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THE BIOLOGY

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THE BIOLOGY

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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 the egg can begin development through the
effect of some change in its environment, is to us still mys-
terious. The findings of experimental parthenogenesis
would help to dispel the mystery if they could serve to indi-
cate how environmental changes effect the initiation of
development in naturally parthenogenetic eggs.

A plausible explanation is suggested by the fact that the
most commonly used means for inducing parthenogenesis
are simple changes in the medium, as changes in salinity,
acidity, and temperature. The normally parthenogenetic
egg probably differs from that which requires fertilization
in being more labile and more responsive; as such it would
react readily to one or another of these most commonly
occurring changes in the environment. That drying, radia-
tions, and puncture also induce development may be sig-
nificant. Certainly, they are environmental changes by
which experimental parthenogenesis can be induced. Since
we know these, we should seek to determine whether they

226

PAR THENOGENESIS

or others start development in the naturally parthenogenetic
egg. Until more data are available, we can not go beyond
such general statements as to the probable cause of natu-
rally occurring parthenogenesis.

As to experimentally induced parthenogenesis, however,
the experimental findings suffice for an attempt at formulat-
ing a theory. And indeed, many theories have been prof-
fered. With respect to one of these which became the most
widely known, one notes a curious, even an anomalous
situation. Although the data on induced parthenogenesis
indubitably show that one means alone suffices to call forth
development in the eggs studied, including those of sea-
urchins, this theory, whose founder is Loeb, runs counter to
these findings, including most of its author's, in explaining
the phenomenon as due to the action of two distinct means
because of the one fact that with a certain method, eggs of
sea-urchins develop in more nearly normal manner after
treatment with two means.

Since the interpretation of the induction of partheno-
genesis in sea-urchins' eggs by the double method came
finally to be the most widely accepted theory of fertilization
embracing all animal eggs, we must examine it here. In
this interpretation it is contended that butyric acid or any
other agent which calls forth membrane "formation" tends
to destroy, i.e., superficially cytolyze, the egg; and that such
an agent acts as do haemolytic agents, those that destroy
red blood cells. The second agent, it is held, corrects the
destructive action of the first. This interpretation of cer-
tain experimental findings on eggs of sea-urchins became
the famous "superficial-cytolysis-corrective-factor" theory
for experimental parthenogenesis and for fertilization. The
question now arises: Do the experimental findings on which
it is based, actually permit this interpretation.^

Eggs in sea-water, no matter from what species of animal
they are, eventually die if they are not fertilized, that is,

22J

THE BIOLOGY OF THE CELL SURFACE

they go to pieces, cytolyze. Hence, for the unfertilized egg
sea-water is normally cytolytic. The length of time which
eggs can remain in sea-water before they begin to cytolyze
depends largely upon the temperature of the sea-water and
the ratio of the volume of eggs to the volume of sea-water;
also it varies with eggs of different species.^ Now the fer-
tilization-capacity of eggs in sea-water diminishes with time;
here also the factors, temperature and volume of eggs come
into play. The rate at which the fertilization-capacity
drops also varies with eggs of different species. But it holds
generally that the fertilization-capacity disappears before
cytolysis begins — obviously, It must have disappeared at
the latest at the moment when cytolysis begins, for a dead
egg can not be fertilized. As a matter of fact, the capacity
of an egg in sea-water to respond to an experimental means
falls off more rapidly than its fertilization-capacity. This is
strikingly brought out by the above mentioned effect of
warm sea-water on the egg of Nereis. Its response to
treatment with warm sea-water is not so good if it has
been in normal sea-water before treatment, whilst its
fertilizability is thereby not lessened. So we consider the
cytolytic action of the sea-water as the antipode of the
initiation of development by spermatozoon or by experi-
mental means.

By the addition of any one of a number of substances to
sea-water the rate at which eggs cytolyze can be increased.
Among these is butyric acid. But the action of butyric
acid in accelerating cytolysis is by no means invariable.
On the contrary, the effects which this acid produces depend
upon its concentration in sea-water and, in the case of
each concentration which is effective for calling forth mem-
brane-separation, upon the duration of time that the eggs

^ In making comparisons of the rate of cytolysis of eggs of different
species one must be careful thai the experimental conditions are
uniform in the compared cases.

228

PAR THENOGENESIS

are exposed to it. If we designate as optimum tlie short-
est exposures which are effective in calling forth complete
membrane-separation, we find, as my own experiments
have so clearly established, that sub-optimum exposures
instead of accelerating, actually retard the cytolysis which
in time normally takes place in sea-water.^ Eggs in sea-
water which show fully separated membranes in conse-
quence of optimum exposure to butyric acid cytolyze more
rapidly than unexposed eggs. Over-exposed eggs cytolyze
at a still more rapid rate. In his experiments Loeb never
used this reagent properly. That is, he exposed the eggs
of both the California and the Woods Hole sea-urchins
beyond the optimum time for membrane-separation. If
he had learned the best exposure, he probably would not
have so greatly emphasized in the butyric acid-hypertonic
sea-water method for the sea-urchin egg the cytolytic action
of the acid. Basing his theory on over-exposure to butyric
acid, he created the term, "superficial cytolysis."^

The superficial cytolysis-corrective-factor theory explains
the action of the two means for inducing parthenogenesis
as follows: the fatty acid treatment causes "superficial
cytolysis" and the hypertonic sea-water treatment follow-
ing "saves" the egg from this impending death. As I
stated above, the order in which the two agents are used is
of no consequence: the exposure of the eggs to butyric acid
may precede or follow that to hypertonic sea-water. That
is, the corrective factor (hypertonic sea-water), according
to the theory, if used first acts to correct where nothing is
to be corrected and the superficial cytolysis factor (the
fatty acid) used at the second place, tends to kill the more
than corrected egg. Thus the sequence in the treatment
so strongly demanded by the superficial-cytolysis-correc-

^ Just, igzo.

- See Just, ig/gc, 1920, ig22a, ig22b, igjoc.

22g

THE BIOLOGY OF THE CELL SURFACE

tive-factor theory not only is not supported b}^ fact but is
contradicted by it.

We may dismiss the superficial cytolysis-corrective
factor theory of parthenogenesis for the following reasons:
All eggs including those of sea-urchins need treatment only
with a single means. Further, for sea-urchins' eggs, the
cytolytic eflFect that the theory stresses, results from over-
exposure to the acid. Finally, the fact that the order of
treatment in the double method can be reversed, makes the
theory even for the single egg of the sea-urchin untenable.

These obvious failures of the theory however did not pre-
vent the exaggeration of its significance to an unusual
degree. The discovery that eggs could be induced to
develop without the spermatozoon gave rise to extravagant
claims by experimental embryologists themselves and
aroused fantastic notions among laymen. Many hailed it
as a demonstration of the creation of life. As if the unfer-
tilized egg were not alive ! According to one report, to
create life is easy since the means for inaugurating the life-
process are at once simple and close at hand: one needs only
two chemicals, vinegar and salt, found in every kitchen, to
start the egg on its long and complicated journey of develop-
ment, the end of which is the adult form. The occurrence
of natural parthenogenesis in the meantime was given scant
attention; indeed, in some quarters it was entirely over-
looked. The one fact alone, that nature dispenses with
spermatozoa for the development of certain eggs — those
of rotifers, aphids, bees and others — this recognized
fact of natural parthenogenesis that we have known for
years, should have saved us from making excessive claims
concerning the significance of experimentally induced
parthenogensis.

As soon as the superficial cytolysis-corrective factor
theory for experimental parthenogenesis had been elab-
orated, the attempt was made to use it as an explanation for

230

PAR THENOGENESIS

the phenomenon of fertilization. The problem seemed to
be especially easy, since the double treatment — and, there-
fore, the factors in the explanation — in these experiments on
inducing parthenogenesis, appears to have parallels in the
two phases in fertilization, the external and the internal
phase. I shortly review these here.

All eggs respond to insemination, as was pointed out,
by some kind of surface or ectoplasmic changes which pro-
duce in some cases very striking results, as the separation of
the vitelline membrane in sea-urchins' eggs or the extrusion
of the superficially located material in eggs of the genus
Nereis which sets as a jelly in the sea-water. Underlying
these changes is always a disintegration of the superficial
cytoplasm upon which follows normally a rapid reconstitu-
tion of the surface. This is the so-called first or external
phase of the fertilization-process.

Once within the egg, the nucleus of the spermatozoon
moves toward the egg-nucleus. Both division-centres arise
near one or the other of the germ-nuclei or one centre
near the egg- and the other near the sperm-nucleus.
The mitotic spindle is established and first cleavage is
initiated. These phenomena constitute the so-called second
or internal phase of the fertilization-process.

With respect to these two phases, it is true that experi-
mental parthenogenesis in the sea-urchin egg initiated by
butyric acid and hypertonic sea-water resembles the fer-
tilization-process inasmuch as the acid calls forth mem-
brane-separation and the hypertonic sea-water initiates the
formation of an amphiastral mitotic figure. Therefore,
the superficial cytolysis corrective factor theory though it
can not be upheld as explanation of experimental partheno-
genesis, may, one might think, nevertheless explain fertili-
zation. Attempting such an explanation, Loeb says that
the "fertilization by the spermatozoon perhaps depended
not upon a single chemical agent, but upon a combination

231

THE BIOLOGY OF THE CELL SURFACE

of two or more^ which were only fortuitously combined in
the spermatozoon" (Italics are mine). According to him
it is a fact which he has proved that "the membrane forma-
tion by the spermatozoon is caused also by a cytolytic
agent- — a lysin."

I have already pointed out that for the sea-urchin's egg
Loeb used butyric acid either too long or in too great con-
centration; such over-exposures very greatly increase
the rate at which this e^^ cytolyzes in sea-water.^ The
optimum exposure to butyric acid, which for the egg of
Arbacia Loeb never knew, does not cause this hastening of
cytolysis. Moreover, neither Loeb nor any one else has
proved the presence of a lysin or of a "fortuitously com-
bined combination" of chemical agents in the spermato-
zoon. Loeb's so-called proof of a lysin in the spermatozoon
is fantastic and wholly specious.

Li calling forth membrane-separation the effect of butyric
acid is, to be sure, somewhat similar to that of the sperma-
tozoon. But there is never membrane-separation in the
butyric acid solution; only after the acid is washed away
by bringing the eggs into a large volume of sea-water does
membrane-separation take place. There is thus an essen-
tial difference between the immediate action of the sperma-
tozoon in calling forth membrane-separation and the
delayed effect of butyric acid. This point raises a further
question. Since according to the author of the superficial
cytolysis-corrective factor theory of fertilization, butyric
acid is only an example of a large class of cytolytic (and
haemolytic) agents, we must ask if the action of butyric
acid is, as we should expect and as the author of the theory
claims, characteristic of the class.

^ The "'or more'' I think is added as a margin of safety since the
theory demands only two.
- Just, I()20.

232

PAR THENOGENESIS

Among the cytolytic (and haemolytic) agents named by
Loeb are i}4 M NaCl and distilled water. Now I find that
either of these induces membrane-separation in Arhacia-
eggs while the eggs are in the solution.^ In a pure 2I2 M
NaCl solution the membranes separate with extreme rapid-
ity,- so that to follow the process one must use a less con-
centrated solution. The separation of the membrane in
hypertonic sea-water is due to the rapid shrinkage of the
egg-plasma. In distilled water, on the other hand, the
eggs distend as water enters but the elastic vitelline mem-
branes distend more rapidly than the plasma so that in five
seconds the membranes are off the eggs, at which time the
plasma shows no sign of disruption. With prolonged expos-
ure the egg-plasma swells and reaches the membrane;
cytolysis follows. The action of butyric acid in causing
membrane-separation resembles neither that of hypertonic
nor that of hypotonic sea-water. In my experience all of
the cytolytic agents named by Loeb fall into two classes:
those that do not call forth membrane-separation directly
but only after they are washed away by transferring the
eggs to normal sea-water; and those that do act directly,
by producing either shrinkage of the egg-plasma or exten-
sion of the elastic membrane. If the spermatozoon's first
duty to the egg be to inject a lysin, which of the three
kinds of cytolysis — that of butyric acid, of hypertonic sea-
water or of hypotonic sea-water — does this behavior of the
spermatozoon resemble t

An agent which induces membrane-separation in the sea-
urchin's egg can not act as a fatty acid, a concentrated neu-
tral salt solution and distilled water at one and the same
time. If we consider all the cytolytic agents which have
been employed, we fail to discover in their mode of action

^ Just, ig22a, ig2Sc.

- Just, unpublished observations.

233

THE BIOLOGY OF THE CELL SURFACE

any one common factor; they act either as butyric acid, as
strong hypertonic sea-water or as distilled water. That
is, the cytolytic agents lack a specific factor, whereas the
fertilization-reaction reveals a high degree of specificity,
borne equally by the components, egg and spermatozoon.
If one adheres to the lysln-corrective factor theory of fer-
tilization, one must assume the addition of a specific factor
in the spermatozoon imposed upon the nonspecific "for-
tuitously combined combinations" of factors, resembling
butyric acid, hypertonic sea-water and distilled water.
From this point of view specificity would reside only in the
spermatozoon, the egg playing no part. We are told that
"the lysins of foreign animals can get into cells by mere
diflFusion, while the lysins of the same species can not get
into the egg by diflFusion. Only through the motile power
of the living spermatozoon which acts as a carrier can the
fertilizing lysin of the animal's own species get into the
egg."^ Here the author of this theory reveals the failure of
well-known physico-chemical agents to act as substitutes
for that "mysterious complex" called the "living sperma-
tozoon." The high purpose, to transfer the problem of the
initiation of development of the animal egg from the realm
of morphology to that of physical chemistry^ is frustrated
at a most crucial point. It is, therefore, needless to discuss
the problem of specificity further in this connection.

The order of the two phases of the fertilization-process
is constant in a normal egg; always the external (ectoplas-
mic) changes come first and the internal (mitotic) come
second. Never does a normal Qgg exhibit the phenomena
of the internal phase before the ectoplasmic changes charac-
teristic of its fertilization-process have taken place. As I
have already pointed out, in the experimental partheno-

1 Loeb, 1913.
- Loeb, ibid.

234

PAR THENOGENESIS

genetic development of the sea-urchin's q^^^ by means of
butyric acid and hypertonic sea-water, the butyric acid is
equally efficient in action preceding or following the treat-
ment with hypertonic sea-water. This notable difference
between "chemical fertilization" and nature's process the
theory discounts. Though Loeb was the first to know that
the order of treatment could be reversed, he made no
attempt to explain this difference from the natural process.

Sharply visible ectoplasmic changes are always pres-
ent in fertilization but not in all cases of experimental
parthenogenesis. As we have seen above, the eggs of sea-
urchins develop with Loeb's original hypertonic sea-water
method, although without membrane-separation. The
double treatment is not essential.^

Because of these considerations we must flatly dismiss the
superficial cytolysis-corrective factor theory as an attempt
to explain fertilization as we have dismissed it as an explana-
tion of experimental parthenogenesis; it fails not only for
eggs generally but also for sea-urchins' eggs specifically.

But the fact remains that parthenogenesis can be experi-
mentally induced. What is experimental parthenogenesis ?

If terms mean anything, we should expect that in experi-
mental parthenogenesis, as in natural, perfect and complete
development should result. Yet, as we have seen, generally
experimental parthenogenetic development does not extend
beyond the larval stage. This is undoubtedly often due to
our failures to overcome the difficulty of rearing animals to
the adult stage from eggs under laboratory conditions. -
Some difficulty also inheres in the methods employed:
means of experimental parthenogenesis often tend to do

^ It is curious that Loeb insisted that the superficial cytolysis factor
is the essential one; he thereby denied his own discovery and deprived
himself of all ground in his whole fight concer?iing priority.

^ I have been able to carry Platynereis megalops through three
generations under laboratorx conditions.

235

THE BIOLOGY OF THE CELL SURFACE

too much — though they initiate development, they also
impair it.

Even if we in time overcome these difficulties, there
still remains doubt that the action of the means of induced
parthenogenesis is identical to that of the spermatozoon.
For while normal, fertilized eggs do, if properly handled,
give at least 95 per cent, perfect larvae, it is difficult if not
impossible to obtain this percentage in experimental par-
thenogenesis. The spermatozoon is more effective than
a means of experimental parthenogenesis; frequently one
finds that a certain lot of eggs, a sample of which will give
more than 95 per cent, fertilization and perfect develop-
ment, will not respond to a means of parthenogenesis that
is usually effective on eggs of this species.

On more general grounds it is still to be doubted that an
experimental means duplicates the action of the normal
stimuli in the intact organism, though experimental
imitation is often possible. Despite these considerations
however, it must be clearly emphasized that complete
development can be induced experimentally.

Even if we do not yet succeed in establishing the par-
ticular way in which either spermatozoa or experimental
means act upon the egg so that it develops, we certainly
can agree that either spermatozoon or experimental means
in initiating the development of the egg produce the same
result — a succession or rhythm of mitoses and cleavages
which finally lead to embryo-formation. As we have
already seen, the experimental means are not specific.
Most probably, the nature of the reaction between experi-
mental means and egg differs also from that between sper-
matozoon and egg, since there is evidence to indicate that
fertilization is a chemical union of an egg-substance with
the spermatozoon, whereas we can assume that the initial
action of the experimental means is physical. But the
end-result, however reached, is the same. The conclusion

236

PAR THENOGENESIS

Is therefore this: the egg-cell like many another living cell —
nerve or muscle, for example — possesses independent irri-
tability. It has full capacity for development. Neither
spermatozoa nor experimental means furnish the egg with
one or more substances without which the initiation of
development would be impossible.

Here lay at the same time the possibilities and the failure
of the work on experimental parthenogenesis. Every
single investigator who erred in "proving" an external
agent (or agents) to be the cause of development neglected
an opportunity to extend our knowledge concerning that
fundamental manifestation of living matter, its independent
irritability. Some theories of fertilization derived from
work on experimental parthenogenesis postulated as the
cause of development a change in the egg, e.g., membrane
separation, that was however merely one consequence
of the ectoplasmic changes, or they related the cause of
development to some concomitant of mitosis. Others, as
had the Loeb theory, merely offered a substitute for the
sperm-borne centrosomes. Instead of these two structural
entities which Boveri had for some time postulated to be
indispensable for fertilization because he thought them to
be either lacking in the egg or present in too enfeebled state, ^
the new school of biology substituted all kinds of means,
like lysins, mysticisms no less mystical because appearing
to belong to the realm of physical chemistry, having the
living condition of the spermatozoon always conveniently
at hand as a refuge finally to be sought.-

I do not wish by seeming to dwell over long on this point
to take unfair advantage of the patent shortcomings of the
work on experimental parthenogenesis. And yet I must
hold it up as an example of what such a large section of the

^ See also Fedjovsky, /SSS-p2.
- Loeb, ip/J.

237

THE BIOLOGY OF THE CELL SURFACE

school of quantitative biology has done for us by over-
reaching facts and drawing unwarranted conclusions as to
the physical chemistry of vital phenomena. One method
used in this work is the " substitution of well-known physico-
chemical agents" for living components. But this substi-
tution is only that of a well-known physico-chemical agent
for another one. One need not be in complete ignorance
of this agent, i.e., the living cell, unless one takes pride in a
disdain for the knowledge accumulated by descriptive
studies. Not hindered by this knowledge one can easily
make great discoveries and, through fecund ignorance, per-
petuate error. And if, in addition, with a paucity of
mathematics, physics and chemistry one elaborates mathe-
matico-physico-chemical theories, then one by no means
wins what quantitative biology so much desires: namely,
security of biology, sitting at the right hand of physics.
Even those who have an adequate knowledge of physics
and chemistry and do appreciate the biological phenomena
which they aim to explain in terms of physics and chemistry,
should, (when at that point beyond which their data do
not warrant conclusions) honestly say: "However much
we desire to establish life as a mechanism, here our present
knowledge comes to its limits." It is sad irony that a
theory of vaunting mechanistic conceptions had, as its
basis, work the true value of which lay in establishing the
fact that the egg as a living cell is self-acting, self-regulating
and self-realizing — an independently irritable system. For
the spermatozoon, the theory's mechanistic conception is
more vitalistic than even the ardent vitalist could desire:
for it says that the living spermatozoon does what it does
only because it is alive. ^

The history of biological research furnishes us with other
examples which illustrate the short-comings of such physico-

1 Loeb^ I.e.

23S

PAR THENOGENESIS

chemical approach in setting up a singly studied property
of living matter as synonymous with the whole complex
of life-processes. Witness the theories of oxidation, per-
meability, electrical conductivity, viscosity and the like.
These all have failed; they leave untouched the cardinal
problem of biology, the structure of a living thing; they do
not relate themselves to the organization which distin-
guishes a living thing from a non-living. Or take the almost
universal fashion in which Hill's work on nerve-conduction
was accepted. By "proving" that a nerve conducts without
heat-loss this work must logically lead to the conclusion
that the nerve-fibre is not living since it gives no evidence
of metabolism. Strictly orthodox morphologists have like-
wise often presented theories of life-processes on the basis of
demonstrations that only emphasize anew the capacity
of the living thing though debased to respond according to
its specific and intrinsic irritability. The now perfect
collapse of the organizator theory is a case in point: all
that remains of it is a name for the well-known power of
protoplasm to respond in the same characteristic, struc-
tural and physiological manner to diverse stimuli.^

True, terms, as inherent irritability and response to
stimuli, are more general than any one denominating a
physico-chemical attribute named above, or than the term,
organizator, even. But the present state of our knowledge
of protoplasmic response does not yet permit the use of
more specific terms with reference to the behavior peculiar
to matter organized in the living state. The reproach that
one retards progress by use of too general terms carries no
serious onus so long as we have not progressed far enough
to warrant the employment of precise physico-chemical
formulation.

^ As an example, cf. Just, 1936c: here ati egg responds zvith the
same structural change to most diverse experimental means.

239

THE BIOLOGY OF THE CELL SURFACE

Experimental parthenogenesis, a problem far from being
solved, invites investigations both for its own sake and for
the implications it possesses for the biology of cells gener-
ally. In the interest of this research it is necessary to
define the problem clearly in order that not all kinds of
observations are considered as belonging to it: Experimental
parthenogenesis must be defined as the development of an
egg that has been initiated and carried to at least the
normal larval stage by an agent other than the living
spermatozoon.

In contrast to this clear definition there exists a great
deal of confusion in the literature on this subject. For
instance, an agent which produces almost no change in an
unfertilized egg, as revealed by the egg's response with
perfect development when inseminated, is often called a
parthenogenetic agent. Or the so-called parthenogenetic
agent induces membrane-separation only, stimulating the
egg to the limited extent that makes fertilization now impos-
sible. In other cases the changes induced may be injurious,
yet the egg does not lose capacity to respond to fertiliza-
tion, though its development is abnormal. Or the pseudo-
parthenogenetic agent induces cytolysis which begins sooner
or later, depending upon the agent's toxicity. Whenever
the changes induced, either because of the nature of the
agents themselves or the method with which they are used,
do not lead to development, they should not be called
parthenogenetic. And certainly, death changes, i.e., cyto-
lysis, should be considered to be beyond the limits of the
term, the initiation of development.

The establishment of the mitotic figure constitutes, as
we have seen, a definite and reliable index for the comple-
tion of the fertilization-process. Normally, development
then proceeds with rhythmical nuclear break-down and
reformation; and in the majority of eggs this division of
the nucleus synchronizes with cleavage of the cytoplasm.

240

PAR THENOGENESIS

In some eggs, e.g., of insects, the first nuclear divisions ensue
without cytoplasmic cleavages, whilst in others, e.g., those
of selachians and of birds, later mitoses proceed without
the splitting up of the cytoplasm. In certain eggs experi-
mentally treated, some kind of development, abnormal
however, can go as far as a swimming form without cyto-
plasmic cleavage. Hence, although development never
completes itself without the earlier or later breaking up of
the cytoplasmic mass of the egg into cells, we can not cate-
gorically define the process of development as related to
the sundering of the cytoplasm. Therefore the end of the
fertilization-process we define by the appearance of the
mitotic figure rather than by the first cleavage of the
cytoplasm.^

Unfortunately, we have neglected to make nice studies
on the differences in the development of two sets of eggs
from the same animal, one set fertilized and the other
treated with some means of inducing parthenogenesis —
such differences, for example, that would reveal themselves
in the size of cells and in the time and the place of their
appearance in an egg whose cell-lineage is known, that
is, one in which the size, order of appearance and loca-
tion of the cells with reference to each other are definite
and invariable. Some differences, as rate of cleavage, we
know; others, indicated for the earlier stages of develop-
ment, are doubtless due to the fact that the experimental
treatment never quite duplicates the action of the sperma-
tozoon. But even if we assume that fertilized and par-
thenogenetically developing eggs, both in the induced and

^ / say mitotic figure because the occurrence of amitotic nuclear
division in developing animal eggs has been doubted. If we include
the -possibilitv of development with amitotsis m our definition., we
substitute in the thesis nuclear division for the term, establishment,
or appearance, of the mitotic figure.

241

THE BIOLOGY OF THE CELL SURFACE

the naturally occurring process, differ in their course of
development, we know certainly that by either fertilization
or parthenogenesis, if development is complete, an adult
animal emerges through a succession of nuclear and cyto-
plasmic divisions. Therefore, despite any differences that
may occur during the developmental process, the beginning
stage is always that of the establishment of the cleavage
figure with consequent nuclear and cytoplasmic division,
the end-stage always being the adult form. The question
thus arises as to the nature of the calling forth of
the nuclear configuration through whose subsequent
rhythmical behavior together with cytoplasmic cleavage
the end-stage is attained. The initial action of spermato-
zoon and of experimental means being different, and dif-
ferences in action obtaining between one means and another,
the question at issue is: Do these differences persist so that
the calling forth of the process of nuclear and cytoplasmic
divisions springs from various causes, or do these means,
whatever they are, elicit the same reaction which sets
up the division-process ? I suggest that they set up the
same reaction.^ Let us briefly review the experimental
findings.

In sea-water sufficiently hypertonic to induce develop-
ment, eggs of any species which respond to such treatment
rapidly and directly lose water. The minimum hyper-
tonicity capable of calling forth development in sea-urchins'
eggs does not bring about a sharply defined shrinkage of the
eggs from their vitelline membranes; only in stronger hyper-
tonic solutions does this egg shrink from its membrane.
Other eggs shrink in hypertonic solutions to such an extent
that the vitelline membranes no longer adhere to the cyto-
plasmic surface. On return to normal sea-water, the eggs
take up water but they do not regain the equilibrium with

1 See Just, ig2Ja.

242

PAR THENOGENESIS

the sea-water which they possessed In their untreated condi-
tion, instead they establish it at a new and different level.

In hypotonic sea-water eggs take up water and increase
in volume. If the degree of dilution is effective, the rapid
intake of water induces break-down of the eggs' surface-
cytoplasm and rapid distension of their vitelline membranes.
Removed to normal sea-water they quickly diminish in vol-
ume but do not regain their previous state because of the
complete break-down at the surface, i.e., complete ecto-
plasmic changes. They come again into equilibrium with
the surrounding sea-water, but this, owing to the altered
surface-structure, is at a new level.

After having been properly exposed to effective acids in
optimum concentration in sea-water, eggs on return to
normal sea-water separate their vitelline membranes. That
is, the acid treatment induces a change which brings about
the subsequent break-down in the surface-cytoplasm.
With the processes of restitution by which a new surface-
layer forms, the eggs come into a new equilibrium with the
sea-water.

Warm sea-water when effective causes Immediate break-
down in the surface-layer. Puncture of the frog's eggs pre-
sumably leads also to ectoplasmic break-down. Radiation,
as of ultra-violet, brings about ectoplasmic break-down.
In these cases as in the others given above, the surface-
structure is altered and thus the equilibrium between egg
and sea-water is different from that which existed before
the treatment.

Also after fertilization an egg having undergone complete
ectoplasmic changes does not return to the state which It
had before fertilization with respect to its equilibrium with
the sea-water. It shows instead a marked difference.
With Its altered surface-layer, the fertilized egg establishes
a new equilibrium with the sea-water. As we shall see In
the chapter that follows, fertilized eggs exhibit in their

243

THE BIOLOGY OF THE CELL SURFACE

reaction to environmental influences rhythmical changes of
resistance and susceptibility that are both difi"erent from
those shown by the unfertilized egg.

The new egg sea-water equilibrium is established by the
structural changes in the ectoplasm; these changes do not
reverse themselves and the egg does not return to the
physiological state it had previous to treatment or to fer-
tilization. Also with respect to water-movements these
same changes sharply set off the devel ping egg from the
untreated and unfertilized. Since the egg loses and gains
water concomitantly with each cleavage-cycle, one can not
say that with break-down of substance in the ectoplasm it
becomes permanently dehydrated. Rather, a momentary
water-loss as a consequence of the stimulus of inducing
means or of the spermatozoon, brings about a change to a
new level with respect to equilibrium with the surrounding
medium, and on this new level the ensuing rhythmical
process of water-entrance and -exit which accompanies the
developmental process takes place.

The rhythmical movement of water, during the cleavage-
cycle, into and out of the egg undoubtedly means a move-
ment of water from place to place within the egg; and this
in turn means local and temporary hydrations and dehydra-
tions. These redistributions of water even in minute intra-
cellular dimensions are favorable for reactions.^ One
visible structure of the cell which in addition to the ecto-
plasm exhibits rhythmical changes during the cleavage-
cycle is the nucleus; its break-down and reformation is
indeed the criterion used for defining the cleavage-cycle.
As we have seen in the chapter on the fertilization-process,
the establishment of the mitotic complex constitutes the
index of the completion of the initial stage of development.
And I suggest that in all modes of initiating development,

^ Just, 1937b.

244

PAR THENOGENESIS

that of experimentally induced and naturally occurring
parthenogenesis as well as that of fertilization, the estab-
lishment of the first mitotic figure is brought about by a
dehydration of some constituent of the ground-substance,
or as the result of a reaction between nuclear and cytoplas-
mic ground-substance brought about by dehydration. The
mitotic figure then is either expressly a dehydration-for-
mation or the result of a reaction rendered possible by
dehydration. In the resting Qgg this material in the
ground-substance is spatially diffused; with the process of
parthenogenesis or of fertilization it becomes aggregated —
in parthenogenesis and in some cases of fertilization around
the egg-nucleus, in most cases of fertilization around the
sperm-nucleus. What we see as asters and as spindle may
be either this material itself or the sign or product of its
activity.

