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Dr. C. R. Austin \[
July 2, 1962 [[
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THE MAMMALIAN EGG
Electron micrograph of a rat egg, showing the head and part of the mid-piece of a sperma-
tozoon shortly after passing through the vitelline surface. The head has entered upon the
changes that lead -to pronucleus formation. Note that the membrane limiting the egg
cytoplasm is folded in around the tip of the sperm head and that the spermatozoon now lacks
any evidence of plasma or nuclear membranes, x 20,000. (By courtesy of D. G. Szollosi
-and H. Ris.)
Frontispiece
THE
MAMMALIAN EGG
By
C. R. AUSTIN
B.V.Sc, D.Sc.
National Institute far Medical Research
Mill Hill London
BLACKWELL
SCIENTIFIC PUBLICATIONS
OXFORD
© Blackwell Scientific Publications Ltd., 1961
This book is copyright. It may not be reproduced by any means in whole
or in part without permission. Application with regard to copyright
should be addressed to the publishers.
First printed September 1961
Printed in Great Britain for Blackwell Scientific Publications Ltd.
by A. R. Mowbray & Co. Limited in the City of Oxford
and bound at the Kemp Hall Bindery
PREFACE
The egg is a unique cell and certainly merits special attention; this
book is an attempt to review in detail available information on
mammalian eggs and to discuss briefly the trends of research from
the point of view of the cytologist.
I am very grateful to my assistant Miss Heather Speer for the
trouble and care that she took in the compilation of the two
Appendices, in the preparation of the diagrams in Figs. 9, 10, 43,
72 and 73, and in the general work involved with the other illustra-
tions. All the colour photographs were taken by Mr. M. R. Young;
those of the fluorescent eggs were made possible by a technique
that he developed for this purpose. My grateful thanks are due to
Professor E. C. Amoroso, F.R.S., for providing the histological
sections of cat eggs illustrated in the colour Figs. 19, 20, 40-45,
67-69 and for the photographs appearing as Figs. 46 and 66, to
Dr. D. G. Szollosi and Dr. Hans Ris for the Frontispiece, to Dr. J. A.
Armstrong and Dr. R. Valentine for making the electron micro-
graphs in Figs. 27, 54 and 70, to Dr. Ruth Deanesly for providing
the sections of bat and hedgehog eggs shown in Figs. 39 and 75, and
to Mrs. Maureen Burke for checking the references. Acknowledg-
ment is gladly made to the publishers for permission to reproduce
the following Figures: Fig. 7, J. B. Lippincott & Co., Philadel-
phia; Figs. 13, 28, 31, 65, Commonwealth Scientific and Industrial
Research Organization, Australia; Figs. 14, 32, 34, 53, Academic
Press Inc., New York; Figs. 24, 29, 58, 59, 61b, 71, Cambridge
University Press; Figs. 48, 49, Blackwell Scientific Publications,
Ltd., Oxford; Fig. 57, Royal Microscopical Society, London. The
blocks for Figs. 1, 6, 10, 12, 15, 16, 19-22, 25, 26, 35, 36, 38-46, 50,
5X> 53. 55-59, 66-69, 74 and 75 were kindly made available by the
Editor of Endeavour. Finally, I should like to acknowledge to the
Medical Research Council my appreciation for being allowed time
to write this book and for the use of the facilities of the National
Institute for Medical Research in its preparation.
C. R. Austin.
National Institute for Medical Research
London
1961
Lu / ^ M
CONTENTS
GENERAL BIOLOGY OF EGGS
Discovery .
Role in Animal Economy .
Life History
Size
i
7
8
STRUCTURE AND FUNCTION IN MAMMALIAN
EGGS
Nucleus
Oocyte Nucleus ......
Maturation ......
Pronuclear Growth and Development .
Properties of Pronuclei .....
Anomalies of Pronuclei: Subnuclei — Rudimentary par-
thenogenesis — Gynogenesis and androgenesis — Aneu-
gamy — Polyandry and polygyny
nucleocytoplasmic relations in fertilization .
Cleavage Nuclei ......
Cytoplasm
Physical Features : Yolk — Fine structure — Changes in
size and form ......
Chemical Components .....
Organelles : Mitochondria — Golgi material — Cortical
granules — Division apparatus — Components of the
spermatozoon ......
16
21
24
30
34
47
48
52
59
63
mnono
CONTENTS
Mechanism of Cell Division
Polar-body Emission
Cleavage of the Fertilized Egg
Fragmentation of Eggs
72
73
78
84
Membranes and Investments
Vitelline Membrane
Zona Pellucida ....
Cumulus Oophorus
Mucin Coat of the Rabbit Egg .
Outer Coats of Marsupial and Monotreme Eggs
86
89
96
100
102
MANIPULATION OF EGGS
Microscopy . . . . . .103
Transfer ....... 109
Studies on Eggs Maintained in vitro : Metabolism — ■ In-
fluence on spermatozoa — Resistance to low tempera-
tures — - Development in culture — Fertilization /// vitro 1 1 1
Appendix No. i
. 125
Appendix No. 2
. 144
References and Author Index
. 149
Subject Index ....
. 177
Index of Organisms
. 182
THE MAMMALIAN EGG
GENERAL BIOLOGY OF EGGS
Discovery
'Omne vivum ex ow'— 'All living things come from eggs' — was a
conclusion reached several centuries ago by the anatomist William
Harvey (165 1), better known for his discovery of the circulation of
the blood. As a generalization, it has proved remarkably true, for
there are few forms of life that arise exclusively by other means
and these are to be found chiefly among the single-celled organisms.
The generalization is remarkable also because it was made when the
nature of eggs of any sort was most imperfectly known and before
those of mammals had even been properly identified. At that time,
what were termed mammalian 'eggs' took most diverse forms:
spherical or ovoid objects, filamentous or membranous structures,
and coagulated masses. These 'eggs' were considered to have been
developed within the uterus from the mingled male and female
'semen'. Galen (a.d. 130-200) had introduced the idea of female
'semen' as a substance separated from the blood stream by the
ovaries and passed into the uterus through the Fallopian tubes.
Later, de Graaf (1672) homologized the mammalian ovary with that
of the bird, maintaining that the eggs originated here and then
passed into the uterus ; he believed that the ovarian follicles, which
now bear his name, were either the eggs themselves or else contained
something analogous to eggs. The former possibility appeared to
be supported by the similarity in general form between the follicle
and the uterine 'egg' — de Graaf worked with rabbits, in which the
blastocyst is a spherical body of about the same size as the pre-
ovulatory follicle. He had also observed how, in the rabbit, the
follicle becomes radically altered after coitus and, a few days later,
blastocysts can be found in the uterus. The Fallopian tubes, how-
ever, were manifestly too narrow to permit the passage of objects
of this size and so de Graaf seems to have preferred the view that the
1
THE MAMMALIAN EGG
contents of the follicle passed, through the tubes in a fluid or un-
organized state, becoming later constituted into the uterine eggs.
