these observations were made on viable cells; I can think of no source of error left unguarded. Now let us follow the process in detail.^ In the hypotonic ^ Although incidental observations had often been made earlier, the first systematic ones were 7nade in ig2^ on which the initial report published the next year zvas based. 130 WATER medium the eggs gradually swell so that finally their bulk greatly exceeds that of the normal q^^. With this increase in volume, the yolk-spheres, so striking in the normal &2,^, seem to disappear, actually they have changed to an unusual degree, losing their refringency and their discrete character; indeed, they may actually merge. These changes in the yolk are worth noting. What happens is this: The indi- FiG. zoa. — Section of a fixed egg of Nereis after 45 minutes' exposure to 4o''per cent, sea-water. The egg is swollen, the ectoplasm thickened and opaque, the yolk spheres are in process of fusion. (Figs. 20^2 to 20^ drawn by Mr. L. A. Hansborough from author's preparations). vidual yolk-spheres increase in size with a liberation of fine drops of oil, thus becoming almost transparent in appearance. If exposure goes farther, the yolk-spheres fuse. Yolk-spheres in fixed normal eggs are homogeneously blackened by iron haematoxylin; the yolk of the swollen egg also stained with iron haematoxylin is seen to be made up of fine threads within a distinct membrane, each yolk- sphere thus resembling a nucleus. The germinal vesicle 131 THE BIOLOGY OF THE CELL SURFACE (nucleus In the resting stage) is also swollen. The ecto- plasm is so narrowed that its fibrillae seem absent; in other o o a ^ %^^ ^ —" . - % *l Hk. IP .■^# (■ "^,M-i* ^M^ Fig. 2ob. — Section of egg, in the same stage as that of Fig. zoa, after having been returned to sea-water. The yolk spheres are regaining normal structure; water in drops moves toward the periphery. Fig. 20C. — Same history as that of Fig. 20b, but a later stage of development. words, the pressure of the egg-contents has reduced the width of the ectoplasm. On return to normal sea-water, the egg shrinks and the water-drops then appear. The 7J2 WATER s :4 R'^ -cfi ■■^;-S^'^> %? #: **'.* ^ «••• * *• fi Fig. 20(^. — Same history as that of Fig. 20c, but a still later stage of development. # -■r«- XL.y c\ M (^.:W^ .^ 'ia 'O W" ^ Fig. 20f. — Egg of Platynereis megalops showing water-drops in cells of a late cleavage-stage on return to sea-water following exposure to 40 per cent, sea-water for one hour. 133 THE BIOLOGY OF THE CELL SURFACE accompanying figure (Fig. 20) illustrates these changes as seen in fixed eggs. First the drops are at or near the egg- centre, then they move outward. As they cross the ecto- plasm they become elongated. Whilst the water-drops form and the egg regains normal shape, the yolk-spheres return to their original condition; they again become dis- tinct spheres in the living egg, appear blackened in the fixed. This change runs parallel with the movement of the fine oil droplets into the yolk. Thus the movement of water from the egg is associated with a marked behavior of the yolk-spheres. As the cells take up water, the yolk-spheres lose oil and increase in size, even fusing to make one mass. That is, the yolk- spheres take up water from the water-logged protoplasm. Then as the cells lose water to the surrounding medium, the yolk-spheres lose water to the cell and regain the oil from the cytoplasm. In the case of the Nereis egg the yolk-spheres possess a very exaggerated water-holding capacity. It would be a mistake, however, to conclude from this description that the yolk-spheres are the sole regulating mechanism for the water control in the cell. Water-drops also form in the nucleus, in the clear cytoplasm in cells of stages of late cleavage which contain no visible yolk-spheres, and likewise in the gut-cells of larvae after all yolk has dis- appeared. Other animal cells than eggs similarly I find show these drops of water. One factor which determines the strong expression of water-drop formation is doubtless the ectoplasm, because eggs and other cells that possess most pronounced differentiated ectoplasm show water-drop formation best. The rate at which the drops appear I have studied. A comparison of this rate in eggs in various stages is inter- esting: Drop-formation is more rapid in fertilized than in unfertilized eggs. It varies in fertilized eggs depending 134 WATER upon the stage in the division-cycle of the egg. The forma- tion of water-drops thus is also a cyclical phenomenon. From these experimental findings we may draw the fol- lowing conclusions concerning the behavior of water in these eggs. First, water leaves the cell as discrete drops. This does not imply that all the water that leaves the cell is in this form. But since, as is shown in these experiments, water leaves the cells in visible drops, a theory concerning the exit of solutions as ions can not apply to these drop-forma- tions. Visible drops of water are of more than even mole- cular size. Second, the change in shape of the drops as they cross the barrier of the ectoplasm suggests that they pass through canals whose diameters are less than theirs. Now the ecto- plasm shows chambers, the spaces between the radial pro- jections. Since the diameter of the drops is less than that of these ectoplasmic chambers, the latter can not be the canals concerned. The canals therefore through which the drops pass though not sub-microscopic are smaller than these ectoplasmic spaces. The experiments thus strongly indicate that under these conditions the ectoplasm is a sieve with extremely small openings. Third, the yolk-spheres take up water from the cyto- plasm. The yolk in the Nereis-egg is a mixture of lipoid and protein.^ This combination breaks down as water accumulates in the cell and enters the yolk-spheres. Lipoid escapes from the yolk-spheres into the cytoplasm. When water leaves the cell, water also leaves the yolk and the lipoid again enters the yolk. In hydration and dehydra- tion of the yolk the lipoid moves out of and into it. Oil moves out of the yolk as water moves into the yolk-spheres leaving them skeins of protein. As water moves out, oil ^ See Konopacki, iQ2g. 135 \ THE BIOLOGY OF THE CELL SURFACE moves in and the yolk-spheres assume their original physical appearance. These changes in the yolk-spheres show one very delicate mechanism by which cells hold water. Fourth, the yolk-spheres are not alone concerned in the water-holding capacity of the cell. The clear, apparently structureless, cytoplasm and the nucleus have the power of holding water and of losing it in the form of drops. It remains now to be decided how far we can apply these conclusions on the findings on eggs under experimental conditions to normal eggs in normal sea-water. Although these eggs of the experiment were viable and capable of recovery and complete development, they nevertheless were subjected to an experimental treatment. However, these experiments of mine described above were made with greatest care within limits definitely set; the processes described can not be attributed to extensive or drastic injury. There is thus less danger in making statements concerning them as explanation for normal conditions than in drawing conclusions concerning normal processes from experimental procedures which killed the eggs; the former only exaggerate, the latter extinguish the process that we wish to explain. If the process of drop-formation of water observed in these experimentally treated eggs is only an exaggeration of a normal process, it should be possible, I thought, to observe it both with other methods of treatment and in the normal untreated Qgg. As a matter of fact, I have found that the drops form as the result of pressure, of exposure to ultra-violet light and after removing the eggs from normal sea-water at 5°C. to that at room temperature (i9°C). During exposure to hypertonic sea-water, the drops are beautifully shown especially by fertilized eggs during every stage of development and by cells in the young worm. In unfertilized eggs in hypertonic sea-water the drops are neither so numerous nor so easily visible. 136 WATER In the untreated normal egg, drop-formation presumably occurs rapidly and the drops are neither so large nor so numerous as in experimental condition. Actually I was able to observe the appearance of drops in normal unfer- tilized eggs. They are much smaller and more evanescent than in the experimentally treated eggs. They appear more clearly in the fertilized than in the unfertilized egg and vary in rate of formation during the cleavage cycle. Drop-formation is thus also in the normal Q^g related to the physiological rhythmical changes coincident with the division-cycle. Since these findings indicate that the experimental means merely prolong and render more easily visible the more fleeting changes in the normal eggs, we may with more confidence use the experimental results for interpreting normal conditions and processes. The experiments have far-reaching significance for cell-biology in two respects: they permit conclusions concerning an important aspect of the problem of protoplasmic structure and concerning the question of the movement of water into and out of cells. Although we have some information on the struc- ture of protoplasm, we are far from possessing adequate knowledge of it. What is known of the chemistry of the cell does not suffice to tell us whether protoplasm is an emulsion, a suspension, a foam or a combination of these. As said above, an advance is made the moment that the ground substance is recognized as the cytoplasm par excel- lence. This appears as a menstruum containing extremely minute bodies. Since water makes such a large part of protoplasmic structure, we ought to seek to learn how it forms part of this structure: whether it encloses the par- ticles or the particles enclose it; or, in the words of the physical chemist, whether water is in the external or dis- persing or in the internal or dispersed phase. On this 137 THE BIOLOGY OF THE CELL SURFACE question the experiments showing that eggs of Nereis lose water in the form of drops throw some Hght. If we think of the water as in the external phase, the effect of increasing the density of the medium — from hypo- tonic to normal sea-water or from normal sea-water to hypertonic — would be either to cause streams of water to form rather than drops, or to free the water generally and equally everywhere from the eggs, so that the water would not be visible as it moves out of the egg. Since however drops of water form and since water can not appear in water as drops, we must assume that the drops are pressed out of structures. We conclude, therefore, that the water which appears in the form of drops is that which was held by solid structures, i.e., was in the internal phase. The formation of drops in normal viable eggs points to the same conclusion. As we have seen, the eg% on return from hypotonic to normal sea-water regains its normal equilibrium as it shrinks. In this process, inasmuch as drops of water leave the yolk-spheres we conclude that the yolk-spheres possess water-holding capacity and thus exercise function in the distribution of water in the cell under the condition of the experiment. Because it is not easy to discern yolk-free areas of the endoplasm in the unfertilized &gg of Nereis, one can not so readily determine to what extent water-hold- ing power resides in the cytoplasm. In the various stages of development — -cleavage, blastula, gastrula, larva (tro- chophore) and young worm — as the yolk becomes segre- gated into certain blastomeres leaving others yolk-free, it becomes easy to learn that yolk-free blastomeres or those in which yolk is not in visible spheres also hold water in the form of drops. Therefore we conclude that the clear homogeneous cytoplasm holds water within its struc- ture which exists in a state of fine subdivision. Moreover, the nucleus reveals water present in drops; these exist apart from chromosomes. Since the water drops can be demon- 138 WATER strated by several experimental methods we may say that slight experimental modifications of the protoplasm cause movement of water out of the structures which hold it. The conclusion is that water as an integral part of the col- loid structure of protoplasm is in the internal phase. This does not mean that no water exists otherwise in living protoplasm. On the contrary, we can only account for the observed initial shrinkage of an Qgg on return from hypotonic to normal sea-water as due to the invisible escape of water which doubtless represents, in part at least, water held as in the external phase. Thus we have answered the first question raised: how is water held in protoplasm 1 I may point out that these experiments as such have some significance for medicine. The distribution of yolk differs with different eggs, for instance in that of Nereis and Platynereis, as I have said above. Likewise, during its development the yolk changes in location as has been shown. Undoubtedly some of the difference in water- holding capacity noted among eggs of several species or in the same egg at different stages is to be attributed to this difference with respect to yolk. What is true of yolk may be true of other bodies in the cytoplasm. In diseased con- ditions as oedema and nephritis in which tissues hold an excessive amount of water, the structure of the cell may be a very important factor in determining this abnormal water content. I turn now to the consideration of the question of the movement of water into and out of cells. This calls for a statement concerning the nature of the cell-surface. When this question of the passage of water and dissolved substances into and out of cells is raised, especially in physio- logical work, by the term, cell-surface, usually is meant a membrane. This membrane is then spoken of as semi-per- meable because it is said to take up certain substances and THE BIOLOGY OF THE CELL SURFACE not others. Much of the work on which the theory of semi-permeability is based is derived from work on artificial inanimate membranes. Many investigators regard the cell membrane as a molecular film; others treat it as such in their physico-chemical and mathematical disquisitions. The chemical nature of the postulated semi-permeable membrane around cells has often been discussed. Depend- ing upon the one or another theory, it is considered protein, lipin, a mosaic of lipin and protein or a more complicated chemical structure. As was pointed out, to some the mem- brane is a dead, inert thing, a precipitation out of the proto- plasm, or merely a surface conditioned by the external medium, etc. For others the membrane is a living part of the cell-structure. In the above given descriptions of eggs, a vitelline mem- brane has been spoken of. This is built by the egg as it develops to maturity and is of measurable width. Beneath this vitelline membrane can be seen in many eggs a very delicate structure, appearing in optical section as an extremely thin line, the plasma-membrane, also built by the egg; it is visible and of measurable thickness and not a molecular film. The surface-structure of the cell is, how^ ever, not to be thought of as comprising only this plasma^ membrane. The deformation of the water drops as they leave the egg takes place in the surface-structure below the plasma-membrane. This cell-structure is the ectoplasm. On these eggs as on so many others it displays amoeboid activity. Changes in a plasma-membrane and certainly those in a molecular film can not account for the rhythmical difference in permeability so often described and postu- lated,^ for which, however, the ectoplasm, both in width and in activity, offers a sufficient basis. 1 For examples of rhythmical changes, see Bayliss, /p/J. 140 WATER In general, while it has been postulated that the differ- ences in permeability noted both in a specific egg during different stages of its development, as before and after fertilization, and in cells of the same type, e.g., red blood cells, but from different animals, are due to the membrane, and that the cytoplasms are the same, the actual and visible changes in the surface-layer of cells have been ignored. Despite the excellent observations made by Mrs. Andrews^ on the spinning activity of the ectoplasmic surface, which others have abundantly confirmed, this type of rhythmical surface-activity no one has sought to correlate with the postulated rhythmical changes in permeability. Though the measurable ectoplasmic layer of cells differs in width, the size of the layer has not entered into the calcula- tions made on the entrance of water. The cell is a bag of watery solution; and being capable of deformation with return to its normal contour and size, this property of elasticity is resident in the surface-cytoplasm. But the elastic property of the cell-surface has been deliberately ruled out in the mathematical calculations on the entrance of water into cells. On the theory that the difference in rate at which water enters a specific cell in different stages depends upon the permeability of the cell-surface, i.e., the plasma-membrane, we should expect a most exhaustive analysis of the structure and structural changes of that surface — certainly we should scarcely expect that in addi- tion to ignoring these the theory would actually discount the elasticity of the cell-surface. Vitelline membranes are often chitin or chitin-like sub- stance; it may be that generally they are protein; the plasma-membrane also may be protein, lipin or what else. These considerations lose in interest in view of the fact that 1 Mrs. Andrews^ 1S96. 141 THE BIOLOGY OF THE CELL SURFACE that cell-structure which controls ingress and egress is the ectoplasm which in large part has chemically the same make-up as the remainder of the cytoplasm since endo- plasm and ectoplasm are one continuous, though differen- tiated system. The question which disturbs the physiologists who say that there must be a membrane in order to keep the cell- contents from flowing out and becoming miscible with the surrounding medium may be answered here. The cell- contents withstand outflow because the cell material is one continuous system. It is not like mercury which is now one mass and then many drops which flow together again; rather, protoplasm is a cohering though extremely thin dilute jelly-like solution, whose biological character is revealed by its strong regional differentiation. Excreted or secreted material, including colloids, gets out of a cell when it is no longer a living part of it. It is broken off and dispelled. Instances in support of this statement are not wanting. In egg cells we have seen that at fertilization the colloids in the surface break down and escape through the vitelline membrane. Material moves out of cells by secretion — that is, the cell breaks off part of itself and washes this part away. The largest question in the permeability problem relates to the entrance of substances into the cell. If we attempt to answer this question we do it on the basis that the cell- surface is living ectoplasm, continuous with the remainder of the protoplasm, and is made up of a brushwork of filaments. In part, the question how substances enter cells is bound up with another, namely, why some substances and not others get into cells. For example, in the gut and in the kidney salts or sugars when present in equimolecular con- centration do not cross the cell-surface in the same amount. These two cases constitute stock arguments of the vitalists 142 WATER against a strictly mechanistic point of view concerning the entrance of substances into cells. The normal living cell carries out certain reactions in which water plays a part; it, therefore, needs either to take in water or to get rid of it. Also in cyclical changes, as those of division, in which the nucleus breaks down and reforms, water moves back and forth between nucleus and cytoplasm as well as between yolk and ground-sub- stance. Whilst the cell-contents may vary from moment to moment with respect to water present, the water within a cell tends to maintain a certain level characteristic of that cell. This level varies with different cells. Thus, the water content of human cells differs: the enamel of the teeth, spermatozoa, and bone cells are poor in water while cells of the liver, kidney, intestine, etc., are rich in water. The level also varies in a given cell with its activity or stage in life-history. Thus, gland cells when actively secreting and when at rest show different levels. An egg-cell will show different levels at different stages of development. Finally, the level varies with changes in the surrounding medium. Every cell tends with respect to water to come into equilibrium with its surround- ings. The level at which this equilibrium establishes itself depends upon the cell's specific composition, stage in its life-history and its activity at a given time. Since the water level within the cell tends to remain con- stant under the changes brought about by reactions, water moves in or out in order to maintain this level. In a way the movement of water between cell and environment is comparable to the movement of oxygen; it is a diffusion- phenomenon for water moves to the region of most concen- trated material, i.e., from the region of more to that of less water. The air which human beings breathe is composed of 79 per cent, nitrogen, 20.96 per cent, oxygen, less than one per 143 THE BIOLOGY OF THE CELL SURFACE cent, carbon-dioxide together with the rare gases, helium, argon, krypton, in traces. The permeability of the cells of the lung for these gases is an important property for without the entrance of them into the lungs and their subsequent conveyance to all parts of the body where oxy- gen is given up to each living cell, life would cease. The air breathed out contains the same 79 per cent, nitrogen, but only 16 per cent, oxygen and an increase of carbon- dioxide amounting to 4.38 per cent. All of us appreciate that the figures reveal how much oxygen the living cells use and need and how much carbon-dioxide they form and get rid of. The passage not only of water into and out of normal cells in normal condition, but also of substances in solution may be compared to the entrance and exit of these gases. Some pass in and out again in equal, others in diminished, and still others in increased amounts. Without the entrance of food-stuiTs and the exit of waste the cells would die. The phenomena of significance are the utilization of incom- ing materials by the cells and the excretion of effete. What has been said concerning water, therefore, may be said of material in solution as salts, sugars, amino-acids. These enter or leave the cells according to the level at which they are present in the cells. They form part of the protoplasmic structure and also take part in reactions going on within the protoplasmic boundaries. If their concentration in the cell is low, the cell retains them as they come in. In order to learn what substances a cell uses, we should not merely inquire what substances get into a cell, but also what of these remain in it. We may imagine that more of a given substance in solution in the surrounding medium passes into cells than remains in them.