This hypothesis — and more can not be proffered in the
present state of our knowledge — is wholly consistent with
the established facts as far as one can reduce them to order.
According to it the most important factor in all modes of
initiating development is a dehydration-process affecting
directly or indirectly the ground-substance.^ In all cases
of the initiation of normal development, dehydration begins
in the ectoplasm.

Ectoplasmic changes alone, as I have shown above, are
not experimental parthenogenesis. They do not of them-
selves determine that development will follow; but they are
a reliable indicator for the quality of the development, if
this ensues. That there are cases of ectoplasmic changes
without subsequent development on the one side and that
the sea-urchins' egg after treatment with weak hyper-

^ Delage in igoib promulgated a dehydration-theory of fertilization;
but this differs from the one here proffered in ascribing dehydration
to the influence of the sperm only.

245

THE BIOLOGY OF THE CELL SURFACE

tonic sea-water develops without complete break-down of
ectoplasmic substance on the other, warns us against
attributing the cause of experimental parthenogenesis
directly to physical changes at the egg-surface, i.e., to
membrane-separation.

Far from being an exception to be explained away, the
case of sea-urchins' eggs in responding to treatment with
the weak hypertonic sea-water without membrane-separa-
tion and with poor quality of development reveals clearly
what other eggs more sensitive in their ectoplasmic response
to experimental means do not: the ectoplasmic changes are
significant for the development in prospect. Again, as in
fertilization, we see that the quality of development depends
upon the quality of these initial ectoplasmic changes.

They are violent eruptions which precede the evanescent
spinning that will accompany cleavages yet to come and
that will build gossamer-like tendrils to bind cell to cell.
Spermatozoon or parthenogenetic means. Nature's or exper-
imenter's, set the life of the egg in quicker motion; the
mitotic spindle comes and goes. The web of life gives a
pattern which we come to know as larval sea-urchin, worm
or clam. But almost with the moment that the egg's
vital activity moves at a quicker tempo we know through
the ectoplasmic behavior the quality of the events to come.

246

lO

Cell-divisiofi

lI2/ith the process of celi>-division, the fertilized
or parthenogenetic egg, a single cell, develops into an adult
organism which may comprise millions of cells. The first
division or cleavage separates the egg (in most cases) into
two portions, blastomeres, which remain attached. Suc-
cessive cleavages give rise to many blastomeres, the egg
becoming a mass of cohering cells. The surface of the
frog's egg, for example, in late cleavage so closely resembles
that of a golf-ball that one gains the impression that sub-
division of its surface by cleavage-planes is the egg's chief
characteristic. If one examines a smaller egg, e.g., of a
sea-urchin, under the microscope, one notes again the
resemblance of its corrugated surface to a golf-ball; looking
closer, one observes within each blastomere a nucleus.
It is a very simple matter to convince oneself by continuous
observation on a living egg throughout its cleavage period
that the "golf-ball" has arisen by successive divisions of
both nucleus and cytoplasm.

The cells which comprise an adult organism are not all
alike in structure. To the naked eye a strand of nerve is
readily distinguishable from a strand of muscle of equal
length and thickness; and a piece of kidney can not be mis-
taken for liver. As en masse, so singly, under the micro-
scope, the cells of nerve, muscle, kidney, and liver are easily
recognized. Since these cells have different functions,
development means something more than mere multiplica-
tion of cells; it implies the cells' differentiation. This
arises in the process of cell-multiplication at an earlier or

247

THE BIOLOGY OF THE CELL SURFACE

later stage, depending upon the species of egg. As we shall
see, this old problem of differentiation is still the major one
of the study of development.

With successive cell-divisions during cleavage, the
blastomeres become progressively smaller. If this process
were to continue indefinitely, the size of the cells would
approximate zero and cell-division would come to a stand-
still. But this never happens, since in later stages the
cells transform food into protoplasm. In many cases the
t^g soon after hatching as a larva takes in food; or
the embryo, as that of the chick, utilizes the yolk which
is present as reserve food material within the egg.

Generally, growth is increase in size. A cell may grow
as such by the intake and transformation of food. In the
initial stages of the development of an egg, cell-multiplica-
tion procedes without any increase of volume. The total
mass of the egg of a sea-urchin, for example, at the end of
its cleavage-period is approximately the same as at the
beginning. In the larval stage, cell-multiplication follows
upon the intake and transformation of food by the cells;
thus here through the combined process of increase in vol-
ume by the single cells and increase in their number, growth
of the embryo is attained.

In the adult organism derived from the egg, cell-division
operates daily in the restitution process. Cells of the
human skin are constantly being replaced. Each day the
blood-forming tissues in bones must throw into the blood-
stream millions of red blood corpuscles to compensate for
the disintegration of older ones. Where there is no more
capacity for cell-division, any injury becomes tragic; thus
the capacity for regeneration in the human central nerve
system is meagre and the possibilities of repair after surgery
slight because its cells have lost the power of division.
Organs which possess reparative capacity have retained
the capacity for cell-division.

248

CELL-DIVISION

Cells in the adult body frequently show a sudden and
exaggerated burst of division-activity which results in
abnormal growth. Thus tumors are built up by riotous
cell-proliferation. The malignant tumor, cancer, in this
respect is not different from the benign and to this extent
any work on cell-division may be of significance for the
cancer problem; this, however, does not mean that every
work on cell-division in various forms of animals from
Amoeba to vertebrate has direct bearing on the problem of
cancer, since here malignancy points to a cause lying beyond
the facts which can be established for tumors in general,
both malignant and benign.

The germ-cells, eggs and spermatozoa, display the most
intense activity of cell-division, especially during their
periods of multiplication. A great deal of our information
concerning cell-division is derived from the study of these
cells particularly in their period of maturation.

It is not only among multicellular organisms as egg or as
adult that cell-division is of consequence; among unicellular
animals and plants it is often the sole mode of reproduction.
These single-cell forms live for minutes, hours or days as
such and then by dividing into two parts bring into being
"new" individuals. Or one cell may divide into several
parts each of which becomes a "new" individual.

In the non-living world, substances may divide, and so
multiply and also differentiate and grow. But these phe-
nomena are quite unlike those exhibited by living things.
Cell-division constitutes a fundamental process which is
never observed outside the world of living things. And
yet, it still lacks an explanation to which biologists agree.
It is, moreover, most often incorrectly defined. The reader
should understand that in the following pages my main
purpose is to derive a definition. Only after having done
this shall I offer an explanation of this phenomenon, the
division of the cell.

24Q

rilE BIOLOGY OF THE CELL SURFACE

Before I begin the discussion I should like again to point
out how necessary it is that we appreciate diflFerences
normally appearing in biological processes before we
attempt to evaluate these processes. With respect to the
process of cell-division, we recognize two kinds. Based
upon the behavior of the nucleus in cell-division as a criter-
ion, two categories of cell-division have been set up: that
with direct (amitotic) and that with indirect (mitotic)
division of the nucleus.

In direct or amitotic nuclear division, the nucleus by
constriction separates into two parts. It first elongates,
taking the form of a dumb-bell or hour-glass, and then
breaks into two equal or unequal portions. Each of these
with cytoplasm constitutes a new cell, if the cytoplasm also
divides. But division of the cytoplasm synchronously
with that of the nucleus is not invariable. The occurrence
and significance of amitosis have been much debated.
Some few biologists hold that amitosis is more widespread
among animals, even in the development of eggs, than is
generally believed and that it may be of equal significance
with mitosis. The majority opinion is that amitosis is of
restricted occurrence and when found among multicellular
organisms is of little significance generally, except as a sign
of decadence or of too highly specialized cells. ^ For the

Protista we meet the view that what is often called amitosis
is a disguised mitosis. Nevertheless, it is generally
admitted that among both multicellular and unicellular
organisms amitosis does occur. It must, therefore, be
embraced by any theory that attempts an explanation of
cell-division, especially if the explanation relates division
of the cytoplasm to that of the nucleus.

As has been pointed out already, mitosis or indirect
nuclear division involves a series of orderly maneuvers of

^ Wilson, 192^.

250

CELL-DIVISION

the chromosomes on a set of "fibres," the mitotic spindle,
beginning with the break-down of the resting nucleus and
ending with the formation of two resting nuclei. If from
the poles of the spindle where the "fibres" more or less
converge, other "fibres," called astral fibres, extend radi-
ally into the cytoplasm, the spindle is said to be of the astral
type; if these radiations are absent, the spindle is called
anastral. In the former type at the centre of the aster may
be found either a single granule, the centriole or centro-
some, or many finer granules; or, the astral centre may be
of such fine structure that it appears to be optically empty.
Mitotic division of the nucleus may normally ensue without
concurrent division of the cytoplasm; experimentally it is
easily possible to suppress or arrest cytoplasmic division
while nuclear divisions continue. Since this is true, the
failure of cytoplasmic division after amitotic nuclear divi-
sion that is sometimes observed, does not deserve the
emphasis which some writers place upon it. The mitotic
complex may arise wholly or in part within or outside of
the nucleus.

What should we demand of any theory of cell-division ^
An explanation of cell-division and not of nuclear phe-
nomena. The theory should cover amitosis and mitosis.^
It should apply with equal force to all types of mitosis
among both Protista and multicellular organisms; to every
variation of the mitotic configuration. I.e., to spindles with
and without asters, with and without centrosomes. Since
cytoplasmic division does not always synchronize with
either direct or indirect division of the nucleus, a theory of
cell-division based exclusively or too strongly on nuclear
division embraces only cells exhibiting synchrony in division
of cytoplasm and nucleus; not only does such a theory fail

^ Delage, i8g^, p. /§S: " Ce qui est essentiel dans la division indi-
recte, c^est la division directe et cette derniere seule est a expliquer."

THE BIOLOGY OF THE CELL SURFACE

to account for cells lacking this synchrony, but it implies
that they are abnormal. The synchrony of nuclear and
cytoplasmic division is an interesting phenomenon but not
an essential characteristic of cell-division; instead of being
over-emphaeized in theoretical considerations, it needs
itself to be explained.

A theory devised to explain division of the cell-body
should also be consonant with the observable phenomena in
the cell both whilst living and when properly fixed. Phy-
sico-chemical speculations however ingenious when incon-
sistent with the observed phenomena lend little to the
solution of the problem. Indeed, we shall notice that in
the attempt to explain cell-division the ignorance of the
actual process and the exaggerated application of physico-
chemical knowledge have together erected serious obstacles
to a proper understanding and that, as the research on
experimental parthenogenesis, so also that on cell-division
can be upheld as an example of the danger which inheres in
this kind of physico-chemical study. Again it should be
emphasized that the first task in the approach to the prob-
lem is to learn as thoroughly as possible the normal process
which is to be explained.

Unfortunately, explanations of the mechanism of cell-
division have frequently been based on abnormal cell-
behavior induced by experimental agents. The chief error
in this type of work does not lie in the use of a strong experi-
mental means that brings about abnormality; it lies in the
fact that the conclusions overlook this result of the treat-
ment. Further, it is clear that experiments, in which the
cells were weak and abnormal at the outset, can not serve
as a basis for the explanation of the normal process.

For a theory of cell-division, as for any problem in bio-
logy, an appreciation of the normal biology of the cells under
their natural conditions is imperative. For example,
important as the work on vertebrate cells in tissue-culture

252

CELL-DIVISION

is for that on cell-division, we must always reckon with the
fact that these cells are not in their natural medium and
also that they have escaped the integration under which
they live while in the intact organism. Similarly, it is
dangerous to interpret the mechanism of cell-division from
the study of living cells after these have been dissected out
of the organism. Those eggs normally shed and insemi-
nated in the sea and free-living protozoa offer the best
opportunities for observations on cell-division in the living
cell under conditions approximating the normal. Since
here we deal with metazoa, I concentrate the discussion
on them, restricting myself largely to animal eggs. This
I do, not only because this book concerns itself with animal
eggs, but also because the most prominent theories on cell-
division are based on the study of eggs, especially those of
the sea-urchins. Nevertheless, we finally encompass cell-
division in all animal forms.

Obtainable in large number, easy to handle, of convenient
size and comparatively simple in structure, eggs of various
species of sea-urchins have been most popular cells for the
study of cell-division. The cytoplasm of some is trans-
parent or nearly so and in them one clearly discerns in the
successive stages of nuclear division the behavior of the
various constituents of the mitotic complex, including
the chromosomes once they appear on the spindle; in others
because some cytoplasmic granules are brightly colored,
one marks by their shift the ebb and flood of the cytoplasmic
tides. The study of the pigmented type supplements that
of the transparent. Also, the transparent eggs one can
color by means of inert dyes that do no harm to the living
cytoplasm, and can in the pigmented ones by innocuous
centrifugal force mass the colored granules so that the
nuclear phenomena more sharply stand out. But the cells
must be normal; when fertilized they should be of the same
specific gravity and almost perfect spheres, all showing

253

THE BIOLOGY OF THE CELL SURFACE

subsequently at each instant in the division-process the
same change in contour and exhibiting a clock-like precision
in the cleavage-rhythm.

Another advantage inheres in the use of sea-urchins' eggs
for the investigation of cell-division. Because of the fact
that these eggs are fertilized after complete maturation, the
process of division is most easily observed, being that of a
single division-cycle and not imposed upon the events of
one or both maturation divisions as in eggs of the other three
fertilization-types. Moreover, the cleavage is of the most
simple type inasmuch as the first three cleavages form
almost equal and close to spherical cells. Since the first
cleavage cycle encompasses the union of the egg- and sperm-
nuclei while all succeeding cleavages are wholly mono-nu-
clear— a fact which we must ever bear in mind — it would
appear necessary to appreciate the sequence of events in
the second or the third cleavage in order properly to evalu-
ate those in the first in which part of the events belongs to
the fertilization-process. One must be fully cognizant of
what events belong to fertilization and what to cell-division.
To obtain a clear picture of the end of the one and the
beginning of the other process is not easy since so much of
the work on cell-division considers the first division only.

The account now given is based upon the Qgg of Arbacia
because of its widespread use by investigators, and not
upon the more favorable but more sensitive egg of Echi-
7iarachnius, equally familiar to me but not so extensively
employed by others. My experience with these eggs as
well as with those of four different species of sea-urchins at
Naples convinces me that, despite certain structural dif-
ferences and some variations in their response to experi-
mental treatment — due to habitat, as, for example,
temperature and salinity of the sea-water — what I here set
forth applies generally to echinid eggs. In order to make
comparisons with the work of others who followed the

254

CELL-DIVISION

events of the first cleavage-cycle only, my account will be
limited similarly. This record of the happenings in the
living egg is supplemented by that obtained through study
of the fixed Q%%.

Let us begin the account of the first cleavage-mitosis in
the Q'g% of Arbacia with the stage when the two germ-nuclei
have disappeared by complete fusion to form a single resting
nucleus, a clear vesicle almost spherical in shape lying in the
polar axis just above the equator of the egg. At this
moment two centrospheres with asters are present at oppo-
site poles of the nucleus; the egg is spherical, oil-drops and
yolk-spheres, lying outside of the spindle-area, are evenly
dispersed, the pigment granules are trapped at the egg-
surface and the hyaline plasma-layer is clearly defined.

In the process of cleavage we shall follow especially these
changes: the growth of the centrospheres and asters, the
varying shape of the eg^, the shifting of the cytoplasmic
inclusions and the activity of the hyaline plasma-layer as
the nucleus goes through its mitotic cycle. These four
changes are easily visible and readily followed in the living
egg. In accordance with the principle hitherto followed, I
shall endeavor to derive a conclusion as to the meaning of
the process by relating the events that can be observed.

The nucleus seen in the living egg does not retain its
spherical form; increasing in size it becomes ellipsoid and
ruptures at the poles of its long axis where the centrospheres
and asters are located. A row of hyaline droplets, the
chromosomes, extends across the equator of the transparent
ellipsoid nuclear region; soon these form two parallel rows
which slowly separate as the ellipsoid nuclear region
by a constriction at the equator becomes dumb-bell
in shape. As each group of droplets approaches the inner
border of the centrosphere (the centrospheres are now hemi-
spheres presenting their plane-surfaces toward the dumb-
bell shaped clear region) they become spherical. Suddenly

255

THE BIOLOGY OF THE CELL SURFACE

they are in the centrospheres where they fuse to form
five or six droplets; these by farther fusion become one
hyaHne mass, the resting nucleus.

Concomitantly with this behavior of the nucleus and the
movement of the hyaline droplets, the centrospheres
steadily increase in size and the astral rays become more
extensive until the moment that the hyaline droplets enter
the centrospheres. Thereupon the centrospheres diminish
in size and the astral rays lose definition and shorten. Dur-
ing their period of growth the centrospheres are spherical;
their change to hemispheres marks the beginning of decrease
in size.

In the meantime the egg exhibits changes in shape.
While the hyaline droplets are moving toward the poles of
the transparent dumb-bell shaped area, the egg maintains
its original spherical form. With the entrance of the hya-
line droplets into the centrospheres, when centrospheres and
asters have begun to wane, the egg quickly elongates in the
long axis of the transparent region. About one minute later
the egg divides by a furrow forming at right angles to the
egg's long axis. Thus the egg at first a sphere becomes an
ellipsoid which divides and then becomes two spheres.

The site of the future cleavage furrow is marked as early
as the stage in which the transparent ellipsoid area is con-
verted into a dumb-bell shape. Already moved by the
fertilization-reaction, the various cytoplasmic inclusions,
especially the yolk, are further distributed by the growth
of the sperm-aster; and later, by the formation and exten-
sion of the amphiastral system. The conversion of the
ellipsoid area to the dumb-bell shape is caused by the move-
ment of yolk-spheres in the equatorial plane of the Qgg
from the periphery toward the centre. The shifting of
the oil-drops can not be easily followed in the living egg.
The pigment granules, after fertilization definitely fixed to
the outer rim of cytoplasm, at the time of cleavage extend

256

CELL-DIVISION

across the e^g in two parallel rows, one on each side of the
cleavage-furrow.

Concurrently with these nuclear and cytoplasmic events,
the clear peripheral cytoplasm, the hyaline plasma-layer,
exhibits changes. During the progress of the sperm-
head toward the egg-centre, the hyaline plasma-layer
is everywhere of equal width; but with the beginning of
the cleavage-process it appears delicately crenated, irregular
in contour. The egg at this time being somewhat darker
because of closer approximation of the yolk-spheres gives
the picture of cytoplasmic contraction, a condition which
passes leaving the egg clearer and the hyaline plasma-layer
of smoother contour. Immediately prior to the egg's
elongation, the hyaline plasma-layer is thinner over the
poles of the long axis of the transparent dumb-bell shaped
area and seems slightly thicker at the equator, the site of
the future cleavage-furrow.^ With cleavage this thickening
apparently increases; actually, the filaments of the hyaline
plasma-layer are stretched, because the plasma-membrane
to which they are attached does not move inward with the
cleavage furrow; they are therefore longer here than
elsewhere.

We can confirm and extend these observations on the
living egg by study of properly fixed normal eggs. The
accompanying figures are from sections of fixed Arhacia

eggs.

Fig. 33«2 is of an egg with intact nucleus, centrospheres
and asters. Fig. 33^ shows an egg with ruptured nucleus
and emerging chromosomes (seen in the living egg as hya-
line droplets); here the centrospheres are larger and the
astral rays more extended. As the heavily stained chro-
mosomes move apart, the centrospheres and asters increase
in size (Figs. 33r, 33^) finally to reach the maximum

1 Cf. Ziegler^ 1^04.

257

THE BIOLOGY OF THE CELL SURFACE

Fig. 33. — For decriptive legend see page 263.

CELL-DIVISION

V*;

■^ '.^f* ■■(■* -.'"if *''' *'■'* "^^

FiG. 33.— For decriptive legend see page 263.

^59

THE BIOLOGY OF THE CELL SURFACE

M

''\^ .

i»" t ^» ***** -

Fig. 33.— For decriptive legend see page 263.
260

CELL-DIVISION

.^Jt^.V^s^i-W'V

%»«t^-

..• -H

' -1.

- 4 . • . . *•

V* » . • • . ••

Fig. 33.— For decriptive legend see page 263.
261

THE BIOLOGY OF THE CELL SURFACE

»•■» • ■ •

^. . ■

■%

':^ .

.. ..AjVV^-.r'/

A^f..

- f • ' "

•

"V II .'('

■"" • • .

■ : " • ■.■.

*>>

•

•

■■■••"•". -•■ ..'. •••% .

•

•'*,••■•, \ • •••

1

•^ ' : ;'^» V ;,.-•'

^

«"

^

■ w

Fig. 33. — For decriptiv'e legend see page 263.
262

CELL-DIVISION

Fig. 33. — Stages in the first cleavage-cycle of the egg of Arbacia. (Drawn by
Mrs. L. G. Hugueley from author's preparations).

(Fig. 331?). As the variously shaped chromosomes are
converted into lightly stained spheres (Fig. 33)), they
are at the inner border of the now no longer spherical
centrospheres; the wide-flung astral rays are losing defi-
nition. When the chromosomes within the centro-
spheres begin to fuse, centrospheres and asters are on
the wane (Fig. 33g). A moment later the egg elongates
(Fig. 33A); cleavage quickly follows (Fig. 331, j, k) to give
rise to two spheres.

For the sake of simplicity and in order that the reader
may not be confused, I omit at this place a description of
the evanescent changes in the hyaline plasma-layer.
Indeed, the figures that I have given here do not show
the layer. A structure concerning which biologists are at
odds, because of lack of knowledge, ought be set off" from

263

THE BIOLOGY OF THE CELL SURFACE

the discussion involving better known structures. The
changes in the hyaHne plasma-layer are therefore taken up
beyond, after its structure has been elucidated.

The foregoing description of the changes in nucleus,
centrospheres and astral radiations agrees with the accounts
given by others for the eggs of several species of sea-urchins.
According to all of these the centrospheres and astral rays
steadily increase in size until the stage of the telophase
whereupon they begin to decrease; later, cleavage occurs.
That is, cleavage does not take place when centrospheres
and astral rays are at their maximum size. In spite of this
mass of well established observations, theories of the
division of the cell-body have been fabricated upon the
false idea of a coincidence of cell-division with the maximum
size of the aster. ^ I do not see any necessity to discuss
such theories.

As stated above, this chapter, whilst it deals with cell-
division primarily in eggs, has to do with the process in its
widest aspects. Thus we are here concerned with all forms
of division of the protoplasmic mass. It is to the phenome-
non as one widely occurring in all living cells that I wish
now to direct attention. I raise the question: Do the phe-
nomena observed in the division-process of sea-urchins'
eggs possess general significance for the problem of cell-
division : In order to answer this question we must estab-
lish those features in the division of the sea-urchins' eggs
which have their parallel in the division both of eggs of
other species and of other cells, and must eliminate whatever
is peculiar to this egg only.

There are other eggs that like the sea-urchins' show that
form of mitotic division of the nucleus in which centro-
spheres and asters are present. But whilst the centro-
spheres of the first clea\'age-figure of eggs of sea-urchins

^ Chambers^ 192^; Gray, 1931.

26^

CELL-DIVISION

lack centrosomes, as we have seen in the chapter on the
fertilization-process, other eggs, indeed most animal eggs,
possess them. The reason for this difference is here beside
the point. The difference exists and its existence warns
us to be wary in drawing too final and dogmatic conclusions

a b c

Fig. 34. — Cell-division, living cells of Triton (after Peremeschko). Note changes

in the ectoplasm.

with respect to these components. Moreover, were we to
compare the first cleavage-stage in other eggs with that in
sea-urchins', we should find no strict correlation of the
appearance of the cleavage-furrow with either the phase of
mitosis or the size of the centrosphere-aster complex. When
the egg of Ascaris^ for example,
divides, the chromosomes in
early telophase are still strongly
stained; no such pronounced
centrospheres as in sea-urchins'
eggs are present. Briefly said,
furrowing of the cytoplasmic
mass of eggs does not occur
always at precisely the same
stage of mitosis in all species
of them. Nor cioes it occur
uniformly In other animal cells.
(Note for example Fig. 34 and Fig. 35.) This is thus my
first point: We must be sure by exact observation as to what
happens in sea-urchins' eggs; but with this knowledge we
are not justified in drawing conclusions concerning division

265

■■■i&

^ - m

my

a b

Fig. 35. — Stages in cell-division,
spermatogenesis of Pyrrhocoris aper-
tus (after Gross).

THE BIOLOGY OF THE CELL SURFACE

in cells generally, if we can base these conclusions only on
the nuclear behavior and state of sea-urchins' eggs.

Besides, as I stated above, many cells lack asters, many
others lack well-defined centrospheres though they possess
asters. Certain eggs, such as those of Ascaris^ of Ciona,
of Phallusia, of Amphioxus, etc., have no asters at the
spindle-ends in the maturation-divisions. Yet they divide.
Many plant-cells go through mitotic division; their mitotic
figures are without asters. Asters and centrospheres are
not essential for mitotic divisions.

Also we should not forget that in many eggs repeated
mitotic divisions ensue without cleavage of the cyto-
plasm. Take eggs of insects, for example: many nuclear
divisions ensue before the cytoplasm divides. Nuclear
division without cleavage of the cytoplasm is not limited
to eggs. It obtains among unicellular animals and among
multicellular, including man, and among plants, in such
protoplasmic systems that contain many nuclei, the syn-
cytia. That nucleus and cytoplasm may divide a-syn-
chronously constitutes the strongest evidence against the
proposition that in mitosis lies the cause for the sundering
of the cytoplasmic mass.

Finally, an explanation of cell-division founded upon the
mitotic complex as a whole or upon any component of it,
falls to the ground for it can not encompass division of
cells whose nuclei divide amitotically. Whatever the
biological significance of amitosis, be it a primitive or a
degenerate process, undubitable cases of amitosis are on
record. Whatever the conditions responsible for its occur-
rence, we must reckon with this as fact. Even if the
mitotic complex were always uniform and cell-division
with mitosis were identical in all cells, we still could not
explain by reference to mitosis the division of the cell-mass
where amitosis prevails.

266

CELL-DIVISION

It becomes obvious in the light of what has been said
that nuclear and cytoplasmic division are separate phe-
nomena.^ Common usage has been loose in giving the term,
cell-division, the meaning of the division of the nucleus.
The statements made above, however, emphasize that we
must define cell-division more clearly and more exactly in
order to avoid the building up of theories on improper
grounds. Cell-division is to be defined as the division of
the cell-body. In what follows I shall adhere strictly to
this clear definition.

Finding it impossible to relate division of the cytoplasmic
mass to the nucleus, we turn to the cytoplasm itself.- Here
we may recall three visible events, accompanying the
division-cycle in eggs: changes in shape of the egg, move-
ment of the cytoplasmic inclusions, and changes in the
behavior of the hyaline plasma-layer. The second and
third are not peculiar to eggs and will be discussed in detail.
Change in shape not being invariable is not given great
emphasis apart from its being a factor in surface-tension-
theories of cell-division taken up beyond. I speak first of
the movement of the cytoplasmic inclusions.

Movements in the cytoplasm constitute a widely occur-
ring phenomenon, some cells exhibiting them to an extreme
degree. The simplest type is Brownian movement, so
extensively studied by physicists in inanimate systems.
This can be beautifully demonstrated in many cells espe-
cially by means of the darkfield in which the suspended
particles appear as bright points which shimmer on the
black background. Quite different from this phenomenon,
which is not peculiar to living cells, are the cyclical move-
ments in cells — often described for plant cells — a streaming

1 Watase, i8gi.

- Cj. Delage, 1895, P- 759-

267

THE BIOLOGY OF THE CELL SURFACE

that always follows a definite path or along preformed
channels; these movements represent specialized conditions.
Many cells, especially egg-cells, exhibit streaming which
is rhythmical but follows no preformed channels. This
streaming can be observed in egg-cells particularly if they
contain easily visible formed bodies or if having been
bathed in a solution containing a non-toxic dye they took up
particles whose movements can be easily followed.

The best of the earliest descriptions of this type of move-
ment in cytoplasm is that by Goette.^ Since his time
many workers, among them von Erlanger,^ have studied
the phenomenon. In living eggs one can of course fol-
low these changes from minute to minute. So-called
resting eggs — i.e., those that remain in a given stage, as for
instance mature but unfertilized eggs — do not show this
type of movement. If such mature resting eggs be fer-
tilized, then the cytoplasm begins to stream. During each
cleavage-cycle there is a period of little movement and a
period of intense movement; these periods appear to parallel
the stages in the cycle of nuclear changes.

With the growth of the asters these movements become
most marked. They begin over the spindle poles and move
toward the spindle equator. As the opposing streams meet,
they have only one possible direction: they can not move
outward because of the resisting surface and thus move
towards the centre of the egg. In this way the curreints
foreshadow the future cleavage plane; before the actual
division of the cell-body the currents have brought about
that disposition of inclusions found when the plane forms. ^

Before fertilization the inclusions maintain constant posi-
tions without reference to their specific gravity. If in such
eggs they are segregated by means of centrifugal force,

1 Goette, i8js.

'- Von Erlanger, iSgj.

^ Cf. Goette, iS/j, loc. cit.

268

CELL-DIVISION

they return in time to their original positions. The oil is
the last to return, I find, while the heavier yolk-spheres
return first and after them the heaviest bodies, the pigment
granules. After normal fertilization the inclusions shift
positions and come to a new arrangement which again has
no relation to their specific gravity. Thus, in the egg of
Arhacia after fertilization the heaviest inclusions, pigment
granules, remain close to the surface; the oil, shifting back
and forth fairly even in distribution except as influenced by
the mitotic spindle, forms clusters which mass and break up
again;^ the yolk is evenly distributed outside the spindle
area. The behavior of the pigment granules is due to the
fact that they are trapped at the surface; they move only
as the cleavage furrows are formed. In some eggs, as those
of Nereis, the yolk and oil come to be massed and are defi-
nitely distributed always to certain cells. These facts lead
us to assume that the flow in the cytoplasm is not every-
where the same and that there are currents which are cir-
cumscribed and limited to definite areas of the cell. If
there were only one current of equal velocity in a medium
everywhere the same, then we should expect a distribution
of particles according to specific gravity, as is for instance
the case in the blood-stream in a blood-vessel.