His search of the Fallopian tubes did, in fact, reveal to him the
much smaller tubal eggs, but the observation was not generally
Fig. 1
'Eggs to be found in all sorts of females.'
A drawing published by Kerckring (1672).
Fig. I depicts the ovaries, uterus and adnexae in the human subject.
Figs. II and III, human ovarian 'eggs'.
Fig. IV, cow ovarian 'eggs'.
Figs. V and VI, human uterine 'eggs', opened to show contents.
credited — the difference in size was incomprehensible and no one
could confirm the finding until Cruickshank did so over a hundred
years later. Cruickshank (1797) identified tubal rabbit eggs as early
as the third day after coitus but could not trace them back further
than this. Other investigators were no more successful and it was
not until thirty years later that the ovarian egg was finally recog-
nized. Von Baer (1827) announced the discovery with a well-
justified air of triumph — 'Led by curiosity ... I opened one of the
follicles and took up the minute object on the point of my knife,
finding that I could see it very distinctly and that it was surrounded
by mucus. When I placed it under the microscope I was utterly
astonished, for I saw an ovule just as I had already seen them in
the tubes, and so clearly that a blind man could hardly deny it'
(translation published by Corner, 1933).
GENERAL BIOLOGY OF EGGS
.T.^
T**y .1*r t//ty
4*t/**
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Fig. 2
Rabbit eggs recovered from the Fallopian tubes and uteri by
Cruickshank (1797). The eggs are shown 'natural size' and enlarged.
Fig. 3
Slightly enlarged portion of von Baer's (1827) plate showing follicular
oocytes surrounded by cumulus-cell masses. On the original, the
magnification was given as: top row, natural size; middle row, X 10;
bottom row, X 30.
THE MAMMALIAN EGG
It cannot be doubted that the choice of experimental animal
played a most important part in the advancement of knowledge of
early mammalian development. Despite the fact that Harvey was
a painstaking and experienced investigator, he quite failed to draw
the proper conclusions from his studies in the deer; ruminant
blastocysts rapidly attain a highly extended state, and Harvey
interpreted this structure as a mass of mucous strands, among which
the embryo was to arise. De Graaf and Cruickshank were fortunate
to select the rabbit as experimental animal, because in this species
ovulation is induced by coitus, the tubal egg is easily visible to the
naked eye owing to its possession of a wide mucin layer, and the
blastocyst is a very distinctive object. Von Baer's discovery, which
was made with the dog egg, must have been facilitated by the fact
that the follicle in the dog ovary is large and comparatively clear,
and the egg stands out in transmitted light owing to its almost
opaque cytoplasm.
Following von Baer's historic announcement, events moved more
rapidly. Studies on the structure of follicles, eggs and developing
embryos were made by Coste (1834) and Barry (1838, 1839) in the
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Fig. 4
Rabbit eggs as described by Barry (1839).
GENERAL BIOLOGY OF EGGS
rabbit, and by BischorT( 1842a, b, 1845, 1852, 1854b, 1863) in several
species. Through the work of Schwann (1839) and Gegenbaucr
(1861; cited by Nordenskiold, 1928), the ovarian egg was shown
to be a single cell. By the middle of the century, it was known that
Fig. 5
Stages of cleavage in the dog egg (BischofF, 1845). Numerous
spermatozoa are shown attached to the zona pellucida.
the mammalian egg consisted of a cytoplasmic mass or vitellus,
containing a nucleus which was termed the germinal vesicle, and
surrounded by a thick transparent membrane, the zona pellucida.
The earliest intimations that spermatozoa enter eggs were provided
by Barry (1843), BischorT (1854a) and Meissner (1855) in the rabbit,
Nelson (1851) in Ascaris, and Newport (1853) in the frog, but the
first worthwhile descriptions of fertilization are those of Van
Beneden (1875) in the rabbit, Hertwig (1876) and Fol (1877, 1879)
in sea urchin and starfish and Van Beneden and Julin (1880) in
bats. From these observations, in the main, the realization came
that fertilization involved the union of egg and sperm nuclei and
represented therefore the cytological mechanism underlying bi-
parental inheritance. Before the close of the century, Sobotta
(1895) published his classical account of maturation, fertilization and
cleavage in the mouse egg, based upon one of the earliest applica-
tions of the histological technique to the study of eggs. The last
THE MAMMALIAN EGG
quarter of the nineteenth century was the Golden Age for gametol-
ogy, marked by the enthusiasm with which an increasing number
of investigators contributed information on an ever- widening range
of animal types, both vertebrate and invertebrate. As early as 1891,
Fig. 6
A few diagrams from the extensive series published by Sobotta
(1895) on fertilization in the mouse egg.
Boveri was able to present a review of knowledge on fertilization
which, through its detail and insight, maintains an authoritative
status to this day. The trend of research in the present century on
the structure and function of gametes has been rather to support
and extend theories founded in the last century than to establish
new ideas — a feature that, as Oppenheimer (1957) points out, is
common to the science of embryology as a whole.
Formal morphological studies on mammalian eggs were soon
accompanied by experimental work on isolated specimens. Schenk
(1878) seems to have been the first to contribute in this field, by
maintaining eggs in vitro and attempting to procure their fertilization
under these conditions. Though his methods were remarkably
advanced for his day, they were not apparently successful. Heape
(1890) holds precedence for the transfer of living eggs from one
animal to another and thus obtaining the birth of young from
unrelated foster-parents. Long (1912) prepared some of the earliest
cinematographic records of the changes shown by living eggs in
GENERAL BIOLOGY OF EGGS 7
vitro, but Lewis and Gregory (1929a, b) seem to have been the first
to obtain the protracted development of mammalian (rabbit) eggs
in culture.
Role in Animal Economy
The ovarian egg, as a single cell, has much in common with the
other cells of the body, but possesses special features. First distin-
guishing traits appear early in embryonic development with the
precursor of the egg, the primordial germ cell, which is marked
out from the other cells of the embryo by its relatively clear cyto-
plasm and large rounded nucleus. This early differentiation has its
parallel in phylogeny, for egg-cells, or the equivalents of egg-cells,
are recognizable in some of the simplest animals : for example, in
members of the Sporozoa, such as the malarial parasite Plasmodium.
In certain other unicellular organisms, such as the Trichonympha, one
cell bodily enters another, in a manner analogous to the entry of
spermatozoon into egg, but the two cells are of much the same
size and general appearance (Cleveland, 1958a, b); here there is a
functional though not an obvious structural specialization of sex cells.
A degree of differentiation of egg-cells is evident, therefore, at least
in some members of all the Phyla of the animal kingdom.