^ ^ The mistake is often made of attempting to learn zvhat can get into cells by too severe treatment of them, injuring them by subjecting them to solutions of too great concentration. 144 WATER If the cell-membrane were permeable only to water, none of us could live because the cells in our bodies would receive only water. The physiologist therefore has finally to postu- late a membrane whose temporary break-down allows sub- stances other than water to pass.^ But I think that there is more to the problem than mere passage across an inert boundary. The complexity of the protoplasmic organiza- tion and the many reactions going on in the protoplasmic system must determine, in part surely, what substances coming in shall remain. What determines the ingress of substances is the cell-surface. By this is not meant a membrane, semi-permeable or otherwise, a dead or living molecular film, but the whole ectoplasmic structure with its innumerable filamentous prolongations of living active cytoplasm. Every cell's ectoplasm is built up of such prolongations, as has been abundantly shown in the chapter on the ecto- plasm. The fact that this discernible, richly filamentous structure exists, taken alone suffices to render untenable ^ Bayliss, 1922, p. J12: " Js a further case of absorption, at all events as it appears to me, the cell-membrane or plasma-membrane may be considered. This is not to be regarded as a fixed permanent structure, but as produced by deposition of cell-constituents which lower surface energy at the interface between protoplasm and sur- rounding medium. Thus, it changes with cell activity and is in equilibrium zvith the cell contents as they alter. Thus, there is no difficulty in the membrane becoming permeable in the active state of the cell to substances to which it is impermeable in rest. Moreover, when a fresh protoplasmic surface is produced by mechanical action, a new membrane is naturally deposited on it. This is no doubt why large particles can be taken up in phagocytosis through a membrane which does not permit even sodium chloride in solution to pass. The particles actually break the membrane, which closes again behind them, in the same tvay as a needle can be passed through a soap film, without bursting it, whereas a gas, such as hydrogen, nearly insoluble in the soap solution, only passes zvith extreme slowness." 145 THE BIOLOGY OF THE CELL SURFACE theories based on a membrane of whatever hypothetical physico-chemical composition. The presence of the fila- ments means that the exposed cytoplasmic surface is enormously larger than if there were only a smooth membrane, and at the same time that it forms a sys- tem of capillary spaces. But this surface is not merely a static structure. The cytoplasmic processes which char- acterize the ectoplasm are indeed fine pseudopodia and as such display constant activity. By means of them the cell has phagocytic power, ingests fine particles. Phago- cytosis is- indeed an activity exhibited by all animal cells and not only by Amoebae, white blood cells and fixed tissue phagocytes as those in the vertebrate liver.^ The moment that we appreciate the normal structure and behavior of the ectoplasm, the problem of the entrance of water and of solutions is placed in a new light. The theories of cell- permeability with their discrepancies and conflicts can with profit be abandoned. Since the ectoplasmic pseudopodia respond actively to the environment they regulate the exchange between cell and external medium. All these considerations and data indicate that the sur- face-cytoplasm can not be thought of as inert or apart from the living cell-substance. The ectoplasm is more than a barrier to stem the rising tide within the active cell- substance; it is more than a dam against the outside world. It is a living mobile part of the cell. It reacts upon and with the inner substance and in turn the inner substance reacts upon and with it. It is not only a series of mouths, gateways. The waves of protoplasmic activity rise to heights and shape the surface anew. Without, the environ- ment plays upon the ectoplasm and its delicate filaments as a player upon the strings of a harp, giving them new forms and calling forth new melodies. But these are too nice for the undiscriminating ear of man. 1 Cf. Geddes, iSSj. 146 7 The Fertilization-process C^yiLL ANIMAL AND PLANT FUNCTIONS CENTER AROUND two processes: nutrition and reproduction. The first is concerned with the blindly egoistic struggle of the individual to preserve itself. The second relates to the altruistic struggle of organisms to perpetuate their kind. Despite their interdependence the processes of nutrition and repro- duction have different values for the organism. Without food or the apparatus for the utilization of food, the organ- ism dies; without the reproductive apparatus (as sys- tems, organs, tissues or cells) sexual organisms can still live. The reproductive (germ) cells are sharply set off from all other (somatic) cells; they have the special burden of the perpetuation of the species. The sex-cell is therefore a thing apart, a tenant housed by mortal somatic cells and like them mortal while the tenancy lasts. House and ten- ant have a common origin. Their separation constitutes the first of the series of those differentiations that mark the development of an individual animal. The adult animal is derived from one cell, the egg, which by the process of cell-division becomes a mass of cohering cells. They form the germ-layers; these give rise to the various organs (or systems of organs) which compose the complex adult individual. In the course of this develop- ment are set off from all the other cells which make up the body of the animal certain ones, the primordial germ-cells, whose function is to produce either eggs or spermatozoa in bisexual animals or both of these in monosexual or herm- aphroditic animals. Thus, the germ-cells, first differenti- 147 THE BIOLOGY OF THE CELL SURFACE • ated from the somatic, are in their turn differentiated from each other. The primordial germ-cells pass through a period of mul- tiplication which ends with the production of many cells called ovogonia (in the female) and spermatogonia (in the male) whose nuclei still possess the somatic number of chromosomes, i.e., the number characteristic for the species. Ovogonia and spermatogonia without increase in their numbers are transformed into primary ovocytes and pri- mary spermatocytes respectively by the pairing of the chromosomes in each nucleus; thus the somatic number of chromosomes is "reduced" to one-half, the gametic or haploid number. Then follows the period of growth which is especially expressed in the egg. The maturation (meiotic) divisions succeed the period of growth and differ somewhat in the male and female sex-cells. In maturation usually each primary spermatocyte divides into two secondary spermatocytes and each of these again into two cells which are called spermatids. Hence a pri- mary spermatocyte gives rise to four spermatids. By a series of cytoplasmic changes together with nuclear con- densation the spermatids become spermatozoa. Only spermatozoa are capable of fertilizing eggs, whilst many eggs, as we shall see soon, can be fertilized before, after or in various stages of their maturation. The maturation (meiosis) of the egg occurs as follows: at the end of the growth-period two divisions of the primary ovocyte follow each other paralleling those in the primary spermatocyte but the cells so arising are markedly unequal in size. The small cells, the polar bodies, contain nuclei enclosed by a minimum of cytoplasm. Of the three ovo- tids (or four, if, as sometimes happens, the first polar body divides) from one primary ovocyte there is only one, the mature e.^%, capable of fertilization; the two (or three) polar bodies are abortive eggs. 148 THE FERTILIZATION-PROCESS This brief account of the history of the germ-cells sub- sequent to their differentiation from the somatic cells teaches us that they pursue diflFerent courses. Without going into details, I call attention further to three facts of general significance for all that follows in my presentation of the problem of fertilization. The first is that there does not exist a single animal whose spermatozoa do not originate from an egg; the second, that no animal spermatozoon ever develops alone; and the third, that fertilization is not indis- pensable for the development of animal eggs, for, as we shall see in the chapter on parthenogenesis, many eggs develop normally or experimentally without the presence of sper- matozoa. These three facts obviously warrant the con- clusion that the egg carries the greater burden in fertilization. That the spermatozoon is itself derived from the egg is a fact to be emphasized. Even where it is indispensable for the egg's development, we can not in the light of its origin regard it as the perfect antipode of the egg. Whatever its highly specialized powers and functions, they rest upon derivatives of the egg-substance whence the spermatozoon came. 7'hat the spermatozoon never develops without the egg, whilst the egg can develop without the spermatozoon is another way of saying that the egg alone carries the bur- den of development. In these differences between the gametes we recognize where lie the powers not only of fertilization, but also of the whole course of the future development; the spermatozoon is a cell reduced to the minimum potency through its differentiation, the Qgg is of all cells known the most potent by virtue of its peculiar differentiation. Structurally, the great difference between spermatozoon and egg relates to the cytoplasm. Out of the egg's cytoplasm the future adult organism is differ- entiated. I propose in the following pages to prove that fertilization, the initial act in the differentiation of the egg, is likewise a cytoplasmic phenomenon. In order to reach 149 THE BIOLOGY OF THE CELL SURFACE this definition, I examine (i) the structure of the sperma- tozoon and of the egg when they normally come together, (2) the changes in both subsequent to union and (3) their behavior at the time of union. The present chapter embraces the discussion of (i) and (2), the succeeding chapter confines itself to (3). For the majority of multicellular animals, the coming together of the male and female germ-cells or gametes, spermatozoon and egg, guarantees the perpetuation of the species. This coming together of the gametes is fertiliza- tion in the widest sense, without which the spermatozoa of all and the eggs of most animals die. In this meaning fertilization marks the beginning of a life though of course both gametes are alive; it is the beginning of a new indi- vidual brought into being through the loss of individuality of each of the two cells which are co-partners in the process. If fertilization in all animals which exhibit it were to occur in the same mode and at the same time, our attack on the problem of fertilization would not be so difficult; but we encounter many differences. Always in biology must we reckon with differences and seek to determine to what extent they are "incidentia" or "differentia." The com- plexity and diversity of animal structure and behavior are either "incidentia" and as such defy reduction to simple terms or, as "differentia," they mask some one common factor to which we can reduce them. That there is one feature common to all fertilization-processes we shall see after we have evaluated the differences in the structure of the gametes as well as those in the time and the mode of their union. The partners in the fertilization-process are as described above markedly different from each other in their develop- ment to the moment of their coming together. In one respect only are they similar: their nuclei contain each one- half the number of chromosomes characteristic of the adult 150 THE FERriLIZATION-PROCESS organisms from which the eggs and spermatozoa come. In this bringing together of the haploid chromosome- garniture from the mother and the haploid from the father, fertilization not only insures the constancy of the species' characteristic chromosome-number, but also, since the chromosomes are concerned in heredity, it maintains equi- librium between paternal and maternal inheritance as far as this is determined by the chromosomes. This similarity of nuclear structure in egg and in spermatozoon has thus significance for the development that results from ferti- lization, but as a static, non-changing factor it can not be related to that radical and far-reaching change in the egg that we denominate fertilization. We, therefore, turn to the discussion of the other characteristics of the partners and shall find in them many differences. Animal spermatozoa exhibit great diversity in structure. In general, they may be classified as flagellated and non- flagellated. The larger number of species of animals have spermatozoa of the former class. Typically one such sper- matozoon is made up of a head (the nucleus) which may be spherical, bullet- or lance-shaped, to whose anterior end generally is fitted a cytoplasmic cap, the so-called per- foratorium, which may be very blunt, as in the starfish- spermatozoon, or more spike-like as in the Nereis- or the Cerebratuhu-speiTaatozoon. The cytoplasm located behind the head is called the middle-piece; it varies greatly but often contains one or more granules of a lipoid nature. The tail or flagellum, the third cytoplasmic structure, is continuous with the cytoplasmic film enclosing the sperm- head and may be of very great length; it is the locomotor appendage of the spermatozoon. Non-flagellated spermatozoa may have the form of a truncated cone. Or they are more definitely amoeboid in form, as in crustacean spermatozoa such as the lobster's, crab's, etc. That is, instead of the blunt contracted form THE BIOLOGY OF THE CELL SURFACE of the ^JT<2ru-spermatozoon, those of these crustaceans present more slender projections resembling the finer pseu- dopodia of an amoeba. These spermatozoa are highly interesting because they are first inverted and explode at fertilization.^ Spermatozoa despite these great variations in structure have certain features in common: they possess very little cytoplasm although the volume of this may be greater than the nuclear volume. Compared to the egg, any species of spermatozoon is extremely minute. One fact already mentioned above is to be emphasized again: sperm-cells capable of fertilizing eggs are always fully matured — that is, they are always spermatozoa and never spermatocytes or spermatids. Whereas most, probably all, species of animal sperma- tozoa are motile, with few exceptions eggs are incapable of locomotion. Whilst, moreover, animal eggs vary in size from a few microns in diameter to several centimeters (e.g., eggs of cursorial birds), the smallest species of eggs are larger than the largest species of spermatozoa. This greater bulk of the egg is due to its cytoplasmic mass with inclusions, oil, yolk, mitochondria, etc. If these are sus- pended in the cytoplasm they may amount to about two- thirds of the egg-volume; if they are pendent to the cyto- plasm, as in birds' eggs, for example, they make up all but an extremely small fraction of the egg. No eggs, including the so-called transparent ones, are entirely yolk-free. In comparison with spermatozoa, animal eggs as a class at the time of fertilization contain more cytoplasm and are richer in reserve or food materials, yolk and oil. As was pointed out, eggs in contrast to spermatozoa can be fertilized before, during or after their maturation, depending upon species. We distinguish four classes of ^ See especially Kolzoff, igio. THE FERTILIZATION-PROCESS eggs according to the stage in maturation in which they are fertilizable. Class I : The &%g reaches the stage just prior to the matu- ration-divisions. This is the so-called germinal vesicle stage characterized by a large nucleus. It there comes to rest and dies unless fertilized. Mere contact of the sper- matozoon is sufficient to cause the break-down of the germinal vesicle and the initiation of the maturation divi- sions. Eggs of various worms, Polys to mum, Gyrodactylus, Ascaris, Sagitta, Nereis, Platynereis, Thalassema, Myzo- stoma, and of the clam, Mactra, are examples of this class. Class 2: Here belong eggs which will not fertilize as long as the germinal vesicle is intact. They develop as far as the stage of first maturation and there remain until death unless fertilized. The eggs of the sea-worms, Thysanosoon, Prostheceraeus, Chaetopterus, Phascolosoma, and of the clams, Cumingia and Mytilus as well as eggs of snails and of the ascidians, Ciena and Phallusia, are examples of this class. Class 3 : Some eggs finish the first maturation before they reach the stage in which they are capable of fertilization. They come to rest with the second maturation spindle formed, one polar body having been extruded. Here belong eggs of many vertebrates; likewise the egg of the worm-like chordate, Amphioxns, regarded by some zoolo- gists as the ancestral form of the vertebrates. Class 4: Eggs of sea-urchins constitute an example of this class in which fertilization is possible only after comple- tion of both maturation divisions. These are called fully matured eggs. It is here necessary to make a sharp distinction between maturation, referring to the phenomena of polar body formation, and physiological ripeness, referring to the capacity of the egg-cytoplasm for fertilization, which as this classification shows reaches its optimum at various stages in the maturation-process (Fig. 21). 