The fact that cytoplasmic streaming in eggs is most
expressed directly following the ectoplasmic changes, which
result from the fertilization-reaction, gives rise to the sug-
gestion that the ectoplasm by its activity sets up a condi-
tion which increases the currents in the cytoplasm. That
increased fluidity of the cytoplasm might be brought about
by the escape of substances from the nucleus is a less likely
assumption for the cause of the increase in streaming. It is
true that streaming is not easily discerned in an e^g like that
of Nereis, fertilizable before maturation, until after break-

1 Just, 1 927 a.

26g

THE BIOLOGY OF THE CELL SURFACE

down of the germinal vesicle; but before this break-down
come the intense ectoplasmic changes incident to fertiliza-
tion. In an egg like that of Chaetopterus after the germinal
vesicle breaks down the spindle goes to the metaphase and
comes to lie at the periphery before fertilization; although
this translocation of the spindle indicates streaming in the
cytoplasm, this soon diminishes; most intense streaming is
resumed after fertilization. The cytoplasm of the unfer-
tilized sea-urchin egg when ripe for fertilization is as fluid
as any that I know, the germinal vesicle having broken
down some time before in the ovary, but cytoplasmic
streaming of measurable degree is demonstrable only after
fertilization. Moreover, this cytoplasmic streaming is
rhythmical: during cleavage the tides ebb and flow. And
always ectoplasmic activity heralds their flood. It may
very well be that in all cells ectoplasmic behavior as a
response to changes in the environment initiates cytoplas-
mic streaming.

In eggs so far used in the study of the currents in cyto-
plasm the two streaming movements from poles to equator
by opposing each other seem to bring about cell-division.
Theories concerning their cause have not been wanting, the
chief of which is that the currents are due to surface-tension.

The theory that the division of the cell-body is caused
by changes in surface-tension is upheld by many investiga-
tors. Some maintain that the cell divides because of
increased surface-tension over the spindle-poles, whereas
just as many workers are certain that the increase is at
the equator. In many quarters what is considered the
strongest proof that the division of the cell is due to changes
in surface-tension comes not from observations and experi-
ments on the cells themselves but from study of oil-drops
suspended in water or solutions. It is a curious fact that
here too the theorists are not agreed: some contend that an
oil-drop divides because of increased surface-tension at the

270

CELL-DIVISION

equator, whilst others insist that the increase is over the
poles. Despite this lack of accord concerning the efficacy
of the infallible principle of surface-tension in the division
of oil-drops, to say nothing of the cells themselves, the
surface-tension theory persists.

More than once I have protested against the widespread
misuse of surface-tension in biological explanations and
theories.^ Drops of oil in water or solution constitute a
liquid-liquid system; a suspension of eggs does not. Whilst
every oil-drop is a homogeneous liquid, each egg, far from
being homogeneous, is itself a suspension of oil-drops, yolk-
spheres, etc. — of materials of diflferent chemical structure
and physical make-up in cytoplasm, the living continuum,
itself heterogeneous. The surface of an oil-drop is a film
of molecular dimension chemically identical with the inte-
rior of the drop; the measurable surface of an egg is thou-
sands of times the width of the film of an oil drop and is a
diflferentiated structure built by the egg during its develop-
ment as a germ cell. The fact that one can induce stream-
ing in oil-drops which closely resembles cytoplasmic
streaming does not warrant the conclusion that since an oil-
drop divides because of changes in surface-tension, division
of the egg or other cells is likewise due to changes in surface-
tension. Models used in biology to prove the cause of
vital phenomena may be interesting but after all they are
literally only models — imitations of the real thing and of
little value for the analysis of the conditions in living
substance.

Equally weak is the support for the surface-tension theory
derived from experiments which demonstrate that during
the cleavage-cycle, eggs of sea-urchins reveal a rhythm of

^ Hercik {igj4) entered a strong -protest against the indiscriminate
adducing of surface-tension for explaining biological processes.
In his opinion the significance of surface-tension is often over-
estimated.

271

THE BIOLOGY OF THE CELL SURFACE

resistance and susceptibility to certain experimental agents.
As I pointed out in the chapter, General Properties of
the Ectoplasm, unfertilized eggs in hypotonic sea-water
disintegrate more slowly than eggs which are exposed to
the same degree of hypotonicity when after insemination
break-down in the ectoplasm takes place. This period of
ectoplasmic changes having passed over, the eggs are more
resistant than unfertilized ones. According to many
observers this resistance persists until just before or at
first cleavage when the eggs become susceptible. It is
held by some that this susceptibility is due to increased
surface-tension over the spindle-poles.

Now sea-urchins' eggs, as we have seen, are first spheres,
then ellipsoids and finally, by cleavage, two spheres. Does
the supposed change in surface-tension take place while the
egg is still a sphere, when it becomes an ellipsoid or as it
forms two spheres .^ Or does the theory disregard entirely
that stage during which the egg elongates in the direction
of the spindle-axis .'' The theory demands the most exact
fixing of the moment in the cleavage-cycle when the egg
reveals its maximum susceptibility in order to relate this
to the act of cleavage. Observations made too far apart
on a lot of eggs developing at the same tempo could easily
miss the moment of maximum susceptibility. If, on the
other hand, closely set observations be made on poor eggs
developing at varying rates and therefore not reaching
first cleavage at the same instant, the moment of maximum
susceptibility would be erroneously fixed as coming in the
stage or stages of the intact eggs present whilst the disin-
tegrated ones would be in other stages.^

^ Degree of hypotonicity should be great enough to give sharp
results; transfer of eggs should be uniform as to number of eggs,
amount of dilute sea-water and amount of normal sea-water into
which eggs are transferred.

272

CELL-DIVISION

In the chapter, General Properties of the Ectoplasm,
was mentioned the method of study of the susceptibility of
eggs by putting them in solutions of dilute sea-water.
In the experiments of R. S. Lillie^ 40 per cent, sea-water
and 60 per cent, tap-water gave a solution that destroyed a
percentage of the eggs at once, others later. In my experi-
ments- on the rhythmical susceptibility of eggs of sea-
urchins to hypotonic sea-water during a cleavage cycle, I
modified this method. Instead of exposing the eggs to a
solution made up of 40 parts sea-water and 60 parts tap-
water, I used much more dilute solutions, mostly one made
up of one part sea-water and 90 parts tap-water. On eggs of
Echinarachnius I have also used 10 cc. of tap-water to one
drop of a sea-water suspension of eggs, an even greater
dilution. The value of this method lies in the sharp
results that it furnishes.

I do not wish to imply that I discount the worth of the 40
per cent, sea-water dilution as a means of revealing the
susceptible periods. On the contrary; for I appreciate the
fact that because it destroys eggs exposed to it during such
periods, whilst It does not immediately arrest development
of eggs exposed during their resistant periods. It Is an excel-
lent experimental means. I have used this dilution as well
as others successfully; but I have found that its use should
be checked by that of the greater dilutions mentioned
above, for the reason that eggs exposed to lesser dur-
ing terminal stages of resistance prior to the onset of the
susceptible period might develop Into this period. In
such cases the eggs do not break down in the stage which
they had reached at the moment of exposure. The dilu-
tions therefore should be so great that they halt develop-
ment abruptly; then the time in seconds from the moment

1 Lillie, R. S., igi6.
' Just, i()22e; ig28d.

THE BIOLOGY OF THE CELL SURFACE

of exposure to disintegration can be attributed wliolly to
the dilution's destructive action and not in part to a pos-
sible anesthetic action.

The use of extremely dilute solutions was not my only
modification of method. I exposed the eggs during a
cleavage-cycle at the short intervals of one and two minutes.
Thus, I was able, using always eggs developing at the same
tempo, to define with sharpest exactness the briefly endur-
ing periods of susceptibility which otherwise would have
escaped observation.

I think that there can be little doubt that my experi-
ments did more than confirm earlier ones which proved the
existence of periods of resistance and susceptibility during
the cleavage-cycle of the sea-urchin &gg\ they demon-
strate exactly the onset and duration of the period of
susceptibility. The significance of my findings for a
theory of cell-division needs no elaboration. If we wish
to base a theory of the cause of cleavage upon the occur-
rence of rhythmical resistance and susceptibility parallel
to the rhythm of cell-division, we first of all need accu-
rately to relate the susceptible period to a definite stage in
the process of cell-division. One finely spun physico-
chemical theory of cell-division falls to the ground because
its author failed to time precisely and to relate definitely
the onset and duration of that period of susceptibility
which precedes the appearance of the cleavage-furrow.^

Perhaps the simplest way to begin the discussion of my
findings is to present the data of a typical experiment, on
the egg of Arhacia punctulata.- Accordingly, these data
in the form of a table (Table III) are herewith given. The
reader will note: First, there are two periods of susceptibility

1 Lillie, R. S., igi6.

^ The egg of Echhiarachiiins which is less resistant than that of
Arbacia reveals ninch more sharply the periods of susceptibility.

274.

CELL-DIVISION

during the cleavage-cycle of the egg. It is the second, the
greater, which comes prior to the appearance of the cleav-
age-furrow, with which we are primarily concerned.
Secondly, during both periods the egg is spherical. The
change in form from spherical to ellipsoid occurs once only
during a cleavage-cycle; It marks the end of the period of
greater susceptibility. These two findings possess especial
significance for an interpretation.

Table III. — The Rate at Which Eggs of Arbacia Disintegrate in Distilled
Water at Intervals after Insemination*

Time, in

Time, in

No.

minutes,
after

seconds, to
complete

Comment

insemination

disintegration

I

3

240

2

6

180

3

10

60

4

15

60

S

17

60

6

18. 5

65

7

21

80

8

23-5

75

Streak indicated

9

26

70

ID

27-5

60

II

29

60

12

31

60

13

33

25

14

35

25

Streak

IS

37

30

i6

39

60

"Dumb-bell stage"

17

41

100

i8

43

60

19

45

60

20
21

47
48

20)
20^

Eggs burst over spindle pole

22

49

60

Eggs elongate

23

51

60

Cleavage furrow indicated

24

S3

120

Cleavage

* Eggs of Arbacia are inseminated, and at intervals after insemination 5 drops of them are mounted
under the microscope in 15 cc. of distilled water. The first column gives the number; the second,
the time after insemination; the third, the time to cytolysis; and the fourth, the stage of the egg at
the time of exposure.

THE BIOLOGY OF THE CELL SURFACE

As regards the first named finding, that there are two
periods of susceptibility in a cleavage-cycle, I should
like to point out that an interpretation based on the
occurrence of the second which overlooks the first is diffi-
cult to defend. If susceptibility as such is the cause for
cleavage, then the egg should cleave after each such period.
This is not the case. Further, it must be remembered that
in many eggs the most pronounced period of susceptibility
occurs within a minute after fertilization — that is, depend-
ing upon the species of egg, anywhere from i^g or M20 of
the total time to first cleavage, at a time long before the
onset of the first cleavage-cycle which, as stated above,
begins only with the stage of apposition of the egg- and
sperm-nuclei. Also if we attempt to correlate the sus-
ceptibility with nuclear phenomena, we find ourselves at a
loss. During this period of susceptibility immediately fol-
lowing fertilization, neither the sperm-nucleus nor the egg-
nucleus Is in active mitotic condition. In that period of
susceptibility which occurs first in a cleavage-cycle, the
apposed nuclei are not in mitosis. Thus only in that sus-
ceptible period immediately prior to cleavage is there
present an active mitosis. Then susceptibility does not
depend upon or run with a definite stage in mitosis. It
follows from this that all the learned disquisitions relating
the origin of the period of susceptibility to the nuclear
state and more specifically to the behavior of the asters
have no basis in fact.

Whilst it is true that the periods of susceptibility can not
be correlated with mitotic phenomena, they can be with
ectoplasmic state or behavior. At the first period of sus-
ceptibilit}' that appears during a cleavage-c}xle, the ecto-
plasm manifests peculiar behavior as it does during the
period of susceptibility immediateh" prior to the act of
clea^'age. The greatest observed susceptibilit}' is that
occurring immediatel)' after fertilization; in this stage the

2y6

CELL-DIVISION

egg exhibits the strongest ectoplasmic changes ever shown
in its development. The periods of susceptibility thus
can be related to structural and behavior-changes in the
ectoplasm. No other constant change being associated
with the period of susceptibility than the visible and easily
demonstrable ectoplasmic state, we can assume that
ectoplasmic state is wholly or in part responsible for the
temporary lowered resistance of the egg to dilute sea-water.

Not only did I fix the period of maximum susceptibility
of sea-urchins' eggs during their cleavage-cycle to hypo-
tonic sea-water; I was able also to localize precisely the
point on the eggs at which they break down during this
period. Properly to appreciate this latter finding, we need
fully to understand the structure of the hyaline plasma-
layer. This, part of the ectoplasm, though often the subject
of discourse by many writers, has never been properly
understood. I give therefore a detailed description of the
origin and structure of the hyaline plasma-layer in sea-
urchins' eggs, thus supplementing the description given in
the chapter on the ectoplasm.

Selenka^ years ago described the surface of various echi-
noderm eggs as composed of a sheath of clear cytoplasm.
Later Hammar- and others also described this external
layer on fertilized sea-urchins' eggs. It is variously known
as the ectoplasmic layer, hyaline plasma-layer, Hammar's
layer, etc. It should not be, as it often is, confused with
the vitelline membrane.^

I have found that in several species of sea-urchins this
hyaline plasma-layer arises in the same way and has much
the same structure as described for the fertilized egg of
Arbacia. Into the periA^itelline space which arises with sep-
aration of the vitelline membrane fine filaments of the cyto-

1 Selenka, 1S78, 1883.
- Hammar, i8g6, i8g/.
' Just, iQjog, igjjc.

277

THE BIOLOGY OF THE CELL SURFACE

plasm project.^ By anastomosis of their free ends a very
fine covering membrane forms. ^ Thus the hyaline plasma-
layer is clear granule-free cytoplasm in the form of finely
spun threads covered by a thin membrane. Other eggs, as
shown in the chapter, The Ectoplasm, possess these ecto-
plasmic filaments. They are present on every marine egg
that I know. Unfertilized sea-urchins' eggs treated with
strong hypertonic sea-water also show that the surface-
cytoplasm is made up of radial strands. Eggs of Nereis
and of Platynereis show especially well after fertilization
immediately below the vitelline membrane a delicate
plasma-membrane, which is the covering film of cytoplas-
mic prolongations. It is these prolongations, of greater
length and diameter than those of sea-urchins' eggs, which
give the surface of the eggs of these worms their striated
appearance. These prolongations constitute the outer
region of the ectoplasm. They and their covering film
compose the hyaline plasma-layer of the egg.

Others before me have described on the fertilized egg
In later stages this layer as made up of filaments. Meves,^
for example, has pointed out that some time after fertiliza-
tion the layer is composed of threads and a thin covering
membrane. This structure of the hyaline plasma-layer
can be demonstrated by placing fertilized eggs in calcium-
free sea-water; the covering membrane becomes destroyed
leaving the filaments free.

During the whole cleavage-period, sea-urchins' eggs
show on the surface of each blastomere these fine cyto-
plasmic projections covered by a delicate membrane which
together make a continuous film enclosing the blastomeres.
The filaments are most easily seen between adjacent blasto-
meres as they are separating at cleavage. Since there is no

1 Cf. also Berthold, i8S6, and Thfcl, iSg2.

2 See also Mrs. Andrezvs.

3 Meves, igi2.

278

CELL-DIVISION

great decrease in width of the hyaline plasma-layer as cleav-
age progresses and since by treatment with calcium-free
sea-water the presence of the filaments can be demonstrated
on eggs throughout cleavage, we reach the conclusion that
the egg builds new hyaline plasma-layers constantly. One
can not say exactly where in the cytoplasm the hyaline
plasma-layer begins, for each filament, the essential struc-
ture of the layer, is a granule-free prolongation of the
cytoplasm. The activity — amoeboid, spinning, and con-
tractile— of the hyaline plasma-layer, so well described by
Mrs. Andrews as early as 1897, is due to the behavior of
these continuations of the egg-plasma. The filaments of
the hyaline plasma-layer are a living part of the living pro-
toplasmic system (see also Fig. 36).^

As a living and integral part of the tgg, these filaments,
we should expect, react as the cytoplasm of which they are
continuations. Because of their tenuous and granule-free
structure they are physiologically different from the remain-
der of the egg. However, any very striking difference in
their physical behavior needs to be fully established. It
was claimed by Goldschmidt and Popoff- first and later by
Gray^ that hypertonic sea-water increases the volume of
the hyaline plasma-layer and decreases that of the egg and
thus reveals a marked difference between the osmotic
properties of the surface-cytoplasm and the remainder of
the egg. If this interpretation were correct, I would call
this difference in osmotic properties — what Gray calls a
fortunate coincidence — a most important discovery. We
should use every available fact concerning the behavior of
colloids in living protoplasm. Is it true that the hyaline
plasma-layer increases in volume whilst the remainder of

^ Ectoplasviic protrusions on living dividing cells have been early
described by ina^iy workers.

- Goldschmidt and Popoff, igoS.
^ Gray, 1931 .

279

THE BIOLOGY OF THE CELL SURFACE

the egg decreases because "the former is freely permeable
to electrolytes whereas the latter is not"?^ The answer is
simple: the existence of such a difference has actually not
been proved.

The correct interpretation of the changes in sea-urchins'
eggs induced by means of sea-water made hypertonic by the
addition of either electrolytes or non-electrolytes is as fol-
lows: placed in hypertonic sea-water, the whole egg, includ-
ing its ectoplasmic filaments, loses water and shrinks. The

Fig. 36. — Cleavage of an egg of Echinus microtuherculatus in calcium-free
sea-water (after Herbst). The covering membrane of the ectoplasm is destroyed,
leaving the filaments free.

filaments decrease in diameter and appear as lines thereby
becoming more easily visible. In every tgg known to me,
the ectoplasmic filaments appear in the same way as an
effect of hypertonicity.-

We can not escape these facts: that after fertilization and
with membrane separation the surface of the egg is studded
with filaments; and that also unfertilized eggs as they shrink
in hypertonic sea-water show these filaments. It is patent,
therefore, that the filaments are not new formations called
forth by the action of electrolytes. The action of electro-
lytes does not demonstrate a difference in permeability
between endoplasm and ectoplasm.

Gray also errs when he says that the hyaline plasma-layer
dissolves in calcium-free sea-water. Rather, eggs in this

^ Gray, I.e.

- Cj. Faure-FreDiiet.

280

CELL-DIVISION

medium show the filaments very beautifully as the figure
taken from Herbst so clearly shows^ (Fig. 36). Herbst
himself thought that he had altered the structure of the
hyaline plasma-layer, changing its homogeneous structure
to a collection of radial threads whereas actually by the
treatment with calcium-free sea-water, he only removed
the covering membrane of the threads. This change in
the eggs' environment prevents the formation of a new
membrane which takes place when the eggs are returned to
normal sea-water. The so-called removal of the hyaline
plasma-layer by microdissection is also a removal only of
the covering membrane.

Goldschmidt and Popoff advanced the idea that the ecto-
plasmic filaments on the sea-urchins' egg are due to astral
radiations from the nucleus. But there are no astral radia-
tions in an unfertilized egg momentarily subjected to the
action of strong hypertonic sea-water; nor are any present
in an egg fifty seconds after insemination. In both cases,
filaments can be observed. Fertilized sea-urchins' eggs
shrink slightly when the astral rays are first most extensive
so that the eggs' surface appears somewhat crenated because
the filaments stand out strongly. It is this crenation that
these authors observed.

Gray and Goldschmidt and Popoff have derived theories
of cell-division from their observations on the behavior of
the hyaline plasma-layer. Since these observations are
incorrect, the interpretation given them is unwarranted.
This is thus another example of the danger of applying
physico-chemical notions to a biological process in the
absence of knowledge of the underlying structural condition.

During a cleavage-cycle of sea-urchins' eggs the ecto-
plasm undergoes changes. Until the stage- when the

^ Herbst, i8gg.

~ See Ziegler, 1^04. Compare Ziegler, iSgS, on egg of Bero'e.

2S1

THE BIOLOGY OF THE CELL SURFACE

chromosomes are at the anaphase and the egg is still spheri-
cal, the hyaline plasma-layer is everywhere of equal width
and the filaments in it are all the same length. In later
anaphase or early telophase the ectoplasm shows a rapid
change, moving from over the spindle poles toward the
equator, the site of the future cleavage-plane, in an amoe-
boid or wave-like fashion. The ectoplasm then begins to
move inward toward the egg-centre, in the direction of the
course of the cytoplasmic currents, and the egg elongates.
As the ectoplasm continues to move inward, the covering
film of the hyaline plasma-layer resists. Thus the moving
ectoplasm exerts a pull on its filaments in the region of the
oncoming cleavage plane so that they are longer here than
elsewhere. With the beginning of the moving of the wave
of amoeboid changes from the area over the spindle poles
to the equator, the filaments in the former region appear
to lose connections with their covering membrane, whilst
those in the latter seem more firmly bound to it.

By the very simple method of treatment with extremely
dilute sea-water, I was able to ascertain that while the
egg is still spherical and when the ectoplasm exhibits
the wave-like movement from the circumpolar areas to the
equator, it has definitely localized weaknesses in this, the
most susceptible, period of its cleavage-cycle. At this time
the egg breaks down in about ten seconds after immersion
in the dilute sea-water by an outflow from the areas over
the spindle-poles. Though eggs of some species of sea-
urchins show this localized disruption more clearly than
others, it can be observed in all; none ever show an exuda-
tion elsewhere than from the circumpolar areas. With
elongation the egg is again resistant.

As we have seen, the eggs' period of greatest susceptibility
to hypotonic sea-water occurs during the process of mem-
brane-separation. At this time the ectoplasmic filaments
appear more sharply because of break-down of material

282

CELL-DIVISION

at the surface of the egg. Though this susceptibility is
not of immediate concern here, since it does not relate to
the cleavage-cycle, we adduce it since it shows susceptibility
as due to ectoplasmic change. In the first period of sus-
ceptibility which occurs during the cleavage-cycle and
which falls in with the stage of union of the egg- and sperm-
nuclei, when there is a normal shrinkage of the egg-contents
and the ectoplasmic filaments are most easily visible, the
egg does not disrupt at definite points. In some other
eggs, those of Crepidula,^ for example, it seems that the
weakness that appears at this time or soon after, persists.
In the period prior to cleavage, there are localized points
of weakness due to ectoplasmic activity; they are limited to
the egg-poles. In all periods, susceptibility is due to
changes in the ectoplasm and where these are local, break-
down of the egg is likewise localized.

In none of these three periods of susceptibility can the
break-down of the egg be ascribed to changes in surface-
tension. It is not possible to regard the complex structure
of the ectoplasm with its palisade arrangements of filaments
and their covering membrane as a simple surface-film.
The width of this covering membrane alone surpasses by
far that of a molecular film. Besides, this membrane is
passive in cell-division; ectoplasm and filaments play the
active role, changing from moment to moment first in one
region and then in another. These changes in structure
and behavior parallel the periods of susceptibility.

The ectoplasm of marine eggs is a highly mobile, con-
stantly changing structure. In eggs as in other cells,
protozoan, nerve, muscle, etc., it is no mere inert sheath.
Instead, it reflects in its spinning, in its suceptibility, the
irritability of the living system in response to environmental
changes. In addition to the significance that its general

1 Conklin, ig/2b.

2S3

THE BIOLOGY OF THE CELL SURFACE

properties have for cell-life, the ectoplasm by its behavior
as here described in the cleavage-cycle, plays a definite
role in the process by which the cytoplasmic mass becomes
two cells.

When a cell divides, the plane of cleavage arises either
from within the cell-mass and extends outward, or from the
periphery and extends inward. The former mode, cleavage
by formation of the so-called cell-plate, is frequently met
with in plant cells. Division of animal cells takes place by
a furrow extending from the periphery inward. Cleavage
by formation of a cell-plate in animal cells is only seldom
found and is a modified process occurring together with
furrowing. The problem of cell-division for animal cells is
that of the origin of the furrow which separates the dividing
cells. The phenomena observed during the process of fur-
rowing in the living sea-urchin eggs by which the single
cell becomes two we summarize as follows:

Currents in the cytoplasm move toward the site of the
future cleavage-plane; meeting there the opposing streams
they move inward. The ectoplasm, in whose behavior lies
the cause of streaming, then actively shifts by amoeboid
movement from the circumpolar areas toward the equator.
With this the egg quickly elongates. The ectoplasm moves
then inward along the plane pre-delineated by the cyto-
plasmic currents. Cleavage in a sea-urchins' egg is there-
fore accomplished by active movement of the ectoplasm.

Neither cytoplasmic movements nor changes in ecto-
plasmic behavior are confined to animal eggs. Cytoplasmic
streaming has been described in plant cells, especially by
Ewart. Movement in the cytoplasm of Protozoa is well
known. Changes in shape and in activity of the ectoplasm
are similarly familiar from descriptions made by the earlier
workers on cells. Recently, investigators using the method
of tissue-culture have, particularly by means of the cine-
matograph, recorded the evanescent changes at the cell-

284

CELL-DIVISION

surface which accompany cell-division. They are also
well defined in other living cells when freshly dissected out
of the organism. We need not, therefore, confine our
explanation of cell-division to eggs of sea-urchins. We
dismiss as cause for cell-division division of the nucleus, by
mitosis with or without asters, and by amitosis, whether
occurring simultaneously with cytoplasmic division or not,
since no constant relation between nuclear division and that
of the cell-body can be established. Since not all cells are
spherical and not all spherical ones elongate during the
cycle, we dismiss also elongation of the cell as a factor.
Movement in the cytoplasm depends upon ectoplasmic
activity; it is thus not a primary factor. There remains
the ectoplasm found in all cells.

Cells do not extend indefinitely into space. They possess
surfaces. Cell-division means that one cell becomes two
by the rise of a new partition. Does this arise within the
cytoplasm or does it come about through the extension of
the ectoplasm inward .^ Until we know more concerning
the origin of cleavage by cell-plates, we may only hazard
conjectures as to the formation of cell-walls within the
cytoplasm as found in plants. For animal cells, generally,
we must seek the cause of cell-division in ectoplasmic
activity.

28'

I I
Cleavage and Differ entiatio?i

!/ HE ESSENTIAL PROBLEM OF ANIMAL DEVELOPMENT LIES

in the question: How does the egg, a single cell, become an
adult organism? If one take the transparent eggs of a
common marine fish like the mackerel which float like
bubbles on the surface of the sea, one can easily follow their
development under low power of the microscope. Before
fertilization a thin film of cytoplasm beneath the membrane
encloses a core of yolk and oil. With fertilization this
cytoplasm flows to one egg-pole to collect there as a disc.^
This disc is next crossed by one furrow and then by another
at right angles to it; so by this cleavage two and later four
cells arise. Cleavage progresses until many cells form,
whilst some of the yolk beneath the cytoplasmic disc is
transformed into cytoplasm. Soon one discerns an opaque
line running the length of the disc. Here there are more
cells than elsewhere, hence the opacity; from some of these
will come the future embryo. Under the microscopic even
one who has very little knowledge of the development of
eggs can follow the origin and formation of the nerve tube
out of which the brain and spinal cord emerge. One looks,
as through an open window, at the very mystery of life,
wondering at the heartbeat, first uncertain and then in
definite, sure rhythm; the bright red blood moving in jerks
with each beat of the heart; the first spasmodic muscular
twitch; the appearance of the purple-black eye-pigment;
the definite fish form; the color of the skin which will give

^ Cy. Ransom, /cS'57.

286

CLEAVAGE AND DIFFERENTIATION

the markings that make the adult mackerel one of the most
beautiful creatures of the sea.

There are other eggs, those of ascidians, which like the
mackerel belong to the highest group in the animal king-
dom, that hatch as swimming larvae in eight hours after
fertilization instead of seventy-two hours, the time the
mackerel Qgg in a warm laboratory requires. From egg to
complex animal in eight hours 1 The problem of the devel-
opment from a single cell to a complex animal is thus a
fascinating one.

In the following exposition we shall see again that exact
description of the process which we aim to explain plays
a chief and significant role. Whilst here as with many
other problems in biology we must realize that a description
does not explain the process described, nevertheless descrip-
tion is the prerequisite of a successful attack on the
problem of development, a problem which doubtless more
than any other in biology is complicated by the interplay
of simultaneously occurring reactions and by rapidly suc-
ceeding events. To determine cause and effect in this
manifold process of differentiation is a task whose difficulty
is so manifest that we must before all else seek to follow
the process as exactly as possible in closely set stages.
We may then be able to relate smallest changes in form and
in expression to general phenomena of development, that
is, to the differentiation which accompanies the cleavage-
process. Such a task is undertaken in this chapter.

Following the classification of Karl Ernst von Baer, one
can separate the development of fertilized or parthenogenetic
animal eggs into four periods: cleavage, formation of the
germ-layers, development of the organs and histological
differentiation. This classification represents a scheme
which holds more nearly true for the higher animals than
for the lower and should not be taken as meaning that the
development of any egg is sharply divisible Into fixed

287

THE BIOLOGY OF THE CELL SURFACE

periods. Development is a catenary process of overlapping
stages and can not be categorically separated by rigid lines.
With periods three and four, which have to do with the
establishment of the organs and the finer differentiation
within them, we have here no concern. Period two includes
the formation of the primary sheets of cells, the ectoderm
and the endoderm in the lowest multicellular animals, the
sponges and the coelenterates, and the ectoderm, endoderm
and mesoderm in all the other multicellular animals.
Period two represents the end-result of period one. It is
in this first period, cleavage, that our problem lies. And
this for the following reasons:

Coming first, this differentiation during cleavage stands
nearest to the condition in the egg at the moment of fer-
tilization, a moment that forms in time the limit between
the condition of the egg as single cell and those conditions
of the egg as multiplying cells which we call the differentia-
tion during cleavage. Hence studying the period of cleav-
age, we approach the source whence emerge the progres-
sively branched streams of differentiation that end finally
in almost quiet pools, the individual cells of the complex
adult organism.