Generally speaking, union of egg and spermatozoon (or of egg-
cell and sperm-cell) is followed immediately by a succession of
divisions of the resulting zygote, with the formation of a number
of new cells, and the process characterizes sexual reproduction. The
new cells represent new individuals in unicellular animals, and,
adhering together, constitute the embryo in Metazoa. In asexual
reproduction, on the other hand, divisions proceed without the
occurrence of conjugation or fertilization. Continuity and increase
can be maintained in a number of animal populations, particularly
in the insect kingdom, by asexual reproduction (see White, 1954),
and this fact serves to emphasize that, notwithstanding its close
temporal and sometimes causative relationship with cell division,
the union of sex cells is not directly concerned with the multiplica-
tion of individuals; indeed, its most direct consequence in unicellular
organisms is a reduction in number. The capacity for population
increase in complex animals depends ultimately upon the poten-
tiality for egg production, and the true process of multiplication in
mammals is the increase in number of primordial germ cells in the
embryonic ovary. The union of the sex cells is primarily of genetic
8 THE MAMMALIAN EGG
significance and has to do with the combination and rearrangement
of genes. Genie reassortment assists adaptive variation within the
species, while combination of genes from different individuals
makes for integration of the race (see Austin, 1959b).
In the female mammal, germ-cell multiplication is intense in the
later phases of embryonic development, and as a result a large
number of oogonia accumulate from which eggs can be derived
(Brambell, 1956). By the time of birth or shortly afterwards, the
oogonia are found already to have differentiated into primary
oocytes in which the nuclei are in the initial stage of the prophase
of the first meiotic division (the dictyate stage). Further germ-cell
multiplication does not appear to take place and the young animal
possesses in its ovaries the stock of oocytes that is to last it for the
whole of its reproductive life (see Zuckerman, i960). The stock is
a very large one, some estimated numbers being: 160,000 in the rat
(Slater and Dornfeld, 1945), 700,000 in the dog (Schotterer, 1928)
and 750,000 in man (Block, 1953); but only a fraction of these
oocytes survives to ovulation, for large numbers degenerate at
various stages of oogenesis and at various times during the animal's
life. Thus, in the Levant vole (Microtus guntheri) the number of
oocytes per ovary, found to be 23,000 at birth, rose to 54,000 on
the 4th day of life and then fell gradually to 14,000 on the 27th day
and 8,000 on the 75th day (Bodenheimer and Lasch, 1957). De-
generation of oocytes can be greatly hastened by treatment of the
animal with ionizing radiations; the degree of effect varies with
dose, type of radiation, species, age of animal and stage of develop-
ment of the oocytes (Brambell, Parkes and Fielding, 1927a, b;
Brambell and Parkes, 1927; Brambell, Fielding and Parkes, 1928;
Geller, 1930; Genther, 1931; Desaive, 1940, 1941; Oakberg, 1958,
i960; Russell and Freeman, 1958; Mandl, 1959; Russell, Stelzner
and Russell, 1959; Russell, Russell, Steele and Phipps, 1959).
Life History
Oogenesis is completed with the differentiation of the primary
oocyte into a mature egg, a process that is characterized by the
occurrence of two co-ordinated chains of events — the development
of the follicle, and the growth and maturation of the oocyte (Fig. 7).
The first evidence of follicle formation is seen when the early
primary oocyte becomes surrounded by a single layer of epithelial
cells. The number of layers of surrounding cells increases as the
GENERAL BIOLOGY OF EGGS
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THE MAMMALIAN EGG
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GENERAL BIOLOGY OF EGGS
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THE MAMMALIAN EGG
oocyte grows and so a wide band of follicle cells is formed. Growth
of the oocyte proceeds until it has increased its original volume,
both of yolk and cytoplasm, many times. Follicular enlargement
continues long after the oocyte has reached its maximum size; this
growth is attributable partly to further multiplication of follicle
cells, but chiefly to the formation of a fluid-filled space or antrum
within the follicle. Throughout all these changes, the oocyte nucleus
remains in the dictyate stage of the first meiotic division. Then, at a
set time before ovulation is due, the meiotic division is suddenly
resumed, the first polar body is emitted and the egg becomes a
secondary oocyte. As a general rule, ovulation occurs spontane-
ously, but in some animals (Table i) it is induced by the act of
coitus. In most species, the egg is ovulated as a secondary oocyte
and does not mature further until it is penetrated by a spermatozoon.
In the dog, fox and possibly the horse,
however, the egg enters the Fallopian
tube while it is still a primary oocyte
(Van der Stricht, 1923 ; Pearson and
Enders, 1943 ; Hamilton and Day,
1945); in the dog, sperm penetration
can occur at this stage, but generally
takes place during the first meiotic
division (Fig. 8) or at the beginning of
the second. (Ovulated oocytes are
known also in rats and mice; they do
not appear to be fertilizable though
spermatozoa may pass through the
zona pellucida: Austin and Braden,
1954c.) After sperm entry, the second
meiotic division proceeds, the second
polar body is emitted and the egg is
now known as an ootid, a term that applies throughout fertilization.
When the chromosome groups deriving from the male and female
pronuclei have come together, fertilization is regarded as complete
and the cell is called a zygote. With successive mitoses, the egg
divides, first into two cells, then into four, eight, sixteen cells, and
so on, until the egg, or embryo as it is now more often called,
comes to consist of a spherical mass distinguished as a morula.
Finally, a space appears within the morula and grows in volume;
this state characterizes the blastocyst, and it is as such that the
Fig. 8
Drawing from an illustration by
Van der Stricht (1923) of a dog egg
with a sperm head lying near the
metaphase first-maturation spindle.
GENERAL BIOLOGY OF EGGS
13
embryo becomes attached to or embedded in the uterine mucosa.
As a rule, fertilization begins and ends in the ampulla of the
Fallopian tube, but there are some exceptions : in the tenrecs (primi-
tive insectivores of Madagascar), sperm penetration occurs while the
eggs are still in the ovary and they pass to the tube during pro-
nuclear development (Bluntschli, 1938; Strauss, 1938, 1950).
Penetration within the follicle has also been said to take place in the
noctule bat (Van der Stricht, 1909), and the shrew (Stratz, 1898,
cited by Strauss, 1954; Pearson, 1944), and even, according to some
early investigators, in the rabbit and dog (Barry, 1839; Bischoff,
1842a). The eggs of most mammals can wait for little more than
12 hr if fertilization and development are to occur in a normal
manner (see Hartman, 1924; Blandau and Young, 1939; Chang,
1952b; Blandau, 1954; Braden and Austin, I954d; Laing, 1957). In
the native cat Dasyimis, the opossum Didclphis, the wallaby Setonix
and the spiny anteater Echidna, the eggs pass into the uterus whilst
still in the pronuclear stage (Hill, 1910; Hartman, 1928; Flynn and
Hill, 1939; Sharman, 1955a, b). Passage through the Fallopian tube
may take only 24 hr, as in the monotremes and marsupials, or 2 to
3 days, as in rodents, but in most other mammals the interval is
Fig. 9
Sizes of animal eggs (vitellus alone).