153 THE BIOLOGY OF THE CELL SURFACE ■ ■ ■ ■ - - ■ ■ — The egg of the starfish is interesting. Whilst other eggs, as we have seen, have a very sharply limited period during which fertilization is possible, the egg of this animal, whose optimum period for fertilization is just after the dissolu- tion of the germinal vesicle, is still capable of fertilization at later stages of maturation and for a time after complete maturation. But inasmuch as the fertilization-process c d Fig. 21. — Diagrams to illustrate the four fertilization classes (after Wilson). a, Class I; b, Class II; c, Class III; d, Class IV. either before or after maturation is below normal, this egg belongs to Class 2. It is evident that an explanation of fertilization must cover these four classes of eggs. A theory that covers the fertilization of the sea-urchin egg and not that of eggs fertilizable in the germinal vesicle stage and in subse- quent stages of maturation would demand a separate explanation for each of the other three classes. This in turn would mean that fertilization diflFers in different eggs — 154 THE FERTILIZATION-PROCESS that, widespread though the phenomenon is, it would reveal no common factor. This point of view is often maintained for all animal biology, some workers going so far as to say- that every animal and every egg is a law unto itself. How- ever, such a point of view does not properly envisage a phenomenon as widespread as fertilization. It is our pur- pose to look beyond differences, to seek a common factor. This can not be related to a particular stage in maturation, as our classification shows. It thus becomes necessary to describe briefly the process of fertilization as it occurs in a representative of each of the classes in order to learn if any feature is common to eggs of all classes. Said other- wise, having examined the structural make-up of the co-partners in the moment when they come together in the act of fertilization, we address ourselves to an exposition of the events that follow this coming together. At this point one general word concerning the way in which egg and spermatozoon meet may not be amiss. Eggs of animals, in which the sexes are separate, may be laid before the spermatozoa reach them. In such cases the eggs are shed into the sea — or fresh water — with or without copulation between male and female. In other cases, eggs are reached within the female's body by sperma- tozoa deposited within her genital tract; here copulation between the sexes is the rule. Eggs in these cases may be deposited at once to undergo development outside the ani- mal's body. Or they may remain within the female where they undergo development completely or in part. The fore- going is true also of normally hermaphroditic animals — i.e., those in which the sexes are united. It should be added that among these, eggs and spermatozoa produced by one individual may unite, so-called self-fertilization; sometimes this is brought about by the presence of some structure that prevents the access of spermatozoa from another indi- vidual to the eggs. In many hermaphroditic animals self- 155 THE BIOLOGY OF THE CELL SURFACE fertilization can not occur because eggs and spermatozoa are not ready for fertilization at the same time. The failure of self-fertilization among hermaphroditic animals is more often assumed than proved.^ None of these modes by which &gg and spermatozoon are brought together can be correlated with the stage in maturation at which the egg- cytoplasm is "ripe" for fertifization. Nor can we say that fertilization and development within the organism are peculiar to higher animals. Among sponges, for exam- ple, the lowest form of animals that produce eggs and spermatozoa, the eggs pass through early development in the parent organism. That tapeworms, members of the third lowest group of multicellular animals, are character- ized by their strong male copulatory organs, is a fact which discredits a popular notion that copulatory organs are found only among the highest animals. Thus, the mode by which the union of egg and spermatozoon is accomplished has no special significance for the events that follow this union. In the exposition of these events, which now is given for each class, we shall begin with contact of egg and sperma- tozoon and end with the first cleavage of the egg. These are common points. Between lie those events whose differ- ences call for evaluation. I request the reader to note them in order the better to appreciate the discussion of their sig- nificance. Named in order, these events are: the initial changes at the surface of the &%%'■, the arising of two star- like formations, the asters, associated with sperm- or egg- nucleus (with or without a discrete body, the centriole, at the centre of each aster); the coming together of the egg- and sperm-nuclei (sometimes called pronuclei); and the formation of the cleavage-spindle. ^ Cf. Just., ig^^b, and earlier workers. THE FERTILIZATION-PROCESS The statement made in the chapter, Life and Experi- ment, that we lack many details concerning the happenings in normal biological processes, can be proved when the history of fertilization in the various examples given below is reviewed. Although I have endeavored to choose a representative for each class whose fertilization is best known, we shall see that in no one have all the steps been completely followed. Fertilization is far from being a sterile field of research; there is no single animal egg for which the events, from the moment of contact of egg and spermatozoon to first cleavage, have been so adequately described in closely set stages that we can say that we pos- sess full information of the chain of events as a continuous process. The description of fertilization in four eggs, that now follows, will give us merely the chief outlines of a pic- ture which we shall try to make more complete by adding lines and details from the process in other eggs. Only then shall we draw conclusions and enter upon the discus- sion of fertilization. I describe fertilization as it occurs in the egg of Nereis limbata found along the Atlantic shores of America; it represents eggs of Class i.^ The fertilization of the egg of this easily obtainable marine worm can be controlled since the eggs are discharged freely into the sea where they are immediately mixed with the spermatozoa; one needs merely to collect the mature males and females separately and then to place them together in pairs at timed intervals to obtain fertilized eggs in a series of as closely set stages as one desires. One draw-back exists, namely, that sexually mature animals can not be found throughout the summer months when the ^ This account is based in large part on that given by L^llte, igil and IC)I2. THE BIOLOGY OF THE CELL SURFACE animals breed. Like many other forms, Nereis exhibits a lunar periodicity in its breedins; behavior and is sexually mature only during the period from full to new moon of each lunar cycle from June to September (at Woods Hole, Mass.).^ Although "ripe" eggs of Nereis limbata are available only during this particular moon-phase, their abundance and the clock-like precision of their development make them ideal objects for observation and experiments on fertiliza- tion. The fertilization-pro- cess as seen in the living egg is as follows: When discharged or re- moved from the female the egg measures about lOO by 80 microns. It reveals in optical section at the centre a large formation, the germinal Fig. 22.-Drawings from photo- vesicle. Around this are graphs of Nereis eggs in a suspension of Chinese ink in sea-water (after Lillie). greenish spheres, the yolk, a, before insemination; Z), three minutes among which are larger re- after insemination, c- . IT 1 •! 1 irmgent bodies, the oil drops. Beyond the area of yolk and oil is a rim, the ectoplasm, made up of coarse strands disposed in a somewhat radial fashion which extend to the clearly discerned vitelline mem- brane. The eggs die in this stage with germinal vesicle and ectoplasm intact, unless fertilized or experimentally treated by means of inducing parthenogenesis. ^Lillie and Just, igi^; Just, /p/^, igzga. THE FERTILIZATION-PROCESS Within three minutes after the addition of a drop of active spermatozoa to the eggs, remarkable changes take place in each Q^g to which a spermatozoon has become attached: a jelly flows out of the ectoplasm; the dull slightly turbid ectoplasm in the unfertilized egg gives way to a shining space crossed by strands. These changes can best be observed under the microscope by adding spermatozoa to eggs in sea-water which contains fine particles of Chinese ink. The two photographs appended show living eggs before and after insemination (Fig. 22). c d Fig. 23. — The fertilization-cone in the egg of Nereis (after Liliie). After the spermatozoon has become attached to the egg- membrane, the egg beneath the site of attachment forms a nipple-like projection, the cone, which shows itself well developed twelve minutes after the mixing of eggs and sperm, as can be noted in the Figs. I'^a and h. (Com- pare these figures with those taken from the egg of Rhyn- chelmis, Fig. 24.) Fig. 25 (from a drawing) pictures a living egg in Chinese ink and sea-water fifteen minutes after spermatozoa had been added to it. One marks easily the germinal vesicle (too strongly drawn), the yolk spheres and larger oil drops in the endoplasm, the ectoplasmic strands, the entrance cone and the spermatozoon, plasma membrane, vitelline membrane, the jelly and the surround- 159 THE BIOLOGY OF THE CELL SURFACE ing ink which extends into the jelly hull where the sper- matozoon is located. The entrance-cone below the spermatozoon now gradually recedes and pulls the membrane along with it so that the PiiiP V 5 • • • '•• to Fig. 24. — The fertilization-cone and sperm-entr\' in the egg of Rhynchelmis (after Vejdovsky and Mrazek). spermatozoon lies in a depression. During these minutes the egg undergoes the irregular changes of form and darken- ing already described in the chapter on the general prop- erties of the ectoplasm. When it regains regular form and clears, twenty-five minutes after insemination, the cone is 160 THE FERTILIZATION-PROCESS no longer visible in the living egg; the spermatozoon, seen with difficulty in the period during which these changes take place, becomes again visible after they pass over. About fifty minutes after insemination, the sperm-head disappears within the egg, leaving the tail and middle-piece outside. ^ 1^ ^ HP ^m mm ^^^■■:|^^ ■ ^^ 8 ^'fl ^^^^^^^^^^^^KV ^S i^^^ .^^ ^H| hi^-- ,"? W^t ■^m:M^K ^^^^1 mk^ Wi: '^n^tt^^^^m ^ 1 k ■ Is^j^^^^^^^^^^^^^^^^ Fig. 25. — Living egg of Nereis in a suspension of Chinese ink in sea-water, fifteen minutes after insemination (after Lillie). About five minutes later follows the extrusion of the first polar body. The second is extruded about fifteen minutes later. The egg cleaves into two unequal blastomeres one hour twenty-five minutes after insemination. Many details of the fertilization-process in the egg of Nereis can be observed only by fixing the eggs with a suit- 161 THE BIOLOGY OF THE CELL SURFACE able reagent which faithfully preserves the oil and yolk as well as the nuclear structures, cutting them into thin sec- tions (three or four microns thick) and coloring them with a dye which stains both cytoplasmic inclusions and chroma- tin material. In sections of eggs fixed after insemination, the following facts can be ascertained: The ectoplasmic breakdown has occurred. The germinal vesicle has broken down and the first maturation spindle has formed. To the fertilization- cone when fully formed the spermatozoon is fixed by the sharp anterior spike, the perforatorium, which traverses the perivitelline space whilst the head, middle-piece and tail remain external to the egg. The fertilization-cone as in the living egg projects beyond the egg-surface and later recedes. What one learns in addition to these details, verifying the observations on the living egg, concerns the change of the perforatorium of the spermatozoon and of the cone. As the spermatozoon enters deeper into the cone, further granules appear at its tip, whilst it itself gains in staining capacity. The cone is sharply marked off from the remain- der of the egg-cytoplasm; it is homogeneous in appearance, free from yolk and actually different in physical make-up. At about fifty minutes after insemination, the sperm- head disappears within the egg. The cone, as can be recog- nized by its staining and by its maintenance of form, acts as a solid body which sinks into the tgg exerting tension upon the sperm-head which stretches like a ductile strand. Fi- nally the strand breaks at the surface of the egg so that the external portion remains outside. This Is the middle-piece; hence, only the sperm-nucleus enters the egg of Nereis. Cone and attached sperm-head, acting as one complex, now revolve through an angle of i8o degrees. Soon there- after a star-shaped formation, the sperm-aster, with a minute granule, the centrosome or centriole, at its centre, 162 THE FERTILIZATION-PROCESS appears in front of the sperm-head (now designated the sperm-nucleus). This minute granule arises from within the sperm-nucleus^ (Fig. 26). As the sperm-nucleus advances toward the centre of the egg, the aster grows in size and it and the centrosome or centriole then divide; parallel with the growth of the aster the sperm-nucleus t''-:M^^^J^<i'^':K ^ -I'^^P^-fc:^ '■.■»:: ■■•f\—.: ls',«' ■- •^^■.■•.a'.:-.V-> ~' * ■'-"■ a b Fig. 26. — Sperm-nucleus within the egg of Nereis (after Lillie); a, with cone attached and h, separated from cone. enlarges, losing its staining power. In the meantime, the entrance-cone has disappeared. The egg-nucleus during the progress of these events has undergone changes. First, as germinal vesicle, it breaks down. The first maturation spindle forms near the centre of the egg and moves to the animal pole where the first polar body and, after the formation of the second matura- tion spindle, also the second is given off. The chromosomes remaining in the egg constitute the egg-nucleus. The union of egg- and sperm-nucleus follows and thus arises the first ^ Just, 1933b. But cf. Lillie, igi2. 163 THE BIOLOGY OF THE CELL SURFACE cleavage or zygote nucleus about which form two asters of unequal size. The larger of these which can be traced continuously is derived from the larger sperm-aster; the smaller, it is believed, though it can not be followed continuously, represents the smaller sperm-aster. Thus in the egg of Nereis both cleavage-asters are derived from the sperm-asters, if it be true that the smaller sperm- centrosome and -aster persist. Since the middle-piece remains outside of the €:gg, the cleavage-centres held to be genetically continuous with those of the sperm-nucleus can not be derived from the middle-piece. The history of fertilization in the egg of another marine worm, Chaetopterus pergamentaceus, as given by Mead,^ may be taken to represent eggs of Class 2 which reach the fertilizable stage after break-down of the germinal vesicle. These worms inhabit U-shaped tubes thirty to forty centimeters long, only the ends of which project above the mud. In the laboratory, these bizarre-looking animals, having been removed from their tubes, may be kept for some days in running sea-water. The sexes are readily distinguished: the eggs give the posterior segments of the female a pale yellow color, whilst the similar segments in the male which contain spermatozoa appear milky-white. It is best to use eggs and spermatozoa removed from ani- mals kept each in a separate container of gently flowing sea-water within twenty-four hours after the animals have been collected. When laid or removed from the animal, the eggs of Chaetopterus are in the germinal vesicle stage, but when they come into the sea-water the germinal vesicle breaks down, and the first maturation spindle forms near the centre of the egg. This moves to the animal pole of the egg and in this stage the egg remains until death unless fertilized or 1 Mead, 189S. 164. THE FERTILIZATION-PROCESS treated by some means to stimulate parthenogenetic devel- opment. At fifteen minutes after removal from the animal, every egg shows the first maturation spindle, its chromo- somes at the metaphase firmly anchored by means of the oo ok^ is o o''°Q.'°o 0^6 o Fig. 27. — For descriptive legend see page 166. > ^» 5/1 I L oS Spindle's outer pole to the egg-periphery. At any time after dissolution of the germinal vesicle the egg is fertilizable. 'The egg before rupture of the germinal vesicle while in the ovary is an irregular pear-shaped body whose upper two-thirds are covered by a delicate vitelline membrane ' ^6S THE BIOLOGY OF THE CELL SURFACE under which lies the ectoplasm, showing one or two rows of granules. As the germinal vesicle breaks down, membrane and ectoplasm move over the vegetal pole and thus enclose the whole egg which has assumed an ellipsoid form. Usu- ally the spermatozoon enters the egg at the vegetal pole o h OcTo o O O 00 °^0%07 Fig. 27. — Stages of sperm-penetration, egg of Mytilus (after Meves). after this has been covered by the moving ectoplasm. (Fig. 27 from the fertilization-process in the egg of Mytilus shows ectoplasmic changes after fertilization for this class of egg.) Entrance of more than one spermatozoon is rare. Within the egg, the spermatozoon after having moved some dis- tance shows an aster with two centrioles; these move apart, 166 THE FERTILIZATION-PROCESS each carrying a separate aster. This configuration, the sperm-amphiaster, together with the sperm-nucleus comes to lie near the centre of the egg. During this change in loca- tion the small solidly staining sperm-nucleus becomes con- verted into a lightly staining larger mass. In other words, the compact mass of chromatin composing the sperm- nucleus is resolved into diffuse faintly staining threads. The exact origin of the sperm-centrosome (centriole) is not known. According to Mead,^ The behavior of the sperm-centrosomes is in harmony with Boveri's theory of fertilization, but is not necessarily a confirmation of it; for the karyokinetic activities which are revived upon the entrance of the sperm are those leading to the formation of the polar globules. The machin- ery for these mitotic divisions is already organized, and it is quite as likely that the stimulus which starts it going emanates from the sperm-nucleus as that it emanates from the sperm-centrosomes. The sperm-amphiaster develops into the cleavage-amphi- aster. The eggs of Amphioxus — Class 3 — are laid toward eve- ning during the breeding season of this animal. If sexually mature animals are removed from the sea-sand, in which they live, to clean sea-water, they will deposit their eggs.^ Both Sobotta and Cerfontaine^ confirm an old observation to the effect that these eggs develop best when shed into sea-water already containing spermatozoa; if they lie in sea-water before spermatozoa are added, they are suscep- tible to polyspermy and develop abnormally. The eggs form the first polar body while in the body of the female I Mead, 1S9S. ~ Lwoff, 1 8^2, reported that he was able several times during the season at Naples to obtain eggs and spermatozoa shed in the labora- tory. On two occasions during i()2g I was able to induce animals to shed. ^ Sobotta, I.e.; Cerfontaine, I.e. 167 THE BIOLOGY OF THE CELL SURFACE and when laid are in the stage of second maturation. They possess a strongly marked ectoplasm which remains wholly colorless after treatment with a dye which stains the yolk-spheres. This ectoplasm is made up of spheres lying among a fine net-work composed of radially projecting \-:-V;;;;/^ 'yi'yvv-v:-:' ^ / Fig. 28. — For descriptive legend see page 169. Strands of the egg-plasma. (See figure in the chapter, The Ectoplasm, p. 99.) On entrance of the spermatozoon, which takes place at the vegetal pole, the ectoplasm breaks down; according to Cerfontaine it begins to go into solution at the site of sperm- entry. The ectoplasmic spheres seem to liquefy and become confluent. Both Sobotta and Hatschek affirm that 168 THE FERTILIZATION-PROCESS the membrane separates with extreme rapidity. The lat- ter also says that separation begins at the point of sperm- entry, whilst according to Cerfontaine, actual lifting begins at the animal pole.^ Appended figures (Fig. 28) of surface- changes are of the egg of Petromyzon, a member of this class. After sperm-entry the bulk of the spermatozoon is car- ried inward leaving a remnant at the entrance-point which k I Fig. 28. — Surface changes, egg oi Petromyzon, during stages of sperm-penetration (after Calberla). is still visible as late as the four-cell stage. Two equal asters arise from the division of the single aster found in the vicinity of the spermatozoon. Whether or not this aster contains a centrosome is not certain. From an excel- ^ Hatschek has also recorded some interesting observations con- cerning elasticity of the membrane. i6g THE BIOLOGY OF THE CELL SURFACE lent description of fertilization in the egg of Amphioxus this statement is taken: "Very close to the head of the spermatozoon one often sees a small intensely stained point, which could very well be the centrosome, but in view of the many yolk-granules in the neighborhood that have the same appearance, I do not dare to decide this."