But just as the source of a river though single may not
be simple but compounded of rivulets, so the fertilized egg
though single is not simple, being itself a complex of many
contributary streams of differentiation. For the fertilized
egg is already a differentiated system. Indeed, the whole
history of the egg from the moment when it became dis-
tinguished as such and separated from body cells is a suc-
cession of diiTerentiations. In form, growth, mode of
building up of food reserves, and in nuclear structure, a
young egg-cell shows itself different from every and any
other cell of the animal's body. The egg-cell is subjected
to the same environmental influences as other, fully special-
ized cells of the animal's body. The changes and processes

288

CLEAVAGE AND DIFFERENTIATION

by which it becomes an embryo thus spring from an initial
differentiation, an intrinsic organization of the egg that
distinguishes it from all other cells.

Consider the chick at the moment when it emerges from
the egg-shell: it has all the organs that it will ever have
and it had the precursors of these at the moment that it
was fertilized. Nothing of organic material did it gain
during the 21 days of incubation. It took in water, oxygen
and heat. But not one single milligram of living proto-
plasm is in the hatched animal but what was within the
unincubated egg. Early the embryo formed blood with
a red pigment; this pigment contains iron. But not the
most minute fraction of this iron was gained during devel-
opment; all present in its body at hatching was already
there before the egg was laid.

True, a mammalian embryo, like that of the human,
develops by the material of the blood which comes to it.
But in the minute human Qgg must be present all that from
which comes a human embryo for the blood which nourishes
it is the same as that which supplies any other cell in the
mother's bod}', no one of which becomes a human embryo.
Hence, though the human egg differs from the bird's, at
basis the same is true: the egg at beginning of development
is a differentiated and a differentiation-capable system.

Following its period of primary differentiation the egg
cell grows, reaching a stage prepared for fertilization but
unfertilizable. Then it becomes fertilizable — another phase
of differentiation. If fertilization ensues, it becomes dif-
ferentiated again. Thus differentiation flows on as a stream
without breaks and if later for purposes of convenience I
use words that seem to connote sharply discontinuous
phases, the reader should keep in mind that I regard these
phases, periods or stages, only as moments in time when
the stream alters its course. Although we can not yet
assert what in all this history is cause and what effect,

289

THE BIOLOGY OF THE CELL SURFACE

nevertheless we may say in general terms that each suc-
ceeding differentiation is conditioned in part by extrinsic
and in part by intrinsic factors, the latter of which are
conditioned by the preceding differentiation.^ One great
task in the study of the differentiation occurring during
cleavage is the analysis of these factors, a task especially
difficult because we have no known point from which we
may start, for in this respect the egg also must be regarded
as an unknown system.

In the second place we concentrate attention on the
cleavage-period in our effort to trace the differentiation
of development, because cleavage is a process common to
all animal eggs. Every species of animal egg passes through
cleavage, but not every animal Qg^^ e.g., of sponges and
of coelenterates, passes through the three other periods
enumerated above. Then the differentiation that takes
place during cleavage we regard as that embracing in
a wider sense those characteristics common to animal
development and most readily reducible to general terms.
As we shall later see, whatever the period of development,
in which differentiation reveals itself to us, the mode of
differentiation is always the same. This being true, study
of the differentiation in the most generally appearing stage
has distinct advantage.

In the third place we have the practical reason that, as
experience has taught, of all the periods of development the
cleavage period lends itself most readily to resolution of
the processes into closely set stages; thus by experiment it
is possible to analyze the factors which set up the conditions
for differentiations in a more normal or natural manner
than, for example, in experiments with transplantations
involving conceptions of "organizators" and the like.

1 Cf. Delage, 1S93, PP- 765-766.

2go

CLEAVAGE AND DIFFERENTIATION

For these three capital reasons, then, I consider it best
to limit my discussion of differentiation to the cleavage-
period. Since the fertilized uncleaved egg is itself dif-
ferentiated, and in this respect an unknown system, a
distinct advantage accrues in pushing back our inquiry as
far as possible to it as a single cell whence originate the many
different kinds of cells that make up the embryo. For the
great question is: how out of a single cell do these manifold
differences arise .^ I repeat: singleness does not mean sim-
plicity. And yet, inasmuch as the complex organism has
its genesis in a single protoplasmic system, the unknown
complexity of this system may in some measure become
known by means of a resolution of the events by which a
single cell organism becomes one of many cells. It follows
from this that we should define very exactly the cleavage-
period. I turn therefore to a brief resume of cleavage
processes in various animal eggs. Such a resume will
further be valuable, because in the later discussions I
shall need to refer to the various cleavage-types; and
because by evaluating differences in the mode of cleavage
we may arrive at a basis of similarity.

Every developing animal egg normally passes through a
period of successive cell-divisions or cleavages. The
cleavage-cells or blastomeres always arise by binary fis-
sion, that is, two "daughter-cells" arise from the division
of one cell. In this fashion is established a cleavage-pattern
which differs with different eggs. Upon these differences
depends the classification of eggs according to their type of
cleavage. Cleavages are classified as total and partial:
total, if the whole egg cleaves; partial, if only a part of the
egg cleaves — this cleaving portion is always either the whole
surface or a definite surface-area.

In total cleavage the blastomeres of first cleavage and
of several subsequent divisions may be approximately equal

2gi

THE BIOLOGY OF THE CELL SURFACE

in size; they may be at first equal in size and early in the
succeeding cleavages show marked size-differences or they
may from the first cleavage show striking disparity in size.
At the end of the cleavage-period the blastomeres are of
unequal size in all totally cleaving eggs.

The position of the blastomeres also plays a part in
making the cleavage-pattern in totally cleaving eggs. If
they stand directly above each other the final pattern is
radial; if the four smaller cells, instead of lying directly
over the larger ones, lie above the furrows of the first and
second cleavage, the pattern is spiral.

The derivation of the blastomeres may be so regular
that one can determine exactly in the cleavage stages the
time and place of the origin of each. For many eggs it
has been shown that the blastomeres in late cleavage can
be traced unerringly back to the stage of first division.
Thus, for these eggs, a cell-lineage, as it is called, can be
traced. In other eggs for several cleavages the blasto-
meres arise always in the same way and hence show con-
stancy in origin; after this period, they vary both as to
time and place of origin. In other words, the cleavage
pattern of animal eggs may be constant or not. If their
cell-lineage can be traced, the pattern so far as the origin
of the blastomeres is concerned, is constant. It is to be
noted that the cleavage of eggs of clams, worms, ascidians,
showing cell-lineage is closely similar. Hence this type of
cleavage is not restricted to any one group of animal eggs.
If the lineage of the cells can not be traced, this means that
the cleavage-pattern is composed of cells whose positions
are not determined by fixed origin.

Partial cleavage is of two forms, discoidal and superficial.
In the former, cleavage is confined to a disc at only one pole
of the egg, whilst in the latter the entire superficial cyto-
plasm cleaves. Discoidal cleavage Is found in eggs of
cuttle-fishes and their allies, in many bon}' fishes and in

2Q2

CLEAVAGE AND DIFFERENTIATION

reptiles and birds; superficial cleavage in eggs of some
coelenterates, of many insects and of some other arthropods.
Superficial cleavage might be said to be the mode among
arthropod eggs for with few exceptions in the eggs of the
members of this group that embraces the crabs, spiders,
insects etc., only the surface-located cytoplasm shows
cell-boundaries.

In figures of the discoidal cleavage in the e^g of an ink-fish
or squid, Loligo, as seen in section, one notes at the upper
pole of the egg a heavily drawn line which represents the thin
sheet of superficial cytoplasm. It is in this sheet that the
cleavages ensue. In the unfertilized egg of a fish a thin
band of cytoplasm encloses the yolk. After fertilization
the superficial cytoplasm moves to the upper pole of the
egg to form a disc where cleavage now takes place. In
eggs of reptiles and of birds the process is the same: cleavage
Is limited to a small area of the total egg. As this disc
becomes cleaved, the underlying yolk is converted into
active cytoplasm which by cleaving adds to the area of
the original disc.

Superficial cleavage was first clearly described by Weis-
mann in 1864. Since the appearance of this classic memoir
on the development of the insect egg, the clear superficial
cytoplasm of the egg, which encloses the egg-yolk and to
which during cleavage cell-boundaries are confined, has
been spoken of as the blastema. Following entrance of the
spermatozoon into the egg through a canal, the micropyle,
located at one pole of the ellipsoid egg, the egg- and sperm-
nuclei come together and form the cleavage nucleus located
below the egg surface. This nucleus divides mitotically sev-
eral times without cleavage of the egg. Later most of these
daughter nuclei become located in the blastema, a few
remaining in the central yolk-mass. With the arrival of
the nuclei in the blastema, cleavage planes appear in it,
the interior of the egg remaining uncleaved.

293

THE BIOLOGY OF THE CELL SURFACE

The foregoing general statement on the cleavage-process
in animal eggs,, though it makes no pretension to be exhaus-
tive, nevertheless covers the essential points on a process
which has been the subject of much admirable and pains-
taking investigation. In a book like this a comprehensive
treatment of cleavage would be out of place, because it
demands more space than we can give it. Moreover, a
certain advantage obtains here, as with other biological
processes, in setting forth a plain and simple statement of
salient features that stand as accepted and established facts.
Without further discussion we may note the following
points concerning the cleavage-process in animal eggs:

1. Cleavage may involve the whole or only a part of the
animal egg. Hence, the pattern varies depending upon
the area of the egg which undergoes cleavage; the pattern
at the termination of the cleavage-period resulting from
total cleavage, as in the egg of a snail, is markedly different
from that of its near relative, the squid, which undergoes
partial (discoidal) cleavage.

2. The size of the blastomeres contributes a distinguish-
ing feature to the cleavage pattern. Where in developing
insect eggs the cell-size has been investigated, it was found
that the blastomeres in the superficial cytoplasm show
equality in size. This is not true for the cleavage-cells in
the eggs of the ink-fish and in other eggs exhibiting discoidal
cleavage. In total cleavage the blastomeres may be mark-
edly unequal in size. Hence, not only the extent of
the egg undergoing cleavage, but also size-differences of
the blastomeres in the cleavage-area constitute a factor
which determines an egg's cleavage pattern.

3. In all forms of cleavage, except superficial, the initial
splitting up of the egg-substance takes place at that pole
from which the polar bodies have formed or will form. In
total cleavage the first and second cleavage planes cut
through this pole at or nearly at riglit angles to each other.

294

CLEAVAGE AND DIFFERENTIATION

In discoidal cleavage the cleaving disc is located at this
tgg pole. In superficial cleavage, the initial nuclear divi-
sions are confined to the region directly below the point
whence the polar bodies form, whilst the cleavage itself
involves the whole egg-surface.

Because hitherto some writers have preferred to assume
a simplicity for the uncleaved egg, thus disregarding its
previous history as a succession of differentiations, fruit-
less attempts have been made to interpret the cleavage-
patterns of the animal &gg^ diverse though they are, as the
mode for or even the cause of the setting-up of those dif-
ferences whose sum-total defines the differentiation made
manifest during cleavage. It is, however, more cor-
rect to see in the cleavage-pattern a display of the differ-
entiations present before cleavage set in. The separa-
tion of the eg^ as egg from other cells of the organism,
its position in the ovary, its relation to food-supply, its
intake and elaboration of food both in quantity and quality
— in short all those processes that long before fertiliza-
tion and cleavage endowed the eggs with those character-
istics to which many authors relate cleavage as total or
partial and as variants of each of these — these are the proc-
esses which condition the respective cleavage-patterns.
Thus, a cleavage-pattern is established by earlier occurring
differentiations; It does not express differentiation occurring
during cleavage. We therefore consider the cleavage-
pattern as only the frame-work within which we try to
find the answer to our question. By analysis of the events
which occur during cleavage, we may reach an agreement
upon a proposition as to the primary and basic factor which
underlies all forms of differentiation. For the cleavage
these chief events are six.

(i) The egg is split up into cells; (2) the embryonic axis
and the plane of bilateral symmetry are revealed; (3) the
cytoplasmic inclusions shift positions; (4) nuclear material

295

THE BIOLOGY OF THE CELL SURFACE

increases; (5) water Is redistributed; (6) the ectoplasm
increases.

Before I begin the discussion of these events, I must deal
with the loss by an egg of capacity to produce more than
one embryo — i.e., with the change of the egg from a pluri-
potent to a unipotent system. This loss of plurlpotency,
often spoken of as a chief problem in development, in my
judgment, is only a revelation that embryogenesis is a series
of progressive restriction.

Loss of pluripotency is revealed at that stage in cleavage
when the blastomeres having been experimentally separated
develop into defective embryos. In the eggs of snails,
for example, this loss can be demonstrated to have taken
place at first cleavage; blastomeres separated at this time
develop each to a swimming form, which is an incomplete
embryo made up of those structures which In the normal
embryo would have arisen from this blastomere. In eggs
of echinlds, on the other hand, loss of pluripotency occurs
later because only after the third cleavage do the blasto-
meres when separated develop into defective embryos;
blastomeres Isolated after first and second cleavage develop
into perfect though dwarf embryos, one-half and one-fourth,
respectively, the size of the normal. Briefly, we find that
animal eggs vary with respect to the time after fertilization
when they lose capacity for multiple embryo-production.

Cleavage has been classified as determinate and inde-
terminate. In eggs with determinate cleavage, the history
of each cell or blastomere in a late period of the cleavage-
process can be traced back to the two cell stage; that is,
some eggs as early as the two cell stage reveal an organiza-
tion which fixes the destiny of the blastomeres derived
from the products of the first cleavage. In so-called inde-
terminate cleavage, although for a time the blastomeres
show constant origin, they later arise in undetermined
fashion. This classification of the cleavage of eggs as

206

CLEAVAGE AND DIFFERENTIATION

determinate and indeterminate I omit from the discussion
for two reasons. First, since in an indeterminate egg, as
that of an echinid, the initial cleavages are determinate,
it can not be called strictly indeterminate. Second, since,
all monoembryonic eggs finally become unipotent, we can
not say that only determinate eggs are unipotent.

The evidence at hand shows clearly that in one respect
eggs are the same: I refer to the fact, never sufficiently
emphasized, that before fertilization all eggs, amenable to
the experiment of merogony, have capacity to produce
many embryos.^ If eggs in the fertilizable stage be broken
up into fragments and these be inseminated, each fragment
produces an embryo. The developmental capacity of such
egg-fragments is as great in the egg of Chaetopterus, in which
pluripotency is lost early during cleavage, as in that of
echinoderm eggs, in which pluripotency persists longer.
Some eggs normally give rise to many embryos, as that of
the Texas armadillo that always forms four, and those of
some insects which give rise each to hundreds. The fer-
tilizable egg thus is a system of multiplex embryonic
potency. This pluripotency however, except for normally
polyembryonic eggs, is never realized in the normal process
of development. Instead, the egg develops as a mono-
embryonic system which becomes successively restricted
within the domain of monoembryogenesis. It is in this
domain that our problem of differentiation during cleavage
lies.2

The primary event of the cleavage period is the act of
cleavage, a rhythmical phenomenon. This sundering
involves both nucleus and cytoplasm; for although in some
cases the nucleus divides without cytoplasmic division at
one time or another during cleavage, normally no egg ever

^ For literature on merogony see Deluge^ ^Sq8, i8gga and b.
- See also Just, ig^/b.

297

THE BIOLOGY OF THE CELL SURFACE

develops throughout this period without cytoplasmic
division. The furrowing of the cytoplasm, that is the act
of cleavage itself, has been thought of as the cause of
differentiation. This assumption however, is untenable,
as I shall now show.

By means of cleavage the cytoplasmic mass, the egg,
is successively sundered into smaller parts, the blastomeres.
This sundering, even if regarded as a means for the separa-
tion of constituents, does not bring about a new distribution
of them by which arise embryonic parts. True, the divi-
sion into smaller parts will facilitate distribution, but it
can not be its cause. It is not the mere division into cells
that makes the head, neck, arms of a human being or that
distinguishes the liver as an appendage of the gut from
which it is derived. Rather, concomitantly with the
act of cleavage changes take place by which the embryonic
areas subsequently arise.

From yet another consideration we conclude that cleav-
age as such is not differentiation. Even if we should
assume that the uncleaved egg were an undifferentiated
system, cleavage-partitions would only set off undifferen-
tiated areas and the differentiation which arises during
cleavage would be due to some other cause — as for instance,
chemical reactions induced in a previously homogeneous
mixture by the act of cleavage in setting up separate reac-
tion-chambers of minute capillary dimensions. But these
reactions and not the act of cleavage would then be the
cause for differentiation. Or were we to suggest that by
the establishment of cell-boundaries brought about by
cleavage, the increase of surface renders possible surface-
reactions characteristic of capillary spaces, we should still
not support the proposition that the mere act of cleavage in
sundering the protoplasmic mass is responsible for the pro-
gressive differentiation which can be followed throughout
the cleavage-process.

2g8

CLEAVAGE AND DIFFERENTIATION

Finally, consider the experimental evidence. This,
although not embracing all eggs, Indicates clearly that
fertilized eggs may reach a certain degree of differentiation
without cytoplasmic cleavage.

Differentiation without cleavage has been noted by sev-
eral observers to take place in eggs of annulated worms and
of ascldlans having been subjected to experimental treat-
ment. The most thorough-going description of this type
of development is that given for eggs of Chaetopterus having
been exposed to hypertonic sea-water.^ These eggs develop
into most abnormal swimming forms made up of either
uninucleated or multinucleated undivided cytoplasm due
either to complete failure of cytoplasmic cleavage or to the
disappearance of cleavages and fusion of the blastomeres.

As development progresses, the cytoplasmic Inclusions
take up new positions in almost the same way and at the
same time as in fertilized normally developing (untreated)
eggs. But the ectoplasm behaves in a quite different
manner. A portion of it moves to the animal pole, a
smaller amount rests at the opposite pole. Later the ecto-
plasm at the animal pole moves toward the vegetal pole
so that the endoplasm Is covered, thus simulating the over-
growth of the larger yolk-containing blastomeres by the
smaller ones in normal development.

Another characteristic feature in differentiation without
cleavage In the egg of Chaetopterus Is the behavior of the
nuclei in uninucleated eggs. In these eggs when the nucleus
reaches the size of the germinal vesicle of the normal egg
there appears a strong mutual attraction between It
and the ectoplasm: the ectoplasm Is drawn Into the egg
to form a mass that lies close to the nucleus or the chromatic
part of the nucleus Is drawn out to the ectoplasm. Eggs
which show ectoplasm drawn Into the interior do not

^ Lillie, F. R., igo6.

2Q9

THE BIOLOGY OF THE CELL SURFACE

develop as far as those in which the chromatic substance
of the nucleus is drawn into the ectoplasm.

We conclude therefore that although cleavage and dif-
ferentiation normally go hand in hand, the relation is not
one of cause and effect.

The second event enumerated above as occurring during
cleavage which has been held as a cause of differentiation, is
the appearance of the embryonic axis and of the plane of
bilateral symmetry, the so-called median plane.

For egg-cells the term, polarity, means that with reference
to an axis the egg in some way, as arrangement of pigment,
location of the nucleus, site of polar body formation, etc.,
shows structural polar arrangement. Now it is often held
that the polar axis of the egg bears some constant relation
to the axis or the median plane of the embryo and on this
assumption are elaborated theories of differentiation as
determined by the egg's polar axis, of a differential axial
gradient, and the like.

In order to clear this problem, we must first distinguish
between the polar axis in eggs of radially symmetrical ani-
mals and that in eggs of the bilaterally symmetrical with
respect to the axis of the embryo in the former eggs and to
the median plane in the latter. Although this distinction
is easy if only we appreciate certain simple geometrical
truths, nevertheless we must give it prominence here
inasmuch as so many writers have most unfortunately
used axis and plane interchangeably in their theories of
differentiation.

Polar and embryonic axes coincide in eggs whence radially
symmetrical embryos arise. A line drawn through the egg
from the site of polar body extrusion to the pole opposite
is also the gastrular and the embryonic axis. In the eggs
of these organisms — Porifera and Coelenterata — the gas-
trula forms in such wise that its axis is along traces of the
polar axis, no matter by which of the several modes of
gastrulation that obtain in these eggs the gastrula arises.

300

CLE A FACE AND DIFFERENTIATION

The gastrula may develop from a morula (a solid mass of
cells) or a blastula (a mass of cells that contains a cavity);
the gastrulation may be that of invagination, delamination
or epiboly; finally the gastrula is a two-layered radially
symmetrical structure enclosing a cavity whose polar axis,
now the embryonic, for the most part traverses empty space,
and at whose poles only are cells located which are part of
the two-layered covering. Components of an axial gra-
dient, if there be such, can become effective only in the
cell-layer.^

Eggs of most bilaterally symmetrical animals begin their
development as radially symmetrical structures and there-
fore show a polar axis. But at the moment after fertiliza-
tion when bilaterality appears in such an egg, we can no
longer speak of an axis. In a bilaterally symmetrical
organism — egg or adult — there exists no line common to
planes as in a radially symmetrical one. Here, accurately
speaking, we can use only the term, plane of symmetry.

Certain eggs out of which develop bilaterally symmetrical
animals — as, for example, those of Loligo, Hydrophilus,
Amia — reveal bilateral organization before fertilization.
In these, obviously, we can not speak of a polar axis. For
even if we know that at some time in their history the eggs
were radially symmetrical, the moment that bilaterality
appears in them there can be no axis with respect to which
the parts are symmetrically arranged.

But even if we allow the incorrect use of the term, axis,
in bilateral embryos, we find no constant relation between
this axis and that of the egg. A survey of embryogenesis

^ /. JV. Wilson and I were never able to repeat Child's observations
on the effect of KCN on eggs of Arbacia which he interpreted as
demonstrating an axial gradient. My own experiments {lQ28c)
on this egg, which gave results similar to Child's, strongly indicate
that these are due to changes in the ectoplasm. Child's ozvn work,
as that on protozoan forms, can be explained in terms of effects on
the superficial cytoplasm.

301

THE BIOLOGY OF THE CELL SURFACE

In eggs whence develop bilaterally symmetrical embryos
does not permit the conclusion that "the axis of the egg
shows a definite relation to that of the gastrula, of the later
embryo, and of the adult body"; and that "this relation,
broadly considered, appears to be constant throughout the
Bilateralia."^ In many radially symmetrical eggs that
subsequently show bilaterality it is true that a line drawn
from the site where the polar bodies lie to the opposite
pole is perpendicular to one drawn along the length of the
embryo. In others, — e.g., the Qg^ of Amphioxus"^ — the
intersection of these lines forms an acute angle. In eggs
that are bilaterally symmetrical before fertilization, the
medium plane of the embryo lies in the egg's plane of bilat-
eral symmetry.

A survey of embryogenesis in the entire animal kingdom
permits the conclusion that the embryo, with the possible
exception of the mammalian, arises from the egg-surface.
In the present state of our knowledge, the mammalian &gg
does not seem to fall in with our generalization, since its
surface-cells form trophoblast. In all other eggs embryonic
axis or median plane is normal not to the core of the egg
but to its surface where the embryo lies. Axis or plane
appears, due to new configurations In the protoplasmic
system;^ the embryo-axis or the plane of bilaterality is an
expression and not the cause of the dIfTerentiation which
unfolds Itself as cleavage progresses.

We turn now to another of the events occurring during
cleavage which were enumerated above, the shift of
the cytoplasmic inclusions, again seeking the cause of
dlflFerentiation.

' Wilson, 1924.
' Cerfontaine.

•' One such configuration is undoubtedly set up by the entrance of
the spermatozoon into the egg — see Just, 1912.

302

CLEAVAGE AND DIFFERENTIATION

The positions especially of the oil and the yolk in the
egg-plasma change after fertilization, as comparisons of the
unfertilized with the fertilized eggs of either Nereis or
Chaetopterus reveal. In the egg of Arhacia as of any other
echinid the shift in oil and yolk is not so well marked.
That is, in eggs with determinate cleavage there is a more
clear-cut change of the cytoplasmic inclusions to new posi-
tions than in indeterminately cleaving eggs. The progres-
sive differentiation from the single cell, the egg, to a complex
organism, the embryo, was once thought of as due to the
distribution of the visible materials in the cytoplasm:
embryo-formation was ascribed to the segregation of these
fat- and fat-containing materials which were therefore
denominated organ-forming substances. To-day, we can
appreciate the naivete of this theory, wondering how we
could have given it such serious consideration.

Eggs differ^ with respect to content of cytoplasmic inclu-
sions. This is especially true of yolk. Indeed, eggs are
sometimes classified on the basis of yolk-content. Once
we spoke of yolk-less (alecithal) eggs; now we know that
all eggs, even the human, contain yolk. Thus it is better to
classify eggs not as yolk-free and yolk-rich, but rather as
telolecithal (yolk in one part), centrolecithal (yolk in the
center) and homolecithal (yolk evenly distributed). \ oik
also differs physico-chemically in different eggs; and I have
some evidence which indicates that it differs in a given egg
before and after fertilization. What is true of yolk, the
food material for the embryonic organism, is true of mito-
chondria, pigment-granules, etc.: they vary as to amount,
distribution and physico-chemical make-up. Whilst all
of these cytoplasmic inclusions of which I here speak differ

^ The reader will have noted thai I ojten emphasize the differences
in animal eggs. In this my general purpose is to sift from the
differences that which is similar and equivalent and thus to order
and to define the biological problems.

THE BIOLOGY OF THE CELL SURFACE

from each other, they are, so far as I know, wholly or In part
composed of lipin (fat or fat-like substance). Though there
Is no reason against the postulate that the organ-forming
substances are composed of fats, nevertheless, I believe
that we are on unsafe ground in attempting to relate the
formation of the many various organs to fat-containing
compounds.

Sometimes the cytoplasmic Inclusions show diiferential
distribution to very definite blastomeres. Consider the
egg of a low form of chordate, for example, that of the tuni-
cate, Cynthia. One substance (yellow) maintains a defi-
nite distribution to given blastomeres. This is an egg
with determinate cleavage. From the blastomeres contain-
ing the yellow substance always in the normal Qg^ muscle
and mesenchyme develop. Apparently, therefore, here Is
a very clear case of an organ-forming substance definitely
localized In the Qgg before first cleavage. But experimental
findings do not permit such a conclusion. To these I shall
later turn.

Other eggs with determinate cleavage for the most part
do not exhibit prelocalizatlon so clearly as that of Cynthia^
though their cytoplasmic Inclusions shift from their original
positions before fertilization to new ones after fertilization
and at first cleavage; thus the yolk and oil come to lie in
cells which are destined to form the gut. This Is not evi-
dence that oil and yolk "determine" gut-formation. Also,
there are many other eggs that do not show any special or
constant disposition of the cytoplasmic inclusions.

On other grounds the theory that the visible cytoplas-
mic Inclusions are organ-forming substances Is untenable.

In the first place, I call attention to the egg of Strongylo-
centrotus lividns, a sea-urchin commonly found in the
Mediterranean, which may exhibit a beautiful superficial
band of orange pigment below the equator In the hemisphere
opposite the animal pole. Boverl used the pigment band

304

CLEAVAGE AND DIFFERENTIATION

of this egg as a means of orientation in experiments. But
the occurrence of this band is inconstant; not every speci-
men of Strongylocentrotus gives eggs containing it. There
is no virtue in colored particles as such suspended in the
cytoplasm for differentiation, though they may be useful
indicators in other respects.

Another cogent argument against the theory that the
visible cytoplasmic inclusions are organ-forming substances
lies in the fact that cleavage in many eggs may ensue for
several cell generations apart from the underlying yolk and
oil; this is true of all eggs with discoidal cleavage. In them
the cytoplasm is at first a transparent and apparently
homogeneous sheath and later a disc below the vitelline
membrane. Cleavage is confined to this inclusion-free
cytoplasm. When in later stages of development — after
the differentiation occurring during cleavage has taken
place — the embryo grows at the expense of the yolk-laden
region of the egg, the yolk is transformed into clear cyto-
plasm. In other words, in these as in all animal eggs,
yolk and oil play no direct part in differentiation.^

Let us recall another fact: many living eggs under the
microscope appear to be transparent. Rigorously to adhere
to the theory of visible organ-forming substances is to deny
organ-formation in transparent eggs. By ascribing a
primary role to the visible inclusions in the cytoplasm, we
have had the tendency to reckon the flood by its burden,
taking the traffic for the stream.

Finally, certain experimental results set up an unsur-
mountable obstacle to acceptance of the theory of organ-
forming substances as an explanation for differentiation.

Above I have pointed out that due to the ectoplasmic
changes caused by fertilization, eggs exhibit cytoplasmic

^ In trematode eggs exists the strongest separation between egg and
yolk, because the egg alone is a product of the ovary; the yolk is
produced by accessory glands and spun around the egg.

305

THE BIOLOGY OF THE CELL SURFACE

currents which move the inclusions. This movement is
most striking in eggs with determinate cleavage — especially
if the inclusions be colored and hence easy to identify.
They come to lie constantly in definite regions of the egg
at first cleavage. If these fixed positions of the inclusions
be responsible for differentiation, then altering them should
modify or hinder development.

By centrifugal force the normal positions of the cytoplas-
mic inclusions can be altered. The materials, which as
spherules and granules are suspended in the cytoplasm
normally without reference to their specific gravity, are
when the eggs are centrifuged moved to new positions and
stratified according to their respective specific gravity.
Thus oil, yolk and pigment form layers in the order named
with a zone of clear cytoplasm beneath the oil. Here our
interest focuses on two results obtained from the centri-
fuging of eggs.i

The first deals with the fact that centrifugal force in
altering the position of the inclusions does not alter the
direction of the cleavage planes. Hence, the new arrange-
ment of the inclusions induced by centrifugal force, which
shifts them to blastomeres in which in normal development
they would not come to lie, has no effect on the develop-
ment of the embryo. It should be emphasized that this
effect of centrifugal force is the same in eggs with deter-
minate and indeterminate cleavage. Investigators are
agreed that the altering of the position of the cytoplasmic
inclusions has no effect on embryo-formation: the eggs
develop normally although the inclusions come to lie in

^ Although centrifuging may be a violent method of treating eggs,
capable of causing their destruction, it can, if used with discretion,
leave them quite uninjured. One can soon learn the least amount
of centrifugal force necessary to stratify the cytoplasmic materials;
there is here no point in using a greater, as Conkhn did on the
ascidian egg.