The horizontal lines show the upper and
lower limits for the eggs of marsupials
and placental mammals, (a) Outline of
the monotreme egg. (b) Some of the
largest invertebrate eggs, such as those of
the squid Loligo, the gastropod Bitsycon,
the starfish Henricia, and the crabLibinia.
(c) The smallest frog eggs, (d) The smallest
fish eggs, (e) The Australian native cat
Dasyurus and also the sea-squirt Amarou-
cium. (/), (g) and (//) The sizes of the
majority of mammalian eggs and also of
those of many echinoderms, tunicates,
molluscs, polychaets, nemcrtines, platy-
helminths and coclenterates. Sheep, cow,
dog and horse eggs are represented by 'f ',
human, rabbit and cat eggs by 'g' and
most rodent eggs by 'h'. (/) The smallest
mammalian egg, that of the field vole
Microtus agrestis; also the egg of the clam
Spisitla. (j) The smallest animal eggs,
including that of the bryozoan Crista.
\00ju
I mm
between 4 and 8 days. Species differences are seen in the rate of
cleavage of the early embryo, p. 83, and in the time of implantation
14
THE MAMMALIAN EGG
or attachment (mouse 5 days, rat 5-6 days, guinea-pig and man 6-7
days, rabbit and ferret 7-8 days, monkey 9-1 1 days, pig about 11
days, dog and cat 13-14 days, sheep 17-18 days, cow 30-35 days,
horse 8-9 weeks, animals with delayed implantation 8-9 months
or longer) (see Pincus, 1936a; Amoroso, Griffiths and Hamilton,
1942; Amoroso, 1952; Beatty, 1956a; Eckstein, 1959).
100 200 300 400 500 600 700 800 900 1000
Fig. 10
Sizes of mammalian eggs; pronuclear eggs except for Nos. 3 and 6. 1. The spiny anteater
Tachyglossus. 2 and 3. The Australian native cat Dasyurus. 4. The American opossum
Didelphis, at 24 hr after coitus; the disposition of the albumen layer and shell membrane at
72 hr is also indicated. 5 and 6. Rabbit eggs at 10 hr and 72 hr after ovulation, respectively.
7. Sheep. 8. Man. 9. Golden hamster. 10. Field vole. A = albumen layer. M = mucopro-
tein layer. S = shell. SM = shell membrane. Zp = zona pellucida.
Size
The sizes of mammalian eggs are by no means proportional to
the sizes of the adult mammals: the horse's egg is rather less than
twice the diameter of the mouse egg and about the same size as the
GENERAL BIOLOGY OF EGGS
15
rabbit egg (Figs. 9 and 10). Variation in egg size is considered to be
attributable largely to differences in the content of non-living yolk
materials, but differences in nuclear size suggest that the amount of
active cytoplasm also varies. The eggs of the placental mammals
measure 60 to 180 \x in diameter (vitellus alone), those of rodents
occupying the lower part of the range. The egg of the field vole
Microtus agrestis (Fig. 24) is the smallest mammalian egg so far
recorded (Austin, 1957b). Very occasionally, 'giant' eggs are found,
which are 30 to 40 per cent larger in diameter
than normal ; these have been described in the
rabbit, rat, mouse (Austin and Braden, 1954c;
Austin and Walton, i960) and cotton-rat
(Austin and Amoroso, 1959) (Fig. 11). The
egg of the Australian native cat Dasyurus is
of notably larger dimensions, namely 240 /x in
diameter, but much the largest mammalian
eggs are those of the oviparous monotremes,
the spiny anteater Tachyglossus and the duck-
billed platypus Ornithorhynchus, in which the
vitellus at ovulation measures 3*5 to 4 mm. in
diameter (Flynn and Hill, 1939). Sea-urchin
eggs (Arbacia) are much the same size as rodent
eggs, the vitellus having a mean diameter of
74 jit (Harvey, 1956). By comparison, fish eggs
vary between 400 \jl and 150 mm., and frog eggs between 700 fi
and 10 mm. (Bcatty, 1956a). On the other hand, the egg of the
bryozoan Crista is only about 18 it in diameter and the oval eggs
of the parasitic worms Ascaris and Clouorchis have diameters of
about 60 and 45 jit, and 28 and 14 /x, respectively. Further informa-
tion on egg size is given by Hartman (1929), Boyd and Hamilton
(1952), Beatty (1956a), Costello et a\. (1957), Austin (1961a).
The eggs of placental mammals, with volumes between 100,000
and 3,000,000 /x3, and that of Dasyurus, with a volume of about
7,000,000 ft3, are very big compared with most tissue cells, of which
the volumes lie between 200 and 15,000 /jl3. A motor neurone in a
large mammal, however, would have a volume of the order of
10,000,000 /x3, mainly on account of its remarkably long axon. The
smallest mammalian cells are probably the red blood cells and
Fig. 11
Normal and 'giant' eggs
of the cotton-rat. X 220.
spermatozoa,
respectively.
the volumes of which are about ioo /x3 and 30
STRUCTURE AND FUNCTION IN
MAMMALIAN EGGS
Nucleus
Oocyte Nucleus
Primary oocytes exist in large numbers in the ovarian cortex of
young animals. They themselves seem incapable of division and
their abundance is owing to the earlier multiplication of the oogonia
from which they have differentiated. As a feature of differentiation,
the oocyte nucleus starts upon the early prophase changes of the
first meiotic division, the chromosomes become somewhat con-
densed, and the nucleus then passes into the dictyate stage. Those
oocytes that are not destined, as many are, for early degeneration
remain in this stage until meiosis is suddenly resumed shortly before
or soon after ovulation. The precise form assumed by the chromo-
somes in the dictyate stage is uncertain though they clearly lose their
earlier partially condensed appearance. In oocyte nuclei in fish,
amphibians, reptiles and birds, the chromosomes take on the form
of fine long threads bearing numerous lateral loops, and are referred
to as lampbrush chromosomes. Their special significance is still
conjectural — they may play a part in yolk synthesis. Equivalent
structures have yet to be demonstrated in mammalian oocytes.
Recent observations of Ohno, Kaplan and Kinosita (i960) showed
that the two X chromosomes in rat oocytes are isopycnotic, both
at the first meiotic prophase and the second meiotic metaphase.
This is in contrast to the positively heteropycnotic state of the XY
bivalent in spermatocytes, as previously demonstrated by these
workers (Ohno, Kaplan and Kinosita, 1957, 1958), and they suggest
that the condition in spermatocytes represents an evolved mechan-
ism that prevents crossing-over and ensures isolation of the female-
determining chromosome from the male-determining chromosome.
Crossing-over between the two X chromosomes in oocytes, on the
other hand, would not impair the sex-determining mechanism.
The early oocyte is distinguished from the other cells of the
ovarian cortex by its larger size, and correspondingly larger nucleus,
and by the presence of yolk materials in the cytoplasm. As seen in
16
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS
17
histological preparations, the nucleus is more or less spherical in
shape and contains one or a very few nucleoli and either irregular
chromatin masses or bodies recognizable as incompletely condensed
chromosomes.
