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FERTILIZATION

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Plate I

A live fertilized mouse egg, showing the whole spermatozoon in
the cytoplasm. Positive phase contrast, ■ 770. Photograph by

J. Smiles.

ERTILIZATION

LORD ROTHSCHILD

G.M., Sc.D., F.R.S.

LONDON : METHUEN & CO. LTD
NEW YORK : JOHN WILEY & SONS, INC

First published in 1956

CATALOGUE NO. 5807/u (mETHUEN)

PRINTED AND BOUND IN GREAT BRITAIN BY
RICHARD CLAY AND COMPANY, LTD., BUNGAY, SUFFOLK

PREFACE

This book is intended for those who have read, or are reading,
Gray's Experimental Cytology, Heilbrunn's Outline of General
Physiology, Fruton & Simonds' General Biochemistry, Hober's
Physical Chemistry of Cells and Tissues and similar textbooks. The
subject is the life of the egg from the attachment of the fertilizing
spermatozoon to the fusion or apposition of the male and female
pronuclei. This process, except in mammalian eggs, usually takes
a little less than one hour. Even so, several important subjects have
had to be omitted: some of these are: (i) Fertilization in the decapod
Crustacea and in sponges. Both of these are too far removed from
'normal' fertilization to be included in a comparatively short book ;
but there are excellent accounts of them by Bloch (1935) and Tuzet
(1950). (2) Asters and the origin of the first cleavage amphiaster.
Much has been written recently on these, apart from the relevant
sections in some of the textbooks mentioned above. (3) Andro-
genesis and Gynogenesis. (4) Merogony. (5) Parthenogenesis. A
comprehensive review of parthenogenesis has been published by
Tyler (19416); but reference to the General Index will show that
the subject is occasionally mentioned. (6) Fertilization in the plant
kitigdojn. Although two chapters are devoted to this subject, its
treatment is far from systematic.

The scope of this book precludes any discussion of cleavage,
which is frustrating; not only because cell division is such a
dominatingly important subject, but also because important papers
such as Brachet's Constitution anorniale du noyau et metabolisme de
Vemhryon chez les Batraciens (1954) cannot be considered.

References. Modern reviews sometimes consist mainly of a list
of papers with little or no attempt at evaluation. Even if this
practice served some useful purpose it would be inappropriate in
a short book. The papers referred to represent a limited selection
from the immense number on fertihzation written during the last
hundred years and, as a rule, I have excluded the following: (i)
references to work which has recently been repeated, under more
modern conditions. But attention is sometimes called to early
papers on subjects in which there has been a revival of interest,
such as cortical granules and the effect of calcium on the hardening

VI PREFACE

of the fertilization membrane, it having been forgotten or ignored
that these were first described some forty years ago. (2) References
to brief and scrappy papers which have not been followed up.
Some exceptions to this rule will be found in chapter 7, Metabolic
and Other Changes at Fertilization. (3) References to papers which
I do not think good. Where work has been, or might be, wrongly
accepted as true, I have drawn attention to the errors in it. But, in
general, such papers have not been mentioned.

Every writer of a book on fertilization must be uncomfortably
aware of his sins of omission and commission, so great is the
labour imposed by the literature on the subject. The author is no
exception and proffers his apologies.

Index of Plants and Animals. There are three columns in this
index. The first gives the name of the organism, some of the
familiar synonyms and the English or American names, when
known. The second column states the order and class to which
the animal or plant belongs. When I was a child, my father
expected my sisters and myself to know the Latin names of
the plants, bees and butterflies which we had to collect. It
was inevitable therefore that an Index of Plants and Animals
should figure in this book. But there was a more cogent
reason. Reference will be found in several places to the specificity
of fertilization, to the alleged specificity of the polysaccharides
in egg jelly, and to interspecific, intergeneric and interphyletic
cross-fertilization. We cannot think clearly about such subjects,
nor describe and compare experiments relating to them, unless
we are reasonably sure of the identity of the organisms concerned.
Reference to the Echinoid synonyms shows that this is not always
easy. A diverting example of the confusion which springs from
careless nomenclature is to be found in a paper by Mitchison &
Swann (19546), which is discussed, for other reasons, in chapter
8. These authors measured the elastic modulus of the cortex of the
unfertilized egg of the sea-urchin Arhacia lixula (Linn.). With the
aid of their own and E. N. Harvey's measurements (193 1), they
calculated the tension at the surface of the unfertilized egg of an
'American species of Arhacia' (p. 469), which they refer to as
Arbacia pustidosa. Arbacia lixula (Linn.) and Arbacia pushilosa
(Leskc) arc synonyms for the same sea-urchin, although Harvey
actually used the eggs of Arbacia punctulata (Lamarck) in the
experiments in question.

PREFACE Vll

Some Latin names of organisms are abbreviated after they have
once been mentioned. If a reader is in doubt, reference to the
Index of Plants and Animals will provide the full name. Its prepar-
ation was made very much easier by the assistance of Sir Gavin de
Beer, F.R.S., and Dr H. W. Parker, of the Natural History
Museum, London.

^Theories' of fertilization and activation. The desire to formulate
a new theory of fertilization seems almost to be an occupational
disease of the gametologist. Such theories have been connected,
at one time or another, with the names of Boveri, Bataillon, Dalcq,
Delage, Ileilbrunn, F. R. Lillie and Loeb. At the present time we
have gone too far — and yet not far enough — to formulate theories,
or even to make an 'Attempt at a Comprehensive View', as Runn-
strom tried to do in 1949. One function of a new theory is to pro-
voke further experiments and, although I have no new theory of
fertilization to offer, I hope that this end, at least, will be achieved.

Acknowledgmetits. I am particularly indebted to Professor Sir
James Gray, F.R.S., who has been my mentor for twenty-five
years; to Dr George Beadle, Chairman of the Biology Division of
the California Institute of Technology, for his help and for his
hospitality at 'Caltech', where most of this book was written; to
Professor Albert Tyler for valuable advice and criticism, not all of
which has been taken; and to the Medical Research Council for
financial aid. In addition I am glad to record my thanks to the
following scientists and non-scientists for their help and advice:
Dr R. D. Allen; Dr C. R. Austin; Dr J. Beament; Miss G. Bend-
ing; Prof. J. Brachet; Miss M. Brewster; Dr R. R. A. Coombs;
Prof. E. G. Cox, F.R.S.; Dr G. Fankhauser; Prof. L. V. Heil-
brunn; Prof. A. L. Hodgkin, F.R.S.; Mr A. F. Huxley, F.R.S.;
Dr G. W. Kenner; Dr M. E. Krahl; Dr T. R. R. Mann, F.R.S.;
Dr R. Markham; Dr R. E. F. Matthews; Dr J. M. Mitchison; Dr
F. Moewus; Prof. A. Monroy ; Prof. C. Niemann; Prof. L. PauUng;
the Hon. Miriam Rothschild; George Rylands; Prof. E. C. Slater;
Prof. M. M. Swann; Dr E. Vasseur; and Prof. L. Zechmeister.

CONTENTS

Preface v

1 Morphology of Fertilization i

2 Sperm-egg Interacting Substances, I 21

3 Sperm-egg Interacting Substances, II 39

4 Sperm-egg Interacting Substances, III 51

5 Metabolism of Eggs, I 56

6 Metabolism of Eggs, II 69

7 Metabolic and Other Changes at Fertilization 80

8 Structural Changes at Fertilization 91

9 Polyspermy 103

10 Bioelectric Measurements 124

1 1 Specificity 136

12 Conclusion 141
References and Author Index 143
Index of Plants and Animals 154
General Index 162

Vlll

PLATES

I A live fertilized mouse egg, showing the whole sperma-
tozoon in the cytoplasm. Photograph by J. Smiles.

frontispiece

II Fertilization of the egg of Urechis caupo. Tyler (1932).

facing page 18

III Agglutination of a 2% suspension of spermatozoa of

Megathtira cremdata in the presence of homologous

egg water. Tyler (19406). 22

IV Dissolution of egg membrane of Megathura crenulata

by an extract of a i % homologous sperm suspension.
Photograph by A. Tyler. 36

V The two sorts of flower of Forsythia X intermedia.

Photograph by Royal Botanic Gardens, Kew. 52

CHAPTER I

THE MORPHOLOGY OF FERTILIZATION

Fertilization is the incitement of an egg to development by a
spermatozoon, together with the transmission of male hereditary
material to the egg. At fertilization the spermatozoon contributes
a, the stimulus for development; b, a set of chromosomes em-
bodying the paternal contribution to the genetic make-up of the
zygote ; and c, a central body which gives rise to, or is concerned
with, the machinery for cell division. In some cases the sperma-
tozoon, according to its point of entry into the egg, also determines
the plane of bilateral symmetry of the embryo. Fertilization is
specific and crosses between different sorts of animals are almost
always impossible. Apart from a few exceptional cases to be dis-
cussed later, fertilization is irreversible. Once an egg has been
fertilized, it cannot be re-fertilized, and once an egg has been
stimulated to develop parthenogenetically, fertilization cannot be
superimposed on parthenogenesis.

Fertilization can be divided into two phases. The first occurs
when the homologous spermatozoon collides with and becomes
attached to the egg surface. This is sufficient to set off a train of
reactions in the egg which may lead to development. This first
phase is called activation and one talks about a spermatozoon
activating an egg or an egg being activated by a parthenogenetic
agent. The spermatozoa of the worm Rhahditis monohystera
Biitschli activate eggs of the same species so that they develop
'parthenogenetically', without containing any male hereditary
material (Peacock, 1944). This phenomenon is known as pseudo-
gamous fertilization and it can be achieved experimentally, by
mixing homologous eggs and spermatozoa and separating them
after a short time (F. R. Lillie, 19126; Rothschild, 1953), or by
heterologous insemination. Bataillon, for example, observed in
1909 that the spermatozoa of Triturus alpestris (Laurenti) activated
the eggs of Pelodytes punctatiis (Daudin) pseudogamously and it
was this observation which led him to carry out his famous ex-
periments on the parthenogenetic activation of frogs' eggs by
puncturing them with fine glass needles. Both Loeb (1913) and

2 FERTILIZATION

Godlewski (191 2) made similar observations following heterologous
insemination.

The second phase of fertilization is concerned with the events
which take place after the spermatozoon has entered the egg,
culminating in the disappearance of the sperm head and the egg
nucleus as separate entities. Strictly speaking, therefore, fertiliza-
tion begins with the sperm-egg collision and ends with syngamy,
the fusion or apposition of the germ nuclei, when the spermatozoon
loses its individuality. This series of reactions may take less than
an hour; but the student of fertilization inevitably finds himself
asking questions about the pre-fertilization behaviour of eggs and
spermatozoa, the domain of the gametologist, and about the
activity of the egg after syngamy, the domain of the embryologist.
Examination of the pre-fertilization behaviour of the gametes must
accompany any study of fertilization and this may well seduce the
student away from his intractable problem. Mention has been
made of spermatozoa colliding with eggs ; why should they be any-
where near each other ? Nature answers this question in bewilder-
ing and fantastic ways : the archegonia of plants produce chemicals
which attract spermatozoa; dogfish and human beings rely on
copulation to ensure sperm-egg collisions. Provided there is no
moon, the male Platynereis megalops (Verrill) deposits spermatozoa
in the mouth of the female, which bursts in about six seconds,
liberating fertilized eggs into the sea (Just, 1914). Some further
aspects of this problem, the liberation of spermatozoa and eggs in
the right place and at the right time, are discussed in later chapters.

Maturation. The condition of the egg before fertilization, par-
ticularly as regards the stage of maturation it has reached, should
always be borne in mind when trying to gain some understanding
of fertilization. It has been insufficiently emphasized that echino-
derm eggs, on which so many experiments have been carried out,
are in an exceptional condition from the point of view of matura-
tion, at the time of fertilization. Fig. i, which is adapted from
Dalcq (1952), explains this point. In sea-weeds, coelenteratcs,
and echinoderms, and not all of them, the egg is fertilized after
maturation (Class 4 fertilization). In all vertebrates and Branchio-
stoma, fertilization takes place at the second maturation meta-
phase (Class 3 fertilization), though there is some evidence that
fox, dog and horse eggs may be fertilized in the germinal
vesicle stage (Pearson & Enders, 1943; van der Stricht, 1923;

THE MORPHOLOGY OF FERTILIZATION 3

Hamilton & Day, 1945). In the eggs of Ciona, Chaetopterus,
Cumingia and Mytilus, fertilization occurs at the first maturation
metaphase (Class 2); while in sponges, Nereis, Spisula, Urechis
caiipo Fisher & MacGinitie (Plate II), Ascaris and Sagitta, the egg

Class 3

Class 2

Class /

Oocyte Ut. Maturation 2nd.Maturation

metaphase metaphase

Ootid

Fusion of
pronuclei

FIG. I. — The four stages of egg maturation at which fertilization occurs in the
animal kingdom, after Dalcq (1952).

is fertilized before the germinal vesicle of the oocyte has broken
down, that is before either maturation division (Class i).* These
four classes should be remembered when studying fertilization,
as we are sometimes tempted to make generalizations based on

* Needless to say, there are exceptions to this classification, e.g. starfish eggs,
which come into Classes i, 2, 3 and 4.

4 FERTILIZATION

the behaviour of eggs in one class when those in other classes be-
have differently. There is, admittedly, a tendency for the sperm
nucleus to remain relatively quiescent within the egg until after
the formation of the second polar body; but this is not a sufficient
reason for ignoring the fact that in the majority of phyla, fertili-
zation does not occur at the same time in the life-history of the
unfertilized egg as it does in sea-urchins. We shall return to this
question when considering the metabolism of fertilized and un-
fertilized eggs.

Jelly and membranes. In some cases, there are what appear to be
barriers between the egg and the spermatozoon. In echinoderms
and frogs, for example, this barrier takes the form of a gelatinous
shell round the unfertilized egg, through which the spermatozoon
must bore or burrow to reach the egg surface. The egg of the
salmon is surrounded by a rather tough chorion, which is im-
permeable to spermatozoa except at one point, the micropyle; this
is a narrow channel in the chorion, through which spermatozoa
must pass to reach the egg. When an unfertilized salmon egg is
put into fresh water, the chorion hardens, the micropyle becomes
occluded and the egg is unfertilizable. This is one of the reasons
why breeders of trout and salmon mix eggs and spermatozoa 'dry',
before dilution with fresh water, though not all of them realise
that the success of 'dry' insemination is due to the egg micropyles
remaining open in the presence of seminal plasma as opposed to
fresh water. The other reason for mixing salmon or trout eggs with
their respective semen in the 'dry' condition is because the sperma-
tozoa of these fish only live for a few minutes after dilution with
fresh water.

Many insect eggs are surrounded by hard and, one would have
thought, impermeable egg shells, which contain several so-called
micropyles. Insect spermatozoa, however, do not necessarily make
use of these structures, which are often too small for the passage
of a spermatozoon, and in many species, fertilization occurs before
the egg shell is laid down. Insect spermatozoa are sometimes said
to contain enzymes capable of dissolving or softening up egg shells.
They can penetrate thin wax layers round the egg (Beament, 1946) ;
but a careful perusal of Cragg's interesting paper (1920) on copula-
tion in Cimex lectiilarius Linn, shows that the claim that bed bug
spermatozoa can 'burrow' through chitin is less certain than has
sometimes been thought. E. B. Wilson (1928) noticed that the

THE MORPHOLOGY OF FERTILIZATION 5

spermatozoa of Cerebratulus [lacteus Verrill ?) pay no attention to the
so-called micropyle present in the membrane round the eggs of this
nemertine, and can reach and fertilize the egg at any point on the
surface. The unfertilized egg of Megathura crenulata (Sowerby) is
surrounded by a tough membrane which is distinct from the
surface of the egg proper, and the same applies to human eggs, in
which the enveloping membrane is called the zona pelhicida.
Spermatozoa get through these barriers with the help of enzymes
located in their heads. Finally, most mammalian eggs are sur-
rounded by follicle cells, in two layers; the innermost of these con-
sists of densely packed, radially arranged cells and is known as the
corona radiata. Outside this there is a layer of sparsely distributed
cells, the cumulus oophonis. The enzyme hyaluronidase, contained
in or on the surfaces of most mammalian spermatozoa, assists in
the dissolution or depolymerisation of the intercellular cement,
hyaluronic acid, by which the follicle cells are stuck to the un-
fertilized egg surface.

Cortical change. Having got through these 'barriers', the sper-
matozoon becomes attached to the surface of the unfertilized egg.
After attachment, the sperm tail may continue to move quite
vigorously, though in other cases, it sticks out from the egg sur-
face, motionless. The first visible reaction of the egg to the attach-
ment of the fertilizing spermatozoon — spermatozoa quite often
become attached to eggs but fail to fertilize them — is a change in
cortical structure, which, starting at the point of sperm attachment,
passes completely over the egg surface. The time relationships of
this reaction are discussed in chapter 9, Polyspermy. According to
J. C. Dan (1950^, p. 402), this change in cortical structure is 'a
visible wave which travels around the egg at speeds varying with
the species, . . .'. 'In the relatively fluid eggs oi Mespilia [globulus
(Linn.)) the passage of this wave is especially striking; it causes a
slight deformation of the surface layers of the egg, which gives the
impression that some sort of tension is being progressively re-
leased, or that a local band of contraction and expansion is passing
around the egg.' This wave of so-called contraction has been
observed by numerous students of fertilization, but it is doubtful
whether the word 'contraction' is apposite or even desirable, except
in special cases such as that of the brook lamprey, Entosphenus
lamot t enii (Lesueur), which does contract after fertilization (Okkel-
berg, I9i4)> or in the case of mammalian eggs. The German word

6 FERTILIZATION

Schrumpfung (wrinkling), roughening, granulation, or simply
cortical change are nearer the facts. Moser (1939a) examined this
reaction in the eggs of Arbacia punctulata. He found that a layer of
cortical granules immediately below the plasma membrane,
diameter o-8/x, disappeared at fertilization, the disappearance start-
ing at the point of attachment of the fertilizing spermatozoon and
passing progressively over the egg surface, in about 10 seconds at
26° C. A breakdown of cortical granules in the eggs of Sahellaria
vulgaris Verrill, 5-10 minutes after fertilization, was described in
the same year by Novikoff (1939). Moser's studies were followed
up by Endo (1952), who observed that at fertilization, the cortical
granules, of which there are about o-Sjfjr in the eggs of Clypeaster
japoniciis Doderlein, doubled their diameters and then exploded.
Just before they disappear, sea-urchin egg cortical granules, which,
according to Monne & Harde (1951), contain polysaccharides
esterified with sulphuric acid residues, exhibit Brownian move-
ment, which suggests that at this time, the cortex becomes more
fluid (Allen, 1954). A similar phenomenon occurs when fish eggs
and those of the marine worm Nereis succinea (Leuckart) are
fertilized, though in these, alveoli in the cortex break down pro-
gressively after fertilization (Yamamoto, 1944; Kusa, 1953;
F. R. Lillie, 1919). In addition, Kusa (1954) has shown that the
cortical alveoli in the egg of the dog salmon, Oncorhyiichus keta
(Walbaum), contain mucopolysaccharides esterified with sulphuric
acid residues. As regards the cortical response to fertilization,
there is, therefore, a marked chemical and morphological re-
semblance between fish and echinoderm eggs. But, as we shall see
later, it would at present be dangerous to ascribe too important or
dominating a role to exploding cortical granules or discharging
cortical alveoli in fertilization.

There has been some misunderstanding (Allen, 1954), perhaps
of a verbal nature, about the disappearance of the cortical granules
and the change in the light-scattering properties of the egg surface
at fertilization, when viewed with dark-ground illumination. There
is no doubt that the cortical granules disappear, but at the same
time, the cortex becomes more granular, or roughened. Rothschild
& Swann (1949) suggested that this granulation, which is associated
with an increase in light scattering, might be due to the formation
of microscopic or sub-microscopic particles at the egg surface.
The appearance of this granulation naturally does not imply that

THE MORPHOLOGY OF FERTILIZATION 7

the cortical granules remain unchanged after fertilization; Both
phenomena occur and are intimately related to each other. The
disappearing cortical granules are concerned in the formation of a
structure which appears round some eggs after fertilization, the
Fertilization Membrane (q.v.).

Fertilization cone. After attachment of the spermatozoon, a
conical hyaline protuberance, the fertilization or entrance cone,
appears at the egg surface. Fig. 2. In the eggs of Psammechiniis
miliaris (P. L. S. Muller), the fertilization cone disappears in less

FIG. 2.

Fertilization cone

-Entry of the spermatozoon into the egg of Patiria pectinifer (J.
Muller & Troschel), after J. C. Dan (1950a).

than 20 seconds at 18° C, but in other eggs it may persist for much
longer. In the case of the egg shown in Fig. 2, for example, the
fertilization cone is visible until the tail of the spermatozoon has
passed into the egg cytoplasm, after which it is more or less re-
sorbed into the egg.

Sperm-egg filaments. In 1877 Fol reported that the starfish
spermatozoon became connected to the surface of the egg by a
long, exceedingly thin filament, which he believed was an extension
of the fertilization cone. This observation was confirmed by R.
Chambers in 1923, contradicted by Just in 1929 and reaffirmed by
Horstadius in 1939. Similar claims, that filaments derived from

8 FERTILIZATION

the egg pull the spermatozoon towards the egg surface, have been
made elsewhere; for example, Colwin & Colwin (1949) reported
that a thread-like structure connected the fertilizing spermatozoon
to the fertilization cone in the egg of Saccoglossiis kowalezoskyi (A.
Agassiz), while Monroy (1948) refers to the fertilizing spermato-
zoon of Pomatoceros triqueter (Linn.) being connected to the egg
surface by a thread. In the case of the starfish spermatozoon,
J. C. Dan (1954) has shown that in certain circumstances, a thin
filament, about 25 yu, long and 0-13 /x in diameter, can be observed
protruding from the front end of the head. Although immature
eggs respond to insemination, and therefore to sperm-egg collisions,
by emitting filament-like structures (E. B. Harvey, 1938), Dan's
work leaves little doubt that Fol, R. Chambers and Horstadius
were wrong in thinking that the starfish egg responds to a nearby
spermatozoon by emitting a filament which joins the egg to the
spermatozoon and pulls the latter towards the egg surface. The
presence of long filaments on the front ends of sperm heads may
be of wider incidence than has hitherto been realised. Rothschild
& Tyler (1955), for example, have reported their occurrence in the
spermatozoa of Echinocardiimi cor datum (Pennant), Mytilus edulis
(Linn.), Strongylocentrotus purpuratus (Stimpson) and Lepido-
chitona cinerea (Linn.). There are, however, some spermatozoa,
e.g. those of the bull and ram, in which such filaments do not exist.
The subject of acrosomal filaments and their role in fertilization is
still very much in its infancy. In a recent paper, J. C. Dan (1955)
has adduced convincing evidence that the spermatozoa of Japanese
sea-urchins eject acrosomal filaments in the presence of sea water
in which eggs of the same species have been standing, though the
reaction does not occur if the calcium content of the medium is
reduced. Do some spermatozoa always have acrosomal filaments
on their heads and others only after responding to some stimulus ?
Further experiments are needed to resolve this interesting and
important question, which has been brought into prominence
mainly through the work of J. C. Dan.

Fertilization membrane. Unfertilized echinoderm eggs are sur-
rounded by a vitelline membrane outside the plasma membrane.
Fig. 3, At fertilization and shortly after the cortical change, the
vitelline membrane separates from the egg surface, the separation
starting at the point of sperm attachment and passing progressively
over the egg surface (Kacscr, 1955). After this, the vitelline mem-

THE MORPHOLOGY OF FERTILIZATION 9

brane becomes known as the fertilization membrane, which is
about 500 A thick (Mitchison, 1953). As will be seen from an
examination of Fig. 3, the cortical granules which disappear at

^i^arrr?:;■:{■^;^^>.~^^■■;^:v■^*-;u■•^''■'^'vt??S?''V

(a) imsn^§Mm

:CKXiaO£H>Q.

■g

(b) l:^:^ff^fp}p^^W§M

(c)

(d)

(e)

-^ ^

-/
-h

FIG. 3. — Formation of the fertilization membrane in the sea-urchin egg, after
Endo (1952). a. Unfertilized egg; b, extrusion of cortical granules; c, ad-
hesion of cortical granules to vitelline membrane; d, further transformation
of fertilization membrane ; e, completely transformed fertiUzation membrane.
V, vitelline membrane; /), plasma membrane; g, cortical granules; h, hyaline
layer;/, fertilization membrane. Note. Diffraction effects at the surface of a
large egg make it extremely difficult to distinguish by optical methods
closely apposed layers which are less than 1-2 n thick.

fertilization in fact fuse with the inner surface of the vitelline
membrane, a phenomenon which was first systematically examined
by Motomura (1936, 1941), though Just observed the escape of
granules from the cortex, their appearance in the perivitelline
space, and possibly their incorporation into the fertilization

10 FERTILIZATION

membrane, as early as 19 19.* Endo (1952) has published some re-
markable photographs of cortical granules adhering separately to
the inner surface of the fertilization membrane of Clypeaster eggs.
The space between the fertilization membrane and the surface of
the egg is called the perivitelline space, an unfortunate term as the
vitelline membrane is outside this space, not inside it. Globular
isotropic cortical granules can sometimes be seen in the perivi-
telline space, where they may undergo a spontaneous transforma-
tion into positively birefringent rod-shaped particles. If unfertilized
eggs are treated with trypsin and then fertilized, these rod-shaped
particles are clearly visible. The fusion of the transformed cortical
granules with the vitelline membrane is responsible for its harden-
ing and transformation into the fertilization membrane, which
takes place during the first ten minutes after fertilization. Calcium
ions and a third factor which can be extracted from eggs are also
concerned in the hardening or 'tanning' of the fertilization mem-
brane (Motomura, 1950, 1954; Runnstrom, 195 1). The properties
of the fertilization membrane have been studied in great detail
under a variety of environmental conditions by Runnstrom and
his colleagues. A detailed review of this subject will be found in
The Cell Surface in Relation to Fertilization by Runnstrom (1952).
One interesting property of the fertilization membrane, which has
not received sufficient mention, was described in some detail by
Pasteels in 1950. He observed that the fertilization membranes of
the eggs of Chaetopterus variopedatus (Renier), Nereis succinea, and
of Spisula solidissima (Dillwyn), are contractile. More accurately,
at certain times after fertilization the membrane 'expands', thereby
becoming creased or folded. The effect is soon reversed and the
membrane re-assumes its usual smooth (contracted ?) and spherical
appearance. The region on the fertilization membrane where this
folding and unfolding phenomenon first occurs, transiently, 20
minutes after fertilization in Chaetopterus eggs, is at the vegetative
pole, i.e. 180° away from the point of expulsion of the first polar
body. The same happens after the expulsion of the second polar
body, 30 minutes after fertilization, while 5 minutes later, the

* Cortical granules and their behaviour at fertilization are much in the lime-
light at present; it is therefore only right to mention that some forty-five years
ago, E. N. Harvey (191 1, p. 523), said that in the eggs oi Arhacia punctulatn, there
were 'numerous minute stained granules, (juite unmoved by the centrifuge. At
the time of fertilization these disappear, apparently going to form the substance
which passes out of the egg and hardens to a fertilization membrane' ! The stain
used was neutral red.

THE MORPHOLOGY OF FERTILIZATION II

fertilization membrane is smooth and spherical again. The
phenomenon is observed again just before the first division. There
is Httle doubt that these changes in the structure of the fertilization
membrane are caused or triggered off by the diffusion of certain
substances from the cortex into the perivitelline space. It is an
open question whether the fertilization membrane has the power
of self-propagation once it has been activated, or whether the
propagation of the effect is due to the progressive diffusion of a
substance out of the cortex, the escaping substance simply causing
a localised expansion of the membrane.

The mechanism responsible for the elevation, as opposed to the
formation, of the fertilization membrane is not clear, though it has
always been assumed that release of substances between the
vitelline and plasma membranes, to which the former is imperme-
able, causes an influx of water with consequent formation of the
perivitelline space. The escaping (and exploding?) cortical
granules could conceivably be responsible for this allegedly
osmotic phenomenon. Apart from these considerations, the con-
tents of the perivitelline space, in which the concentration of
solids is about 0-07% (Mitchison & Swann, 1953), require further
investigation, as in spite of the observations of Gray (1927) and
R. Chambers (1942), which suggest that the perivitelline fluid is
liquid, Hiramoto (1954) has recently claimed, as Fol did in 1879,
that it has viscous-elastic properties which can be removed by
treatment with calcium-free sea water.

Structures somewhat similar to the fertilization membranes of
echinoderm eggs are found in other eggs, for example in those of
Branchiostoma, prototherian mammals, Ascaris, the frog and trout.
In the frog's egg the membrane in question is called the vitelline
membrane and in that of the trout, the chorion. In both cases the
membrane separates or peels off from the egg surface, after ferti-
lization; but the same thing happens, though much more slowly,
in the frog's egg, if the unfertilized egg is left in tap water, from
which one might conclude that these eggs sustain an abortive
parthenogenetic stimulus by immersion in fresh water. Bialas-
zewicz (19 1 2) produced rather convincing evidence that the growth
of the perivitelline space in frogs' eggs was an osmotic phenomenon
associated with the presence of proteins, derived from the egg, in
the perivitelline space.

There is no phenomenon comparable to the elevation of the

12 FERTILIZATION

fertilization membrane in mammalian eggs; these eggs are, how-
ever, surrounded by a membrane, the zona pellucida, whose
structure changes shortly after fertilization (Smithberg, 1953;
Braden et al., 1954), in a way which is somewhat reminiscent of
the 'tanning' of the fertilization membrane. As a result of this
change, the zofta pellucida becomes less permeable to spermatozoa.
Braden et al. (1954) believe that the change in the permeability of
the zona pellucida to supernumerary spermatozoa is a reaction
propagated within the zona; but their experiments do not entirely
rule out an alternative hypothesis, that a substance progressively
released from the egg surface into the perivitelline space is re-
sponsible for the 'tanning' of the zona (see chapter 9).

Entrance of sperm tail and middle-piece. In Anthocidaris crassis-
pina (A. Agassiz) the sperm tail lies motionless outside the ferti-
lization membrane for about 2| minutes after fertilization. It then
straightens out, radially to the egg surface, and starts moving
vigorously. According to J. C. Dan (1950a), this movement makes
the sperm tail enter the egg in 15-30 seconds. There seems to be
no fixed rule as to whether the tail and middle-piece of a sperma-
tozoon enter the egg at fertilization. In Nereis succinea, neither
the tail nor the middle-piece go in with the head (F. R. Lillie, 19 126),
while in Nyctalus noctula (Schreber), the whole spermatozoon is
found in the cytoplasm (van der Stricht, 1902). In spite of the
classical and painstaking research of Meves (191 2) on the fate of
the middle-piece in those cases where it does enter the egg, and
in spite of the fact that the whole spermatozoon often (or perhaps
always) enters the mammalian egg (Blandau & Odor, 1952;
Austin, 1953; Shettles, 1954), the existing evidence does not per-
mit the conclusion that either the middle-piece or the tail of the
spermatozoon has an important function in fertilization, after
attachment of the fertilizing spermatozoon. This is to be expected
in the case of the sperm tail, which is obviously an organ of locomo-
tion, at any rate in animal spermatozoa.* Recent studies suggest
that the middle-piece may contain mitochondria-like material
concerned with locomotion, and endogenous substrates (Rothschild
& Cleland, 1952). Austin would probably not agree with these
views, as he has recently said (1953, p. 196), in regard to fertilization

* It may be that some of the tails of plant spermatozoa have a sensorj' function,
apart from being locomotor organs; but there is no evidence to support this idea
at present.

THE MORPHOLOGY OF FERTILIZATION I3

in mammals, that 'It seems clear that, in all the species in-
vestigated, the whole sperm enters the egg and that the cytoplasmic
components of the mid-piece mingle with the egg cytoplasm and
thus contribute something to the embryo.' Before any progress
can be made in resolving this question, we shall have to try and
find out what this 'something' is, and what its importance is to
the embryo.

To turn from the echinoderms and examine the early phases of
fertilization in a different phylum is both interesting and instruc-
tive. In the unfertilized egg of Spirocodon saltatrix (Tilesius),
the egg nucleus lies at the base of a slight depression on the
egg surface, this depression appearing after the extrusion of the
second polar body. J. C. Dan says (19506) that the sperma-
tozoon invariably enters the egg in the immediate neighbourhood
of the female nucleus; also that other spermatozoa accumulate
round this part of the egg. These observations raise the possibility
that sperm chemotaxis, for which there is very little reliable evi-
dence in the animal kingdom, may occur in the coelenterates.
Immediately after the sperm head has penetrated into the cortex,
a tubular structure develops round its tail. The growth and
degeneration of this structure are shown in Fig. 4, at various times
after the beginning of fertilization. There are no membranes
round the egg, before or after fertilization. The sperm tail passes
completely into the egg cytoplasm in about 15 minutes and during
this time it is in continual but slight movement.

The earliest phases of fertilization have now been observed in
mammalian eggs; Shettles (1954), for example, has reported the
successful fertilization of human eggs in vitro. Earlier claims of
success in this field have been sympathetically but firmly reviewed
by Austin (19516) and Smith (1951).

Hyaline Layer. Soon after the elevation of the fertilization
membrane, the so-called hyaline layer or Hyaloplasm appears on
the surface of sea-urchin eggs. This is a thin, extracellular and
gelatinous layer which dissolves in calcium-free sea water. As
this layer can be removed without affecting the viability of sea-
urchin eggs for a considerable time, it used to be thought that this
structure was an extraneous membrane whose main function was
to hold the blastomeres together after cleavage. The suggestion
has, however, recently been made that the hyaline layer may play
some part in preventing more than one spermatozoon entering an

FERTILIZATION

(a)

id)

(e)

(f)

FIG. 4. — Entry of spermatozoon into the egg of Spirocodun saltatrix, after J. C.
Dan (19506). o. Unfertilized egg showing female nucleus at periphery of
egg and depression in egg surface; b, 30 sec. after fertilization (/.); r, 3 min.
35 sec. after/. Development of 'fertilization tube'; d, 7 min. 15 sec. after/.
Swelling and incipient disintegration of fertilization tube; e, 1 1 min. 20 sec.
after/. Further disintegration of fertilization tube and only tip of sperm tail
visible outside egg;/, 15 min. 20 sec. after/. Spermatozoon completely en-
gulfed, fertilization tube disintegrated and swollen fusion nucleus moving
away froin the periphery towards centre of egg.

egg (Hagstrom & Hagstrom, 1954c; Hagstrom & Allen, 1956).
We shall examine this idea more closely in a later chapter, speci-
fically concerned with polyspermy.

Movements of the male and female pronuclei. After passing
through the cortex, the head of the spermatozoon rotates through

THE MORPHOLOGY OF FERTILIZATION 15

180 degrees so that the anterior end points outwards, in the direc-
tion from which the spermatozoon entered. According to E. B.
Wilson (1928, p. 423) the rotation of the sperm head is 'a very wide-
spread if not universal phenomenon'. This is an exaggeration as
it definitely does not occur in rat or mouse eggs. Rotation of the
sperm head occurs rather quickly, in 2-3 minutes, in the egg of
Lytechinus variegatus (Lamarck). Nothing is known about the
mechanism underlying the phenomenon.

The subsequent movements of the male and female pronuclei
present the following problem: in the unfertilized egg the female
pronucleus may be situated almost anywhere in the cytoplasm; it
may be in the centre of the egg, or at the periphery, as in Fig. 4.
Usually, the spermatozoon may enter the egg at any point on the
surface and after passing through the cortex, becomes the male pro-
nucleus with its surrounding aster, a prominent structure in many,
but not all, eggs, formed in the cytoplasm under the influence of the
sperm head. Ultimately, the male and female pronuclei meet at
about the centre of the egg. How do they get there ? The first stage
in trying to answer this question is to examine the morphology of
the reaction. One of the most detailed descriptions of the move-
ments of the pronuclei in normal, uncompressed eggs is that of E. L.
Chambers (1939). Chambers says that, in spherical eggs, the male
pronucleus moves at a uniform speed * towards the centre of the
egg, along a straight line at right angles to the egg surface. Fig. 5.
In the older literature, summarised by E. B. Wilson (1928) and also
by Chambers, the male pronucleus was said to travel to the centre
of the egg along a curved path which could be resolved into two
components, a penetration path at right angles to the egg surface ;
and a copulation path, towards the female pronucleus. Chambers
thinks, probably rightly, that this curved path is abnormal, at any
rate in Class 4 fertilization, and due to the compression of the egg,
or to the egg being non-spherical. It seems likely that the movement
of the male pronucleus to the centre of the egg is caused by the
growth of the sperm aster round it. If so, when the rays of the grow-
ing sperm aster come up against the inside of the egg surface, their
elongation simply pushes the male pronucleus towards the centre
of the egg. According to E. B. Wilson (1902), this explanation is

* According to Allen (1954), the velocity of the male pronucleus is far from
uniform; but his experiments were done on eggs which had been sucked into
narrow capillaries and which were therefore deformed in shape.

FERTILIZATION

invalidated by an experiment in which the development of the
sperm aster was inhibited by treating eggs with ether shortly after
fertilization. He claimed that in these circumstances the move-
ments of the male pronucleus were unaffected, Wilson makes no
reference to this experiment in his famous textbook The Cell in
Development and Heredity, and the ether experiment should be

Sperm aster

Female pronucleus

FIG. 5. — Path of male pronucleus from periphery of egg, labelled sperm aster,
towards centre of egg, the path being shown by ■ • • ■. Four alternative
. paths of female pronucleus to centre of egg, according to original position in
the unfertilized egg, are also shown. After E. L. Chambers (1939).

treated with reserve unless it is repeated. At the same time, Conk-
lin's examination (1905) of fertilization in Styela partita (Stimpson)
shows that in some cases, at any rate, protoplasmic streaming may
affect the movements of the male pronucleus. As a general rule,
however, and until experiments of the Chambers type are done on
eggs other than those of echinoderms, and particularly on mam-
malian eggs in which there is nothing comparable to the sea-
urchin or frog egg sperm aster, the movement of the male pro-
nucleus must be assumed to be a straightforward mechanical
phenomenon, caused by the growth of the sperm aster (only, of

THE MORPHOLOGY OF FERTILIZATION 17

course, in those cases where a sperm aster exists). In di- and tri-
spermic eggs the paths of the male pronuclei are clearly curved ;
but there is no need to postulate the existence of fancy forces as
an explanation. When the pronuclei get near enough to each
other, they are pushed apart by their own growing asters.

The movements of the female pronucleus are not so straight-
forward. Fig. 5 shows the various paths followed by the female
pronucleus, according to its original position in the unfertilized
egg. If these movements are connected with the sperm aster, the
influence of the latter must extend far beyond its visible boundaries.
While the female pronucleus is moving through the cytoplasm,
granules can be seen moving with it and at the same rate ; but once
it reaches and starts moving through the sperm aster, always
radially with respect to the astral rays, no synchronous granule
movements can be observed. The velocity is markedly non-
uniform, which is understandable, first because of the inertia of
the female pronucleus, which will affect its initial movements, and
secondly, because of the gel-like consistency of the sperm aster
through which it travels to the male pronucleus. There can be no
doubt that the male pronucleus 'attracts' the female pronucleus.
This remark is nothing more than the verbal equivalent of what
one can see in Fig. 5. If the male pronucleus were not present, the
path of the female pronucleus would be different. But there is no
implication that the male pronucleus attracts the female pro-
nucleus by electrostatic or electromagnetic forces; nor that long
range forces, which were almost as popular as hyaluronidase a few
years ago, have anything to do with the phenomenon. The male
pronucleus might, purely for example, be responsible for cyto-
plasmic currents which move the female pronucleus in the re-
quired direction. Chambers made the interesting observation that
if the female pronucleus happened to be in the centre of the egg,
it moved away from this position to enter the sperm aster. He be-
lieves that in the initial stages of its movement, the female pro-
nucleus is subjected to an attractive stimulus towards the centre
of the sperm aster; the direction of this attractive stimulus con-
tinually changes as the sperm aster moves towards the middle of
the egg. When close to the male pronucleus, Chambers suggests
that a further stimulus ('component of force', p. 418) acts on the
female pronucleus so that, although approaching the male pro-
nucleus, it also travels parallel with it. This second component.

l8 FERTILIZATION

which is responsible for the two pronuclei taking up a central
position within the egg, is what used to be called the Cleavage
Path of the fusion nucleus. The movements of the female pro-
nucleus towards the male pronucleus are thought by Chambers to
be caused by cytoplasmic streaming towards the middle of the male
pronucleus.

This description of the movements of the pronuclei may seem
complicated, but in fact it is over-simplified in several respects;
for example, little or no attention has been paid to the somewhat
conflicting results obtained by earlier workers such as Fol (1879)
or to the path of the male pronucleus in Classes 1-3 fertilization.
A more serious difficulty concerns the behaviour of the female
pronucleus in the absence of a male pronucleus. As is well known,
activation makes the egg nucleus swell. But, as Moore showed in
1937, the female pronucleus not only swells after parthenogenetic
activation but also moves to the centre of the egg under its own
steam. The same occurs after pseudogamous fertilization. Roths-
child (1953) reproduced a photograph of several pseudogamous
sea-urchin eggs, in which a swollen female pronucleus can be
clearly seen in the centre of the egg, though in this species,
Paracentrotus lividus (Lamarck), the nucleus of the unfertilized egg
is often eccentrically placed in the cytoplasm.

To sum up, the movement of the male pronucleus may be caused
by the growth of the sperm aster, at any rate in Class 4 eggs ; the
movement of the female pronucleus is influenced by, but not en-
tirely dependent on, the male pronucleus. Further experiments
on the morphology and mechanics of the reaction are clearly needed
before much will be learnt from chemical and biochemical studies.

Austin (1951a) has given an excellent account of the formation,
growth and conjugation of the pronuclei in the rat egg. The most
striking features of the process on the male side are the 'dissolu-
tion' of the sperm head 10-60 minutes after entering the egg, and
the appearance of numerous male nucleoli which swell and ulti-
mately coalesce to form the male pronucleus. On the female side,
fertilization catalyses the completion of maturation and the
development of female nucleoli which also swell and coalesce, to
form the female pronucleus. As mentioned earlier, asters are far
less prominent than in the eggs of the frog or sea-urchin.

Changes in the shape and volume of the egg. One of the inost con-
sistent features of fertilization is that eggs change their shape at

(a)

•i'Ei

(b)

(c)

4>t. J

/>"•:

(d)

(e)

(f)

(hy

Plate II

Fertilization of the egg of Ureckis caiipo. a, unfertilized, with intact
germinal vesicle and invagination; b-h, i, 2, 3, 5, 12, 30 and 35 min.
after fertilization. Note changes in shape and polar body extrusion.

Tyler (1932).

THE MORPHOLOGY OF FERTILIZATION 19

fertilization, such changes always resulting in the egg becoming
more spherical. For example, the unfertilized egg of Urechis caupo
has a large indentation in its surface, which disappears a few
minutes after fertilization, the egg becoming spherical, Plate II

32'A

32 y^

35 '/s

FIG. 6. — Changes in the shape of the egg oi Ascidiella aspersa (O. F. Miiller) after
fertihzation (external membranes removed), after Cohen & Berrill (1936).
The numbers below each drawing are minutes after fertilization.

(Tyler, 1932). The eggs of Cumingia tellitioides (Conrad) are not
spherical before fertilization, become transiently ovoid thirty
seconds after fertilization and round up in about a minute (Morgan
& Tyler, 1930), while every student knows that some batches of
sea-urchin eggs are pear-shaped before, and completely spherical

20 FERTILIZATION

a few minutes after, fertilization. The same occurs when the
eggs of Saccoglossus kowalewskyi and Thalassema neptiini Gaertner
are fertiHzed (Colwin & Colwin, 1953; Hobson, 1928). Changes in
the shape of the eggs of Ascidiella aspersa after fertihzation are
shown in Fig. 6. Rounding up after fertihzation is not due to the
influx of water, but to changes in the physical properties of the
cortex. These are discussed in a later chapter.

According to Glaser (1913, 1914, 1924), there is an 8% reduction
in the volume of Arbacia eggs at fertilization, but this has been
denied by R. Chambers (1921). An 8% change in volume would
not be easy to establish with any certainty by measurement of egg
diameters; but I do not believe that any reduction occurs in the
eggs of Echinus esculentus Linn, or Psammechinus miliaris. A re-
duction in volume definitely occurs at fertilization in the eggs of
the brook lamprey, of Ascaris equorum Goeze (Faure-Fremiet,
19 1 3), of Hydroides norvegicus Gunnerus (Monroy, 1954), of
Chaetopterus variopedatus (Monroy, 1954), and of a number of
mammals (Pincus, 1936).