306

CLEAVAGE AND DIFFERENTIATION

blastomeres other than those that normally hold them.
For the cleavage planes bear any relation to the axis of
stratification.

Even more significant is the second result, namely, that
fragments of clear inclusion-free cytoplasm taken from
centrifuged eggs, whether these eggs cleave determinately
or indeterminately, develop when fertilized as whole eggs.
Many unfertilized eggs — e.g., those of sea-urchins and those
of other species whose germinal vesicles have broken down
— can be strongly deformed by pressure, centrifugal force,
etc. This is due to the fact that the fluid protoplasm is
enclosed by a highly elastic membrane. Such eggs when
centrifuged are pulled out into strands, a fact known already
many years ago. With a greater degree of centrifuging
the strands break up into fragments. Some of these frag-
ments, with or without the egg nucleus, are composed of
the clear hyaline cytoplasm only. Such clear fragments
devoid of inclusions are capable of fertilization and develop-
ment; they cleave as whole eggs.-^

These two experimental findings alone render untenable
the theory that considers the visible inclusions as organ-
forming substances. The differentiation from the single
cell, the Qgg, to the complex multi-cellular embryo can not
be related to the distribution of visible materials suspended
in the cytoplasm. That these materials have functions,
no one would deny. Most of them, oil and yolk, are food
for the embryo or larva; the remainder are doubtless for
respiration, secretion, etc. They move with the cyto-
plasmic ebb and flow but are not themselves the tide of life.

The orderly progressive shifting of the visible multi-
colored spherules and granules in a living egg, especially
in one with determinate cleavage, is an entrancing spec-
tacle: in defiance to gravity, oil, yolk, mitchondria and

1 Lillie, F. R., igo6.

307

THE BIOLOGY OF THE CELL SURFACE

pigment granules, each at a different rate, shift to new posi-
tions, always the same in every undisturbed egg of a given
species and definite at its every cleavage. In the order
and amplitude of this shifting we recognize the power of
the so little known cytoplasmic currents. Since these in
turn are directed by ectoplasmic changes, as I have shown,
we may by study of the shifting of the inclusions come to
know more about the ectoplasm and its interaction with
the inner cytoplasm.

In this inner cytoplasm is situated that fixed and little
changing cell-component, the nucleus, to which has been
assigned the role of maintaining intact the inheritance of
the species. What is its role in differentiation?

Along with the successive acts of cleavage by which the
egg substance is divided up into blastomeres and as the
embryo-axis or plane of symmetry becomes recognizable,
the original single nucleus of either the fertilized or the
parthenogenetic egg undergoes division so that each blasto-
mere receives a nucleus. In discussing the probable role
of the nucleus as a causal factor in the differentiation during
cleavage, I shall now deal with the following points: the
quantity of nuclear material finally found at the close of
the cleavage-period, the origin of this material, and how
the nuclei, with special reference both to chromosome-
content and to the genes making up the chromosomes, may
be conceived as taking part in determining the development
of the egg.

Although with each successive nuclear division during
cleavage of the egg the nuclei progressively diminish in
size, no individual nucleus in any blastomere at the end of
cleavage ever vanishes. On the other hand, the total
quantity of nuclear substance, where this has been deter-
mined, is greater at the end than at the beginning of the
cleavage-period. The quantitative increase in nuclear
substance continues through the whole embryonic period

308

CLEAVAGE AND DIFFERENTIATION

and becomes therefore a definite characteristic not only
for cleavage but also for the whole course of develop-
ment. The body of a newly hatched chick contains
nuclear matter far in excess of that in the uncleaved
egg which gave rise to the animal. Indeed, as long as the
chick lives and is capable of producing germ-cells it elab-
orates nuclear substance. Moreover, every organism that
regenerates lost parts, every one whose tissues exhibit
pathological growths, as tumors, builds nuclear substance.
Hence, the building up of the chemical constituents of the
nucleus represents a basic property of protoplasm as a self-
reproducing system. In other words, as protein-synthesis
represents that fundamental chemical characteristic of
living matter that distinguishes it from non-living, so
nuclear synthesis (in part a protein-synthesis also) stands
as one of the primary chemical activities of the self-regula-
tion and the self-differentiation exhibited as special attributes
of cells capable of reproducing themselves. The course
of development is marked by syntheses. By these arise
secretions as those of the thyroid, the pancreas, etc., and
such compounds as haemoglobin, the iron-containing respir-
atory pigment of the blood, as found in the embryos of
animals which possess these substances. By synthesis also
nuclei arise.

The question now Is: out of what are the nuclei synthe-
sized .^ With the progressive increase in amount of nuclei
runs during the period of cleavage a decrease in that of the
cytoplasm without any change in the amount of the yolk
and oil. This fact, holding for all animal eggs, is especially
clearly shown in totally cleaving eggs. In the egg of Nereis,
for example, I have followed very exactly the composition
of the blastomeres throughout cleavage and can state that
in the blastula-stage the total amount of cytoplasm Is
less than that in the egg at first cleavage, whereas the oil
and yolk do not change in amount. The clearest evidence

309

THE BIOLOGY OF THE CELL SURFACE

that nuclei arise out of the cytoplasm is furnished by the
development of those clear hyaline pieces of protoplasm
already referred to, for in the cleavage-process of such frag-
ments also the nuclear matter increases and the cytoplasm
decreases. It has been shown that the nuclei in yolk-
free blastomeres are larger than those in yolk-rich blasto-
meres and that the size of the nuclei is proportional to the
hyaline content of a blastomere rather than to the total,
encompassing the inclusions.^ These facts prove that in
totally cleaving eggs during cleavage the nuclei are synthe-
sized from the cytoplasm. In partially cleaving eggs as well
the yolk takes no part in development during cleavage.
When, as happens in these eggs, at the end of cleavage new
cells are added to the embryonic area from the yolk region,
the yolk, as the evidence indicates, is transformed into cyto-
plasm and does not go directly into the nucleus.

The increased nuclear content at the end of cleavage
therefore can only mean that the nuclei are elaborated out
of nuclear stuffs or their precursors; it can not mean an
automatic activity of the nucleus capable of building nuclei
out of nothing. Nuclei are built up from the cytoplasm.
So too their constituents, the chromosomes.

In preparation for fertilization both egg and sperm-cell
go through a process by which the number of chromosomes
characteristic for the species is halved; at fertilization this
number is restored, half from the father, half from the
mother. This is the strongest evidence in favor of the
chromosomes as the means by which the off-spring inherits
qualities from both parents. The number of chromosomes
of the fertilized egg is the somatic or so-called diploid
number characteristic for the species. During development
this number remains constant. Every somatic cell of the
adult organism no matter how complex contains in its

^ Conklin, igi2.

310

CLE A FACE AND DIFFERENTIATION

nucleus the number of chromosomes equal to that of the
fertilized Qgg. The germ cells of the adult in their terminal
stages after the process of reduction again possess half the
somatic number. Thus the cycle from germ cell to germ
cell is complete.

During the cleavage stages, the chromosomes are halved
in quantity at each cell-division. Since, as we know, they
never reach the zero-point, they must increase in mass
during the period of cleavage. Says Morgan^: "During
development, especially during early cleavages, the amount
of chromatin steadily increases in amount, giving an expo-
nential curve resembling the first half of a curve of a mono-
catalytic reaction." What evidence we possess definitely
warrants the statement that the chromatin material
increases in amount during cleavage.

Suppose we consider the egg of a large fish, a carp or
salmon. Such an egg after fertilization has two groups of
chromosomes, one from the sperm-nucleus and one from
the egg-nucleus, which united give the total number of
chromosomes characteristic for this particular species of
carp or salmon. Eventually from this single fertilized egg
the adult fish develops, which is composed of millions and
millions of cells constituting all of the organs of the fish;
if it is a female, it produces thousands or even millions of
eggs, if it be a male, billions of spermatozoa. Eggs or
spermatozoa of an adult fish have chromosomes equal in
number, size, form and, presumably, in weight to those
found In the egg- or sperm-nucleus of the fertilized egg from
which this adult fish was developed.

If the chromosomes grow — and It is Inconceivable that
they do not — they grow at the expense of something; they
can not grow directly out of themselves without obtaining
the new materials for themselves. The synthesis of chro-

^ Morgan, ig2j.

311

THE BIOLOGY OF THE CELL SURFACE

matin demands the utilization of raw materials out of which
chromatin is constituted. Then the question arises: Is
chromatin synthesized directly from the elements of
which it is composed or from compounds? We know
that the synthesis, upon which directly or indirectly all
living stuff depends, comes from CO? and HOH which in
the presence of chlorophyl and sunlight make the simplest
sugars. From these by polymerization polysaccharides are
derived and, with N, proteins and fats are built up. In
other words, the protoplasmic synthesis of organic com-
pounds like proteins is never from elements directly but
from compounds in the cell. If such protoplasmic syn-
theses are from compounds, why not chromatin, itself a
conjugated protein.^ Chromatin grows because more of
it is made; and it is made from compounds available in the
cell. Even if we assume that it is made out of that material
which is already in the nucleus as non-chromatin, we must
conclude that this non-chromatin in being part of the
nucleus has come from the cytoplasm.

It is clear, first, that chromatin grows; second, that It
can not grow in some magical way but most probably
grows as other cell-stuff grows — i.e., according to principles
governing protoplasmic syntheses as far as we to-day know
these, that is, from preexisting and simpler compounds.
The fact that during the cleavage stages of most eggs the
cytoplasm decreases as the chromatin Increases further
Indicates that chromatin is synthesized directly out of the
cytoplasm. Oil and yolk as such play no part in this
process since they tend to be segregated and the cells con-
taining them are the least actively dividing and those free
of them most productive in the elaboration of new chro-
matin substance.

Thus we deal with the definite increase of a chemical
stuff, nuclein; a measured weight of this must mean a
definite weight of precursors that produce it. Briefly, in

312

CLEAVAGE AND DIFFERENTIATION

dealing here with this conversion of cytoplasm into nuclei
we are concerned with the formation of a known chemical
stuflf. If now we relate differentiation to this chemical
process, a process whereby a definite chemical stuff is
removed from the cytoplasm so that cytoplasmic reactions
are rendered possible, we may have close at hand an
explanation of differentiation as a series of chemical changes.
In this wise it may be possible to detect certain chemical
stuffs as potencies. Surely, the more we can substitute for
such terms as "potency" chemical reactants in chemical
reactions, the closer shall we come to the solution of the
problem of differentiation of development. In relating
differentiation in part to the synthesis of nuclear material,
we take the first step in this direction.

The progressive differentiation of the egg during cleav-
age according to this conception is brought about neither
by the pouring out of stuffs by the chromosomes into the
cytoplasm nor by segregation of embryonic materials as
postulated by those who uphold the theory of embryonic
segregation, but by a genetic restriction of potencies
through the removal of stuff from the cytoplasm to the
nuclei.

Consider a fertilized egg, ABCD, with determinate cleav-
age, which at first cleavage forms two blastomeres, AB and
CD; there must be differentiation, since the AB and the
CD blastomeres when separated give rise to partial larvae.
This would mean according to the theory of segregation
that AB is minus CD's material and CD is minus AB's.
AB thus would be a cell in which the prospective ^5-poten-
cies are present; the same would be true in the CD cell for
the CZ)-potencies. After the second cleavage, A-, B-, C-
and Z)-potencies would be present in blastomeres A, B, C
and D respectively. Similar segregation would happen at
each succeeding cleavage. For eggs with indeterminate
cleavage the presence of material — and conversely, the loss

3^3

THE BIOLOGY OF THE CELL SURFACE

of other material — would take place at that stage in the
cleavage-process when the blastomeres on separation lose
the potency for development into complete though dwarf
larvae. But is an egg-cell (in this case with determinate
cleavage) JBCD = AB + CD, as we would express it
according to the theory of segregation ? Or can we say that
AB = AB + i-CD); CD = CD + {-AB) ? Can we say
that ABC Dh really AB + {-CD) + CD + {-AB) and do
we gain thereby ?

Restriction is, as I shall show, preferable to segregation
for it more nearly expresses the process. Restriction
implies a loss, in this case of developmental potencies,
without necessarily a rearrangement of materials. Segre-
gation connotes new arrangement or sorting out of mate-
rials without any implication of change in them. If as
cleavage progresses cytoplasmic materials for embryo-
formation are merely shifted to new positions, then reversal
of the cleavage process should be possible. Stages of dif-
ferentiation then would be repetitive. I do not wish to
indulge in hair-splitting definitions; nevertheless I must say
that I for my part can not see how the materials of an egg by
mere acts of separation can account for differentiation.
Against the assumption of a mere transposed order of
materials stands the fact that as development proceeds the
individual blastomeres lose potency.

At first cleavage blastomere AB becomes such because
it loses CD-material, and blastomere CD becomes such
because it loses .^5-material. A separation into AB-
and CZ)-blastomeres means that AB somehow removes
all CZ)-material and CD all .-/^-material. So does gene-
tic restriction begin and so it continues. This takes
place through the absorption of CD-material by the
nucleus of the ^^^-blastomere-to-be and of the ^5-material
by the nucleus of the CD-blastomere-to-be. The subtrac-

314

CLEAVAGE AND DIFFERENTIATION

tion of the cytoplasmic materials by the nucleus takes place
with each mitotic cycle.

We know, as has been pointed out, that as cleavage pro-
gresses the nuclei increase in number and en fnasse in vol-
ume. That is, they grow. We know also that in this same
period the total volume of cytoplasm decreases and that the
growth of the nuclei is at the expense of the cytoplasm.
Genetic restriction then depends upon the removal by the
nucleus of certain materials from the cytoplasm, leaving
others free. The free materials determine the character of
the cell — as ectoderm, mesoderm or endoderm and later as
any one of the organs arising therefrom.

Something leaves the cytoplasm with each cell-division;
then why not the materials of the cytoplasm which are no7i-
specific for the given blastomere .? Certainly, the mecha-
nism for the removal of material is present in this growth
of the amount of nuclear substance as development pro-
gresses. With each cleavage each nucleus fixes all material
other than that which makes the blastomere what it is,
AB or CD; A or B, C or Z), etc., to the end of cleavage.
Then the nucleus of cell AB is different from that of the
CD cell since the ^5-nucleus contains bound CD-material
and vice versa for the CD-nucleus.

Then the potencies for embryo-formation are all present
in the uncleaved tgg\ cleavage serves to remove these and
this removal is fast or slow, early or late depending upon
the species of egg. The question arises: When does the
cytoplasm of the egg gain its potencies for differentiation
during cleavage t

Pluripotency becomes demonstrable through merogony,
as was pointed out, with the onset of the fertilizable condi-
tion inasmuch as now fragments of the egg are when fer-
tilized each capable of development. From this we might
reason that the potencies that were stored up in the nuclei

315

THE BIOLOGY OF THE CELL SURFACE

during the previous development of an ^gg are restored to
the cytoplasm at this time, that is, to the cytoplasm of
the new egg. However, the demonstration that pluri-
potency is present gives no evidence of the time when it
arose. Merogony may be only an indicator of an earlier
established pluripotency. Having no other criterion than
fertilizability for the demonstration of pluripotency, we
can not exactly define the moment when the egg becomes
pluripotent.

An attractive hypothesis is that pluripotency arises with
breakdown of the germinal vesicle when there escapes into
the egg-cytoplasm residual nuclear stuff greatly in excess
of what remains to form the mitotic complex of the first
maturation-division. The potencies might be regarded as
identical to, or associated with, this extra-nuclear material.
According to this suggestion, the rise of pluripotency would
be separated from the fertilizable period, for the fertiliz-
ability does not depend upon a particular stage in matura-
tion. But since many species of eggs are fertilizable whilst
their germinal vesicles are intact, not a single one of these
should be pluripotent if we are to relate the rise of pluri-
potency to substances escaping into the cytoplasm after
breakdown of the germinal vesicle. Where, however, frag-
ments taken' from such eggs develop when fertilized, it
must be ascertained that the fragments are devoid of stuff
having escaped from the germinal vesicle. The lack of
data on merogony in such eggs does not warrant a decisive
conclusion in these questions.

There remains another possibility. After the germ-cells
have become differentiated from somatic cells through the
loss to their nuclei of all potencies, with only the potencies
for germ-cells left free in their cytoplasm, they become
isolated from the soma. This isolation brings about the
escape of all potencies that were up to that time bound in
the nuclei, into the cytoplasm. Thus the eggs would

316

CLEAVAGE AND DIFFERENTIATION

become pluripotent. The condition of the spermatozoon
is discussed later.

Any theory offered to account for the differentiation that
takes place during cleavage should be consistent for both
the cleavage-period and the succeeding periods of develop-
ment during which differentiation occurs, since in essen-
tials, every period of differentiation is alike.

Certain phenomena occurring in the organism as egg, as
cleaving mass, as embryo and as adult are based on some
differentiation. A theory of differentiation during cleav-
age should hold for these phenomena. They are: (i)
polyembryony and experimental twinning; (2) merogony;
(3) the development of diploid fragments; (4) the develop-
ment of isolated blastomeres; (5) haploid parthenogenesis;
(6) experimental and natural polyploidy; (7) asexual repro-
duction by budding and fragmentation, and alteration of
generations; (8) regeneration of lost parts; and (9) the origin
of tumors. I shall now endeavor to show that these phe-
nomena are better explained on a theory of differentia-
tion as genetic restriction than on one that postulates
segregation.^

I. The capacity of some eggs, as those of certain insects,
normally to produce many embryos from one egg as well as
the possibility of producing twins experimentally from
normally mono-embryonic eggs including those of the
determinate type, as the eggs of Nereis, Chaetopterus,
Tubifex, etc., shows that eggs have more latent potency
than that required for producing one animal. A theory
postulating a restriction of potencies seems to meet these
facts better than one suggesting that materials for the
embryo are segregated.

1 Cf. Perez i^igiz) who lists much the same experimental evidence
against the mosaic-theory (aji earlier form of the segregation-theory)
of development, which he justly considers a modern version of the
preforynation doctrine.

THE BIOLOGY OF THE CELL SURFACE

2. This same conclusion holds for the data on merogony.
It may be regarded as proved that all eggs which can be
fragmented before fertilization have the capacity for the
production of many embryos. Merogonic development
Indicates strongly, especially in determinate eggs, that
dliTerentlation ensues as a restrictive process — restricting
potency for multiple embryo-formation to one. The theory
of segregation In these cases would imply telescoping of
several embryos.

3. Some fragments of eggs contain the egg-nucleus and
hence when fertilized develop with two nuclei constituting
a diploid nucleus. In such fragments restriction obtains
as in whole eggs, except that the chromosomes remove less
cytoplasmic stuff since less is available. From the point
of view of embryonic segregation, we would have to assume
that since perfect though dwarfed embryos result from the
fragments, the segregates in whole eggs are pluralistic.
From this it should follow that all eggs when separated Into
blastomeres should develop into entire organisms or Into
embryonic regions, each containing parts reduplicated.

4. The development of blastomeres Isolated during
cleavage Into perfect though dwarf embryos stands as
strong evidence that restriction and not segregation under-
lies differentiation. For how from already segregated
areas could perfect embryos arise .^ Or, if it be postulated
that the areas are not yet segregated at the stage of Isola-
tion, why do the blastomeres in an Intact egg not develop
each into an embryo.^ On the basis of our theory of
restriction, potencies bound in the cytoplasm In the Intact
Qgg become free in the Isolated blastomeres because of the
new conditions set up by Isolation.

5. If we put forward the theory of segregation to account
for the diiTerentlation occurring In a parthenogenetic egg
containing a nucleus with half the number of chromosomes
found In the eggs of this species when they are fertilized,

3iS

CLEAVAGE AND DIFFERENTIATION

we encounter the difficulty of explaining how under the
domination of this "half" nucleus the cytoplasm is ordered
into regions whence the organs arise. The theory of restric-
tion by means of the nucleus removing potencies from the
cytoplasm does not encounter this difficulty for accord-
ing to it the nuclei extruded as polar bodies relieve the
cytoplasm of potencies.

6. Similar reasoning applies to eggs developing with
polyploid nuclei — i.e., with more chromosomes than those
contained in the united egg- and sperm-nuclei. In the egg
of Nereis, for example, fertilized after treatment with
ultra-violet light, ^ the polyploid nucleus, made up of the
three nuclei from the suppressed polar bodies plus the egg-
and the sperm-nuclei, remove cytoplasmic potencies pre-
cisely as these are removed in normal fertilization by polar
bodies and egg- and sperm-nuclei. But from the point of
view of segregation, conditioned by nuclear material or
power escaping into the cytoplasm, development should
not take place because of the over-powering influence of
the super-abundant nuclear matter.

7. Certain eggs develop into animals which have the
capacity for asexual reproduction by the formation of buds
or by the process of breaking up into two or more fragments.
Both the buds and the fragments develop into complete
organisms similar to the form whence they arose. If sexual
and asexual phases regularly succeed each other, they con-
stitute a life-history said to reveal alternation of generations.

Asexual reproduction can be explained by assuming that
potencies in the egg, present in excess of those for the forma-
tion of a single individual, remain free. The reduplication
of the animal by bud or fragmentation though it occurs
late in the life-history is thus comparable to poly-embryony.
In those cases of alternation of generations, where egg- and

1 Just, 1933 c.

THE BIOLOGY OF THE CELL SURFACE

sperm-producing areas as motile swimming organisms
arise, restriction has taken place by removal of these free
potencies.

8. Regeneration of lost parts is encountered in all ani-
mals; In some, as the flatworms, it reaches the level of
asexual reproduction; in others, capacity for regeneration
is limited. In some forms regeneration is said to be due
to the presence of formative cells, cells in which it is assumed
that differentiation has been arrested; in others regenera-
tion of structures occurs from tissues that normally never
give rise to them.^ In either case the capacity for regenera-
tion may be thought of as a reorganization under the influ-
ence of the liberation of potencies, previously bound by
the chromosomes, into the cytoplasm induced by the
changed condition of the cells as a result of the injury or
loss.

9. Abnormal growths or tumors can be explained in the
same way. Some change In the environment of the cells
stimulates the throwing out by the nuclei of potencies into
the cytoplasm where a new type of development is set up.
A tumor-cell is one which has escaped the domination exer-
cised by contiguous cells. It becomes physiologically
isolated.

In view of all of these facts that support my theory of
differentiation as genetic restriction, I state this again:
As development progresses, the egg-potencies are restricted
through their withdrawal from the cytoplasm by the
chromosomes with each successive cell-division. Thus the
cytoplasm forms functional areas. At some time in the
history of the egg's development the potencies, having
been previously taken out and stored by the chromosomes
during cleavage and succeeding stages of differentiation,
escape into the cytoplasm. The cytoplasm of the fertilized
or parthenogenetlcally developing t^g restores them again

' Reed, 1904.

■ 320

CLEAVAGE AND DIFFERENTIATION

to the chromosomes. This theory of differentiation as a
genetic restriction is consonant with the nine phenomena
enumerated above.

This conception of the role of the chromosomes in the
process of differentiation stands in contrast to the attempt
of genetics to explain differentiation.

The last thirty years have seen as an outgrowth of the
well-known Mendelian laws that flourishing branch of
biology, genetics, nourished by the knowledge of chromo-
some behavior, develop almost to the proportions of a
separate science^at least It has a very rich vocabulary of
Its own. Mendel's laws are not causal, but statistical con-
clusions concerning the regularity with which offspring
show the characters of parents. The chromosome theory
of Mendelian heredity attempts a causal explanation.

According to the current conception each chromosome is
a string of units, the genes, which are the carriers of hered-
ity. The gene theory has been developed by work done on
the small fruit-fly, Z)rojo^Az7a, raised in the laboratory under
fairly constant conditions of temperature, of food, of per-
iodic subjugation to heavy doses of ether, etc. Thus
divorced from the rigors of nature these animals imprisoned
in large numbers In small glass containers have undergone
changes which endure for so many generations that they
are without doubt constant. Animals exhibiting these
enduring changes are mutants. For each mutation genetics
has assigned a place on one or another chromosome; some-
times determiners of the same mutation are located on
several chromosomes. Among geneticists the gene-theory
Is widely accepted. Among biologists, on the other hand,
even of those who do not accept the gene theory of the
geneticists, the majority agree that the chromosomes are
the bearers of heredity.

According to the geneticists the chromosomes In every
cell of the most complex organism are identical with those
of the fertilized egg. That Is, as they express It, the chro-

321

THE BIOLOGY OF THE CELL SURFACE

mosomes maintain their individuality. The hnear arrange-
ment of the genes in each chromosome^ is the same for the
given chromosome whether it is in a fertihzed egg, in a kid-
ney-cell, a muscle-cell or any other cell of the adult organ-
ism. Now at each cell-division though the chromosomes
are split they must somehow maintain their identity or
individuality; this maintenance is the prevailing doctrine
among geneticists.

Speaking of the individuality or genetic continuity of
the chromosomes, Wilson says":

In any general account of the history and genetic relations
of the chromosomes in the life-cycle, we inevitably find our-
selves speaking of them as if their identity were really lost
when they disappear from view in the resting or vegetative
nucleus. The vast literature of the subject is everywhere
colored by the implication that chromosomes, or something
which they bear, have a persistent individuality that is
carried over unchanged from generation to generation. This
view has met with some determined opposition; but with tlie
advance of exact studies on the chromosomes escpticism
has gradually yielded to the conviction that the chromo-
somes must, to say the least, be treated as if they were per-
sistent individuals that do not wholly lose their identity at
any period in the life of the cell but grow, divide and hand
on their specific type of organization to their descendants.
This does not mean that chromosomes are to be thought of
as fixed and unchangeable bodies. Beyond a doubt they
undergo complex processes of growth, structural transfor-
mation and reduction, in some cases so great that no more
than a small fraction of the substance of the mother-
chromosomes at its maximum development is passed on to
the daughter-chromosomes. Whether we can rightly speak
of a persistent "individuality" of the chromosomes is a
question of terminology. What the facts do not permit us
to doubt is that chromosomes conform to the principle of
genetic continuity; that every chromosome which issues
from a nucleus has some kind of direct genetic connection

^ Delage, iSg^, p. 731, very clearly expressed the idea of a linear
arrangement in the chromosomes.
2 Wilson, 1925.

322

CLEAVAGE AND DIFFERENTIATION

with a corresponding chromosome that has previously
entered that nucleus.

What here is individuality? If I am able to keep alive
all the descendants which arise from a single slipper ani-
malcule by cell-divisions at the rate of three divisions a day,
in seven days I shall have 2,097,152 organisms, each of
which has 1/2,097,152 part of the original Paramoecium.
Only this fraction, then, of individuality is maintained.
And this is a much simpler case than that of individuality
of each member in a chromosome-complex from fertilized
egg to adult of a complex organism. I think that the
question of the persistent individuality of the chromosomes
is more than a question of terminology.

If we turn from the chromosomes to the genes themselves
that compose each chromosome, we find the same difficulty.
It has been suggested that the gene is a molecule. The
two daughter-cells arising from first cleavage would then
possess chromosomes made up of half molecules; in the four-
cell stage there would be quarter molecules; and in each
succeeding division there would be a corresponding reduc-
tion of the original gene-molecules found in each chromo-
some of the fertilized egg. But in chemistry we know of no
fractional molecules. Then, let us start with the adult
organism. On the assumption that each gene in each
chromosome of every cell of the whale, for example, is a
molecule, it is necessary to assume that each gene of the
fertilized egg is a complex of molecules whose number
equals that of the sum total of all the descendants from
this gene which are In each and every cell of the adult. It
would be necessary further to assume that the gene-mole-
cules vary in size in stages of development from egg to
adult. The gene therefore can not be a molecule according
to physico-chemical usage. The proponents of the gene
theory should be the last to postulate any non-physico-
chemical molecule.

323

THE BIOLOGY OF THE CELL SURFACE

Thus the term, Individuality of the chromosomes or of
their constituent genes, Is of restricted meaning. I can
not see how a given chromosome or a gene in the fertilized
egg should be the same individual in every cell of the body
of an adult male including every one of the billions and
billions of spermatozoa that such an animal, like the trout,
for example, sheds during its life-time. With regard to the
"individuality" of the genes or the chromosomes genetics
has offered us nothing except a term, which, as we have
seen. Is not even clear. From this point of view the gene
theory certainly can not give an explanation of the develop-
ment of an egg into an adult organism.

Let me hasten to state that in company with the majority
of biologists I consider the weight of evidence sufficient
for the assumption that the chromosomes have to do with
heredity. As I have pointed out already, I see in the
nucleus that component of the cell which tends to maintain
the specificity of the cells by Its change-resisting character.
Then the chromosomes maintain Individuality or identity.
This maintenance however, as we have seen, can only come
through growth at the expense of the cytoplasm. This fact
allows us to see In a new light this maintenance : It is brought
about by growth, the up-take of material that carries or
assumes the characteristics of that which Is already present
in the chromosome. Further, this maintenance of Identity
must stand In some relation to the source that furnishes
the material, namely, the cytoplasm.

Counter to this fact of the growth of the chromosomes
at the expense of the cytoplasm runs the postulate of
those geneticists who seek to explain differentiation
by the gene-theory of heredity, namely, that the genes
are factors which order the developmental processes
by giving up substance to the cytoplasm. But even
If the upholders of the gene-theory of heredity accept
the fact that the chromosomes grow at the expense of

324

CLEAVAGE AND DIFFERENTIATION

the cytoplasm, their conception of the action of the genes
as unalterably fixed entities can not explain differentiation
of development. For how could the genes be responsible
for differentiation, if they are the same in every cell?^
Unless the geneticists assume that their genes are omnipo-
tent, we can not understand how the problem of differentia-
tion can be solved by the gene-theory of heredity.-

The gene-theory of heredity is an ultra-mechanical rig-
orously bound concept. This mechanistic rigidity renders
it inadmissible as an explanation of the process of develop-
ment, a process marked by the egg's inherent capacity and
by its mobile responses to external influences. On the other
hand, the gene-theory, postulating factors in chromosomes,
fits in well with the stable and change-resisting character
of chromosomes. But in thus accentuating the relative
non-mutability of the chromosomes as carriers of heredity,
the most stable possession of the organism, the gene-theory
sets off the process of heredity from differentiation of
development; hence, as we have seen, it offers us no help
in our attempt to explain differentiation.