(e)
Fig. 12
Relative nuclear size (rat) in (a) early primary oocyte, (b) late primary
oocyte, (c) egg in late stage of fertilization, (d) 2-cell egg, (e) 4-cell egg,
(/) 8-cell egg, (g) follicle cell, and (h) spermatozoon.
The oocyte undergoes considerable enlargement before it is ready
for ovulation, the increase in volume in the rat being of the order
of ninety fold. The volume of the nucleus increases proportionately;
in the living rat oocyte, it reaches about 18,000 /x3, which is more
than the entire size of most tissue cells (Fig. 12). When the nucleus
is examined by phase-contrast microscopy, it is seen to be spherical
in shape and to contain generally a single large, excentrically placed,
highly refractile nucleolus and some small granular masses of
irregular form. Within the nucleolus, there is often a spherical
vacuole which may be quite large and appears to contain nucleo-
plasm. Examined by ultra-violet and fluorescence microscopy
(pp. 107-108), it is evident that material containing a high concen-
tration of dna exists as a thick shell about the nucleolus and in the
irregular granular structures nearby (Austin and Braden, 1953c;
Austin and Bishop, 1959a) (Figs. 13 and 15). The nucleolus itself
appears to contain some rna but the nuclear sap is virtually devoid
is
THE MAMMALIAN EGG
of nucleic acids. Histological studies yield similar results; tests with
ribonuclease show specifically the presence of rna in the nucleoli
(Vincent and Dornfeld, 1948). The total amount of dna in the
oocyte nucleus throughout oocyte growth has been shown to be
Fig. 13
Rat oocyte nucleus photographed by (a) phase-contrast
microscopy, and (/>) ultra-violet microscopy (at 2,600 A). X
800. (From Austin and Braden, 1953c.)
constant at the tetraploid level, the concentration falling during
growth, presumably through dilution with increasing nuclear
volume (Vincent and Dornfeld, 1948; Alfert, 1950; Van de
Kerckhove, 1959). Experiments with glycine-2-14C show that the
tracer accumulates particularly in the nucleolus and its shell, in
accordance with current ideas on protein and nucleic-acid synthesis
(Edwards and Sirlin, 1958). The material composing nucleoli
appears to have a higher specific gravity than the other constituents
of nucleus and cytoplasm (Dalcq and Van Egmond, 1953).
By electron microscopy, the oocyte nucleus in the mouse and
rat was found to be occupied chiefly by a finely granular mass
representing the nucleoplasm and limited by a double membrane,
in which the characteristic pores could be discerned (Yamada,
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 19
Muta, Motomura and Koga, 1957; Sotelo and Porter, 1959; Odor,
i960). Scattered irregular aggregates of denser granular material
were observed within the nucleus, lying free and also in contact
with the nuclear membrane and the nucleolus. It was agreed, too,
that the nucleolar substance consists of closely-packed small dense
granules and bears no evidence of a limiting nucleolar membrane.
Descriptions of the general structure of the nucleolus varied, how-
ever. According to Yamada et ah, most of the nucleoli they saw in
mouse eggs were made up of a coarse irregular framework, the
meshes having ovoid profiles and being occupied by finer granular
material like the bulk of the nucleoplasm. The structure is strongly
reminiscent of the nucleoloneme as seen in oocytes of non-mammals
and in tissue cells (see De Robertis, Nowinski and Saez, 1954). In
addition, there was often found, attached to the nucleolus, an
irregular mass of lower density which also presented some indica-
tion of a network. Sometimes this body extended towards, and
even became attached to, the nuclear membrane. The authors
suggested that this represents the nucleolus-associated chromatin.
By contrast, Sotelo and Porter, who worked on rat eggs, reported
that oocyte nucleoli lack obvious organization, except for a broad
subdivision of nucleolar substance into a finely granular core
surrounded by a thick outer layer or wall of much denser con-
sistency. The wall substance resembled the material composing the
chromosomes that were found in sections of a secondary oocyte,
and it is possible that the thick wall may have represented the dna
shell referred to above. Differences in nucleolar structure are pro-
bably due to differences in the stage of oocyte development. Sotelo
(1959) described in the nuclei of rat primary oocytes the presence
of pairs of ribbon-like threads twisted around a thinner medial
element; often these structures appeared to be associated end-on to
the nuclear membrane as though attached to it. They evidently
represent the form taken by chromosomes in the oocyte nucleus.
It has often been maintained that, in the oocytes of amphibia and
other non-mammalian forms, nucleoli pass bodily into the cyto-
plasm, possibly through a pinching-off of the nuclear membrane
(see Vincent, 1955, and Brachet, 1957). Migration is said to occur,
too, in mammalian oocytes (Makino, 1941) and in eggs undergoing
fertilization (Kremer, 1924, who also reviews the earlier literature;
Izquierdo, 1955; Dalcq, 1955a). Sotelo and Porter (1959) report
finding an object like a nucleolus in the cytoplasm by electron
20 THE MAMMALIAN EGG
microscopy, and there is no doubt that small structures of this kind
can sometimes be found by phase-contrast microscopy, but this
does not necessarily imply that they have migrated from the
nucleus or, indeed, that they are really forms of nucleoli. If migra-
tion does take place, it seems unlikely to involve a pinching-off
process, for this would surely have been seen in all its phases during
any of the more extensive investigations on mammalian eggs ; no
such records appear to have been made. It is possible, however,
that the nucleolus could pass through the nuclear membrane in a
physically divided state and reconstitute on the other side. Accor-
ding to Anderson (1953), substances with a molecular weight of
15,000 can traverse nuclear-membrane pores and evidence of actual
transfer of material through the pores into the cytoplasm has been
obtained by electron-microscopic observations on insect nurse-cells
(Anderson and Beams, 1956). Another possible mode of transfer,
and one that presumably would permit the passage of more highly
organized substances, is suggested by the finding of Gay (1956) of
minute but distinct outpocketings of the nuclear membrane which
she believes become detached and move into the cytoplasm.
Anomalies involving oocytes include chiefly the presence of two
and sometimes more in a single follicle, the presence of two nuclei
and sometimes more in a single oocyte, and the occurrence of
'giant' oocytes. Polyovular follicles and multinuclear oocytes have
been described in a wide variety of mammalian species (Hartman,
1926, who reviews the earlier literature; Engle, 1927; Mainland,
1928; Evans and Swezy, 193 1; Ota, 1934; Dederer, 1934; Stockard,
1937; Lane, 1938; Pankratz, 1938; Waterman, 1943 ; Harrison, 1948;
Bacsich, 1949; Davis and Hall, 1950; Fekete, 1950; Dawson, 1951;
Skowron, 1956; Kent, 1959, i960). Both are common in the
opossum Didclphis and dog. Fekete found polyovular follicles at an
unusually high incidence (6.1 per ovary) in an inbred strain of
mouse (C58), and inferred that this showed an important influence
of heredity. Polyovular follicles are found more often in immature
ovaries and involving immature oocytes. Kent considers that the
incidence of both anomalies varies with oestrogen level. Informa-
tion on the ultimate fate of these anomalies is fragmentary.