CHAPTER 2

SPERM-EGG INTERACTING SUBSTANCES, I

The spermatozoa and eggs of many animals and plants contain or
produce substances which have well-defined effects on the gametes
of the opposite sex and, in some instances, on those of the same
sex. These substances are sometimes known as Gamones (=
gamete hormones), though this word is not in general use. They
have also been given other names: F. R. LilHe (1912a) published
the first systematic account of one of these substances, derived
from unfertilized eggs, which he called Fertilizin. In certain
circumstances a solution of fertilizin agglutinates spermatozoa
of the same species. The substance on the head of the spermato-
zoon with which fertilizin reacts is known as Antifertilizin ; it was
first extracted from spermatozoa by Frank (1939) and Tyler
(1939), while later, Tyler (1940a) also extracted it from eggs. The
biological and chemical characteristics of sperm-egg interacting
substances are summarized in Table i, which also contains a list
of their various names.

Reciprocal induction of spawning. Among aquatic organisms it
has been known for many years that a spawning female often
induces males and other females of the same species to shed their
gametes, and vice versa. For example, spawning females of the
acorn worm, Saccoglossus horsti Brambell & Goodhart, induce
males of the same species to spawn (Burdon-Jones, 195 1), while
the spawning of a male sea-urchin may stimulate every other male
urchin in an aquarium tank to start shedding its spermatozoa, to
the dismay of the biologist hoping to work with this material. This
phenomenon is put to commercial use in the oyster industry, the
gonads of some hundreds of oysters being thrown into oyster-beds
to stimulate mass spawning, and thereby increase the oyster popu-
lation (Quayle, 1940). So far as the induction of spawning is con-
cerned, the nature of the responsible substances has been mainly
investigated in oysters, in particular by Nelson & Allison (1937),
Galtsoff (1940) and Nelson (1941). Although the results of these
studies are not particularly encouraging from the point of view of
isolating one compound with specific stimulatory activity, there

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Plate III

Agglutination of a 2% suspension of spermatozoa of Mcgathiira crenu-

lata in the presence of homologous egg water, a, untreated; b, 15

sees, after addition of egg water; c, after 30 sees.; d, after 10 min.

Magnification, x|. Tyler (19406).

SPERM-EGG INTERACTING SUBSTANCES, I 23

is evidently something in the ejaculates of many male and female
invertebrates which stimulates other members of the same species
to shed their gametes ; and it is in part due to the existence of this
something that such intensive investigations have been made into
the nature and properties of sperm-egg interacting substances.

Agglutination of spermatozoa by egg water. In the presence of
water in which unfertilized eggs of the same species have been
standing, 'egg water', spermatozoa come together in clusters
(Plate III), which consist of dense aggregates of spermatozoa,
usually stuck together by their heads, but sometimes by their
tails and even, on occasions, head to tail. The phenomenon has
been observed in echinoderms and annelids (F. R. LilHe, 1919),
molluscs (Tyler, 19406; von Medem, 1942), ascidians (Minganti,
1951), cyclostomes (Schartau & Montalenti, 1941), fish (Hart-
mann, 1944; von Medem et al., 1949) and amphibia (Glaser, 1921)
and, in some forms, is quite similar to the agglutination of bull
spermatozoa in the presence of antisera (Henle et al., 1938). The
agglutination of sea-urchin spermatozoa is often spontaneously
reversible, the time for reversal depending on the concentration
of spermatozoa and of the compound in the egg water responsible
for the effect. This compound is called fertilizin or Gynogamone
II (G.II). It used to be thought that the unfertilized egg secreted
fertilizin into the surrounding sea water. But in 1939 and 1940
Tyler & Fox showed that fertilizin was derived from the jelly
which surrounds the unfertilized eggs of a number of aquatic
organisms. This jelly, which Vasseur (195 1) believes is secreted
by follicle cells in the ovary, swells and slowly dissolves in sea
water. As the dissolution proceeds, the sperm-agglutinating
power, or titre, of the egg water becomes stronger. The process
of dissolution can be greatly accelerated, without injury to the eggs,
by acidifying the sea water, the degree of acidification required
varying in different species. The sperm-agglutinating power of
fertilizin so obtained is equal to, or greater than, that from un-
treated suspensions in which the unfertilized eggs have been
allowed to remain in sea water for a sufficient time for the jelly
round them to dissolve. Eggs do not usually yield any more
fertilizin once the jelly round them has been removed or has dis-
solved. These observations have led to the view that egg jelly is
fertilizin and, in support of this contention, Tyler (1942a) re-
ported that virtually all the organic material from an electro-
c

24 FERTILIZATION

phoretically homogeneous solution of egg jelly could be absorbed by
spermatozoa. In spite of this evidence, Motomura (1950, i953«, b)
has claimed that a sperm-agglutinating factor, Cytofertilizin, can
be extracted from jelly-free unfertilized eggs of Hemicentrotus
pulcherrimus {A. Agassiz) and from the perivitelline fluid of ferti-
lized eggs of the same species ; also, that cytofertilizin is secreted,
transiently, by fertilized eggs of Strongylocentrotus nudus (A.
Agassiz) and Temnopleurus hardwicki (Gray). In the last two cases,
the vitelline membrane was removed before fertilization by treat-
ment of the unfertilized eggs for 10-15 minutes, with o-5M-NaSCN,
so that there was no fertilization membrane and, therefore, no
perivitelline fluid at the time when the cytofertilizin was secreted.
In spite of being queried by Byers (1951), Motomura's results
cannot be ignored or explained away on the basis of species diflfer-
ences. The only way of resolving the impasse created by this work
is to repeat Motomura's experiments, using his techniques but
eggs of Arbacia pimctidata, Arbacia lixula, Paracentrotus lividus,
Echinocardium cordatum, Strongylocentrotus purpuratus, or Lyte-
chinus pictus (Verrill). Although the agglutination of sea-urchin
spermatozoa by homologous egg water is often spontaneously re-
versible, this is not always the case. A difficulty therefore arises in
distinguishing between irreversible agglutination by homologous
fertilizin or cytofertilizin, and irreversible agglutination caused by
'unnatural' substances (e.g. cerium ions, Gray, 1920), of which
cytofertilizin could conceivably be an example, though this is un-
likely.

The procedure for extracting fertilizin in powder form is given
in Table 2 (Tyler, 1949). Defining agglutination titre as the
maximum dilution at which agglutination is observed when added
to an equal volume of a 1% suspension of spermatozoa (about
2.10® sperm /ml.), the egg water from a 20% suspension of eggs,
as in Table 2, has an agglutination titre of about 1,000. Fertilizin
is a hexosamine-free glycoprotein or mucopolysaccharide contain-
ing 20% protein and 80% polysaccharide esterified, according to
Vasseur (1952), with one sulphate group per monosaccharide
residue; * it is highly acidic, the electrophoretic mobility, about
18 X io~^ cm-/sec./volt, towards the anode, changing little as the
pH is lowered from 8-6 to 2-0 (Runnstrom et al., 1942; Tyler,

* The amounts of sulphate found in the molecule are not always consistent
with this arrangement.

SPERM-EGG INTERACTING SUBSTANCES, I 25

1949). These acidic properties are due to the large amount of acid-
labile sulphate, which may amount to 25% or more, in the mole-
cule. Fertilizin's sedimentation constant is 2-9-6-3 X lO"^^.
Measurements of diffusion coefficients are difficult to make on
fertilizin solutions, because they gelate at relatively low concen-
trations ; but on the basis of a sedimentation constant of 6-3 X io~^^
and the value 2-i X lO"^ cm^/sec, which Tyler (1949) and Tyler
et al. (1954) considered the most accurate of their estimates of the
diffusion coefficient, the molecular weight of fertilizin from
Arbacia pmictulata is about 300,000. A reciprocal density of 0-65,

TABLE 2
Preparation of powdered fertilizin from sea-urchin egg water

(i) Prepare 20% suspension, by volume, of unfertilized eggs.

(2) Bring pH to 3-5 with o-i N-HCl.

(3) Centrifuge and remove supernatant.

(4) Add 40 ml. N-NaOH per litre of supernatant.

(5) Suspend precipitate in 3-3% NaCl and dialyze against 3-3% NaCl.

(6) Remove insoluble particles and precipitate with ij vols. 95% ethanol.

(7) Dissolve in 3*3% NaCl, re-precipitate with ethanol or saturated

(NHJaSOj, and dry.

Yield, ca. 250 mg./L. {S. purpuratus)

which is of the right order for a polysaccharide, is assumed in the
calculations. The corresponding axial ratio (unhydrated prolate
ellipsoid) is 28: i, and, with a reasonable value for water of hydra-
tion, the ratio becomes about 20: i.

Fertilizin with a high sulphur content, i.e. from egg suspensions
of Strongylocentrotus droebachiensis (O. F. Miiller) (9%), and Para-
centrotus lividus (8 %), gives a good agglutination reaction with homo-
logous spermatozoa. Egg jelly oi Brissopsis lyrifera (Forbes), which
has a small sulphur content, 2-7%, does not cause agglutination of
homologous spermatozoa, from which Vasseur (1952) concludes
that agglutination is dependent on fertilizin containing an adequate
number of sulphate groups. But we shall see, when considering
the serological aspects of agglutination, that non-agglutinating
fertilizin can be rendered agglutinating by the addition of adjuvants,
while agglutinating fertilizin can be rendered non-agglutinating by
various treatments, including trypsin (Tyler & Fox, 1940) and
periodate (Immers & Vasseur, 1949), which could be interpreted
as meaning that both the protein and the polysaccharide part of the
molecule are necessary for agglutination, though this is not

FERTILIZATION

proved. It follows that the presence or absence of the necessary
number of sulphate groups may not be the only factor which
determines the agglutinating powers of fertilizin towards homo-
logous spermatozoa.

Interesting studies have recently been made, notably by Vasseur,
on the specificity of the carbohydrate components in fertilizin ob-
tained from different sea-urchins. The results are summarised in
Table 3, from which it is unfortunately clear that one should not

TABLE 3
Carbohydrates in the egg jelly of certain Echinoderms

E. cordatum

Fucose

Vasseur, 1952

E. esculentus

Galactose

Vasseur, 1950

S. droebachiensis

Fucose and galactose

Vasseur, 1948;

Vasseur & Immers, 1949

P. lividus

Fucose and glucose

Vasseur, 1948;

Vasseur & Immers, 1949

E. paritia

Fructose

Bishop, 1 95 1

S. purpuratus

Galactose and fucose

Tyler, 1949

A. lixula

Fucose

Monroy et al., 1954

H. pulcherrinms

Fucose

Nakano & Ohashi, 1954

P. depressus

Fucose

Nakano & Ohashi, 1954

A. crassispina

Fucose

Nakano & Ohashi, 1954

try to make any generalisations about the genus or species speci-
ficity of the carbohydrate components of sea-urchin egg jelly.

Hetero- agglutination. The agglutination of spermatozoa by
homologous egg water has several features in common with
serological reactions. For example, the reaction is specific. It is
true that spermatozoa are also agglutinated by heterologous
fertilizin, but this occurs mainly with closely related organisms.
The reaction occasionally takes place between distantly related
organisms, as in the case of the agglutination of Nereis spermatozoa
by Arbacia egg water. F. R. Lillie believed that a distinction could
be made between hetero- and iso-agglutination on the grounds
that the former does not exhibit the phenomenon of spontaneous
reversibility. This view is now known to be incorrect because iso-
agglutination may be irreversible, as in the case of the spermatozoa
of Megathura cretiulata described by Ty^^^ i^94^^)- Hetero-
agglutination can, however, be distinguished from iso-agglutina-
tion in other ways. Tyler (1946, 1948) has studied the hetero-
agglutination of vertebrate and invertebrate spermatozoa by blood,
body fluids and sperm extracts. When fertilizin of one organism.

SPERM-EGG INTERACTING SUBSTANCES, I 27

such as Arbacia punctulata, agglutinates the spermatozoa of a
distantly related organism, such as Nereis succinea, the same re-
action is elicited by blood, body fluids and sperm extracts; but
when the organisms under examination are closely related, these
latter substances do not agglutinate spermatozoa. A serum or body
fluid may contain a large number of independently absorbable
agglutinins, each of which exhibits group specificity, in the sense
of reacting with particular spermatozoa. In the serum of Panulirus
interruptus (Randall), for example, there are ten serologically dis-
tinguishable agglutinins, eight of which react specifically with the
blood or spermatozoa of scyphozoans, phanerocephalid polychaets,
holothurians, asteroids, ascidians, amphibians, reptiles and birds,
but not with the blood or spermatozoa of, for example, anthozoans,
cryptocephalid polychaets or turbellarians (Tyler & Metz, 1945).
These hetero-agglutinins are all present in a protein fraction which
is homogeneous on the basis of electrophoresis and ultracentrifuge
measurements. In the same way, parts of the fertilizin molecule
may carry such hetero-agglutinating properties. F. R. Lillie also
based his view that the hetero-agglutinating power of egg water
was due to the presence in it of a substance distinct from iso-
agglutinin on the observation that the hetero-agglutinin in the
egg water of Arbacia punctulata could be removed by sperma-
tozoa of Nereis succinea, though after the removal, the egg water
would still iso-agglutinate Arbacia spermatozoa. According to
Tyler (1948), the correct interpretation of this experiment is that
the fertilizin molecule has both hetero- and iso-agglutinating pro-
perties and that the inactivation of some molecules in a solution
through a hetero-agglutination reaction would not make much
difference to the iso-agglutination titre of the solution. A similar
situation exists in human blood sera which contain hetero-
agglutinins for the red blood cells of other vertebrates, though they
may not be distinct from the iso-agglutinins which distinguish the
blood groups.

To sum up this section, the reactions of fertilizin are dominantly
species specific ; cross reactions occur mainly with spermatozoa of
closely related organisms. The occasional cross reactions with
spermatozoa of remotely related organisms are due to the similari-
ties between certain molecular configurations in the fertilizin
molecule and those in other proteins in the body fluids or blood of
the organism in question.

28 FERTILIZATION

Multivalent and univalent fertilizin. Tyler (1948) has put for-
ward the view that agglutination is caused by the combination of
fertilizin molecules which, like antigens and antibodies, are 'multi-
valent' * with respect to their combining groups, with spermatozoa
which are multivalent with respect to the number of receptor
groups on their surfaces. If, therefore, a fertilizin molecule is to
cause sperm agglutination, it must have at least two combining
groups so that two spermatozoa can become attached to it. If one
spermatozoon with two or more receptors on its surface combines
with two or more fertilizin molecules and each multivalent fertilizin
molecule combines with a number of spermatozoa, the observed
macroscopic agglutination will occur. Once spermatozoa have
reversibly agglutinated, they cannot be re-agglutinated and their
fertilizing capacity is reduced, Tyler (19410) has suggested that
the reversal of agglutination may be due to the fertiHzin molecules
being split into 'univalent' fragments by an enzyme in the sperma-
tozoa, or possibly by the movements of the spermatozoa themselves.
If the former hypothesis is correct, each spermatozoon will be
covered by univalent fragments and therefore cannot be re-
agglutinated by fresh fertilizin. In spite of certain obvious differ-
ences, the agglutination of vertebrate blood cells by influenza and
other viruses is a rather similar phenomenon as the reaction is
spontaneously reversible, after which the blood cells cannot be
re-agglutinated (Hirst, 1942). This haemagglutination reaction is
inhibited by a number of cell-free extracts and tissue fluids and
there is some evidence that the reactive groups of the inhibitors
and the blood cells are mucopolysaccharides. The receptor groups
are sensitive to periodate, as is fertilizin (Vasseur, 1952). In sup-
port of the multivalent fertilizin theory, Tyler (1941a, i942«)
showed that treatment of fertilizin with proteolytic enzymes, ultra-
violet light and heat made it incapable of causing sperm agglutina-
tion, though spermatozoa treated with this modified fertilizin could
not be agglutinated by normal fertilizin and sustained a loss of
fertilizing capacity without impairment of motility. (Untreated
fertilizin also reduces the fertilizing capacity of homologous
spermatozoa.) Tyler suggested that the above treatments of fer-
tilizin converted it into a univalent form. The spermatozoa com-

* The Lattice theory of antigen-antibody combination requires the existence
of divalent or multivalent antibodies ; but the existence of such antibodies has not
yet been unequivocally demonstrated.

SPERM-EGG INTERACTING SUBSTANCES, I 29

bine with univalent fertilizin and therefore sustain a loss in fertiliz-
ing capacity because a certain number of combining groups on
their surfaces, which would otherwise be used during the union of
the spermatozoa with homologous eggs, are covered by fertilizin
molecules. No agglutination occurs because the fertilizin molecules,
being univalent, only have one combining group per molecule.
The univalent fertilizin concept provides an explanation of the
apparent absence of fertilizin in many organisms. The egg water
of Cumingia tellinoides (Sampson, 1922), of Urechis caupo (Tyler,
i94i«) and of the starfish Patiria miniata (Brandt) (Metz,
1945) does not cause agglutination of homologous spermatozoa.
Metz was able to make these starfish spermatozoa agglutinate in
the presence of egg water by the addition of an adjuvant, hen's
egg-white, which, in the absence of starfish egg water, did not
cause sperm agglutination. Again, there is an analogy in the field
of Rh antibodies. These are quite often found in a non-agglutinat-
ing, univalent form and can be made to agglutinate Rh-positive
cells by such substances as serum albumin (Race, 1944; de Burgh,
et al., 1946). Furthermore, multivalent immune antibodies can be
converted into univalent forms by treatments similar to those
which have been used on fertilizin (Tyler, 1945).

Zone phenomenon. The resemblance between the behaviour of
spermatozoa in the presence of fertilizin and serological reactions
is strengthened by the occurrence of the zone phenomenon (Tyler,
1940a). In serological reactions the zone phenomenon refers to
the fact that maximum or most rapid agglutination or precipita-
tion only occurs when antigens and antibodies are mixed in par-
ticular proportions. The zone phenomenon provides an interpre-
tation of the conditions in which feeble sperm agglutination is
observed, for if fertilizin molecules are present in great excess,
there will be enough of them to saturate all the combining groups
on individual spermatozoa without the necessity for sharing of
fertilizin molecules by numbers of spermatozoa, with consequent
agglutination. Alternatively, the intensity of the agglutination re-
action may be reduced if univalent as well as multivalent fertilizin
molecules are present in egg water and the former are nearly able
to saturate the combining groups on the spermatozoa. Zone
phenomena have been observed and studied in the spermatozoa
of Lytechinus pictus by Spikes (1949a).

Role of egg jelly in fertilization. Although the description of the

30 FERTILIZATION

reaction between spermatozoa and egg water as a typical serological
reaction is plausible — fertilizin can even combine with comple-
ment (Tyler, 1942/^)— criticisms of the analogy have not been lack-
ing, while some of the difficulties will not have escaped the reader.
Rybak (1949), for example, said that reversal of agglutination was
not due to the splitting of fertilizin but to a change in the sperma-
tozoa. Popa made a similar claim as long ago as 1927 and more
recently, J. C. Dan (1952, 1955) has published electron micro-
graphs showing morphological changes which are believed to take
place after treatment of sea-urchin spermatozoa with egg water.
Rybak also disbelieves in univalent fertilizin, but his experiments
in support of this view are not convincing. A further difficulty is
the fact that fertilizin appears to impair the fertilizing capacity of
spermatozoa by masking some of the combining groups on the
sperm head surface. It seems curious that an unfertilized egg
should be normally surrounded by a substance which positively
interferes with fertilization. At the same time Tyler (i94i«)
showed that following complete removal of jelly from unfertilized
eggs of Strongylocentrotus purpuratiis, by methods which did not
harm the eggs from the point of view of subsequent development,
more spermatozoa were needed to achieve a particular percentage of
fertilized eggs than when the jelly was present. This observation
was confirmed and put on a quantitative basis, in terms of the
probability of successful sperm-egg collisions, by Rothschild &
Swann (1951).

An interesting question has been raised by Monroy et al. (1954)
— whether the reaction between a spermatozoon and egg jelly in
solution in sea water, or between a spermatozoon and egg jelly
surrounding an egg, are necessarily the same, a subject which re-
quires further investigation. As regards the former reaction, these
workers, together with Hultin et al. (1952), have shown that living
spermotozoa of Arbacia lixula and Echinocardium cordatum remove
fucose from egg water (Table 4), which confirms that surface
groups on the spermatozoa react with and bind fertilizin. (Mudd
et al. (1929) found that the ^-potential of the spermatozoa of
Arbacia punctulata increased from —22 to —25 mV. in the presence
of homologous egg water.) Monroy, in the paper referred to above,
and Vasseur (1952) suggest that, as alkylation of amino groups on
spermatozoa inhibits agglutination (Metz & Donovan, 195 1), the
reaction given in Table 4 is between amino groups on sperm

SPERM-EGG INTERACTING SUBSTANCES, I

surfaces and sulphate groups in fertilizin. Attractive as this idea
may be, it is probably an over-simplification, if only because steric
factors play a dominating role in the specificity of antigen-antibody
reactions and, by analogy, might be expected to be important in
sperm agglutination.

There are, of course, aquatic organisms whose unfertilized eggs
are not surrounded by an obvious shell of jelly. The best known
example is that of Nereis siiccinea, though there is no doubt that a
substance with similar properties to sea-urchin egg jelly diffuses

TABLE 4

Effect of homologous spermatozoa ofifucose content, in [xgltnl.,

of egg water (A. lixula), Monroy et al. {1954)

Without sperm

After treatment
with sperm

2340
9-8

1200
198
42-0

44-0

4-2

490

2-2

256

out of unfertilized Nereis eggs. These eggs extrude jelly, which
has fertilizin-like properties, after fertilization.

One obvious function of egg jelly in fertilization is to prolong
the life of the spermatozoon. This effect is not very specific and
can be reproduced by adding albumin to the sea water in which
the spermatozoa are suspended (Wicklund, 1954). As chelating
agents such as versene have the same effect (Tyler, 1953; Roths-
child & Tyler, 1954), it may be that, in this context, egg water
protects the spermatozoa from the adverse effects of small quan-
tities of heavy metals in sea water. Two lines of attack on the
problem of the reactions between spermatozoa and fertilizin might
repay further investigation: first, a careful investigation with the
electron micrograph into the morphology of spermatozoa before
and after treatment with multivalent and univalent fertilizin.
Care must be taken in such experiments to resist the temptation
to select and generalise from photographs which show interesting
structural changes, when others from the same suspension do not.
The spermatozoa of Nereis succinea would be particularly suitable
for such a study as Lillie (19 19) noted that the heads of agglu-
tinated Nereis spermatozoa are swollen and spherical. Secondly,

32 FERTILIZATION

the fertilizing capacity of spermatozoa after subjection to different
concentrations of multivalent and univalent fertilizin could be
quantitatively examined with advantage.

Activation of spermatozoa by egg water. Egg water makes
homologous spermatozoa move more actively, respire at a greater
rate, and continue their movement, and therefore their respiration,

=1
I

800
400

t (minJ

FIG. 7. — Effect of egg secretions on the O, uptake of sea-urchin spermatozoa
{Psommechinus miliaris). •, with egg secretions; O, without egg secretions.
After Gray (1928).

for longer than they do in ordinary sea water. The effect is rather
variable, though when it is present, there is no doubt about its
existence. Gray (1928) was the first to examine this phenomenon
quantitatively, using the spermatozoa of Psammechiniis miliaris
(Fig. 7). He found that in some cases, the initial rate of O2 uptake
was four times as great in egg water as in sea water. The pro-
longation of sperm movement has obvious advantages from the
point of view of fertilization, though without a detailed knowledge

SPERM-EGG INTERACTING SUBSTANCES, I 33

of the morphology of the movement, it is not so obvious that an
increase in speed is beneficial in increasing the chance of an en-
counter between a spermatozoon and an egg. This question,
which involves consideration of the statistical mechanics of sperm
movement, is gone into in greater detail in chapters 4 and 9. An
increase in sperm respiration in the presence of egg water does not
always occur. Hayashi (1946), Spikes (19496) and Rothschild
(1952) observed a decrease in Og uptake on addition of egg water,
which was not due to agglutination, while Carter (1931) found
that fully ripe spermatozoa of Echinus esculentus and Psammechinus
miliaris appeared to be unaffected by egg secretions. On the other
hand, Vasseur (1952) has shown that egg water undoubtedly in-
creases the Oo uptake of ageing sperm suspensions of a number of
different genera and species of sea-urchins. In the Mediterranean
sea-urchin, Arhacia lixiila, the substance, sometimes called Gyno-
gamone I (G.I), responsible for sperm activation, was said by
Hartmann et al. (1940) to be the substituted naphthoquinone,
echinochrome (2-ethyl-3, 5, 6, 7, 8-pentoxinaphthoquinone-i, 4),
probably in equilibrium with isomeric quinones. According to
Hartmann and his co-workers, echinochrome, which is responsible
for the pink colour of these sea-urchin eggs, is bound to a protein
carrier within the eggs. In this form echinochrome is biologically
inactive and before being able to activate spermatozoa, it must
become attached to a second carrier derived from the egg jelly, or
be separated from its original protein carrier. The ternary com-
plex is said to 'activate' spermatozoa at a dilution of 1/3.10^^. The
significance of the word 'activate' is not clear in this context and
if it merely means that the spermatozoa seemed to move more
quickly in the presence of the ternary complex, the observation
should be taken with a grain of salt. No quantitative information,
based on O2 uptake, is available. A similar claim, that astaxanthine,
which is normally present in trout eggs, increases the activity of
trout spermatozoa, was made by Hartmann et al. (1947). As this
is not a book about spermatozoa per se, the differences of opinion
which exist about the effects of echinochrome and many other
substances on the activity and metabolism of spermatozoa will not
be further discusssed. Further information will be found in Mann's
recent book, The Biochemistry of Semen (1954), an important
paper by Bielig & Dohrn (1950) which casts a good deal of doubt
on the echinochrome story, and reviews by Tyler (1948) and

34 FERTILIZATION

Rothschild (195 1). To sum up, there is no doubt that egg water
does in certain circumstances stimulate spermatozoa to increased
and more prolonged movement and metabolism. Whether this is
due to a substance which is not the same as fertilizin is an open
question. The protein carrier and ternary complex story should
not be accepted until it is independently confirmed.

Inhibition of sperm agglutination by egg and sperm extracts. F. R.
Lillie suggested in 19 14 that a substance which could neutralise
fertilizin was present in sea-urchin eggs. This substance, called
antifertilizin or egg-antifertilizin, was extracted from sea-urchin
eggs by Runnstrom (19356) and Tyler (1940^). Using the gamone
terminology, antifertilizin would be called G.III or, if one wanted
to be pedantic, Gynandrogamone I, as it can be extracted from
eggs and spermatozoa. Both egg- and sperm-antifertilizin inhibit
the action of egg water and, in suitable conditions, precipitate
fertilizin ; they make unfertilized eggs of the same species agglu-
tinate and induce the formation of a membrane-like structure,
usually known as a precipitation membrane, on the surface of egg
jelly. The latter is somewhat similar to the Neufeld reaction of
pneumococci and other encapsulated bacteria reacting with
specific antisera. If fresh eggs are extracted by freezing and thaw-
ing, no antifertilizin is obtained, because it is neutralised by the
fertilizin in the jelly present round such eggs. Injection of egg-
antifertilizin into rabbits induces the production of antibodies
which precipitate antifertilizin and agglutinate homologous
spermatozoa (Tyler, 1948, p. 202).

A substance with very similar properties can be extracted by
heating (Frank, 1939), freezing and thawing (Tyler, 1939), and
acidification (Tyler & O'Melveny, 1941) from spermatozoa,
Table 5 ; it also is called antifertilizin and is the substance with
which fertilizin is believed to combine, causing agglutination. It
is evident from what has been said immediately above that there
is an antigenic complementarity between sperm-antifertilizin and
the antibodies formed in rabbits following injection of egg-
antifertilizin.

There has been some discussion as to whether sperm-anti-
fertilizin is an acidic protein, as stated above, or, as Hultin (1947)
thinks, an unspecific basic protein derived from sperm nuclei.
The latter induces the formation of a precipitation membrane on
egg jelly and causes homologous sperm agglutination (Metz, 1949),

SPERM-EGG INTERACTING SUBSTANCES, I

while sperm-antifertilizin does not cause homologous sperm
agglutination. This complicated question, which has been dis-
cussed at great length by Runnstrom (1949), is not yet resolved,
and further work is needed to clarify the chemical properties of
sperm-antifertilizin, which is certainly of biological importance,
both in eggs and spermatozoa. If a decision had to be taken now,
it would probably be in favour of sperm-antifertilizin being an
acidic protein, and not an unspecific basic protein.

F. R. Lillie believed that the function of egg-antifertilizin was to
neutralise fertilizin which would otherwise diffuse out of the egg

TABLE 5
Electrophoresis of purified solutions of sperm-antifertilizin ofE.
cordatum {Runnstrom et al., 1942) and L. pictus {Tyler, 1949)

Buffer

Mobility in cm.^lsec.lvolt . 10^

E. cordatum

L. pictus

4-6

4-9
5-0
60

6-9
7-3

Acetate

Phosphate

Barbital

30
37
41
6-4

4'2

4-8
61
93

at a time when there would be no point in it doing so. This idea
loses much of its value now that we believe fertilizin is in general
derived from egg jelly and not from eggs; and it is therefore not
surprising that Monroy & Runnstrom (1950) were able to extract
antifertilizin from fertilized eggs. Runnstrom (1952) believes that
egg-antifertilizin is a chromoprotein which reacts with the vitelline
membrane on the unfertilized egg at the moment of fertilization,
hardening and converting it into the fertilization membrane. If
this view is correct, egg-antifertilizin might be located in the cortical
granules of the egg, as these are known to have a hardening or
tanning effect on the fertilization membrane (see pp. 9-10).

Sperm lysins. Spermatozoa contain compounds which can break
down or dissolve the membranes and jelly which so often surround
unfertilized eggs, and a similar substance may have a lytic effect
on the plasma membrane itself. Some such action seems to be
required at fertilization, as the spermatozoon has got to get through

36 FERTILIZATION

the plasma membrane after attachment to the egg surface. Two
different compounds with these properties can be extracted from
spermatozoa, though there is no evidence that both can be ob-
tained from sperm of the same species. The existence of these two
classes of compounds, both of which are called sperm lysins in
the literature, makes the terminology confusing. One of them,
sometimes called Androgamone III (A.III), but more often sperm
lysin, was extracted with methanol from lyophilized sea-urchin
spermatozoa by Runnstrom et al. (1945). It diffuses through
cellophane and is therefore probably not a protein. It may be a
fatty acid. A similar substance, which is believed to be an 18-
carbon fatty acid with four double bonds, can be extracted from
mackerel testes. This lysin, which is found in the supernatant
fluid after spermatozoa have been centrifuged, is haemolytic and
activates unfertilized eggs, though it inhibits fertilization. Its
effects can be reproduced by detergents and bee venom. Osterhout
(1950, 1952) has obtained a similar substance by heating the sperma-
tozoa of Nereis succinea for ten minutes at 55° C. The compound
released into the sea water by this treatment is highly surface active
and activates eggs of the same species. Similar results are obtained
with the detergent Duponol, which is mainly composed of sodium
dodecyl sulphate. Some caution is necessary in the interpretation
of such experiments, as Osterhout (1953) has also shown that the
same treatment displaces protamine from Nereis spermatozoa,
though it appears from his brief note that the detergent-like com-
pound is responsible for the activation of eggs of the same species,
rather than protamine.

The other compounds with lytic properties, which can be ex-
tracted from spermatozoa, are proteins. The most famous of
these is the enzyme hyaluronidase which causes dispersal of the
follicle cells that surround unfertilized mammalian eggs. Some-
thing similar to hyaluronidase is believed to exist in the spermatozoa
of Discoglossus pictus Otth (Hibbard, 1928; Parat, 1933), and of
marine invertebrates. Hartmann & Schartau (1939) were the first
to make this claim as regards sea-urchin spermatozoa and Monroy
& Ruffo (1947) said that they also could extract a substance from
these spermatozoa, by treatment with o-iN-acetic acid followed
by precipitation with acetone, which 'completely dissolved' (p.
604) the jelly round unfertilized eggs. There is some doubt about
these claims because sperm-antifertilizin sometimes has a curious

(el

(f)

Plate IV

Dissolution of egg membrane of Megathura cyemilata bv an extract
of a 1% homologous sperm suspension, a, i min., b, 1-75 min.,
r, 2-00 min., d, 2-25 min., e, 3-00 min.,/, 3-25 min. after addition
of extract. Photograph by A. Tyler.

SPERM-EGG INTERACTING SUBSTANCES, I 37

effect on egg jelly, apart from forming a precipitation membrane
at its surface. After the precipitation membrane has formed, the
jelly contracts to such an extent that its surface becomes contiguous
with that of the egg proper and is, consequently, invisible. On the
basis of this phenomenon Tyler & O'Melveny (1941) believe that
the substance claimed to have hyaluronidase activity is in reality
sperm-antifertilizin and that sea-urchin spermatozoa do not contain
a separate depolymerase. The experiments of Krauss (1950) con-
firm that claims for the existence of a hyaluronidase-like enzyme in
sea-urchin spermatozoa should not be accepted. Vasseur (1951)
also reported that sea-urchin spermatozoa contained an enzyme
which splits egg jelly. But in this case again, the claim has been
refuted, by Monroy & Tosi (1952), who also cast doubt on the
earlier work on this subject, mentioned above, by Monroy &
Ruffo (1947). Ishida (1954) has recently claimed that sea-urchin
spermatozoa release a jelly-dissolving substance at the moment of
fertilization. The situation is sufficiently confused to merit
systematic re-examination.

A protein which is, however, distinct from sperm-antifertilizin
can be extracted from limpet spermatozoa. The unfertilized egg
of Megathura crenulata is surrounded by a tough membrane which
is not dissolved by concentrated acid applied over a period of hours.
Yet suspensions of homologous spermatozoa dissolve the mem-
brane in a matter of minutes, Plate IV, while extracts obtained by
freezing sperm suspensions to —80° C and thawing achieve the same
result. These eggs also have jelly round them, through which the
spermatozoa must get to reach the membrane (Tyler, 1939). Berg
(1950a) has made similar observations on the eggs of Mytiliis edulis
and Monroy (1948) on those of Pomatoceros triqiieter. One cannot
help feeling that, although enzymes of this type may dissolve mem-
branes external to the egg surface, they must also have a function in
softening up the plasma membrane, so that the spermatozoon is
able to enter the egg and get on with the business of syngamy.

Inhibitors of sperm movement derived from spermatozoa or semen.
A substance which inhibits sperm movement and might, therefore,
be said to antagonize fertilizin, can be extracted from sea-urchin
spermatozoa. This substance, Androgamone I (A.I), can be ex-
tracted from these spermatozoa by a variety of treatments such as
centrifugation (Southwick, 1939; Vasseur & Hagstrom, 1946;
Rothschild, 1948), warm water (Frank, 1939), or extraction with

38 FERTILIZATION

methanol (Runnstrom et al., 1944a). Whether this substance
normally diffuses out of sea-urchin spermatozoa and therefore has
a function in fertilization is much more debatable. According to
Hartmann et al. (1940), A.I. is responsible for the lack of sperm
movement in undiluted sea-urchin semen. This is wrong as the
lack of movement has been proved to be due to lack of oxygen ;
sea-urchin spermatozoa can be induced to move in undiluted
semen by increasing the oxygen tension, while the effect can be
reversed by replacing the oxygen with nitrogen (Rothschild, 1948).
Spermatozoa in salmon semen cannot be induced to move by in-
creasing the oxygen tension (Rothschild, 195 1); there is no doubt
that salmon semen contains an inhibitory substance and that it has
a genuine biological function (Runnstrom et al., 1944a). When
ejaculated salmon semen is diluted with water, the concentration
of the inhibitory substance, which is not a protein and diffuses
through cellophane, falls below the level at which it inhibits move-
ment, and the spermatozoa become motile.

Although the systematic study of sperm-egg interacting sub-
stances was started by F. R. Lillie (1919) * and Just (1939),* in-
tensive investigations into their biological and chemical properties
only started in about 1939. Like all young subjects, this one is still
confused and therefore difficult to expound in an aesthetically
satisfying way. Some of the more 'straightforward' aspects are
discussed in the succeeding chapters. The reader may be surprised
that, apart from studies on hyaluronidase, so much of this work
has been done on marine invertebrates. Is there, for example, any
similarity between the causes of sperm inactivity in the mammalian
epididymis and the causes of sperm inactivity in undiluted salmon
semen? Apart from Glaser's preliminary observations (1921) on
frog spermatozoa, are there biological similarities between the jelly
round frogs' eggs, whose chemistry has been intensively studied
(Folkes et al., 1950), and fertilizin? Does it, for example, inhibit
the clotting of blood ? Such questions might repay investigation.

* These contain references to early work on this subject.

CHAPTER 3

SPERM-EGG INTERACTING SUBSTANCES, II

Morphology of chemotaxis. This chapter is concerned with the
chemotaxis of spermatozoa towards eggs, as a result of the secretion
of substances from eggs or the cells near them. Except possibly in
the case of the coelenterate Spirocodon saltatrix (J. C. Dan, 19506),
the phenomenon almost certainly does not occur, and certainly has
not been shown unequivocally to occur, in the animal kingdom.
But in plants and, in particular, in the ferns, mosses, horse-tails,
liverworts and quillworts, the chemotaxis of spermatozoa towards
egg secretions is an established fact. Chemotaxis means that
spermatozoa are attracted towards eggs through the medium of
some substance produced by the eggs or cells near them. The most
famous case of sperm chemotaxis, discovered by Pfeffer (1884),
occurs in the ferns. For example, the archegonia of bracken,
Pteridium aquilimim (Linn.), are said to produce L-malic acid *
which diffuses into the external aqueous medium. Bracken
spermatozoa are sensitive to the malic acid gradient produced by
the diffusion of this acid out of the eggs, and swim towards the
source. If there were no gradient, that is if the spermatozoa were
suspended in a uniform solution of malic acid, the spermatozoa
could not swim preferentially in any particular direction and would
remain uniformly distributed. Before considering the chemistry
of chemotaxis, it is worth making a brief examination of the mor-
phology of the reaction. The behaviour of bracken spermatozoa in
ordinary tap water is shown in Fig. 8fl. This diagram was obtained
by taking a cinematograph film of the spermatozoa swimming in
tap water, projecting the film frame by frame on to paper, and
joining up the consecutive positions taken up by each spermatozoon
by straight lines, so that every spermatozoon in the field is asso-
ciated with a 'track'. The movements of the spermatozoa will be
seen to be random, in the sense that the tracks do not point in any

* Although it is virtually certain that the substance is L-malic acid, chemical
identification has not yet been achieved. Prof. E. C. Slater and I tried, un-
successfully, to inhibit the reaction with malic dehydrogenase; but there was
evidence that the enzyme preparation was not sufficiently strong to decompose
the malate at the required rate.

D 39

FERTILIZATION

particular direction. After the photographs on which the tracks
were based had been taken, a glass pipette, filled with a i % solu-
tion of sodium L-malate in tap water (containing i% agar), was
inserted into the sperm suspension. Malate ions immediately
began to diffuse out of the pipette into the external medium;
capillary effects or hydrodynamic flow, which would have con-
fused the issue, were prevented by the agar gel. The response of
the spermatozoa to the malate gradient is shown in Fig. 8b, which

. 16 /■ 2'4

0-1

0 2

T
0-3

0-4

0-5 0-6

f (mm.)

0-7

0-8

0-9

FIG. 8a. — Movements of bracken spermatozoa in tap water. The circles indicate
where each track begins. Numbers at the beginning and end of each track
refer respectively to time of start and duration of track in seconds.

requires two comments. First, the spermatozoa move in a re-
markably purposeful way towards the source of malate. They do
not drift statistically towards the source, as might be expected if
the mechanism of attraction were of the type known as Klino-
kinesis with Adaptation, which occurs when Ullyott's 'Turning
Worm' is subjected to a light stimulus (1936). The conclusion is
almost inescapable that these spermatozoa have 'sense organs', or
their functional equivalents; but in this case the sense organs must
be efficient, because, in such a system, the differences in concen-
tration between the front and back ends of the head of a sperma-

SPERM-EGG INTERACTING SUBSTANCES, II 41

tozoon are only about 0-5% (Rothschild, 195 1). If the tails of the
spermatozoa are concerned with the identification of gradients,
the perception system need not be so efficient, as they may be ten
times as long as the head. A defect in the experiment depicted in
Fig. 8b concerns the behaviour of the spermatozoa immediately
after the pipette is inserted into the suspension. If the directions
of movement of the spermatozoa are random before the pipette is

0-9

"1
10

0-5
t (min. )

FIG. 8b. — Movements of bracken spermatozoa after insertion of pipette (dia-
meter 30 microns), containing 1% sodium L-malate in a 1% agar-tap water
gel, into the same sperm suspension as in Fig. 8a. Numbers at the beginning
of tracks indicate time in seconds after insertion of pipette, Rothschild (1952).

inserted, as in Fig. 8a, they should be random immediately after
the pipette is put in, before the malate has had time to diffuse
significantly into the external medium. Unfortunately, the in-
sertion of the pipette into the suspension inevitably causes macro-
scopic disturbances in the fluid, so that the earliest phases of the
reaction are lost. When someone can devise a method of intro-
ducing a source of malate into a suspension without causing such
disturbances, we shall learn more about the morphology of chemo-
taxis. One interesting question which may be resolved by such a
study concerns the morphology of turning. Although there are
references in the literature to spermatozoa turning and bending

42 FERTILIZATION

towards regions of higher concentration of an attractive substance,
it is not at all clear how they do it. Does the head move to the right
or the left; or can the waves which travel along their tails be
bilaterally asymmetrical when necessary ? Or in the multiflagellate
spermatozoa of ferns and mosses, do some of the flagella move
more quickly than others under the appropriate conditions of
stimulation? Once the spermatozoa have arrived at the source,
i.e. at the tip of the pipette filled with agar and i% sodium malate,
they must be continually turning; otherwise they would not buzz
round the tip like a swarm of bees, something which every student
of the phenomenon has seen. The second point to notice in Fig.
8^ is that one spermatozoon at least, at about 2-30 o'clock (ii*i),
was completely unaffected by the malate gradient.

Several workers have wrongly assumed that a substance which
makes spermatozoa swim more quickly, according to its concentra-
tion, will act as an attractive agent. The argument is that if a
spermatozoon happens to be swimming in the direction of ascend-
ing concentration of the stimulating substance, it will swim more
quickly and get nearer the source of the substance. If, on the other
hand, a spermatozoon happens to be swimming in the direction of
descending concentration of the stimulating substance, it will
swim more slowly and therefore get less far from the source. This
argument is fallacious, as can be seen from the following over-
simplified example. Suppose we have a suspension of spermatozoa,
moving at random (Fig. 8a), and we suddenly point a death ray at
a small region in the suspension. Any spermatozoon which happens
to swim into this region will be killed, that is to say its movements
will be greatly slowed up and stopped. As the movements of the
spermatozoa are random, most of them will sooner or later enter
the lethal area. In due course, therefore, nearly all the spermatozoa
will be found in this region, where they move most slowly. Con-
versely, if the region in question makes the spermatozoa swim
more quickly, it will on the average contain less spermatozoa than
there are outside. This example is over-simplified, in that the
lethal or stimulating region is assumed to end abruptly, and not to
give rise to gradients in the extra-regional space. But given that
this mechanism (Orthokincsis) will not achieve the desired etfect
and that Klinokinesis with Adaptation * is inconsistent with the

* Orthokincsis with Adaptation can also act as an attractive mechanism, though
no-one has so far postulated its existence in biological systems. It suffers from

SPERM-EGG INTERACTING SUBSTANCES, II 43

morphology of the phenomenon, we are driven to postulate the
existence of sperm sense organs not fundamentally different from
those which cause a moth to fly towards a light. Another example
of the 'repulsive effect' of an increase in speed is a sealed test-tube
containing a gas, one end of which is heated. The highest con-
centration of gas molecules will be found at the cold end of the
test-tube, where the molecules will be moving more slowly than
at the hot end.