Now the gene-theory as formulated may not be the only
way of interpreting the vast amount of reliable data accu-
mulated by the numerous geneticists the world over.
These many data, let us say at once, we accept. If, how-
ever, we can offer another interpretation of them, can for
example substitute for the factorial concept in the gene-
theory another concept which is less rigid, more consonant
with our knowledge of physiological processes and one

^ Cf. Lillie, F. R., 192/; Conklin, 1924^ et. al.

- Untutored savage man made his god as big as possible because
his god could do everything. It remained for the geneticists to
make one of molecular size, the gene. Here obviously infinite
minuteness means infinite capacity. According to one geneticist,
Demerec, the gene has almost magic power. This is physico-
chemical biology with a vengeance!

325

I

THE BIOLOGY OF THE CELL SURFACE

substituting a means of protoplasmic reactions for a non-
physical concept of molecules, we should be able to do what
so far has been attempted in vain in biology, namely, to
envisage differentiation and heredity as merely two expres-
sions of development.

It has been said that differentiation of development
and genetics must remain forever separated.^ And when
and wherever geneticists have attempted a union of the
two, they have failed.- But since heredity is expressed
during the process of development and indeed is a kind of
differentiation, biology must attempt to find ground com-
mon to both. Up to now this common ground has not been
located. I here propose a suggestion concerning the role
of the gene in heredity and in differentiation.

The moment that we postulate that differentiation dur-
ing cleavage turns upon the taking up of potencies leaving
free in the cytoplasm those that give the blastomeres their
characters as such, we see that an organ becomes such
because in the cytoplasm of its cells are those potencies free
which make it a special organ. Every cell in an organism
becomes what it is because its cytoplasm has free its par-
ticular potencies whilst its nucleus binds all others. These
latter would, if left unbound in the cytoplasm, act as
obstacles to the display of the special potencies. Thus,
the removal of potencies at the same time means removal
of obstacles to cytoplasmic reactions.

My fundamental thesis is that all the differences, i.e.,
differentiation, that appear during development, rest upon
cytoplasmic reactions. These are made possible through
removal of obstacles by nuclei, hence, by chromosomes and
genes. The nuclei by removal of substances release the
activity of the cytoplasm in one direction. The genes also

^ Lillie, F. R., 192/, p. j6/.
- Morgafi; Golds chmidt.

326

CLEAVAGE AND DIFFERENTIATION

act by removing impediments to cytoplasmic reactions.
Let us examine this proposition more closely,

I begin with the assumption that with the onset of the
fertilizable condition of the egg there are in the cytoplasm
all the potencies — chemical reactants — whence arise the
future organs. Indeed, as experiments on fragments of
eggs obtained during this period show, such cytoplasm has
capacity to produce several embryos. The chromosomes
in such an t^g now begin to remove from the cytoplasm
potencies so that others remain free to initiate reactions
responsible for differentiation. Thus progressively restric-
tion ensues.

Contrast this condition with that in the history of the
spermatozoon. With the two rapidly ensuing maturation-
divisions the cytoplasm of the spermatocyte is divided
among four spermatids giving rise to mature spermatozoa
with less cytoplasm than the mature Qgg possesses. Egg
and spermatozoon thus are markedly different with respect
to the amount of their cytoplasm at the moment of fertiliza-
tion. We may recall a further difference mentioned earlier
in this book. Whilst eggs can develop in some cases with-
out the spermatozoon, no spermatozoon has ever been
found capable of development without the cytoplasm or
at least a fragment of the cytoplasm of an egg. In
capacity for development, thus, the egg is superior to the
spermatozoon. This superiority is related to the egg's
cytoplasm since this can develop either with egg- or sperm-
nucleus. Now, according to our conception, the egg-
nucleus gives up its potencies to its cytoplasm at some
moment before the fertilizable stage. The sperm-nucleus,
being likewise capable of taking up potencies again in the
development of the Q%g which it fertilizes, must have lost
its own potencies also before it takes up those residing in
the egg-cytoplasm. When this loss occurs we do not know.
A probable assumption is that as early as the moment when

327

THE BIOLOGY OF THE CELL SURFACE

the sperm-cell as such is differentiated from all other cells
and retains in its cytoplasm only the potency for sperm-
ness, this conditions the nullification in some way of those
potencies borne by the chromosomes. Or they may be
lost during the process of the ripening of the sperm-cell,
when, as is known, concomitant with morphological changes
amino-acids are lost. A less likely assumption is that
potencies of the sperm-nucleus are lost and nullified at
fertilization.

In any case, both egg- and sperm-chromosomes are pre-
pared for removing stuffs from the cytoplasm of the egg.
I bring an example: If a spermatozoon from a Drosophila
possessing pure red-eye fertilizes an egg of an also purely
red-eyed female, in the cells which show redness, the egg-
and sperm-genes remove from the cytoplasm the hindrance
to the reaction leading to redness. The "factor," redness,
is resident in the cytoplasm and expresses itself because the
genes remove stuff opposing this reaction. If on the other
hand the sperm-chromatin is descended from a white-eyed
animal and the egg-chromatin from a red-eyed one, the
sperm-chromatin, when in those cells which give the color to
the eve removes stuff which releases whiteness-reaction, and
the egg-chromatin stuff which releases redness-reaction,
with the result that the cytoplasmic reaction is now no longer
r -f r = i? as in the first case, but r -\r w — R{zv) where
R{w) gives either dominant red-eye color or an interme-
diate color between red and white. Thus this conception,
whilst consonant with the experimental findings of Men-
delian genetics, differs from the theory of the gene for it
places the determination of characters in the cytoplasmic
reactions. The active "factors" for Mendelian characters
do not reside in the genes; rather, the genes by extracting
definite materials from the cytoplasm render possible the
reaction of the cytoplasm-located hereditary factors. Onl)'
so far as they take out substance do the genes determine
heredity.

328

CLEAVAGE AND DIFFERENTIATION

Thus finally every cell in the most complex organism
has in its nucleus all the potencies bound except that one
which is free in the cytoplasm and which makes the cell
specific. The egg-cell, for example, the moment it becomes
differentiated from other cells of the body is such because
it has in its nucleus all bound potencies except that which
makes it an egg-cell. And it is this potency in the cyto-
plasm which determines the future growth and differentia-
tion of the egg to that moment, when these bound potencies
are thrown again into the cytoplasm. The sperm-cell is
such because in its nucleus are all the potencies bound except
that which determines the sperm-character of the cell.
This potency determines the further differentiation of the
spermatozoon and most probably is responsible for the
nullification of the potencies in the sperm-nucleus.

This conception of mine concerning the action of the gene
offers a far better interpretation for several phenomena
than the hitherto proffered conceptions. Thus, for exam-
ple, can we state that sex-differentiation is resident in the
cytoplasm. For the majority of animals, the taking out
from the cytoplasm of more sexual potencies by the chro-
mosomes results in femaleness. So in those experimental
conditions, where more than one set of chromosomes is
present, the organism becomes female. The taking out of
fewer sex-potencies by the chromosomes gives rise to a
male. Thus an egg with half the somatic number of chro-
mosomes, the so-called haploid number, is always, so far
as we know, a male. Finally, hermaphroditism means
equal abstraction of sex-potencies by the chromosomes,
leaving in the cytoplasm both male- and female-factors.

These considerations warrant the assumption that the
genes play a part in heredity but are not the factors of
heredity in the sense of the geneticist; the genes act through
their binding of potencies in such wise as to free the action
of the cytoplasm-located factors of heredity — that is,

329

THE BIOLOGY OF THE CELL SURFACE

chemical reactions in the cytoplasm underlie both differen-
tiation and heredity.

In thus relating heredity and the action of the genes to
reactions in the cytoplasm, we come to a more physio-
logical conception for heredity than the theory of the gene
and one which instead of running counter to the facts of
differentiation of development is consonant with them.
From fertilized egg to the least active cell in the adult
organism, the visible manifestations of life are outside the
nucleus. It is the cytoplasm therefore that we consider to
be the field of activities be they concerned with heredity
or differentiation.

With reactions in the cytoplasm deals the fifth event dur-
ing cleavage enumerated above with which our discussion
of the cause of differentiation is concerned: the redistribu-
tion of water during cleavage.

From fertilization through cleavage eggs of marine
animals certainly and all others probably show a visible
altered distribution of water. This is best observed in
experimentally treated eggs, but it can also be seen in
normal development, as I stated In the chapter on water.
After the initial ectoplasmic dehydration which occurs when
eggs are fertilized or subjected to experimental means that
Initiate development, the egg establishes a new equilibrium
with the sea-water. On this level, water, as discrete drops,
moves from place to place within the egg or from egg to
external medium. The formation of water-drops Is a
rhythmical phenomenon which accompanies each division-
cycle of the cleavage-period.

The addition or removal of water In a reversible reaction
determines hydrolysis or synthesis, respectively. Thus,
water plays a role as a component In chemical reactions;
In addition, it is both a solvent for other components and
a part of the cytoplasmic structure, the chamber of the
reactions. Then the demonstration that water is inter-

330

CLEAVAGE AND DIFFERENTIATION

mittently present as discrete drops in viable cells having
had any one of several kinds of treatment — whose action
is not deleterious since one can prove that the cell after
treatment returns completely to the untreated state — has
far-reaching significance: it becomes an index to the nature
and rate of reactions. In both normal egg and blastomeres,
the rhythmical appearance of water-drops allows us to
correlate their occurrence with the rhythm in a cleavage.
DiflFerences in the formation of the water-drops in diflferent
blastomeres we may regard as an index to chemical changes
underlying the progressive restriction that runs with
cleavage.

With each succeeding cleavage the egg is further sub-
divided into chambers of capillary dimensions. At the
end of the cleavage-period the total surface-area of the
blastomeres is much greater than that of the uncleaved egg.
Thus, during cleavage the ectoplasm increases in amount.
This increase of ectoplasm constitutes the last of the six
enumerated changes that occur during cleavage. The
cleavage-period may be defined as one in which the cyto-
plasm diminishes through its conversion on the one side
into new nuclear substance and on the other into new cell-
partitions. Having examined the role of nuclear growth
in differentiation, I seek now to evaluate the evidence
from which I derive a postulate concerning the role of that
part of the ground-substance located at the cell-surface,
the ectoplasm, in differentiation.

In the first line stands the fact that the ectoplasm
increases in amount with the progressive sundering of the
egg into blastomeres, regardless of the type of cleavage by
which this result is reached. Whilst no animal egg is a
perfect sphere and never by cleavage gives rise to blasto-
meres which are perfect spheres, nevertheless the ratio of
total surface to total mass which obtains for a sphere when
sub-divided into spheres is closely approximated by the Qgg

331

THE BIOLOGY OF THE CELL SURFACE

during cleavage; the mass of protoplasm does not increase
but the total surface-area does. Increased surface-area
is best seen in an egg with total cleavage which develops
into a blastula. It is clearly shown by the egg of the star-
fish for during early cleavage the blastomeres are at first
separated from each other. In totally cleaving eggs which
give rise to morulae, the increased surface area appears in
the covering blastomeres; they are more flattened and the
ratio of surface to mass thus is greater. For eggs with
discoidal cleavage, it has been established that new cells
are added to the embryonic disc from the underlying yolk
where nuclei undergo mitotic division without being par-
titioned off into cells; only when around those nuclei lying
next to the disc cell-boundaries form, are new cells added
to the growing embryonic disc. Cleavage-planes arise in
superficially cleaving eggs only after the nuclei reach the
ectoplasm. The difference between totally and partially
cleaving eggs lies in the fact that the initial cleavages in
the latter are confined to an ectoplasmic region which is
utilized in the formation of cell-walls before additional
ectoplasm forms; in the former, cleavage and increase in
ectoplasm run more synchronously.

Since the ectoplasm is a part of the living system and
since during cleavage no living substance is added to the
egg from the outside world, the source of new ectoplasm is
from the egg-substance itself: ground-substance alone
constitutes this source.

But the interpretation of the role of the ectoplasm in
differentiation does not rest only upon its amount and its
source; in some of the events named as occurring during
the cleavage-period, the ectoplasm plays a part.

In the first place, consider the fact that a fertilized egg,
which possesses pluripotency, develops normally only into
one embryo. This means that only if the ectoplasm of the
blastomeres is removed from its normal contact with the

332

CLE A FACE AND DIFFERENTIATION

other blastomeres — as happens in the experiment of sep-
arating the blastomeres — do they develop separately. In
the intact cleaving egg the ectoplasm interacts with that
of the neighboring blastomeres and thus insures the normal
development into one embryo.

Secondly, in normal development the egg cleaves into
blastomeres, that is, new cell-partitions arise. When exper-
imentally eggs are induced to differentiate without cleavage,
abnormal development results. In such cases of differ-
entiation without cleavage there is always an abnormal
accumulation of ectoplasm.

In the third place: the embryonic axis in eggs of radiate
animals and the plane of bilateral symmetry of the
embryo in eggs which develop into bilateral animals, arise
in the surface and not within the bulk of the egg-substance.
If there be gradients of development these are ectoplasmic
and not axial, polar or otherwise.

Fourthly, the ordering and shifting of cytoplasmic
inclusions, in terms of primary causes, depend upon the
ectoplasm, since the changes in the ground-substance,
responsible for these movements, are themselves dependent
upon activity in the superficial cytoplasm, as was shown
in the chapter on cell-division.

The importance of the ectoplasm as a causal factor in
differentiation is in, addition predicated upon its general
properties, its role in the exchange of water between egg
and outside world, its necessity for fertilization, its signif-
icance in parthenogenesis, its part in initiating division of
the cell. These attributes alone would suffice to establish
the ectoplasm as a leading factor in differentiation. But
there are other and more general considerations.

The establishment of new surfaces during cleavage alters
the physical properties of the protoplasm. With the
separation of the egg-substance into blastomeres the origi-
nal physical state of the uncleaved egg is altered since some

333

THE BIOLOGY OF THE CELL SURFACE

of it goes to make up new nuclei and another portion forms
the new cell-boundaries. Thus, the viscosity of the cyto-
plasm, for example, in the individual blastomere can not be
the same as that of the cytoplasm of the uncleaved egg.
Furthermore, the diflferential distribution of the cytoplas-
mic inclusions during cleavage certainly alters the original
physical state of the egg.

With subdivision of the egg into blastomeres, changes in
the chemical reactions take place. Each blastomere repre-
sents a separate reaction-chamber of capillary dimensions
favoring especially those reactions which are confined to
surfaces.

Cleavage means a change in the space-relations of the
original egg-substance. With the formation of new cell-
partitions, especially in totally cleaving eggs which form
blastulae, the subdivision into cells means new disposition
of materials which at first were in more intimate contact.
The orderly arrangement of blastomeres may be looked
upon as one of the most characteristic attributes of the
cleavage-period. The cell-surfaces are not simple ones
but are made up of prolongations of unequal distribution,
length, and activity.

With cleavage, then, by virtue of the ectoplasm, the
original egg-substance is separated into blastomeres and
the blastomeres are integrated by means of intercellular
connections, that is, the ectoplasmic prolongations. It is
well known that cells in a strip of tissue, like a strand of
ciliated epithelium, behave differently when part of the strip
and when isolated. The beating of the cilia can be observed
in an intact strip to run in order from one cell to the next;
whereas when the cells are isolated the cilia beat irregu-
larly. The same phenomenon has been observed in sus-
pensions of spermatozoa.^ In a normal sperm-suspension

1 Lillie, F. R., /g/J.

334

CLEAVAGE AND DIFFERENTIATION

each spermatozoon moves by the lashing of its tail. If the
spermatozoa are caused to form balls by the addition of
some substance which agglutinates them with their heads
sticking together, the tails now beat one after the other
in succession. It is also well known that cells of a tissue
when isolated and grown in tissue-culture manifest form-
changes and behavior which never occur when they are
part of the tissue.^ Finally, blastomeres when isolated
reveal behavior arising out of their isolation. In brief,
therefore, the behavior of blastomeres during cleavage
must be in part due to an integration brought about by
the intercellular connections arising from the ectoplasm.

The movement of blastomeres is an undoubted factor in
development and this movement is a function of the ecto-
plasm. The rise of cells out of which is formed an embry-
onic area in a specific region is to be attributed to a
movement on the part of the cells which is closely akin to
amoeboid movement. True, physical factors may be con-
cerned in the displacement and new alignment of blasto-
meres, but these are to be regarded as subsidiary. No
purely physical theory has as yet accounted for the process
of invagination by which a hollow blastula is converted into
a gastrula. On the assumption, however, that cells take
up a certain position with reference to others through their
active movement we may approach the explanation of
many processes, as invagination and evagination, epiboly,
etc.

Finally, there is clear experimental evidence that during
cleavage the ectoplasm reveals structural changes and
activities which parallel cleavage-rhythms and which indi-
cate that the ectoplasm plays a role in determining the
direction of differentiation. Some simple observations of
my own may be cited, ^

^ Harrison, Lewis and Lewis, et al.
- Just, 192SC.

335

THE BIOLOGY OF THE CELL SURFACE

If eggs of Arbacia be exposed to hypotonic sea-water dur-
ing the time after fertiUzation when the vitelline membrane
is being separated and are then returned to normal sea-
water, they develop throughout the cleavage-period much
as untreated fertilized eggs. A sharp difference is noted,
however, in the blastula stage, for then, whereas the normal
blastula is made up of cylindrical cells enclosing a cavity,
the eggs which have had treatment with hypotonic sea-
water are made up of cells approaching the cuboidal form;
the cilia of their cells are longer than those on the normal
blastula.

If the eggs of the same species of sea-urchin are treated
three minutes after fertilization, that is, two minutes later
than those used in the observation described above, part
of the egg protrudes beyond the membrane and these eggs
develop as twin embryos. In some cases the twins remain
joined, in others they become separated. I have found by
exposing eggs from the same females at different intervals
of fifteen or thirty seconds after fertilization that the most
favorable time for the production of these twins comes
immediately following the separation of the membrane
from the entire egg. Thus, the same treatment if given in
a different stage of the egg's development, yields a different
result. This statement holds generally for the different
periods during the cleavage-process; to treatment with
hypotonic sea-water, eggs in different stages of development
respond differently as shown by the difference in the types
of embryos resulting.

Now the variety of the results obtained in these observa-
tions can be related to ectoplasmic behavior. Beginning
with the moment of fertilization the ectoplasm goes through
changes in behavior which are easily followed under the
microscope. One needs, therefore, only to treat the Qgg
during periods when by observation one notes a difference
in the quality of ectoplasmic behavior in order to obtain an
alteration in the development.

336

CLE A FACE AND DIFFERENTIATION

The evidence that the treatment affects the ectoplasm
alone is derived from the following considerations: First,
there are no other noticeable or as striking changes elsewhere
in the egg. For instance, the time for obtaining blastulae
with cuboidal blastomeres and long cilia comes when the egg-
surface is breaking down in the process of separating its
vitelline membrane. On the other hand, the best time for
the production of twins comes when the ectoplasmic surface
is being reconstituted, or, one might say, a new surface
arises. Now at these moments, separated by a time inter-
val of about two minutes, no profound changes either in
the egg- or sperm-nucleus or in the cytoplasm below the
ectoplasm can be noted. Secondly, the method employed
in these simple observations is that of treating the eggs
experimentally for fifteen or at the most thirty seconds.
An exposure to hypotonic sea-water of this duration is
most certainly to be regarded as affecting the egg-surface
only. I conclude, therefore, that the various alterations
in the development called forth by treating eggs with
hypotonic sea-water at intervals after fertilization are due
to an effect on the ectoplasm. As development ensues,
the ectoplasm undergoes definite changes correlated with
the definite periods in development. If these surface
reactions are modified, development is modified. I may
refer again to the phenomenon of differentiation without
cleavage. Certainly this represents an extreme type of
developmental modification. As certainly it indicates
that the alteration is due to modification in the behavior of
the egg-surface.

Finally I cite an observation on the egg of Asterias^.
Normally during early cleavage the blastomeres of this
egg lie within the vitelline membrane apart from each
other; later, regaining contact they develop into one
embryo. When after the second cleavage the four blasto-

' Just, igjT.

337

THE BIOLOGY OF THE CELL SURFACE

meres lie apart, they may with care by puncture of the
vitelline membrane be removed as four independent cells.
If brought together again and kept in close contact, by
surrounding them with fibres of lens paper, they unite and
develop into a single embryo. Also, two blastomeres from
one egg when brought together with two from another in
some cases united and developed into one embryo; often
however union failed to take place. I found that this
failure resulted whenever the transferred blastomeres were
not in exactly the same moment of development. For
example: two lots of eggs from the same female were fer-
tilized at an interval of one minute apart from each other.
At second cleavage, the four blastomeres were removed
from one egg of each fertilized lot. Two blastomeres of the
one were next brought together with two blastomeres of
the other; no union was established. Observation revealed
that the surface-changes in the transposed blastomeres
were different. This observation furnished the clue for
the failure of union between transposed blastomeres taken
from a lot of eggs which had been fertilized at the same time :
For the establishment of the interconnection of blastomeres,
the ectoplasmic condition must be identical. Therefore, the
union of blastomeres can be made certain if by direct obser-
vation their surface changes have been found to be in the
same stage. Since in any lot of eggs in best condition fer-
tilization takes place in every egg at almost the same instant
and the development ensues at much the same rate, at
second cleavage the difference in the ectoplasmic behavior
between the most slowly and the most rapidly developing
egg is never as great as that in eggs fertilized at an interval
of one minute. The failure of blastomeres from two eggs
of the same fertilized lot to unite therefore is to be attrib-
uted to changes in ectoplasmic behavior resulting from a
difference in rate of development which is of seconds only.
To mv knowledge, there exists no observation which so

338

CLEAVAGE AND DIFFERENTIATION

clearly and so beautifully shows the quickly occurring
changes in ectoplasmic activity.

The ectoplasm stands not simply as a barrier of the cell
against the outside world; it is also the medium of exchange
between cytoplasm and environment. As such, it is the
first cell-region to receive impressions from the outside
world; through its delicacy of adjustment and fineness of
reaction, it constitutes the first link in the chain of cyto-
plasmic reactions and sets the path for the orderly suc-
cession of events comprising the course in the difi"erentiation
of development.

The moment to moment changes in the ectoplasm in
its response to the cell's milieu set up conditions in the
cytoplasm which are favorable to the mechanism of the
synthesis of nuclein, i.e., of the building up of nuclei out
of cytoplasm. This synthetic reaction constitutes the
removal of a barrier to those cytoplasmic reactions leading
to differentiation. Alongside this function of setting up
conditions favorable to the releasing-reaction of nuclear
syntheses, is the growth of ectoplasm through the transport
of ground-substance to the cell-surface. The ectoplasm
impresses the cytoplasm, and this ectoplasm-induced cyto-
plasmic activity brings about the nuclear behavior which
underlies genetic restriction. Ectoplasmic behavior is thus
the direct and so far the only visible manifestation of the
cause of the differentiation of development which takes
place during cleavage.

339

12

C/iromoso^nes a?id Ectoplasm

Jn a large measure our knowledge of the effects
of experimental means on cells Is the result of studies on
eggs. But in these studies the primary effect of the experi-
mental means on the protoplasmic system was often mis-
interpreted. Enthralled by the dramatic manoeuvres of
the chromosomes in mitosis investigators assumed for the
chromosomes an independence from the remainder of the
cell; this point of view easily led to the assumption that
external agents directly affect the chromosomes. So also,
the origin of mutations has been predicated upon inherent
activity of the chromosomes.

The effects on marine eggs of changes in temperature, in
salinity or in hydrogen-ion concentration vary, depending
upon the eggs employed. For a species of eggs these effects
vary before and after insemination. For the fertilized egg
again there is a differential effect of the means which runs
with the mitotic cycle. But there is here no evidence to
indicate that this effect is primarily or only on the nucleus
or its chromosomes; what the experiments do show is that
the effect is first on the ectoplasm.^

These changes in the surrounding medium bring about
changes in the nucleus only secondarily. Always the ecto-
plasmic changes come first. Indeed, within optimum
range, the magnitude of the ectoplasmic changes determines
that of the nuclear. There is also a relationship between
the duration of the exposure of the egg to these changed

1 Just, 1932.

340

CHROMOSOMES AND ECTOPLASM

environmental factors and the quality and character of
the ectoplasmic reaction. Again the Intensity, that is, the
strength of the change In the environment, determines the
rate of the ectoplasmic response; and within optimum
range, this rate determines the degree of the nuclear
response.

Specific examples to support these statements one may
find In the literature on fertilization, experimental par-
thenogenesis, experiments on cell-division and on develop-
ment. I may mention again the observations on the &^g
of Chaetopterus which after treatment with hypertonic sea-
water develops and differentiates Into an abnormal swim-
ming form; here clearly ectoplasmic activity conditions
nuclear behavior. In sea-urchins' eggs treated with potas-
sium cyanide, the failure of mitosis runs with an exagger-
ated activity of the ectoplasm.^ Also, In sea-urchins'
eggs, the experimental prolongation of the monaster
stage is conditioned by an exaggerated ectoplasmic
activity.

Egg cells have been subjected not only to changes in
temperature, In salinity and in hydrogen-Ion concentration
of the surrounding sea-water; they have also been exposed
to radium, Roentgen and ultra-violet rays. The first
noticeable effect of such exposures Is on the ectoplasm.

Some years ago Packard"' reported results of exposing
eggs of Nereis to radium. The first effect observed Is
cytoplasmic. In consequence or at least in sequence to
this follow modifications in nuclear behavior. Rediield^
especially has analyzed the ectoplasmic response displayed
by this egg after exposure to various rays. In this same egg
I found that ultra-violet rays induce profound local ecto-

1 Just, 192 jb.
- Packard, 1^14.
^ Redfield, igiS.

THE BIOLOGY OF THE CELL SURFACE

Fig. 37. — Egg of Nereis with first maturation spindle held near the centre of the
cell as an effect of ultra-violet irradiation.

0#

♦»•

^ -4$ ^•

^•'^#

^ 1

■■■■■ ♦./•/

Hi..'';

*?^ ti^

Fig. 38. — Irradiated egg of Nereis showing nuclear division without extrusion of

polar bodies.

342

CHROMOSOMES AND ECTOPLASM

plasmic changes which are followed by abnormal nuclear
behavior.^

The effect of the rays is limited to the superficial cyto-
plasm, whose altered condition brings about the remarkable
result that the eggs develop with seventy chromosomes
instead of the normal number, twenty-eight. The pro-
cess is as follows : Consequent to the injury of the ectoplasm,
the normal cytoplasmic movements are inhibited. As an
effect of this inhibition the first maturation-spindle formed

/

<k-

Fig. 39. — Irradiated egg of Nereis with Fig. 40. — First cleavage spin-
four groups of chromosomes resulting die, irradiated egg of Nereis,
from division of the two spindles in showing some of the seventy
Fig. 38. chromosomes.

after break-down of the germinal vesicle fails to move to
the surface of the egg. Instead of at the periphery — to
which normally the spindle moves and where after separa-
tion of the chromosomes their outer group is extruded with
the first polar body — the first maturation mitosis takes
place at or near the centre of the egg (Fig. 37), two nuclei
arise and from each of these a spindle forms, each giving
rise to two groups of chromosomes (Fig. 38). In other
words, the first and second maturation mitoses take place
near the centre of the egg and four egg-nuclei arise (Fig.
39). These four nuclei with the sperm-nucleus, each con-
taining fourteen chromosomes, give rise in some cases to

^ Just, ig26a, 1933b and c.

343

THE BIOLOGY OF THE CELL SURFACE

a cleavage-spindle with seventy chromosomes (Fig. 40),
In other cases multi-polar (two, three or more) spindles

-»

>»

^^H.

>

Fig. 41. — A multipolar first cleavage spindle, irradiated egg of Nereis.

^. , -^J

Fig. 42. — Another type of multipolar first cleavage spindle, irradiated egg of

Nereis.

arise as the appended figures (Figs. 41 and 42) of the radi-
ated Nereis egg show.

344

CHROMOSOMES AND ECTOPLASM

The striking similarity between these figures of abnormal
mitoses and those found in cells of human cancer deserves
some comment.

Almost fifty years ago Galeotti described and figured
abnormal mitoses in human cancerous cells. Later he
showed that poisons, acting on skin cells of the salamander,
induce pathological mitoses closely resembling those found
in cancer. Subsequently various investigators have con-
firmed Galeotti's findings. Cancer cells of man and of
other mammals in tissue culture likewise show abnormal
mitoses. If one compares under the microscope my prepa-
rations of these abnormal mitoses in eggs of Nereis with
those of human cancer cells, one notes at once that, except
for their greater clearness and sharpness due to more excel-
lent technique, the mitotic figures in the eggs bear the
closest resemblance to those in the cancer cell. How far
this resemblance has common cause, it is hazardous to
assert. And this for the following reasons.

In the first place, eggs of a worm, freshly discharged
into the sea, are far removed from the cells of warm blooded
animals, which in the intact organism stand in a quite
different relation to their environment, conditioned by
nervous and humoral integrations. Conclusions for human
cells derived from studies on cells of other mammals even
are not always safe. Hence we are wary in making final
statements on the basis of animal experimentation concern-
ing human disorders, the more so since man varies so much
one with the other, and since in one individual the har-
monious adjustment of cells, their capacity for self-regula-
tion, so greatly depends upon that so little understood
principle of individuality, which in the final analysis goes
back to the chemical make-up of the ground-substance of
every single cell;^ from it emanate what we designate indi-
vidual resistance and susceptibility.

1 Just, 1936b.

345

THE BIOLOGY OF THE CELL SURFACE

In the second place, the fact that abnormal mitoses
arise in response to most diverse means needs to be con-
sidered. The Nereis egg which portrays them as an effect
of radiations, responds in like manner to other means. ^
Then the reaction is an expression of independent irritability
which reveals specific structure. So with somatic cells
which react similarly. Thus, abnormal mitosis does not
as such mean that the cell is one of cancer. Moreover,
not every human cancer cell displays abnormal mitosis.