O'Donoghue (1912) reported finding a mature polyovular follicle
in a specimen of Dasyurus and such a finding is rare; nevertheless,
Allen, Brambell and Mills (1947) and Fekete (1950) maintain that
at least some polyovular follicles must undergo ovulation and yield
Fig. 15
Rat primary oocyte and surrounding follicle cells showing
fluorescence induced by treatment with acridine orange and ultra-
violet irradiation. (The fluorescence shown by the eggs in Figs. 16,
25, 26, 35 and 36 was induced bv the same method.) X 500.
Fig. 16
Rat tubal oocyte with second maturation spindle at metaphase.
: 500.
Facing page 20
Fig. 19
Cat secondary oocyte with part of the metaphase group of
chromosomes seen in polar view. X 700. (Zenker formol;
Weigert H and E ; processing has removed the fat droplets.)
(E. C. Amoroso.)
Fig. 20
Syngamy in the cat egg; chromosomes beginning to condense
in apposed regions of the pronuclei. X 700. (Flemming;
Hcidenhain haematoxylin. Fat droplets stained.)
(E. C. Amoroso.)
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 21
eggs capable of normal fertilization and development. Ovulated
eggs with two second maturation spindles have been described, but
these could have arisen through first-polar-body suppression (p. 23) ;
Dempsey (1939), however, records a binuclear (guinea-pig) oocyte
which appeared clearly to be undergoing maturation. Giant eggs
are known in several different groups of animals (Wilson, 1928,
p. 972) and their occurrence in mammals has already been referred
to (p. 15). In non-mammalian animals, giant eggs are generally
binuclear and the embryos resulting from their fertilization triploid.
In mammals, both binuclear and mononuclear giant eggs have been
found undergoing fertilization, and giant 2-cell eggs have been
reported (Fig. 11), but their ultimate fate is unknown. Binuclear
oocytes may arise during multiplication of oogonia, from nuclear
division unaccompanied by cytoplasmic division, or from fusion of
two oogonia. The former possibility seems to be the more likely,
but, in either case, the cells would probably be tetraploid.
Maturation
Before it takes part in fertilization, the oocyte undergoes ripening
or maturation. This involves a reduction of the chromosome
number to half, which is brought about in the course of two
maturation, reduction, polar or meiotic divisions, and the extrusion
of two polar bodies (Fig. 14). In the first meiotic division, the
nucleus passes out of the dictyate stage — the nucleolus fades and
vanishes, the chromosomes condense into small, rounded bodies
scattered through the nucleus, and the nuclear membrane disappears.
The chromosomes become arranged at the equator of the first
meiotic spindle, either directly from their scattered positions
(Makino, 1941) or first forming a dense mass of chromatin (Odor,
1955) (Figs. 15, 16 and 19). During the prophase, the chromosomes
are brought together in homologous pairs, chiasmata develop and
parts of corresponding chromatids are exchanged in the process
known as crossing-over. At the first meiotic anaphase, the mem-
bers of the homologous chromosome pairs are separated again, their
component chromatids now having a different constitution than
they had at the start of prophase. The division advances to telophase
and the chromosomes form compact groups at the poles of the
spindle. Since the oocyte nucleus was tetraploid in respect of
chromatids, each of these groups has a diploid number of chromo-
somes ; one group is expelled in the first polar body while the other
22
THE MAMMALIAN EGG
remains within the vitellus. The vitelline group of chromosomes
now arranges itself as the equatorial plate of the second meiotic
spindle, the centromere of each chromosome is split in half and,
Fig. 14
Stages of maturation in the rat egg. In (i) to (1), the first polar body is shown with broken
outline because it often disappears before ovulation. In (j) to (1), lines in zona pellucida and
thickened outline of vitellus indicate occurrence of zona reaction and block to polyspermy,
respectively. Shrinkage of vitellus takes place about the time of first-polar-body emission,
(f) to (i), and again shortly after sperm entry which is supposed to have happened between
(i) and (j). (From Austin, 1959c.)
at anaphase, the component chromatids are separated to opposite
poles of the spindle. Again, one group is expelled, this time within
the second polar body, and the other retained in the vitellus. Each
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 23
of these groups has a haploid number of chromosomes. In many
non-mammalian animals, the first polar body passes through a
division equivalent to the second meiotic division of the oocyte ;
thus, four haploid cells can be formed, one ootid and three polar
bodies. This is analogous to the formation of four haploid sperma-
tids from each primary spermatocyte in the course of spermato-
genesis. With both systems, the final cells each have a genotype
that can be considered unique, because the pattern of chiasma
formation is not fixed and each homologous chromosome pair may
form from one to ten chiasmata — under these circumstances the
number of possible genie recombinations is very large indeed
(White, 1954). This means that the hereditary characters contributed
by the female will vary in detail with each egg. Beatty (1956b),
Beatty and Napier (i960), Beatty and Sharma (i960) and Sharma
(i960) have produced evidence that the genotype of the spermato-
zoon influences its phenotype, and so the possibility presents itself
that variations in the genotype of eggs might also be recognizable
from their visible features. To some extent this has been found to
be so: the eggs of some inbred strains of mice can be distinguished
from those of other strains by the appearance of the cytoplasm
(Braden, 1959, 196 1). (An excellent discussion on the genetic
individuality of spermatozoa is given by Bishop, i960.)
As a spontaneous anomaly or through experimental treatment,
either of the meiotic divisions may be inhibited (see Beatty, 1957).
If the first anaphase separation is blocked, the chromosomes remain
together, still constituting a tetraploid group; when the second
division takes place and the chromatids separate, two diploid
chromosome groups are formed, one passing into the polar body
and the other remaining within the vitellus. The fertilization of
such an egg gives a triploid embryo. If the first meiotic division is
inhibited after anaphase separation of the chromosomes, it is
possible that two second maturation spindles will develop; the
presence of two such spindles, occasionally reported in the literature
(Pesonen, 1946a, b; Vara and Pesonen, 1947; Braden and Austin,
1954b; Austin and Bishop, 1957b; Braden, 1957), can therefore be
ascribed not only to the maturation of a binucleate oocyte but also
to the form of inhibition just referred to. The second meiotic
division may likewise be blocked at either of two points; the
outcome in this case could be the development of a single diploid
female pronucleus or of two haploid ones, both conditions possibly
24 THE MAMMALIAN EGG
leading to a triploid embryo. Eggs with single female pronuclei
that could have been diploid were recovered from rats after colchi-
cine treatment (Austin and Braden, 1954b). The presence of two
female pronuclei may clearly come about through any of three
mechanisms : maturation of a binuclcar oocyte, or blockage at the
appropriate point of either the first or the second meiotic divisions.