Chemistry of chetnotaxis. This subject has been most sys-
tematically examined in the ferns, horse-tails and quillworts; the

TABLE 6
Effect of certain organic acids on the spermatozoa of Equisetum

arvense

•i

'r*

<-»

^Vi

•i

-c.

.•0

■-1

COOH

-OOH

COOH

OOH

COOH

HO-C-H
1

H-(

•OH

H-C-H

H-C

-H

H-C-Br

H-C-OH

H-C-H
1

c»

H-C-H

H-(

;-0H

H-C-H

H-(

;-Br

H-C-Br

Br-C-H

HO-C-H

1
H-C-NH,

COOH

principal results are summarised in Table 9 at the end of this
chapter. In this table, ' + ' means 'attracts', 'o' means 'does not
attract', and '— ' means 'no information'. The organic anion is
responsible for the attraction, because in those cases where it
occurs, the same effects are obtained, for example, with the sodium
salt. The case of Equisetum arvense Linn, is in some respects the
simplest, from the point of view of the specificity of the perception
mechanism. The relevant information is extracted in Table 6, from
which the following conclusions can be drawn : to attract the sperm
oi Equisetum, Xho. suh?,t2inQ,Q must (i), be a 4-carbon dicarboxylic
acid; (2), have an OH group on C^ or C^ (Geneva system of

the same defects as Klinokinesis with Adaptation so far as sperm chemotaxis is
concerned. A few observations about the possibility of Orthokinesis with
Adaptation will be found in a paper by Rothschild (1952).

44 FERTILIZATION

numbering the carbon atoms); and (3), have OH groups in the
cis and not the trans position * on C^ and C^, if both C^ and C^
have OH groups attached to them. This information suggests other
experiments to enable the perception mechanism to be examined
in greater detail. Some of the more obvious substances to try are
shown in Table 7. Compounds i and 2, Table 7, deal with the

TABLE 7
Organic acids to be tried on spermatozoa o/Equisetum arvense

COOH COOH

COOH

COOH COOH

COOH

HO-C-OH H-C-OH

HO-C-Me

HO-C-H HO-C-H.

Me-C-Me

HO-C-OH HO-C-OH

H-C-Me

H-C-Me Me-C-H

HO-C-Me

COOH COOH

COOH

COOH COOH

COOH

I. 2.

4. 5-

question: does attraction occur if there are more than two OH
groups on C^ and C^; Compound i is in equilibrium with diketo-
succinic acid but the doubly hydrated form is believed to pre-
dominate :

COOH

HO-C-OH

-H2O

HO-C-OH

-H2O

1
CO

HO-C-OH

+H2O

+H20

1
CO

COOH

Compound 2 is equivalent to dihydroxyfumaric acid (which does
not attract bracken spermatozoa), because of the following
equilibria :

COOH

COOH
H-C-OH

COOH

H-C-OH
HO-C-OH
COOH

-H,0

i..

+ H.0

— i>.

C-OH
HO-C

COOH

Compound 3 deals with the question: is the configuration
HO-C-H necessary for attraction, given that the introduction of

* The use of the words cis and trans is improper, but convenient, in this con-
text. Strictly, one should say 'have the meso configuration' instead of 'OH groups
in the cis . . . position'.

SPERM-EGG INTERACTING SUBSTANCES, II 45

a methyl group does not inhibit attraction; Compound 4 deals

HO-C
with the necessity or otherwise for the configuration I

C-H
HOC
The role of the configuration I is dealt with by Compound 5.

If an OH group is sufficient, without an H atom on C^ or C^,
Compound 6 should be effective. It would, perhaps, be preferable
to introduce bromine atoms rather than methyl groups in Com-
pounds 3-6 in Table 7. Unfortunately, Compounds 3 and 6 with
Br atoms replacing the Me groups are too unstable to exist in
aqueous solution. This type of experiment is capable of extension
in many interesting directions provided the required substances
can be made; but although the experiments are simple, certain pre-
cautions, which have not always been observed in the past, must
be taken. As mentioned earlier, the experimental procedure is to
fill a glass pipette with the test substance, put the pipette or one
end of it into the sperm suspension, and see what happens. If the
solution in the pipette is not a gel, spermatozoa may swim into the
pipette by chance and be unable to get out, because the diameter
of the pipette is too small to allow random sperm movement.
Failure to appreciate this difficulty has undoubtedly been respon-
sible for some of the claims for the existence of sperm chemotaxis
in the animal kingdom. Furthermore, convection currents and
hydrodynamic flow, or even a pumping and suction action, can
and do occur, and may cause spurious results, both of a positive
and negative kind. When using solutions of organic acids in agar
gels, attention must be paid to the possibility of the acid decom-
posing when the agar is dissolved by heating. An alternative
method of preparing the pipettes is to fill them with i % agar in
water and, after cooling, allow both ends of the pipette to dip into
aqueous solutions of the organic acid. The normal process of
diffusion will ultimately make the concentration of the acid in the
pipette equal to that in the solutions in which the two ends of the
pipette are immersed. For a pipette of fength 5 cm., it will take
three or four days for the average concentration in the pipette to
become half of what it is in the external solutions. The process
will not be seriously slowed up if carried out in a refrigerator to
avoid bacterial contamination.

Returning to Table 9, a further interesting feature is the ability

46 FERTILIZATION

of all spermatozoa except those of Equisetum arvense to distinguish
between cis and trans unsaturated dicarboxylic acids. The sperm-
atozoa of Isoetes japonica A. Braun react towards the trans, but are
indifferent to the cis, configuration ; while those of Sahinia natans
AUioni, Osmiindajavanica Blume, Pityrogramma sulphurea (Schwartz)
and Pteridium aquilimim, which, with the exception of Sahinia
natans, are ferns, are attracted by the cis but not the trans forms.
Unfortunately the specificity of the reaction is not so clear in fern
spermatozoa as it is in those of Equisetum arvense. If, for example,
we examine the structure of those acids which attract the sperm-
atozoa of Pteridium aquilinum (Table 8), there is evidently no single

TABLE 8
Organic acids zvhich attract bracken spermatozoa

.VI

.Vi

6
8

••*

s
2

4**

COOH

H-C-OH

HO-C-H

H-C-OH

H-C

CHs-C

H-C-OH

H-C-H

H-C-OH

H-C

COOH

configuration which stimulates their perception mechanisms, as
there seems to be in Equisetum arvense. In spite of these difficulties,
some predictions can be made from Table 8. Other substituted
and unsaturated 4-carbon m-dicarboxylic acids will probably
attract bracken spermatozoa. As an OH group on C^ or C^
appears to be essential if saturated acids are used — succinic and
malonic acids do not attract — chloromalic acid would be an in-
teresting compound to try. It is, however, difficult to predict the
effect of chloromalic acid in view of the reputed behaviour of
Sahinia natans to succinic and monobromosuccinic acids (Table 9).
In addition to their responses to organic acids, fern spermatozoa
are reported to be attracted by gradients of many other substances,
of which calcium, strontium, lithium, morphine and yohimbine
are a few examples (Shibata, 1911). These claims must be accepted
with reserve until the experiments have been repeated under care-

SPERM-EGG INTERACTING SUBSTANCES, II 47

fully controlled conditions, particularly as a number of the re-
ported cases of attraction are said to be associated with repulsion
at the same time. It is of course possible that strong concentrations
of a substance may repel and weaker concentrations of the same
substance attract, organisms, as in the case of certain flagellate
protozoa (Fox, 1921). But interpretation of experiments is more
difficult in such cases.

This chapter is not intended to review all work on the chemo-
taxis of spermatozoa towards eggs, but rather to indicate future
lines of research which might be profitable. There is, therefore,
no discussion of sperm chemotaxis in mosses, for which sucrose,
according to Pfeffer (1884), is the only attractive substance. In
recent years little work has been done on the chemotaxis of plant
spermatozoa, except for a few observations by the author which have
been mentioned earlier, one paper by Wilkie (1954), and the work
of Cook et al. (1948, 1951). These workers examined the chemotaxis
of seaweed spermatozoa [Fucus serratus Linn., Fiicus vesiculosus
Linn., and Fucus spiralis Linn.) towards the secretions of eggs of the
same species. This work is interesting from two points of view;
first, the egg secretions and other substances which attract thal-
lophyte spermatozoa are chemically quite diflFerent from those
which attract fern and moss spermatozoa ; secondly, in spite of an
intensive investigation by organic chemists, which included the
examination of sea water containing egg secretions with the mass
spectrometer, it was not possible to identify the substance or sub-
stances which are produced by seaweed eggs and which attract
spermatozoa of the same genus. The mass spectrometer evidence
suggested that the naturally occurring substance had a molecular
weight of 74; that it contained an Me group which was rather
easily lost under the influence of the ion gun; that it had a carbon
chain which contained three or less carbon atoms ; and that it did
not contain an easily lost hydrogen atom, as in an alcohol group.
These considerations raised the possibility that the compound
might be diethyl ether; but this compound, though it attracted
seaweed spermatozoa, did not do so at the required dilution.
Other substances which attract these spermatozoa are «-hexane,
i-hexene, sec.-h\ity\ alcohol, and w-propyl acetate. At one moment
the normally occurring compound seemed most likely to be n-
hexane, but the data obtained with the mass spectrometer made
this possibility remote.

TABLE 9

The reactions of certain plant spermatozoa towards organic acids ;
'-}-' = attracts ; 'o' = does not attract ; ' — ' = no i?iformation

Acid

K
CO

5
a

L-malic

COOH
HO-C-H
H-C-H
COOH

D-malic

COOH
H-C-OH
H-C-H

COOH

D-tartaric

COOH
H-C-OH
HO-C-H
COOH

Mesotartaric

COOH
H-C-OH
H-C-OH

COOH

Racemic

I
H-C-OH

1 +
HO-C-H

HO-C-H
H-C-OH

—

Succinic

COOH
H-C-H
H-C-H

COOH

TABLE 9 (continued)

Acid

5
a

>-;

Monobromosuccinic

COOH

1
H-C-H

H-C-Br

COOH

Dibromosuccinic

COOH
H-C-Br
H-C-Br

COOH

Isodibromosuccinic

COOH
H-C-Br
Br-C-H

COOH

Fumaric

HOOC-C-H

H-C-COOH

Maleic

H-C-COOH
H-C-COOH

Dihydroxyfumaric *

HOOC-C-OH
HO-C-COOH

—

• —

—

Mesaconic

CHg-C-COOH
HOOC-C-H

Citraconic

CH3-C-COOH
H-C-COOH ^

—

Itaconic

CH2 = C-COOH
H^-C-COOH

^—

—

* Until 1953, a bottle labelled dihydroxymaleic acid, which probably would
attract some plant spermatozoa, actually contained dihydroxyfumaric acid
(Hartree, 1953), which does not attract.

TABLE 9 (continued)

1,1,1-Ethane-
tricarboxylic

Agaricic

Tartronic

Suberic

Sebacic

Glutaric

Acid

Ha-C

HOOC-C-COOH

1
COOH

CH , C(OH)— CHCieH :

COOH COOH COOH

COOH

I
H-C-OH

COOH

I
(CH^),

COOH
COOH

(CHa),

COOH

(CH2)3

COOH

c/i- Camphoric

frani-Camphoric

COOH
COOH

COOH

S
o

§-

Co
o

3
to

Oh an

O — —

Notes: The following acids are inactive: aconitic, oxalic, malonic, formic,
glycoUic, lactic, acetic, saccharic, mucic, aspartic, glutamic, phthallic, terephthallic
and a-truxil!ic acids. Citric acid was reported by Bruchmann (iQog) to attract
the spermatozoa of Lycopodiiun clavatum Linn., but it has no effect on the
spermatozoa of the plants mentioned in this table.

CHAPTER 4

SPERM-EGG INTERACTING SUBSTANCES, III

The shrubs of Forsythia X intermedia Zabel have two sorts of
flowers, which are not found growing together on the same bush.
In one the styles are short and the stamens long, while in the
other, the styles are long and the stamens short (Plate V). In nature,
fertilization only occurs when pollen from a flower with long
stamens comes into contact with a stigma on a long style (on
another flower), or when pollen from a flower with short stamens
comes into contact with a stigma on a short style (on another
flower). In addition, and for reasons which are obvious from what
has been said, Forsythia is self-sterile. Not only does the pollen
from one flower never fertilize the same flower (self-sterility); it
also never fertilizes a flower of the same type whether on the same
bush or another. Moewus (1949, 1950) claimed to have found that
when water extracts of Forsythia pollen and stigmata were mixed,
the flavonol, quercetin, was found in some cases and not in others.
Table 10 shows which combinations of pollen and stigmata produce

quercetin and which do not. As the amounts of quercetin formed
were small and could not be identified by chemical analysis,
Moewus used a biological method of identification, which, being
of considerable gametological interest, will be described later.

Further investigations by Kuhn & Low (19490:) appear to have
revealed that pollen from long stamens contained rutin, while that
obtained from short stamens contained quercitrin, the yields of
these substances from pollen being about 10% in each case.
Rutin is found in the petals of both types of flower (Kuhn & Low,
19496), in approximately equal quantities; it is only in the pollen

FERTILIZATION
TABLE ID

Stigma from

Pollen from

Quercetin

Long style
Short style
Long style
Short style

Long stamen
Short stamen
Short stamen
Long stamen

Present
Present
Absent
Absent

that the different flavonol glycosides are found. These observa-
tions, if they are correct, are important in that, for the first time,
self-sterility and the specificity of fertihzation can be related to

rhamnose

HO.

Rutinose
Rutin (long stamens)

Qit Q rhamnose

Quercitrin (short stamens)

specific chemicals occurring in the sexual organs. The produc-
tion of quercetin, the aglucone of rutin or quercitrin, following
the interaction of the 'right' sort of pollen with the 'right' sort of
stigma, is achieved by hydrolysing enzymes, presumably acting in
a similar way to rhamnodiastase, which is obtained from Chinese
buckthorn. These hydrolysing enzymes are said to be located in a
thin layer of cells covering the top of the stigma. The two types of
enzyme are specific, the one in 'short' stigmata hydrolysing quer-
citrin to quercetin, while that in 'long' stigmata hydrolyses rutin
to quercetin.

Moewus' biological identification of quercetin has been men-
tioned. Three clones of the alga Chlainydomonas eugametos Moewus
are used: Clone i, which is obtained by subjecting female cells to
temperature shock, consists of mutants which, unlike the original
organisms, do not produce wo-rhamnetin. The inability of these

Plate V

The two sorts of flower of Forsvtbia ; intermedia. Left, long styles
and short stamens; right, short styles and long stamens. Photograph
by Royal Botanic Gardens, Kevv.

SPERM-EGG INTERACTING SUBSTANCES, III 53

mutants to produce wo-rhamnetin is due to their inability to

OCR

OH O

Z50-Rhamnetin

synthesize the intracellular precursor of this substance, quercetin.
In the presence of added quercetin they can produce wo-rhamnetin
and then become able to copulate with male gametes. Clone 2 is
a homothallic strain which normally produces female and male
gametes. When Clone 2 cells are treated with peonin or 4-hydroxy-
^-cyclocitral, which induce male characteristics, all of them
copulate with female cells if these are available; if, however,
homothallic cells are treated with wo-rhamnetin, which induces
female characteristics, all of them pair with male cells. Clone 3
consists of normal male cells.

OCH,

Y"-^^o^

HjC CH,

"\ V CHO

elucose

glucose

H— C

\/-/^\.

/ ^C'

OH /\
H H

Peonin

CH,

4-hydroxy-^- cyclocitral

When a solution is believed to contain quercetin, it is added to
a suspension of Clone i ; if quercetin is present, the Clone i cells
produce wo-rhammetin. Clone 2 cells are then treated with the
solution containing /^o-rhamnetin. This causes them to react as
female cells so that Clone 3 cells will copulate with them. The test
procedure can therefore be summarised as follows:

Test solution — > Clone i — > Clone 2 < — Clone 3

If Clone 3 cells copulate with Clone 2 cells, quercetin is present in
the test solution.

As often happens, other scientists have been unable to con-
firm Moewus' findings. Lewis (1954, p. 266), for example.

54 FERTILIZATION

says that 'T. O. Dayton, working in this laboratory, has made
chemical tests on pollen of a number of different plants from two
species, including Forsythia intermedia var, spectahilis. In none of
the pollen from either pin or thrum plants could quercitrin be
detected, and all contained rutin. I have tested the effect of boric
acid on pollen-tube growth * on incompatible stigmas but with
negative results.' Although Lewis suggests a possible explanation
of these negative results, the grave doubts which have been ex-
pressed about Moewus' Chlamydomonas work (see for example
Forster & Wiese, 1954), coupled with these recent findings on
Forsythia, must leave all scientists with very uncomfortable
feelings.

Apart from the work of Moewus & Kuhn on the role of flavanone
derivatives in plant gametology, claims have been made that

OCH,

rhamnosc-
glucose — O

rutinosc

hcsperitin
Hesperidin

phosphorylated hesperidin, another flavanone derivative, inhibits
fertilization in mammals. Work published on this subject does not
make clear at what points in the hesperidin molecule phosphoryla-
tion occurs. In 1952 Martin & Bciler reported that oral or intra-
peritoneal administration of phosphorylated hesperidin inhibited
conception in 44 out of 54 rats. The substance was said to exert
its effect on the female and not the male rats. Previously, these
two workers had found that phosphorylated hesperidin inhibited
the enzyme hyaluronidase and it was this fact which prompted
them to investigate its effect on conception in rats. Later in 1952,
Sieve claimed that out of 300 married couples he had persuaded
to take phosphorylated hesperidin (orally) as a possible contra-
ceptive measure, only two had conceived during the experimental
period and that these two couples were unreliable. Unlike Martin

* Moewus (1950) stated that quercitrin and rutin at a dilution of 1:10" were
inhibitors of pollen germination in sugar solution and that this inhibition was
counteracted by 0.01% boric acid.

SPERM-EGG INTERACTING SUBSTANCES, III 55

& Beiler in their rat experiments, Sieve said it was essential for
the men to take phosphorylated hesperidin, as well as the wom^en.
Sieve's claims should not be accepted until they have been in-
dependently confirmed, but this may be difficult, as in his paper
he does not reveal the structure of the phosphorylated hesperidin
used in his experiments, though he says that only one of the
possible phosphorylated compounds is efficacious. More recently
Chang & Pincus (1953) have repeated and failed to confirm Martin
& Beiler's results. They say (p. 275) that 'phosphorylated hesperi-
din does not inhibit fertilization when deposited into the Fallopian
tubes of rabbits at the time of sperm penetration, nor does it
inhibit ovulation, implantation, or normal development of the
embryo when administered intraperitoneally or orally to rats,' In
a 1% solution, phosphorylated hesperidin did, however, impair
the fertilizing capacity of rabbit spermatozoa, but, as Chang &
Pincus point out, such a high concentration would be unlikely to
be achieved after oral or intraperitoneal administration.

CHAPTER 5

THE METABOLISM OF EGGS, I

Oxygen uptake. In 1908 Warburg did some famous experiments
showing that fertiHzation caused a sharp and immediate increase
in the respiration of sea-urchin eggs. The resuhs of a more
recent, manometric experiment on this subject, with KOH in the
centre well of the flask, are given in Fig. 9. The difference be-
tween the pre- and post-fertilization rates of O2 uptake is about
600%. During the first five minutes after the addition of sperm-
atozoa to the egg suspension, respiration appears to have com-
pletely stopped; but this is an illusion caused by the transient pro-
duction of egg acid which takes place at fertilization (q.v.). This
acid displaces CO2 from the bicarbonate in the sea water at such a
rate that the KOH in the centre well cannot absorb it immediately.
As a result, the negative pressure in the manometer, due to the
disappearance of oxygen, is temporarily masked by the positive
pressure of the evolved COg.

In spite of the experiments of Loeb & Wasteneys (1913) on
starfish eggs, in which no comparable rise in respiration after
fertilization was found, the belief used to be widely held that
fertilizafion was associated with an increase in oxidative activity,
and that since an embryo would have to do more work, it would
obviously require more oxygen than an inert, unfertilized egg. As
we shall see, this led to further attractive but untenable ideas, for
example that 'cytochrome' is thrown into circulation at fertiliza-
tion. A new light was shed on the famous phenomenon of in-
creased O2 uptake at fertilization when Whitaker (1931^,6)
pointed out that though O2 uptake increased when the eggs of
Arbacia punctidata, Fucus vesiculosus and Nereis succmea were
fertilized, there was a negligible increase in the case of Sabellaria
alveolata (Linn.), and a decrease in the eggs of Cumingia tellinoides
and Chaetopterus variopedatus (Fig. 10). Nor is there any increase
in O2 uptake when the eggs of Saxostraea commercialis (Iredale &
Roughley) are fertilized (Cleland, 1950^), in the frog's egg
(Brachet, i934Z»), nor in some batches of eggs of Urechis caupo
(Tyler & Humason, 1937). The next discovery of interest in this

THE METABOLISM OF EGGS, I

-70

tq-60

•6
^-50

e-40

c;
o

"o -30
o

"2-20

c
o

-10

=1.

+10

+20

I /

11^

■^

30
t (min.)

FIG. 9. — Metabolism of eggs of Paracentrotus lividus, before and after fertiliza-
tion. F, addition of spermatozoa. Gas phase, air. /, with 0-2 ml. 10% KOH
in the centre well of the manometer flask; //, without KOH. Both curves
corrected for sperm respiration. T° C, 20.

field was due to Holter & Zeuthen (1944), who found that the
respiration of unfertilized eggs of Ciona intestinalis (Linn.) de-
clined with time after removal from the female. A few years later,
Borei (1948, 1949), using the Cartesian diver technique, examined
the variation in the Oo uptake of unfertilized sea-urchin eggs
{Psammechinus miliaris) with time after removal from the ovary.
His results are shown in Fig. 11. If it were possible to measure the
respiration of unfertilized sea-urchin eggs immediately after

58 FERTILIZATION

shedding and to fertilize them at once, it seems probable that no
increase in O2 uptake would be observed after fertilization. In
fact, reference to Fig. 1 1 shows that there might well be a decline
in O2 uptake, though at a later stage in embryonic development
there would, of course, be the well-known increase in respiration.
In nature, sea-urchin eggs are fertilized soon after shedding, for
reasons which are gone into at length in chapter 2, Sperm-Egg In-
teracting Substances, I. Claims that cytochrome is 'thrown into
circulation' at fertilization, or that, before fertilization, there is
limited contact between respiratory enzymes such as cytochrome

■t~i
%
0^90

t (hours)

FIG. 10. — Rate of O2 uptake of eggs of Chaetopterus variopedatm. F, addition of
spermatozoa. T° C, 21. The post-fertilization rate is expressed as a
percentage of the pre-fertiHzation rate (100), which is —24 /il02/hour/io /tl
eggs. After Whitaker (1933a).

and substrates in the egg (Runnstrom, 1930); or that there is a
block in the chain of carriers at this time (Runnstrom, I935«);
or that unfertilized eggs are in a biochemically similar condition to
insect embryos in diapause (Needham, 1942), lose some of their
value in the light of Borei's experiments. It is, of course, interest-
ing to know that unfertilized eggs preserve their substrates by a
reduction in metabolic activity when allowed to remain unfer-
tilized for hours rather than minutes, a frequent concomitant ot
their preparation for manometric experiments; but generalisations
and interpretations based on a study of biological material in
abnormal conditions are apt to be misleading, particularly if the
abnormal features have escaped the notice of the experimenter.

THE METABOLISM OF EGGS, I 59

Cleland (ig^oa) was unable to observe any decline in O^ uptake
with time after removal from the ovary, in unfertilized oyster eggs,
in Warburg manometers. But sooner or later, being shaken in
manometers has an injurious effect on unfertilized eggs, which is
reflected in pathological increases in O2 uptake. Alternatively, the
apparent increases in the O2 uptake of unfertilized eggs may be

o»
o

POST-FERTILIZATION RATE

PRE -FERTILIZATION RATE

400

Minutes after removal from ovary

500

FIG. II. — Comparison of pre- and post-fertilization O2 uptake of eggs of Psani-
mechinus miliaris, after Borei (1949). The asymptotic O., uptake of the un-
fertilized eggs, 5-10 ~ * /Ltl02/hour/egg, is only about yth of the O^ uptake in
Fig. 13-

due to the growth of bacteria in the suspension, though, admittedly,
it takes some while for this effect to become significant. Tyler et
al. (1938) carried out some interesting experiments, Fig. 12, com-
paring the respiration of unfertilized eggs of Arbacia punctulata in
sterile and non-sterile media. The experiments show that con-
tamination of cultures with bacteria and the metabolism of the
latter are factors which require careful attention in experiments of
this sort. Some of these factors may explain Cleland's results; but

FERTILIZATION

Borei's findings are of sufficient importance to warrant their being
repeated and it is to be hoped that this will soon be done.

We must now enquire into the reasons for the different respira-
tory responses of eggs to fertilization. Three explanations have
been put forward, involving respectively the state of maturation
of the egg at fertilization, the pre-fertilization level of Oo uptake,
and changes in the nature of unfertilized egg metabolism, accord-
ing to their and their parents' history. Reference was made in
chapter i to the four different states of egg maturation at which
fertilization occurs. Table 1 1 gives some details of the ratio (O2

FIG. 12. — O2 uptake of unfertilized eggs of Arbacia puiirtiilota under sterile and
non-sterile conditions. The figures against each curve refer to the number
of bacteria per ml. of egg suspension. After Tyler ct al. (1938).

uptake, fertilized eggs)/(02 uptake, unfertilized eggs) in each of the
four classes. Further information will be found in papers by Ballen-
tine (1940) and Cleland (1950a). The ratio will be seen to be about
I, or less, in Classes 2 and 3, not in general much more than i in
Class I, and markedly more than i only in Class 4. There are a
few exceptions ; but the different temperatures at which the experi-
ments were done and the variations in time after removal from the
ovary, may be responsible for these. The table shows, however,
that when the nucleus is in what is sometimes called the 'kinetic'
state, there is no increase in O2 uptake at fertilization, but that
when it is quiescent, Oo uptake increases at fertilization. The in-
crease may, in fact, be caused by sperm penetration or the onset
of meiosis. The Class 2 situation, in which the ratio may be less

THE METABOLISM OF EGGS, I

than I, suggests that the increased respiration at the onset of the
meiotic divisions is not merely what might be expected in a divid-
ing cell, but that breakdown of the germinal vesicle is the re-
sponsible factor. This latter point is confirmed by the experiments

TABLE II
The ratio (O2 uptake, fertilized eggs)/{02 uptake, unfertilized eggs)

in various organisms

Class

. at germinal ^

reside stage

02„

N. succinea
M. laterialis
U. caupo

1-3
1-8

1-2

Barron, 1932
Ballentine, 1940
Tyler & Humason,

1937

Class

f. at first maturation metaphase

C. intestinalis
C. tellinoides
S. commercialis
C. variopedatus
M. glacialis
S. alveolata
C. intestinalis

0-4S

053

I'l

02„

Holter & Zeuthen, 1944
Whitaker, 193 16
Cleland, 1950a
Whitaker, 1933^7
Borei, 1948
Faur^-Fr^miet, 1922
Tyler & Humason, 1937

Clasi

f. at

second maturation metaphase

R. temporaria
B. bufo
F. heteroclitus
F. heteroclitus

-0,e/-

I
I

i6-7

Zeuthen, 1944
Stefanelli, 1938
Boyd, 1928
Philips, 1940

Clasi

•4

f. after maturation

F. vesiculosus
S. purpuratus
P. miliaris
P. lividus
A. punctulata

1-9

3-7
3-6

4-7

4-5

02„

Whitaker, 1931a
Tyler & Humason,
Borei, 1948
Brock et al., 1938
Ballentine, 1940

1937

of Borei (1948) on the eggs of Marthasterias glacialis (Linn.) in
which respiration increases during the breakdown of the germinal
vesicle and the meiotic divisions. In the case of the latter, the in-
crease persists after the meiotic divisions are completed. Cleland
(1950a) also found a difference in the respiration of oyster eggs,
before and after the breakdown of the germinal vesicle.

62 FERTILIZATION

The weird case of the eggs of Fimdulus heteroclitus (Linn.) re-
quires re-investigation, particularly since Nakano (1953) found no
changes in Og uptake after fertilization of the eggs of Oryzias
latipes (Temminck & Schlegel). Very little respiratory increase
occurs in this egg until about two hours after fertilization.

Whitaker (19336) has put forward the view that the post-ferti-
lization level of respiration, and therefore the change in respiration
at fertilization, is dependent on the pre-fertilization level, as many
fertilized eggs tend to have the same respiratory rate, — (i-2)fil02/
hour/io/xl eggs at 21° C, while the pre-fertilization rate may be
well above or below this level. Whitaker has adduced convincing
evidence in favour of this contention, which Brachet supports in
Chemical Embryology. One might summarise Whitaker's hypo-
thesis by saying that the respiration of the unfertilized egg is
regulated by fertilization, many eggs which, before fertilization,
have widely different rates of O2 uptake, approaching the same
rate after fertilization.

The idea that the past history of eggs and the females from which
they were obtained may influence the respiratory response to fer-
tilization was first put forward by Tyler & Humason (1937),
following their experiments on the eggs of Urechis caupo. Fertiliza-
tion of these eggs may produce an increase, a decrease, or no change
in O2 uptake, depending on how long the animals have been kept
in the aquarium tanks. When eggs are obtained from a female
which has not been kept long in the aquarium, the unfertilized
rate is relatively high; but in eggs from a female which has been
in the aquarium for a considerable time, and which is therefore
starved, the unfertilized rate is low. Kavanau (19546), in his
studies on the amino acid metabolism of sea-urchin eggs, claims
that the high respiratory rate of eggs freshly removed from the
ovary, and of oocytes, is due to the high energy requirements of
yolk protein synthesis, which he believes goes on at this time.
Kavanau's results are discussed in more detail in the next chapter,
but it seems doubtful whether this can be the whole explanation of
the wide variation in respiration observed in eggs of diftcrent
species. It is, however, clear that in future, more attention must
be paid to the history both of the eggs and of the females from
which they were obtained, before the experimental period.

When the rate curves immediately after fertilization are ex-
amined, Fig. 13, it will be observed that, apart from any steady

THE METABOLISM OF EGGS, I 63

increase in the level of O2 uptake, fertilization is followed by a
transient increase in respiration, even though rate curves tend to
over-emphasize such effects. According to Boyd (1928), the same
thing happens in the eggs of Fundulus heteroclitus, though the time

\
w

720

640

560

480

4nn

n
1 1
1 1

F J

▼1

3 20

240

160

o\J

t (min.J

FIG. 13. — Total CO2 production and O2 uptake in lA gas/hour, before and after
fertilization of eggs of Psammechinus miliaris. F, addition of spermatozoa.
Warburg indirect method. Gas phase, 03% CO2 in Oj. T° C, 20. (Laser
& Rothschild, 1939).

scale is longer. Whether this always occurs and what its signifi-
cance is are questions which require further investigation.

Respiratory quotient. After straightforward measurements of
O2 uptake, the next stage in a metabolic investigation often takes
the form of an examination of the respiratory quotient (R.Q.), i.e.

64 FERTILIZATION

(CO2 produced)/(02 consumed). Such measurements are technic-
ally rather difficult to do immediately after fertilization, when sea-
urchin eggs are the biological material, for the following reasons:
first, the primitive method of measuring R.Q. by having two mano-
metric flasks containing eggs and absorbing the evolved CO2 in
one of them (which gives — O2), but not absorbing the evolved
CO2 in the other flask (which gives +CO2 — O2), does not work;
because sea-urchin eggs, unlike those of the oyster (Cleland,
1950a), respire at higher rates when the bicarbonate content of the
sea water is normal than when it is low, following the absorption
of CO2 from the gas phase. Secondly, CO2 retention must be
measured. This is troublesome and necessitates the use of mixtures
containing known tensions of CO2 in the gas phase. Thirdly, un-
fertilized sea-urchin eggs which have been prepared for mano-
metric experiments and may, therefore, have been out of their
ovaries for more than an hour, have a low rate of metabolism, as
can be seen from Figs. 9, 11 and 13, and are easily damaged by
being shaken in manometers. These difficulties in the systematic
repetition of experiments on unfertilized eggs may drive the ex-
perimenter to the dangerous expedient of selecting experiments in
which 'everything went well'. The apparent R.Q. of unfertilized
sea-urchin eggs is about 1-4 (Laser & Rothschild, 1939); earlier
workers, such as Ashbel (1929) and Borei (1933) obtained somewhat
lower figures, i-i and 1-1-2. These experiments only tell one that
the ratio (CO2 produced)/(02 consumed) is greater than unity in un-
fertilized eggs. They provide no information about the nature of the
endogenous substrate being metabolized, because the experiments
do not enable any distinction to be made between respiratory CO2
and CO2 displaced from the sea water by acid diffusing out of the
eggs. An overall R.Q. of more than i very probably indicates that,
in these circumstances, acid is being produced by the eggs ; but in
the absence of information as to how much, we cannot tell what
the true R.Q. is, and, therefore, what substrates are being utilised.
The delicacy of unfertilized sea-urchin eggs has already been
mentioned. The possibility that shaking them in manometers in-
duces a pathological formation of acid requires further investiga-
tion.

In rock-oyster eggs, the R.Q. is 0*8 before fertilization, which is
interpreted by Cleland (1950^) as being due to the eggs metabolis-
ing a mixture of carbohydrates and lipids. The R.Q. is also lower

THE METABOLISM OF EGGS, I 65

than I, 0-69-0-89, in the unfertilized eggs of Urechis caupo (Horo-
witz, 1940); but Brachet (1934a) obtained the value 0-99 for the
unfertilized frog's egg. These results certainly do not suggest a
common endogenous substrate in unfertilized eggs of different
species.

Intuitively, one feels that profound changes in metabolism
should occur at fertilization and that these may be reflected in the
R.Q. of the egg. In the period 5-20 minutes after fertilization of
the sea-urchin egg, the R.Q. is o-66, but in the first 2-10 minutes,
even lower values are obtained. The average R.Q. for the first
30 minutes after fertilization is 0-84. These results were obtained
by Laser & Rothschild (1939) using the Warburg indirect method.
Low values for sea-urchin eggs at somewhat longer times after
fertilization have been obtained by Ephrussi (1933), Borei (1933)
and Ohman (1940), while Brachet (1950) has reported that the
R.Q. of the frog egg falls from 0-99 to 0-66 after fertilization; these
latter measurements were done over a very long period, 10-15
hours, and are not, therefore, strictly relevant to the issue of what
metabolic changes occur at fertilization. Cleland (1950a) found
no change in the low R.Q. of rock-oyster eggs at fertilization, but
Horowitz (1940) states that the low R.Q. which is characteristic
of unfertilized Urechis eggs disappears at fertilization, and that
during the first two hours of development, the R.Q, is i. These
results are probably not so confusing as they superficially seem to
be, when one remembers the diff'erent times after fertilization that
measurements have been made and the difference in the mor-
phology of a fertilized egg, 10, 30 and 120 minutes after fertiliza-
tion. In this book we are only concerned with the early phases of
reproduction, the first 60 or so minutes of the egg's existence
following fertilization. Metabolism after the fusion of the pro-
nuclei and energy sources during development (Needham, 1942;
Brachet, 1950) are not, therefore, considered.

Acid production. The sudden evolution of acid, about 20 /x-
moles/ioo mg. N, when the sea-urchin egg is fertilized, was first
mentioned in 1929 by Ashbel and examined in detail by Runn-
strom (1933). Reference to Fig. 13 shows that the evolution of this
acid, or at any rate the bulk of it, is of very short duration, about
five minutes. The nature of the acid is unknown; it is neither
lactic, pyruvic nor malic acid and, according to Yeas (1950), it is
unlikely to be any of the Krebs cycle acids. In spite of Cennamo

66 FERTILIZATION

& Montella's claim (1947) that the acid formed in cytolysing sea-
urchin eggs is phosphoric acid, and the inhibitory action of phlor-
rhizin on this reaction (Rothschild, 1939), the acid which diffuses
out of these eggs at fertilization is not phosphoric acid. Perhaps
we should take account of the possibility that no organic acid
diffuses out of the egg at fertilization, but that some ion exchange
reaction, involving H3O+, occurs. According to Cleland (1950^,
p. 314), who did not observe any acid production when rock-oyster
eggs were fertilized, 'experiments on cytolytic acid production . . .
suggested beyond reasonable doubt that most of the acid produc-
tion was non-metabolic' The word non-metabolic means non-
glycolytic; an exchange reaction would presumably come into the
'non-metabolic' category. Alternatively, the reaction

hexokinase

ATP + glucose > ADP + glucose-6-phosphate + H+

at pH 7, is one suggestive example of a means of producing acid
in the form of hydrogen ions.*

It is a remarkable and disappointing fact that acid production
at fertilization has not been observed, assuming that anyone ex-
cept Cleland has looked for it, in eggs other than those of the sea-
urchin.

The cytochrome system. After measurements of O.^ uptake and
R.Q., the next step might well be to investigate the activity of the
cytochrome system in fertilized and unfertilized eggs, as cyto-
chrome oxidase is the terminal enzyme in aerobic catabolism. We
can be virtually certain that cytochrome will be present, because
it has been found in all aerobic organisms which have so far
been examined ; but confusion about the existence of cytochrome
in sea-urchin eggs and its role, if any, in fertilization, has
arisen for two reasons. First, because many workers have failed
to observe cytochrome spectroscopically in sea-urchin eggs; it
was not until these eggs were examined spectroscopically at the
temperature of liquid air (Rothschild, 1949a), a technique which

* When considering increases in O2 uptake at fertilization and reactions
involving ATP, the student should note that it", at fertilization, ATP is broken
down with liberation of ADP, oxidations may be facilitated, as reactions of the
type

AH2 + B — >A + BH,

very often cannot take place, unless they occur in the following way

AH2 + B + ADP + P — > A + BHa + ATP

THE METABOLISM OF EGGS, I 67

Keilin & Hartree discovered in 1939 and which markedly inten-
sifies the various bands of cytochrome, that cytochrome was clearly
seen. The sea-urchin egg spectrum is atypical, as although band a
is present, the normal bands of b and c are replaced by one which
can be called b^. Yeas (1954) confirmed these observations. The
band of CO-cytochrome oxidase, or COa^, is too faint to be seen
with certainty, though cytochrome oxidase has been identified
by Krahl et al. (1941). The apparent absence of cytochrome
c — according to Borei (195 1), sea-urchin eggs must contain less
than 5 X io~* y/mg. dry matter — is surprising. But in view of
the reactions of fertilized and unfertilized eggs to inhibitors
of cytochrome oxidase and the increased O2 uptake observed in
the presence of dimethyl-^-phenylenediamine (Runnstrom, 1930),
we can be certain that cytochrome c, or a carrier which is function-
ally indistinguishable from it, is present in the sea-urchin egg.
Cytochrome c is present in the eggs of the rock-oyster (Cleland,
i%ob). According to Horowitz & Baumberger (1941), the eggs of
Urechis caupo do not contain cytochrome but an oxidizable and
reducible haem pigment, Urechrome, believed to act in a similar
way to cytochrome. In spite of this observation, a further examina-
tion of these eggs at the temperature of liquid air should be re-
warding.

The second reason for the confusion about cytochrome in sea-
urchin eggs concerned the inability of early workers to recognise
that cyanide, which inhibits the reduction of ferri-«3, and CO,
which in the dark or green light inhibits the oxidation of ferro-%,
inhibit the respiration of unfertilized eggs. These early failures
were for the time being partly responsible for the seductive but
false idea that the cytochrome system does not function in the un-
fertilized egg but is 'thrown into circulation' following fertihzation
or parthenogenetic activation. It is now known, through the work
of Robbie (1946) and Rothschild (1949(2), that both cyanide and
CO reversibly inhibit the respiration of unfertilized sea-urchin
eggs. These observations prove that the cytochrome system is
present and functioning in these eggs. One of the difficulties en-
countered in the use of these inhibitors is that, at certain concen-
trations but not at others, both of them increase the Oo uptake of
unfertilized eggs, as Lindahl observed in 1940. The mechanism
of this eflPect is not clear in the case of cyanide. In the case of CO,
the eggs may oxidize CO as in heart muscle (Fenn & Cobb, 1932),

68 FERTILIZATION

though Lindahl (1938) does not think this Hkely; alternatively,
their respiration may be stimulated by CO, which is known to
occur in other tissues (Daly, 1954). A further complicating factor
in CO-inhibition studies is that the respiration of unfertilized sea-
urchin eggs is depressed in the presence of strong light, which
must be used to obtain photo-reversal of CO-inhibition.

In the light of what has been said above, I am inclined to think
that, for historical reasons, Needham placed too much emphasis
in Biochemistry and Morphogenesis on the alleged change-over at
fertilization from a non-ferrous to a ferrous type of respiration.
In other respects his section on Respiration, pp. 562-605, is ex-
cellent and contains a good deal of information omitted, for reasons
of space, from this chapter.

To sum up, changes in Oo uptake at fertilization depend on the
time after shedding or removal from the ovary when fertilization
occurs; on the state of maturation of the egg at fertilization; and
on the previous history of the animals from which the eggs were
obtained. These factors may result in there being an increase, no
increase, or a fall in respiration at fertilization. The transient in-
crease in O2 uptake, which occurs immediately after fertilization,
has only been observed in sea-urchin eggs (apart from an early
experiment, which should be repeated, on Funduliis eggs) ; the same
applies to acid production and the low R.Q. at this time. The lack
of systematic examination of other eggs, particularly in regard to
acid production, is a serious lacuna in our knowledge. The cyto-
chrome system is present and functioning in unfertilized and ferti-
lized eggs. No qualitative changes in the behaviour of this system
occur at fertilization.

CHAPTER 6

THE METABOLISM OF EGGS, II

Carbohydrate metabolism. One of the objects of studying the meta-
bohsm of eggs is to translate the observed biological results of
fertilization into quantitative chemical language. This will probably
not be achieved in the next twenty-five years and, as always
happens when subjects are in their infancy, our knowledge of the
metabolic changes induced by fertilization is disordered and con-
tradictory. We can follow up the investigations described in the
previous chapter by asking the following questions: what are the
principal sources of energy in the egg and what are the metabolic
changes which make this energy available for such purposes as
maintenance of structure, pumping sodium out of the egg if that
is necessary, rotation of the sperm head, cytoplasmic movements
when they occur, aster formation, and movements of the pro-
nuclei? The R.Q. experiments already discussed show that it is
far from certain that carbohydrate is always the principal endogen-
ous substrate of eggs; but because our knowledge of the pathways
of carbohydrate catabolism is greater than that of lipid or protein
catabolism, more attention has naturally been paid to the former
than the latter. Tables 12 and 13 summarise the evidence that sea-
urchin eggs have a normal anaerobic carbohydrate metabolism, or
at any rate the machinery for it. The reason for the qualification
is that, because of the impermeability of eggs (unlike spermatozoa,
Mann, 195 1), experiments to investigate metabolic pathways in
eggs almost always necessitate the use of homogenates, with or
without the addition of various cofactors; in these experimental
conditions, the normal egg structure and, therefore, conceivably,
the normal egg metabolism, is destroyed.

Cleland (1950a, b) has obtained evidence which is very similar
to that given in Tables 12 and 13 for the existence of a classical
glycolytic system in the eggs of the rock-oyster.

In spite of what has been said above, opinions are not unanimous
about the principal pathways of carbohydrate metabolism in the
sea-urchin egg. Lindberg & Ernster (1948) were the first to suggest
that the mechanism often known as the hexose monophosphate

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THE METABOLISM OF EGGS, II 71

. TABLE 13
Further evidence for the existence of a classical glycolytic cycle
in sea-urchin eggs (Cleland & Rothschild, I952h)

1. Accumulation of pyruvate and lactate anaerobically and in presence of

HCN.

2. Inhibition of O2 uptake by fluoride and reversal by pyruvate.

3. Accumulation of pyruvate after addition of glucose-6-phosphate.

4. No accumulation of pyruvate after addition of phosphogluconate.

5. Active oxidation of hexose diphosphate with pyruvate accumulation.

6. Stimulation of endogenous O2 uptake by DPN.

7. Stimulation of fructose oxidation by DPN.

8. Inhibition of 6 and 7 by fluoride and iodoacetate.

shunt, or an oxidative pathway, was mainly responsible for carbo-
hydrate breakdown in sea-urchin eggs. As these pathways may
not be so famihar to some readers as the usual glycolytic one, they
are reproduced on p. 72.