None the less is the resemblance striking. Far removed
though any cell — egg of worm or cell of an onion root tip —
is from a human cell of cancer, it might not be too far fetched
to see in the similarity of response a manifestation of a
fundamental structure common to all protoplasmic systems.
It is as imperative for the problem of cancer as for any
problem in biology discussed in this book that we add to
our knowledge of this structure.

The fact that the egg of a worm and a human cell show
comparable configuration in abnormal condition points to
a similarity in a basic pattern of organization in living mat-
ter upon which the characters of speciation are imposed.
If therefore we could designate in the egg one characteristic
deformation as diagnostic of its response to changes in the
external medium, we should have at hand a point of depar-
ture for study of the cancer cell. Now such diagnostic is,
as I shall show, available: All means that elicit the rise of
abnormal mitosis do so through alteration of the ectoplasm.
A more exact cytology of human cancer cells with reference
to ectoplasmic behavior is thus indicated.

The effect of the radiation on the ectoplasm of the egg
of Nereis is shown in the following ways. First, the normal
egg after fertilization extrudes a jelly throughout the whole
ectoplasm so that every fertilized egg is enclosed in a hull

1 Just, 1936c.

346

CHROMOSOMES AND ECTOPLASM

of jelly of the same consistency throughout and of equal
width. The ectoplasm whence the jelly escaped shows no
persistent localized inequality. The changes taking place
in it are not confined to any particular region. The egg
fertilized after radiation, on the other hand, extrudes jelly
of lesser degree of consistency so that the eggs tend to fall
out of their jelly hulls. Always is there present in the
ectoplasm thereafter an injured region marked by the
change in the structure of the ectoplasm as well as by
the greater distance of the vitelline membrane at this site.
This change in the physical make-up of the jelly is an index
of the ectoplasmic injury induced by radiation. In the
second place, in the radiated eggs the first cleavage plane
passes through the mark of injury revealed after the extru-
sion of the jelly. Third, a localized area of injury in the
ectoplasm persists through the egg's development and can
be traced into the larval worm.

This observation on the effect of ultra-violet rays on
the Nereis egg is supported by my findings on the egg of
Chaetopterus; in a similar, but perhaps not so striking a
manner, ultra-violet rays affect the surface cytoplasm of
this egg.^

Since in some cases radiations, especially radium and
Roentgen rays, are most effective after nuclear breakdown,
it is often held that irradiation does not affect the cell while
its nucleus is intact. From this it is argued that radiation
directly affects the chromosomes. Indeed, some workers
consider that the action of Roentgen rays, for example, is
limited to the chromosomes alone. Against this position
arguments may be adduced.

For many cells it has been learned that the display of
normal mitotic activit}' depends upon some condition in
the cytoplasm which often may even be visible. It is well

1 Just, iQjod, 1934c. /^^^^L /

THE BIOLOGY OF THE CELL SURFACE

known that a certain complex of physical and chemical
conditions in the cytoplasm is necessary for the initiation
and completion of mitosis. The rhythm of nuclear division
is itself parallel with the rhythm of structural and physico-
chemical changes in the cytoplasm. Abundant evidence
indicates that parallelling the rhythm of mitosis is a rhythm
of susceptibility and resistance of the cytoplasm to many
and diverse experimental means. In some cases, notably
that of the egg of Nereis, the experimental means including
radiations are as effective on the egg in the condition of the
resting nucleus as in stages of mitosis. The susceptibility
displayed by a nucleus is to be correlated with changes
in the cytoplasm; and since radiations are not alone among
means which may be more effective on cells in stages of
mitosis than on those with the so-called resting nuclei, we
can not denominate Roentgen or radium rays as specific
chromosome-affecting means.

What is true is rather this: radiations are far nicer means
than, for example, hypotonic and hypertonic solutions,
heat and cold — all of which give effects similar to those of
radiations. Radiations produce effects which are more
rapid, more exactly measurable and more widespread in a
given population of cells. This, in my judgment, is the
chief value of radiations as experimental means. ^ Failure
to appreciate that in experiments with radiations the cyto-
plasm is affected and that radiations belong to a large class
of experimental means has given birth to the fertile error
that Roentgen, radium and other rays are causative factors
for inducing directly profound changes in chromosomes.
The offsprings of this notion, nursed by the gene-theory
of heredity, are theories concerning the origin not
only of species but also of the whole realm of living
things.

1 Just, ic/^6c.

34S

CHROMOSOMES AND ECTOPLASM

The fact that in Roentgen therapy there is a difference
in the susceptibility of human cells does not vitiate the
argument. Among these cells those most highly endowed
with division and growth capacity are most susceptible — a
fact which does not prove that the rays are chromosome-
specific in their action. Rather, because of such division-
and growth-capacity, the cells have cytoplasm whose con-
dition renders them more susceptible to radiation than
other cells.

As we have seen, the effect of the feebly penetrating
ultra-violet rays is a sharply localized ectoplasmic injury.
Hence, it is not the penetrating power of the rays and so
their power to reach the more deeply lying chromosomes
which is responsible for their effects. Even the more deeply
penetrating rays. Roentgen and radium, as Redfield has
shown, also affect the ectoplasm; we therefore can not
assume an effect of these radiations that is limited to chro-
mosomes only.

Further, materials entering the cell — gases, water, etc. —
come into relationship first not with the nucleus but with
the ectoplasm. We may here reason from analogy. Take
oxygen consumption, for example.

There was a time when biologists assumed the nucleus
to be the seat of cellular oxidations. The presence of iron
in the nucleus was postulated as part of the oxidation
mechanism. On a priori grounds one would assume that
oxygen entering the cell would combine with cellular constit-
uents lying in the cytoplasm between the cell boundary
and the nucleus. Now we know that this is at least nearer
the truth: oxygen consumption is a function of cytoplasmic
structure. Moreover, the attempt to demonstrate the
presence of iron in the nucleus of spermatozoa failed though
impeccable methods were used.^ What is true of oxygen is

^ Cf. Lynch, ig22.

349

THE BIOLOGY OF THE CELL SURFACE

doubtless true of other substances that enter cells: they
react first with the cytoplasm.

Experimental cell-study gives instances of aberrant
behavior of the chromosomes in egg cells. All of this work
Indicates that the experimental means first affects the
ectoplasm. According to the theory here advanced this
ectoplasmic effect Is responsible for secondary effects on
the whole egg cell. Among these Is the aberrant behavior
of the chromosomes.

In the early days of the modern work on genetics, the
experimental analysis could not have got very far without
the descriptive studies on the behavior of the chromosomes.
In the first line of this classic period for cytology stands
Boveri's work. According to his analysis of experiments
on dispermic fertilization, normal development depends not
on the number but on the proper combination of chromo-
somes. These experiments furnished part of the basis for
the theory of the individuality of chromosomes.

To secure dispermic or polyspermic fertilization of an
echinid egg, one must first weaken its ectoplasm or employ
heavy insemination. Where polyspermy ensues without
experimental weakening of the eggs, we must assume that
they were weak at the outset. The aberrant development
of the blastomeres in his experiments Boveri related to the
wrong combinations of chromosomes. But these combina-
tions themselves depend upon the weakened conditions
in the cytoplasm which make dispermy possible. In the
normal egg the unimpaired ectoplasm protects against dis-
order of the chromosomes.

Aberrant behavior of paternal chromosomes Is revealed
In cross-fertilized echinid eggs when in certain crosses the
paternal chromosomes, failing to take part in the mitotic
process, are eliminated from the spindle. The possibility
for cross-fertilization in all these cases is rendered greater
by Injuring the ectoplasm of the eggs. Here again, there-

350

CHROMOSOMES AND ECTOPLASM

fore, the condition of the cytoplasm determines the behavior
of the chromosomes. To induce cross-fertilization espe-
cially between widely separated species, for most eggs at
least, one must impair the integrity of the eggs' ectoplasm.
Such impairment means a weakened cytoplasm. As with
the aberrant behavior of the chromosomes in experimental
polyspermy, so with that in cross-fertilization: it follows
injury to the egg's ectoplasm.

A point here must be emphasized. Too frequently bio-
logists speak of the incompatibility of chromosomes to
account for the elimination of chromosomes in cross-fer-
tilization. As a matter of fact, chromatin may be elimi-
nated in straight fertilized eggs. I have found in straight
fertilized eggs of Echinarachnius that the whole egg-nucleus
may fail to take part in the cleavage-mitosis.^ Such eggs
are injured.- Here there can be no question of the "incom-
patibility" of chromosomes to foreign cytoplasm. Rather,
the chromosomes fail to take part in the ensuing mitoses
because of the weakened condition of the cytoplasm pre-
viously treated. Work on the effect of temperature and
of ether on the sperm-nucleus in the echinid egg are ame-
nable to the same interpretation. The behavior of monaster
eggs is also a case in point: the abnormal behavior of chro-
mosomes is clearly due to injury of the superficial cytoplasm
brought about by vigorous shaking at a time after insemina-
tion when the ectoplasm is very susceptible to experimental
treatment. The effect of radiation referred to above on
straight fertilized eggs may be recalled. Here again the
aberrant behavior of the chromosomes is in consequence
of a definite ectoplasmic injury. Finally, Dubois has shown
for the egg of Sciara that chromosomes are normally elimi-

1 Just, 1924.

- See also J. Gray on Echinus-egg.

THE BIOLOGY OF THE CELL SURFACE

nated during cleavage and that this elimination is deter-
mined by the ectoplasm.^

In all the foregoing it is safe to conclude that the injury
to the cytoplasm is an ectoplasmic injury. We may there-
fore proflfer the hypothesis that in ectoplasmic behavior
lies the cause of the behavior of the chromosomes. Normal
chromosome-distribution and -combinations depend upon
the integrity of the ectoplasm; their aberrant behavior is
the eflFect of the loss of this integrity. Such behavior may
manifest itself in chromosomal elimination, fragmentation,
and the like.

To me the conclusion seems inescapable: ectoplasmic
behavior determines the cytoplasmic reactions that lie at
the basis of nuclear activity in both normal and abnormal
mitoses. Where experimentally induced mutations are
related to visible changes in the chromosomes, as their
fragmentation, translocation, etc., these are not to be
regarded as direct effects of the experimental means
employed, — e.g., temperature, radiations — but as express-
ions of the antecedant altered cytoplasmic reactions brought
about by changes in the ectoplasm that evolved in response
to the action of the means.

If one observes under a microscope, equipped with a
dark-field condensor, a suspension of Chinese ink ground
up in water, one sees these inanimate particles scintillating
back and forth in Brownian movement. One knows that
the shining particles are moved and are not themselves
motile. It is a far cry from the dance of ink-particles to
the orderly movements of chromosomes in a living cell.
And yet, it is warrantable to assume that as we look upon
the shifting of chromosomes in cells, their constant distribu-
tion during cell-division, generation after generation, we
behold, not a motility inherent in these bodies themselves

^ Dubois, ig33.

352

CHROMOSOMES AND ECTOPLASM

but a behavior which expresses a force in the cytoplasm
enclosing them. In normal mitosis, in experimentally
induced mutations, the chromosomes express the underlying
behavior of the medium in which they lie. They mark for
us the ebb and flood of the cytoplasmic tides. These in
turn are under control of ectoplasmic activity.

353

13
Kctoplasni and Evolution

J HE PRINCIPLE OF EVOLUTION IS AS FIRMLY ESTABLISHED

as any in biology. The evolution-theory constitutes a
fundamental postulate of the science of biology and has
proved a guiding principle of uncalculable value for bio-
logical research. Among biologists exists the almost unani-
mous verdict that evolution took place. According to
the prevailing opinion, the world of living things was
evolved from a unicellular organism.

It should however be emphasized that this first form of
life was not that of some now existing protozoon. The
word, Protozoa, literally means first animals; but we should
bear in mind that among the Protozoa themselves evolution
has taken place. We therefore assume that the first form
of life was a simpler unicellular structure than the proto-
zoan. From this both the Protozoa as we now know them
and multicellular animals came, the former evolving in one
direction, the latter in another.

We encounter two questions: How did this first living
thing arise ? What was (and is) the cause of evolution ?

With the first question most biologists will not concern
themselves — they deem it unanswerable and thus only
provocative of fruitless speculation. And yet such specula-
tion will always be alluring. The drama of the universe in
the act now before us is a tremendously moving spectacle,
but the prologue to its pageantry is also capable of moving
the dullest imagination. One need, therefore, make no
apology in voicing a note concerning the origin of the world
of living things. The answers to the second question are

3S4

ECTOPLASM AND EVOLUTION

innumerable, as is attested by the many and various theories
of evolution. Those biologists who, after having weighed
the evidence, accept the theory that animals and plants
exist as products of evolution and reject the theory of a
special creation for every living being that ever was and now
is, by no means agree concerning the cause of evolution.

To take up our first question: How did the first living
thing arise.'' To put it otherwise: How out of non-living
matter did life arise .^

The combination of chemical compounds from the envi-
ronment to make up the first living thing must obviously
have meant a separation from the environment, that is,
the combination must have been peculiar, both physically
and chemically; otherwise, there never could have come
about its separation and the maintenance of its integrity
apart from the environment. Now the moment that this
peculiar combination of compounds arose, there must have
begun reactions or responses of it to the environment —
especially to temperature, to gases and to electrolytes.
The chief characteristic of this original substance was its
peculiar and complex organization, which set it apart
from its environment; but at the same time it must have
been responsive to environmental changes. Environmen-
tal changes must in the first instance have brought about
the combination of compounds peculiar to living substance,
and in the second place must have conditioned its activity.

This original mass of primitive protoplasm at first per-
haps showed no high degree of differentiation, but we can
scarcely imagine it as a homogeneous structure through-
out; as such it could not have endured for any great length
of time. The moment that we assume that a combination
of chemical compounds was separated out from the envir-
onment as a peculiar system, we must postulate some dif-
ferentiation in the mass — which differentiation served to
keep the combination of compounds intact.

355

THE BIOLOGY OF THE CELL SURFACE

If, however, we assume that this combination of com-
pounds separated from the environment was first purely
homogeneous throughout, then there must soon have come
a time when factors in the environment played upon this
structure and so modified, if they did not determine, its
behavior. It would be difficult to imagine a structure
made up of the same elements or compounds found in the
environment and maintaining its separateness from the
environment without some structural difference from it.

The moment that such a peculiar combination of com-
pounds arose, it assumed life; it had response to its
environment, both because it arose from it and had to exist
in it.^

Living substance can not be considered abstracted either
from time or from space. The organism can not be separated
from its environment; they form together one inter-acting
system. Two predominating characteristics exhibited by
living organisms are: first, those changes which are time-
ordered; and second, those which are environment-condi-
tioned. Thus, the organism — a single cell, for example —
changes from moment to moment and the rate of such
changes becomes its differentiating characteristic; within
the organism these changes tend to run in one direction —
the building up of protoplasm from simpler compounds
while life lasts. External to it the environment plays a
part in yielding up the raw material for these changes
and setting the conditions for the reactions in the living
matter. In a certain sense we should not speak of the "fit-

^ Although there is the possibility of life below that size that can
be made visible by the microscope, zve must admit that we know little
concerning the organization or structure of such ultramicroscopic
organisms. Therefore, zve might most profitably begin with a
hypothetical organism whose size is within the range of resolution
by the microscope. Nevertheless, what is said of such a hypothetical
structure might also hold for one of ultramicroscopic size.

ECTOPLASM AND EVOLUTION

ness of the environment" or the "fitness of the organism";
rather, we should regard organism and environment as
mutually adapted.

The play of factors in the environment — of temperature,
of gases and of electrolytes — upon the living organism
must be first on the cytoplasmic surface. Even if we
assume that the primordial living thing was a mass of
homogeneous protoplasm structurally the same throughout,
there must have early arisen a differentiation between sur-
face and interior.^ In the constant interchange between
environment and organism reactions must have taken
place first in the more superficially located cytoplasmic
structure; these reactions would condition succeeding ones
In the endoplasm. The first step In the evolutionary proc-
ess, then, was a diiferentiation of the cytoplasm into ecto-
plasm and endoplasm. The second step, according to
this theory, was a nucleo-cytoplasmic differentiation.

We have thus a picture of the primordial living thing as a
mass composed of the prototype of the ground-substance
in cells as we know them to-day which limited itself in
space by a changed surface, its ectoplasm. In time this
primordial thing showed a farther differentiation of a sub-
stance which by opposing its more fixed character to the
ever-changing mobile character of the ectoplasm tended to
maintain the stability of this primitive protoplasm. Thus,
nuclear substance arose.

In thus postulating for the nucleus only a secondary
origin I reject the theory that the first form of life was a
chromatin-granule.- However attractive this latter theory
may be to those who regard the ultra-filterable virus as
living and to those who believe that the gene represents the
fundamental living unit,^ my speculations concerning evolu-

^ Cf. Child igiS on "Surface-interior patterns.^'
- Cf. Minchin, 1915.
^ -£".§., Jennings., ^93^-

357

THE BIOLOGY OF THE CELL SURFACE

tion derive their plausibility from their consistency with
known facts of cellular phenomena: one of these indicates
the elaboration of chromatin out of cytoplasm. More
than once have I in the foregoing chapters referred to the
origin and growth of chromatin out of the cytoplasm. On
the other side, there exist no data to indicate that chromatin
builds up c}'toplasm.

The same emphasis that I place upon the differentiation
of ectoplasm out of the ground-substance in the genesis
of the primordial living thing, I place upon it as a cause in
the evolution of the animal and plant kingdom.

Protozoa are classified into ascending orders on the basis
of their ectoplasmic structure and behavior. Eggs develop
into adults by virtue of ectoplasmic changes during differ-
entiation. Among multicellular adult organisms, grades
of complexity can be recognized : the more complex the
organisms, the richer are their modes of integration as
shown by comparative studies on nerve-systems. What
makes man's brain the greatest among animals, is not the
number of Its nerve-cells — indeed, in a given area, the pri-
mate brain has fewer cells than that of other mammals —
but the richness of their connections. Other forms of inter-
cellular integrations in multicellular organisms are also
ectoplasmic.

Let us recall once more the fundamental functions of
living protoplasm. These are contraction, conduction,
respiration and nutrition. The primordial contraction, let
us say that exhibited by the egg or protozoan cell, involves
the cell interior to a secondary degree only. The cilia of
ciliated Protozoa are ectoplasmic structures. Muscle-
contraction in the highest organisms is a phenomenon of
the cell-surface.^

Also conduction, we found, is an ectoplasmic function.
The transfer of the effect of a stimulus is ectoplasmic, as

1 Hill et al.

358

ECTOPLASM AND EVOLUTION

for instance in the fertilization of the egg. In tissues highly
endowed with conductivity, as in those highly endowed
with contractility, the predominating characteristic is rela-
tively large surface area. In nerve cells, conduction travels
over the nerve fibre (ectoplasmic prolongation), and is
transferred by means of ectoplasm from one unit to another.
By and large, it is reasonable to assume that both contrac-
tility and conductivity are greater in surface-rich than in
surface-poor protoplasm.

The subsequent differentiation from a hypothetical first
form of life into animal or plant we may suppose came about
through the higher development of contraction and of con-
duction by that form which evolved as animal. A deviation
or less emphasis on these brought plants as such into being.
It is surely on the side of the nervous tissue that animals
and plants differ most.

Cellular oxidation is a function of cytoplasmic structure.
Again it is reasonable to assume that oxygen coming into
the cell makes first some union with the superficial cyto-
plasm, as we have seen.

Now of three fundamental life-processes — contraction,
conduction and respiration — respiration may be regarded
as primary: on it depends all vital activity. Respiration,
the same in both animals and plants, may have been largely
responsible for the early separation between plant and
animal. That is, those primordial individuals possessing
most rapid rates of oxygen-consumption tended to oxidize
themselves, or as cannibals, their like. Some, because of
lesser intake of oxygen, tended to accumulate CO2 and thus
developed photosynthetic power as a means of protection.
With this went also the building up from COo and water of
carbohydrate polymers, including cellulose.^ The presence
of cellulose determined the disposition of the cytoplasm
found in higher plants — that is, a cytoplasm of peripheral

Cf. also Geddes.

359

THE BIOLOGY OF THE CELL SURFACE

location enclosing a vacuome. Thus, respiration may
have determined the difference between animal- and
plant-nutrition.

Animals evolved slowly or rapidly, farther and farther,
depending upon the degree to which ectoplasmic behavior,
Its faculty for contraction and conduction, developed. The
greater became these two forms of behavior, the more
Imperative became the need of exquisite means for respira-
tory-exchange. Respiration, according to our assumption,
played a part in establishing the difference between anlmal-
and plant-nutrition. Animal nutrition advanced showing
progressively increased utilization of large surface-areas for
the play of reactions — in digestion and for absorption of
the digested end-products. With this evolved in increasing
complexity a circulating system which gained steadily in
structural perfection whilst its correlation with the respira-
tion-system increased; and a more exact Integration by
nerves and finally by Internal secretions arose.

Thus all forms of behavior by which we recognize that
a thing is alive express themselves In response to the envi-
ronment in the activity either of the ectoplasm itself —
as in unicellular organisms — or of structures which are rich
In ectoplasm. The fineness and nicety of ectoplasmic
organization Increase progressively in the animal kingdom
from the lowest to the highest organisms and thus parallel
the course of evolution. This course, from the emergence
of life out of non-life to the separation of animals from plants
and farther to the unfolding of progressive complexity of
animal-form, makes manifest the role and Importance of
the ectoplasm in evolution.^

This hypothesis of the evolution of the living world holds
also for the more restricted problem of evolution, the
origin of species. As I see it, the most valid criterion for

1 Just, 1933 a.

360

ECTOPLASM AND EVOLUTION

determining that animals of a genus belong to the same
species is the capacity of normal fertilization and the pro-
duction of fertile offspring. As has been abundantly
shown, fertilization of an egg by a foreign spermatozoon is
only possible if the ectoplasm has been debased. The
unimpaired ectoplasm is a barrier to fertilization by non-
specific spermatozoa. Then species arose through changes
in the structure and behavior of the ectoplasm.

In the differentiation of ectoplasm from the ground-sub-
stance we thus must seek the cause of evolution.

361

Conclusion

7 HE IMPOSING MASS OF EVIDENCE PRESENTED IN THE

chapter, The Ectoplasm, can not be gainsaid. In all
cells, of unicellular and multicellular animals, the existence
of the ectoplasm can be demonstrated. We have seen what
is its role in the phenomena of conduction, contraction,
respiration, the intake and output of water — these all being
general properties of all animal cells. More specifically for
the animal ^%<g^ we have seen that without the ectoplasm,
fertilization can not take place, that in both fertilization
and parthenogenesis the response of the ectoplasm to the
inciting means for development is prognostic for the quality
of the future development; that in cell-division the ecto-
plasm initiates the event by regulating the movements
within the cytoplasm and that by redistribution of its
structure and relocalization of its activity, it establishes
new cell-surface; and that during differentiation the ecto-
plasm increases in amount and reveals a differential activity.
This behavior of the ectoplasm was shown to be one causa-
tive factor in differentiation of development, the other
being the building up of nuclear material out of the cyto-
plasm. Thus the reactions underlying both differentiation
and heredity were shown to be under the domination of
cytoplasmic reactions, resulting from an interplay of both
ectoplasm and nucleus with the cytoplasm. On this basis
an interpretation of the action of the gene was offered.
The behavior of the chromosomes themselves in normal
cells and in experimentally induced mutations was shown
to be dependent upon ectoplasmic activity. It was finally

362

CONCLUSION

suggested that ecto-endoplasmic differentiation is a factor
in evolution.

Thus briefly the argument in favor of my theory of the
ectoplasm. Let it be clearly understood, however, that
the theory in no wise invalidates the conception of the
protoplasmic system as the unit of the state of being alive.
Rather, the whole argument upon which the theory rests
aims at encompassing this conception in a fuller and more
complete fashion than hitherto was possible. Because of
the up to now existing failure to assign to ectoplasmic
behavior a role in the integrative action of the living system,
it has been necessary to emphasize what this role is. I
wish very clearh' again to state my position: life resides in
the whole of the protoplasmic system taken as a unit — the
phenomena of life are not to be dissociated from the integra-
tion of the system's constituent regions, the integration
which is the basic manifestation of the state of being alive.

While life rests in the protoplasmic system as a unit,
life is not static, is not structure only, but is the sum-total
of activities; protoplasmic organization or integration,
therefore, means orderly reactions as surely as it means
space-distributions of the protoplasmic components. We
may never come to know what life really is, but if we do
approach such knowledge, it will be through appreciation
of activities which constitute the protoplasmic integration;
these we learn by their expression. Life is not confined to
nucleus only and certainly not to constituent genes; nor
yet does it reside in the ectoplasm alone. The significance
of the ectoplasm is that it gives visible expression to these
activities.

On the one side ever}' living thing tends to fixity and
rigidity, resistant to change. This tendency aids to pre-
serve individual integrity which hands over intact from
generation to generation the character of the organism.
In the protoplasmic system the nucleus represents this

3<^3

THE BIOLOGY OF THE CELL SURFACE

conserving static Moment. On the other side, the living
thing is highly mobile, changing with every change in the
environment, accommodating itself and thus evincing
capacity for self-regulation. In the protoplasmic system
the ectoplasm is the region of active momentary changes in
response to environmental conditions.^ Within the proto-
plasmic system is a stability that is derived from the inter-
play of the more static and the more mobile factor. This
stability thus brought about determines both that organiza-
tion of matter called living thing and that specificity which
sets off one living thing from another.

To the varying states of the protoplasm due to the con-
comitant and succeeding reactions we need especially to
address ourselves. The region of the protoplasmic system
in which these changes are most strongly revealed is the
ectoplasm. Its most important characteristic is its change
in time, its rapid response to outside conditions. No other
cell-component exhibits this characteristic in a like degree.

By re-evaluating structure and function of the proto-
plasmic system, my theory of the ectoplasm has signifi-
cance not only for the advance of biological investigation;
it offers a point of view also for medicine — the highest
form of applied biology — whence medical problems can be
surveyed.

Medicine, as human biology, a welter of complex rela-
tions, even less than the biology of other animals can depend
upon the ultimate particles of physical science for the
solution of its problems.^ As protoplasmic systems, human
cells are capable of study by comparable cytological tech-
nique and experimental methods which used on eggs have
furnished the particulars that give basis for my general-
ization. An insistent demand for medicine today is,
therefore, a far-flung attack on human cells as such. We

^ Cf. Montgomery, igo4.

~ See Sauerbruch, 1^26, 1937.

CONCLUSION

need more and more to supplement that histology of normal
and pathological states which although it suffices for dia-
gnosis nevertheless remains the study of tissues — and more
often of organs — by most exhaustive study of the minute
structure of every single type of human cell both in its
normal and in its every available pathological state.

The study in every type of cell of its ground-substance —
i.e., of the protoplasm minus the inclusions and the products
of metabolism — may win a new understanding of the func-
tions of the liver, the kidney, the gut, the pancreas, etc.
Extension of the descriptions of the ectoplasm in these
various cells given by the older medical histologists may
serve for a closer correlation of structure and function in
health and disease. In spite of the view that cancer, for
example, is caused by external agents and that purely micro-
scopic investigations here are fruitless, nevertheless it may
be a problem intrinsic to the cell. In oedema, forms of
nephritis and all other pathological states in which the
balance of water is disturbed, careful microscopic investi-
gation aiming at the fullest possible description of the
minutiae in the cell is called for. The white blood-cells used
every day in the diagnosis of disease have not been suffi-
ciently investigated beyond diagnostic needs; studies made
by means of new methods of cell fixation and staining might
yield results that would throw additional light on the
changes both in number and in quality which white blood-
cells show in various human diseases. In these three prob-
lems of medicine are strong indications that the ectoplasm
is involved.

From consideration of the theory of the ectoplasm as a
working principle in the attack of general problems in
biology and its application to problems in medicine, let
us turn to its philosophical implications.

Biology being limited to protoplasmic organization and
not being able to go below it without that life is destroyed,

365

THE BIOLOGY OF THE CELL SURFACE

must establish Its philosophy on this basis. Any philos-
ophy of biology must take Into account the activity of the
superficial cytoplasm.

The living thing Is part of the natural world; It grows
and lives on the stuff of which It Is made and whence It
came. Then living thing and outside world constitute one
Interdependent unity, as evolution teaches, as the develop-
ment of an animal egg reveals. As the boundary, the living
mobile limit of the cell, the ectoplasm, controls the Integra-
tion between the living cell and all else external to It. The
ectoplasm Is the means of exchange for Incoming and out-
going substances. It Is keyed to the outside world as no
other part of the cell. It stands guard over the peculiar
form of the living substance. Is buffer against the attacks
of the surroundings and the means of communication with
It.

If we trace the development of the brain In higher ani-
mals. Including man, we find that always It arises from
ectoderm cells, those cells that possess most ectoplasm.
The whole nervous system arises in animals always on the
outer surface of the embryo; only later in development does
it become enclosed within the body by other cells. Then
the functions of the brain represent highly developed general
properties of primitive ectoplasm. The brain can not be
fully appreciated unless we bear this in mind. As with
brain, so with sense organs.

The origin and development of the brain show that a
conception which assumes that either the individual alone
or only the outside world is real, has no biological basis.
The interdependence between individual and outside world
is a postulate which has its sanction not from any abstract
philosophical principle, but is true because of the biological
basis here set forth. The best system of philosophy, then,
Is that which recognizes living thing and outside world as
one interdependent continuum. Instead of building our

366

CONCLUSION

philosophical theories of life on the behavior of electrons,
it is safer to erect them on a biological basis. We conceive
human behavior in terms of the history, the evolution, of
the differentiation of the cytoplasm, as this differentiation
appears in the course of development of the living world,
attaining its highest degree in the human race and in the
human individual.

The activity of the brain means a manifestation of ecto-
plasmic properties which we may regard as evolved from
primitive ectoplasmic relation to outside world. Every
mental state may thus be conceived as having behind it
this old relation. Perhaps it is because of this that man is
able to trace the evolution of the universe. Our minds
encompass planetary movements, mark out geological eras,
resolve matter into its constituent electrons, because our
mentality is the transcendental expression of the age-old
integration between ectoplasm and non-living world.