Further consideration of the consequences of inhibition of meiotic
divisions is given particularly by Beatty (1951a, 1957), and also by
Austin (1960b), in Table 2 and on p. 40.
The effect of sperm entry upon the egg, the first evidence of
which is the resumption of the second meiotic division and the
emission of the second polar body, is known as activation ; other
changes associated with this process are a reduction in vitelline
volume and a rearrangement of the cytoplasmic granules. If, on the
other hand, sperm penetration does not take place, the second
meiotic division may eventually be resumed spontaneously, marking
the beginning of parthenogenetic development — this is particularly
liable to happen in the golden hamster (Austin, 1956a; Chang and
Fernandez-Cano, 1958). In rats, mice and rabbits, the chromosome
group generally breaks up, chromosomes scatter through the cyto-
plasm and apparently later lead to the development of subnuclei.
The initiation of parthenogenesis may be achieved much more
commonly in these animals' eggs if they are subjected to certain
artificial stimuli (see p. 38).
Pronuclear Growth and Development
Two pronuclei take part in the normal process of fertilization,
the male pronucleus originating from the nucleus of the sperm head,
and the female pronucleus from the group of chromosomes that
remain within the vitellus after the expulsion of the second polar
body. The sperm-head nucleus consists principally of deoxyribo-
nucleoprotein which appears to be disposed in a compact state
resembling that of a crystal lattice (see Bishop and Walton, i960);
the chromosomes must presumably be there in a form appropriate
to the preservation of gene relations, but they are difficult to recog-
nize. The transformation of the sperm-head nucleus into a male
pronucleus involves loss of the characteristic shape, increase in
volume, apparently by a form of hydration, and a change in state
of the ground substance from solid to fluid (Fig. 17). At an early
stage, minute nucleoli make their appearance and grow, coalescing
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 25
when they come into contact with each other. By the time nucleoli
are evident, a distinct nuclear membrane can be seen. In the deriva-
tion of the female pronucleus, nucleoli appear in the irregular mass
of aggregated chromosomes, and an encircling nuclear membrane
soon makes its appearance.
C
Fig. 17
Transformation of the rat sperm head into a male pronucleus. (Drawn
from photographs taken of the changes as they proceeded in vitro.)
The pronuclei grow rapidly, and this involves not only increase
in nuclear volume and total nucleolar volume, but also increase, at
least at certain stages, in the number of nucleoli (Austin, 1952a). In
the living rat egg examined by phase-contrast microscopy, the new
nucleoli appear to be generated at the nuclear membrane, often
seeming at such times to indent the membrane quite distinctly. These
nucleoli are themselves distorted from the spherical, and the whole
effect suggests that the nucleoli have surface tension and are capable
26 THE MAMMALIAN EGG
of 'wetting' the nuclear membrane. This relationship between
nucleolus and nuclear membrane has also been noted by Sotelo and
Porter (1959) in electron-microscope studies of rat eggs. They
maintain that both layers of the nuclear membrane are continued
around the indenting part of the nucleolus which is therefore fully
within the nucleus and not projecting into the cytoplasm. When
the pronuclei have reached their maximum size, they move together
and come into intimate contact with each other in the centre of the
egg. After a pause, syngamy is initiated: the pronuclei begin to
decrease in size and some of the nucleoli undergo coalescence.
Reduction in volume then affects both pronuclei and nucleoli and
continues until the pronuclei reach about half their maximum size.
The nuclear membrane now disappears, as the last of the nucleoli
fade out, and the nuclear sap assumes the consistency of a gel,
within which the condensing chromosomes become visible. The
two chromosome groups move together making a single group
which resolves itself into the metaphase plate of the first cleavage
spindle. The gathering together and possible intermingling of the
chromosome groups deriving from male and female pronuclei is
the consummation of the fertilization process (Figs. 18 and 20). It
is characteristic of mammals that intermingling does not occur until
this point, the final phase of syngamy ; the formation of a zygote
nucleus by union of male and female pronuclei, which takes place
to varying degrees in invertebrates (see Wilson, 1928), is not known
in mammals, with the possible exception of the monotremes.
According to Flynn and Hill (1939), when the pronuclei of Echidna
become apposed the nuclear membranes over the area of contact
disappear and a single cleavage nucleus is formed.
In the rat, the volumes of the pronuclei and the numbers of
nucleoli reach their maxima in about half the pronuclear life-span,
and the levels are maintained until the start of syngamy. Nucleolar
volume increases more rapidly so that the maximum is reached in
about a quarter of the pronuclear life-span; in the early male
pronucleus, the increase in nucleolar volume initially outstrips that
of nuclear volume so that coalescence and reduction in number of
nucleoli occur, but later the enlarging pronucleus is able to accom-
modate extra nucleoli. Pronuclear growth involves an enormous
increase in volume : the nucleus of the rat sperm has a volume of
the order of 10 /x3 and the male pronucleus at full development
about 5,500 jit3, an increase of 550 times (Fig. 12). The mean and
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 27
largest number of nucleoli recorded in one series of observations on
the rat male pronucleus were 17 and 36, respectively (Austin, 1952a) ;
the second figure is well in excess of the number of chromosomes
that would be present (N = 21). If pronuclear nucleoli are formed
J
k l
Fig. 18
Pronuclear development and syngamy in the rat egg. (a)-(d) Later phase in the growth
of the male pronucleus, (e) Male pronucleus (below) and female pronucleus (above) at the
start of syngamy. (/)-(/) Condensation and conjugation of chromosome groups. (Drawn
from photographs; the changes from (e) to(g) and from (h) to (/) were observed as continuous
processes that occurred in vitro in separate eggs.)
at specific nucleolus-organizing loci on chromosomes, as is the case
in tissue cells, it must be surmised either that pronuclei possess
numerous nucleolus organizers (more than one per chromosome),
28 THE MAMMALIAN EGG
or else that nucleoli can become detached from their loci, leaving
them free to generate further nucleoli. Neither of these alternatives
is consistent with the generally accepted idea of the mechanism of
nucleolus formation. Total nucleolar volume is about 10 per cent
of the nuclear volume; by contrast, the proportion is only about
i per cent in most tissue-cell nuclei (Vincent, 1955). The male
pronucleus of the rat egg maintains a volume of about two-and-a-
half times that of the female pronucleus, and approximately the
same relationship holds also for number and total volume of
nucleoli (see also Blandau and Odor, 1950; Odor and Blandau,
1951b; Dalcq, 1955b).