^ When the egg- and sperm-nuclei unite, the sperm-centres continue as the cleavage centres. The eggs of sea-urchins, fertilizable only after complete maturation, represent the fourth class to be discussed. We may take the egg of Arhacia as an example. The normal,- living, unfertilized egg of Arbacia is of 76 microns in diameter and bounded by a thin elastic mem- brane below which is the delicate ectoplasm. The nucleus in the living unfertilized egg appears as a clear space lying in any position with reference to the polar axis of the egg.^ On fertilization the vitelline membrane separates beginning at the point of sperm-entry where a nipple-like protrusion — the fertilization-cone — from the egg-surface forms to pull in the sperm-head. The surface of the egg seen under high power of the microscope appears delicately crenated because 1 Sobotta, iSgy, p. 40. - Normal, living, unfertilized eggs of Arhacia are spheres of approximately the same size and specific gravity, each enclosed in a jelly hull. They possess bright red pigment granules evenly dis- tributed and never clumped. Eggs that do not satisfy these criteria should be rejected for experiment especially if they color the sea-water in which they lie; such discharge indicates the presence of moribund or cytolyzing eggs. ^ This axis is an imaginary line passing through the centre of the egg and the point at which the polar bodies are given off. In sea- urchins'' eggs the polar bodies are always given off at the point opposite a tube in the jelly-hull of the egg. Because in these eggs the polar bodies are usually lost before the eggs are shed, the location of the tube in the egg's jelly hull is important for determining the egg's polar axis. I JO THE FERTILIZATION-PROCESS of thread-like projections of granule-free cytoplasm into the space between the vitelline membrane and the egg- plasma. Later by the anastomosis of the free ends of these threads a very thin sheath forms; the threads and their enclosing sheath constitute the hyaline plasma-layer. Fifteen minutes after fertilization (temperature of the sea- water around 2i°C.) the hyaline plasma-layer stands out very sharply even under low power of the microscope. During this same period the sperm-head evolves as the sperm-nucleus with attendant cytoplasmic changes. Although I have found it easy to follow the history of the sperm-head in the egg of Arbacia, I prefer at this point to base the following description on the egg of the flat sea- urchin or "sand-dollar," Echinarachnius. This I do because the &^g of Echinarachnius being larger and not so highly colored as that of Arhacia lends itself readily to exact observation in the living state. For both, except for minor variations, the process is the same. Because of the significance ascribed to the middle-piece of the sperma- tozoon in echinids by Boveri's theory of fertilization, it is necessary to take up the history of the middle-piece in some detail. During the stages of sperm-attachment and penetration, the middle-piece reveals the same structure and position found in the free-swimming spermatozoon. That is, it is closely fitted to the basal end of the sperm-head and shows prominently a bipartite granule. In fixed preparations treated with a dye, haematoxylin, the nucleus of the sperm- atozoon stains bluish gray; the middle-piece, the outer limits of which are continuous with the nuclear membrane, is also gray. The middle-piece granule is seen as a sharply defined black body, lying — as in the free-swimming sperma- tozoon— in various positions in the middle-piece. Though in some views it shows up as a continuous horse-shoe shaped body and in others as two rods, it is in reality a body com- 171 THE BIOLOGY OF THE CELL SURFACE posed of two curved rods joined together by a delicate but clearly revealed bridge. Once the spermatozoon together with the middle-piece is within the egg — the tail does not enter — it rotates through } an angle of i8o degrees. This rotation may, however, take place while the spermatozoon is still In the entrance- cone. At this stage occur the following changes: the sperm-head may reveal a more sharply stained outline, thus giving the appearance of a hollow tube; the outline of the middle-piece is no longer distinguished; and the middle- piece granule is closely stuck to the base of the sperm-head. After rotation, the sperm-nucleus is directed toward the centre of the egg. The middle-piece granule, hitherto a closely fitting cap over the base of the sperm-head, slips off carrying with it a thread of sperm-substance. Or It may be that the sperm-head swells except In the region at which the middle-piece granule is clamped to Its base. Figure 29^ shows a sperm-head at this stage. Certainly the sperm-head now shows well defined difference in volume and form; these changes take place with the separation of the middle-piece granule.^ The sperm-aster, never found before the stage at which the middle-piece granule draws away from the sperm-head, Is now well defined and distinct. Its spherical central portion, the astrosphere, appears in fixed sections as clear, homogeneous substance which does not stain. The astral rays are paths of clear granule-free ground-substance lying between rows of the cytoplasmic constituents — mitochon- dria, yolk spheres and oil drops. The astral configuration noted in properly fixed eggs bears the closest resemblance to that observed in the living egg, ^ These changes in the sperm-head are not unlike those found in free-swimming spermatozoa fixed after agglutination with specific egg-sea-water that will later be discussed. 172 THE FERTILIZATION-PROCESS Within the astrosphere the middle-piece granule is bril- liantly revealed — a black eccentrically placed body, in sharp contrast to the clear substance immediately around it and the grayish blue cytoplasmic constituents farther beyond. The granule has never been found at the centre of the astrosphere. This is easily established in those stages, like Fig. 29/^, in which the astrosphere is well developed. During the ensuing stages of sperm-penetration up to the stage of apposition of the nuclei, the sperm-nucleus increases in size. The greater the distance, and therefore the time, which the sperm-nucleus must travel before reach- ing the egg-nucleus, the greater is its increase in size. This distance, of course, depends upon the site of sperm entry, which may be at any point on the egg-surface, with refer- ence to the location of the egg-nucleus, which has no fixed position. It is therefore very easy to obtain a large number of stages during penetration. During these stages I have found a behavior of the mid- dle-piece granule which differs from that described by Meves for the egg of Parechinus — a difference which may be due to technique, though I doubt that since the his- tory of the granule in fertilized eggs of Arbacia fixed as those of Echinarachnius is like that of Parechmus. This difference is that the middle-piece granule in the egg of Echiiiar achnuis separates from each of its free limbs a more minute granule. Thus each limb of the original bipartite granule in turn becomes bipartite. These two new forma- tions move away from the parent granule, each maintaining connection by means of a delicate thread. Figs. {2gb), (29c) and show this formation. As the sperm-nucleus increases in size, its basal thread, at the tip of which is the middle-piece granule, becomes longer, an indication of the ductility of the sperm-nucleus during these stages. In form, therefore, it resembles a ^73 THE BIOLOGY OF THE CELL SURFACE 5': ^"'^ % i^> *IV^ V.' ' ' 'J. '■ at* N. To ^.^» *'«. •Si.' Fig. 29. — For descriptiv^e legend see page 175, ^74 THE FER T I LIZA TION-PROCESS i ^ t ♦ 6 iv"^ •*►• ^' <— middle-piece .\- tl / middle-piece -» >■ y* Fig. / 29. — History of the middle-piece of the spermatozoon within the egg, Echinarachnius parma. (Original.) THE BIOLOGY OF THE CELL SURFACE round or oval flask with a long neck. In earlier stages this neck appears solid; in later stages it is really a tube, as Figs. 2()d and 2()e show. Now in this stage the structure of the middle-piece gran- ule and its relationship to the sperm-nucleus are most clearly discerned. The sperm-nucleus and the middle-piece granule form one continuous complex. The tip of the tube arising from the base of the sperm-head spreads out slightly as very delicate threads to connect with the minute granule which arose from the middle-piece body. Thus, the middle- piece granule resembles the top of a parachute, the threads of which come together at the tip of the sperm-tube. At this junction another granule is often found. With the apposition of the nuclei, the sperm-nucleus loses connection with the middle-piece granule. This is shown in Figs. 29/ and g. There is never a re-establish- ment; in the succeeding stages in mitosis leading to first cleavage, the granule may lie as a discrete single inert body at any point in the cytoplasm with reference to the spindle. I have never found any evidence of its division or of its taking up a position at either spindle pole. By the time that the nuclei come into apposition, the more minute granules can no longer be traced. Within the egg the sperm-head, a structure notable for its low water-content, at first maintains its shape and size but as it is carried from the periphery of the egg it slowly approaches spherical form, increasing in volume and its outline losing definition. Observations on eggs fixed during this period reveal what the living egg does not so clearly show: namely, that the transformation of the sperm-head into a vesicular nucleus is the resolution of a greatly con- densed mass of chromatin — an exaggerated and rapid evolu- tion of chromosomes from telophase condition to that of a resting nucleus. Though this process is more easily visible before the sperm-nucleus unites with that of the &g%, never- iy6 THE FERTILIZATION-PROCESS theless it can be discerned after this union. Finally, the sperm-nucleus loses its visible identity through complete fusion with the egg-nucleus. Soon thereafter two asters arise, presumably from the single sperm-aster. The foregoing accounts of fertilization, embracing eggs of the four classes made with respect to the period in matu- ration when eggs are in the stage for reception of sperma- tozoa, reveal two phenomena as common to all animal eggs. First, after attachment of the spermatozoon to the egg- surface, the egg-surface undergoes a change with the result / C^ /W y; i^^ Fi G. 30. -Surface changes in the egg of Mitrocoma attending sperm-entry (after MetschnikofF). that the vitelline membrane becomes separated. The sur- face-changes differ in quality : in eggs of Nereis a superficially located jelly is extruded; in the Chaetopterus-egg the obser- vable changes are less striking; in the egg of Amphioxus discrete bodies flow together and liquefy before the mem- brane separates widely from the egg] in sea-urchins' eggs the briefly enduring surface-changes are most violent. In order further to elucidate such surface-changes, I include pictures of a Medusa-egg (Fig. 30) described by Met- schnikofT.^ It should also be mentioned that the entrance- ^ Metschnikoif, 1S86. 177 iJ^^U L \ THE BIOLOGY OF THE CELL SURFACE cones may be exaggerated in unripe echinoderm eggs, i.e., those incapable of fertilization and development (Fig. 31). Fig. 31. — Ectoplasniic changes in unripe eggs in response to sperm-entry, a, egg of a starfish (after Schneider); b, egg of a holothurian (after Iwanzoff). The cones may be made more expressed under experimental conditions.^ In the second place, the sperm-nucleus having reached the egg-nucleus forms with it the zygote- or ^ Fol, iS/Q; Schneider, i8g^; Iwanzoff, i8g8; Just, Tg2Qb. \.^^^^ THE FER T I LIZA TION-PROCESS cleavage-nucleus, about which the first mitotic figure arises. Thus the fertilization-process in these four examples resolves itself into two phases — an external, that concerns the ecto- plasm, and an internal, that concerns the nuclei. Fertilization means in the strict sense of the word the coming together of egg and spermatozoon, for without it there is no fertilization. Hence obviously, the initial act in the whole chain of events is the contact between the two partners. If we endeavor to state the definite result of these events, we can say that it is the development of the egg. But it would certainly be impractical to define fertilization as some have defined it, namely, that it is complete only when the developed organism has reached that stage in its development when in its germ-cells the chromosomes pair. Since in all animal eggs a mitotic figure is established for the first division-nucleus, it is far more practicable to take this feature, common to all eggs after development has begun, as the end-point of the series of events that begins with the coming together of egg and spermatozoon. These events we speak of as the fertilization-process; this resolves itself, as said above, into an external and an internal phase. I shall now endeavor to determine which of these must be regarded as the fundamental phase in fer- tilization, that is, during which takes place the funda- mental happening, event, in fertilization. I first discuss the internal phase of the fertilization-process, since it involves more generally known phenomena and since pre- vailing opinion among biologists places the basic event in this phase. I begin with the question of the union of the egg- and sperm-nuclei. Were we to conclude from the descriptions of fertiliza- tion of eggs of the four classes as given above, we could say that fertilization is the union of the egg- and sperm-nuclei. However, cases exist in which egg- and sperm-nuclei norm- 179 THE BIOLOGY OF THE CELL SURFACE ally never fuse, although the presence of the sperm-nucleus within the egg is necessary for the egg's development. Indeed, in extreme cases the cleavage-nucleus is distinctly bipartite and remains so during many successive cleavage- stages. In the egg of the water-flea, Cyclops, for example, according to Haecker such a bipartite nucleus persists from one generation to the next.^ Bipartite nuclei have been described for other eggs — it shows clearly in those of the large tailed amphibian, Cryptobranchus. The egg of Pediculopsis presents an interesting condition: some of the cells resulting from its first division after fertilization contain single nuclei whilst in others each chromosome per- sists as a separate entity with its own spindle. Between the two extremes of complete fusion at the time when the egg- and sperm-nuclei appose and the persistence of these nuclei as revealed by the bipartite character of the cleavage- nuclei, intermediate grades exist, even in one egg-genus.^ In eggs of Rhabditis, though they must be fertilized in order to develop, the sperm-nucleus remains inert and never unites with that of the egg.^ Briefly, actual fusion with immediate loss of identity of the egg- and sperm-nuclei is no sine qua non of the fertilization-process. Experimentally, it can be shown that fertilization may take place without the egg-nucleus. The presence of either the Qgg- or sperm-nucleus alone suffices for the egg's development: the former is concerned in parthe- nogenesis, the latter in an Qgg whose nucleus has been actually or virtually removed by experimental means. The fertilization of an egg-fragment without the egg-nucleus is known as merogony."* Such fertilizable fragments have been obtained from eggs of sea-urchins, starfishes and ^ Haecker, i8go. See also Heberer for literature. - Cf. Boveri^s observatiojis on eggs of the genus, Echinus. '■'' Kriiger, 191 3. ■* Delage and others. 180 THE FERTILIZATION-PROCESS worms. Fertilization of eggs whose nuclei are hindered from taking part in development has been accomplished on eggs of sea-urchins and of amphibia. Experimentally it is thus shown that neither fusion nor even mere apposition of the sperm- with the egg-nucleus is essential for the fertiliza- tion-process. Some observations of my own bear on this point. The first of these shows that in eggs of Echinarachnius, fertilized after having been treated with dilute sea-water — or after having lain in sea-water for several hours — the egg-nucleus takes no part in the subsequent cleavages but remains as a "resting nucleus" in every way similar to its condition in the unfertilized egg.^ In the meantime, the sperm-nucleus divides in the typical manner of the zygote-nucleus. The second observation relates to eggs of the sea-urchin, Arbacia, fertilized In various stages of mitosis Induced by treatment with hypertonic sea-water.^ In some of these eggs the egg- and sperm-nuclei divide independently though in dif- ferent tempo, as one would expect. Here then the stage in the mitotic activity of the egg-nucleus does not inhibit the independent activity of the sperm-nucleus. The evidence derived from observations on normal fer- tilization-processes indicates that fertilization can occur without fusion, union, apposition, or even approach of the sperm- and egg-nuclei. The experiments cited show that development ensues when the egg-nucleus is absent or when though present it is rendered inert. We are therefore not justified In retaining the old definition of fertilization as fusion of the egg- and sperm-nuclei. To speak of the RJiabciitis-egg, for which sperm-penetra- tion is essential to development, as "nature's bridge between parthenogenesis and fertilization,"''^ because the 1 Just, I()24. ' Just, I<)22h. ^ Bracket, 191 J. 181 THE BIOLOGY OF THE CELL SURFACE sperm-nucleus once in this egg lies inert whilst the egg- nucleus alone takes part in development, is a pretty state- ment without much scientific value. If an Qgg can not develop without fertilization, the criterion of its having been fertilized is its development. If the spermatozoon is essential to the initiation of development, fertilization has been efTected, if this egg develops. To speak of this case and of experimental conditions in which the egg-nucleus though present is inhibited from taking part in cleavage as partial fertilization is to ignore the essential problem. If an egg-fragment without a nucleus develops after having been entered by a spermatozoon, it has been fertilized. To retain the old definition of fertilization as the union of egg- and sperm-nucleus is to violate both fact and logic. But whatever the situation with respect to a union of the egg- and sperm-nuclei, always a division-spindle arises and in this the fertilization-process reaches its culmination. At the poles of this spindle are star-like formations, the asters. These beautiful formations are especially striking in many living eggs and since they are always present in the nuclear division of cleaving fertilized eggs, they have been held to be the cause of fertilization. Thus Boveri postulated a theory of fertilization which, though he himself later abandoned it, is even to-day defended by man}' writers: namel}', that the essential feature in fertilization is the introduction into the tg^ of two centrosomes by the spermatozoon, about which two asters form which persist as the asters of the egg's divi- sion-spindle. Were it not for the fact that many writers still uphold this definition, we could dismiss it at once inas- much as we have seen in the descriptions of the fertilization- process of the four types of eggs given above that the cleav- age-centres can not be always shown to have arisen from sperm-centres. Moreover, whilst in the majority of eggs the cleavage-centres probably are continuations of both sperm- centres, there are eggs in which the centres come one from the egg-nucleus and one from the sperm-nucleus, as in eggs 1S2 THE FERTILIZATION-PROCESS of many snails. In addition, there are eggs in which the asters of the division-spindle arise entirely around the egg- nucleus, the sperm-nucleus never showing any trace of astral radiations, as in eggs of trematodes. Finally, as we have seen, the division-spindle in eggs of sea-urchins shows asters devoid of centrosomes. What in these Boveri iden- tified as centrosomes have been proved beyond doubt to be bodies located in the middle-piece of the spermatozoon that are cast off into the egg-cytoplasm and have no causal relation to the asters.