Life is not only a struggle against the surroundings from
which life came; it is also a co-operation with them. The
Kropotkin theory' of mutual aid and co-operation may be
a better explanation of the cause of evolution than the
prevailing popular conception of Darwin's idea of the strug-
gle for existence. The means of co-operation and adjust-
ment is the ectoplasm. But we can go farther.

Man with his highly complex nervous system constitutes
a species apart from the rest of the animal kingdom.
Nevertheless he maintains communion both with animate
and with inanimate nature. Still closer is his relationship
with fellow man. These relationships rest upon a purely
biological principle. The foregoing pages have established
this thesis. Here, then, is indicated where we may seek
the roots of man's ethical behavior.^

^ See also Ch. IV of Darwin's ''''Descent of Man.'''

- In a forthcoming essay, I deal with this point at greater length.

THE BIOLOGY OF THE CELL SURFACE

Nature is both continuous and corpuscular. In the
former sense, we pass from lower to higher revelations of
organization almost insensibly and with scarcely a break.
Every form of matter follows upon another. In the latter
sense, we recognize breaks in natural states from electron
to atom, from atom to molecule, from molecule to com-
pound, and from compounds in association to living matter.
But even conceived of as corpuscular, matter, as we know it,
is never purely discrete and absolutely independent from
the remainder of nature. Whether we study atoms or
stars or that form of matter, known as living, always must
we reckon with inter-relations. The universe, however
much we fragment it, abstract it, ever retains its unity.

The Q^g cell also is a universe. And if we could but
know it we would feel in its minute confines the majesty
and beauty which match the vast wonder of the world
outside of us. In it march events that give us the story
of all life from the first moment when somehow out of
chaos came life and living. That first tremendous upheaval
that gave this earth its present contour finds its counter-
part in the breaking up of the surface of the egg which
conditions all its life that is to follow. The sundering of
the egg into many parts, to be woven again into a whole
is no less wonderful than the breaking up of the primeval
unit out of which the sun and the stars, the earth and the
moon were made separate and brought together again in
the pattern of the heavens and the earth.

The lone watcher of the sky who in some distant high
tower suddenly saw a new planet floating before his lens
could not have been more enthralled than the first student
who saw the spermatozoon preceded by a streaming bubble
moving toward the egg-centre. And as every noviate in
astronomy must thrill at his first glance into the world of
stars, so does the student to-day who first beholds this
microcosm, the egg-cell. For the student of Nature there

368

CONCLUSION

is always that which moves him, that something which
he can not reduce. And out of this intuition he comes to
know what otherwise would remain hidden.

We feel the beauty of Nature because we are part of
Nature and because we know that however much in our
separate domains we abstract from the unity of Nature,
this unity remains. Although we may deal with particu-
lars, we return finally to the whole pattern woven out of
these. So in our study of the animal egg : though we resolve
it into constituent parts the better to understand it, we
hold it as an integrated thing, as a unified system: in it
life resides and in its moving surface life manifests itself.

3^9

15
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Svedberg, T. 1925. KoUoid-Chemie. Leipzig.
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under laboratory conditions. J. exp. Zool., 9.

. 1920. Chromatic material and hybridization. Anat. Rec.

Theel, H. 1892. On the development of Echinocyamus pusillus. Roy. Soc.

Ups.
Torrey, H. B. 1907. Biological studies on Corymorpha. Univ. Calif. Publ.
Torrey, J. C. 1900. The early embryology of Thalassema tnellita (Conn.).

Trans. New York Acad. Sci.
Treadwell, a. L. 1902. Notes on the nature of artificial parthenogenesis in

the egg of Podarke obscura. Biol Bull., 3.
Vejdovsky, F. 1888-1892. Entwicklungsgeschichtliche Untersuchungen.

Prag.
Vejdovsky, F. and A. Mrazek. 1903. Umbildungendes Cytoplasmas wahrend

der Befruchtung und Zellteilung. Nach den Untersuchungen am Rynchel-

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Verworn, M. 1899. Geneial Physiology. Macmillan and Co., New York.
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Oxydationsgeschwindigkeit in Zellen. Ergeb. Physiol.. 14.
Warren, E. 1903. On the anatoni}- and development of Distomum cirrigerutn,

V. Baer. Q.J.M. Sci., 47.

379

THE BIOLOGY OF THE CELL SURFACE

Watase, S. 1 891. Studies on Cephalopods. J. Morph., IV.
Watson, D. L. 1931. Biological organization. Quart. Rev. Biol.
Weismann, a. 1864. Zur Embryologie der Insecten. Arch. f. Anat. u.

Physiol.
Wertheimer, E. 1934. Die physiologische Bedeutung der Zellstrukturen im

besonderen der Struktur der Zellgrenzschicht. ProtopL, 20.
Whitaker, D. M. 1931. On the conduction of the cortical change at fertiliza-
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Whitman, C. O. 1893. The inadequacy of the cell-theory of development.

J. Morph., 8.

1895. Evolution and epigenesis. J. Morph, 10.

Wilson, C. B., 1899. The habits and early development of Cerehratulus.

QJ.M.S., 43-
Wilson, E. B. 1892. The cell-lineage of Nereis. J. Morph., 6.
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WooDGER, J. H. 1929. Biological principles. London.
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3S0

Indt

ex Oi

Auth

ors

Agassiz, 91

Allman, 2

Altmann, 63

Andrews, E. A., 93

Andrews, G. F., 96, 103, 117, 141. 278,

279
Appelof, 91
Armstrong, 43

V. Baer, 287

Barcroft, 225

Bataillon, 225

Bataillon et Tchou, 225

Bayliss, 140, 145

Bensley and Gersh, 56

Bergmann, 96, 97

Bernard, CI., 225

Berthold, 96, 117, 278

Bohm, 100

Bonnevie, 203

Boveri, 26, 180, 182, 237, 304

Brachet, 181

Bradley, 41

Brauer, 208

Brucke, 2

Buchner, 212, 213

Bijtschli, 63, 88. 93

Calberla, 100

Cerfontaine, 99, 167, 302

Chambers, 264

Child, 301, 357

Chun, 91

Ciamician, 91

Coe, 93

Conklin, 66, 91, 283, 306, 310. 325

Delage, 34, 180, 186, 212, 245. 251,
267, 290, 297. 322

Demerec, 325
Derbes, 20, 104, 105
Descartes, 34
Doflein, 88
Dubois, 351, 352
Dujardin, 2

Erlanger, 93, 268
Ewart, 284

Faure-Fremiet, 102, 280
Fischer, A., 65, 66
Fischer, E., 5
Fiemming, 49, 77
Fol, 91, 96, 105, 199

Galeotti, 345

Gatenby, 89

Geddes, 146, 359

Gerould, 96

Goette, 43, 97, loi 268

Goldschmidt, 326

Goldschmidt u. Popoff, 279, 281

Gray, 264, 279, 280, 281. 351

Greef, 213

Groom, 98

Gutstein, 88

Haeckel, 75, 88, 89

Haecker, 180

Halkin, 92

Hammar, 96, 122, 277

Hardy, 65, 66

Hargitt, 91

Harm, 91

Hartridge, 119

Harrison, 82, 83, 84, 85, 120, 335

Hatschek, 168, 169, 203

Heberer. 180

S8i

INDEX OF AUTHORS

Heider, 98

Heilbrunn, 44, 222

Heisenberg, 13, 14

Hempelmann. 97

Henle, 76

Herbst, 281

Hercik, 271

Herfort, 100

Hertwig, O., 96, 214, 215

Hertwig, O. and R., 117, 203

Hibbard, 57

Hill, A. v., 239, 358

His, loi

Hobson, 217

Hopkins, 5

Iwanzoff, 178, 201

Janicki, 92
Jennings, 7, 357
Jones, W. and Folkoff, 31
Jorgensen, 97

King, loi
Koelliker, 76
Kolzoff, 152
Komai, 91
Konopacki, 63, 135
Korotneff, 91
Kossel, 5

Kostanecki, 97, 217
Kowalewsky, 91, 97
Kowalewsky et Marion, 91
Kruger, 180
Kiihne, 64

Lang, 92

Lavoisier, 4

Lefevre, 97, 217

Lewis and Hartmann, loi

Lewis and Lewis, 63, 79, 81, 335

Leydig, 57

Lillie, F. R., 97, 117, 157- 163,

299> 307, 32s, 326, 334
Lillie, R. S., 116, 126, 213, 216,

273> 274
List, loi

Loeb, II ff., 216, 219 ff.
Lucke and McCutcheon, 127

I95>

^24,

Ludwig, 96
Lwoff, 167
Lynch, 349

Maas, 91

MacBride, 203

Mathews, 61, 212

McMurrich, 66

Mead, 164, 167, 216

Mellor, 25

Metschnikoff, 87, 89, 91, 92, 117, 177

Meves, 92, 96, 97, 99, 173, 183, 278

Minchin, 357

Montgomery, 364

Moore, 2

Morgan, 21, 27, 190, 311, 326

Packard, 341
Pasteur, 5
Patten, 97
Pereyaslawzewa, 92
Perez, 317
Ponder, 119
Prouho, 93

Ransom, loi, 117, 286

Redfield and Bright, 341

Reed, 320

Reighard, 189

Retzius, 213

Rogers and Cole, 114, 115

Rohde, 33

Sauerbruch, 364

Schaffer, Folkoff and Bayne-Jones, 31

Scheikewitsch, 66

Schneider, 178

Schuberg, 77

Schumacher, 88

Scott, 216

Sedgwick, 33

Selenka, 92, 93, 96, 277

Shaerer, 113

Sobotta, 99, 167, 170

Spallanzani, 188

Spek, 91

Spengel, 97

Storch, 93

3S2

INDEX OF AUTHORS

Studnicka, 33
Svedberg, 44

Tennent, 21, 57
Theel, 96, 278
Torrey, H. B., 91, 108
Torrey, J. C, 97
Treadwell, 216

Van Beneden, 92
Vejdovsky, 96, 237
Vejdowsky and Mrazek, 96
Verworn. 2

Warburg, 113, 118
Warren, 92

Watase, 267

Watson, 12

Weismann, 293

Whitaker, 194

Whitman, 12, 33, 122

Wilson, C. B., 93

Wilson, E. B., 21, 61, 97, 250, 302, 322

Wilson, J. W., 301

V. Wistinghausen, 97

Wohler, 2

Woodger, 34

Yatsu, 91, 93

Zelinka, 93

Ziegler, 91, 96, 257, 281

3S3

Index of Subjects

B

Agglutination of spermatozoa, 196
Ah'eolar theory of cytoplasmic struc-
ture, 59-63
Amia, egg of, 301
Amino-acids, 39
Amitosis, 250, 266

Amoeboid changes in eggs, 89, 93, 108
Amoeboid movements, 87

of nerve-cells, 82-85
Amphibians, ectoplasm of eggs of, 99
Aynphioxus, ectoplasm of egg of, 99

egg of, 153, 177, 201, 266, 302

fertilization in egg of, 167-170

Anastral mitotic figure, 266

Arbacia, egg of, 23, 48, 64, 108, 112,

116, 117, 126, 127, 173, 187,

192, 200, 201, 222, 224, 233,

269, 274, 27s, 277, 303, 336

fertilization in egg of, 170-171

spermatozoa of, 104
Arthropods, ectoplasm in eggs of, 98
Ascaris, egg of, 49, 153, 266
Asexual reproduction, 319
Asteracanthion, egg of, 213, 214
Asterias, egg of, 186, 188, 201, 212,

213, 214, 215, 337
Asters in cell-division, 255

of first cleavage, 182
Astral fibres, 69, 215

rays, 69
Astropecten, egg of, 215
Asynchrony of nuclear and cyto-
plasmic division, 266
Autolytus varians, eggs of, 57
Axial gradient, 301
Axis of embryo, 300

Bacteria, 31, 32, 35

ectoplasm in, 88
Bacterium, 8
Beroe, egg of, 91
Bilaterality, 301
Biogen-molecule, 5
Biological system, 17
Biologists, physico-chemical, 14
Biology, '"physico-chemical," 11
Birds, ectoplasm of eggs of, loi
Blastomeres, movement of, 335
Blastula, 301
Blue-green algae, 31, 32
Body-fluid, role of, in fertilization,

196, 198
Bonellia, egg of, 97
Brain, development of, 366
Butyric acid, in experimental partheno-
genesis, 213, 222

Cancer, 345-346, 365
Capillary spaces, 298, 344
Carbohydrates, 40

Carbon-dioxide, means for experi-
mental parthenogenesis, 212
Cell, 35

chemical composition of, 38 ff.

forms of, 37

size-variations of, 37
Cell-division, change in shape of
echinid egg during, 272

definition of, 267

in egg of Arabacia, 255 ff.

in sea-urchin eggs, 253 ff.

3^"^:

INDEX OF SUBJECTS

Cell-division, movements in the cyto-
plasm during, 267 ff.

resistance and susceptibility in,
272

surface-tension in, 270 ff.
Cell-lineage, 241, 292
Cell-membrane, 70-74
Cells, fixation of, 65-67

physical properties of, 42
Cellular oxidation, 349
Centres of first cleavage spindle, origin

of, 182
Centrifuged eggs, 306
Centriole, 69, 162, 166
Centrosomes in fertilization, 182, 183
Centrosphere, 69, 162

in cell-division, 255
Cerehratulus, egg of, 93
Chaetopterus, egg of, 64, 97, 117, 153,
177, 191, 223, 270, 297, 299,

303, 317, 341, 347
fertilization in egg of, 164-167
Chemistry of the cytoplasm, 58
Chiton, egg of, 97
Chromatin, increase of, 311
Chromidia, 56

Chromosomes, 6, 49, 50, 310
aberrant behavior of, 350-352
degeneration of, 56
incompatibility of, 351
individuality of, 321-324
number of, in parthenogenesis, 210
role of, in differentiation, 321 ff.
Ciona, egg of, 195, 201, 266
Cleavage, classification of, 291
determinate, 269
indeterminate, 269
Cleavage-amphiaster, 167
Cleavage-asters, 164

origin of, 182, 183
Cleavage-centres, 164
origin of, 182, 183
Cleavage-cycle, ectoplasmic changes
during, 281
maximum susceptibility in, 277
periods of susceptibility in, 274-
277
Cleavage-nuclei, types of, 180
Coelenterata, 300

Coelenterates, ectoplasm in eggs of, 90
Colloid, 43, 44
Colloid chemistry, 59
Conduction, 358

in living cells, 119-121
Contractility in eggs, 116, 117
Contraction, 358

in living cells, 123
Copulation, 155

in Platynereis, 189
Corymorpha, egg of, 283
Crepidula, egg of, 283
Criteria for normality, 22, 23
Cross-fertilization, 21, 57, 198
Cryptobranchus, egg of, 180
Crystals in eggs, 57
Cumingia, egg of, 98, 153, 223
Cyclops, egg of, 49, 180
Cynthia, egg of, 304
Cytolysis in eggs, 228
Cytolytic agents, 227, 232
Cytoplasm, 8

chemistry of, 58

ground-substance, 57

movements in, 43, 52, 267-270,
282, 284, 305, 306, 308

physical properties of, 58

role of, in heredity and differen-
tiation, 326-330
Cytoplasmic inclusions, 8, 52, 53 ff".,
269
role of, in differentiation, 302 ff.
Cytoplasmic reactions, 326
Cytoplasmic structure, alveolar theory
of, 59-63

filar theory of, 63

granular theory of, 63-64

D

Dehydration, 125, 244, 245

Delamination, 301

Dentalium, egg of, 97

Description, role of, in biology, 10, 25

Differentiation, in cells, 247

ecto-endoplasmic, 8, 47-49, 363

nucleo-cytoplasmic, 7, 47

surface-interior, 357

without cleavage, 216, 217, 218,
299

386

INDEX OF SUBJECTS

Dilute sea-water, unfertilized eggs in,
126

Dinophilus, egg of, 187
Drosophila, 321, 328

Echinarachnius, egg of, 57, 69, 106,
108, 109, 115, 116, 126, 173,
181, 187, 192, 199, 201, 222,

254, 273
fertilization in egg of, 171-177
Echinocardium, egg of, 192
Echinoderms, ectoplasm in eggs of,

93-96
Echinus, eggs of, 106, 192
Ectoderm, 122, 288, 366
Ectoplasm, 8

defined, 75

in bacteria, 88

in cells in tissue-culture, 83-86

in cells of multicellular organisms,

76
in eggs, 89 ff.

of amphibians, loi

of Jmphioxus, 99

of arthropods, 98

of birds, loi

of Cerebratulus, 93

of Coelenterata, 90

of echinoderms, 93-96

of flatworms, 92

of insects, 98

of lamprey, 1 00

of Lingula, 93

of mammals, loi

of nematodes, 92

of Phallusia, 98

of reptiles, loi

of Rotifera, 93

of segmented worms, 96

of selachians, 100

of sponge, 89

of teleosts, loi
in leucocytes, 86
in muscle-cells, 78-81
in nerve-cells, 81-85
in plant-cells, 88
in protozoa, 87, 88

Ectoplasm, in red blood cells, 86
in sponge cells, 75
increase of during cleavage, 331-

332

necessity of, for fertilization,
192-194

philosophical implications of
theory of, 365-366

relation of, to cell, 9

relation of, to environment, 9

role of, in differentiation, 332-339
Ectoplasmic changes due to sperm-
attachment, 104
Ectoplasmic structure of eggs revealed

experimentally, 101-102
Egg, mammalian, 302
Egg-cell, 36

Egg-fragments, development of, 53,
180, 297, 307

diploid, 318
Egg-jelly, role of, in fertilization, 19
Egg-secretions, effect of, in initiating

development, 20-21
Eggs, centrifuged, 306

colors of, 57

ectoplasm in, 89 ft".
Electron-supermicroscope, 26
Endoderm, 288
Endoplasm, 8, 48

definition of, 76
Endoplasmic buds, 192
Ensis, egg of, 98
Entrance-cone, 177, 178
Epiboly, 301
Ethics, 367
Exoplasm, 75, 88, 89
Experiments, control of, 22

in biology, categories of, 16

on eggs, 17

on the living system, 16

on tissues in vitro and in situ, 19
Extra-ovates, 24, 336, 337

Fertilizability of egg of Amphioxus, 188
of Arbacia, 187
of Asterias, 186
of Fundulus, 189

3^7

INDEX OF SUBJECTS

Fertilizability of egg of Nereis, i86

of Platynereis, 189
Fertilizability, seat of, in eggs, 191
Fertilizable condition of eggs, 186
Fertilization, as chemical reaction, 203

Boveri's theory of, 182

classes of eggs in, 153

inhibition of, 196

phases of. 179, 231

phenomena common to, 177

specificity in, 197-198

straight, 21

superficial cytolysis — corrective
factory theory of, 23c
Fertilization-cone, 159 ff., 178
Fertilizin, 195-197
Filaments, ectoplasmic, 79, 80, 85,

102, 105, 145, 277-280
Filar theory of cytoplasmic structure,

63
Fixation, 16

of cells, 65-67
Flatworms, ectoplasm in eggs of, 92

Gastrula, 213, 300

Gastrulation, 193, 301

Gene, 7

role of, in heredity and differentia-
tion, 326

Gene-molecule, 6—7, 323

Genes, linear arrangement of, 322

Gene-theory of heredity, 7, 325

Genetic restriction, 313, 320

Genetics, 8, 321

Germ cells, 147, 249, 316

Germinal vesicle, 131, 153, 316

Golgi bodies, 55

Gradient, axial, 301

Granular theory of cytoplasmic struc-
ture, 63-64

Ground-substance, 37, 57-58, 64, 67,

365

dehydration of, 245

in nucleus, 70

radiating structures in, 68-70

structure of. 64
Growth, 248
Gyrodactylus, egg of, 153

H

Haemolytic agents, 227, 232
Heat-liberation of eggs, 115
Heat-production in eggs, 1 14-11 5
Hermaphroditism, 155, 156, 329

in starfish, 213
Hetero-agglutination of spermatozoa,

197
Heteronereis phase, 186
Hormones, integration by, 122
Hyaline plasma-layer in sea-urchin
eggs, 95-96, 107, 171, 257,
277
Hydration and dehydration, of yolk-
spheres, 135
role of lipoid in, 135
Hydrogen-ion concentration, effect of

changes in, on eggs, 340
Hyd-rophilus, egg of, 301
Hypotonic sea-water, changes of eggs
in, 130
development of eggs in, 130
means for experimental parthe*

nogenesis, 129
susceptibility of eggs to, 108-

113

Hypotonicity, limits of, 127-129

I

Independent irritability, 237

Indicia, physiological, for normality,

23

Individuality of the chromosomes,
322-324

Inhibition of fertilization by body-
fluid, 198

Initiation of development in eggs, 206-
207

Insects, ectoplasm in eggs of, 98

Integration of cells, 121-123, 363

Intercellular connections, JJ, 120, 335
fibres, 7J, 78

Invagination, 193, 301

Isolated blastomeres, 318

K

Kropotkin theor}-, 367

3S8

INDEX OF SUBJECTS

Lamprey, ectoplasm of egg of, loo

Life-molecule, 5

Linear arrangement of the genes, 322

Lingula, ectoplasm in egg of, 93

Lipins, 39, S3, 54

Living and non-living, similarities in,

IS
Living things, limitation of investiga-
tion, 3
organization of, 2
physico-chemical systems, i
Loligo, egg of, 301
Lunar periodicity, 158
Lysin, 232

M

Mactra, egg of, 97, 153, 223

Mammals, ectoplasm of eggs of, loi

Mathematics, use of in science, 25

Maturation, 148

Mayonnaise, 62

Measurements in biology and physics,

3
Mechanistic, use of term in biology, 14

Median plane, 300

Medicine, 364

Medusa-egg, 177

Membrane, 70-74, 140

nuclear, 50

plasma, 72, 140

precipitation, 73

semi-permeable, 140

synaptic, 120

vitelline, 72, 140
Mevibranipora, ectoplasm in egg of, 93

egg of, 203
Mendel's laws, 321
Merogony, 180, 297, 315, 318
Mesoderm, 288
Methods of investigation in biology

and physics, 3
Micropyle, 201 \—[X>
Middle-piece of spermatozoon, 171 ff.
Migration of cells in tissue-culture,

85-86
Mitochondria, 56

Mitosis, 250

Mitotic figure, 59, 240

Models in biology, 16, 62, 271

Monaster-stage, 341, 351

Morula, 301

Multicellular organism, history of, 36

Muscle-cells, ectoplasm in, 78-81

Mutations, 321, 340, 352

Mytilus, egg of, 97-98

middle-piece of sperm of, 183
Myzostoma, egg of, 153

N

Nematodes, ectoplasm in eggs of, 92

Nephritis, 139, 365

Nereis, egg of, 24, 53, 54, 55, 56, 59,
64, 97, 102, 108, 113, 116,
129, 134, 135, 138, 139. 153,
177, 186, 194, 19s, 233. 231,
269, 278, 303, 309, 317,

341, 347

Nerve-cells, ectoplasm in. 81-85

Nerve-systems, 358
Non-mechanistic, 14
Normal egg, 177
Normal living organism, 17
Nuclei, size of, 49
Nucleic acid in bacteria, 31
Nuclein, 312
Nucleolus, 50
Nucleo-protein, 41, 51
Nucleus, 8, 31, 51

chemistry of, 51

form of, 49

ground-substance in, 70

origin of, 357

resting, 51

role of, in differentiation, 308 ff.
Nutrition, 139, 358

animal and plant. 360

O

Oedema, 139, 365

Oil, function of, in cell, 54

Oniscus, 66

Opalina, 33

/

J)

'■^#

3S9

INDEX OF SUBJECTS

Organism, relation to environment,

226, 356
Organismal conception, 33
Organism-as-a-whole, 33
Organization, 14

of living matter, 2, 7

of protoplasm, 7
Organizator theory, 239, 290
Origin of species, 360-361
Osmotic method for experimental

parthenogenesis, 219
Oxidation, cellular, 118-119, 349
Oxygen-consumption in eggs, 113-114

Pancreas, 122
Paramoecium, 71, 323
Parechinus, egg of, 173
Patella, egg of, 21, 97
Parthenogenesis, experimental, butjT-
ic acid in, 221
chromosomes in, 210
definition of, 240
effect of hypertonic sea-water

in, 218
effect of hypotonic sea-water

in, 218
effect of strong hypertonic

solutions in, 224
effect of temperature in, 218
effect of ultra-violet radiation

in, 218
In egg of Amphitrite, 216
of Arbacia, 219
of Asterias, 2 1 2 ff .
of Chaetopterus, 216
of Cumingia, 216
of frog, 211
of Mactra, 2 1 7
of Nereis, 217
of Podarke, 216
of Strongylocentrotus, 219
of Thalassema, 217
lysin-corrective factor method

in, 221
osmotic method for, 219
superficial cytolysis theory of,
227

Parthenogenesis, natural, chromo-
somes in, 208
facultative, 208
fixed, 208

stages of maturation of eggs in,
211

Parthenogenetlc egg, haploid, 318

Pedicellina, egg of, 203

Pediculopsis, egg of, 180

Perivitelline space, 72, 105, 108, 162,
203, 277

Petromyzon, egg of, 100, 169

Phagocytosis, 86-87, I4S> H^

Phallusia, egg of, 98, 153, 266

Phascolosoma, egg of, 96, 153

Physical properties of the protoplasm,
42 ff.

Physical science, 364

Physico-chemical biology, 1 1
means, 15

Physics and chemistry, in biology, 17

Physics, concepts of, 12

Pigment granules, 57, 269

Plant-cells, ectoplasm in, 88

Platynereis, egg of, 20, 24, 54, 55, 56,
69, 72, 102, 139, 191, 19s,
200, 201, 278
description of egg of, 47

Pluripotency, 296, 315

Polar bodies, 148

nuclei of, in parthenogenesis,

210
suppression of, 342 ff.

Polarity in eggs, 300

Polyembryony, 297, 317

Polyenergid, 35

Polyploidy, 20, 319

Polyspermy, 199-203

Polystomum, egg of, 153

Porifera, 300

Potencies, of the egg, 327

of the spermatozoon, 327

Primitive protoplasm, 355

Propagation of effect of sperm-attach-
ment in eggs, 1 16

Prostheceraeus, egg of, 153

Protein, 39

Proteins and specificity, 41

Protoplasmic organization, 9

390

INDEX OF SUBJECTS

Protoplasmic structure, theories of, 59
Protoplasmic system as life-unit, 37
Protozoa, 33, 36, 358

ectoplasm in, 87. 88
Psammechinus, egg of, 57
Pseudopodia, ectoplasmic, 81, 82-85,
89, 103

r~\

Q

Quantitative biology, 3, 5, 25, 238

R

Radiations as experimental means, 348
Radium, 341
Red blood cells, 86

Red blood corpuscles, 36, 54, 71, 119
Refractive index, 44
Regeneration, 248, 320
Reptiles, ectoplasm in eggs of, loi
Resistance and susceptibility in cell-
division, 272
Respiration, 358, 359
Resting nucleus, 51
Rhabditis, egg of, 180, 181, 206
Rhynchelmis, egg of, 97
Roentgen-rays, 341, 349
Rotifera, ectoplasm in eggs of, 93

Sahellaria, egg of, 102

Saccocirrus, egg of, 187

Sagitla, egg of, 153

Salinity, effect of changes In, 340

Sciara, egg of, 351

Sea-urchin, skeleton of larva of, nor-
mal variation, 21

Segmented worms, ectoplasm in eggs
of, 96

Selachians, ectoplasm in eggs of, 100

Self-differentiation, 9, 309

Self-fertilization, 155

Self-regulation, 9, 104, 238 364

Semi-permeability, 139-146

Sex-differentiation, 329

Somatic cells, 147

Specific gravity, 44

Specificity. 41

Specificity in fertilization, 197-198,

2C4

Sperm-amphiaster, 167
Sperm-aster, 163
Spermatozoa, origin of, 149

structure of, 151
Spermatozoon, middle-piece of, 171 ff.

of Ascaris, 152

of Cerebratulus, 151

of Nereis, 1 5 1
Sperm-centrosome, 163
Sperm-nucleus, 163
Sperm-rotation, 162, 172
Spindle-threads, 69
Sponge, ectoplasm in egg of, 89
Starfish, egg of, 154
Strongylocentroius, egg of. 106, 192,

304, 30s

Sub-normal eggs in experiment, 19

Surface-cytoplasm, 8

Surface-tension, 283

in cell-division, 270 ff.

Susceptibility during membrane-sepa-
ration, 109 ff., 282

Synaptic membrane, yj, 120

Syncytia, 33

T

Teleosts, ectoplasm of eggs of, loi
Temperature, as experimental means,
20, 218

effect of change in, on eggs, 340
Thalassema, egg of, 97, 153, 223
Thysanosoon, egg of, 153
Tissue-culture, cause of migration of
cells in, 85-86

cells in, 252

ectoplasm in cells in, 83-86
Tissues, behavior of, under laboratory

conditions, 18
Trematode eggs, 183, 305
Trophoblast, 302
Tubifex, egg of, 317
Tumors, 43, 249, 320

U

Ultra-centrifuge, 39
Ultra-filterable viruses, 32

301

INDEX OF SUBJECTS

Ultra-microscope, 59
Ultra-microscopic organisms, 356
Ultra-violet light, 20

effect of, on egg of Nereis,
218,341-344, 34^347
Unicellular organism, 35, 249
Union of egg- and sperm-nuclei, modes
of, 179 ff.

V

Viscosity, 44-46

Vitalism, 14

Vitelline membrane, 140

W

Water, component in vital reactions,
125
distribution of, during cleavage,

330-331
in cells, 41

movement into and out of cells,

139

structural part of living system,
124

Water-drops, 126, 132

and protoplasmic structure, 137
effect of temperature in producing

them, 136
formed by ultra-violet light, 136
hypertonic sea-water and, 136
in normal eggs, 137
in nucleus, 134
rate of formation of, 134
rhythmical phenomenon, 137

Water-level in cells, 143, 243-245

White blood cell, 365

X

X-ray spectroscopy, 39
Y

Yolk, S3, 54

role of, in development, 54-55
Yolk-spheres, fusion of, 131

polyphasic, 55

Zygote nucleus, 164

392

iQf