The evidence in favour of the operation of a scheme along these
lines in sea-urchin eggs is: (a) egg homogenates actively metabolise
phosphogluconate. In addition, Krahl et al. (1955) have shown
that Arbacia homogenates contain glucose-6-phosphate and 6-
phosphogluconate dehydrogenases, and can form ribose from
either substrate. These workers found that the rate of TPN re-
duction with glucose-6-phosphate as substrate was about 3 /x,-
moles/min./g. eggs, enough to permit an O2 uptake six times that
of fertilized and twenty-four times that of unfertilized eggs. On the
other hand, the rate of DPN reduction by egg extracts with
fructose- 1, 6-diphosphate as substrate was o- 1-0-2 ju,-moles/min./g.
eggs. At the same temperature, the Oo uptake of the fertilized eggs
would require 0-5 ju,-moles of DPN to be reduced if all the Og up-
take were associated with the glycolytic breakdown of carbohydrate.
Although all the Oo uptake of fertilized eggs is almost certainly not
associated with carbohydrate breakdown, the amount of carbo-
hydrate which can pass the aldolase-oxidizing enzyme step appears
to account for only 20-40% of the oxygen actually consumed in
the eggs of Arbacia punctulata. (b) Low concentrations of iodo-
acetate do not inhibit the endogenous Oo uptake of egg homo-
genates ; there are differences of opinion on this point. Cleland &
Rothschild (1952a) found that iodoacetate did inhibit glycolysis
in egg homogenates of Echinus esculentus, and so did Yeas (1954),
using egg homogenates of Stro?igylocentrotus purpuratus and
Lytechinus pictus. Lindberg & Ernster's experiments were done in

72 FERTILIZATION

TABLE 14

A, hexose monophosphate shunt ; B, oxidative breakdown of carbo-
hydrate, after Horecker (1953). One mole of CO2 is evolved, two
moles of TPN are reduced, and one mole of O^ is consumed, per cycle.
Two complete cycles will regenerate one mole of glucose-6-phosphate
and will produce one mole of tetrose phosphate, which, for the com-
pletion of the carbohydrate oxidation, must be converted to glucose-
6-phosphate.

Glycogen

I
TPN+ GIucose-6-phosphate

TPNH 6-phosphogluconic acid

6-phospho-2-ketogluconic acid

Ribulose-5-phosphate

Ribose-5-phosphate ^ Ribose- 1 -phosphate

D-glyCeraldehyde-3-phOSphate

t
Lactic acid

Glucose-6-P

-2H

\ Mg

Fructose-6-P -f Tetrose P

f h

6-P-Gluconate

ceraldehyde-3-P

Sedoheptulose-7-P
B

CO,

Ribulose-5-P

Ribosc-5-P

THE METABOLISM OF EGGS, II 73

the presence of o-iM-NaF, which would make it most unUkely that
any glycolysis would have been observed. In any case, rather low
concentrations of iodoacetate were used and little time was allowed
for the development of the inhibition. The hexose monophosphate
shunt. Table 14A, will, of course, be just as sensitive to iodo-
acetate as the glycolytic pathway. If, on the other hand, the hexose
monophosphate shunt is not involved in the terminal part of the
glycolytic pathway, iodoacetate should not have any inhibitory
action, (c) The addition of hexose monophosphate caused a greater
stimulation of O2 uptake than hexose diphosphate. This observa-
tion also is the subject of dispute ; (d) significant amounts of the
phosphorylated intermediates of glycolysis, e.g. alkali-labile
phosphate (triose phosphate), could not be identified by Ba
fractionation of acid egg extracts; (e) triose phosphate dehydro-
genase was said to be absent by Jandorf & Krahl (1942). Table
12 shows that this is probably incorrect.

To sum up, there are evidently two pathways by which carbo-
hydrate is broken down in the sea-urchin and oyster egg, the nor-
mal anaerobic and also an oxidative pathway ; except in the case of
Arbacia eggs, where the 'strength' of the latter mechanism appears
to be greater than that of glycolysis, at any rate in homogenates, we
do not know which mechanism predominates, though both prob-
ably function to a greater or lesser extent.

Quite apart from being involved in the degradation of poly-
saccharides to provide phosphate bond energy, the hexose mono-
phosphate reaction may be concerned with entirely different
cellular activities. The reaction

Nicotinamide riboside + phosphate ^ Nicotinamide + ribose-i -phosphate,

discovered by Rowen & Romberg (195 1), suggests that the hexose
monophosphate reaction may be involved in nucleic acid meta-
bolism, on the synthetic and not the catabolic side. Some interest-
ing speculations about the role of this reaction in determination,
and, in particular, animalization, will be found in Hultin's review.
Studies on the Structural and Metabolic Background of Fertilization
and Development {ig ^2^).

Transient changes in carbohydrate metabolism at fertilization.
ZieHnski (1939) and later Orstrom & Lindberg (1940) showed that
the 'glycogen' content of sea-urchin eggs decreases at fertilization ;
in terms of glucose, the breakdown is equivalent to 26 /x-moles/ioo

74 FERTILIZATION

mgN. The effect is a transient one, the rate of 'glycogenolysis'
nearly returning to the pre-fertiHzation level ten minutes after
fertilization. In spite of the acid production at fertilization, this
breakdown is not associated with the accumulation of lactic acid
or any of the tricarboxylic acids; moreover, the amounts of in-
organic phosphate, pyrophosphate and hexose phosphates have
been said not to change at fertilization (Runnstrom, 1933; Zie-
linski, 1939; Orstrom & Lindberg, 1940), though we shall see in
the next chapter that recent experiments, in which more refined
methods were used, do not entirely support these findings. If
the low R.Q. during the first few minutes after fertilization is
linked to carbohydrate metabolism, which seems unlikely, we must
conclude that an obscure and transient reaction, involving the in-
complete oxidation of carbohydrate and about which, at present,
there are practically no clues, must occur, transiently, at fertiliza-
tion. Both Brachet, in Chemical Embryology (1950), and Runn-
strom, in The Mechanism of Fertilization in Metazoa (1949), seem
to favour the view that the disappearance of polysaccharide at
fertilization has something to do with the hexose monophosphate
pathway. If this system is only acting as a partial by-pass of normal
glycolysis, lactic or pyruvic acid should be formed and oxidized in
the usual way. If, on the other hand, the hexose monophosphate
reaction is concerned with nucleotide metabolism, why should it
only last for ten minutes after fertilization ? The lack of answers to
these questions indicates, as usual, the need for more experiments.
Lipid metabolism. The low R.Q. at fertilization suggests the
transient oxidation of fats at this time, quite apart from the possi-
bility of fats being an important endogenous substrate before fer-
tilization. The most systematic study of this subject is due to
Ohman (1945), who found that the total lipid content of the eggs
of Echinocardiiim cor datum, which is about 200 mg/ioo mg N,
decreases for some seven hours after fertilization. Of these lipids,
the choline-containing phosphatides or lecithins decrease as de-
velopment proceeds, while the cephalins or non-choline-containing
phosphatides increase. Ten minutes after fertilization, 29% of the
free cephalin in the egg becomes bound to proteins. The difference
between free and bound cephalin is based on the solubility
of the former in ether-chloroform mixtures and the solubility of
the latter in chloroform-ethanol mixtures. In an earlier paper,
Ohman (1942) reported that, at fertilization, there was a reduction

THE METABOLISM OF EGGS, II 75

in free (i.e., extractable with ether-chloroform mixtures) 'chole-
sterol'; but in his 1945 paper, Ohman says that his previous ob-
servation was unreliable and that there is no change in the 'chole-
sterol' content of the eggs at fertilization. Monroy & Ruffo (1945),
on the other hand, reported an increase in free 'cholesterol' at
fertilization in sea-urchin eggs. The word cholesterol is placed in
quotation marks above, because there is some doubt whether the
substance whose concentration was measured by Ohman, Monroy
& Ruffo, and earlier workers such as Mathews (1913) and Page
(1923), was actually cholesterol or a related steroid.

2-O-CO-R

•CO-O-CH

a-O-PO-O-CHa-CHj

OH
CL-Lecithin

CHa'O-CO-R
•CH

•N(CH3)30H

•co-o

CHg-O-PO-O-CHa'CHg-NHa

OH
Phosphatidylaminoethanol, a simple cephalin
R-COOH and R'-COOH are fatty acids

Nitrogen metaholism. Important work in this field was done by
Orstrom in 1941. He reported that, at fertilization or parthogenetic
activation, there was a transient production of ammonia, lasting
for about ten minutes, and that simultaneously, a substance which
liberated ammonia upon heating and which Brachet (1950) thinks
may be a glutaminylpolypeptide, disappears. Orstrom believed that
the ammonia production resulted from the deamination of nucleic
acid. However, this view was based on the deamination of muscle
adenosine and adenylic acid by unfertilized egg homogenates, a re-
action which he said was accelerated by the addition of cytolysed
spermatozoa. Orstrom also claimed that fertilized eggs differed
from unfertilized ones in being able to synthesize glutamine from
glutamic acid and ammonia, though both unfertilized and fertilized
eggs can effect the reverse reaction, the hydrolysis of glutamine
to glutamic acid and ammonia. xA.part from transiently producing

76 FERTILIZATION

ammonia, Orstrom observed that the NHg-binding capacity of
fertiHzed sea-urchin eggs was higher than that of unfertiHzed eggs.
This observation has been confirmed by Hultin (1950c) who says
that the maximum rate of ammonia uptake occurs during the
formation of the fertiUzation membrane. It may seem curious
that, in the absence of added ammonia, this is precisely the time
at which the eggs are producing the maximum amount of ammonia.
When sea-urchin eggs are allowed to develop in the presence of
^^N-glycine or ^^N-DL-alanine, and subsequently extracted with
KCl or sucrose solutions, insoluble proteins, which are probably
derived from the microsomes, are found to incorporate the isotope
rapidly in the early phases of development, while the soluble
proteins do not (Hultin, 1953a). Hultin interprets these results as
showing that soon after fertilization, there is a 'rebuilding or multi-
plication of small, cytoplasmic granules, containing ribonucleic
acid' (p. 18), a process which is intensified during determination.
In spite of the advances made in recent years in accumulating in-
formation about nucleotide, protein, and amino acid metabolism in
developing sea-urchin eggs, it is unfortunate that Orstrom's
original work has not been systematically repeated and, therefore,
remains largely unconfirmed. Even the transient production of
ammonia at fertilization has not been observed by other workers
and if this experiment could be done fifteen years ago, it could
easily be repeated now.

Kavanau (1953, 1954^, b) has made interesting observations
on the amino acid metabolism of sea-urchin eggs. He observed
cyclical changes in amino acid concentration (free, peptide bound,
and derived from proteins), in fertilized eggs of Paracentrotus
lividus, but not in the eggs of Strongylocentrotus purpuratus ; the
failure to detect these changes in the latter may have been due
to unavoidably poorer experimental conditions. The experi-
ments were done rather a long time after fertilization, from
the point of view of this book, though the fall in protein in-
soluble in a mixture of o-iM-KCl and o-oiiM-acetic acid, in total
protein, and in total amino acid concentration, two to three hours
after fertilization, is striking. Kavanau believes that an abrupt
change in amino acid metabolism occurs at fertilization, yolk
synthesis stopping and intense yolk-protein degradation starting,
the latter being a major source of energy. In the case of unfer-
tilized eggs Kavanau observed an increase in protein and a decrease

THE METABOLISM OF EGGS, II 77

in non-protein amino acids after ageing in sea water. These changes
are said to reflect the fact that unfertilized eggs synthesize their
own yolk proteins, free amino acids and peptides being supplied to
them from other cells in the ovary; though this is not in accord
with the fairly prevalent view that large molecules such as proteins
are supplied to the growing oocyte in finished form. Unfertilized
eggs from freshly collected Strongylocentrotus purpuratiis had a high
free amino acid content which dropped after fertilization, but this
was not so in unfertilized eggs obtained from urchins kept under
conditions inhibiting spawning. In the latter, the free amino acid
content increased after fertilization. Kavanau states that a high
free amino acid content is characteristic of eggs in which yolk
synthesis is proceeding rapidly, which suggests under-ripeness;
a low free amino acid content means that yolk synthesis has ceased,
free amino acids and small peptides are no longer being absorbed,
and the eggs are ripe (or perhaps even over-ripe), and ready to
be spawned. A different interpretation of the facts is, however,
possible. The low free amino acid content of unfertilized eggs
obtained from animals kept under conditions which inhibited
spawning and feeding may be correlated with the nutritional state
of the animal, rather than with changes in the synthetic activities of
the egg. Freshly collected and, presumably, well fed animals might
be expected to be relatively rich in free amino acids, while stored
and probably semi-starved animals might have a rather low content
of them, the difference being reflected in all the body tissues, in-
cluding the ovaries and eggs. According to Kavanau (1954a, p.
566), 'the early post-fertilization changes in non-protein amino
acids (and probably other metabolites) must apparently be viewed
as adjustments which bring an initially flexible system to a more
rigidly defined state from which normal development can proceed.'
These views recall those of Whitaker (19336) on the 'regulation' of
respiration by fertilization. The idea that metabolic changes at
fertilization may be positive or negative according to the past
history of the unfertilized egg may seem startling, but there is
evidence to support this idea. The fact that the increase in O, up-
take after fertilization of Psammechinus eggs depends on the delay
between spawning or removal from the ovary (Fig. 11) is one
example, as is the paradoxical behaviour of Urechis eggs after
fertilization (Tyler & Humason, 1937). Another concerns the
contradictory results of Infantellina & La Grutta (1948) and

yS FERTILIZATION

Bolognari (1952) on the glutathione content of sea-urchin eggs both
before and immediately after fertihzation. There are two morals to
be drawn from these new 'difficulties' in interpreting metabolic
changes at fertilization. First, the spectre of unreproducible or
contradictory results, which often haunts the biologist, may not
be so frightening as it sometimes seems ; secondly, in investigating
the various changes which occur at fertilization, we must not only
bear in mind species differences, but also the past history of the
material, for it is evident that the pre-fertilization history of eggs
influences metabolism during early development.

Tricarboxylic acid cycle. The tricarboxylic acid cycle un-
doubtedly functions in sea-urchin, oyster (Cleland, 19506), and cer-
tain fish eggs. Oxaloacetate, succinate, a-ketoglutarate, glutamate
and citrate are rapidly oxidized (Crane & Keltch, 1949), while
Keltch et al. (1950) showed that a particulate cell-free system ob-
tained from unfertilized Arbacia eggs esterified orthophosphate
during the oxidation of Krebs cycle intermediates. These results
have been confirmed and extended by Yeas (1954), who also
demonstrated the inhibition of Oo uptake by malonate, and by
Cleland & Rothschild (19526), who showed that egg homogenates
oxidize pyruvate, the effect on O2 uptake being more pronounced
when the formation of pyruvate from endogenous substrate was
blocked by fluoride. The complete oxidation of pyruvate is cogent
evidence in favour of the tricarboxylic acid cycle, as the latter is
believed to be the only mechanism which will combust pyruvate
to CO2 and water in animal tissues. The oxidation of pyruvate
by sea-urchin eggs was also noted by Goldinger & Barron (1946)
and Krahl et al. (1942).

Hishida & Nakano (1954) found that addition of the usual
Krebs cycle intermediates stimulated the Oo uptake of egg homo-
genates of Oryzias latipes; succinate had the greatest efi^ect, in-
creasing the endogenous Oo uptake by 500%. The Oo uptake of
these homogenates, with or without the addition of substrates, in-
creases twenty-four hours after fertilization. The authors interpret
this as showing that there is a synthesis of Krebs cycle enzymes at
this time, though they point out that this view conflicts with that
of Spiegclman & Steinbach (1945), who found that the O2 uptake
of frog's egg homogenates decreased as development went on.
The latter concluded that, at the beginning of development, the
respiratory enzymes in the egg were not saturated by their appro-

THE METABOLISM OF EGGS, II 79

priate substrates, although at a later stage in development they
were. This does not seem to be true in the case of the eggs of
Oryzias lattpes, in which the Japanese workers have found a pro-
nounced synthesis of cytochrome oxidase and phosphothiamine
during development.

Fluoroacetate has only a small inhibiting effect, about 20% at
io~^M, on sea-urchin egg homogenates. As it is a powerful inhibitor
of acetate metabolism in some animal tissues, acetate metabolism
may only play a small part in the overall oxidative metabolism of
such systems ; but the student should remember that fluoro-
acetate inhibits the Krebs cycle at the citric acid stage, owing to
its conversion to fluorocitric acid. Fluoroacetate will, therefore,
inhibit any oxidation which is mediated through the Krebs cycle.
The acetate inhibition hypothesis is confirmed by Hultin's ex-
periments (19536) on the metabolic utilization of i-^^C-acetate in
sea-urchin embryos. Utilization is low in the early phases of
development, though the position is not the same later, when
visible diflFerentiation starts. This suggests that except perhaps
during the first few minutes after fertilization, fatty acid meta-
bolism may be of secondary importance in the early phases of
development.

CHAPTER 7

METABOLIC AND OTHER CHANGES AT
FERTILIZATION

This chapter consists of a list, with occasional notes, of the principal
changes (or lack of changes) which have been reported to occur at,
or soon after, fertilization. Morphological, structural and certain
other changes are dealt with in separate chapters. The references
are not intended to be all-inclusive and little attention has been
paid to priority of discovery. References with an asterisk after
them are brief notes, f. stands for fertilized or fertilization; u. for
unfertilized.

(i) Increase in permeability to sparingly ionized solutes, e.g.,
water, /j = o-i (u.), 0-2-0-4 (f.). A. punctulata {R. S. Lillie, 1916;
Lucke et al., 193 1).

The permeability constant k is the amount of water ((j.^), entering
the cell in unit time (i min.), through unit area {[x^), under unit differ-
ence of osmotic pressure (i atm.). The change in k occurs 2-4 min.
after fertilization and is complete in 10 min. (Lucke & McCutcheon,

^932).

Measurements of permeability changes after fertilization or par-
thenogenetic activation of sea-urchin eggs have recently been made by
Ishikawa (1954), Fig. 14. When the eggs of Hemicentrotus pulcherri-
mus are activated by treatment for 12 min. with sea water contain-
ing butyric acid (50 ml. sea water + 3 ml. N/io butyric acid), there is,
according to this worker, no breakdown of cortical granules, no mem-
brane formation and no increase in permeability to water. This casts
some doubt on the importance of granule breakdown (see pp. 9-10),
which Ishikawa thinks is responsible for the increase in permeability,
in activation. Similarly, Kusa (1953) was able to activate salmon eggs,
parthenogenetically, without the cortical alveoli breaking dov/n.
Sugiyama (1953) says that butyric acid treatment does cause granule
breakdown and membrane formation in the eggs of Hemicentrotus
pulcherrimiis. The shape of the normal fertilization curve in Fig. 14 is
similar to that obtained by Hobson (19320), using the eggs of Psam-
mechinus miliaris.

(I'l) Osmotically inactive fraction, or 'non-swcllable volume',
7-3% in u. and 27-4% i^i f. eggs. A. punctulata (Shapiro, 1948).

In this paper, Shapiro states that there is a 2-7% increase in the
volume of this egg at fertilization, see p. 20.

METABOLIC AND OTHER CHANGES AT FERTILIZATION 8l

lO 3

=^ 2

/\J ^ _,_— — i-» "* '

10 20 30 40 50 60 70

Time after fertilization orparthenogenetic
activation in minutes

FIG. 14

Change in volume of sea-urchin egg {Heniicetttrotus pulclierrimus), in
/Li" ~ 10*, after three minutes' immersion in 40% sea water + 60% distilled
water, at various times after fertilization. The point whose co-ordinates on
the middle curve are app. 2-5 X 10"^ ^^ and 5 min. therefore refers to an
egg which was placed in hypotonic sea water (for 3 min.), 5 min. after
fertilization. The rate of swelling at this time is lower than it is in eggs
which are examined earlier, or later, than 5 min. after fertilization. O,
normal fertilization; C.unfertilized eggs previously treated for 3 min. with
M-urea ; # , unfertilized eggs previously treated with butyric acid-sea water
for 30-50 sees. T^ C, 17. After Ishikawa (1954).

(1-2) Negligible increase in permeability to water, ^ ^^^ 0-5
(u.), 0-6 (f.). C. variopedatus (Shapiro, 1939*).

Compare the decline in Og uptake after fertilization in these eggs.

(1.3) 8% reduction in vapour pressure 30 min. after f. R.
temporaria (Picken & Rothschild, 1948).

(2) Rate of P entry from external medium (to which ^^p had been
added) 40 times greater in f. than in u. eggs. A. punctulata
(Abelson, 1947*, 1948*).

The increase started 7-10 min. after f. i-6 X io~^M-4-6-dinitro-
o-cresol inhibited P uptake by a factor of 6 though it doubled Og up-
take and virtually inhibited division.

(2.1) Rate of P entry from external medium (to which ^^P had
been added) 130-160 times as great in f. as in u. eggs (accumula-
tion, not exchange). S. purpiiratus (Brooks & Chambers, 1948*).

(2.2) No increase in rate of P entry from external medium (to

82 FERTILIZATION

which ^-P had been added), after f., until after second cleavage,
and then slowly. U. caupo (Brooks & Chambers, 1954).

These eggs are very variable in their respiratory response to
fertilization (Tyler & Humason, 1937).

(2.3) Rate of P entry from external medium (to which ^^p had
been added) and incorporation in acid labile fraction much faster
after f. (accumulation, not exchange). P. miliaris (Lindberg,
1948).

(2.4) f. eggs accumulate P fast, o-oo3-o-oo4 mg P/ml. f. eggs/
min., from external medium (to which ^-P had been added), u.
eggs do not, though P exchange occurs. Increase in ATP content
70-90 min. after f. at expense of inorganic P. S. purpuratus,
S. franciscanus, L. pictiis (Chambers & White, 1949*, 1954)-

(2.5) No change in ATP content after f. (1-20-1 -45 mg/ml. in
A. forbesi). S. droebachiensis, A. forbesi (Chambers & Mende,

i953«)-

Increased synthesis after fertilization might be balanced by
increased utilization.

(2.6) ATPase activity in f. egg homogenates twice that in u.
egg homogenates. S. purpuratus (Connors & Scheer, 1947).

This may be due to Ca release at fertilization (see 4).

(2.7) Increase in arginine phosphate and decrease, 32%, in
inorganic phosphate, app. 5 min. after f., the latter being mainly
accounted for by the former. No change in ATP. S. droebachiensis
(Chambers & Mende, 19536).

This paper contains a discussion of the contrary results obtained by
Runnstrom (1933), Zielinski (1939), and Orstrom & Lindberg (1940),
using the eggs of Faracentrotus lividiis. The authors conclude that the
change in inorganic P might have taken place when these eggs were
fertilized, but that, because of their higher inorganic P content
(x 15-30), it might have been missed. The synthesis of arginine
phosphate occurs precisely at the time of the transient increase in Go
uptake and acid production, see Fig. 13. Although the authors suggest
that the energy for the synthesis is derived from carbohydrates, the
low R.Q. at this time is difficult to reconcile with this view. The
authors also made one interesting observation on an abnormally con-
centrated egg suspension, in which the eggs developed pathologically.
In this case, there was no decrease in inorganic P and no synthesis of
arginine phosphate after fertilization.

"Ps

METABOLIC AND OTHER CHANGES AT FERTILIZATION 83

(2.8) Intracellular distribution of ^-P before and after f. L.
pictus (Whiteley, 1949).

When the egg is centrifuged (E. B. Harvey, 1941), it separates into
two fragments: a large light part containing an oil cap, a small hyaline
layer, the nucleus or mitotic figure and yolk; and a smaller heavier
part, containing mitochondria and an optically empty layer. In un-
fertilized eggs which have been allowed to accumulate ^^P for six
hours, there is 1-2 times as much ^-P in the light, as in an equal
volume of the heavy part. In fertilized eggs, the ratio (^^P light) /(^-P
heavy) = 0-5, implying that a good deal of the P goes into the mito-
chondria, about 20 minutes after fertilization. This paper contains a
review of the work on P metabolism in eggs up to 1949.

(3) Readily exchangeable K, 20% of total in u. eggs; 80% in f.
eggs. S. purpuratus (Chambers et al., 1948*).

(3.1) K exchange 16 times faster in f. than in u. eggs. S. pur-
puratus (Chambers, 1949*).

(3.2) Cyclical changes in K permeability after f. Uptake, o-io
min. ; release, 10-40 min.; uptake, 40-60 min. 60% of K in eggs
non-exchangeable. P. lividus, A. lixula (Monroy-Oddo & Esposito,

1951)-

These results are statistically significant.

(4) Bound Ca decreases at f. A. punctulata (Heilbrunn et al.,
1934; Mazia, 1937).

(4.1) Ca diffuses out of eggs at f . P. lividus (Orstrom & Orstrom,
1942).

(4.2) Ca and Mg diffuse out of eggs at f. (first measurements
15 min. after f.). A. lixula (Monroy-Oddo, 1946).

Lindvall & Carsjo (1951) were unable to confirm (4.1) and (4.2)
using the eggs of Echinus esculentus; neither were Barnes, Cleland and
I (unpublished). The presence of small amounts of jelly, which con-
tains Ca, round unfertilized eggs, may be a confusing factor in experi-
ments of this type. The uptake of Ca by sea-urchin egg jelly has been
described by Rudenberg (1953); (4-i) and (4.2) should not be
accepted unless they are confirmed. There is a further discussion of
the role of Ca in fertilization in chapter 8.

(5) The following changes in inorganic constituents occur at f.

84 FERTILIZATION

(or laying?): Na, -10%; Ca, -8%; CI, -8%; inorganic P,
— 16% (conversion to organic P ?). S. salar (Hayes et al., 1946).

Manery & Irving (1935) found no change in CI when the eggs of
Salnto gairdneri Richardson were fertilized.

(6) Increase in rate of disappearance of pyruvate, added to sea
water, after f. ; u., 64)/, f., 448y/hour/g. dry weight. A. piinctulata
(Goldinger & Barron, 1946).

This experiment does not, in fact, tell one anything about the differ-
ences between u. and f. eggs in regard to pyruvate metabolism. The
authors do not refer to the changes in permeability which are known to
occur when sea-urchin eggs are fertilized. Nor did they examine the
possibility of different effects on f. and u. eggs of lithium, which was
added to the sea water as lithium pyruvate. This paper contains
several interesting but speculative observations about fertilization,
some of which are now known to be wrong. The earlier negative
results on pyruvate metabolism of Runnstrom (1933) and Orstrom
& Lindberg (1940) are said to be due to inadequate techniques.

(7) No change in diphosphothiamine at f. A. pimctulata
(Krahl et al., 1942; Goldinger & Barron, 1946).

The first authors- found 8-5 y/g. dry weight and the second 16-2 yjg.
dry weight. Diphosphothiamine is concerned in pyruvate metabolism,
as it acts as a coenzyme in the reaction:

carboxylase

CHo-CO-COOH -> CH3CHO + CO,

diphosphothiamine "

(7-1) Fall in free thiamine and phosphothiamine synthesis after
f. O. latipes (Hishida & Nakano, 1954).

(8) DPN, u., 385 y/g. wet eggs; f. (30 min.), 345; f. (600 min.),
242. S. franciscanus, A. piinctulata (Jandorf & Krahl, 1942).

(9) Decrease in ribonuclease activity after f. A. piinctulata
(Bernstein, 1949*).

(9-1) DNA of f. eggs not formed from RNA of u. eggs. A.
pimctulata (Schmidt et al., 1948; Villee et al., 1949).

(9.2) DNA of f. eggs formed from RNA of 11. eggs. P. lividus
(Brachet, 1933).

It is now generally agreed (Abram?, 1951) that (9.2) is wrong and
(9.1) is right. RNA fragments may, however, be utilized in DNA
synthesis.

METABOLIC AND OTHER CHANGES AT FERTILIZATION 85

(9.3) Surface of oi! droplets Feulgen-negative in u. eggs and
Feulgen-positive in f. eggs. A'', succinea (Lovelace, 1949*).

This experiment requires confirmation. A Feulgen-positive re-
action in the presence of lipids is a common artifact. However,
Costello (1938) found that the oil droplets can be made to coalesce
after fertilization, but not before, in eggs of the same species.

(9.4) Doubling of DNA content of pronuclei after f. and before
fusion. M. differ entialis, P. variegatus, M. musculus (Swift, 1953;
Swift, unpubl.).

(9.5) Appearance of RNA 'granules' in the cytoplasm of the
spermatozoon and their dispersion in the egg cytoplasm by the end
of maturation. A. equorum (Panijel, 1947; Pasteels, 1948).

(9.6) 'Dissolution' of Ascaridin, previously surrounding the
sperm head. A. equorum (Pasteels, 1948).

Note on (9.i)-(9.6). The problem of RNA and DNA variations and
their inter-relationship in eggs and spermatozoa is extremely compli-
cated, if not confused ; the non-specialist might be wise to wait a few
years before tackling this question. Pasteels & Lison (1951) for
example, made a detailed examination of the DNA content of the eggs
and spermatozoa of SabeUaria alveolata. Contrary to expectation,
they claimed first, that the DNA content of eggs and spermatozoa was
not the same ; and secondly, that the DNA content of diploid cells was
not double that of haploid cells, the ratio being i : 12 in the case of
spermatozoa compared with the first blastomere, and i : 5-5 in the
case of the mature egg compared with the first blastomere. After
fertilization, Pasteels & Lison found that DNA was synthesized in the
male pronucleus, its content increasing by a factor of 6. The factor
for the egg during this period was 2-75. Further information will be
found in a paper by Alfert & Swift (1953), who could not confirm the
results of Lison & Pasteels; in a review by Swift (1953); and in a
paper by Hoif-Jorgensen (1954), who reported that the total DNA
content of sea-urchin eggs {Paracentrotus lividus) and of frogs' eggs
(Rana temporaria Linn.) remained constant for a considerable time (3
and 18 hours, respectively) after fertilization.

(9-7) D-usnic acid inhibits the fusion of the male and female pro-
nuclei, though they approach each other in the usual way. It also
inhibits cleavage and the uptake of ^^P, though Og uptake is un-
affected. A. punctulata (Marshak & Fager, 1950).

86 FERTILIZATION

COCH,

HO.
Usnic acid, T f 1 ^^ believed to inhibit

H3C y CH3 I COCH,

OH OH

DN-ase when cobalt is present, though it is also thought to interfere
with oxidative phosphorylation. As several workers have put forward
the view that the biological activity of unsaturated carbonyl com-
pounds is due to their additive interaction with thiol compounds
(Sexton, 1949), the effects of cysteine and thioglycollic acid on usnic
acid inhibition would be of interest. The fusion of the pronuclei is
also inhibited by anaerobiosis and HCN (Runnstrom, 1935a).

(10) Increase in alkaline phosphatase after f. P. lividiis (Wick-
lund, 1948).

(10. 1 ) No change in alkaline phosphatase until 10 hours after f.
P. miliaris (Gustafson & Hasselberg, 1950).

(10-2) No change in acid or alkaline phosphatase until gastrula-
tion. A. punctulata (Mazia et al., 1948*).

(10.3) No sudden change in alkaline phosphatase after f. X.
laevis, S. mexicanum (Krugelis, 1950).

(11) Transient NH3 production at f. P. lividtis (Orstrom,
1941).

(ii.i) Non-transient NH3 production after f. A. punctulata
(Hutchens et al., 1942).

This work has been criticised on technical grounds by Lindberg
(1945)-

(11.2) f. eggs form glutamine from added NH3 and glutamic
acid, u, eggs do not. P. lividns (Orstrom, 1941).

(12) No change in 'free' hypoxanthine and guanine, present in
the ratio 2:1. R. temporaria (Steincrt, 1952).

(13) No change in free 'cholesterol' at f. E. cordatum (Ohman,
1945)-

METABOLIC AND OTHER CHANGES AT FERTILIZATION 87

(13. i) No change in free 'cholesterol' for 15 min. after f. ; then
gradual decrease. P. lividiis (Lindvall & Carsjo, 1948).

(13.2) Increase in free 'cholesterol' at f, A. lixiila (Monroy &
Ruffo, 1945*).

(13.3) Fall in free cephalin at f. E. cordatum (Ohman, 1945).

(13.4) Decrease in lipids after f. A. punctiilata (Hayes, 1938).

(13.5) No change in lipids or lipo-proteins after f. A. punctulata
(Parpart, 1941*).

(13.6) Increase in lipids after f. ^. e^'Morwrn (Panijel, 1951).

(14) Decrease in carbohydrate after f. P. lividus (Zielinski, 1939) ;
P.Uvidus, E. cordatum (Orstrom & Lindberg, 1940); sea-urchin
(Lindberg, 1945); A. eqtiorum (Panijel 195 1); R. temporaria (Panijel,
1 951); P. miliaris (Lindberg, 1943).

(14. i) No decrease in carbohydrate after f. A. punctulata
(Hutchens et al., 1942).

This work has been criticised by Lindberg (1945).

(15) No change in dipeptidase activity after f. P. miliaris,
(Doyle, 1938); E.parma (Holter, 1936).

(15. 1 ) Slight decrease in dipeptidase activity after f. U. caupo
(Linderstr0m-Lang & Holter, 1933).

(15.2) Fall in amino-nitrogen, principally glycine, during first
two hours after f. and accumulation of free glutamine immediately
after f. P. lividus (Gustafson & Hjelte, 195 1).

(^5"3) No change in free amino acids after f. S. purpuratus
(Berg, 19506).

(15-4) Amino acid metabolism and, in particular, Kavanau's
recent results, are discussed in chapter 6.

(15.5) Transient activation of proteolytic enzymes. P. lividus,
A. lixula, E. cordatum, E. esculent us, B. lyrifera (Lundblad, 1949-
1954)-

FERTILIZATION

Lundblad has published an interesting series of papers showing
that proteolytic enzymes are activated transiently at fertilization (Fig.
15). Two of these enzymes, EI and EIII, can be activated in un-
fertilized egg homogenates by ribonuclease. Lundblad & Hultin
(1954) beHeve that EI and EIII are activated at fertilization by ribo-
nuclease, derived from the fertilizing spermatozoon or from the egg,
following the activation of egg ribonuclease by the fertilizing spermato-
zoon. EI and EIII are also activated by SH groups, which have an
inhibitory effect on EI I, as does heparin. These observations are not

FIG.

15. — Activity of proteolytic enzymes in sea-urchin eggs. A, before fertiliza-
tion; B, at fertilization; C, at membrane elevation; D, immediately after
membrane elevation; E, ten minutes after membrane elevation. Three
enzymes, EI, EII and EIII, are involved in each period. After Lundblad
(1954^).

incompatible with the ribonuclease hypothesis, as SH reagents
increase the activity of ribonuclease in liver (Roth, 1953), while
heparin inactivates sea-urchin egg ribonuclease (Lindvall & Carsjo,
1954). The most dominating feature of Lundblad's experiments is
the sharp activation of proteolytic enzymes during the elevation of the
fertilization membrane. It would, therefore, be of interest to investi-
gate the course of events in eggs treated with trypsin before fertiliza-
tion, so that membrane formation is suppressed.

(16) Solubility of proteins in M-KCl decreases by 12%, 4-10
min. after f. A. piinctulata, S. purpiiratus (Mirsky, 1936).

(16.1) New electrophoretic component appears in water ex-
tracts shortly after f. and disappears after 30 min. Ammonium

METABOLIC AND OTHER CHANGES AT FERTILIZATION 89

sulphate extracts show a decrease in solubiHty of one component
immediately after f. P. lividus (Monroy, 1950).

In a later paper by Monroy & Monroy-Oddo (1951, p. 246), it is said
that 'the electrophoretic patterns of the water extracts of unfertilized
and fertilized eggs of Arhacia lixula are essentially identical. . .'

(16.2) No change in solubility of proteins extractable with
M-KCl after f. A. piinctidata (Monroy & Monroy-Oddo, 195 1).

The eggs were treated with trypsin before fertilization to prevent
the formation of the fertilization membrane, it being thought that the
salts in the perivitelline fluid might cause ambiguous results. Al-
though the authors found a change in the M-KCl-soluble fraction
after fertihzation of Arhacia lixula eggs, they say that further experi-
ments are needed to decide whether the postulated coagulation 'is an
actual occurrence under natural conditions.' (p. 253). Similar doubts
about Mirsky's results have been expressed by Lindvall & Carsjo
(1951), using the eggs oi Echinus esculentus.

(17) Production of acetyl choline or acetyl choline-like substance
at f. P. depressus (Numanoi, 19536).

In the same paper the author states that spermatozoa of Pseudo-
centrotus depressus (A. Agassiz) and Clypeaster japonicus contain cholin-
esterase, as do mammalian spermatozoa (Sekine, 1951). Scheer (1945)
showed that acetyl choline and physostigmine, at concentrations of
i/io^, inhibited the activation of eggs of Urechis caupo and Strongy-
locentrotus purpuratus. In a later paper, Scheer & Scheer (1947) found
that the inhibitory action of acetyl choline was extremely complicated,
at any rate in Urechis eggs, inhibition being most marked when activa-
tion was affected by subjecting unfertilized eggs to potassium- and
magnesium-free, but calcium-enriched, sea water. In view of these
results, it would be unwise to attach too much importance to Nu-
manoi's results until they have been confirmed. Heilbrunn (1952, pp.
635-642) makes some cogent remarks about the necessity for caution
in interpreting experiments designed to demonstrate the existence of
acetyl choline in tissues.

(18) Release of sulphate due to splitting of heparin-like poly-
saccharide sulphate at egg surface (jelly?) by sperm sulphatase.
H. pulcherrimiis (Numanoi, 1953a).

(19) No change in hexokinase activity at f. A. punctulata (Krahl
et al., 19546).

(20) No change in urease activity at f. S. purpuratus (Brook-
bank & Whiteley, 1954).

90 FERTILIZATION

(21) Negligible change in catalatic activity after f. E. esculentus
(Rothschild, 1950).

(22) Increase in glutathione content immediately after f; fall
after 45 min. P. lividus (Infantellina & La Grutta, 1948).

(22.1) Fall in glutathione content immediately after f. ; rise after
45 min. P. lividus (Bolognari, 1952).

(23) No change in refractive index of cytoplasm, 1*375, ^o'' o^
egg nucleus, 1-360, after f. P. miliaris (Mitchison & Swann,

1953)-

(24) O2 uptake, see chapter 5.

(25) Acid production, see chapter 5.

CHAPTER 8

STRUCTURAL CHANGES AT
FERTILIZATION

Birefringence. Chapter i, The Morphology of FertiHzation, con-
tains a description of various structures which can be seen in
fertiHzed and unfertilized eggs, with an ordinary microscope.
Studies with the polarising microscope provide information from
which a few inferences can be made about the sub-microscopic
morphology of eggs, the scale changing from microns to angstroms.
The most interesting of these studies concerns the egg cortex. In
the unfertilized sea-urchin egg, this structure is negatively bi-
refringent with respect to the tangent and has a radial optical axis
(Runnstrom et al., 19446). The birefringence is very weak but as
the cortex also scatters and depolarises a considerable amount of
light, the egg surface appears quite bright under the polarising
microscope. At fertilization this negative birefringence disappears,
at about the same time as the light-scattering properties of the egg
surface change (chapters i & 9). Some minutes later, the cortical bi-
refringence re-appears, reaching a maximum by anaphase. Accord-
ing to Mitchison & Swann (1952), the intrinsic birefringence is
more strongly negative than the total birefringence, so that the
form birefringence must be positive. This suggests that the mole-
cular structure of the cortex is radial, but that the micellar organisa-
tion is tangential. The fall in total birefringence at fertilization is
probably due to a decrease in radial molecular order.

Both the fertilization membrane and the hyaline layer are bi-
refringent. The former is virtually isotropic when first formed, but
by the time it is fully tanned (pp. 9-10), its form and intrinsic
birefringence are positive (Runnstrom, 1928; Runnstrom et al.,
1946). The birefringence of the hyaline layer is positive with
respect to the tangent (Runnstrom et al., 19446).

Mechanical properties of the cortex. Many of the methods of in-
vestigating changes in the physical state of the cortex and the
cytoplasm suffer from the disadvantage that they do not enable an
estimate to be made of the degree to which measurements of
cortical viscosity, surface tension, or rigidity are influenced by the

92 FERTILIZATION

physical state of the cytoplasm, and vice versa. Cole, for example,
attempted in 1932 to measure the internal pressure of a sea-urchin
egg, and its surface tension, by observing the changes in the shape
of the egg when compressed by a small gold beam. Reliable
measurements by this method require rather large deformations
of the egg. One then measures the resistance to deformation of
internal structures, such as the amphiaster, as well as the resist-
ance to deformation of the cortex. Mitchison & Swann (1954a, b;
1955) invented an instrument, which they call the Cell Elastimeter,
though it is commonly known as the 'Sucker', to overcome such
difficulties. The instrument consists of a micropipette, filled with
water, one end of which is connected to a device for producing

Pipette

ci\

FIG. 16. — Diagram showing measurements involved in estimating cortical stiff-
ness with the Elastimeter. After Mitchison & Swann (1954a).

negative pressures; the other end, which has a terminal diameter
of about 50 /x, is placed against the surface of an egg. When a
negative hydrostatic pressure is applied, part of the egg surface
bulges into the pipette, Fig. 16. From a knowledge of the diameter
of the pipette, the negative pressure applied, and the degree to
which the egg bulges into the pipette, an estimate can be made of
the tension at the surface or, more accurately, the stiffness, of the
cortex. The measurements involved in estimating the degree of
bulging are shown in Fig. 1 6. The units for stiffness are dynes. cm~''
per /x deformation. By comparing the observed behaviour of sea-
urchin eggs and large-scale models such as balloons, Mitchison
& Swann came to two conclusions, apart from quantitative data
referred to later ; first, as was to be expected, that the cortex was
elastic; and secondly, that its thickness was appreciable. Mitchi-
son (1956) has adduced other, optical, evidence in favour of
this view. The permeability barrier may still, of course, be fairly
thin, the capacitance measurements mentioned in chapter 10

STRUCTURAL CHANGES AT FERTILIZATION 93

suggesting a layer of the order of 100 A thick for this part of the
cortex. If magnified, a sea-urchin egg would therefore be more like
a tennis ball, which resists deformation because of the rigidity of
its 'cell membrane', than like a rubber balloon, which resists de-
formation because of its internal pressure. Measurements are ex-
pressed in terms of corrected stiffness, which involves correcting
observations for variations in egg and pipette diameters. Full
details are given in Mitchison & Swann (1954a). Experiments
with the Elastimeter show that the Young's modulus of the cortex
of the unfertilized sea-urchin egg is 1-2. 10* dynes. cm"^, and that
the internal pressure of the egg is less than 95 dynes. cm"^ (Mitchi-
son & Swann, 19546).* These values agree rather well with those of
E. N. Harvey (193 1), who measured the centrifugal force necessary
to split a sea-urchin egg into two halves.

What happens, structurally, to the cortex at and after fertiliza-
tion? Mitchison & Swann (1955) found that, at fertilization, there
was a sudden rise in stiffness, from the value 9 dynes. cm~2./x~^ for
the unfertilized egg, which was followed by a fall, during the early
sperm aster stage, to the lowest value, 4 dynes. cm"-./x~^, which
occurs during development. There is a steep rise, to about 61
dynes. cnr-.ju,"^, in late anaphase, just before cleavage. Normal eggs
cannot be used for direct measurements of stiffness at fertilization,
because of the elevation of the fertilization membrane. Runnstrom
et al. (19446) discovered, however, that treatment of unfertilized
sea-urchin eggs with trypsin prevented the elevation of the fer-
tilization membrane, perhaps by digesting the vitelline membrane.
Treatment of unfertilized eggs of Psammechinus microtuberculatus
(de Blainville) with o-i% w/v. trypsin in sea water reduced the
stiffness of the cortex of these eggs, which normally is 9 dynes.
cm~^.ju,~^, by a factor of 4 or 5. At fertilization, the cortical stiffness
increased by the same factor. Fig. 17.

In their most recent paper, Mitchison & Swann (1955) give a
comprehensive review of the earlier methods of investigating the
mechanical properties of the cortex. More or less serious objec-
tions can be raised against all the classical methods. Cole's experi-
ments have already been discussed ; in the same way, measurements
of the ease with which eggs can be separated into two halves by

* Young's modulus can be derived from the corrected stiffness values, assum-
ing a cortical thickness of i 6 ft and zero internal pressure. The internal pressure
is very low.