^ For other eggs it has also been shown that granules in the middle-piece of the sperma- tozoon can not be identified as centrosomes.- In one egg only, that of Nereis, has it been shown that the centrosomes arise out of the sperm-nucleus. The conclusion is patent: as it is the case with the stage in maturation when eggs reach the fertilizable condition, and with the mode of union of the egg- and sperm-nuclei, so with the origin of the cleavage-centres — all possible varia- tions exist. Hence we find no constancy with respect to the origin of the cleavage-centres and therefore can not define fertilization as the importation into the egg of cen- trosomes by the spermatozoon. Failing to discover in any of the events that happen dur- ing the internal phase of the fertilization-process the funda- mental act in fertilization, we turn to the external phase, to the changes at the egg-surface incident to the attach- ment of the spermatozoon. All animal eggs in response to sperm-attachment show some visible change in the ecto- plasm. In the following chapter I present my hypothesis that underlying these changes at the egg-surface is the fundamental act in the fertilization-process. I denominate this act the fertilization-reaction. ^ See Meves, 191 2; Just, 192'ja. ^ According to Meves there are not Vivo hut jive such granules in the sperm-middle-piece of Mytilus. 1S3 8 The Fertilization-reactio/i It is a curious fact that by far the largest body of data, both of observation and of experiment, on fertiliza- tion relates to the end results and final consequences of the coming together of eggs and spermatozoa. The phenomena embracing the union of egg- and sperm-nuclei and the estab- lishment of the first cleavage-figure are end-events of a chain of happenings in a complex, heterogeneous and little understood system and not the end-point of a single one- way reaction taking place in a simple, homogeneous, fully understood system. Many biological processes doubtless may be explained on the assumption of an underlying sim- ple, even mono-molecular, reaction so far as such in chemical experiments with pure reactants be known or postulated. Nevertheless, to ignore the polyphasic nature of the cyto- plasm needlessly obscures the problem; comparisons of cytoplasmic processes with simple or even with complex reactions in test-tubes may cause serious retardation in the solution of the problems of biological behavior. Chem- istry, in so far as it relates to end-points, offers little help. As in chemistry more information regarding the onset of even the simplest reactions is desired, so here in the problem of fertilization: we stand in need of more exact knowledge as to the initial reaction which leads to the catenary pro- cesses culminating in establishing the cleavage-figure with which by rhythmical reduplication the development of the egg ensues. Hence, the study of the happenings immedi- ately ensuing after the mixing of eggs and spermatozoa 184 THE FER T I LIZA T ION-RE AC TION assumes great significance for the understanding of the process, fertilization. Moreover, as we shall see, in this study inheres another value, since these happenings are of prospective significance for the whole range of consecutive form-changes by which from the e^^ the animal emerges, changes which taken together we speak of as the egg's development. The evidence summarized in the preceding chap- ter makes it clear that in fertilization it is the egg-cyto- plasm that reacts with the spermatozoon. A recital of this evidence is here unnecessary; one fact noted we may recall. This is that whilst no animal sperm-cell is capable of fertilizing an egg except as a spermatozoon — ■ a sperm-cell which has completed both maturations and has become transformed from a spermatid into a spermato- zoon by nuclear condensation and remarkable cytoplasmic changes — the egg, depending upon the species, has capacity for fertilization before, during or after maturation. Thus the fertilizability of all animal eggs hangs together with some condition in the cytoplasm of the egg and is independ- ent of its nuclear state, as germinal vesicle, as first or second maturation-nucleus or as a completely matured nucleus. This fact would still stand were the events in the ensuing fertilization-process the same in all eggs; it becomes of paramount significance since we can not reduce these events to a common underlying principle which holds for all eggs. Some change, then, supervenes in the egg-cytoplasm which transforms it from a condition of non-fertilizability into one of fertilizability. Since we intend to examine the reaction between egg and spermatozoon at the moment of insemination, and since we know that spermatozoa are at this time alike with respect to fertilizing capacity, our task concerns itself with the fertilizable condition of the egg-cvtoplasm. 185 THE BIOLOGY OF THE CELL SURFACE The fertilizable condition in animal eggs arises suddenly. Delage' demonstrated that with break-down of the germinal vesicle the Qgg of a starfish undergoes a change which ren- ders it fertilizable, a finding which I have confirmed in the same and in two other species of the same genus, Asterias. Further, not only are these eggs unfertilizable while the germinal vesicle is intact; attachment and entrance of the spermatozoa during this stage actually inhibit completely the break-down of the germinal vesicle. Other eggs which normally are extruded into the sea-water in the stage of intact germinal vesicle are fertilizable only after its disrup- tion, an event which occupies a few minutes. The fertilizable condition can not, however, in all eggs be correlated with break-down of the germinal vesicle, since, as we have seen, eggs like that of Nereis, for example, and of many other animals, are fertilizable only in the germinal vesicle stage. The egg of Nereis offers another example of the sudden onset of fertilizability. This is shown by the following: Eggs of this worm are Jiormally shed when the animals are in the so-called heteronereis phase, i.e., when during a period of full-moon they swim actively at the sur- face of the sea, at which time all the eggs are in the optimum fertilizable condition. Now I have reared in the laboratory to the heteronereis phase many of these worms which had been collected while immature (i.e., in the nereid phase). On five successive days before full-moon I have inseminated eggs removed from these worms without obtaining develop- ment despite the fact that under the microscope the eggs resembled fertilizable ova.- On the day of full-moon, eggs taken from the same animal, from which others had been removed during previous days and inseminated without ^ Delage, igoia. ~ If a fully ripe female {one in the sztumming stage) be punctured all of her eggs are usually extruded. After the same degree of puncture of an immature female only fetv eggs exude from the site of injury. i86 THE FERTILIZATION-REACTION success, fertilized normally and gave normal development. Comparable results were obtained with eggs of Platynereis; never did I observe copulation of the animals and the con- sequent laying of fertilized eggs before full-moon. Often during the early days of its breeding season, the sea-urchin, Arbacia, contains eggs with polar bodies attached, a condition which indicates that maturation was only then ending; usually, normally shed eggs show no polar bodies. Such eggs with polar bodies either are not fertiliz- able at all or only in small numbers. During one season I followed the development of the ovaries of this sea-urchin daily beginning in April through to the first of-^July. Only after the eggs had passed the maturation-stages which occupy a brief period, are they fertilizable. The fertilizable condition hangs upon or comes with a change which occupies a mere point of time in the egg's history: before it the egg is unfertilizable and after, fertiliz- able. This is doubtless true also for eggs like Otomesostoma, Dnwphilus, Saccocirrus. The spermatozoa enter these eggs when they are very young ovocytes, i.e., before the eggs' growth-stage. Only when these eggs are fully grown and matured, which state they attain some time after the precocious sperm-entry, can these eggs be said to be fer- tilized. Presumably here also something happens in the egg so that it passes from the condition of unfertilizability to that of fertilizability. The fertilizable condition may endure for hours or even for three days as in eggs of sea-urchins, depending upon the species and for any given species upon the temperature; it persists longer at lower than at higher temperatures. In eggs of the flat sea-urchin, Echinarachnius^ its duration is very short compared with that of Arhacia at the same temperature.^ Eggs of other animals, fertilized outside ^ By frequently changing the sea-water of the same temperature in which the eggs lie, the duration of the fertilizable condition becomes shorter. 187 THE BIOLOGY OF THE CELL SURFACE of the female's body, as those of echinoderms other than sea-urchins, of worms, of molluscs, of ascidians and of vertebrates may retain their fertilization-capacity for hours. With one exception species of animal eggs undergo no change in maturation whilst the fertilizable condition per- sists but remain unless fertilized until death in that stage in which fertilization normally occurs. Eggs of the genus, Asterias, (starfish) make the exception, as pointed out. When normally laid by the females, these eggs are in the stage of the breaking-down germinal vesicle, their normal stage for fertilization. The steadily accumulating evidence to show that in normal conditions for breeding the males and females of starfishes closely congregate prior to the shedding of spermatozoa and eggs also indicates that the optimum stage for fertilization is that found in the eggs when shed. But if shed eggs or those removed from an ovary with their germinal vesicles intact are brought into normal sea-water without spermatozoa, they may undergo complete maturation. Such eggs fertilized during stages of first maturation develop normally; thereafter, fertiliza- tion-capacity steadily falls off. After complete maturation only low percentages both of fertilization and of subsequent development are obtained and are highly abnormal. The Abbe Spallanzani made interesting observations on the duration of the fertilizability of frog's eggs after deposi- tion. Sobotta points out that fertilization of the eggs of Amphioxus succeeds best when the eggs exude from the female into sea-water containing spermatozoa; whereas the addition of spermatozoa to eggs already in sea-water tends to result in abnormal fertilization — an observation indi- cating the short duration of fertilizability in this tg^. Fertilization of the eggs of a fish, the wall-eyed pike, drops from 40 per cent, for the eggs that have lain in water for two minutes to zero per cent, for eggs that have been in 188 THE FER T I LIZA TION-REACTION water for ten minutes.^ Where copulation takes place between male and female before egg-laying and insemination outside of the female's body, the fertilizable condition may pass off rapidly. Thus, in eggs of the minnow, Fundulus heteroclitus, a bony fish, the fertilizable condition is at its height as the eggs extrude during the time, when as the male clasps the female, spermatozoa are shed. If one removes the female during the act of copulation, then gently presses her in order to obtain eggs and places these in sea-water, one finds that with residence in sea-water the eggs' capacity for fertilization diminishes. The normally shed and insemi- nated eggs show one hundred per cent, fertilization. The most interesting case illustrative of a brief duration of ferti- lizability is that of the eggs of Platynereis, fertilized within the female's bodv. The male and female of this marine worm go through a most peculiar type of copulation. ^ The sexually mature animals swim at the surface of the sea at night during the period from full to new moon of the breeding season (sum- mer months) and are easily captured. In the laboratory, by bringing a male and a female together in a vessel of sea- water, one can observe more closely the normal behavior displayed at the surface of the open sea. The rapidly swimming male entwines the less active and larger female and thrusts his tall into her jaws. She thus takes up the spermatozoa which pass from buccal cavity to pharynx and through lesions in the wall of the pharynx into the coelom where they become attached to the eggs. The eggs are Immediately laid. In one set of observations on 87 females, I found, with the aid of fellow workers who recorded the time, that the whole process, from the moment that the male entwined the female to the beginning of egg-laying, 1 Reighard, iSgj. - Just, 191 4. 189 THE BIOLOGY OF THE CELL SURFACE \ consumed only about five seconds. And yet every egg laid showed an attached spermatozoon and only one. These observations were made soon after capture of the worms: even so, the delicate males are apt to suffer some- what in handling. In nature, therefore, this period is presumably not longer than five seconds and may very well be shorter. Only a very brief fraction of this period of five seconds can be concerned with insemination per se. Most of the time is consumed by the act of copulation, by the movement of the spermatozoa to reach the eggs, and by the wave- like muscular contraction of the female by which the eggs are laid. That the fertilizable condition is here of extremely short duration is further proved by the fact that eggs can not be removed from the virgin female to a volume of sea- water greater than that of the mass of eggs and the sea-water in turn removed quickly enough to insure fertilization. In other words, since it can be shown that sea-water does not impair the fertilization-capacity of the spermatozoa, it must impair very quickly that of the eggs. But the instant that the spermatozoon becomes affixed to the egg, the sea- water is harmless.^ The cytoplasm of eggs, then, at one or another stage of maturation becomes suddenly fertilizable and remains so for a longer or shorter period of time. Eggs die unless fertilized, without exhibiting any change in nuclear state. With fertilization, they pass beyond the fertilizable condi- I , tion. I know of only one report,^ based on insufficient .^yj^ evidence, which claims that fertilized eggs can be re-fer- tilized. In my experience, eggs having been fertilized lose capacity for fertilization for neither they nor their frag- ments can be fertilized again. ^ ' Just, 191 Sh, 1915c. ' Morgan, /S95. •■' Just, /92ja. IQO S^ THE FERTILIZATION-REACTION Fertilizability being resident in the cytoplasm, we may ask whether or not it has definite location. As we have seen, in the egg of Platynereis, for example, loss of fertiliz- ability may occur very quickly. Since an egg is either fertilizable or not, it is easy to assume that for eggs generally the loss, like the onset, of fertilizability is the event of a moment. Now the rapidity of the loss strongly indicates the superficial location of that upon which fertilizability depends. Two cases show that with the onset of fertilizability the ectoplasm of the egg exhibits visible alteration in structure. At Naples I often noted that eggs of sea-urchins after com- plete maturation but before they are fertilizable show very minute projections from their surfaces. Fertilizable eggs, in contrast, present a smooth contour; the ectoplasm is homogeneous in appearance. The egg of Chaetopterus undergoes a series of remarkable changes, its ectoplasm flowing from the animal pole to cover the hitherto exposed vegetal pole, before it becomes fertilizable by spermatozoa entering at the vegetal pole. With the flowing movements in the ectoplasm, fertilization may take place at other points. No one has to my knowledge made a systematic study of a great number of species of eggs with respect to the occur- rence of structural changes in the ectoplasm which might be correlated with fertilizability. Until this has been done, we can not reject the possiblity that such changes generally intervene. Also, these changes may be of such extreme delicacy that they are overlooked. Finally, the change to the fertilizable condition may not always express itself as visibly structural. The rapidity with which fertilizability arises and disap- pears points to the conclusion that it is a condition of the ectoplasm. Then, also, we may assume that fertilization is concerned with the ectoplasm. But we need not rest 191 THE BIOLOGY OF THE CELL SURFACE content with mere assumption; evidence at hand amounts to proof that the chief event in fertilization is a reaction between the egg's ectoplasm and the spermatozoon. This evidence will now be set forth. The proposition that the main event in fertilization is a reaction between egg-ectoplasm and spermatozoon is supported by the following: (i) The ectoplasm is necessary for fertilization. (2) The onset and loss of fertilizability is correlated with the appearance and disappearance of an ectoplasmic substance. (3) Specificity in fertilization depends upon the integrity of the ectoplasmic layer. (4) Polyspermy obtains when the ectoplasm is slow in reacting, I first cite an observation of my own.^ If uninseminated eggs of the fiat sea-urchin, Echinarach- nins, stand in shallow dishes of sea-water, they undergo a change due to the increasing salinity of the sea-water caused by evaporation, a change which manifests itself by an alteration of the eggs' surface. This same change is induced by placing the eggs in sea-water made hypertonic by the addition of sodium chloride (6 parts of 232 M NaCl plus 50 parts sea-water). In either case the surface-layer of the eggs on return to normal sea-water is seen to be of a thickened and translucent jelly-like nature. Many of these eggs, having thus been exposed to hypertonic sea-water, under pressure, as by forceful ejection from a pipette, form each a protrusion. These vary in size. By other methods eggs may be induced to form protrusions; often they show them after having lain in normal sea-water for some time. Eggs of other sea-urchins, Arbacia, Strongylocentrotus, Echi7iiis, Echinocardium, treated with hypertonic sea-water likewise form these protrusions when they are brought into normal sea-water. The explanation of the formations is 1 Just, 1923a. ig2 THE FERTILIZATION-REACTION this: they represent extruded endoplasm escaping tlirough the ruptured surface-layer of the egg, and occur when this layer has been so altered that it loses the elasticity so char- acteristic of it on the normal unfertilized egg. The resi- dence in concentrated sea-water brings about abstraction of water from the eggs as revealed by their shrinkage and the closer apposition of their cytoplasmic inclusions. The surface-layer also loses water; its greater visibility is owing to its altered structure: on the normal unfertilized egg it appears homogeneous, on the treated egg it appears as a system of fine cytoplasmic prolongations attached to the vitelline membrane. When these treated eggs are brought suddenly into normal sea-water, this altered ectoplasmic surface ruptures; and the endoplasm flows out as a cohering bud. This outflow is checked as the eggs come into equi- librium with their normal medium. Every egg with a protrusion, then, is made up of two com- ponents, one possessed of the altered surface-layer, the other, an endoplasmic bud, devoid of it. Following insemi- nation, that portion of every egg enclosed by ectoplasm separates a membrane, goes through cleavage and develops into a swimming larva. The endoplasmic bud undergoes no change; it can not be fertilized. As an Qgg with a bud of endoplasm develops, the bud persists as a mass of undif- ferentiated intact material which finally disintegrates. The size of protruded endoplasm is without significance; it may be extremely minute, of the size of a polar body, or it may contain most of the egg-substance. In the latter case only the minute component with ectoplasm separates the membrane and cleaves, the cleavage resembling the so-called disocidal cleavage which is normal for the eggs of many animals; but such eggs never form the typical inver- sion, invagination, of cells by which the egg becomes a cup- like swimming form, composed of two cell-layers. It is interesting to note that this invagination (a form of gas- ^93 THE BIOLOGY OF THE CELL SURFACE trulation) when it occurs — In eggs with buds equal to or smaller than the egg-component having ectoplasm — takes place at the pole where the bud is formed. This fact might mean that the endoplasm is always extruded from the area in the uninseminated egg which is destined to be the site of invagination. But it is also probable that the endoplasm extrudes from any region of the egg, which means that the site of invagination becomes pre-determined by extrusion of the endoplasm. Whilst sea-urchins' eggs lend them- selves beautifully to this mode of endoplasmic extrusion, other eggs do not because of the nature of their surfaces and the changes taking place in them after experimental treatment. In these the vitelline membranes must be punctured before the endoplasm can protrude. Such injury leads at once to break-down of the egg-cytoplasm, as happens when one cuts into the tough membrane enclosing the egg of Nereis. In the Q^^ of the starfish one can demonstrate that the egg-surface plays in fertilization a role similar to that of the surface of sea-urchins' eggs. Observations reported by Whitaker^ make it clear that in fertilization of the star- fish Qg%, the effect of the spermatozoon is conducted only by the egg-surface. The conclusion reached from these observations, that the ectoplasm is necessary for fertilization, though it is defi- nitely proved for only a few eggs, may nevertheless hold for eggs generally, since in them all some kind of surface- change follows sperm-attachment. We should not, to be sure, make a virtue of the necessity imposed upon the spermatozoon that to effect fertilization it must first make contact with the egg-surface; we must know that this par- ticular surface is something peculiar because of special endowments which set it apart from the endoplasm. ^ Whitaker^ igji. 194 THE FERTILIZATION-REACTION Mere location does not make the ectoplasm and its behavior peculiar, nor the fact that in all cells cytoplasm becomes converted into ectoplasm. If the ectoplasm plays this role in fertilization, for egg-cells this conversion of cyto- plasm into ectoplasm must take place in such wise that new and stimuli-receiving substance comes to the surface of unfertilized eggs as they pass from the unfertilizable to the fertilizable condition. We possess some evidence for the appearance of such a substance in the surface-layer of the egg with the moment that it becomes fertilizable. According to the fertilizin-theory of Lillie, eggs (of sea- urchins and of Nereis) in the fertilizable condition contain a substance, fertilizin, located at their surfaces which is necessary for fertilization. Thus, the statement, no fertili- zation without ectoplasm can be amended to read in these cases, no fertilization without ectoplasm-located fertilizin. Whilst the presence of fertilizin has not been demonstrated for all eggs, its occurrence is not limited to those of the forms studied by Lillie. I have detected its presence in eggs of other sea-urchins and of two other worms (Platynereis megalops and P. diimerilii). It has been found in the ^g^ of Ciona, an ascidian. I have elsewhere reviewed the work of fertilizin;' to this review together with Lillie's original papers the reader is referred.^ Here I give only a resume with special reference to the ectoplasm-located fertilizin. Fertilizin is held to be a mid-body in the reaction between egg and spermatozoon. Without it, in the eggs named, fertilization does not take place. No cells other than eggs possess it and these only when fertilizable. Neither in immature nor in fertilized eggs can its presence be detected. Eggs treated with means that induce the surface-changes identical to those induced by spermatozoa likewise do not 1 Just, /pjoc. - Lillie, 1913, 1919. THE BIOLOGY OF THE CELL SURFACE contain It. Repeated changing of the sea-water in which eggs lie can wash them free from it so that fertilization is no longer possible. Also, by bathing the eggs in sea-water containing the animals' body fluid, the fertilizin present in them can be bound: fertilization is inhibited. That washing the eggs removes the fertilizin is evidence of its ectoplasmic location. Further, I found that after eggs In sea-water containing body-fluid have been mixed with spermatozoa, the spermatozoa attach themselves to the ectoplasm and even penetrate it but do not fertilize the eggs.^ If however these eggs are soon thereafter thor- oughly washed, they develop. From these facts it can be concluded that the inhibition to fertilization, by the block- ing of fertilizin by body-fluid, takes place in the ectoplasm. Moreover, the rapid loss of fertilizing capacity following the ectoplasmic changes which underlie the separation of the vitelline membrane whether induced by spermatozoon or by other means, Indicates that the fertlHzIn is located in the ectoplasm. The presence of fertilizin can be detected only if some of It escapes from the egg into the surrounding sea-water. The means of detection is simple. Sea-water in which fertllizable eggs have been lying has marked effects on sper- matozoa of the same species. If a drop of this sea-water be added to a dense suspension of spermatozoa, they are first stimulated to intense activity and rushing together adhere in masses by means of their heads. This agglutina- tion endures for some seconds — or minutes. If the sea-water is highly charged with the agglutinating substance, fer- tilizin— and then passes off". This agglutination-reaction Is a striking and Interesting phenomenon capable of nicely quantitative study. Its value is that of an indicator of the presence of the fertilizin that escaped from the eggs; it may itself have no significance for fertilization although ' Just, ic}22c, ig2jb. ig6 THE FERTILIZATION-REACTION it may suggest that the initial stage in the reaction between egg and spermatozoon is the agglutination of spermatozoon to egg-substance. The agglutination here discussed is never induced by treating spermatozoa of one species with sea-water charged with the sperm-agglutinin from eggs of another. This, hetero-agglutination, offers a different pic- ture qualitatively and quantitatively and is due to a sub- stance in the body-fluid.^ Thus fertilizin, as indicated by the agglutination-reaction which it induces, is specific. No question in the whole problem of fertilization has more profound significance than this of specificity. Fre- quently students of fertilization have asked themselves the question: how is it that in the sea, eggs of one species are fertilized by specific and not by foreign spermatozoa t In other words, how do eggs maintain fertilization-specificity? In the laboratory fertilization by foreign spermatozoa of most marine eggs almost never occurs as a normal event. If one mixes eggs of a sea-urchin and of a worm and insemi- nates them with spermatozoa of either sea-urchin or worm, only specific fertilization occurs. Also, a mixture of two kinds of spermatozoa added to eggs belonging to only one species of them induces species-fertilization. It has gen- erally been found that before cross-fertilization takes place among sea-urchins, starfishes, etc., the eggs need either a previous treatment with alkali or heat, or long residence in sea-water, so that they become "stale," or an insemina- tion with an excessive number of spermatozoa. All these methods for inducing cross-fertilization can be shown to injure the egg-surface, and so to decrease the egg's vitality. As long as the integrity of the ectoplasm remains unim- paired, cross-fertilization fails. Specificity in fertilization closely resembles that in immu- nity-reactions, where a specific anti-toxin combines with specific toxin. It is far more specific than an enzyme- ^ Just, igigb. IQ7 THE BIOLOGY OF THE CELL SURFACE reaction; besides, there is no evidence that the sperma- tozoon initiates fertilization by means of an enzyme, though enzyme-reactions certainly follow after fertilization. As I see it, specificity in fertilization depends in part upon the chemical constitution of the blood or body-fluid of the species which produces the eggs and spermatozoa. Whilst the chemical constitution of the egg of a species differs from that of all other cells comprising the animal's body, nevertheless there is a chemical similarity among all cells of the animal's body including the Q^g^ inasmuch as they are all nourished by the same body-fluid. Species- spermatozoa have the identical chemical structure as eggs to the extent that they are products of the same species' body and blood; at the same time they differ chemically in other respects from eggs. Blood (or body fluid) is different from all cells including eggs and spermatozoa. Now where blood inhibits species-fertilization, this inhibition is owing to its being present in such amount that it makes inert the specific substance in the egg's ectoplasm. A smaller amount blocks foreign spermatozoa and this amount can not be wholly removed except by injury of the ectoplasm. In nature, owing to the breeding habits of animals, eggs and spermatozoa of one species tend to be emitted simul- taneously within a fairly restricted breeding ground. Thus the chances for specific fertilization are enhanced. Where copulation takes place, specific fertilization is still more highly insured. On the other side, laboratory conditions for inducing cross-fertilization could scarcely obtain in the open sea. If nevertheless cross-fertilization should happen in nature, we can relate it to an injured state of the egg's ectoplasm. i As stated above, spermatozoa do not enter such fertilized eggs, or fragments thereof, which are normally mono- spermic, i.e., into which only a single spermatozoon enters. The prevailing opinion concerning the block to poly-sperm- igS THE FERTILIZATION-REACTION entry has supported the conclusion reached by Fol in his study of sperm-entry into the eggs of the starfish and of the sea-urchin, that the separation of the vitelline mem- brane prevents the entrance of supernumerary spermatozoa. The physical and chemical changes which take place in the membrane after separation from the egg-surface are cer- tainly such as would bar sperm-entry. But if the block were purely mechanical, removal of the vitelline membrane should make possible the entrance of extra spermatozoa into the egg. It has been shown for the eggs of other ani- mals, in addition to those of echinoderms, that disruption or removal from fertilized eggs of the vitelline membrane does not render the eggs any more liable to poly-sperm- entry. ^ With membrane-separation the eggs undergo some change and it is this change — not Its result, membrane- separation — which constitutes the block to the entrance of additional spermatozoa. Thus this block, which is more subtle than the mechanical obstacle interposed by the presence of a separated membrane, is established before membrane-separation occurs. A rather exact indication as to the moment when the block intervenes is furnished by my observations on the &^g of Echinarachnius . Here one can follow the wave in the ectoplasm which begins at the point of sperm-entry and sweeps over the egg. As this wave progresses, it renders the egg immune to the entry of supernumerary attached spermatozoa. The behavior of these latter sper- matozoa can be observed to change with the entrance of the "fertilizing" spermatozoon into the egg: as the wave ^ By -putting inseminated eggs in the best fertilizable condition through bolting-silk at the 7noment zvhen they separate their mem- branes one can most easily and with the least harm deprive them of their membranes. Spermatozoa may be added inunediately or at any time thereafter but each egg remains fertilized only by the single spermatozoon of the first insemination. igg THE BIOLOGY OF THE CELL SURFACE moves from the site of entry, the movements of those sper- matozoa nearest the site slacken and cease; next those farther away become immobile; finally those situated i8o° away come to a standstill. These events take place before membrane-separation. Often, however, the process is much too rapid to allow one easily to follow it. Neverthe- less I ha^'e under a good apochromatic lens followed it in eggs in best condition taken from hundreds of specimens. The eggs of Arbacia^ in my experience, if in best condition are never polyspermic. It is possible to fix eggs one second after spermatozoa have been added to them; examined under the microscope, each egg shows a spermatozoon attached to it. If a thick sperm-suspension be added to the eggs as early as one second after the first insemination, no polyspermy occurs. I have also made the initial insemi- nation with the heaviest sperm-concentration procurable, i.e., "dry" sperm as it exudes from the male, and have obtained only mono-spermic fertilization. Thus, the block to polyspermy is most rapidly interposed. The eggs of Platynereis when laid have each one sperma- tozoon attached. And yet in the body-cavity of the worm, the eggs, especially those in the anterior segments, are in the presence of supernumerary spermatozoa, as sections of the worms made immediately after copulation reveal. In all my slides of normally laid Platynereis eggs I have rarely seen one egg on which two spermatozoa are attached. I have never seen more than one spermatozoon within an egg. Surelv under the conditions of the normal insemination of eggs of Platynereis one can not postulate a mechanism so precise that it would distribute the spermatozoa in such wise that each egg would receive only one spermatozoon. Rather, within the narrow closed space of the body cavity packed with eggs — even the head segment contains them — prevails a situation most favorable for the aggregation of supernumerary spermatozoa around each eg^. And yet 200 THE FERTILIZATION-REACTION polyspermy does not occur. Eggs in the anterior segments of the worm, where most spermatozoa are found, are as free from polyspermy as those which first issue from the anal segment. ]\Iy preparations of starfish eggs which show more than one spermatozoon entering are of eggs fertilized either before break-down of the germinal vesicle or after second matura- tion. Never in the stage of first maturation when the eggs are in the stage of optimum fertilizability is there poly- sperm-entry. If, however, fertilizable eggs be injured, supernumerary spermatozoa enter. These observations lend themselves to the conclusion that the block to polyspermy is rapidly established with the attachment or entry of one spermatozoon. Now into all these eggs — of Echinarachnius, Arhacia^ Platy7iereis, Asterias — the spermatozoon may enter at any point. They thus differ from some other monospermic eggs, ~as^ those of the trout or the ink-fish, which possess a tube, the micropyle, through which the spermatozoon gains entrance; or such eggs, as of Ciona and of Amphioxus, into which spermatozoa enter always at the vegetal pole. Theoretically, therefore, these eggs under discussion are polyspermic; until they establish the block against super- numerary spermatozoa any part of the eggs' surface is responsive to sperm-attachment — leaving aside the unlikely assumption that one site alone admits the spermatozoon and that this would vary even in eggs of the same female. From this point of view the interposition of the block to polyspermy becomes highly significant for it oflFers a means by which we can set the termination of the initial reaction between normal egg and normal spermatozoon of any of the species named: it ends with sperm-attachment.^ The ^ This refers to normal eggs, it must be understood, since sperma- tozoa may enter unfertilizable eggs — i.e., immature ones. See Iwanzoff and others. 201 THE BIOLOGY OF THE CELL SURFACE process to all Intents and purposes is instantaneous, and therefore ectoplasmic. And this reaction that brings about a fundamental change in the egg which renders it incapable of reacting to other spermatozoa present and initiates the egg's development is the fertilization-reaction. The rapidity with which the complete fertilization-reac- tion terminates, obviously depends upon the speed at which the reaction runs. If the egg's ectoplasm is highly reactive the reaction will proceed at a rapid rate. In another egg the rate normally may be slower and yet sufficiently rapid to inhibit polyspermy under natural conditions where eggs and spermatozoa are fully normal. In these, experimental polyspermy may be induced with greater facility. The reaction will be slowest in normally polyspermic eggs. If the block to polyspermy also in normally polyspermic eggs is taken as the indication that the fertilization-reaction is complete, this end would come in such eggs when finally they can receive no more spermatozoa. Normally monospermic eggs can be rendered polyspermic by experimental treatment as staling (long residence in sea-water), treatment with alkali, acids or poisons, subjec- tion to low temperature, in short, by methods for inducing injury or weakness. The eggs also tend to be polyspermic when below optimum condition as, for example, those obtained toward the end of the breeding season or from moribund animals. Polyspermy in them thus is a sign of weakness and hence pathological. In all conditions fav- orable to poly-sperm-entry the ectoplasmic response differs from that in normal fertilization. If, for example, eggs of a sea-urchin after having been kept at low temperature, around 5°C., are inseminated, they show polyspermy and the vitelline membrane separates only very slightly, which ■ Indicates abnormal ectoplasmic activity. If treated witlr an organic acid, e.g., 2 cc. Ko normal butyric acid plus 50 cc. of sea-water, for one minute or more, eggs of sea- 202 THE FERTILIZATION-REACTION urchins are rendered highly susceptible to poly-sperm-entry whilst their ectoplasm becomes thickened and the mem- brane only slightly separated. The same result obtains after exposing the eggs to the alkaloids, strychnine, nicotine, etc., as the Hertwigs^ showed in their now classic experi- ments. If one repeats their observations one marks the striking responses of the egg-surface to insemination, among others, the longer duration of the many "fertilization- cones." The ectoplasm need not be severely injured in order that polyspermy succeeds. If the ectoplasmic response to insemination be slowed down, polyspermy may ensue. Thus, if one knows that eggs of a given lot are below normal by having learned through trial insemina- tions on some of them that the surface-changes underlying membrane-separation proceed at an abnorrrially slow rate, one can by inseminating with a heavier sperm-suspension than that usually employed secure polyspermy. Some animal eggs, as those of cartilaginous fishes, of some amphibians and of birds, are normally polyspermic. Poly- spermy has also been reported in insect eggs. All normally polyspermic eggs are large. ^ But since there exist also some large eggs which are not polyspermic, it is, presum- ably, not so much the size of the Qgg but the slow reaction of its ectoplasm which makes polyspermy possible. The suddenly arising fertilizable condition of animal eggs thus resides in the ectoplasm. The indications are that this is a chemical reaction. Consider the problem of spe- 1 Hertwig, 0. and R., 1SS7. ~ Bonnevie^s conclusion {igoy) that polyspermy obtains in eggs of Membranipora is incorrect as careful study of her paper reveals. See also my failure to find polyspermy in this egg — Just, 1934. MacBride'' s statement that polyspermy occurs in eggs of Pedicellina is undoubtedly due to his error in interpreting Hatschek^ s statement that he often observed numerous 7notile spermatozoa in the perivitelline space. 