94 FERTILIZATION

centrifugation are open to the criticism that the measurements are
sensitive to changes in cytoplasmic gelation as well as to the struc-
tural changes in the cortex, which are the object of the enquiry.
In spite of difficulties in estimating the internal pressure of eggs,
upon which interpretation of Elastimeter measurements depends
to a considerable extent, this technique provides a pov^erful and

lA
Hj
C

V,
-to

5 10

t (min.) after fertilization

Fic. 17. — Changes in cortical stiffness (corrected, in dynes. cm~^ per fi deforma-
tion), at fertilization. The four curves refer to four eggs of Psammechinus
ftiicrotuberculatus, previously treated with tr>'psin to prevent elevation of the
fertilization membrane. A, value for stiffness of normal unfertilized egg; B,
value for stiffness of trypsin-treated unfertilized egg; C, stiffness of
fertilized egg at sperm aster stage. After Mitchison Sc Swann (1955).

sensitive new method of investigating the structural characteristics
of the cortex.

Calcium and protoplasmic consistency. Heilbrunn (19 15, 1952)
has for many years been the principal proponent of the view that
cellular reactivity in general and activation in particular are 'caused'
by the release into the cell interior of calcium, previously bound in
the cortex in Ca-protein complexes. Once released, calcium pro-
motes a protoplasmic clotting reaction or gelation. The release of

STRUCTURAL CHANGES AT FERTILIZATION 95

calcium from the egg cortex is associated with the so-called lique-
faction or decrease in rigidity of this structure, which follows
fertilization or parthenogenetic activation. Heilbrunn believes that
this rigidity is due to the presence of calcium in the cortex; but
according to Mitchison & Swann (1955), removal of calcium from
the external medium makes very little difference to the rigidity of
the cortex of the fertilized sea-urchin egg (bearing in mind that
this treatment removes the hyaline layer), while in the case of the
unfertilized egg, calcium lack causes a slight, but insignificant, in-
crease in rigidity. W. L. Wilson ( 1 95 1 ) examined the post-fertiliza-
tion changes in the cortical properties of the eggs of Chaetopterus
variopedatus. He expressed rigidity in terms of the centrifugal
force, which, applied for i minute, was necessary to break the
continuous layer of granules which exists in the cortex of this egg,
in not less than i6 and not more than 19 eggs, out of a sample of
20. The experiment was done at different times after fertilization.
Wilson found that the rigidity of the cortex decreased after fertiliza-
tion, for about 4 minutes. After 6 minutes, the rigidity began
to increase and in lo-ii minutes, it had reached the pre-fertiliza-
tion level, where it stayed until 35 minutes after fertilization, when
it again declined. These observations do not fit well with the re-
sults of the more refined measurements of Mitchison and Swann,
though this may be explained by the different maturation states
of the eggs of Chaetopterus and of the sea-urchin at fertilization.
Alternatively, Wilson may have been measuring the Shear mo-
dulus, and not Young's modulus, which Mitchison and Swann
measured. Until 30 minutes after fertilization, Wilson's results
are to a certain extent reflected in the changes in cytoplasmic
viscosity * which occur in the same egg during the same time.
Heilbrunn & Wilson's results (1948) on this latter subject, which
are, perhaps, less open to doubt than those of Wilson on cortical
changes, are reproduced in Fig. 18. Similar curves were obtained
for the eggs of Arhacia punctulata and Cumingia tellinoides by
Heilbrunn ini92i. It is not quite clear how the observed reduction
in cytoplasmic viscosity, immediately after fertilization, fits in with
Heilbrunn's thesis that one of the important characteristics of egg
activation is cytoplasmic clotting or gelation, due to the release of
calcium from the cortex. According to Heilbrunn, a wide variety

* The cytoplasmic viscosity of an unfertilized egg is about 3 times that of
water (Heilbrunn, 1952).

96 FERTILIZATION

of agents, such as heat, cold and electric shocks, which cause par-
thenogenetic activation, induce a release of calcium from the cortex,
which in turn starts a cytoplasmic clotting reaction. In confirma-
tion of this view, Shaver (1949) found that the foreign agents which
must be present as 'needle contaminants' for successful traumatic
parthenogenesis in frogs' eggs, favour blood-clotting, while the
introduction of the anticoagulant heparin * into the egg inhibits
parthenogenesis. Harding (1949) claimed to have confirmed
Shaver's results and stated, in addition (1951), that Shear's bacterial

1 ,

8
•^ in

'■\

•■^ 1 u

,./

■^ 5

<b
0:

10 20 30 40 50

timin.) after fertiLizat'ion

FIG. 18.^ — Changes in the viscosity of the egg of Chnetopterus variopedatus, at
various times after fertilization. The viscosity units are arbitrary, being the
number of seconds of centrifugation at 2325 g. required to achieve a particu-
lar accumulation of fat granules at the centripetal pole of the egg. A, first
polar body; B, late anaphase; C, 2nd maturation prophase; £), 2nd polar
body; E, apposition of pronuclei. After Heilbrunn & Wilson (1948).

polysaccharide, which inhibits blood-clotting, also inhibited par-
thenogenesis in the frog's egg. Calcium is a co-factor in that part
of the blood-clotting reaction which is concerned with the pro-
duction of thrombin and Heilbrunn believes that the release of
calcium into the egg cytoplasm promotes the production of a
thrombin-like substance. Although there is a little doubt that
fertilization and parthenogenetic activation induce changes in the
distribution of calcium in the egg and that some form of cortical
liquefaction (see also the reference on p. 6 to Allen's work),
followed by cytoplasmic gelation, occurs when an egg is activated,
the comparative significance of these reactions is not yet clear.
Are they, as Heilbrunn believes, the important feature in fertiliza-

* Heparin is not only an anticoagulant, but is, for example, also concerned
with the dissolution of chylomicrons in blood plasma (Florey, 1955).

STRUCTURAL CHANGES AT FERTILIZATION 97

tion, in the way that the action potential might be said to be the
important feature in nervous reactivity; or are calcium release,
cortical liquefaction and cytoplasmic gelation just some of the
many manifestations of the differences between fertilized and un-
fertilized eggs, which will only be seen in correct perspective many
years hence ? In any case, is it legitimate or logical to talk about the
important feature in any biological reaction ? Is the action potential
more important than the liberation of acetyl choline at a nerve
junction? Such questions are meaningless. The most we can do,
and all we need to do, in describing some biological phenomenon,
is (ultimately) to make a time-sequential analysis of the pheno-
menon in the language of physical chemistry. If calcium release
comes first in the fertilization reaction, we can, if we wish, define
coming first in terms of prime importance.

The idea that gelation is one of the characteristics of the early
phases of fertilization is supported by the work of Immers (1949)
on the inhibitory action of fertilizin on the coagulation of fibrinogen
by thrombin. This, together with the observation that periodate
counteracts the anticoagulating action of fertilizin and heparin
and, in some circumstances, facilitates fertilization (Runnstrom &
Kriszat, 1950), has from time to time led to the expression of the
view that fertilizin inhibits fertilization and that the spermatozoon
introduces into the egg, or releases in the egg, a factor which over-
comes the inhibition imposed by fertilizin. This sort of argument
does not bear much examination. In so far as an unfertilized egg
is not a fertilized one and that morphological observations for more
than a hundred years have shown that fertilization unleashes all
sorts of anabolic reactions which are obviously catalyzed by
enzymes, the mere statement that the unfertilized egg is in an in-
hibited condition and that enzyme systems are activated at fer-
tilization is only a re-statement of the morphological facts. What
we must do is to identify the activated enzyme systems and the
chemical nature of the so-called inhibition. As regards the former,
a little progress has been made ; in the case of the latter, the idea
that fertilizin is responsible is a little difficult to swallow.

Apart from being concerned in cortical liquefaction and cyto-
plasmic gelation, calcium is important in other ways in fertiliza-
tion and in the physiology of the gametes. In marine invertebrates,
it must be present in the external medium for fertilization or, for
that matter, the fertilizin-antifertilizin reaction, to take place at

98 FERTILIZATION

all, as Loeb pointed out in 19 14. Calcium is also involved in the
maturation of marine eggs ; this has been noted in several different
sorts of immature eggs, as Table 15 shows.

TABLE IS
Presence of Ca in the external medium necessary for the induction

of maturation

Organism

Phylum

Reference

M. glacialis

Echinodermata

Dalcq, 1924

T. neptuni

Annelida

Hobson, 1928

P. strombi

Annelida

Dalcq et al., 1936

H. uncinatus

Annelida

Pasteels, 1935

P. triqueter

Annelida

Horstadius, 1923

N. succinea

Annelida

Heilbrunn & Wilbur, 1937

B. Candida

MoUusca

Pasteels, 1938

S. solidissima

Mollusca

Heilbrunn & Wilbur, 1937

Calcium is necessary for the discharge of the cortical granules
in the sea-urchin egg (Moser, 1939^) ; for the transformation of the
isotropic cortical granules into birefringent rods while in the peri-
vitelline space (Endo, 1952); and for the tanning of the fertiliza-
tion membrane (Hobson, 1932^), though for reasons discussed in
chapter i, this may be another and less detailed way of express-
ing Endo's observation. In addition, Hultin (1949, 1950^,6) has
made an interesting series of studies on the effects of adding calcium
to sea-urchin egg homogenates prepared in Ca-free media. His
main findings were that, after adding CaCU, there was :

(i) A rapid uptake of Oo, without a corresponding increase in
CO2 evolution, for twenty minutes. Cyanide did not inhibit the
reaction.

(2) No breakdown of carbohydrate.

(3) Inhibition of the reaction after treatment of homogenates
for thirty minutes with 0-005 M-monoiodoacetate.

(4) Granular components in the homogenates, including yolk
globules, disintegrated explosively and there was a marked in-
crease in the viscosity of the whole system. Note, Echinochrome
granules, which are freely distributed in the cytoplasm of the un-
fertilized egg of Arbacia punctulata, move into the cortex after
fertilization, arriving there about ten minutes after fertilization
(E. N. Harvey, 19 10). Heilbrunn (1934) showed that these pig-
ment granules disintegrate explosively in the presence of free
calcium or magnesium, the former being a hundred times as

STRUCTURAL CHANGES AT FERTILIZATION 99

efficacious as the latter. When equal quantities of sea water and
of isotonic potassium chloride, oxalate, or citrate are added to
fertilized eggs of Arbacia punctulata, eleven, but not less than
eleven, minutes after fertilization (i.e. at the monaster stage), the
echinochrome granules explode and echinochrome is found in
the surrounding sea water (Churney & Moser, 1940). The eggs
subsequently cleave in the usual way. Heilbrunn says that this
experiment demonstrates the release by potassium of calcium,
previously bound in the cortex. These observations may have
some bearing on the 'clotting reaction', as Donnellon (1938)
observed that, during the clotting of sea-urchin perivisceral fluid,
the red granules in the red amoebocytes and the colourless ones
in the white amoebocytes explode. Isotonic KCl has the same
eff"ect (cf. exploding platelets in mammalian blood-clotting.)

(5) Acid production, which was not inhibited by iodoacetate
to the same extent as Oo uptake. The addition of papain, in-
stead of CaClg, also induced acid formation.

These striking observations indicate once more the importance
of calcium in the metabolism of eggs. Moreover, they confirm, as
Heilbrunn has so often insisted, that changes in the distribution of
calcium have profound effects on protoplasmic structure.

The ejfects of changes in the external environment on the cortex.
An interesting contribution to our knowledge of the cyclical
changes in cortical structure which occur after fertilization and
parthenogenetic activation was made by Herlant in 1920, using the
eggs of Paracentrotus {lividus}) and Sphaerechinus (granularis
(Lamarck)?). Herlant examined two things: the incidence of
plasmolysis (shrinking) and cytolysis in eggs exposed to various
hypertonic solutions ; and the variation in the susceptibility of eggs
to a number of cytolytic agents, at various times after fertilization.
As Herlant's paper is extremely long and does not contain a sum-
mary, its contents are summarised below:

(i) Fertilized sea-urchin eggs undergo cyclical variations in
their susceptibility to hypertonic sea water (100 ml. s.w. + 20-25
ml. 2-5M-NaCl), as indicated by plasmolysis. Unfertilized eggs
plasmolyse, the surface of the egg becoming wrinkled, but this
characteristic progressively disappears after fertilization, until
there is a zero plasmolysis from 5-25 minutes after fertilization.
From 25-70 minutes, plasmolysis re-occurs, becoming maximal

100 FERTILIZATION

after 45 minutes and remaining so until 75 minutes after ferti-
lization, when mitosis begins (17° C). During mitosis there is
no plasmolysis. This description of the reactions of fertilized
sea-urchin eggs to hypertonic sea water is somewhat surprising
and does not agree with the descriptions of Hobson (ig^^a) and
Monroy & Montalenti (1947); the experiments, which are
technically simple, could be repeated with advantage. Inter-
pretation, however, is not easy, because of the delay between
treatment and the onset of symptoms.

(2) In grossly hypertonic sea water (60 ml. s.w. + 40 rnl.
2-5M-NaCl), the eggs either plasmolyse or cytolyse, the in-
cidence of the former being inversely proportional to that of the
latter.

(3) If it is assumed, as Herlant does, that changes in the ex-
ternal environment directly and exclusively affect the properties
of the cortex, NaCl- and KCl-enriched sea water increase the
permeability of the cortex, while CaClg and MgClg have the
reverse effect. According to Herlant, an increase in the incidence
of plasmolysis implies a decrease in permeability to salts, it being
assumed that permeability to water is unaffected by the experi-
mental treatment; conversely, that an increase in cytolysis is
caused by an increase in permeability to ions. Tracer work will
clarify these questions in the next few years.

(4) Herlant also investigated the effects of various agents on
the incidence of cytolysis in fertilized and unfertilized sea-
urchin egg suspensions; his results are summarised in Fig. 19.

Herlant's experiments show without doubt that fertilization
induces a series of cyclical changes in the properties of the cortex.*
For example, the low resistance of the egg to digitonin in the in-
terval 10-35 minutes after fertilization suggests that, just before
this period, cholesterol or a cholesterol-like compound becomes
more accessible to the digitonin molecule, at the cell surface. These
and other results which may bear on the same subject are sum-
marised in Tabic 16. When examining this table, it must be re-
membered that, with the possible exception of those of Mitchison
& Swann (1954a), the methods for investigating changes in the

* Herlant was not, of course, the first person to make this discovery, as a
perusal of R. S. Lillie's papers or Needham's Cheviical Embryology shows. But
his work was far more comprehensive than anything done before, or, probably
since, (iQSS)-

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102

FERTILIZATION

consistency of the cortex may not be unequivocal, in the sense of
being able to separate cortical from cytoplasmic changes. Table i6
is of interest in several different fields of cell physiology. First, it
presents biological evidence of structural and, probably, metabolic
changes in the cortex, induced by activation ; secondly, it provides
a number of clues for the further investigation of the cortex; and

Permeability
Hypotonic s.w.
Salts

Strong bases

Ammonia

Strong acids
Fatty acids
Ether
ALcotiol
Cloroform
Chloral Hydrate
Acetone
Saponin

Digitonin ...^^_____^_^^___________^^

Zbmin. ^75min,,,

Fertilization Amphiaster

Metaphase

Cleavage ends

FIG. 19. — Changes in the susceptibility of the sea-urchin egg to various reagents.
Each horizontal section shows the percentage cytolysis at different times
after fertilization. The hatched regions at the top are periods of high
permeability. After Herlant (1920).

thirdly, the information may not only apply to the fertilized egg,
but to other dividing cells. Now that we understand a little more
about cell membranes and, with the aid of labelled atoms, can in-
vestigate sodium, potassium, phosphorus and, perhaps, calcium
fluxes across the cell surface, more rapid progress should be
possible in this field, in spite of the complications introduced by
the possibility of active transport. Among other things, it would
be interesting to examine the effects, if any, of saponin and digi-
tonin on the form and intrinsic birefringence of the cortex, in the
ways described by Mitchison & Swann (1952).

CHAPTER 9

POLYSPERMY

After fertilization the haploid egg nucleus normally fuses with a
single haploid sperm nucleus : in the rare event of it uniting with
more than one sperm nucleus, development is almost invariably
abnormal. Two mechanisms exist to prevent polyandrous syn-
gamy of egg and sperm nuclei. One of these, Type I Inhibition,
prevents all but one spermatozoon from entering the egg. In the
second mechanism. Type II Inhibition, several spermatozoa
enter the egg but only one sperm nucleus unites with the egg
nucleus.

Type II Inhibition of Polyspermy. The eggs of selachians
(Riickert, 1899), urodeles (Jordan, 1893), reptiles (Oppel, 1892),
Polyzoa (Bryozoa) (Bonnevie, 1907), birds (Blount, 1909; Hamil-
ton, 1952), some molluscs (Bretschneider, 1948), and many insects
(Richards & Miller, 1937) are normally polyspermic in the sense
that several spermatozoa enter the egg at fertilization. Only one
of these fuses with the female nucleus, the remainder degenerating
near the surface of the egg, though abortive divisions of the super-
numerary sperm nuclei sometimes occur. Fankhauser (1925-1948)
has made a most interesting study of polyspermy in the eggs
of Triturus helveticus (Razoumowsky) and of Diemictylus viri-
descens (Rafinesque), in which several spermatozoa usually enter
the egg after insemination. In spite of polyspermy, cleavage is
normal and monospermic, provided less than ten spermatozoa
enter the egg, although for three hours after fertilization the egg
appears to be typically and pathologically polyspermic. At the
time when the sperm nucleus (the one which happens to be nearest
the egg nucleus after maturation) fuses with the egg nucleus, the
supernumerary sperm nuclei, which may have proceeded as far as
prophase or even to the release of chromosomes (particularly in
Diemictylus viridescens), begin to degenerate, those nearest the fusion
nucleus degenerating first. If, after fertilization, an egg is ligatured
so that one half contains the egg nucleus and sperm nuclei, the
half in question develops normally. The other half, which con-
tains sperm nuclei but no egg nucleus, cleaves as frequently as a

H 103

104 FERTILIZATION

normal egg, but the cleavages are usually abnormal and retarded.
Nuclear and astral cycles are often out of phase in dividing super-
numerary sperm complexes. In the half of a previously fertilized
egg which contains the egg nucleus but no sperm nucleus, cleavage
is incomplete and anastral. When a half contains no egg nucleus,
cleavage is frequently abortive. If one half has neither egg nor
sperm nucleus, there is either no cleavage or abortive cleavage.
When a fertilized egg is constricted, not ligatured, so that it be-
comes shaped like a dumb-bell, the situation becomes more com-
plicated, but can be summarized as follows, calling one half of the
dumb-bell L and the other R.

L, female nucleus and sperm; R, sperm; thin bridge betzveeti L
and R. Cleavage in L and R.

L, female nucleus and sperm; R, sperm; thick bridge between L
and R. Cleavage in L but not in R.

L, female nucleus; R, sperin. No cleavage in L, cleavage in R.
When the bridge is thick, the male or female nucleus sometimes
moves across the bridge and joins the other nucleus, in which
case a normal first cleavage furrow occurs in L or R.

These experiments suggest that substances may diffuse out of
the female nucleus or the sperm nucleus under the influence of
proximity to the female nucleus, which cause degeneration of
supernumerary spermatozoa. Some information about the charac-
teristics of this substance might be obtained by continuing Fank-
hauser's studies with different bridge widths between dumb-bells
and by observing the variations in the times at which supernumer-
ary sperm nuclei degenerate according to their distance from the
fusion nucleus. These remarks apply particularly to the eggs of
Triturus helveticus. In those of Diemictylus viridescens, super-
numerary sperm nuclei which are at different distances from the
fusion nucleus begin to degenerate at the same time, a phenomenon
which seems to be incompatible with a diffusion mechanism. The
experiments of Allen (1954), on the behaviour of the egg nucleus
after fertilization of sea-urchin eggs sucked into narrow glass
capillaries, suggest that some substance diffuses out of the sperm
head or male pronucleus into the cytoplasm, where it affects the
female pronucleus. Allen found that the latter elongated or
bulged in the direction of the sperm head, which at the time was
located at the periphery of the elongated egg. To make distinctions

POLYSPERMY IO5

between physical and chemical mechanisms in biological systems,
except in the case of such phenomena as electrotonus, is of ques-
tionable value; but for what this distinction is worth, Ziegler
(1898), who also did constriction experiments with cotton threads
on sea-urchin eggs, found that if he constricted the fertilized egg
in such a way that the spermatozoon was in one part, connected by
a narrow bridge of cytoplasm to the rest of the egg, which contained
the female nucleus, that part of the egg which contained the female
nucleus failed to divide though the nucleus showed signs of
activity, while the part containing the sperm head did divide. A
somewhat similar situation occurs in the eggs of Crepidula plana
Say, in which only that part of the egg which contains the sperm
nucleus divides, the other part being called, rather tautologically,
a polar body. Evidently one of the essential features in fertiliza-
tion is the introduction of a division centre into the egg by the
spermatozoon, 'rather than a diffuse chemical action of the sperm'
(T. H. Morgan, 1927, p. 512). In recent years little work has been
done on the problems raised by Type II Inhibition of polyspermic
development. Further experiments on newt eggs, which are easily
obtained, would bring their own reward.

Failure of the Inhibition. Type I and Type II Inhibition may
fail or be induced to fail, and an immense amount of work was done
on the consequences of such failures in the nineteenth and early
twentieth centuries (see, for example, Boveri, 1907). When more
than one sperm nucleus engages in syngamy, abnormal cleavages
occur, followed by the early death of the pathological embryo.
Some cases of dispermic adults are, however, known in insects
(Goldschmidt & Katsuki, 193 1 ; L. V. Morgan, 1929), and in birds
(Hollander, 1949), in which large patches of feathers, coloured
differently from the rest of the animal, are found on occasions. In-
teresting accounts of two 'mosaic' cocks, as such animals are called,
bred from the same sex-linked cross. Light Sussex $ X Rhode
Island 3, and of a mosaic daughter of a mosaic cock, will be found
in papers by Greenwood & Blyth (195 1) and Blyth (1954). These
mosaics may sometimes be caused by dispermic fertilization, in
which case part of the tissue of the animal has a set of genes derived
from one spermatozoon and part from a different set of genes
derived from the other spermatozoon. The evidence for the ex-
istence of dispermic adult humans, or indeed any mammals, is not
conclusive.

I06 FERTILIZATION

Polyspermy in Mammals. Except in the case of Ornithorhynchus
anatinus (Shaw & Nodder), in which it may be that polyspermy
is the rule rather than the exception (Gatenby & Hill, 1924),
the incidence of polyspermy in mammalian eggs is low, being
of the order of 1-2% under normal mating conditions. They
should therefore be classified as Type I eggs, in which only one
spermatozoon enters. Austin & Braden (1953) published an im-
portant paper, which includes a critical review of earlier work,
on polyspermy in rats and rabbits. They found that rat and
rabbit eggs go through a critical period, depending on the time
after ovulation when they come into contact with spermatozoa, as
regards their ability to prevent polyspermy. If mating is delayed
so that the eggs remain unusually long in the female reproductive
tract before fertilization, the incidence of polyspermy goes up
quite sharply, which recalls the increases in polyspermy observed
in ageing marine invertebrate eggs. These observations, together
with the small numbers of polyspermic eggs normally found, con-
firm that mammalian eggs belong to the Type I class. Although,
for obvious reasons, Austin & Braden were unable to obtain any
information about the ultimate fate of polyspermic mammalian
eggs, they observed that the supernumerary male pronuclei did
not induce the formation of separate spindles near the periphery
of the egg, but approached the female pronucleus and contributed
to the first cleavage spindle. The spindle, however, was normal
and not multipolar. These observations raise a number of questions
about the fate of accessory chromosomes, but the answers must
await the results of further experiments. A valuable summary of
the information available about the occurrence of polyspermy in
mammalian eggs has recently been published by Braden et al.

(1954)-

Type I Inhibition of Polyspermy. Type I Inhibition, often called

the Block to Polyspermy, involves a change in the egg surface,

after the fertilizing spermatozoon has become attached, such that

further spermatozoa cannot enter the egg. It has been known since

the nineteenth century that the fertilizing spermatozoon initiates

some alteration in the egg which prevents re-fertilization, and many

workers have observed changes in the morphology of the egg

surface immediately after fertilization, such that a point on the

surface opposite to where the fertilizing spermatozoon became

attached is the last to be affected. The wave of granule breakdown

POLYSPERMY 107

in the eggs oi Arbacia puuctidata at fertilization, observed by Moser
(i939«), and the similar phenomenon observed by Endo (1952) in
Japanese sea-urchins, are examples of such morphological changes.
Some workers, for example Runnstrom (1928, p. 395), have re-
ported that when the egg is examined with dark ground illumina-
tion, a colour change passes over the egg surface in four to six
seconds. But they did not realize that unless the egg is fertilized
at the equator, as seen by the observer who is looking down the
microscope, estimates of the time taken for the cortical change to
pass completely over the egg have no meaning. The observer,
whose eye is directly above the north pole of the egg, sees it in
optical section, the periphery of the section being the circum-
ference of an equatorial great circle. When a spermatozoon fer-
tilizes an egg, a cortical change spreads out in all directions over
the egg surface, from the point of attachment of the spermatozoon

oo«

FIG. 20. — Development of cortical change in an egg fertilized at 1.30 o'clock

(Rothschild & Swann, 1949).

(Fig. 20). If the spermatozoon fertilizes the egg exactly at the
north pole, the whole periphery of the optical section of the egg
will alter instantaneously, because the cortical change reaches all
points on the equatorial great circle simultaneously. This is shown
in Fig. 21, where the periphery of the alteration in surface structure
is shown as a series of 'isochrones' depicting the position of the
leading edge of the change at various times after fertilization
has started. If the spermatozoon fertilizes the egg at the equator,
the periphery of the optical section changes colour at the true
rate of propagation. At points intermediate between the north
(or south) pole and the equator, the propagation rate, as judged by
the time taken for the periphery of the optical section to change
colour, will seem to be faster than it actually is. The form of the
conduction-time curve will also be affected by these considerations.
The best way to make a serious examination of the conduction
time of the surface change is by taking dark-ground cinemicro-
graphs of eggs, immediately after insemination, and noting in
which eggs a fertilization cone can be seen at the equator. When

I08 FERTILIZATION

the cone is seen at the equator, the egg was fertiHzed at the equator.
An experiment of this sort shows that the conduction time is about
twenty seconds at i8° C in the eggs oi Psamtnechinus miliaris (Roths-
child & Swann, 1949), with quite a significant induction period
before the cortical change begins to pass over the egg surface (Kacser,
1955). It is natural to ask whether the change in surface structure
underlying this colour change or increase in light scattering is the
block to polyspermy, but this at once raises another question —
how many spermatozoa collide with the egg and, in particular, with

FIG. 21. — Passage of the cortical change over the egg surface. The observer at
O sees the egg in optical section, the periphery of the section being the
equatorial great circle E. (a) Fertilization at the north pole. The whole of
the cortical great circle E changes colour instantaneously, (b) Fertilization
at the equator. The cortical great circle E changes colour at the true rate at
which the change is propagated, (c) Fertilization at 2.0 o'clock. The change
affects the cortical great circle E more quickly than the whole egg and the
conduction rate appears to the observer to be higher than it is. The con-
centric circles Cj, C2, etc. represent the leading edge of the propagated change
at times ^1, (2, etc. (Rothschild & Swann, 1949).

parts of the egg surface unaffected by the surface change until the
end of the twenty second period ? When eggs are inseminated with
fairly dense sperm suspensions, 10'^ /ml., swarms of spermatozoa
are normally seen round every egg in the suspension ; yet only one
spermatozoon fertilizes each egg. This has led people to believe
that the block to polyspermy passes over the egg in an incredibly
short time. But this presupposes that every spermatozoon which
collides with an egg is capable of fertilizing it, and this presupposi-
tion requires examination.

The number Z of spermatozoa, moving in random directions —
sea-urchin spermatozoa do move in random directions, even when
near homologous eggs (Rothschild & Swann, 1949) — which will
collide with an egg each second is given approximately by the
equation

Z = Tra^nc . . . • (0

POLYSPERMY IO9

where a = radius of the egg, n = no. of sperm/ml., and c = mean
speed of the spermatozoa. When the appropriate values are sub-
stituted in this equation, Z, the number of sperm-egg colUsions
per second, is found to be o-i6, i-6, and 16 for sperm densities of
10^, 10^, and lo'^ per ml., respectively. These values for Z are
probably too high because no account is taken of dead spermatozoa
in the suspensions. Although the percentage of dead sperm in
mammalian suspensions can be estimated by the live-dead stain-
ing technique (Lasley et al., 1942), and is often about 10%, no
method is at present available for doing this in suspensions of sea-
urchin spermatozoa. As the cortical change takes twenty seconds
to pass over the egg surface, there will in that time be respectively
3-2, 32 and 320 collisions at the three sperm densities in question.
About half of these will collide with parts of the egg surface already
covered by the cortical change, so that at a sperm density of 10'^ /ml.,
only I /i 60th of the spermatozoa colliding with the egg surface are
capable of fertilizing it, if the cortical change is the block to poly-
spermy. How can the probability, p, of a sperm-egg collision
being successful, be estimated? Elementary probability theory
shows,* and experiments confirm, that if unfertilized eggs and
spermatozoa are left in contact with each other for a series of known
sperm-egg interaction times t^, ta, . . . tn sees, (n > 45), and the
number of fertilized and unfertilized eggs are later counted in each
case, the proportion of unfertilized eggs, u, is given by the equation

log u = — at . . . (2)

where a is the sperm-egg interaction rate or fertilization parameter
with dimensions [T]~^.

Assuming that the spermatozoa do not change their fertilizing
capacity during the experimental period, a is a measure of the
receptivity of the egg surface to spermatozoa and its value may
reflect the fact that, on a submicroscopic scale, an egg surface is

* Divide the interval (o, t) during which the eggs and spermatozoa are in
contact into r sub-intervals, each of duration t. Let the probability of an egg be-
ing fertilized during t be p, and of not being fertilized, q, where p + q = i •
There being r sub-intervals of duration r during (o, t), the probability of an egg
not being fertilized throughout (o, t) is, by the Product Theorem

q'' = exp(r log q) = exp(t/T. log q)
= exp(^at)

where a = — i jr. log q

The theoretical proportions of unfertilized and fertilized eggs are, therefore,
u = exp(— at) and f = i — exp(— at).

no FERTILIZATION

probably a mosaic of sperm-receptive and non-receptive regions.
It can be expressed, approximately, in the form a = Zp, where p
is the probability of a successful sperm-egg collision. Some bio-
logists have felt uncomfortable about treating spermatozoa as gas
molecules colliding elastically with spheres. In defence of this
feeling, a, though more abstract, has more 'depth' than p and Z.
For if we only make the plausible and weak assumption that the
chance of an egg being fertilized in a time interval §t is propor-
tional to that interval of time, the proportionality constant being
denoted by a, Equ. (2) automatically follows. Another advantage
of oc over p and Z is that it does not involve such considerations as
the random movements of spermatozoa, chemotaxis, and the trap
action of egg jelly on spermatozoa. Excluding chemotaxis, these
factors interfere, though perhaps not to a great extent, with the
*sperm-gas molecule' analogy.

Calculations based on Equ. (2) show that at sperm densities of
7-4 X lo'^/ml., p = 0-23 and at a density of 9-6 X lo^/ml., p =
o-oi. Further enquiries into the conduction time of the block to
polyspermy can be made by means of the following pair of experi-
ments, run at the same time (Rothschild & Swann, 195 1). In Exp.
I unfertilized eggs were mixed with spermatozoa at t = o and
functionally separated after 25 seconds, by killing the spermatozoa
but not the eggs. The same procedure was adopted in Exp. 2,
but, at the time when the spermatozoa were killed in Exp, i, more
spermatozoa were added, the sperm density being increased by a
factor of 100. From the experiment described immediately above
Equ. (2), it is known what proportion of eggs will have been fer-
tilized in 25 seconds (Exp. i ), at any given sperm density, and there-
fore what proportion of the eggs will have started their blocks to
polyspermy during that time. If, therefore, instead of killing the
spermatozoa at t = 25, the number of sperm-egg collisions is
greatly increased by the addition of more spermatozoa, there should
be a negligible incidence of polyspermy unless a considerable
number of successful sperm-egg collisions do occur during the block
to polyspermy. Table 17 shows that, in such an experiment, there
are three times as many polyspermic eggs in Exp. 2 as there were
unfertilized eggs in Exp. i . In other words, nearly half of the eggs
which were fertilized in 25 seconds had not finished propagating
their blocks to polyspermy in that time, and became polyspermic
because of the new lot of collisions they received after the initial

POLYSPERMY

III

25 second period was terminated. This experiment provides strong
evidence for the conduction time of the block to polyspermy being
of the order of seconds rather than small fractions of a second, as
used to be thought. At the same time there remains the question
as to why, if the block to polyspermy takes seconds to pass over
the egg surface, so few eggs are normally polyspermic even at
comparatively high sperm densities. This difficulty can be resolved
by experiments based on the following self-evident proposition.
Suppose, for example only, that a number of eggs are all fertilized at
t = o and that the block to polyspermy is complete at t == 5 seconds.

TABLE 17
Conduction velocity of block to polyspermy {Rothschild & Swann,

1951)

Time, sec.

Exp. I

Exp. 2

Sperm added, 3 X 10^ /ml.
Sperm killed

Sperm added, 3 X 10* /ml.

More sperm added,

3 X io*/ml.

Unfertilized, %
Monospermic, %
Polyspermic, %

54
44

After 5 seconds there will be a a certain number of polyspermic
eggs in the egg population. The eggs will not all be polyspermic
because some of them will not have sustained more than one suc-
cessful sperm-egg collision during that 5 seconds. As, however,
all the blocks to polyspermy are complete by t = 5, the number of
polyspermic eggs will never be greater than it is at t = 5. If the
percentage of polyspermic eggs is 50 after 5 seconds, the per-
centage will still be 50 after 6, 60, or 600 seconds. It follows from
this argument that, if we take a series of egg suspensions and fer-
tilize all the eggs in them at t = o, and then 'remove' the sperm-
atozoa (Rothschild & Swann, 1951; Hagstrom & Hagstrom,
i954«) from these suspensions at various times after t = o, for
example, at 5, 10, 15 and 40 seconds, the time after which there is
no increase in the incidence of polyspermy will be the conduction
time of the block to polyspermy. Conversely, any decline in the
incidence of polyspermy at t = r, as compared with t = s, where
r > s, will be due to sampling errors or mistakes in deciding

112

FERTILIZATION

whether an egg is polyspermic or monospermic. Fig. 22 (Roths-
child & Swann, 1952) shows the sort of curve obtained when this
experiment is done. In fourteen experiments of this type, the
average figure for the conduction time of the block to polyspermy
was 63 seconds. This type of experiment not only enables one to
measure the time taken for the egg to become completely imperme-
able to spermatozoa after fertilization ; it also enables estimates to

0-75

.y 0-5

c
o

0-25

o
o

^ — 9 •

0 510 20 40 50 65

T 100105

150

200

FIC

tfsec.)
22. — Proportion of polyspermic eggs {Psammechinus miliaris) in a suspension
after various times of contact between eggs fertilized at / = o, and spermato-
zoa. Sperm density, g- 1 1 < 10' /ml. #, experimental points; O, theoretical
points. T, 85 sec. The thick line from 65-105 sec. on the time axis is the
interval within which T lies with a fiducial probability of 09 (Rothschild &
Swann, 1952).

be made of a during the passage of the 63-second change. During
this time, a is only i/20th of what it is before the first fertilization,
which means that the receptivity of the egg surface is twenty times
higher before than after the first fertilization. After the 63 seconds,
the receptivity of the egg surface is zero. The implications of these
observations are shown in Fig. 23, from which it will be seen that
the block to polyspermy is probably diphasic, in the sense that a
partial block to polyspermy sweeps over the egg surface in a
second or so, and is followed by a slower mopping-up process
which makes the egg completely impermeable to spermatozoa.

POLYSPERMY II3

The cortical change which can be seen under the microscope is,
therefore, not the block to polyspermy, but a reflection or phase of
the slow part of it. The recent experiments of Nakano (1954)
support this analysis.

Professor M. Sugiyama told me about an interesting experiment
he had done, the details of which will be published. It supports the
view that fertilization induces the propagation of an invisible
change, distinct from the cortical change, round the egg surface or
through the cytoplasm. He sucked an unfertilized sea-urchin egg
into a glass capillary whose diameter was slightly less than that of

t=60

FIG. 23. — Diagram of the block to polyspermy in a sea-urchin egg, showing rapid
partial block (grey), and slow complete block (black). Time, t, in seconds
(Rothschild, 1953).

the egg. Part of the egg, the 'proximal' part, protruded from the
end of the capillary, while the other, 'distal', part was within the
capillary and in contact with sea water. When the capillary and egg
were placed in M-urea in distilled water, there was a breakdown of
granules in the cortex of the proximal part, but no membrane
formation, because of the absence of divalent cations in the medium.
A normal membrane formed round the distal part. Sugiyama con-
siders that, in these conditions, both the invisible and cortical
changes occur. When the capillary and egg were placed in sea
water containing butyric acid, cortical granules broke down and
a membrane formed at the distal end ; but no breakdown of cor-
tical granules occurred at the proximal end. Sugiyama's inter-

114 FERTILIZATION

pretation is that butyric acid treatment by itself induces the
invisible change, but inhibits the cortical change. When the
capillary and egg were placed in sea water containing detergent,
a membrane appeared round the proximal, but not the distal,
part of the egg. The detergent induced the breakdown of cortical
granules without the invisible change.

Type I Inhibition seems to involve more complicated mechan-
isms in mammalian eggs. When a rat spermatozoon passes through
the zona pellucida of a rat tgg, a minute slit or hole remains where
the spermatozoon penetrated (Austin, 195 1 6). Braden et al. (1954)
examined the relative positions of these slits in dispermic rat eggs,
and obtained the distribution shown in Table 18, which shows that

TABLE 18

Angle, subtended at the centre of a dispermic rat egg, by two sperm
slits in the zona pellucida {Braden et al., ig54 )

Angle, degrees

No. of eggs

20-30

40-50
80-90

lOO-IIO

120-130

140-150

160-170

3
I

the most probable place for a second spermatozoon to penetrate
the zona is in the opposite hemisphere to that which the first
spermatozoon penetrated. Braden and his co-workers conclude
from this and other information about the number of spermatozoa
entering eggs and the perivitelline space that the first spermato-
zoon to penetrate the zona initiates a self-propagating structural
change, analogous to the cortical block to polyspermy, in the zona.
By a probabilistic analysis which is somewhat similar to that used
in the earlier calculations to do with the conduction time of the
cortical block to polyspermy, they estimate that the conduction
time of the change in the structure of the zona, which makes it
impermeable to spermatozoa (see p. 12), is between 10 and 90
minutes. If this interpretation of the facts is correct, mammalian
eggs have two blocks to polyspermy, one propagated round the
cortex and the other round the zo?ia pellucida. The latter appears
to be a rather inefficient mechanism, if the minimum conduction

POLYSPERMY II5

time is 10 minutes; but the implications of this inefficiency depend
on the sperm density in the neighbourhood of the eggs and, there-
fore, on the sperm-egg colHsion frequency. If the colHsion fre-
quency is low, a fast block to polyspermy is unnecessary and a
simple calculation shows that if we assume an average sperm speed
of I GO /x. /sec. and the value o-oi for the probability of a successful
sperm-egg collision, a sperm density of the order of ^/fA, in the
neighbourhood of the eggs, would not be excessive. But why
should a mammalian egg need two distinct blocks to polyspermy ?

An alternative interpretation of the experiments is worth con-
sideration. In a number of non-mammalian eggs the cortical block
to polyspermy is associated with the outward diffusion of substances
into the perivitelline space, where they react with and tan the
vitelline membrane (which is not, of course, always homologous
with the zona). These substances are progressively released from
the egg, following the progressive change in the egg cortex known
as the block to polyspermy. It follows that the vitelline membrane
is progressively tanned, though it is not excited, by the penetrating
spermatozoon. The tanning is a passive process which results
from the excitation, by a spermatozoon, of the egg cortex.

There are the following arguments against this hypothesis and
in favour of the zona being capable of propagating its own tanning
reaction in the rat egg :

(i) Cold-shock induces contraction of the egg and the release
of fluid into the perivitelline space, both of which are charac-
teristic of activation ; but the zona remains permeable to sperm-
atozoa (Austin & Braden, 19546).

(2) Heat-shock inhibits the cortical block to polyspermy, but
has little effect on the zona reaction (Austin & Braden, 19546).

(3) After penetrating the zona, spermatozoa may remain for
as long as thirty minutes in the perivitelline space before fertiliz-
ing the egg (Austin & Braden, 1954a). There should, therefore,
be little relationship between the point of penetration through
the zona and the point of attachment or fertilization on the egg
surface.

The last argument seems the most cogent; but a more direct
confirmation of the existence of this zona reaction would be wel-
come.

Induced Polyspermy. The possibility of interfering with the

ii6

FERTILIZATION

block to polyspermy by chemical treatment of eggs was examined
in detail by the Hertwigs in 1887. In recent years Clark (1936) is
the only person who has made a systematic study of this subject.
Although a number of different agents, which appear to have no
common denominator, such as heat, cold, acid sea water, excess
magnesium, alkaloids, fat solvents, chloral hydrate, and, in some
circumstances, extracts from eggs and spermatozoa, cause poly-
spermy, nicotine and magnesium are probably the most efficacious

Proportion of isotonic MgCl^ in sea water iv/v)

FIG. 24. — Effect of addition of isotonic MgCL to sea water on incidence of
polyspermy in eggs of Arbacia punctulata (after Clark, 1936).

agents. Fig. 24, showing the effect of increasing the magnesium
content of sea water, is adapted from Clark's paper. As regards
nicotine, Clark makes the interesting observation that the degree
of polyspermy is a function of the time of exposure and the con-
centration of nicotine in the sea water. I have confirmed this
observation, but it would be of interest (and comparatively easy) to
examine the form of this strength-duration curve in greater detail.
The existence of this relationship means that neither the block to
polyspermy nor the receptivity of the egg surface is an all-or-none
phenomenon ; they are capable of being varied in a continuous way

POLYSPERMY II7

over wide limits, quite apart from differences in threshold suscepti-
bility between different egg batches. The question as to which of
these two, a or the block, is influenced by nicotine can be examined
by the method of known sperm-egg interaction times. The pro-
cedure is shown diagrammatically in Fig. 25. Such experiments
show (Rothschild, 1953) that nicotine abolishes or slows up the
fast partial block to polyspermy. According to the concentration
of nicotine used and the duration of its application to the un-
fertilized egg, the conduction time of the nicotine-modified block
to polyspermy can be extended to an almost indefinite extent.

2,25

4,25

FIG. 25. — Experimental procedure for investigating effect of nicotine on the
block to polyspermy. The curved lines with arrows show which vessels
are emptied into which. The numbers by the curved lines refer to the times
of emptying, e.g. 2, 25 means that vessel c was emptied into vessel d 2 min.
25 sec. after the beginning of the experiment, which started when b was
emptied into c at t — o, a, 10 ml. of sperm suspension; b, 2 ml. nicotine in
sea water (i/iooo, v/v); c, 2 ml. egg suspension; d, 90 ml. hypotonic sea
water (28 %) ; e, 700 ml. sea water + 2 1 ml. 11% NaCl in sea water (Roths-
child, 1953).