203 THE BIOLOGY OF THE CELL SURFACE cificity. The phenomenon of specificity generally we relate to chemical constitution and not to physical properties. No valid reason can be proffered for assuming that spe- cificity in fertilization is unlike that met with in other bio- logical processes. Unless such reason is forthcoming, we must assume specific fertilization as chemical. Where, as in eggs of some species, a substance has been isolated upon which fertilization depends, we are warranted again in postulating that the fertilization-reaction is chemical — one between this substance and the spermatozoon. Finally, the changes, so strikingly visible in many eggs, whereby the vitelline membrane is separated, can not, as we have seen, be regarded as the initial reaction in fertilization: membrane-separation, a physical process, though common to all eggs, is the result of the fertilization-reaction and not the reaction itself. What happens is this: the spermatozoon sets off a first explosion in the narrow area of the egg-surface which it touches, and kindles the spark which leads to a chain of explosions. The fertilization-reaction is thus a trigger- reaction. A large portion of the surface is shattered by the explosive effect with gas-exchange and heat-liberation. One can in many eggs see this break-down of the surface by which the egg loses substance and the membrane is separated. The pressure between egg and membrane, in the perivitelline space, probably is considerable as the ectoplasmic colloids disintegrate and go into solution. Water rushes in and further distends the still ductile mem- brane which then sets as a stiff structure. The fully separated membrane then becomes brittle; one can more easily remove it immediately after its separation than later when it becomes tough. But these changes in the mem- brane are due to its separation from the Qgg, for it no longer forms part of the living system. What therefore are impor- tant for the fertilization-reaction are the underlying surface- 20.p THE FERTILIZATION-REACTION changes that result in membrane-separation and neither the consequent physical act of separation nor the changes in the membrane itself. Nevertheless this separation of the membrane constitutes an easily visible indicator of the underlying surface-changes initiated by the fertilization-reaction. It tells us quickly whether or not fertilization has taken place. Also, it gives us information concerning the quality of the eggs fertilized. Membranes that separate incompletely and are not equi- distant from the egg at all points and are slow in rate of separation mean eggs of poor fertilizability that subse- quently develop abnormally. Let me emphasize that here I speak only of fertilized eggs which have favorable conditions for subsequent develop- ment. If after fertilization conditions are deleterious, the eggs will not develop normally no matter how perfect the membranes. Moreover, as we shall learn in the next chap- ter, membrane-separation alone, though most perfect, does not guarantee development. Indeed, even in those cases in which perfect membranes are separated by experimental agents, they are not wholly identical with those called forth by sperm. What here looms large is the value of the quality of the surface-changes for foretelling the future course of the egg's development. Quite apart there- fore from their value for the study of the fertilization-reac- tion, the ectoplasmic changes by which the membrane becomes separated have greatest significance. Develop- ment embraces a series of surface-changes which vary as the surface-area increases with the march of development. Those occurring at the very outset are most striking; they mark out the course, direct the way toward the final out- come of fertilization, the formation of the complex organism out of a single cell, the egg. 205 9 Parth en og en esis jDiological processes often reveal themseles as themes with many variations. Fugitive incidental nuances embellish the process of fertilization, as we have seen; but though they run the whole range of variation, they never obscure the motive: fertilization as the union of egg-plasma and spermatozoon. That the egg- and sperm-nucleus are equipotent in the developmental process, since with either alone the egg's development can successfully proceed, has been pointed out. Thus, whilst the development of the egg of any one of sev- eral marine invertebrate animals can be initiated by fer- tilizing it after its nucleus has been removed, the egg-nucleus of the tg^ of Rhahditis aherrans alone takes part in devel- opment, for the sperm-nucleus within the Q^g remains Inert. Neither of these examples is a violation of the state- ment that for the majority of multicellular animals the life of the new individual begins with the coming together of the two living gametes, egg and spermatozoon. Rather, both demonstrate very clearly this essential fact: fertiliza- tion Is not the fusion of the egg- and sperm-nuclei but the union of egg-plasma and spermatozoon. Now we turn to the discussion of phenomena which reveal that even this union Is not always necessary for the elevation of the life- process in the egg from the level of a single cell to that of an individual of multicellular organization. There are eggs which normally develop without spermatozoa. This type of development, encountered among many animal species. Is called parthenogenesis. Of the theme, the Inltia- 206 PAR THENOGENESIS tion of the development In the egg, therefore, fertilization and parthenogenesis are variations. Investigations in the realm of inanimate nature have in the last decades led to such a wealth of new discoveries, have so shattered the foundations of the old point of view that we are warned against beholding the picture of inani- mate nature now offered us as final. Full well we know that every day holds the possibility of new discoveries which must make everything known appear in a new light. Also in the investigation of living nature we may be certain that we stand only on the threshold of the knowledge of its deeper principles and we should avoid too quick and final conclusions. Even when we know clearly the exact goals which living nature reaches, and when we survey the chief paths which it traverses to reach these goals, always does the fact that there are by-paths — detours to our conditioned human comprehension — take us by surprise. We should not look upon this endless richness of nature's creative capacity as an opportunity for us to confirm a preconceived notion or theory. Rather with wide open mind should we be recep- tive to this richness In all its manifold expressions. In calling forth a new individual, nature has not limited itself to one mode; both fertilization and parthenogenesis initiate development of the multicellular animal. When it was found that parthenogenetic development could be initiated also in the laboratory by experimental means, we should have taken this discovery as a sign of the manifold possibilities that inhere in an egg-cell to respond to stimuli. Instead, this discovery was held to mean that the final solu- tion of the problem of the initiation of development in animal eggs had been reached, that the final word concern- ing one biological phenomenon had been spoken — and even, that man here had mastered nature, that he himself could create life. The discovery of isotopes has taught us 207 THE BIOLOGY OF THE CELL SURFACE that with respect to even simpler material structure nature does not always limit itself to one mode, that we must amend our natural laws as we learn more about nature. Such discoveries also suggest that more complex natural processes wear many guises. Had the parthenogenesis that occurs in nature received due attention — if in fact it had come within the knowledge of those who so greatly busied themselves with experimental parthenogenesis — the discovery of experimental parthenogenesis never would have had the palm awarded it. Parthenogenetic development is found naturally occur- ring especially in the groups of rotifers and arthropods. This natural parthenogenesis may be fixed (obligatory) — i.e., the eggs develop normally only parthenogenetically. Experimentally, a change either in the environment or of the food of the mother may render the eggs capable of receiving the spermatozoon. In such cases the period of fertilizability comes during first maturation. Natural parthenogenesis is also variable (facultative) — these eggs develop normally either parthenogenetically or by fertiliza- tion: as, for example, eggs of the honey-bee. In fixed parthenogenesis, the first polar body is extruded, the second is not; hence the egg develops with the double (somatic) number of chromosomes. The chromosomes of the second polar body may unite with those of the egg- nucleus, thus acting as a substitute for the sperm-nucleus.^ An egg of the variable type of parthenogenesis extrudes both polar bodies and develops with only a single set of chromosomes. A double set of chromosomes is not, there- fore, a prerequisite for the onset of naturally occurring parthenogenetic development; nor does the presence of only a single set of chromosomes imply defective develop- ment: the male honey bee (or drone) for instance, partheno- ^ Cf. Artemia, Brauer, /S^j. 20S PAR THENOGENESIS genetic in origin, is a normal organism though he lacks the sperm-borne chromosomes of his sisters. With the aid of chemical solutions, changes in tempera- ture, mechanical shock or radiations, eggs which normally never develop without fertilization can be induced to develop parthenogenetically. Up to the present we have succeeded in inducing this artificial or experimental par- thenogenesis in eggs of echinoderms, worms, molluscs, a spider, fishes, and frogs. As we shall later learn, the only period in which experimental parthenogenesis is possible coincides with the stage in which the egg is fertilizable. Since, for the eggs of the animals named, this fertilizable period falls, for some of them, in the germinal vesicle stage, for others during first, for others during second, and for the remainder after complete maturation, we can not regard only one of these stages as the prerequisite for induced parthenogenesis. Unfortunately, of the eggs of the various species of ani- mals that have been treated with means for inducing par- thenogenesis only those of a sea-urchin, of a starfish and of frogs have been reared to sexual maturity. The composition of the nucleus can generally be given only for early stages of development. With respect to the end- result of development our knowledge of induced partheno- genesis falls far short of that of the natural. Of the few cases known the sea-urchins' and the starfish eggs show throughout development a single set of chromosomes. The adult frog derived from a parthenogenetic ^gg is different; such an individual has either cells with single or such with double sets of chromosomes or cells of both types. Noth- ing indicates that the normality of these adults varies with chromosome-garniture.^ The same can be said for the ^ We must not overlook the fact that there are variations in the chromosome number in cells of adults from fertilized eggs. 2og THE BIOLOGY OF THE CELL SURFACE larval stage of those eggs whose induced parthenogenetic development has not been followed farther. After successful treatment with a parthenogenetic means eggs whose optimum period for fertilization follows com- plete maturation develop parthenogenetically with one set of chromosomes (example: echinids). The egg of the frog after puncture (the method used for inducing partheno- genesis in this egg) made after extrusion of the first polar body, which is the fertilizable stage for this egg, extrudes the second polar body and begins development always with only a single garniture of chromosomes, while in later devel- opment this chromosome-number has been found doubled. The situation is different with eggs physiologically ripe for fertilization in either the stage of the intact germinal vesicle or that of first maturation. After successful treatment with a means of experimental parthenogenesis, they develop with or without one or both polar bodies — hence, their blastomeres contain single, double or more garnitures of chromosomes. It is thus clear that development induced by experimental means does not depend upon the presence of two or more sets of chromosomes. In this respect experimental par- thenogenesis resembles the normal. An explanation of either type of development can not, therefore, be based upon a bipartite make-up of the first cleavage-nucleus. Here we note, however, the following difference between the natural and the experimental process: in natural par- thenogenesis the egg always has the nuclei of both polar bodies available although it develops with or without the union of the second of these with the definite egg-nucleus. In experimental parthenogenesis the polar-body nuclei are not always available for fusion with the egg-nucleus. This difi"erence is due to the fact that in eggs which possess the capacity for natural parthenogenesis, development, either with or without spermatozoon, begins in the stage of first maturation, i.e., before polar body extrusion — whilst eggs 210 PAR THENOGENESIS capable of developmental response to experimental means are distributed among all of the four categories made above with respect to the stage in the maturation process, in which animal eggs are physiologically ripe for fertilization. Eggs which are fertilizable in the stage of first matura- tion are more widespread in occurrence among animals than those of any other class. Since both the facultative and the fixed parthenogenetic eggs belong to this large class, we might expect that experimental parthenogenesis is more easily elicited in this class of eggs than in any other. This expectation, however, is not fulfilled. Although the Qg% of the starfish, fertilizable in the stage of first matura- tion, and the first in which was discovered the capacity of eggs to respond to experimental treatment with develop- ment, is of all animal eggs the most readily responsive to experimental means for parthenogenesis, other eggs of the same class, as we shall learn, may fail wholly to respond to means effective upon the starfish egg or they respond with extremely abnormal development. Also, the greatest number of positive results has been obtained on completely matured eggs, i.e., those of sea-urchins. Finally, eggs of the two remaining classes when properly treated develop normally; frogs' eggs, for example, reach the adult stage. We can not therefore correlate the ease with which experi- mental treatment elicits parthenogenesis with the nuclear state of the egg. Whether or not the restriction of natural parthenogenesis to eggs fertilizable in the stage of first maturation bears a causal relation to this mitotic phase of the nucleus, has not yet been determined. As to the cause of experimental parthenogenesis, however, there are only two possibilities: it rests either in the egg's cytoplasm or in the means that brings about the parthenogenetic development. The latter view one often meets, namely, that the means carries into the egg something which initiates its develop- ment. The brief review of the response of individual eggs 211 THE BIOLOGY OF THE CELL SURFACE to experimental means for eliciting development which now follows will give evidence against this view and prove that the egg's response like that in fertilization is cytoplasmic. Above I called attention to the interesting case of the starfish egg. Like that of many other eggs, its germinal vesicle breaks down when the egg comes from the female into sea-water. But whereas other eggs of this class do not develop farther unless they are fertilized, the unfertilized egg of the starfish completes both maturation-divisions. This fact suggests that this egg is extremely unstable and lies close to being normally parthenogenetic. It is not astonishing, therefore, that the starfish egg is found to be most easily induced to parthenogenetic development by one of several means. The knowledge that sea-water charged with carbon-diox- ide brings about the development of eggs of a starfish, Asterias glaciaiis, we owe to Delage^ who was able to rear the parthenogenetic larvae through metamorphosis. This method has been successfully employed by others- on eggs of this as well as on those of other species of Asterias. Development also results if one crowds a large number of eggs in the stage of first maturation in a small volume of sea-water and leaves them there for about an hour — in this case the result is due to carbon-dioxide produced by the eggs. In my judgment the parthenogenetic development obtained by Mathews^ through shaking eggs of Asterias forbesii is likewise to be attributed to carbon-dioxide pro- duced by crowding the eggs; shaking of maturing eggs, in my experience at least, is without avail unless the eggs be crowded. If a thin suspension of maturing unfertilized eggs of this species is placed in large volumes of sea-water in uncovered 1 Delage, igo8, igio. - Buch^ier, igii and others. •■' Mathews.^ igoi. 212 PAR THENOGENESIS shallow dishes so that evaporation takes place, the eggs develop when transferred to normal sea-water. Similarly, sea-water to which solutions of sodium or potassium chloride have been added induces development. Thus, hypertonic sea-water is a means of calling forth parthenogenesis. Butyric acid in sea-water, like carbon-dioxide, also induces parthenogenesis in the eggs of Asterias forbesii. R. S. Lillie has studied the relation of the duration of the treatment with this acid in sea-water to the degree of response elicited. He obtained only membrane-separation with short exposure; membrane-separation and cleavage with a longer exposure, and full development to the swim- ming stage by a still farther increased length of exposure. Increased temperature of the sea-water alone or combined with butyric acid, and two short exposures to the acid sepa- rated by the residence of the eggs in normal sea-water are also successful. Any one of these methods is superior to the hypertonic sea-water method by giving higher percent- age of parthenogenetic development. In 1876 Richard Greef reported that he had observed the parthenogenetic development of the eggs of a starfish, Asteracanthion [Asterias) to the ciliated larval stage (gas- trula). The larvae obtained were vigorous and corre- sponded thoroughly with those developed from fertilized eggs. Since Greef had taken every precaution against the accidental presence of spermatozoa, since further the ani- mals from which the eggs came gave no evidence of being hermaphroditic,^ and since, as he had learned before, ferti- lized eggs reach first cleavage in one to two hours after insemination, whilst these parthenogenetic ones cleaved first only at ten to twelve hours after having come into sea- water, he was certain that he had observed a true case of ^ Hermaphroditism among star^sh is rare. See Retziiis, /(?//, Vol. 16 of his collected zvorks; Buchner, ipil. 2rj THE BIOLOGY OF THE CELL SURFACE parthenogenesis. That he had induced parthenogenesis and had not followed a normally parthenogenetic develop- ment seems to me to be beyond question. Thus without knowing it he was the first to induce parthenogenesis in marine eggs. Evidence may be adduced upon which this judgment is based. '-"' In the first line I place Greef's work itself. The observed great difference between the fertilized and the partheno- genetic eggs with respect to the time when they reach first cleavage, points strongly to an experimental induction of development. This was probably brought about by an altered condition of the sea-water. If the eggs were crowded, carbon-dioxide was pres- ent in high concentration. At the time of year, the beginning of May, when the observations were made, the water in the vessels containing the eggs most probably rose in temperature, in the absence of any precautions taken against such rise. Or, if the eggs were kept in uncovered vessels, evapor- ation took place. Any one of Fig. 32.— Ripe egg of Aster- -i •,• • n n 1 acanthion ruhens after having these possibilities, all Well-known lain two hours in sea-water means for inducing parthenogenesis (after Schneider). • ^ c ^ 1 j ^ in Starfish eggs, would account for Greef's results. Schneider in 1883 pictured an tgg of Aster acanthion which after having lain two hours in sea- water showed many asters. This is clear evidence to any- one familiar with this egg that this change is induced by the residence of the egg in sea-water (Fig. 32). Secondly, later work on this egg may be considered. In reading O. Hertwig's report on his observations made four- teen years after Greef's, on the egg of Asterias glacialis and 214 PAR THENOGENESIS especially on that of Jstropecten, one is struck more by his failures than his success to obtain parthenogenesis.