Hagstrom & Allen (1956) have recently tried to re-examine the
problem of nicotine-induced polyspermy in sea-urchin eggs, using
the method of known sperm-egg interaction times. They con-
clude, on rather slender evidence, that the 20-second cortical
change, described by Rothschild & Swann in 1949, is the block to
polyspermy. Attractive as this idea may be, there is a considerable
body of evidence which makes it unacceptable, quite apart from
experiments with nicotine. Hagstrom & Allen have failed to
appreciate the importance of this evidence. For example, in
attempting to explain the fact that even when eggs are heavily in-
seminated, the proportion of polyspermic eggs is very low, they
postulate that the acid produced by sea-urchin eggs at fertilization
kills or inactivates supernumerary spermatozoa before they can

Il8 FERTILIZATION

eflPect polyspermy. There is an obvious conceptual fallacy in this
suggestion : the speed at which egg acid would have to be produced
to achieve the desired result would be so high as to make egg acid
production equivalent to a fast block to polyspermy. In the same
paper these workers make the startling suggestion that the develop-
ment of the hyaline layer plays a part in preventing polyspermy
in normal eggs. They do not comment on one consequence of this
suggestion: that if it is true, spermatozoa must be able to pass
through the fertilization membrane. Until someone sees this
occur, we need not consider whether a special mechanism (i.e. the
hyaline layer) exists to obviate any ill effects from its occurrence.
Intracortical and intracytoplasmic conduction. There has been
some discussion as to whether the cortical change (or the block to
polyspermy) is conducted round the cortex or through the cyto-
plasm. After comparing the form of the curve showing the rate at
which the sea-urchin egg surface becomes covered by the cortical
change with two 'models', in which this was eifected by intra-
cortical and intracytoplasmic diffusion of a substance with mole-
cular weight 20,000, I tentatively came to the conclusion (Roths-
child, 1949^) that if a diffusion mechanism was involved, the evi-
dence pointed towards the substance diffusing through the egg
cytoplasm and hitting various points on the cortex from the inside,
rather than towards diffusion in the cortex itself. Runnstrom &
Kriszat (1952) came to the opposite conclusion on the basis of
experiments done with damaged sea-urchin eggs. When these eggs
become stuck to a glass surface and are subsequently fertilized, the
part of the cortex which is stuck to the glass surface can be fer-
tilized independently of the rest of the egg, when the egg is un-
stuck. They concluded that the cortex was injured by being stuck
to the glass, that the cortical change was propagated round the
cortex and that consequently the injured part remained unfer-
tilized. The logical flaws in this argument are not difficult to see.
If part of the cortex is upset by being stuck to a glass surface, it
might be equally incapable of reacting to some stimulus from
within as from neighbouring uninjured parts of the cortex. In any
case, Horstadius & Runnstrom (1953) have recently described
experiments which might support the opposite viewpoint, in-
tracytoplasmic conduction; as, if an egg is constricted in a glass
tube so that it is nearly cylindrical in shape, and is fertilized at one
end, a fertilization membrane appears at both free ends but not in

POLYSPERMY II9

the middle. Although it has been known for some years that the
cytoplasm of an egg alters in various ways immediately after fer-
tilization, it seems probable that both mechanisms, a self-pro-
pagating cortical change as postulated by Allen (1954), and intra-
cytoplasmic diffusion, occur. Whether one, both, or neither are
uniquely responsible for the block to polyspermy is a question
which still remains to be answered. Kacser (1955) has recently
published an interesting and detailed examination of this question.
The experiments of Amoroso & Parkes (1947) lend support to
the view that some substance in the sperm head may be concerned
with the establishment of the block to polyspermy. They found a
higher than normal incidence of polyspermy in the eggs of rabbits
inseminated with x-irradiated spermatozoa (>2,500 r). The
irradiation presumably inactivated some 'block-catalyst' in the
sperm head, or modified some substance which normally diffuses
out of the sperm head at fertilization and initiates the block to
polyspermy. These experiments could be repeated with advantage,
on a larger scale.

Reversal of Fertilization. I referred earlier to all-or-none re-
actions. Biologists have often hoped that the reactions of living
matter would conform to this principle, but as time goes on, it is
found that fewer and fewer do. Fertilization used to be thought of
as an irreversible reaction. Once activated, the egg could not be
reactivated. Tyler & Schultz (1932) were the first to cast doubts on
this concept, when they found that fertilization could be inhibited
and reversed in the eggs of Urechis caiipo by treatment of the
fertilized eggs with acid sea water. Reversal in this species, which
is characterized by the egg reassuming its unfertilized appearance
(Plate II) in spite of containing a spermatozoon, could only be
achieved if the eggs were exposed to acid sea water within three
minutes of fertilization. When such eggs are re-inseminated, a
second spermatozoon penetrates and a normal block to polyspermy
is estabhshed. More recently, Allen (1953), using the eggs of
Spisula solidissima, reversed fertilization during the first four to
five minutes after fertilization, by putting the eggs into calcium-
free sea water, sea water acidified to pH 5, or sea water containing
o-3-o*5% ether. The most interesting experiments on this subject
are those of Sugiyama (195 1), using sea-urchin eggs, in particular
those of Hemicentrotus pulcherrimiis. Refertilization was achieved
by subjecting fertilized eggs to calcium- and magnesium-free sea
I

120

FERTILIZATION

water or molar urea, pH 7, with or without removal of fertilization
membranes. Urea was found to be the more effective agent, and
some of Sugiyama's results are given in Table 19. If eggs are re-

TABLE 19
Refertilization of sea-urchin eggs (Hemicentrotus pulcherrimus)
inseminated in normal sea water after washing in M-urea for
2 min. Membranes not removed before application of urea.
Polyspermic eggs in cofitrol inseminations, o. T° C, 11.

Time from insejuination

to i?n?uersion in urea

solution, sec.

Monospermic eggs

Polyspermic eggs

120

97
95
96

fertilized before anaphase, polyspermic divisions take place at the
time of first cleavage ; but if they are refertilized after the full growth
of the amphiaster, first cleavage proceeds normally and poly-
spermy becomes evident at second cleavage. Eggs can even be
refertilized after second cleavage. These experiments are of such
interest that they deserve to be repeated, when some obscure points
in Sugiyama's work could be cleared up. For example, no in-
formation is given in Table 19, which is extracted from Sugi-
yama's paper (p. 341), about refertilization sperm densities; but
from other data in his paper, the final sperm dilutions must have
been i/ioo or i/iooo. If the semen of this Japanese sea-urchin is
similar to that of British varieties, these dilutions correspond to
sperm densities of 2 X 10^ and 2 X 10" per ml. The first of these
is a fairly thick soup, and one wonders what would have happened
if untreated fertilized eggs had been re-inseminated at this sperm
density (cf. Table 17). What, if any, is the effect of supernatants
from dense sperm suspensions on fertilized and treated eggs?
Doubts may be entertained whether Sugiyama or Rothschild &
Swann (1952), in their experiments involving insemination with
dense sperm suspensions, have paid sufficient attention to Samp-
son's work (1926a, b) on the effects of sperm extracts on fertiliza-
tion and development, though the point is discussed in Rothschild
and Swann's paper (p. 479). This question certainly requires
further investigation in the context of these recent experiments;

POLYSPERMY 121

but interpretation will not be easy. The difficulties imposed by
the use of high sperm densities did not arise in the experiments of
Hagstrom & Hagstrom (19546). They repeated Sugiyama's
experiments but used sea-urchin eggs which had been pre-treated
with trypsin, to remove the vitelline membrane, before fertiliza-
tion. In these conditions refertilization can be achieved at a com-
paratively low sperm density, 2-6 X lo^/ml.

Conclusions regarding Type I Inhibition of Polyspermy. The
experiments described in this chapter make it possible to construct
a tentative picture, about which there will be disagreement, of the
operation of the block to polyspermy (Type I). At the moment of
attachment of the fertilizing spermatozoon to the egg surface, a
change in cortical structure passes over the egg in less, but prob-
ably not much less, than two seconds. This reduces the chance of
refertilization by a factor of twenty, and catalyses the production
of a sperm-impermeable layer at the egg surface. Exploding
cortical granules, discharging alveoli, or their equivalent near the
egg surface may contribute to the formation of this layer, which is
estabUshed in about sixty seconds, though this will undoubtedly
vary from species to species. The integrity of the layer depends,
inter alia, on the existence of divalent cations in the external
medium. If they are not present, the layer may 'dissolve' and
refertilization may be possible.

An action potential may pass over the egg surface before these
changes, as so many cell physiologists have believed or hoped; but
experiments to establish the existence of such action potentials, or
electrical depolarization of the egg surface, are exceedingly difficult,
and claims to have observed them must be examined with caution,
if not scepticism. This subject is discussed in detail in the next
chapter.

The block to polyspermy and the 'fertilization impulse', as the
early phases of the fertilization reaction are sometimes called, are
being actively investigated at the present time. Partial fertilization,
for example, first systematically examined by Allen (1954), will
undoubtedly shed light on these phenomena, and in a few years'
time the picture referred to above will be less hazy, though it may
require modification.

'Heterologous' Polyspermy and Somatic Fertilization. An account
of polyspermy would be incomplete without some mention of
recent Russian work on this subject and, in particular, of a very

122 FERTILIZATION

interesting review by Kushner (1954). Kushner reports that if
hens are inseminated on consecutive days by different and un-
related cocks, their offspring exhibit a higher growth rate, a higher
live weight, a higher blood haemoglobin content, and more vigour
than control chicks obtained by 'normal' insemination. This is
interpreted as being caused by 'heterologous' polyspermy (though
only one male pronucleus fuses with the female pronucleus) and
not, as might be thought, by heterosis. The supernumerary
spermatozoa which enter the egg are said to influence its meta-
bolism,* though it is also stated that the offspring resulting from
mixed inseminations on occasions exhibit characteristics of both
fathers. Similar experiments on pigs, sheep, cattle, trout, silk-
worms and silver foxes are reported, though in these cases, the
accent is more on increased fertility and better post-natal perform-
ance after insemination with semen from different sires than on
the appearance in the offspring of characteristics derived from
both male parents. But it is almost as difficult to understand why,
if one inseminates an animal with a mixture of semen from two
sources, the offspring should be more vigorous than those obtained
by insemination with either sort of semen singly, as it is to under-
stand the phenomenon of tri- or poly-parental inheritance. Double
inseminations would, of course, increase the number of sperm-
atozoa in the female reproductive tract and this might increase the
probability of fertilization. But this has nothing to do with the
subsequent performance of the offspring unless polyspermy is a
normal and, from the point of view of the future offspring, a
valuable event. Similarly, it is possible, as Kushner says, that
double matings increase the number of eggs ovulated, which might
cause increased litter sizes in pigs. But again, in the absence of
polyspermy, this has nothing to do with the later performance of
the offspring.

Kushner also reports that the adverse effects of excessive in-
breeding in rabbits can be counteracted by adding bull or ram
semen to rabbit semen before its introduction into a doe.

It is hardly necessary to mention that these experiments, if con-
firmed, must have a profound effect on the whole subject of fer-
tilization, quite apart from their impact on agriculture. The im-
portant thing is to repeat the experiments, which, in themselves, are

* Warburg (iQii) and Brachet (iQ34/») have reported that the O2 uptake of
polyspennic eggs is slightly higher than that of monospermic eggs.

POLYSPERMY I23

simple enough. Evaluation of the results, however, may not be so
easy and it is interesting to find that Kushner, apart from inter-
preting the results of his colleagues on the basis of polyspermy,
raises the question of somatic fertilization, a subject which, as he
rightly points out, has fallen into disrepute in recent times, except
in the special case of fertilization in sponges (Tuzet, 1950). Somatic
fertilization implies that spermatozoa enter somatic cells in the
female reproductive tract and exert an influence on them. Kushner
quotes several Russian workers who have claimed that somatic
fertilization of the mother exerts a beneficial eff"ect on her oflFspring.

CHAPTER 10

BIOELECTRIC MEASUREMENTS

'Action potentials.' Cell physiologists have often hoped — and
sometimes persuaded themselves to believe — that there is some
common denominator in the responses of cells to stimuli. Heil-
brunn (1952), for example, is a proponent of the 'calcium-release'
theory of stimulation. According to this theory, such varied cells
as muscle fibres, plant cells and unfertilized eggs respond to their
specific stimuli by the intracellular release of calcium. R. S. Lillie
(1924) placed the emphasis elsewhere. He believed that the first
and most important response of the unfertilized egg to activation
was the propagation of an action potential over the egg surface.
Such an action potential would probably diff"er from those which
occur in nerve fibres and cylindrical plant cells, in not having a
recovery phase. A nerve fibre can propagate action potentials
along its length repeatedly and at a very high frequency. The
ability to do this depends upon the membrane reconstitution or
recovery travelling along the nerve immediately behind the elec-
trical depolarization which is one of the characteristics of the
action potential. We can predict, on purely biological grounds,
that if an egg responds to fertilization by propagating an action
potential over its surface, the form of the electric change will be
quite different from that observed in active nerve or muscle fibres,
because fertilizatio7i is, under ordinary cotiditions, irreversible. The
action potential would not, therefore, be expected to look like the
normal one in Fig. 26a, but more like that shown in Fig. 26b,
which has no recovery phase. Before considering the possibility
of action potentials being propagated over egg surfaces at fertiliza-
tion or activation and the claims which have been made to this
effect, one should be clear as to what an action potential is, and
how it is recorded. Two electrodes, connected to a voltmeter, are
shown on the intact surface of a nerve fibre in Fig. 27a, with an
action potential coming towards them from left to right. At this
stage, the electrodes are equipotential as they are both on inactive
parts of the nerve surface. When the action potential reaches
electrode 1 , Fig. zyb, a transient potential difference develops be-

124

BIOELECTRIC MEASUREMENTS

125

tween the electrodes, because the 'breakdown' of the nerve mem-
brane, which characterizes the action potential, causes electrode i

FIG. 26a. — ^Action potential recorded between inside and outside of squid giant
axon. The vertical scale shows the potential in millivolts of one electrode
inside the axon, relative to a second electrode in the external medium,
assumed to be at zero potential (after Hodgkin & Huxley, 1945).

FIG. 266. — The same action potential as in a, without any recovery phase. In
both cases, the dots below the action potentials are Tooth of a second apart.

to be transiently connected to a 'sodium battery' (Fig. 28). In
Fig. 27c the action potential is between the electrodes,* which

* The wave-length of the action potential is assumed to be short compared
with the inter-electrode distance.

126

FERTILIZATION

Sodium in

Potassium
out

(0)

time

(b)

■N^-'

(c)

(d)

(e)

FIG. 27. — An action potential travelling along a nerve fibre from left to right,
with two electrodes, connected to a voltmeter V, on the nerve surface.

BIOELECTRIC MEASUREMENTS

127

are, therefore, equipotential again; while in Fig. 2']d it has reached
electrode 2, causing a second transient potential difference, which
is the mirror image of Fig. 276, between the electrodes. If the
system under examination has not got a muscle attached to one end
of it and cannot be repeatedly stimulated, a set-up of the type
described above is necessary for a demonstration of conduction.
Even then difficulties in interpretation may arise; the reader may
like to consider the difference between a record of a propagated

Outside

Inside the nerve
membrane

FIG. 28. — Electrical model of a nerve membrane: a, membrane capacitance; b,
K+ channel; c, Na+ channel; Gj, membrane K conductance, 05 mmho/cm-;
G2, membrane Na conductance, 001 mmho/cm^. The batteries are due to
the unequal distribution of Na and K between the inside and outside of the
nerve membrane. In the early phases of the action potential, G2 rises
(resistance falls), so that the recording system measures the potential due to
the sodium battery. A 5-mV. change in the p.d. across the membrane causes
an e-fold change in membrane conductance (adapted from Hodgkin &
Huxley, 1952).

action potential with no recovery phase and a record of a non-
propagated electrical change, with recovery, at one electrode.

If a resistance meter is substituted for the voltmeter in the above
circuit, the resistance of a square centimetre of membrane can be
shown to fall by a factor of 100 or more in the 'active' region,
where there is a potential change of lOO mV. There is no capacit-
ance change.

These are, very briefly, the electrical changes which constitute
the action potential. Chemically, it consists of an influx of sodium
ions during the rising phase, Fig. 26a, possibly due to the transient

128 FERTILIZATION

removal of calcium from sites in the nerve membrane (Franken-
haeuser & Hodgkin, 1955), and an outflow of potassium during
the falling or recovery phase.

What evidence is there that anything like the phenomena de-
scribed in the preceding three paragraphs occurs at fertilization?
Dorfman claimed in 1934 that there was a potential difference of
44 mV. between the inside and outside of the unfertilized frog's
egg, the inside being negative, and that this potential difference
was reversed at fertilization. As, however, the insertion of a
needle or micro-electrode into an unfertilized frog's egg activates
the egg parthenogenetically, the potential changes observed by
Dorfman could have had little if anything to do with the early
phases of fertilization. Apart from this, the reversal of p.d.
took place one hour after insemination. In 1935, Hasama said
he had observed electrical changes in the egg of Hynobiiis nebu-
lostis (Schlegel) at fertilization. There is no reason to suppose
that Hasama's published records are anything but random base-
line fluctuations due to the measuring apparatus. A further claim
that activation of the frog's egg, both by a spermatozoon and by a
glass needle, is associated with electrical changes, was made in the
same year by Peterfi & Rothschild (1935). No records and very
few experimental details were published. When I systematically
repeated these experiments a year or so later, I came to the con-
clusion that though the puncture of an unfertilized frog's egg was
associated with electrical changes or signals which might be of
biological origin, the form of the changes was unpredictable and
the evidence that they were propagated over the egg surface in-
adequate. When, on the other hand, a frog's egg is fertilized, the
evidence that bio-electric changes occur is more convincing,
though such experiments present formidable technical difliculties;
but there is no reason to suppose that such changes are propagated
and to describe them as action potentials is wrong. Recent claims
that 'action potentials' occur when sea-urchin eggs are fertilized
(Scheer et al., 1954) are open to the same criticisms as the ex-
periments referred to above, which were done some twenty years
ago. The most we can say is that when eggs are fertilized, potential
changes of obscure origin are sometimes observed; but in the
absence of further evidence, these should not be called action
potentials, nor thought of as such.

There are several reasons for thinking that these potential

BIOELECTRIC MEASUREMENTS

129

changes may not be of much importance in fertilization. In spite of
the asymmetrical distribution of ions between the inside and out-
side of the sea-urchin egg (Table 20), which, unless the plasma

TABLE 20

Inorganic constituents of unfertilized eggs of Paracentrotus lividus
and of sea water {Rothschild & Barnes, 1953)

Eggs, ttiM

Sea water, tnM

Sodium

485

Potassium

210

Calcium

Magnesium .

Chloride

566

Sulphate

Total phosphorus

[2-1]

In the case of eggs, mM means millimoles per kilogram of water in the eggs
(dry weight, 24%; density, 1-09); in the case of sea water, mM means milli-
moles per kilogram of water, chlorinity i9-21%o- The figure in square brackets
for total phosphorus is in mg./ml. eggs.

membrane is impermeable to sodium and potassium, one would
have expected to cause a potential difference across the egg surface,
no such difference has been observed (Rothschild, 1938). In the
1938 experiments the terminal diameter of the electrode inserted
into the egg was 2-10 yc. On modern standards an electrode of this
size would be considered coarse and liable to tear the egg surface,
with consequent short-circuiting and failure to record any poten-
tial difference. Suppose that there is a potential difference across
the plasma membrane and that the insertion of a micro-pipette
causes such short-circuiting as to make the potential difference so
small as to be unmeasurable. The fact remains that eggs can be
fertilized after the insertion of two such electrodes, which means
that fertilization is not dependent upon the existence of a potential
difference across the egg surface. It is far from clear how an action
potential could be propagated over the egg surface, when, in the
resting state, there is no potential difference across the membrane
under consideration. The idea that no-one has ever got a micro-
electrode, as opposed to an ultramicro-electrode, into a sea-urchin
egg, and that the electrode merely causes an extended invagination
of the plasma membrane, is scarcely tenable. It is, for example,
possible to inject a live spermatozoon into the cytoplasm of a sea-
urchin egg, though fertilization does not occur. Similarly, as is

130 FERTILIZATION

well known, indicators and dyes have been repeatedly injected into
sea-urchin eggs, I have confirmed the apparent lack of p.d. across
the sea-urchin egg plasma membrane, using ultramicro-electrodes,
and so have Scheer et al. (1954). Lundberg (1956) believes that
when such electrodes are used, attempts at insertion are often un-
successful because the electrode does cause an extended invagina-
tion of the plasma membrane. When, however, he did manage to
effect contact with the egg cytoplasm, it appeared to be 5-10 mV.
positive with respect to the external medium. In view of the size
of liquid junction potentials in systems of this sort, a resting
potential of 5-10 mV. is too close to zero to have much significance.
Moreover, it is very difficult to see how the potential of the inside
of the sea-urchin egg can be positive with respect to the outside,
unless the plasma membrane is impermeable to potassium ions
and permeable to sodium ions, the latter being actively pumped
out.

To sum up this section: there is as yet no good evidence that
the irregular potential changes observed in eggs at fertilization
occur at the plasma membrane ; nor that they are propagated over
the egg surface and, therefore, connected with the block to poly-
spermy; nor that they are important in fertilization.

Membrane resistance. Very few estimates of egg membrane
resistances have been made because of the technical difficulties
inherent in such measurements on small spherical cells. The
following calculation shows the origin of these difficulties, par-
ticularly if an a.c. method of measuring resistance is used, with
external electrodes. Suppose that the membrane resistance is 500
ohm-cm.^ (Davson, 195 1). The radius of a sea-urchin egg (Psam-
mechinus miliaris) is 50ju,, so that its surface area is about 3.10^* cm^.
The actual resistance to be measured will, therefore, be of the
order of 10^ ohms, in comparison with which the resistance of the
external medium is negligible. As a result, the only systematic
measurements of egg membrane resistance are those of Cole &
Guttman (1942), using the unfertilized egg of Rana pipiens
Schreber, They obtained a value of 170 ohm-cm.-, with an alternat-
ing current bridge method. If ultramicro-electrodes could be in-
serted into eggs without their tips being broken or clogged, the
most satisfactory method of measuring egg membrane resistances
would be by inserting two electrodes into the egg, flowing current
across the plasma membrane using one of the internal electrodes,

BIOELECTRIC MEASUREMENTS

131

and measuring the resultant ohmic drop of potential across the
plasma membrane with the other internal electrode. Such an
experiment would not be easy and might first be tried on unfer-
tilized trout eggs in oil, before contact with tap water. This would
avoid the complicating factor of the development of the chorion.
Cole & Guttman calculated from Holzer's data (1933) that the
trout egg membrane resistance was about 5,000 ohm-cm^. How-
ever, they failed to notice that Holzer's experiments were done on
trout eggs which had been cut in half. This treatment always kills
the eggs and entirely destroys the resistive properties of the
vitelline membrane; this hypothetical value for the trout egg
membrane resistance should not, therefore, be accepted.

Lundberg (1956), using the 'ohmic drop' method with two

TABLE 21
Egg membrane capacitance, Cm, and egg cytoplasm resistivity, rg

species

Cm,

tiFjcm "

ti, ohm

-cm

Method

Reference

A . pundulala

0-85

3-3

105

133

a.c. (intact eggs)

Cole &. Spencer, 1938

T. ventricosiis

087

2-0

203

349

Cole, 1935

A. forbesi

I-IO

136-225

Cole & Cole, 1936a

R. pipiens

2-0

570

„

Cole & Guttman, 1942

S. trutta

0-57

0-58

202 *

159 *

„

Rothschild, 1946

S. trutta

90 *

a.c, directly on
contents

Gray, 1932

♦ These measurements were on the inner contents of the egg, not the egg cytoplasm (see text).
U, unfertilized; F, fertilized.

ultramicro-electrodes inside the egg (Psammechiniis miliaris),
managed to make a satisfactory measurement on one egg and ob-
tained the value 2,200 ohm-cm - for its membrane resistance. In
his other measurements, great difficulties were experienced, as
mentioned earlier, in establishing electrical contact between the
electrodes and the egg interior.

Membrane capacitance. The most important work on this subject
is that of Cole and his co-workers. His results on egg membrane
capacitance and egg cytoplasm resistivity, together with a few
others, are given in Table 21. Cole made two important dis-
coveries in his capacitance measurements on eggs. First, that there
is a marked increase in membrane capacitance when the eggs of
Arbacia pnnctulata and of Tripneustes ventricosiis (Lamarck) are
fertilized (about 400% and 240%). This has been confirmed by
lida (i943«, 6), using the eggs of Pseudocentrotus depressus and

132 FERTILIZATION

Hemicentrotus pulcherrimus. The approximate time course of the
capacitance change is shown in Fig. 29. The reader should beware
of a somewhat confusing interpretation of this phenomenon,
involving a large capacitance in the fertilization membrane, put
forward at one time by Cole & Cole (19366). This interpretation
is most unlikely to be correct, as Cole & Spencer pointed out
in 1938. The change in capacitance at fertilization is real and
reflects the structural changes in the cell surface which are known

1-6

^ 1-2
06

0-4

^ "^~v

^n/

t {mm.} after fertiLization^
at t=o

FIG. 29. — Changes in membrane capacitance of eggs of Pseudocentrotus depressus
following fertilization. A set of points connected by a line refers to consecu-
tive readings on a sub-sample containing lightly centrifuged eggs, removed
from one parent sample. Cleavage at 80-100 min. in parent sample. T° C,
i7-9-i8-5. Adapted from lida (1943a).

to take place at that time. Apart from what might be called
chemical changes, the cell surface becomes thinner at fertilization,
because of the elevation of the vitelline (= fertilization) membrane.
This reduction in thickness is consistent with the observed increase
in capacitance, for the following reason: the capacitance of a
parallel plate condenser is given by the equation

:A/477l

(3)

where e = dielectric constant of the medium between the plates.

BIOELECTRIC MEASUREMENTS I33

A = cross-sectional area of plates, and 1 = distance between the
plates. If 1 becomes smaller, i.e., the condenser becomes thinner,
the capacitance becomes larger. Chemical changes in the cell
surface may, of course, also be responsible for the observed change,
but with our present exiguous knowledge of the chemistry of the
cell surface, it is difficult even to speculate about the nature of
changes which might cause an increase in capacitance.

It would be of great interest to try to find out at what rate this
capacitance increase, which presumably starts at the site of sperm
attachment, is propagated over the egg surface. Such an experi-
ment would again probably involve difficult experiments with
ultramicro-electrodes.

The second discovery that Cole (1935) made in this field was
that the membrane capacitance of the eggs of Tripneustes ventri-
cosiis is inversely proportional to the surface area of the egg, when
this is varied by diluting the sea water around the eggs with dis-
tilled water. This also has been confirmed by lida (1943c), using
the eggs of Pseiidocentrotus depressus, in which the changes in
membrane capacitance associated with alterations in the hypo-
tonicity of the sea water were found to be reasonably reversible.
This shows that the unexpected sense of the capacitance change is
not due to irreversible injury of the cortex following stretching,
lida's results are given in Fig. 30. Unless an egg in hypotonic sea
water continually synthesizes new membrane material to maintain
its normal thickness (curve B, Fig. 30), a most improbable situa-
tion, the membrane capacitance should increase (curve A, Fig.30),
not decrease, when the egg swells. The reasons are clear, as before,
from a consideration of Equ. (3). These observations may well
have revealed a fundamental, but paradoxical property, of cell
membranes in general, quite apart from those of sea-urchin eggs.
They merit further investigation, particularly in conjunction with
the Elastimeter experiments of Mitchison & Swann (1954^),
discussed in chapter 8. When considering the interpretation
of these capacitance changes, lida (1943c, p. 171) says: 'If an
assumption is made that the membrane is of a mosaic structure
with two intermingling areas, of which one is "effective" and the
other is "ineffective" in manifesting measureable capacitance, and
if the latter area alone is extensible on mechanical stretching, the
capacitance will vary in a manner represented by C. A scheme like
this appears to be a little too artificial, but it may not be altogether

134

FERTILIZATION

physically implausible.' * An alternative interpretation of these
'anomalous' capacitance changes is that in the unswollen condition,
the sea-urchin egg membrane is folded, on a sub-microscopic
scale, and that when the egg swells, the membrane unfolds. Under
these conditions, calculation of the membrane capacitance per unit
area for the unswollen egg will produce too high a value, but the
calculation for the swollen egg will be more accurate. If, in fact,
the membrane is a 'reasonable' one (curve A, Fig. 30), it should be

Ml
0-74
0-37

* •

^___ —

.^^ •

•

2-95 5-90 8-85

FIG. 30. — Change in egg membrane capacitance per unit area with surface area of
egg, the latter being varied by immersing the eggs in sea water of different
degrees of hypotonicity. Fertilized eggs of Psendoccntrotus depressiis were
used. A, theoretical curve showing expected behaviour of the membrane
capacitance when the egg swells; B, theoretical curve if the membrane
thickness remains constant while the egg swells; C, variation in membrane
capacitance as an inverse function of the surface area. Adapted from lida
(1943c).

possible to make an estimate of the minimum degree of folding
from capacitance measurements in normal and hypotonic sea water.
Cytoplasmic resistivity. Table 21 gives the results of experiments
in this field. The differences between unfertilized and fertilized
eggs may or may not be significant ; in any case, the most important
changes which occur in the early phases of fertilization, with which
we are concerned in this chapter, take place at the cell surface f

* Unfortunately, a further paper dealing with the membrane capacitance of
sea-urchin eggs, in Japanese, by lida (1949) Zool. Mag. (Dobutsugaku Zasshi),
58, 122-125, is not available in the United Kingdom.

t K. Dan (1947) reported a small change in i potential after fertilization of the
eggs oi Pseiidocentrotus depressus and Anthocidaris crassispina; but the change is
too small to be of much interest.

BIOELECTRIC MEASUREMENTS I35

and not in the cytoplasm. Later, chemical changes in the cytoplasm
become of profound importance, but it is doubtful whether or not
straightforward measurements of cytoplasmic conductivity will
produce information of great interest. In the case of the column
headed r.^ in Table 21, the values for the trout egg refer to the
globulin-containing solution within the vitelline membrane and
not to the cytoplasm, as in the other cases mentioned in this
Table. There is no cytoplasm to speak of in the unfertilized trout

egg-
To summarize the contents of this chapter, measurements of

bio-electric phenomena at fertilization have so far revealed little
of importance except for the increase in membrane capacitance
when sea-urchin eggs are fertilized. The main reason for this
unsatisfactory state of affairs is the refractory nature of the bio-
logical material and the associated technical difficulties. The
reader should consult Cole's papers for further details of experi-
ments involving alternating current measurements, while the
theory of such measurements when applied to eggs, and such
questions as the meaning of polarization capacitances, charac-
teristic frequencies, and the frequency-dependence of membrane
capacitances are summarized by Rothschild (1946).

Note. Since this chapter was written, the results of new experi-
ments on the bioelectric properties of eggs have been published.
Although they do not in general affect my conclusions, they are
sufficiently important to deserve special mention. Grundfest et al.
(1955)* and Tyler et al. (1955)! observed a potential difference
of 30-60 mV. between the inside and the outside of the starfish
egg, the inside being negative. The p.d. could be reversibly re-
duced to zero by increasing the K content of the surrounding sea
water. The membrane resistance and capacitance were, respec-
tively, 2,000-3,000 ohm-cm^ and 0-5-1 -o /xF/cm^. The p.d.
decreased transiently at about the time of fertilization and, within
a minute of insemination, increased to 10-15 mV. above the "rest-
ing potential".

* Grundfest, H., Kao, C. Y., Monroy, A. & Tyler, A. (1955) Biol. Bull., Wood's
Hole, 109, 346.

t Tyler, A., Monroy, A., Kao, C. Y. & Grundfest, H. (1955) Biol. Bull., Wood's
Hole, 109, 352-353.

CHAPTER I I

SPECIFICITY

The reactions between the gametes exhibit a high, but not total,
degree of specificity, whether the reaction is fertihzation or the
agglutination of spermatozoa by egg water. Quite apart from en-
vironmental barriers in nature, we can, for example, be virtually
certain that bull spermatozoa will be unable to fertilize or even
activate a rabbit egg. In modern language, p, the probability
of a successful sperm-egg collision (i.e. one which achieves
fertilization), or a, the sperm-egg interaction rate, will be ex-
tremely low in such a case ; p will not be zero and might be made
appreciably greater than zero by appropriate treatment of the egg.
One such treatment, which has been extensively but empirically
used in effecting cross-fertilization, is to have an abnormally large
number of heterologous spermatozoa round the eggs in question.
The probabilistic analysis of fertilization, discussed in chapter 9,
Polyspermy, explains why, but not how, an increase in sperm
density improves the chances of cross-fertilization. Equ. (2) in
chapter 9 can be written in the approximate form

u = exp(— Tra^ncpt) . , (4)

where u = proportion of unfertilized eggs in a suspension allowed
to interact with spermatozoa, density n, for a known time t ;
a = egg radius; c = mean speed of the spermatozoa; and p =
probability of a successful collision.

We can make u smaller, i.e. increase the number of fertilized
eggs, by any of the following operations :

(i) Increase c. In general, this is extremely difficult, if not im-
possible, to do, though it is conceivable that Loeb's method of
improving cross-fertilization, which involved making the sea
water more alkaline (1903), worked partly in this way. Sea-urchin
spermatozoa sometimes become more active in alkaline sea water.

(2) Increase t. Although this can always be done to a certain
extent, the fertilizing life span of spermatozoa is limited, and so is
the life of the unfertilized egg. In any case, the technique of known
sperm-egg interaction times was only developed in 1950 and, at the

136

SPECIFICITY 137

time of writing this book, the method has not been appUed to cross-
fertiHzation experiments, the unfertihzed eggs simply being left in
contact with the heterologous spermatozoa for an unknown and
indefinite time.

(3) Increase p. p can sometimes be increased by removal of egg
jelly (Harding & Harding, 1952a), by pre-treatment of the gametes
with glycine + egg water or sodium periodate -\- egg water
(Harding & Harding, 19526), or by interfering with the vitelline
membrane of unfertilized eggs through treatment with such agents
as trypsin(Hultin, 1948). Trypsin alsoinduces polyspermy in homo-
logous fertilization, as Hagstrom & Hagstrom (1954c) have recently
shown. One might envisage the surface of an unfertilized egg as a
three-dimensional jig-saw puzzle, made of rubber and containing
very weak magnets. Of course, the magnets are, in reality, van der
Waals' forces which are inversely proportional to the seventh
power of the distances between the atoms involved, hydrogen
bonds and attractions between oppositely charged groups (Pauling
et al., 1943). The application of trypsin could be likened to a
bunsen burner turned on to the jig-saw puzzle. Some of the pro-
tuberances on the surface will be melted, decreasing the preciseness
of fit with complementary structures on the head of the homologous
spermatozoon, but permitting a reaction to occur with less precisely
complementary structures on the heads of some heterologous
spermatozoa. An alternative interpretation of the effect of trypsin
is, however, possible — that it exposes more or 'deeper' combining
groups. Incomplete antibody, for example, combines with antigens
on red blood cells, though no agglutination takes place. But if the
red blood cells are pre-treated with trypsin, washed, and then sub-
jected to incomplete antibody, agglutination occurs (Coombs,
1954). This suggests that, so far from blunting combining groups,
treatment with trypsin sharpens or exposes them. A similar con-
clusion might be reached from the work of Coffin & Pickles (1953),
who found that periodate destroyed the D P.h antigen and that
subsequent treatment with trypsin brought back the property of
agglutination. The procedure could be repeated, as if combining
sites were obliterated by periodate, but that new ones were exposed
by a second treatment with trypsin.

Runnstrom et al. (19446) found no improvement in cross-
fertilizations between Psammechimis miliaris and Echinocardium
cordatum after pre-treating the unfertilized eggs with trypsin,

138 FERTILIZATION

while Hultin (1948) had the same experience with other sea-
urchins.

(4) Increase n. This- is the classical way of achieving cross-
fertilization. An increase in sperm density involves an increase in
the number of sperm-egg collisions as the latter, Z, is equal to
Tia^nc. For any p, however low, the more collisions there are, the
greater the chance that an egg will be fertilized.* The most im-
portant investigation of cross-fertilization in which n was con-
trolled was made by Tyler (1949), though Fuchs (1914-1915) also

TABLE 22

Comparison betzveen cross-fertilization and cross-agglutination

The upper figures in each pair refer to the dilution of a standard sperm
suspension necessary to achieve 2% fertilization (e.g., 3000 means i /3000). The
lower figures in each pair are the highest dilutions of fertilizin solution at which
visible agglutination occurred.

Eggs or fertilizin of

Spermatozoa of

S.
purpuratus

S.
franciscanus

L.
pictus

excentricus

S. purpuratus

3,000
512

I
64

5
4

S. franciscanus

600

—

512

L. pictus

2
64

4
32

850
64

D. excentricus

40
2

4,400
128

realized the importance of controlling n, in his studies on self-
sterility in Ciona intestinalis. Tyler's results are reproduced in
Table 22. The experiments were done for a particular reason, to
compare cross-fertilization and cross-agglutination; the technique
of known sperm-egg interaction times had not been developed at
that time. Tyler's results are interesting as they show that the
correspondence between the degree of cross-fertilizability and
cross-agglutination is not particularly marked. This means
that there is more specificity in fertilization than in the fer-
tilizin-antifertilizin reaction, or that fertilization is not ex-

* This is not the same proposition as the familiar but fallacious one about red
being 'bound' to turn up after a run of 20 blacks, at roulette.

SPECIFICITY 139

clusively determined by the fertilizin-antifertilizin reaction. On
the other hand, Table 22 shows that in general, cross-fertilization
implies cross-agglutination. Even in the apparently exceptional
cases of eggs or fertilizin from Strongylocentrotiis franciscanus (A.
Agassiz) or spermatozoa from Strongylocentrotiis purpuratiis and
Dendr aster excentricus (Eschscholtz), Tyler has shown that sperm-
atozoa of the latter cchinoderms do combine with fertilizin of the
former.

The partially successful interphyletic crosses which have been
achieved, such as Strongylocetitrotiis $ X Mytilus <S (Kupelwieser,
1909), raise a number of interesting but difficult problems, p will
clearly be very low in such cases ; but there is no particular reason
why a successful hit should not be achieved from time to time,
even if subsequent development is gynogenetic. Kupelwieser's
experiments do not exclude an alternative possibility: that such
crosses can be achieved without specific adhesion between the
surfaces of the egg and the spermatozoon, but as a result of the
action of a non-specific lysin or detergent-like compound, diffusing
out of the sperm head and softening up the egg cortex so that the
sperm can be readily engulfed. F. R. Lillie (1919) favoured this
interpretation of Kupelwieser's results, and there have been refer-
ences elsewhere in this book to the possibility of misinterpreting
experiments involving insemination with very high sperm con-
centrations, because of the possibility of non-specific effects due to
sperm lysins or A. III. But even if, intuitively, we do not like the
idea of there being a sufficient degree of complementariness be-
tween the surface of eggs and spermatozoa of different phyla to
permit activation, we must still admit that the possibility exists,
first because the 'fit' does not have to be complete (see later in this
chapter), and secondly, because the reaction is essentially a proba-
bilistic one, in which p does not equal zero. Whatever one's in-
tuitive feelings may be, cross reactions do occur: a classical ex-
ample is the Weil Felix Reaction, in which B.proteus is agglutinated
by serum from patients with typhus ! -

Apart from certain examples and analogies, what has been said
above about specificity is mainly empirical, or analytical, in the sense
of being based on the laws of probability and the kinetic theory of
gases. When we come to examine the 'how' part of the problem,
almost complete ignorance prevails, in spite of the immense number
of cross-fertilization experiments which have been done during

140 FERTILIZATION

this century. Even in 1900, for example, Vernon could report
twenty-nine different and successful echinoderm crosses; but
though these and subsequent cross-fertilization experiments are
interesting from an embryological and geneticai point of view, they
do not help in gaining an understanding of specificity and self-
sterility. In general terms the specific reaction between a sperm-
atozoon and an egg is due to complementariness of surface struc-
ture which allows close contact to occur over an area which, by
analogy with serological reactions, might be of the order of
100 A^; this enables the weak forces of intermolecular interac-
tion to combine to produce a strong union. The fit of an anti-
body to an antigen is close, the complementariness in structure
being such that an increase of o-8 A in the size of one atom in a
group can cause steric hindrance. On the other hand, cross-
fertilization does not imply that the heterologous spermatozoa in
question have surface combining configurations which are identical
with those on homologous spermatozoa. For example, in antigenic
proteins containing azobenzene arsonic groups, the substitution of
a methyl group by a chlorine or bromine atom causes little change
in serological properties, because the van der Waals' radii of the
methyl group, chlorine and bromine are, respectively, 1-9, i-8i
and 1-95 A. We can imagine that different groups in particular
regions of the folded polypeptide chains, which probably con-
stitute the protein parts of the reacting molecules, sometimes have
sufficiently similar van der Waals' radii in different species to allow
an unexpected cross-reaction to take place. The 'rubberiness' of
the fit, referred to in the jig-saw puzzle analogy, is reflected in the
behaviour of anti-^-(^-azobenzene-azo) benzene arsonic acid
antiserum, in which a radial dilation of about 1-5 A is possible
without interference with the reaction.

To sum up, the only advances that have been made in the
study of specificity in fertilization since F. R. Lillie wrote a
chapter on this subject, which all students of fertilization should
read, in Problems of Fertilization, are: (i) more information about
the immunological nature of the reactions between the gametes,
almost entirely due to the work of Tyler; (2) more information
about the nature of immunological reactions in general, much of
which is due to Landsteiner and Pauling; and, possibly, (3) the
development of techniques for the quantitative study of inter-
specific and intergeneric cross-fertilization.

CHAPTER 12

CONCLUSION

In my Preface I expressed a hope that this book would provoke
further experiments. The question is, what experiments? Pro-
phecies are always dangerous and usually wrong ; but for what it is
worth, I believe that the following subjects would repay further
investigation :

(i) Morphology of pronuclear movements.

(2) Sub-microscopic morphology of spermatozoa in sea water
and egg water.

(3) Physiology of frog's egg jelly.

(4) Turning mechanisms in plant spermatozoa.

(5) Structure-action relationships in the chemotaxis of plant
spermatozoa.

(6) Variations in the receptivity of different parts of the egg
surface.

(7) Oxidative carbohydrate breakdown in eggs.

(8) DNA content of eggs, spermatozoa and pronuclei.

(9) Partial fertilization, using 'cylindrical' eggs.

(10) Irritability of the zona pellucida.

(11) K and Na fluxes across egg surface before and after
activation.

(12) Conduction velocity of capacitance change at fertiliza-
tion.

(13) Membrane resistance before and after activation. This
may be linked with (11).

(14) EflFects of periodate and trypsin on heterologous fertiliza-
tion.

(15) The morphology, physiology and biochemistry of fer-
tilization and parthenogenetic activation in any eggs other
than those of echinoderms. Japanese biologists have realised
the importance of this subject and in their hands it is beginning
to pay dividends. Novikoff's experiments, on the escape of

141

142 FERTILIZATION

cortical granules from the fertilized egg of Sabellaria alveolata,
should cause those who work exclusively on echinoderm eggs
some anxiety.

Other lists could, of course, be made. The reasons why my list
contains what it does are to be found, I hope, in this book.

REFERENCES AND AUTHOR INDEX

The numbers in italics after a reference refer to the pages on which this work
of the author is mentioned, for example :

Endo, Y. (1952) Exp. Cell Res., 3, 406-418. 6, 9, 10, g8, 107.
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Abelson, p. H. (1947) Biol. Bull, Wood's Hole, 93, 203. 81.

Abelson, p. H. (1948) Biol. Bull., Wood's Hole, 95, 262. 81.

Abrams, R. (1951) Exp. Cell Res., 2, 235-242. 84.

Alfert, M. & Swift, H. (1953) Exp. Cell Res., 5, 455-460. 85.

Allen, R. D. (1953) Biol. Bidl, Wood's Hole, 105, 213-239. 119.

Allen, R. D. (1954) Exp, Cell Res., 6, 403-424. 6, 15, 104, iig, 121.

Amoroso, E. C. & Parices, A. S. (1947) Proc. roy. Soc. B, 134, 57-78. iig.

Ashbel, R. (1929) Boll. Soc. ital. Biol, spcr., 4, 492-493. 64, 65.

Austin, C. R. (1951a) y.R. micr. Soc, 71, 295-306. 18.

Austin, C. R. (19516) Aust. J. set. Res., B, 4, 581-596. 13, 114.

Austin, C. R. (1953) Aust. vet. J., 29, 191-198. 12.

Austin, C. R. & Braden, A. W. H. (1953) Aust. J. biol. Sci., 6, 674-692. 106.

Austin, C. R. & Braden, A. W. H. (1954a) Aust. J. biol. Sci., 7, 179-194. 115.

Austin, C. R. & Braden, A. W. H. (1954*) Aust. J. biol. Sci., 7, 195-210. 115.

Ballentine, R. (1940) y. cell. comp. Physiol., 15, 217-232. 60, 61.

Barron, E. S. G. (1932) Biol. Bull, Wood's Hole, 62, 42-45- 6j.

Bataillon, E. (1909) Arch. EntwMech. Org., 28, 43-48. i.

Beament, J. (1946) Proc. roy. Soc. B, 133, 407-418. 4.

Berg, W. E. (1950^) Biol. Bull., Wood's Hole, 98, 128-138. 37.

Berg, W. E. (19506) Proc. Soc. exp. Biol., N.Y., 75, 30-32. 87.

Bernstein, M. H. (1949) Biol. Btdl., Wood's Hole, 97, 255. 84.

BlA-fcASZEWicz, K. (19 12) Arch. EntzvMech. Org., 34, 489-540. 11.

BiELiG, H.-J. & Dohrn, p. (1950) Z. Naturf., 5b, 316-338. 33-

Bishop, D. W. (195 1) Biol. Bull. Wood's Hole, loi, 215-216. 26.

de Blainville, H. M. D. (1825) Dictionnaire des Sciences Naturelles, 37, F. G.
Levrault, Strasbourg & Paris. 154.

de Blainville, H. M. D. (1834) Manuel d'Actinologie, F. G. Levrault, Stras-
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Blandau, R. J. & Odor, D. L. (1952) Fertility and Sterility, 3, 13-26. 12.

Block, F. (1935) Trav. Sta. zool. Wimereux, 12, 183-279. v.

Blount, M. (1909) J. Morph., 20, 1-64. 103.

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BoLOGNARi, A. (1952) Arch. Sci. biol., 36, 40-47. 78, go.

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BoREi, H. (1933) Z. vergl. Physiol., 20, 258-266. 64, 65.

Borei, H. (1948) Biol. Bull., Wood's Hole, 95, 124-150. 57, 61.

BoREi, H. (1949) Biol. Bull., Wood's Hole, 96, 117-122. 57, 59-

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BovERi, T. (1907) jfena Z. Naturzv., 36, 1-292. 103.

Boyd, M. (1928) Biol. Bull., Wood's Hole, 55, 92-100. 61, 63.

Brachet, J. (1933) Arch. Biol., Paris, 44, 519-576. 84.

Bracket, J. (1934a) Arch. Biol., Paris, 45, 611-727. 63.

Bracket, J. (19346) Arch. Biol., Paris, 46, 1-24. 56, 122.

143

144 FERTILIZATION

Bracket, J. (1950) Chemical Embryology, Interscience Publishers Inc., New

York. 62, 65. y4. ys.
Brachet, J. (1954) Arch. Biol., Paris, 65, 1-72. v.
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391-409. J2, 106, 114.
Bretschneider, L. H. (1948) Proc. kon. Ned. Akad. Wet., 51, 3-7. 103.
Brock, N., Druckrey, H. & Herken, H. (1938) Arch. exp. Path. Pharmak, 188,

451-464. 61.
Brookbank, J. W. & Whiteley, A. H. (1954) Biol. Bull., Wood's Hole, 107, 57-

63. 8g.
Brooks, S. C. & Chambers, E. L. (1948) Biol. Bull., Wood's Hole, 95, 262-263.

81.
Brooks, S. C. & Chambers, E. L. (1954) Biol. Bull., Wood's Hole, 106, 279-296.

82.
Bruchmann, H. (1909) Flora, Jena, 99, 193-202. 50.
Burdon-Jones, C. (1951) y. Mar. biol. Ass. U.K., 29, 625-638. 21.
de Burgh, P. M., Sanger, R. A. & Walsh, R. J. (1946) Aust. J. exp. Biol. med.

Sci., 24, 293-300. 29.
Byers, H. L. (1951) Biol. Bull., Wood's Hole, loi, 218. 24.
Carter, G. S. (1931) J. exp. Biol., 8, 176-201. 33.
Cennamo, C. & MoNTELLA, S. (1947) Experientia, 3, 415-416. 66.
Chambers, E. L. (1939) J. exp. Biol., 16, 409-424. 15-18.
Chambers, E. L. (1949) Biol. Bidl., Wood's Hole, 97, 251-252. 83.
Chambers, E. L. & Mende, T. (1953(3) Arch. Biochem. Biophys., 44, 46-56, 82.
Chambers, E. L. & Mende, T. (19536) Exp. Cell Res., 5, 508-519. 82.
Chambers, E. L. & White, W. E. (1949) Biol. Bull., Wood's Hole, 97, 225-226.

82.
Chambers, E. L. & White, W. E. (1954) Biol. Bull., Wood's Hole, 106, 297-307.

82.
Chambers, E. L., White, W., Jeung, N. & Brooks, S. C. (1948) Biol. Bull.,

Wood's Hole, 95, 252-253. 83.
Chambers, R. (1921) Biol. Bull., Wood's Hole, 41, 318-350. 20.
Chambers, R. (1923) jf. gen. Physiol., 5, 821-829. 7.
Chambers, R. (1942) jf. cell. comp. Physiol., 19, 145-150. 11.
Chang, M. C. & Pincus, G. (1953) Science, 117, 274-276. 55.
Churney, L. & MosER, F. (1940) Physiol. ZooL, 13, 212-217. gg.
Clark, J. M. (1936) Biol. Bull. Wood's Hole, 70, 361-384. 116.
Cleland, K. W. (1950a) Proc. Linn. Soc. N.S.W., 75, 282-295. 5^- 59-^ i- ^4.

65. 6g.
Cleland, K. W. (19506) Proc. Linn. Soc. N.S.W., js, 296-319. 66, 67, 6g, yy,

78.
Cleland, K. W\ & Rothschild, Lord (1952a) J. exp. Biol., 29, 285-294. yo.

71-
Cleland, K. W. & Rothschild, Lord (19526) J. exp. Biol., 29, 416-428. yi,

78.
Coffin, S. F. & Pickles, M. M. (1953) J. Immunol., 71, 177-182. I3y.
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Cole, K. S. (1932) Jf. cell. comp. Physiol., i, 1-9. 92.
Cole, K. S. (i935) J- fi<^n- Physiol., 18, 877-887. 131, 133.
Cole, K. S. & Cole, R. H. (1936^) y. gen. Physiol., 19, 609-623. 131.
Cole, K. S. & Cole, R. H. (19366) y. gen. Physiol., 19, 625-632. JJ-'.
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Cole, K. S. & Spencer, J. M. (1938) y. gen. Physiol., 21, 583-590. 131, 132.
Colwin, a. L. & Colwin, L. H. (1953) y. Morph., 92, 401-454. 20.

REFERENCES AND AUTHOR INDEX I45

COLWIN, L. H. & CoLWiN, A. L. (1949) Biol. Bull, Wood's Hole, 97, 237. 8.

CoNKLiN, E. G. (1905) y. Acad. nat. Sci. Philad., 13, 1-119. 16.

Connors, W. M. & Scheer, B. T. u947) J- cell. comp. Physiol., 30, 271-283.

yo, 82.
Cook, A. H., Elvidge, J. A. & Heilbron, Sir I. (1948) Proc. roy. Soc. B, 135,

293-301. 47-
Cook, A. H. & Elvidge, J. A. (195 1) Proc. roy. Soc. B, 138, 97-1 14- 47-
Coombs, R. R. A. (1954) Schweiz. Z. allg. Path., 17, 424-439. 137 ■
Costello, D. (1938) Anat. Rec, 72, Abstracts p. 112. 85.
Cragg, F. W. (1920) Ifidianjf. nied. Res., 8, 32-79. 4.
Crane, R. K. & Keltch, A. K. (1949) jf. gen. Physiol., 32, 503-509- 78.
Dalcq, a. (1924) Arch. Biol., Paris, 34, 507-674. 98.

Dalcq, A. (1952) Initiatiofi aVembryologic generale. Masson & Cie, Paris. 2,3.
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ser. 3, 881-912. g8.
Daly, J. M. (1954) Arch. Biochem. Biophys., 51, 24-29. 68.
Dan, J. C. (1950a) Biol. Bull., Wood's Hole, 99, 399-411. 5, 7, 12.
Dan, J. C. (19506) Biol. Bull., Wood's Hole, 99, 412-415. 13, 14. 39-
Dan, J. C. (1952) Biol. Bull., Wood's Hole, 103, 54-66. 30.
Dan, J. C. (1954) Biol. Bull., Wood's Hole, 107, 203-218. 8.
Dan, J. C. (1955) Biol. Bull., Wood's Hole, 107, 335-349- 8, 30.
Dan, K. (1947) Biol. Bull., Wood's Hole, 93, 259-266. 134.
Davson, H. (1951) A Textbook of General Physiology. J. & A. Churchill Ltd.,

London. 130.
Donnellon, J. A. (1938) Physiol. ZooL, 11, 389-397. gg.
DoRFMAN, W. A. (1934) Protoplasma, 21, 245-257. 12S.
Doyle, W. L. (1938) J. cell. comp. Physiol., 11, 291-300. 8y.
Endo, Y. (1952) Exp. Cell Res., 3, 406-418. 6, 9, 10, 98, loj.
Ephrussi, B. (1933) Arch. Biol., Paris, 44, 1-148. 65.
Fankhauser, G. (1925) Arch. EntwMech. Org., 105, 501-580. 103, 104.
Fankhauser, G. (1932) y. exp. ZooL, 62, 185-235. 103, 104.
Fankhauser, G. (1934) y. exp. ZooL, 67, 159-215. 103, 104.
Fankhauser, G. (1948) Ann. N.Y. Acad. Sci., 49, 684-708. 103, 104.
Fankhauser, G. & Moore, C. (1941) y. Morph., 68, 347-385. 103.
Faure-Fremiet, E. (1913) Arch. ?riikr. Anat., 15, 437-757. 20.
Faure-Fremiet, E. (1922) C.R. Soc. Biol., Paris, 86, 20-23. 61.
Fenn, W. O. & Cobb, D. M. (1932) Amer.y. Physiol., 102, 379-401. ^j.
Florey, Sir H. (1955) Proc. roy. Soc. B, 143, 147-158. 96.
FoL, H. (1877) Arch. ZooL exp. gen., 6, 145-169. 7.
FoL, H. (1879) Mem. Soc. Phys. Geneve, 26, 89-250. 11, 18.
Folkes, B. F., Grant, R. A. & Jones, J. K. N. (1950) J. chem. Soc, Part III,

2 1 36-2 1 40. 38.
FoRSTER, H. von & WiESE, L. (1954) Z. Naturf., 9b, 470-471. 54.
Fox, H. M. (1921) y. gen. Physiol., 3, 501-51 1. 47.
Frank, J. A. (1939) Biol. Bull., Wood's Hole, 76, 190-216. 21, 34, 37.
Frankenhaeuser, B. & HoDGKiN, A. L. (1955) y. Physiol., 128, 40P-41P. 128.
Fruton, J. S. & SiMMONDS, S. (1953) General Biochemistry, John Wiley & Sons,

Inc., New York. v.
Fuchs, H. M. (1914) Arch. EntwMech. Org., 40, 157-252. 138.
Fuchs, H. M. (19 1 5) y. Genet., 4, 215-301. 138.
Galtsoff, P. S. (1940) Biol. Bull., Wood's Hole, 78, 117. 21.
Gatenby, J. B. & Hill, J. P. (1924) Quart, y. micr. Sci., 68, 229-238. 106.
Glaser, O. (191 3) Science, 38, 446-450. 20.
Glaser, O. (1914) Biol. Bull., Wood's Hole, 26, 84-91. 20.

146 FERTILIZATION

Glaser, O. (1921) Amer. Nat., 55, 368. 23, 38.

Glaser, O. (1924) Biol. Bull., Wood's Hole, 47, 274-283. 20.

GoDLEWSKi, Jun., E. (1912) Arch. EntwMech. Org., 33, 196-254. 2.

GoLDiNGER, J. M. & Barron, E. S. G. (1946) J. gen. Physiol., 30, 73-82. yS, 84.

GoLDSCHMiDT, R. & Katsuki, K. (1931) Biol. Zbl., 51, 58-74. 10$.

Gray, J. (1920) Proc. roy. Soc. B, 91, 147-157. 24.

Gray, J. (1927) Brit. J. exp. Biol., 5, 102-111. 11.

Gray, J. (1928) Brit. Jf. exp. Biol., 5, 362-365. 32.

Gray, J. (193 1) Experimental Cytology, University Press, Cambridge, v.

Gray, J. (1932) J. exp. Biol., 9, 277-299. 131.

Greenwood, A. W. & Blyth, J. S. S. (1951) Heredity, 5, 215-231. 105.

GusTAFsoN, T. & Hasselberg, I. (1950) Exp. Cell Res., i, 371-375. 86.

GusTAFSON, T. & HjELTE, M.-B. (1951) Exp. Cell Res., 2, 474-490. 8j.

Hagstrom, B. & Allen, R. D. (1956) Exp. Cell Res., to be published. 14, 117.

Hagstrom, B. & Hagstrom, B. (1954(7) Exp. Cell Res., 6, 479-484. 11 1.

Hagstrom, B. & Hagstrom, B. (1954&) Exp. Cell Res., 6, 491-496. J21.

Hagstrom, B. & Hagstrom, B. (1954c) Exp. Cell Res., 6, 532-534. 14, 137.

Hamilton, H. H. (1952) Lillie's Development of the Chick. Henry Holt & Co.,

New York. 103.
Hamilton, W J. & Day, F. T. (1945) J. Anat., London, 79, 127-130. J.
Harding, C. V. & Harding, D. (1952a) Ark. ZooL, 4, No. 3, 91-93. i37-
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Harding, D. (1949) Proc. Soc. exp. Biol., N.Y., 71, 14-15. 96.
Harding. D. (1951) Nature, Lofjd., 167, 355. 96.
Hartmann, M. (1944) Naturzvisseftschaften, 32, 231. 23.
Hartmann, M., Medem, F. Graf von, Kuhn, R. & Bielig, H.-J. (1947) Natur-

wissenschajten, I, 25. 33.
Hartmann, M. & Schartau, O. (1939) Biol. Zbl., 59, 571-587. 36.
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423- 33, 38.
Hartree, E. F. (1953) J. Amer. chem. Soc, 75, 6244-6249. 49.
Harvey, E. B. (1938) Biol. Bull., Wood's Hole, -js, 170-188. 8.
Harvey, E. B. (1941) Biol. Bull, Wood's Hole, 80, 354-362. 83.
Harvey, E. N. (1910) J. exp. ZooL, 8, 355-376. 98.
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Harvey, E. N. (193 i) Biol. Bull., Wood's Hole, 6l, 273-279. vi, 93.
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Hayashi, T. (1946) Biol. Bull., Wood's Hole, 90, 177-187. 33.
Hayes, F. R. (1938) Biol. Bull, Wood's Hole, 74, 267-277. 87.
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631. 84.
Heilbrunn, L. V. (1915) Biol. Bull., Wood's Hole, 29, 149-203. 94.
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Heilbrunn, L. V. (1952) An Outline of General Physiology. W. B. Saunders

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Abstracts, p. 32. 83.
Heilbrunn, L. V. & Wilbur, K. M. (1937) Biol. Bull., Wood's Hole, 73, 557-

564. 98.
Heilbrunn, L. V. & Wilson, W. L. (1948) Biol. Bull., Wood's Hole, 95, 57-68.

95, 96.
Henle, W., Henle, G. & Chambers, L. A. (1938) J. exp. Med., 68, 335-352-

23-

REFERENCES AND AUTHOR INDEX I47

Herlant, M. (1920) Arch. Biol., Paris, 30, 517-600. 99-102.

Hertwig, O. & Hertwig, R. (1887) Jeiw Z. Naturw., 20, 120-242. 116.

HiBBARD, H. (1928) Arch. Biol, Paris, ■3^,251-32.6. 36.

HiRAMOTO, Y. (1954) y^ip-J- Zool., 2, 227-243. II.

Hirst, G. K. (1942) 7- cxp. Med., 75, 49-64- 28.

HiSHiDA, T. & Nakano, E. (1954) Emhryologia, 2, 67-79. 78, 84.

HoBER, R. (1945) Physical Chemistry of Cells and Tissues. J. & A. Churchill Ltd.,
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HoBSON, A. D. (1928) Brit. J. exp. Biol, 6, 65-78. 20, 98.

HoBSON, A. D. (1932a) y. exp. Biol, 9, 69-92. 80, 100, loi.

HoBSON, A. D. (19326) y. cxp. Biol, 9, 93-106. 98.

HoDGKiN, A. L. & Huxley, A. F. (1945) J- Physiol., 104, 176-195- -^25.

HoDGKiN, A. L. & Huxley, A. F. (1952) J- Physiol., 117, 500-544. 127.

HoFF-JoRGENSEN, E. (1954) Recent Developments in Physiology. Butterworths
Scientific Publications, London. 83.

Hollander, W. F. (1949) J- Hered., 40, 271-278. 105.

HoLTER, H. (1936) y. cell. comp. Physiol., 8, 179-200. 87.

HoLTER, H. & Zeuthen, E. (1944) C.R. hah. Carlsberg, 25, 33-65- 57, 6-r.

HoLZER, W. (1933) Pfliig. Arch. ges. Physiol., 232, 835-841. 131.

HoRECKER, B. L. (1953) Brezv. Dig., 28, 214-219. 72.

Horowitz, N. H. (1940) y. cell. comp. Physiol., 15, 299-308. 65.

Horowitz, N. H. & Baumberger, J. P. (1941) J- biol. Chan., 141, 407-415. 67.

HoRSTADius, S. (1923) Arch. EntwMech. Org., 98, 1-9. 98.

HoRSTADius, S. (1939) Pubbl. Staz. zool. Napoli, 17, 221-312. 7.

HoRSTADius, S. & RuNNSTROM, J. (i953) Exp. Cell Res., 4, 468-476. iiS.

HuLTiN, E., ICriszat, G., Lindvall, S., Lundblad, G., Low, H., Runnstrom,
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HuLTiN, T. (1947) Pubbl. Staz. zool. Napoli, 21, 153-163. 34.

HuLTiN, T. (1948) Ark. Zool, 40A, No. 20. 137, 138.

HuLTiN, T. (1949) Ark. Kemi. Min. GeoL, 26A, No. 27. 98.

HuLTiN, T. (1950a) Exp. Cell Res., I, 159-168. 98.

HuLTiN, T. (19506) Exp. Cell Res., I, 272-283. 98.

HuLTiN, T. (1950c) Exp. Cell Res., i, 599-602. 76.

HuLTiN, T. ( 1 95 3a) Studies on the Structure and Metabolic Background of Fertili-
zation a7id Development. Stockholm. 73, 76.

HuLTiN, T. (19536) Ark. Kemi., 6, No. 15, 195-200. 79.

HuTCHENS, J. O., Keltch, A. K., Krahl, M. E. & Clowes, G. H. A. (1942) J.
gen. Physiol., 25, 717-731- 7^, 86, 87.

IiDA, T. T. (i943<3) y. Fac. Sci. Tokyo Univ., Sectfi. IV Zoology, 6, 141-151.
131, 132.

IiDA, T. T. (19436) y. Fac. Sci. Tokyo Univ., Sectn. IV Zoology, 6, 153-163.

131.
IiDA, T. T. (1943c) y. Fac. Sci. Tokyo Univ., Sectn. IV Zoology, 6, 165-173.

133, 134-
Immers, J. (1949) Ark. Zool., 42A, No. 6. 97.
Immers, J. & Vasseur, E. (1949) Experientia, 5, 124-125. 25.
Infantellina, F. & La Grutta, G. (1948) Arch. Sci. biol., 32, 85-106. 77, 90.
Ishida, J. (1954) y. Fac. Sci. Tokyo Univ., Sectn. IV Zoology, 7, 53-59- 37-
IsHiKAWA, M. (1954) Embryologia, 2, 57-62. 80, 81.

Jandorf, B. J. & Krahl, M. E. (1942) j- gen. Physiol., 25, 749-754. 70, 73,84.
Jordan, E. O. (1893) y. Morph., 8, 269-366. J03.
Just, E. E. (1914) Biol. Bull., Wood's Hole, 27, 201-212. 2.
Just, E. E. (1919) Biol. Bull., Wood's Hole, 36, i-io. 9.
Just, E. E. (1928) Physiol. Zool., I, 26-36. loi.

148 FERTILIZATION

Just, E. E. (1929) Biol. Bull., Wood's Hole, si, 311-325. 7.

Just, E. E. (1939) The Biology of the Cell Surface. The Technical Press Ltd.,

London. 38.
Kacser, H. (1955) J. exp. Biol., 32, 451-467. 8, 108, iig.
Kavanau, J. L. (1953) y. exp. ZooL, 122, 285-337. y6-y8.
Kavanau, J. L. (1954a) Exp. Cell Res., 6, 563-566. 76-78.
Kavanau, J. L. (19546) Exp. Cell Res., 7, 530-557. 62, 76-78.
Keilin, D. & Hartree, E. F. (1939) Proc. roy. Soc. B, 127, 167-191. 67.
Keltch, a. K., Strittmatter, C. P., Walters, C. P. & Clowes, G. H. A.

(1950) y. gen. Physiol., 33, 547-553- 78.
Krahl, M. E., Jandorf, B. J. & Clowes, G. H. A. (1942) y. gen. Physiol., 25,

733-747- 7^, 84.
Krahl, M. E., Keltch, A. K., Neubeck, C. E. & Clowes, G. H. A. (1941) y.

gen. Physiol., 24, 597-617. 67.
Krahl, M. E., Keltch, A. K., Walters, C. P. & Clowes, G. H. A. (1953) Biol.

Bull., Wood's Hole, 105, 377. 70.
Krahl, M. E., Keltch, A. K., Walters, C. P. & Clowes, G. H. A. (1954a)

Biol. Bull., Wood's Hole, X07, 315-316. 70.
Krahl, M. E., Keltch, A. K., Walters, C. P. & Clowes, G. H. A. (19546) J.

gen. Physiol., 3S, 31-39. 70, 8g.
Krahl, M. E., Keltch, A. K., Walters, C. P. & Clowes, G. H. A. (1955) y.

gen. Physiol., 38, 431-439. 71.
Krauss, M. (1950) Science, 112, 759. 37.

Krugelis, E. J. (1950) C.R. Lab. Carlsberg, 27, 273-290. 86.
KuHN, R. & Low, I. (1949a) Chem. Ber., 82, 474-479. 51.
Kuhn, R. & L'^iw, L (19496) Chem. Ber., 82, 479-481. 51.
KuPELWiESER, H. (1909) Arch. EntivMech. Org., 27, 434-462. 139.
KusA, M. (1953) Annot. zool. jap., 26, 72-77- 6,80.
KusA, M. (1954) Amjot. zool.jap., 27, 1-6. 6.
KusHNER, Kh. (1954) Izvestiya Akadetnii Nauk SSSR (Ser. biol.), No. i, 32-52.

122, 123.
Laser, H. & Rothschild, Lord (1939) Proc. roy. Soc. B, 126, 539-557. 63-6^=^.
Lasley, J. F., Easley, G. T. & McKenzie, F. F. (1942) Anat. Rec, 82, 167-174.

log.
Lewis, D. (1954) Advanc. Genet., 6, 235-285. 53.
Lillie, F. R. (1912a) Science, 36, 527-530. 21.
LiLLiE, F. R. (19126) y. exp. ZooL, 12, 413-478. i, 12.
Lillie, F. R. (1914) y. exp. ZooL, 16, 523-590. 34.
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Chicago. 6, 23, 31, 38, 139, 140.
Lillie, R. S. (1916) Amer. y. Physiol., 40, 249-266. 80.
Lillie, R. S. ( i 924) General Cytology (ed. E. V. Cowdry). University of Chicago

Press, Chicago. 124.
Lindahl, p. E. (1938) Naturzvissenschaften, 26, 709-710. 68.
LiNDAHL, p. E. (1940) Ark. Kemi. Min. GeoL, 14A, No. 12. 67.
Lindahl, P. E. & Runnstrom, J. (1929) Acta zooL, Stockh., 10, 401-484. 158.
LiNDBERG, O. (1943) Ark. Keitii. Min. GeoL, i6a. No. 15. 87.
Lindberg, O. (1945) Ark. Kemi. Min. GeoL, 20H, No. i. 86, 87.
LiNDBERG, O. (1948) Ark. Kemi. Min. GcoL, 26n, No. 13. 82.
Lindberg, O. & Ernster, L. (1948) Biochim. biophys. Acta, 2, 471-477. 6g, 71,

73.
Linderstr/)m-Lang, K. & Holter, H. (1933) Z. physiol. Chem., 215, 167-178.

87.
Lindvall, S. & Carsjo, A. (1948) Ark. Kemi. Min. GeoL, 26b, No. 12. 87.

REFERENCES AND AUTHOR INDEX I49

LiNDVALL, S. & Carsjo, A. (1951) Exp. Cell Res., 2, 491-498. 83, 8g.

LiNDVALL, S. & Carsjo, A. (1954) Ark. Kenii., 7, 17-27. 88.

LOEB, J. (1903) Pfliig- Arch. ges. Physiol., 99, 323-356. 136.

LoEB, J. (1913) Artificial Parthenogenesis and Fertilization. University of

Chicago Press, Chicago, i.
LoEB, J. (1914) Science, 40, 316-318. 98.

LoEB, J. & Wasteneys, H. (1913) Arch. EntwMech. Org., 35, 555-557. 56.
Lovelace, R. (1949) Biol. Bull., Wood's Hole, 97, 259. 85.
LucKE, B., Hartline, H. K. & McCutcheon, M. (1931) y. gen. Physiol., 14,

405-419. 80.
LucKE, B. & McCutcheon, M. (1932) Physiol. Rev., 12, 68-139. *o.
LuNDBERG, A. (1956) Exp. Cell Res., to be published. 130, 131.
LuNDBLAD, G. (1949) Nature, Lond., 163, 643. 87-88.
LuNDBLAD, G. (1950) E.xp. Cell Res., i, 264-271. 87-88.
LuNDBLAD, G. (1954a) Ark. Kemi., 7, 127-157. 87-88.
LuNDBLAD, G. (1954&) Ark. Kemi., 7, 159-167. 87-88.
LuNDBLAD, G. (19540 Ark. Kemi., 7, 169-180. 87-88.
LuNDBLAD, G. (1954^) Proteolytic Activity in Sea-Urchin Gametes. Uppsala.

87-88.
LuNDBLAD, G. & HuLTiN, E. (1954) Exp. Cell Res., 6, 249-250. 88.
LuNDBLAD, G. & LuNDBLAD, I. (1953) Ark. Kemi., 6, 387-415. 87-88.
Manery, J. F. & Irving, L. (1935) J- cell. comp. Physiol., 5, 457-464. 84.
Mann, T. (1951) Biochem. Soc. Symposia, No. 7, 11-21. 6g.
Mann, T. (1954) Biochemistry of Semen. Methuen, London. 33.
Marshak, a. & Fager, J. (1950) J. cell. comp. Physiol., 35, 317-329. 85.
Marsland, D. a. (1939) y. cell. comp. Physiol., 13, 15-22. loi.
Martin, G. J. & Beiler, J. M. (1952) Science, 115, 402. 54.
Mathews, A. P. (1913) y. biol. Chem., 14, 465-467. 75.
Mazia, D. (1937) y. cell. comp. Physiol., 10, 291-304. 83.
Mazia, D., Blumenthal, G. & Benson, E. (1948) Biol. Bull., Wood's Hole, 95,

250-251. 86.
Medem, F. Graf von (1942) B/o/. Z6/., 62, 431-446. 23.
Medem, F. Graf von, Rotheli, A. & Roth, H. (1949) Schzveiz. Z. Hydrol., 11,

361-377. 23.
Metz, C. B. (1945) Biol. Bull., Wood's Hole, 89, 84-94. 29.
Metz, C. B. (1949) Proc. Soc. exp. Biol., N.Y., 70, 422-424. 34-
Metz, C. B. & Donovan, J. (1951) Biol. Bull., Wood's Hole, loi, 202. 30.
Meves, F. (1912) Arch. mikr. Anat., 80, 81-123. -^^•
MiNGANTi, A. (1951) Puhbl. Staz. zool. Napoli, 23, 58-65. 23.
MiRSKY, A. E. (1936) Science, 84, 333-334. 88.
Mitchison, J. M. (1953) Exp. Cell Res., 5, 536-538. 9.
MiTCHisON, J. M. (1956) Quart, y. micr. Sci., to be published. g2.
Mitchison, J. M. & Swann, M. M. (1952) y. exp. Biol., 29, 357-362. 91, loi,

102.
Mitchison, J. M. & Swann, M. M. (1953) Quart, y. micr. Sci., 94, 381-389.

II, 90.
Mitchison, J. M. & Swann, M. M. (1954a) y. exp. Biol., 31, 443-460. 92, 93,

100, 133.
Mitchison, J. M. & Swann, M. M. (19546) y. exp. Biol., 31, 461-472. vi, 92,

93.
Mitchison, J. M. & Swann, M. M. (1955) J- exp. Biol., 32, 734-750. 92-95,

lOI.

MoEWUS, F. (1949) Portug. acta biol., Ser. A (R. B. Goldschmidt Volume), 161-
199. 51.

150 FERTILIZATION

MoEWUS, F. (1950) Biol. ZbL, 69, 181-197. 51, 54-

MoNNE, L. & Harde, S. (195 i) Ark. ZooL, i, 487-498. 6.

MoNROY, A. (1945) Experientia, I, 335-336. loi.

MoNROY, A. (1948) Ark. ZooL, 40A, No. 21. 8, 37.

Monro Y, A. (1950) Exp. Cell Res., i, 92-104. 8g.

MoNROY, A. (1954) Pubbl. Staz. zool. Napoli, 25, 188-197. 20.

MoNROY, A. & Monroy-Oddo, a. (1951) J- gen. Physiol., 35, 245-253. 8g.

MoNROY, A. & MoNTALENTi, G. (i947) Biol. Bull., Wood's Hole, 92, 151-161.

JOO, lOI.

MoNROY, A. & RuFFO, A. (1945) Boll. Soc. ital. Biol, sper., 20, 406-407. 75, 87.

MoNROY, A. & RuFFO, A. (1947) Nature, Land., 159, 603. 36, 37.

MoNROY, A. & RuNNSTROM, J. (1950) Biol. Bull., Wood's Hole, 99, 339. 35.

MoNROY, A. & Tosi, L. (1952) Experientia, 8, 393-394- 37-

MoNROY, A., Tosi, L., GiARDiNA, G. & Maggio, R. (1954) Biol. Bidl., Wood's
Hole, 106, 169-177. 26, 30, 31.

Monroy-Oddo, A. (1946) Experientia, 2, 371-372. 83.

Monroy-Oddo, A. & Esposito, M. (1951) J- gen. Physiol, 34, 285-293. 83.

Moore, A. R. (1937) Protoplasma, 27, 544-551. 18.

Morgan, L. V. (1929) Carnegie Inst. Washington Pub., No. 399, 223-296. 105.

Morgan, T. H. (1927) Experimental Ejubryology. Columbia University Press,
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Morgan, T. H. & Tyler, A. (1930) Biol. Bull, Wood's Hole, 58, 59-73. ^9-

Moser, F. (1939a) J. exp. ZooL, 80, 423-445. 6, 107.

MosER, F. (1939^) J. exp. ZooL, 80, 447-471. 98.

MoTOMURA, I. (1936) Zool. Mag., Tokyo, 48, 753-758. 9-

Motomura, I. (1941) Sci. Rep. Tohoku Univ., 4th Ser., B, 16, 345-363. 9-

MoTOMURA, I. (1950) Sci. Rep. Tohoku Univ., 4th Ser., B, 18, 561-570. 10, 24.

Motomura, I. (1953a) Sci. Rep. Tohoku Univ., 4th Ser., B, 20, 93-97. 24.

MoTOMURA, I. (19536) Exp. Cell Res., 5, 187-190. 24.

Motomura, I. (1954) Sci. Rep. Tohoku Univ., 4th Ser., B, 20, 158-162. 10.

Des Moulins, C. (1835-37) Act. Soc. linn. Bordeaux, 3, 126. 154.

MuDD, E. B. H., Mudd, S. & Keltch, A. H, (1929) Proc. Soc. exp. Biol., N.Y.,
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Nakano, E. (1953) Embryologia, 2, 21-31. 62.

Nakano, E. (1954) Jap. J. ZooL, II, 245-251. 113.

Nakano, E. & Ohashi, S. (1954) Etnbryologia, 2, 81-86. 26.

Needham, J. (1931) Chemical Embryology, University Press, Cambridge. 100.

Needham, J. (1942) Biochemistry and Morphogenesis, University Press, Cam-
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Nelson, T. C. (i94i) Atiat. Rec, 81, Abstracts, p. 88. 21.

Nelson, T. C. & Allison, J. B. (1937) Anat. Rec, 70, Abstract p. 124. 21.

NoviKOFF, A. B. (1939) y. exp. ZooL, 82, 217-237. 6.

Numanoi, H. (i953rt) Sci. Papers Coll. Gen. Ed. Univ. Tokyo, 3, 55-65. 8g.

NuMANOi, H. (19536) Sci. Papers Coll. Gen. Ed. Univ. Tokyo, 3, 193-200. 8g.

Ohman, L. O. (1940) Ark. ZooL, 32A, No. 15. 65.

Ohman, L. O. (1942) Naturzvissenschaftcn, 30, 240. 74.

Ohman, L. O. (1945) Ark. ZooL, 36A, No. 7. 74, 75, 86, 87, loi.

Okkelherg, p. (1914) Biol. Bull., Wood's Hole, 26, 92-99. 5.

Oppel, a. (1892) Arch. ?>iikr. Anat., 39, 215-290. 103.

Orstrom, a. (1941) Z. physiol. Chem., 271, 1-176. 75, 76, 86.

Orstrom, a. & Lindherg, O. (1940) Enzymologia, 8, 367-384. 70, 73, 74, 82,

84,87. „
Orstrom, A. & Orstrom, M. (1942) Protoplasma, 36, 475-490. 83.
OsTERHOUT, W. J. V. (1950) Biol. BulL, Wood's Hole, 99, 362. 36.

REFERENCES AND AUTHOR INDEX 151

OsTERHOUT, W. J. V. (1952) Biol. Bull, WoocVs Hole, 103, 305-306. 36.

OsTERHOUT, W. J. V. (1953) Biol. Bull., Wood's Hole, 105, 379-380. j6.

Page, I. H. (1923) J- biol. Chem., 57, 471-476. 75.

Page, I. H. (1929) Brit. J. exp. Biol., 6, 219-228. loi.

Panijel, J. (1947) Btdl. Soc. Chim. bioL, Paris, 29, 1098-1 106. 85.

Panijel, J. (1951) Metabolisme des Nucleoproteiiies. Hermann & Cie, Paris. 8y.

Parat, M. (1933) C.R. Soc. Biol, Paris, 112, 1134-1137. 56.

Parpart, a. K. (1941) Biol. Bull, Wood's Hole, 81, 296. 87.

Pasteels, J. J. (1935) Arch. Biol, Paris, 46, 229-262. 98.

Pasteels, J. (1938) Trav. Sta. zool. Wimereux, 13, 515-530- 9'^.

Pasteels, J. (1948) Arch. Biol., Paris, 59, 405-446. 85.

Pasteels, J. (1950) Arch. Biol., Paris, 61, 197-220. 10.

Pasteels, J. & Lison, L. (1951) Nature, Lond., 167, 948-949. 85.

Pauling, L., Campbell, D. H. & Pressman, D. (1943) Physiol. Rev., 23, 203-

219. 137-
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P^TERFi, T. & Rothschild, V. (1935) Nature, Lo?jd., 135, 874-875. 128.
Pfeffer, W. (1884) Unt. Bot. Inst. Tubingen, I, 363-481. 39, 47-
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PiCKEN, L. E. R. & Rothschild, Lord (1948) j. exp. BioL, 25, 227-236. 81.
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Popa, G. T. (1927) Biol. Bull., Wood's Hole, 52, 238-257. Jo.
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8-9. 21.
Race, R. R. (1944) Nature, Lond., 153, 771-772. 29.
Richards, A. G. & Miller, A. (1937) J-N.Y. ent. Soc, 45, 1-60. 103.
Robbie, W. A. (1946) J. cell. comp. Physiol., 28, 305-324. 67.
Roth, J. S. (1953) Nature, Lond., 171, 127-128. 88.
Rothschild, Lord (1938) J- exp. Biol, 15, 209-216. 129.
Rothschild, Lord (1939) J- exp. Biol, 16, 49-55- "56.
Rothschild, Lord (1946) J. exp. Biol., 23, 77-99- 131, i35-
Rothschild, Lord (1948) J- exp. Biol., 25, 344-352. 37, 38.
Rothschild, Lord (1949a) J. exp. Biol., 26, loo-iii. 66, 67.
Rothschild, Lord (1949^) J- exp. Biol., 26, 177-181. 118.
Rothschild, Lord (1950) jf. exp. Biol., 26, 410-411. 90.
Rothschild, Lord (1951) Biol. Rev., 26, 1-27. 34, 38, 41-
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Rothschild, Lord (1953) J- exp. Biol, 30, 57-67. i, 18, 117.
Rothschild, Lord & Barnes, H. (1953) J- exp. Biol, 30, 534-544. 129.
Rothschild, Lord & Cleland, K. W. (1952) J- exp. Biol, 29, 66-67. ^2'
Rothschild, Lord & Swann, M. M. (1949) J- exp. Biol, 26, 164-176. 6, 107,

108, 117.
Rothschild, Lord & Swann, M. M. (1951) J. exp. Biol, 28, 403-416. 30, no,

III.
Rothschild, Lord & Swann, M. M. (1952) J. exp. Biol, 29, 469-483. 112, 120.
Rothschild, Lord & Tyler, A. (1954) J- exp. Biol, 31, 252-259. 31.
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Rudenberg, F. H. N. (1953) Exp. Cell Res., 4, 116-126. 83.
Runnstrom, J. (1924) Acta, zool, Stockh., 5, 345-392. loi.

152 FERTILIZATION

RuNNSTRoM, J. (1928) Protoplasma, 4, 388-514. gi, loy.
RuNNSTROM, J. (1930) Protoplasma, 10, 106-173. 5^, 67,
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RuNNSTROM, J. (1935a) Biol. Bull., Wood's Hole, 68, 327-334. 58, 85.
RuNNSTROM, J. (19356) Biol. Bull., Wood's Hole, 69, 345-350. 34.
RuNNSTROM, J. (1949) Advanc. Emymol., 9, 241-327. vii, 35, 74.
RuNNSTROM, J. (195 1) Harvey Lectures, Series 46, 1 16-152. 10.
RuNNSTROM, J. (1952) Syrup. Soc. exp. Biol., 6, 39-88. 10, 35.
RuNNSTROM, J. & Kriszat, G. (1950) Exp. Cell Res., i, 355-370. gy.
RuNNSTROM, J. & Kriszat, G. (1952) Exp. Cell Res., 3, 419-426. 118.
RuNNSTROM, J., LiNDVALL, S. & TiSELius, A. (1944a) Nature, Lond., 153, 285.

38.
RuNNSTROM, J., MoNNi, L. & Broman, L. (19446) Ark. Zool., 35A, No. 3. gi,

93, loi, i3y.

RuNNSTROM, J., MoNNt, L. & WiCKLUND, E. (1946) J. Colloid Sci., I, 421-452.

91-

RUNNSTROM, J., TiSELIUS, A. & LiNDVALL, S. (1945) Ark. ZooL, 36A, No. 22.

36.

RuNNSTROM, J., TiSELIUS, A. & Vasseur, E. (1942) Ark. Kemi. Min. GeoL, 15A,

No. 16. 24, 35.
Rybak, B. (1949) Bull. Soc. Chim. biol., Paris, 31, 464-484. 30.
Sampson, M. M. (1922) Biol. Bull., Wood's Hole, 43, 267-284. 2g.
Sampson, M. M. (1926a) Biol. Bull., Wood's Hole, 50, 202-206. 120.
Sampson, M. M. (19266) Biol. Bull., Wood's Hole, 50, 301-338. 120.
Schartau, O. & Montalenti, G. (1941) Biol. Zbl., 61, 473-478. 23.
SCHEER, B. T. (1945) Science, lOl, 645-646. 8g.
Scheer, B. T., Monroy, A., Santangelo, M. & Riccobono, G. (1954) Exp.

Cell Res., 7, 284-287. 128, 130.
Scheer, B. T. & Scheer, M. A. R. (1947) Physiol. Zool., 20, 15-32. 8g.
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Serine, T. (1951) J. Biochem., Tokyo, 38, 171-179. 8g.
Sexton, W. A. (1949) Chet?iical Constitution and Biological Activity. E. & F. N.

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Shapiro, H. (1939) Biol. Bidl., Wood's Hole, 77, 317-318. 81.
Shapiro, H. (1948) J. gen. Physiol., 32, 43-51. 80.
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Shettles, L. B. (1954) Trans. New York Acad. Sci., 17, 99-102. 12, 13.
Shibata, K. (1911) Jb. wiss. Bot., 49, i. 46.
Sieve, B. F. (1952) Science, 116, 373-385. 54-
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Smithberg, M. (1953) Anat. Rec, 117, Abstracts p. 554. 12.
Southwick, W. E. (1939) Biol. Bidl., Wood's Hole, 77, 147-156. 3y.
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78.
Spikes, J. D. (1949a) Biol. Bull., Wood's Hole, 97, 95-99. 2g.
Spikes, J. D. (19496) Amer. Nat., 83, 285-301. 33.
Stefanelli, A. (1938) Arch. Sci. biol., 24, 411-441. 61.
Steinert, M. (1952) Bull. Soc. Chim. biol., Paris, 34, 923-936. 86.
Stricht, O. van der (1902) Verh. anat. Ges. Jena, 21, 163-168. 12.
Stricht, O. van der (1923) Arch. Biol., Paris, 33, 229-300. 2.
Sugiyama, M. (1951) Biol. Bull., Wood's Hole, loi, 335-344. 119, 120.
Sugiyama, M. (1953) Biol. Bull., Wood's Hole, 104, 210-215. 80.
Swann, M. M. (1952) Synip. Soc. exp. Biol., 6, 89-104. loi.

REFERENCES AND AUTHOR INDEX I53

Swift, H. (1953) Int. Rev. Cytology, 2, 1-76. 85.

TuzET, O, (1950) Vie et Milieu, i, 163-177. v, 123.

Tyler, A. (1932) J. exp. ZooL, 63, 155-173. ^9- PI- H-

Tyler, A. (1939) Proc, nat. Acad. Sci., Wash., 25, 317-323. 2J, 34, 37.

Tyler, A. (1940a) Proc. nat. Acad. Sci., Wash., 26, 249-256, 21, 29, 34.

Tyler, A. (19406) Biol. Bull, Wood's Hole, 78, 159-178. 23, 26. PI. III.

Tyler, A. (i94i«) Biol. Bull., Wood's Hole, 81, 190-204. 28-30.

Tyler, A. (19416) Biol. Rev., 16, 291-336. v.

Tyler, A. (1942a) West. J. Surg., 50, 126-138. 23, 28.

Tyler, A. (19426) Proc. nat. Acad. Sci., Wash., 28, 391-395- 30.

Tyler, A. (1945) J. Immunol., 51, 157-172. 29.

Tyler, A. (1946) Biol. Bull., Wood's Hole, 90, 213-219. 26.

Tyler, A. (1948) Physiol. Rev., 28, 180-219. 26-28, 33, 34.

Tyler, A. (1949) Amer. Nat., 83, 195-219. 24-26, 35, 138.

Tyler, A. (1953) Biol. Bull., Wood's Hole, 104, 224-239. J-t.

Tyler, A., Burbaxk, A. & Tyler, J. S. (1954) Biol. Bull., Wood's Hole, 107,

303; 304. 25.
Tyler, A. & Fox, S. W. (1939) Science, 90, 516-517. 23.
Tyler, A. & Fox, S. W. (1940) Biol. Bull., Wood's Hole, 79, 153-165. 23, 23.
Tyler, A. & Humason, W. D. (1937) Biol. Bull., Wood's Hole, 73, 261-279.

56, 61, 62, 77, 82.
Tyler, A. & Metz, C. B. (1945) J. e.xp. ZooL, 100, 387-406. 27.
Tyler, A. & O'Melveny, K. (1941) Biol. Bull., Wood's Hole, 8i, 364-374. 34,

37-
Tyler, A., Ricci, N. & Horowitz, N. H. (1938) J. exp. ZooL, 79, 129-143.

59, 60.
Tyler, A. & Schultz, J. (1932) J. exp. ZooL, 63, 509-530. iig.
Ullyott, p. (1936) y. exp. Biol., 13, 265-278. 40.
Vasseur, E. (1948) Acta chem. scand., 2, 900-913. 26.
Vasseur, E. (1950) Acta chem. scand., 4, 1144-1145. 26.