^ Were the eggs of these two starfishes normally parthenogenetic, he would have secured a higher per cent, of development; also, the development would have gone farther and would have more closely resembled that of fertilized eggs. Three years earlier, when he attempted to observe parthenogenesis in the eggs of Asterias and so to repeat Greef's work, he had either changed or aerated the sea-water, so that the sea-water neither increased in hypertonicity nor in tempera- ture and did not become charged with carbon-dioxide. At that time he failed completely to secure development beyond the establishment of the definite egg-nucleus. In other words, the eggs, as is normal for them, merely com- pleted maturation in sea-water. In the later experiments where he observed parthenogenetic development, he does not mention having protected the eggs against changes in the sea-water. The one egg that reached the blastula- stage, though seemingly normal in outward appearance, lacked the separated membrane so characteristic of the fertilized egg and of that which has been induced to develop by means of treatment with CO2 or with increased tempera- ture. This observation on the single blastula obtained, strongly indicates in the light of my experience that Hert- wig induced parthenogenesis by means of sea-water made hypertonic through evaporation. The strongest evidence that both Greef and Hertwig induced parthenogenesis experimentally in these eggs lies in the fact that although starfish eggs are extremely responsive to experimental means, no one since has suc- ceeded in demonstrating that they are normally partheno- genetic. My experience with the egg of Astropecten ^ Hertwig, 0., iSgo. 215 THE BIOLOGY OF THE CELL SURFACE convinces me that it resembles those of three species of Asterias which I have studied, and that its classification as a normally parthenogenetic egg is unwarranted. Eggs of the marine worm, Chaetopterns, like those of the starfish fertilizable in the stage of first maturation, when treated with hypertonic sea-water develop into bizarre and wholly abnormal swimming forms through repeated nuclear divisions in the cytoplasm which fails to divide, a condition known as differentiation without cleavage.^ Following exposure to hypertonic sea-water, other eggs fertilizable in the stage of first maturation, e.g., those of Podarke'^ and of Amphitrite,^ both worms, develop without cytoplasmic cleavage. For the egg of Chactopterus and for that of the small marine clam, Cumingia, increased temperature of the sea-water, induces the best type of parthenogenetic development — with cytoplasmic as well as nuclear divisions. The Q^g of Chaetopterus merits attention because Mead used it in his interesting experiments. Previously in his paper on the normal fertilization-process in this egg he had set forth a strong argument against the theory that fertilization is due to the introduction of centrosomes into the egg by the spermatozoon. His work on the effect of salt solutions in initiating development was the logical outcome of his main conclusion in the work on fertilization, a later statement of which reveals his clear conception of the initiation of development as a chemical process. His work on inducing parthenogenesis, therefore, was no mere acci- dental experiment; rather, at its basis was a well defined working hypothesis. His was the first work based on the assumption that development can be experimentally induced. Why Mead so suddenly dropped this promising line of research is strange. It was justly said by Loeb, ^ Lillie, /po6. ~ Treadwell, igo2. ^ Scott, igo6. 216 PAR THENOGENESIS that Mead's work is too often overlooked and deserves greater appreciation than it has received. Turning to that class of eggs which are fertilizable in the stage of the intact germinal vesicle, I may cite the results of experiments to induce parthenogenesis in three eggs: those of Mactra, of Thalassetna and of Nereis. Eggs of Mactra, a marine clam, exposed to lo cc. of 23-2 N KCl plus 90 cc. of sea-water for 45 minutes complete maturation and develop as far as the six-cell stage. Longer exposures, i^-? to 4 hours, and to stronger solutions, give development without formation of polar bodies. KCl- treated eggs may also develop into swimming forms by nuclear divisions only, i.e., they differentiate without cleav- age as do the eggs of worms mentioned above that are similarlv treated.' By the action of either mineral or organic acids, including carbon-dioxide, in sea-water, one can induce the develop- ment of 50 to 60 per cent, of unfertilized eggs of Thalas- sema mellifa,'' a marine worm, to the larval stage; this is scarcely to be distinguished from that of the normal, i.e., developed from fertilized eggs. Because of these positive results obtained with acids, the effects of hypertonic sea- water were not thoroughly investigated. Those reported are not only too incomplete but also too contradictory to permit the drawing of a conclusion. In view of the effi- ciency of hypertonic sea-water on so many other eggs, how- ever, one may hazard the opinion that it is efficacious on this egg also. One is strengthened in this opinion by recent work on the eggs of another species of Thalassema, T. neptuni.^ In the egg of Nereis by shaking or centrifugal force ecto- plasmic break-down, with extrusion of jelly, and matura- ^ Kostanecki, igii and earlier. ' Lefevre, igoi. ^ Hobson, ig2S. 217 THE BIOLOGY OF THE CELL SURFACE tion are brought about. Prolonged exposure to weak or short exposure to stronger hypertonic sea-water gives the same results. With still longer exposure to hypertonic sea- water, differentiation without cleavage is called forth. By treatment with hypertonic sea-water I have never been able to induce development with cytoplasmic cleavage in this egg. Hypotonic sea-water also gives differentiation without cleavage.^ I find that the percentage of swimming forms may be increased by the addition of an acid or an alkali to the dilute sea-water. Hypotonic sea-water in my experience is an inferior means of inducing parthenogenesis in this egg. Ultra-violet radiation also induces a qualita- tively poor development.^ If eggs from a female from whose body the sea-water has been removed, are placed on a dry glass plate for a few moments, and are then put in normal sea-water, a small percentage develops.'^ Eggs having been kept at a temperature around 5°C. extrude jelly at once when removed directly to normal sea-water at room temperature (around 20°C.). Of these some will develop farther. The best method for inducing the devel- opment to the stage of larval worms, which can scarcely be distinguished from those developed from normal, fer- tilized eggs, is to expose eggs to sea-water at a temperature of from 30 to 33°C.'* In this method it is necessary that the eggs come directly from the female into the warm sea-water without having lain in sea-water at room tempera- ture. If eggs are taken from sea-water at room tempera- ture, they need an exposure to a higher temperature, around 40°C., for about a minute to be stimulated to develop.'' In ^ Just, ig2Se. -Just, 1933c. 3 Just, 1915a. ^ * Ibid. " Just^ unpi(bluhed observations. ~ • 21S PAR THENOGENESIS this case the percentage of development is not so high as that obtained by use of the method just mentioned. Eggs of sea-urchins, fertilizable after complete matura- tion, early became the favorite objects for experiments on induced parthenogenesis. Although Morgan made experiments which indicated that by means of treatment with hypertonic sea-water, eggs of Arbacia can be induced to develop parthenogenetically, he failed to extend his studies sufficiently and thus to obtain the production of larval forms from unfertilized eggs. Despite this failure his work demonstrated that treatment with hypertonic sea-water can call forth the establishment of the mitotic complex, the sign that development has been initiated. By extending Morgan's work, J. Loeb was able to induce eggs of Arbacia to develop parthenogenetically to swimming forms.' He deserves the full credit because he appreciated his findings, whilst it appears that of his pre- decessors, Greef, O. and R. Hertwig, Mead and Morgan, only Mead experimented with the definite purpose of induc- ing development by chemical means. Loeb found that, whilst after exposure for two hours to a solution of 50 cc. of sea-water plus 50 cc. i^g M MgCU, the eggs of the common sea-urchin, Arbacia, found at Woods Hole, Mass., developed into swimming larvae, the use of other salt solutions gave no results. The next year, work- ing on the California coast with eggs of the sea-urchins, Strongylocentrotus purpuratus and .S, franciscanus, he was able to induce development not only with MgCU, but also with NaCl or KCl, either of these salts being added to sea-water, in the proportions 10 cc. of a 2.5 gram molecular solution to 90 cc. of sea-water. Later, other salts as well as cane sugar and urea were also found to be effective. This is the so-called old or original method of parthenogenesis. ' See Loeb /p/J and earlier. 2ig THE BIOLOGY OF THE CELL SURFACE sometimes called the osmotic method for inducing develop- ment of the unfertilized sea-urchin egg. This early work established that sea-water, if made suffi- ciently hypertonic by the addition of electrolytes or non- electrolytes, is capable of initiating development of sea-urchin eggs. It shows that the effective agent is not specific; the original failure to induce development with the chlorides of sodium and potassium was obviously due to an error which Loeb made in preparing the solutions. It was soon learned that sea-water concentrated by boiling is capable of inducing the development of this egg. I have been able to induce experimental parthenogenesis simply by allowing the eggs to remain uncovered in a glass dish with sea-water; in this way, sufficient evaporation takes place to bring the sea-water to that degree of hypertonicity which is effective for stimulating the eggs to develop.^ But there are certain shortcomings to this osmotic method. As we have seen, the eggs of sea-urchins separate their vitelline membranes after insemination. The final distance of the membrane from the egg and the rapidity of this separation are indices of the physiological state of the egg: an egg in best condition separates its membrane at a uniform and rapid rate, with the result that it is equidistant from the egg-surface at all points.- After the above men- tioned treatment with hypertonic sea-water, sea-urchin eggs do not show a separated membrane. Instead, the vitelline membrane present on the egg before treatment remains closely stuck; the hyaline plasma-layer beneath it swells.^ Furthermore, the fertilized egg, if it be in opti- mum condition, cleaves at a regular tempo and the blasto- meres adhere to each other. After the treatment with the hypertonic sea-water here discussed the blastomeres 1 Just, igzSa. - Just, igzSc. ^ Just, 1919c, 1922b. 220 PAR THENOGENESIS cleave irregularly, both as to tempo and as to size, and they tend to fall apart. In the next place, normally the fertilized egg develops into a form which swims at the surface of the sea-water; subsequent to treatment with this hypertonic sea-water, those eggs which happen to develop as far as the larval stage, never become top-swimming forms. Finally, if sea-urchin eggs are in best physiological condition at the time of fertilization, close to one hundred per cent, of them reach the larval stage; on the other hand, with the osmotic method of initiating development, the percentage of developing eggs lies far below that obtained from fer- tilized eggs of the same female; if the worker is careless, using eggs which are not in best condition, he may obtain no developing forms. Because Loeb noted that the vitelline membrane did not separate, and that the larvae failed to swim at the surface of the sea-water, he next endeavored to find a method which would overcome these deficiencies. The result of his investigations was the so-called improved method of arti- ficial parthenogenesis for sea-urchins' eggs. This is the famous fatty acid plus hypertonic sea-water method, some- times called the Ivsin-corrective factor method. In this method the fatty acid mostly employed is butyric acid. Loeb first studied the eggs of the California sea-urchin, Strongylocentrotiis purpuratus. He placed unfertilized eggs in a solution of 50 cc. of sea-water plus 2.8 cc. of 1 10 normal butyric acid and left them in this solution for one and one- half to two and one-half minutes. On removal of these eggs to normal sea-water, they separated membranes. Eggs removed earlier than the minimum time did not separate membranes nor did those eggs which remained in the acid solution longer than optimum time, because prolonged exposure to the acid is injurious. After the eggs had been in normal sea-water for fifteen to twenty minutes, they were immersed in a solution of 50 cc. of sea-water plus 8 cc. of 221 THE BIOLOGY OF THE CELL SURFACE 2.5 gram molecular NaCl. From this solution, beginning thirty minutes after immersion, thev were transferred to normal sea-water at intervals of either two and one-half or live minutes. This double treatment of fatty acid and hypertonic sea-water results in a development that closely approximates the normal, because the eggs not only show separated membranes, but the larvae swim at the surface of the sea-water. It is to be especially emphasized that in this method the order in which the means are used is of no consequence to the results obtained: the butyric acid may be used before or after the hypertonic sea-water. For the eggs of Arbacia at Woods Hole, the method is somewhat different. According to Loeb, 2 cc. of ^^f 0 normal butyric acid in 50 cc. of sea-water acting from one and one- half to three minutes must be employed. Even so, Loeb did not succeed in inducing the eggs of Arbacia to separate their vitelline membranes; they only showed what he called a fine gelatinous layer which was not easily visible. It remained for Heilbrunn^ to show that the butyric acid treatment for this egg must be shortened; then the vitelline membranes are separated in much the same form as from fertilized eggs. This I have confirmed for the eggs of Arbacia. Also, I have obtained with butyric acid perfect membrane-separation in the tg<g of another echinid, Echinarachnius."^ Of this resume, although it is by no means complete, we can make the following summarized statement which holds for all cases of parthenogenesis experimentally induced in marine eggs. 1. Only when they are in their normal fertilizable period do eggs respond to experimental means. 2. For all eggs, one means only — as heat, cold, acids, hypotonic sea-water, hypertonic sea-water, etc, — is suffi- ^ Heilbrunn., 191 5- 2 Jusi, 1919c, 1920. 222 PAR THENOGENESIS cient. We shall soon learn that eggs of sea-urchins which, with the above discussed osmotic method, develop abnor- mally, are no exception to this rule. 3. Hypertonic sea-w^ater is most generally effective although the result is not the same qualitatively for each species of eg^. 4. No experimental means is limited in its action to eggs of one fertilization-class only. No. 4 requires comment. If we arrange eggs into four classes, on the basis of their fertilization-moment, we do not find that for each of these classes one special treatment is alone successful. Heat, for example, is as successful on the egg of Nereis in the germinal vesicle stage as on that either of the worm, Chaetopterus, or of the clam, Cumingia, both in the stage of first maturation. On the other hand, eggs of the same fertilization-class do not always respond to the same means. Hypertonic sea-water does not induce cytoplasmic division in the egg of Nereis, but does in the egg of Alactra, which like that of Nereis is fertilizable in the germinal vesicle stage. Acid-solutions in sea-water fail to Initiate development in the egg of Nereis; they call forth development in the egg of Thalassema^ fertilizable like that of Nereis in the germinal vesicle stage. Moreover, consider the egg of the starfish. It responds best when in that stage, first maturation, which is optimum for its fer- tilization. Carbon-dioxide, butyric acid or heat alone is sufficient for eliciting complete response: membrane-separa- tion, cleavage and vigorous larvae. Butyric acid alone, however, does not initiate development in eggs of other species in whatever stage of maturation they are fertilizable. Shaking, which suffices for the induction of development of the starfish egg, is without effect on other eggs fertilizable in the same stage, though In the egg of Nereis it does cause the break-down In the ectoplasm and the dissolution of the germinal vesicle. THE BIOLOGY OF THE CELL SURFACE Thus, as far as we have seen, a single inducing means is effective in eliciting complete development in all eggs named except in the sea-urchins'. The two exposures to the solu- tion of butyric acid in sea-water used on the eggs of the starfish are not to be regarded as a double treatment since development is initiated by one exposure if it be sufficiently prolonged; in the double or intermittent exposure the effects are additive. Treatment with two different means, such as butyric acid and hypertonic sea-water, is unnecessary for all eggs named above, except for the sea-urchin's egg which does not develop normally with the original osmotic method, as we have seen. This point is of fundamental significance since a theory of experimentally induced parthenogenesis has been based on the exceptional case of the sea-urchin's egg. Upon the experience that the double treatment brings about good development in the sea-urchin's egg a theory of general application was built. And this despite the fact that for all other eggs the double treatment is not necessary. But, as I was able to prove, also the sea-urchin's egg can be made to develop normally by a single parthenogenetic means. ^ Using a strong solution made up of 20, 22 or 24 parts of 23^^ M NaCl (or KCl) plus 80, 78 or 76 parts respectively of sea-water on the eggs of Arhacia instead of the solution employed by Loeb (8 parts 232 M NaCl plus 50 parts of sea-water), I was able to induce the development of a high per cent, of Arbacia eggs. The eggs separate beautiful membranes while in any one of these solutions, but need to remain in it for some time thereafter to give farther develop- ment.'^ On transfer to normal sea-water after the opti- 1 Just, ig22a. - It should be noted that R. S. Lillie found thai to induce complete development of the starfish egg — i.e., to the larval stage — the acid must act for a longer time than that sufficient to cause membrane- 224 PAR THENOGENESIS mum exposure, they cleave regularly, later becoming larvae which swim at the surface of the sea-water. This method is far simpler than the butyric acid plus hypertonic sea- water method; and in my experience it is also superior. Batallion and Batallion and Tchou working with several species of sea-urchins have confirmed my findings.^ Thus, the method of double treatment is unnecessary for sea-urchins' eggs. They, like all other animal eggs so far studied, respond to treatment with a single agent as the author of the method of the double treatment himself has shown in his original studies on three species of sea-urchins. The fact that treatment with a single means initiates development of unfertilized eggs renders less difficult the approach toward an understanding of naturally occurring parthenogenesis. Every organism, unicellular or multicellular, lives in and depends upon a world of its own. Whilst some have all the oceans, all the earth, as home, or freely roam all the sky, most dwell in a more circumscribed sphere; yet all sense quickly changes in the environment that hems them in. As with individuals, so with the cells in multicellular organisms — to changes in alkalinity, acidity, salinity and temperature they are acutely sensitive. Within only nar- row ranges of alkalinity, acid-, salt-content and temperature can the cells of the human body, for example, exist.- Vary any one of these factors beyond a certain limit and human life becomes impossible, as we all know. The grand wonder of the human body is the maintenance of these factors as constant. separation. This finding is true for all the marine eggs named. The inducing of development in the frog's egg by puncture seems to be an exception. 1 Batallion, 1926; Batallion and Tchou, igsS. See also these workers {/Qjf) on Bombyx eggs. - See Barcroft, /pj2; and earlier., CI. Bernard. 225 THE BIOLOGY OF THE CELL SURFACE Now although other organisms and their cells do not so narrowly depend upon environmental changes, as do man and other warm-blooded animals, nevertheless they quickly respond to changes in their peculiar world, the environment to which they are keyed and with which they are in accord. For practical purpose, we abstract organisms and cells from their surroundings — we could scarcely do otherwise in most cases for we can not always study whole organisms or entire cells at once but only their parts, each in turn. Still we need ever to remember that we indulge in abstracting solely for the purpose of comprehension and that an organism or cell has a dependent relation to its environment. Life can not exist apart from its external world. Nowhere, I think, in all biology does this most strong relationship, unity, really, of organism or cell and environ- ment more forcibly reveal itself than in the problem of the parthenogenetic development of the animal egg. The problem, how