Vasseur, E. (195 1 ) Acta Borealia, A. Scientia No. 2, Tromso Museum. 23,37.
Vasseur, E. (1952) Emil Kihlstrom's Tryckeri A.-B. Stockholm. 24-26, 28,

30, 33-
Vasseur, E. & Hagstrom, B. (1946) Ark. ZooL, 37A, No. 17. 37.
Vasseur, E. & Immers, J. (1949) Ark. Kemi., i, 39-41. 26.
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70.
Whiteley, A. H. (1949) Amer. Nat., 83, 249-267. 83.
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Company, New York. 4, 15, 16.

154 FERTILIZATION

Wilson, W. L. (1951) J. cell. comp. Physiol., 38, 409-415. 95, 10 1.

Yamamoto, T. (1944) Amiot. zool.jap., 22, 109-136. 6.

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87.

INDEX OF PLANTS AND ANIMALS

The authors of new combinations, and dates, have been omitted; for example,
Limnatis nilotico (Savigny, 1820) Moquin-Tandon, 1826, would be abbreviated
to Limnatis nilotica (Savigny). When an author's name is in parentheses after
the Latin name of an organism, that author was the first to describe the organism,
but under a different name, which has been discarded. Synonyms are restricted
to those which occur in gametological papers.

NAME OF ORGANISM, SYNONYMS AND COMMON
NAME, WHEN KNOWN

(i) Amblystoma mexicanum (Shaw), see (125)

(2) Amphioxus lanceolatus (Pallas), see (17)

(3) Anthocidaris crassispina (A. Agassiz)

Heliocidaris crassispifia (A. Agassiz)
Heliocidaris tuberculata (A. Agassiz)
Strongylocentrotus tuberculata A. Agassiz
Sea-urchin

(4) Arbacia aequituberculata (de Blainville), see

(S)

(5) Arbacia lixula (Linn.)

Arbacia pustulosa (Leske)

Arbacia aequituberculata (de Blainville)

Echinus aequituberculatus de Blainville *

Sea-urchin

(6) Arbacia punctulata (Lamarck)

Sea-urchin

CLASS, ORDER,
ETC.

Echinoidea
Diadematoida

PAGES

12, 26, 134.

Echinoidea vi, 22, 24, 26,
Diadematoida 30,31,33,83,
87, 89.

Echinoidea vi, 6, 10, 20,
Diadematoida 24, 25, 27, 30,
56,59-61,71,
73,78,80,81,
83-89,95,98,
99, 107, 116,
131.

Phasmida 11,20,85,87.
Rhabditida

(7) Arbacia pustulosa (Leske), see (5)

(8) Ascaris equorum Goeze

Parascaris equorum (Goeze)
Ascaris megalocephala Cloquet
Horse Round-worm

(9) Ascaris megalocephala Cloquet, see (8)

(10) Ascidiella aspersa (O. F. Miiller) Ascidiacea 19, 20.

Sea-squirt Enterogona

(11) Asterias forbesi (Desor) Asteroidea 82,131.

Starfish Forcipulata

(12) Asterias glacialis Linn., see (70)

(13) Asterias miniata Brandt, see (93)

(14) Asterina pectinifer (J. Miiller & Troschel),

see (94)

* Echinus equituherculatus de Blainville, 1825, p. 86, and Echinus aequitubercu-
latus Des Moulins, 1837 p. 126, are synonyms for Sphaerechinus granularis
(Lamarck). Echinus aequituberculatus de Blainville, 1825 p. 76, and Echinus
equituberculatus de Blainville, 1834 p. 226, are synonyms for Arbacia lixula
(Linn.).

L 15s

156

FERTILIZATION

NAME OF ORGANISM, SYNONYMS AND COMMON
NAME, WHEN KNOWN

(15) Balanoglossus kowalevskii A. Agassiz, see

(117)

(16) Barnea Candida (Lamarck)

White Piddock, Angel's Wing

(17) Branchiostoma lanceolatum (Pallas)

Amphioxus lanceolatus (Pallas)
Lancelet

(18) Brissopsis lyrijera (Forbes)

Heart-urchin

(19) Bufo biifo (Linn.)

Bufo vulgaris Laurenti
Toad

(20) Bufo vulgaris Laurenti, see (19)

(21) Cerebratuliis lacteus Verrill

Ribbon Worm, Proboscis Worm

(22) Chaetopterus pergamentaceus Cuvier, see (23)

(23) Chaetopterus variopedatus (Renier)

Chaetopterus pergamentaceus Cuvier
Tube-worm

(24) Chlamydomonas eugametos Moewus

(25) Cimex lectularius Linn.

Bed-bug

(26) Ciona fascicularis Hancock, see (27)

(27) Ciona intestinalis (Linn.)

Ciona fascicidaris Hancock
Sea-squirt

(28) Clypeaster japonicus Ddderlein

Sand-dollar, Cake-urchin

(29) Crepidula plana Say

Flat Slipper Limpet, Boat shell

(30) Ciimingia tellinoides (Conrad)

Clam

(31) Cynthia partita Stimpson, see (137)

(32) Dendrastcr excentricus (Eschscholtz)

Sand-dollar

(33) Diemictylus palniatus (Schneider), see (153)

(34) Diemictylus viridescens (Rafinesque)

Molgc viridescens (Rafinesque)
Triturus viridescens Rafinesque
Triton viridescens (Rafinesque)

Newt

(35) Discoglossus pictus Otth

Painted Toad

(36) Dolichoglossus kowalevskii (A. Agassiz), see

(117)

(37) Echinarachnius parma (Lamarck)

Sand-dollar

class, order, pages

etc.

Bivalvia 98.

Eulamellibranchiata

Cephalochordata 2, ii.

Echinoidea
Spatangoida

Amphibia
Salientia

25, 87.
61.

Anopla
Heteronemertea

S-

Polychaeta 10,
61,

20, 56, 58,
81,95,96.

Chlorophyceae
Volvocales

52, 54.

Insecta
Hemiptera

Ascidiacea 57, 61, 138.
Enterogona

Echinoidea
Clypeastroida

6, 10, 89.

Gastropoda 105.
Mesogastropoda

Bivalvia 3,19,29,56,
Eulamellibranchiata 61, 95.

Echinoidea
Clypeastroida

138, 139-

Amphibia
Caudata

103, 104.

Amphibia
Salientia

36.

Echinoidea
Clypeastroida

26, 87.

INDEX OF PLANTS AND ANIMALS

Marsipobranchii
Hyperoartia

NAME OF ORGANISM, SYNONYMS AND COMMON CLASS, ORDER,

NAME, WHEN KNOWN ETC.

(38) Echinocardium cordatum (Pennant)

Heart-urchin

(39) Echinus aequituberculatus de Blainville, see

(5)

(40) Echinus brevispinosus Risso, see (126)

(41) Echinus droebachiensis O. F. Miiller, see (129)

(42) Echinus equituberculatus de Blainville, see

(126)

(43) Echinus esculentus Linn.

Sea-urchin

(44) Echinus miliaris P. L. S. Miiller, see (104)

(45) Entosphenus lamottenii (Lesueur)

Petromyzon appendix De Kay
Entosphetms uilderi (Gage)
Lampetra wilderi Gage
Brook-lamprey

(46) Entosphenus wilderi (Gage), see (45)

(47) Equisetum arvense Linn.

Field Horsetail

(48) Forsythia X intermedia Zabel

Golden Bell

(49) Fucus serratus Linn.

Serrated Sea-weed, Serrated Wrack

(50) Fucus spiralis Linn.

Sea-weed

(51) Fucus vesiculosus Linn.
Twin-bladder Wrack, Sea-ware

(52) Fundulus heteroclitus (Linn.)

KiLLIFISH

(53) Gymnogramma sulphurea (Schwartz), see

(98)

(54) Heliocidaris crassispina (A. Agassiz), see (3)

(55) Heliocidaris tuberculata (A. Agassiz), see (3)

(56) Hemicentrotus pulcherrimus (A. Agassiz)

Psammechinus pulcherrimus A. Agassiz
Strongylocentrotus pulcherrimus (A. Agassiz)
Sea-urchin

(57) Hipponoe esculenta A. Agassiz, see (148)

(58) Hydroides norvegicus Gunnerus

(59) Hydroides uncinatus (Philippi)

(60) Hynobius nebulosus (Schlegel) ^

(61) Jsoetes japomca A. Braun

QmLLWORT

(62) Lampetra wilderi Gage, see (45)

PAGES

Echinoidea 8,24,26,30,35,
Spatangoida 74, 86, 87, 137.

Echinoidea 20,26,33,71,
Diadematoida 83,87,89,90.

5, 20.

Pteridophyta
Equisetales

43-46, 48-50.

Angiospermae
Contortae

51-54-

Phaeophyceae
Fucales

47.

Phaeophyceae
Fucales

47.

Phaeophyceae
Fucales

47, 56, 61.

Pisces

61-63, 68.

Microcyprini

Echinoidea 24, 26, 80, 81,
Diadematoida 89, 119, 120,
131-

Polychaeta

20,

Polychaeta

98,

Amphibia
Caudata

128.

Pteridophyta
Isoetales

46, 48-50

158

FERTILIZATION

NAME OF ORGANISM, SYNONYMS AND COMMON
NAME, WHEN KNOWN

(63) Lepidochitona cinerea (Linn.)

Grey Mail Shell

(64) Lycopodium davatum Linn.

Club-moss

(65) Lymnaea stagnalis (Linn.)

Great Pond Snail

(66) Lytechinus pictiis (Verrill)

Sea-urchin

(67) Lytechinus variegatus (Lamarck)

Toxopneustes variegatus (Lamarck)
Sea-urchin

(68) Mactra laterialis Say, see (76)

(69) Mactra solidissima (Dillwyn), see (128)

(70) Marthasterias glacialis (Linn.)

Aster i as glacialis Linn.
Starfish

(71) Megathura crenulata (Sowerby)

Giant (Great) Keyhole Limpet

(72) Melanopus dijferentialis (Thomas)

Grasshopper

(73) Mespilia globulus (Linn.)

Sea-urchin

(74) Molge palmata (Schneider), see (153)

(75) Molge viridescens (Rafinesque), see (34)

(76) Mulinia laterialis (Say)

Mactra laterialis Say
Dwarf Surf Clam

(77) Mus musculus Linn.

House Mouse

(78) Mytilus edulis (Linn.)

Edible Mussel

(79) Nereis limhata Ehlers, see (80)

(80) Nereis succinea (Leuckart)

Nereis limbata Ehlers

(81) Nyctalus noctula (Schreber)

Vespertilio noctula Schreber
Vesperugo noctula (Schreber)
Common Noctule

(82) Oncorhynchus keta (Walbaum)

Dog Salmon, Chum

(83) Ornithorhyncus anatinus (Shaw & Nodder)

Platypus anatinus Shaw & Nodder
Ornithorhynchus paradoxus Blumenbach
Duck-billed Platypus

(84) Ornithorhynchus par adoxus'QXumQnhach., see

(83)

class, order, pages

ETC.

Loricata 8.

Lepidopleurida

Pteridophyta 50.

Lycopodiales

Gastropoda 103.

Basommatophora

Echinoidea 24,29,35,71,
Diadematoida 82, 83, 138.

Echinoidea
Diadematoida

IS-

Asteroidea
Forcipulata

61, 98.

Gastropoda 5, 26, 37.

Archaeogastropoda

Insecta
Orthoptera

85.

Echinoidea
Diadematoida

Bivalvia
EulamelHbranchiata

61,

Mammalia
Rodentia

85.

Bivalvia 3, 8,
Anisomyaria

37,

139-

Polychaeta 6, 10, 12,
36,56,61

. 27, 31,
,85,98.

Mammalia
Chiroptera

12.

Pisces
Isospondyli

Mammalia
Monotremata

106.

INDEX OF PLANTS AND ANIMALS

159

PAGES

CLASS, ORDER,
ETC.

Pisces 62, 78, 79, 84.

Microcyprini

Pteridophyta 46, 48-50.

Filicales

Decapoda
Palinuridae

27-

Echinoidea 18,24,25,26,
Diadematoida 57,61,76,82-

87, 89,90,99,

129.

NAME OF ORGANISM, SYNONYMS AND COMMON
NAME, WHEN KNOWN

(85) Orysiias latipes (Temminck & Schlegel)

Medaka, Killifish

(86) Osmunda javanica Blume

(87) Ostraea comniercialis Iredale & Roughley, see

(123)

(88) Palinurus interruptiis Randall, see (89)

(89) Pamdirus interriiptus (Randall)

Palinurus interriiptus Randall
Senex interruptus Ortmann
Spiny Lobster, Sea Cra^tish

(90) Paracentrotus lividus (Lamarck)

Strongylocentrotus lividus (Lamarck)
Toxopneustes lividus (Lamarck)
Sea-urchin

(91) Parascaris equorum (Goeze), see (8)

(92) Parechinus miliaris (P. L. S. Muller), see (104)

(93) Patiria miniata (Brandt)

Asterias miniata Brandt
Webbed Sea Star, Sea Bat

(94) Patiria pectinifer (J. Muller & Troschel)

Asterina pectinifer (J. Muller & Troschel)
Starfish

(95) Pelodytes punctatus (Daudin)

Mud Diver

(96) Petromyzon appendix De Kay, see (45)

(97) Phascolion strombi (Montagu)

(98) Pityrogramma sulphtirea (Schwartz)

Gymnogramma sulphurea (Schwartz)
Golden Fern

(99) Platynereis megalops (Verrill)

(100) Platypus anatinus Shaw & Nodder, see (83)

(loi) Pnetimonoeces variegatus (Rudolphi)
Lung-fluke

(102) Pomatoceros triqueter (Linn.)

(103) Psammechinus microtuberculatus (de Blain-

ville)
Sea-urchin

(104) Psammechinus miliaris (P. L. S. Muller) *

Echinus miliaris P. L. S. Muller
Parechinus miliaris (P. L. S. Muller)
Psammechinus miliaris L. Agassiz & Desor
Psanunechinus pustidatus (L. Agassiz)
Sea-urchin

* References are sometimes found to 'Psammechinus miliaris Z' or 'Psamme-
chinus jniliaris S'. These are private designations in which Z and S refer re-
spectively to littoral and deeper (35 m.) habitats. There are morphological
differences between the two forms (Lindahl & Runnstrom, 1929).

Asteroidea

29.

Spinulosa

Asteroidea

Spinulosa

Amphibia
Salientia

Sipunculoidea

98.

Pteridophyta
Filicales

46, 48-50.

Polychaeta

Trematoda

85.

Digenea

Polychaeta

8, 37. 98.

Echinoidea
Diadematoida

93, 94-

Echinoidea
Diadematoida

7, 20, 32, 33,
57,59,61,63,
77,80,82,86,
87, 90, 108,

112, 130, 131,

137-

i6o

FERTILIZATION

CLASS ORDER,
ETC.

NAME OF ORGANISM, SYNONYMS AND COMMON
NAME, WHEN KNOWN

105) Psamtnechinus pulcherrimus A. Agassiz, see

(56)

106) Psammechinus pustulatus (L. Agassiz), see

(104)

107) Pseiidocentrotus depressus (A. Agassiz)

Sea-urchin

108) Pteridium aquilinum (Linn.)

Bracken

109) Rana fusca Roesel, see (112)

1 10) Rana pipietis Schreber

Leopard Frog

111) Rana platyrrhina Steenstrup, see (112)

112) Rana temporaria Linn.

Rana fusca Roesel

Rana platyrrhina Steenstrup

Common Frog

113) Rhabditis tnonohystera Butschli

114) Sabellaria alveolata (Linn.)
Tube-worm

1x5) Sabellaria vulgaris Verrill
Tube-worm

116) Saccoglossus horsti Brambell & Goodhart

Acorn Worm

1 1 7) Saccoglossus kowalewskyi (A. Agassiz)

Balanoglossus kozvalevskii A. Agassiz
Dolichoglossus kozvalevskii (A. Agassiz)
Acorn Worm

1 1 8) Sagitta Quoy & Gaimard

Arrow Worm

119) Salmo gairdneri Richardson

Rainbow Trout

120) Salmo salar Linn.

Salmon

121) Salmo trutta Linn.

Brown Trout

122) Salvinia nutans Allioni

123) Saxostraea commercialis (Iredale & Rough- Bivalvia
ley)

Ostraea commercialis Iredale & Roughley
Rock Oyster

page s

Echinoidea
Diadematoida

26,89,131-
134.

Pteridophyta
Filicales

39-42, 46,
48-50.

Amphibia
Salientia

130, 131-

Amphibia 61
Salientia

,81,85-87.

Phasmida
Rhabditida

Polychaeta 56

61,85, 142-

Polychaeta

Enteropneusta

21.

Enteropneusta

8, 20.

Chaetognatha

Pisces
Isospondyli

84.

Pisces
Isospondyli

84.

Pisces

Isospondyli

131.

Pteridophyta
Filicales

46, 48-50.

Bivalvia
Anisomyaria

56, 61, 64-
67, 69.

124) Senex interruptus Ortmann, see (89)

125) Siredon mexicanum Shaw

Amblystoma mexicanum (Shaw)

AXOLOTL

Amphibia
Caudata

86.

INDEX OF PLANTS AND ANIMALS

NAME OF ORGANISM, SYNONYMS AND COMMON
NAME, WHEN KNOWN

(126) Sphaerechmus granulans (Lamarck)

Echinus brevispinosus Risso
Toxopneustes brevispinosus (Risso)
Echinus equituberculatus de Blainville *
Sea-urchin

(127) Spirocodon saltatrix (Tilesius)

CLASS, ORDER,
ETC.

Echinoidea
Diadematoida

Hydrozoa
Anthomedusae

161

PAGES
99.

13, 14. 39-

Bivalvia 3. 10, 98,

Eulamellibranchiata 119.

(128) Spisula solidissima (Dillwyn)

Mactra solidissima (Dillwyn)
Solid Surf Clam

(129) Strongylocentrotus droebachiensis (O. F. Echinoidea

Miiller) Diadematoida

Echinus droebachiensis O. F. Miiller

Toxopneustes droebachiensis (O. F. Miiller)

Strongylocejitrotus granularis (Say)

Toxopneustes pallidus G. O. Sars

Toxopneustes pictus Norman

Sea-urchin

(130) Strongylocentrotus franciscanus {A. Agassiz) Echinoidea

Sea-urchin Diadematoida

(131) Strongylocentrotus granularis (Say), see (129)

(132) Strongylocentrotus lividus (Lamarck), see (90)

25, 26, 82.

(133) Strongylocentrotus nudus (A. Agassiz)
Sea-urchin

(134) Strongylocentrotus pulcherrimus (A. Agassiz),

see (56)

(135) Strongylocentrotus purpuratus (Stimpson)

Sea-urchin

Echinoidea
Diadematoida

82, 84, 138,
139-

24.

Echinoidea 8, 24-26, 30,

Diadematoida 61, 71, 76,

77, 81-83,
87-89. 138,
139-

(136) Strongylocentrotus tuberculata A. Agassiz,

see (3)

(137) Styela partita (Stimpson)

Cynthia partita Stimpson
Tethyum partitum (Stimpson)
Sea-squirt

(138) Temnopleurus hardwicki (Gray)

Sea-urchin

(139) Tethyum partitum (Stimpson), see (137)

(140) Thalassema neptuni Gaertner

(141) Toxopneustes brevispinosus (Risso), see (126)

(142) Toxopneustes droebachiensis (O. F. Miiller),

see (129)

(143) Toxopneustes lividus (Lamarck), see (90)

(144) Toxopneustes pallidus G. O. Sars, see (129)

* See footnote under (5).

Ascidiacea
Pleurogona

Echinoidea
Diadematoida

Echiuroidea

16.

24.

20, 98.

1 62

FERTILIZATION

NAME OF ORGANISM, SYNONYMS AND COMMON
NAME, WHEN KNOWN

(145) Toxopneustes pictus Norman, see (129)

(146) Toxopneustes variegatus (Lamarck), see (67)

(147) Tripneiistes esculentus (A. Agassiz), see (148)

(148) Tripneustes ventricosus (Lamarck)

Hipponoe esculenta A. Agassiz
Tripneustes esculentus (A. Agassiz)
Sea-urchin

(149) Triton alpestris Laurenti, see (152)

(150) Triton palmatus Schneider, see (153)

(151) Triton viridcscens (Rafinesque), see (34)

(152) Triturus alpestris (Laurenti)

Triton alpestris Laurenti
Alpine Newt

(153) Triturus helveticus (Razoumowsky)

Diemictylus palmatus (Schneider)
Molge palmata (Schneider)
Triturus palmatus (Schneider)
Triton palmatus Schneider
Palmate (webbed) Newt

(154) Triturus palmatus (Schneider), see (153)

(155) Triturus viridescens (Rafinesque), see (34)

(156) Urechis caupo Fisher & MacGinitie

Inn Keeper, Horse Cock

(157) Vespertilio noctula Schreber, see (81)

(158) Vesperugo noctula (Schreber), see (81)

(159) Xenopus laevis Daudin

Clawed Toad

class, under,

ETC.

PAGES

Echinoidea
Diadematoida

131. 133-

Amphibia
Caudata

Echiuroidea

Amphibia
Caudata

103, 104.

3, 19, 29, 56,
61, 62, 65, 67,
77,81,82,87,
89, 119.

86.

GENERAL INDEX

Except when there might be a misunderstanding, the word 'eggs' has
been omitted, e.g. hexokinase means hexokinase in eggs. The word
'spermatozoa' has not been omitted in comparable cases.

Acetate metaboHsm, 79.

Acetone, as cytolytic agent, loi,
102.

Acet>'lcholine, 89.

Acid production, of eggs, 56, 63,
64, 65-66, 117; of egg homogen-
ates, 69-73, 98-99-

Acrosomal filaments, 7-8, 30.

Action potential, 131, 124-130;
conduction of, 124-127.

'Action potential', in sea-urchin
egg, 128-129.

Activation, of eggs, definition, i ; of
spermatozoa, 22, 32-34.

ADP, 66.

ATP, 66, 70, 82.

ATPase, 82.

Adjuvants, effect on non-agglutin-
ating fertilizin, 25, 29.

Agglutination, of spermatozoa, 22-
35, Plate III; of trypsin-treated
red blood cells, 137; of un-
fertilized eggs, 34.

Agglutination titre, 23-24, 138.

Agglutinins, in sera and body fluids,
26-27.

Albumin, addition to sea water, 3 1 .

Aldolase, 70, 71.

Alkaline sea water, effect on c, 136.

a. See Fertilization rate.

Amino acids, free, in eggs, 76-77,

87.

Ammo acid metabolism, 76-78, 87.

Ammonia production, 75-76, 86.

Amphiaster, resistance to deforma-
tion, 92.

Androgamone I (A.I), 22, 37-38.

II (A.II). See Sperm-anti-

fertilizin.

Ill (A.III). See Sperm

Lysin.

Anticoagulating action of fertilizin,

97-
Antifertilizin, 21, 22, 34-35. 37-
Arginine phosphate, 82.
Ascaridin, in sperm head, 85.
Astaxanthine, 22, 33.
Asters, 15-18.

Bacterial contamination, of egg

suspensions, 59, 60.
Bee venom, 22, 36.
Bicarbonate, effect on O2 uptake of

eggs, 63, 64.
Bioelectric measurements, 124-135.
Birefringence, of cortex, 91, loi ; of

fertilization membrane, 91; of

hyaline layer, 91, 10 1.
Blister formation, in eggs, loi.
Block to polyspermy, 13-14, 106-

115; in mammalian eggs, 114-

115-
Blood clotting, 96.

Body fluids, agglutinins in, 26-

27.
Boric acid, effect on pollen tube

growth, 54.
Brownian movement, of cortical

granules, 6.
Butyric acid, 80-81.

c. See Mean speed of sperm sus-
pension.

Calcium, 10, 11, 13, 82-84, 94-99,
119, 128, 129; changes in distri-
btition during action potential
(nerve), 128 ; effect on egg homo-
genates, 98-99; in eggs, 83, 129;
in egg maturation, 98; in ferti-
lizin-antifertilizin reaction, 97 ;

163

164

FERTILIZATION

in sea water, 129; in tanning of
fertilization membrane, 10.

Calcium, lack, effect on cortex,
95 ; effect on hyaline layer, 1 3 ;
reversal of fertilization, 119.

release, 82-83, 94-97,

Capacitance, decrease in hypotonic
sea water, 133-134; increase after
fertilization, 1 31-133; lack of
change during action potential
(nerve), 127; of plasma mem-
brane, 92-93, 131-134-

Carbohydrate, metabolism, 69-74,
87; oxidative breakdown of, 69-
73 ; specificity in fertilizin, 26.

Carbon monoxide, effect on O2 up-
take of eggs, 67-68.

Catalase, 90.

Cell Elastimeter, 92-93, 94, 10 1.

membrane. See Cortex,

Centrifugation, of eggs, 83, 93-95,

lOI.

Cephalin metabolism, 74-75, 87.

Chelating agents, 31.

Chemotaxis, of spermatozoa, 13,
22, 39-50; structure-action rela-
tionships, 43-47.

Chinese buckthorn, enzymes in,
52.

Chitin, 4.

Chloride, in eggs, 84, 129; in sea
water, 129.

Cholesterol, in cortex, 100; meta-
bolism, 75, 86-87.

Cholinesterase, in spermatozoa, 89.

Chorion, 4, 11.

Classification of fertilization, ac-
cording to maturation state of
egg, 2-4, 60-61.

Cleavage path, of fusion nucleus,
14, 16-18.

Clotting, of protoplasm, 94-99;
of sea-urchin perivisceral fluid,
99.

Cold-shock, effect on zona pellu-
cida, 115,

Combining sites, exposure by tryp-
sin, 137; obliteration by perio-
date, 137,

Complement, 30.

Conductance change. See Resis-
tance change during action po-
tential (nerve).

Constriction experiments, 103-105.

Contraceptives, oral, 54-55.

Contractility, of fertilization mem-
brane, lO-II,

Contraction, of egg surface, 5-6 ; of
jelly, 36-37.

Copulation path, of male pro-
nucleus, 14-18.

Corona radiata, 5.

Cortex, birefringence of, 91, 10 1 ;
cholesterol in, 100; cyclical
changes in, 99-100, 102; effect
of calcium lack on, 95 ; gelation
of, 94-97 ; light scattering by, 6,
107-108; liquefaction of, 6, 95-
96; rigidity of, 91-95, loi ; Shear
modulus of, 95 ; stiffness of, 92-
94, loi ; thickness of, 92-93 ;
Young's modulus of, 93, 95.

Cortical alveoli, 80, 121; polysac-
charides in, 6.

change, 5-7, 91, 106-108, 109,

113, 114, 121.

granules, 6-7, 9-^1, 35, 80,

95, 106-107, 121 ; Brownian
movement of, 6 ; effect of trypsin
on, 10; polysaccharides in, 6.

Cross-agglutination, relationship
with cross-fertilization, 138, 139.

Cross-fertilization, 136-140; effect
of n, 138-139; effect of trypsin,
137-138 ; relationship with cross-
agglutination, 138, 139,

Cumulus oophorus, 5.

Cyanide, effect on metabolism of
egg homogenates, 71, 98; effect
on O., uptake of eggs, 67,

Cyclical changes in cortex, 100, 102.

Cytochrome, in eggs, 56, 58, 66-68,

79-
Cytofertilizin, 24.
Cytolysis, relationship between

plasmolysis and, 99-102.
Cytolytic agents, susceptibility of

eggs to, loi, 102.
Cytoplasm, resistivity of, 131, 134-

135-

GENERAL INDEX

D Rh antigen, effect of periodate on,

137.
Delayed mating in mammals, 106.

DNA, 84-85.

Detergents, 22, 36.

Diffusion coefficient, of fertilizin,

25-
Digitonin, as cytolytic agent, 100.

lOI.

Dihydroxyfumaric acid, 44, 49.
Dihydroxymaleic acid, 49.
Dimethyl-/)-phenylenediamine,

effect on O2 uptake of eggs, 67.
Dipeptidase activity, 87.
DPN, 71, 84.

Diphosphothiamine, 79, 84.
Dispermic adults, 105.
Dispermy, in rat eggs, 114.
Double matings, 121-123.

Echinochrome, 22, 33-34, 98-99;
granules, 98-99.

Egg-antifertilizin, 22, 34-35.

Egg homogenates, acid production
of, 99 ; effect of calcium on, 98-
99 ; effect of cyanide on, 98 ; effect
of fluoracetate on, 79; effect of
iodoacetate on, 99 ; effect of
papain on, 99 ; viscosity of,
98.

maturation, classification of

fertilization according to matura-
tion state, 2-4, 60-61 ; role of
calcium in, 98.

membrane Lysin. See Sperm

Lysin.

w^ater, 23-34.

Electrophoretic mobility, of anti-
fertilizin, 35 ; of fertilizin, 24.

Endogenous substrates, in eggs,
64-65, 69-79.

Enolase, 70.

Entrance cone. See Fertilization
cone.

Ether, effect on male pronucleus,
15-16 ; reversal of fertilization by,
119.

Expansion, of fertilization mem-
brane, lO-II.

165

Fertilization cone, 7, 107-108.

impulse. See Cortical change.

membrane, 8-12, 35, 88, 91,

98, 118; birefringence of, 91;
contractility of, lo-ii; effect of
trypsin on, 88, 93, 121 ; expansion
of, lo-i I ; role of calcium in tan-
ning of, 10.

rate, (a), 109-113, 136.

tube, 13, 14.

Fertilizin, 21-38, 138, 139; as anti-
coagulant, 97 ; axial ratio, 25 ;
carbohydrates in, 26; composi-
tion, 24-25 ; diffusion coefficient,
25; effect of periodate on, 25,
28 ; effect of trypsin on, 25 ; elec-
trophoretic mobility, 24; as in-
hibitor (of fertilization), 28-30,
97 ; modification by proteolytic
enzymes, 28 ; molecular weight,
22, 25 ; morphological effects on
spermatozoa, 7-8, 30; multi-
valent and univalent, 28-29;
non-agglutinating, 25, 28-29;
pow^der, 24, 25 ; sedimentation
constant, 25 ; specificity of, 26-
27; sulphate content, 24-26, 31 ;
univalent fragments, 28-29.

Fertilizin-antifertilizin reaction, 34-

35, 97, 138-139.

Flavonols, 51-54.

Fluoracetate, effect on egg homo-
genates, 79.

Fluoride, inhibition of endogenous
metabolism, 71, 73, 78.

Follicle cells, 5, 36.

Forsythia, 51-54, Plate V.

Fructose, in egg jelly, 26.

Fucose, in egg jelly, 26, 30-31 ; in
egg water, 30, 31.

Fusion nucleus, cleavage path, 14,
16-18.

Galactose, in egg jelly, 26.

Gamones, 21-38.

Gelation, of cortex, 94-97 ; of pro-
toplasm, 94-99.

Glucose-6-phosphate, in eggs, 71,
72.

1 66

FERTILIZATION

Glutamine, hydrolysis, 75 ; syn-
thesis, 75, 86, 87.

Glutathione metabolism, 78, 90.

Glycine, effect on p, 137; metabol-
ism, 87.

Glycogen, in eggs, 70-74, 87.

Glycogenolysis, in eggs, 73-74.

Glycolysis, in eggs, 69-74.

Glycolytic enzymes, in eggs, 70.

Glycolytic intermediates, 70.

Guanine, 86.

Gynogamone I (G.I), 22, 33-

34-

II (G.II). See Fertilizin.

Gynogenetic development, 139.

Haemagglutination, effect of peri-
odate on, 28 ; effect of trypsin on,

137.

Heat-shock, effect on block to
polyspermy, 115.

Heavy metals, in sea water, 31.

Heparin, 88, 96.

Hesperidin, 54-55.

Hetero-agglutination, 26-27.

Hetero-agglutinins, in human blood
sera, 27 ; physico-chemical prop-
erties of, 27.

Heterologous insemination, 1-2,
136-140.

'Heterologous' polyspermy, 121-
123.

Hexokinase, 66, 70, 89.

Hexose monophosphate shunt, 69-

73-
Hirst phenomenon, 28.
Hyaline layer, 13-14, loi, 118;

birefringence of, 91, loi ; effect

of calcium lack on, 13.
Hyaloplasm. See Hyaline layer.
Hyaluronic acid, 5.
Hyaluronidase, 5, 22, 36-38, 54.
Hydrogen bonds, 137.
Hydrolysing enzymes, in Forsythia,

52.
4-Hydroxy-P-cyclocitral, 53.
Hypertonic sea water, variations in

sensitivity of eggs to, 99-101.

Hypotonic sea water, effect on eggs,
80-81, 133-134; variations in
sensitivity of eggs to, loi.

Hypoxanthine, 86.

Immature eggs, response to in-
semination, 8.

Incomplete antibody, 137.

Induced polyspermy, 115-118.

Induction period, between sperm-
attachment and cortical change,
108.

Inhibition of fertilization, by ferti-
lizin, 28-30, 97 ; bv Sperm Lysin,
36.

Inhibition of pollen germination,
by quercitrin, 54; rutin, 54.

Inhibition of polyspermy. Type I,
106-115.

, Type II, 103-105.

Inhibitors, of sperm movement, 22,

37-38.

Internal pressure, of eggs, 92-94.

Interphyletic crosses, 139.

lodoacetate, effect on egg homo-
genates, 71, 73, 99.

Ions, concentrations of, in sea-
urchin eggs, 129.

Irreversibility, effect on action
potential, 124-127.

/50-rhamnetin, 53.

Jelly, 23-26, 29-32; contraction of,
37 ; effect of removal on fertiliza-
tion, 30 ; effect of removal on p,
137; fucose in, 26, 30-31; gal-
actose in, 26 ; precipitation mem-
brane, 22, 34, 37; removal of,
23, 24-25 ; sulphate in, 24-25, 31,

Klinokinesis with Adaptation, 40,
42.

Lactic acid, 70, 71.
Lecithin metabolism, 74-75, 87.
Light, effect on 0.> uptake of eggs,
67-68.

GENERAL INDEX

Light-scattering, by cortex, 6, 107-

108.
Lipid metabolism, 74-75, 86, 87.
Liquefaction, of cortex, 6, 95-97.
Live-dead staining, of spermatozoa,

109.

167

effect of bicarbonate on, 63, 64;
effect of CO on, 67-68 ; effect of
cyanide on, 67 ; effects of fertiliza-
tion on, 56-65 ; effect of light on,
67-68 ; effect of malonate on, 78 ;
effects of polyspermy on, 122.

Magnesium, in sea-urchin eggs, 83,
129; in sea water, 129.

Male pronucleus, effect of ether on,
15-16; movements of, 14-18, 85,
86, 104, 106, 122; penetration
path of, 15-18.

Malic acid, 39-43, 44, 46, 48.

Malonate, inhibition of O, uptake,
78.

Mammalian eggs, block to poly-
spermy in, 114-115; fertilization
of, Plate 1,2,5,11-13,18,36,54-
55, 106, 114-115, 119, 121-123.

Maturation, of eggs, 2-4, 60-61, 98.

Mean speed of sperm suspension,
(c), 108-109, 136.

Metabolic inhibitors, 67-68, 71, 78-

79. 98-99-
Micro-electrodes, insertion into

eggs, 128-131, 135.
Micropyle, 4-5.

Mitochondria, phosphorus in, 83.
Molecular weight, of fertilizin, 22,

25.
Mosaics, 105.
Multivalent fertilizin, 28-29.

n. See Sperm density.
Nicotine, 116-117.
Nitrogen metabolism, 75-78, 87.
Nucleic acid metabolism, in eggs,

73, 84-86.
Nucleoli, in mammalian eggs, 18.
Nutritional state of parent, effect on

eggs, 76-78.

Oral contraceptives, 54-55.
Orthokinesis with Adaptation, 42.
Osmotically inactive fraction, 80.
O, uptake of eggs, 56-63, 67-68;

p. See Probability of successful
collision.

p, effect of glycine on, 137 ; effect of
jelly removal on, 137; effect of
periodate on, 137; effect of tryp-
sin on, 137.

Papain, effect on egg homogenates,
99.

Parthenogenesis, i, 80-81, 96, 128.

Parthenogenetic activation, by bu-
tyric acid, 80-81; by needle, i,
128.

Penetration path, of male pro-
nucleus, 14-18.

Peonin, 53.

Periodate, as anti-anticoagulant, 97 ;
effect on combining sites, 137;
effect on D Rh antigen, 137;
effect on fertilizin, 25, 28; effect
on haemagglutination, 28; effect
on^, 137.

Perivisceral fluid of sea-urchins, 99.

Perivitelline fluid, 11, 24.

space, 9-1 1, 114-115.

Permeability, 80-81, 100, 102.

Petals of Forsythia, rutin content,

51-

Phlorrhizin, 66.

Phosphatases, 86.

Phosphogluconate, metabolism of,
by eggs, 71-73-

6-Phosphogluconate dehydrogen-
ase, 71.

Phosphoric acid, 66.

Phosphorus, distribution in eggs,
81-83; entry, 81-83, 85; ex-
change, 82 ; in mitochondria, 83 ;
total, in eggs, 129.

metabolism, 70-73, 81-83,

86.

Phosphorylated hesperidin, as oral
contraceptive, 54-55.

1 68

FERTILIZATION

Phyla, in which polyspermy norm-
ally occurs, 103.

Physostigimine, 89.

Plasma membrane, 8, 9, 128-134.

Plasmolysis, 99-101 ; polyhedral,
loi ; spherical, loi.

Polar bodies, 3, 10, Plate II, 105.

Pollen, quercitrin content, 51, 54;
rutin content, 51, 54.

germination, inhibition of, 54.

tube growth, effect of boric

acid on, 54.

Polyhedral plasmolysis, loi.

Polysaccharides, in cortical alveoli,
6 ; in cortical granules, 6.

Polyspermy, 17, 103-123; effect on
O2 uptake, 122; induction by
trypsin, 137; in particular phyla,
103.

Potassium, in eggs, 129; outflow
during action potential (nerve),
126, 127, 128; permeability of
eggs to, 83 ; in sea water, 129.

Potential change, after activation,
128, 135 ; during action potential
(nerve), 124-127.

difference, across surface of

frog's egg, 1 28 ; across surface of
sea-urchin egg, 129, 130, 135.

Precipitation membrane, on egg
jelly, 22, 34, 37.

Probability of successful collision,
p, 110-113, 136-139.

Pronuclei, DNA content, 84-85 ;
movements of, 14-18, 85, 104,
106.

Protamines, in spermatozoa, 36.

Protein synthesis, by unfertilized
eggs, 76-78.

Proteins, solubility of, 88-89.

Proteolytic enzymes, activation of,
in eggs, 87-88 ; effect on fertilizin,
28.

Protoplasmic clotting. See Proto-
plasmic gelation.

gelation, 94-99.

streaming, 16,18.

Pseudogamous fertilization, i, 18.

Pyruvate, metabolism, 70, 71, 78,
84 ; permeability of eggs, 84.

Quercetin, 51-53 ; biological identi-
fication of, 53.

Quercitrin, 51, 52, 54; inhibition of
pollen germination, 54.

Random motion of spermatozoa,

39-42, 45, 108, no.
Recovery phase in action potential

(nerve), 124-127.
Red blood cells, agglutination of,

137-

Refractive index, of cytoplasm, 90;
of nucleus, 90.

Regulation of metabolism, by ferti-
lization, 62, 77.

Resistance, of frog's egg cortex, 130,
131; of sea-urchin egg cortex,
130-131, 135.

change during action potential

(nerve), 127.

Resistivity, of cytoplasm, 131, 134-

135-
Respiratory quotient, of eggs, 63-

Reversal of fertilization, 119-121.
Rh antibodies in non-agglutinating

form, 29.
Rhamnodiastase, 52.
Ribonuclease, 84, 88.
RNA, 76, 84-85.
Rigidity of cortex, 92, 95, 10 1.
Rotation of sperm head, 14-15.
Rutin, 51, 52, 54; inhibition of

pollen germination by, 54.

Saponin, as cytolytic agent, loi.

Sea water, calcium in, 129 ; chloride
in, 129 ; effect on c of alkalinity of,
136; effect on cortex of calcium
lack in, 95 ; effect on eggs of
hyper- and hypotonicity of, 99—
10 1 ; effect on membrane capaci-
tance of hypotonicity, 133-134;
effect on spermatozoa of addition
of albumin to, 31 ; heavy metals
in, 31; magnesium in, 129; po-

GENERAL INDEX

169

tassium in, 129; sodium in, 129;

sulphate in, 129.
Sedimentation constant, of fertili-

zin, 25.
Self-sterility, in Ciona, 138; in

Forsythia, 51-52.
Sense organs, in spermatozoa, 40,

43-

Sera, agglutinins in, 26-27.

Shape changes, in eggs, 18-20,
Plate II.

Shear modulus, of cortex, 95.

Sodium, in eggs, 84, 129; in sea
water, 129.

Sodium influx, during action poten-
tial (nerve), 126, 127.

Solubility of proteins, 88-89.

Somatic fertilization, 123.

Spawning, reciprocal induction of,
21-23.

Specificity, 52, 136-140.

Sperm density (n), 108-115; effect
on cross-fertilization, 138-139.

head, Ascaridin in, 85 ; rota-
tion of, 14-15.

Lysin, 22, 35-37. Plate IV,

139-

— middle-piece, entrance into
egg, Plate I, 12-13.

— tail, entrance into egg, Plate I,

12-13.

Sperm-antifertilizin (A. II), 22, 34-
35. 37; nature of protein, 34-35-

Sperm-egg collision frequency (Z),
108-110, 138.

filaments, 7-8, 30.

interaction rate. See Fertiliza-
tion rate.

interaction time {t), 109-113,

117, 136.

Spermatozoa, activation of, 22, 32-
34; agglutination of, 21-31, Plate
III; chemotaxis of, 13, 22, 39-
50; effects of fertilizin on mor-
phology of, 7-8, 30, 31; lack of
movement in semen, 22, 37-38;
live-dead staining, 109 ; morphol-
ogy of turning, 41 ; Oj uptake of,
32-34; protamines in, 36; ran-
dom motion of, 39-42, 45, 108,

no; repulsive effect of sub-
stances on, 47.

Spherical plasmolysis, loi.

Stamens, long and short, in For-
sythia, 51-54, Plate V.

Steric hindrance in antigen-anti-
body reactions, 31, 140.

Stiffness, of cortex, 92-94, loi.

Stigmata of Forsythia, hydrolysing
enzymes in, 52.

Styles, long and short, in Forsythia,
51-54, Plate V.

Sucker. See Cell Elastimeter.

Sulphatase, in spermatozoa, 89.

Sulphate, in eggs, 89, 129; in
fertilizin, 24-26, 31 ; in sea water,
129.

Supernumerary spermatozoa, be-
haviour of, 103-106, 121-123.

Surface tension, of egg, 92.

Tanning of vitelline (fertilization)
membrane, 9-10, 12, 35, 91, 98,

IIS-
of sona pellucida, 12, 11 4- 115.

Thiamine, 84.

Thrombin, production of, in eggs,
96.

Tricarboxylic acid cycle, in eggs,
78-79.

Trypsin, effect on agglutination of
red blood cells, 137; effect on
combining sites, 137; effect on
cortical granules, 10; effect on
cortical stiffness, 93, 94; effect
on cross-fertilization, 137-138;
effect on elevation of fertilization
membrane, 88, 93, 121 ; effect on
fertilizin, 25 ; effect on incidence
of polyspermy, 137; effect on p,
137.

Univalent fertilizin, 28-29.

Urea, reversal of fertilization by,

120.
Urease, 89.
Urechrome, 67.
Usnic acid, 85-86.

1 70

FERTILIZATION

Van der Waals' forces, 137, 140;

radii, 140.
Vapour pressure of egg contents,

81.
Viscosity, of cytoplasm, 94-97 ; of

egg homogenates, 98.
Vitelline membrane, 8-12, 115.
Volume changes, in eggs, 5, 18-20,

80-81.

Weil Felix reaction, 139.

Yolk protein synthesis, 62, 76-78.

degradation, 76-78.

Young's modulus, of cortex, 93, 95.

Z. See Sperm-egg collision fre-
quency.

^-potential, of eggs, 134; of sperma-
tozoa, 30.

Zona pellucida, 5, 12, 11 4- 115;
block to polyspermy in, 1 14-1 15 ;
effect of cold-shock on, 115.

Zone phenomenon, 29.

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