The pronuclei of other mammalian eggs have not been studied
in such detail as those of the rat egg, but certain similarities and
differences are evident. Mouse pronuclei tend generally to resemble
rat pronuclei, though they usually have fewer nucleoli and often
show a single nucleolus at presumed full development. In the
wcw*
Fig. 21
Rabbit pronuclei. X 1,500.
mouse, as in the rat, the male pronucleus is much larger than the
female. A moderate pronuclear disparity is seen in the eggs of the
guinea-pig, rabbit (Fig. 21), multimammate rat, Chinese hamster
and Libyan jird (Fig. 22), but it is uncertain whether it is the male
29
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS
or the female pronucleus that is the larger. In the spiny antcatcr
Echidna, the opossum Didelphis. the native cat Dasyurus, the wallaby
Setonix, the armadillo (Fig. 23), the bat, the ferret, the pig, the
golden hamster (Fig. 22), the field vole (Fig. 24), and man (Hvatov,
Fig. 22
Pronuclei of the Libyan jird (above) and golden hamster (below). X 1,200.
Fig. 23
Pronuclei of the armadillo Dasypus
novemdnctus. (Drawn from an illustration
by Newman, 1912, which was based on
sections passing through the animal pole
of the egg.)
30
THE MAMMALIAN EGG
Fig. 24
The egg of the field vole Microtus agrestis. X 900. (From Austin, 1957b.)
1959), the two pronuclei often do not differ appreciably in size.
The pronuclei of rodent eggs in general seem to be characterized
by being relatively big (nucleocytoplasmic ratio about i : 30) and
having relatively large nucleoli ; by contrast, the rabbit egg shows a
ratio of about 1 : 90 and nucleolar volume constitutes only about
1 per cent of the pronuclear volume.
Properties of Pronuclei
During its formation, and before nucleoli become visible, the
incipient pronucleus appears by all tests as a dense accumulation
of dna (Fig. 25), but the concentration soon diminishes as the
pronucleus grows (Alfert, 1950; Braden and Austin, 1953; Ludwig,
1953, 1954; Austin and Bishop, 1959a; Austin and Amoroso, 1959).
It is reasonable to suppose that the diminution is owing to a dilution
effect attributable to the great increase in volume that occurs during
Fig. 25
Early fertilization in a rat egg. The sperm head and the telophase
second-meiotic chromosome groups fluoresce green, x 500.
Fig. 26
Early pronuclear rat egg; the female pronucleus is above and the
male below and to the right. X 500.
Facing page 30
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 31
pronuclcar growth. In living eggs recovered during the early part
of pronuclear growth, when the dna can still be detected (by
ultra-violet absorption and induced fluorescence — Fig. 26), it seems
to be distributed evenly in the nucleoplasm, thus presenting a clear
difference from the nuclei of oocytes, cleaving eggs and tissue cells
in which most of the dna appears in aggregate form. In histo-
logical sections of early pronuclear eggs stained with Feulgen's
reagent or methyl green, the dna is found especially around the
nucleoli and lining the nuclear membrane — in view of the appear-
ance in living eggs, this distribution seems likely to have been
produced by fixation. When the pronuclei reach their full size,
dna cannot be detected with certainty by ultra-violet absorption
or by Feulgen or methyl-green staining, but there is still visible in
the nuclear sap a faint green fluorescence following treatment with
acridine orange. Later, as the time of syngamy approaches, the
green fluorescence is found to have become distinctly stronger and
dna can once more be demonstrated by histological methods.
Measurements of total dna content show that the amount doubles
during the pronuclear life-span, the complement in individual
pronuclei ranging from the haploid quantity to the diploid (Alfert,
1950). In mice injected a few hours before ovulation with
adenine-8-l4C, the earliest synthesis of dna by the pronuclei, as
detected by labelling, was evident about 13 hr after ovulation
(or about 11 hr after the estimated time of sperm penetration)
(Sirlin and Edwards, 1959). Later, chromosome condensation in
the prophase of the first cleavage division is apparent in the local-
ization of dna near the nuclear membrane in each pronucleus,
particularly in the region where the pronuclei are in contact.
Finally, the condensed chromosomes gather in the single large
tetraploid group from which the metaphase plate of the cleavage
spindle develops.
In histological preparations, differences have been observed in
the staining reactions of male and female pronuclei: in the pig
(Pitkjanen, 1955; Thibault, 1959), rabbit (Dauzier and Thibault,
1956), hamster (Hamilton and Samuel, 1956). In the hamster, the
larger paler-staining female pronucleus is said to be readily distin-
guished from the smaller darker-staining male pronucleus. Late-
phase female pronuclei in the rabbit and pig are described as being
asymmetrical, owing to the gathering of chromatin near the nuclear
32 THE MAMMALIAN EGG
membrane on the side nearest the male pronucleus. Such a distribu-
tion of chromatin in pig pronuclei has been recorded also by Han-
cock (1961).
The highly refractile nucleoli are striking features of the pro-
nuclei. Centrifugation of pronuclear eggs causes the nucleoli to
coalesce and makes it clear, too, that they are appreciably denser
than most other components of the egg (Dalcq, 195 1, 1952). If a
living egg is ruptured whilst under examination, the nucleoli are
often set free into the surrounding medium and can then be seen to
behave rather in the manner of oil droplets (again suggesting that
they have distinct surface tension, c.f. p. 25). Constrained by
movement of the medium to pass through a narrow space between
cell fragments, the nucleoli readily deform and break up into
smaller bodies which immediately resume the spherical shape.
Quite often, a nucleolus is found to contain a spherical inclusion
(Fig. 24); these inclusions vary greatly in diameter, as do those
of the nucleoli in cleavage nuclei (see p. 49 and Fig. 33). The
material within the inclusion resembles nucleoplasm in appearance;
occasionally, nucleoli with large inclusions are seen to 'break',
releasing the contents of the inclusion, which mixes freely with the
nucleoplasm. In the field vole, pronuclear nucleoli may show the
presence of a small body within an inclusion, the arrangement
suggesting a 'bull's-eye' in appearance. The small inner body seems
likely to be a fragment of nucleolar material. Throughout pro-
nuclear life, the nucleoli appear to be free of nucleic acid: they show
negligible ultra-violet absorption (Austin and Braden, 1953c), no
detectable fluorescence (Austin and Bishop, 1959a; Austin and
Amoroso, 1959) and are acidophilic and not basophilic when tested
under controlled ionic conditions (Braden and Austin, 1953). It
seems likely, therefore, that they consist largely of basic protein.
They stain with pyronine (Odor and Blandau, 1951b), but this reac-
tion is of uncertain significance. They stain orthochromatically
with toluidine blue and often contain metachromatic inclusions
(Izquierdo, 1955) which also give a positive reaction with the
periodic acid-Schiff test (Dalcq, 1955a) and which can be regarded
as consisting probably of mucopolysaccharides. Nucleoli have been
reported to contain phospholipid (Dalcq, 1954a, b) and alkaline
phosphatase (Mulnard, 1955).
Early investigators, using the older histological methods, often
described pronuclear nucleoli as being of two or three different
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS
33
kinds. There were said to be strongly basophilic 'nucleinic' nucleoli,
which became deeply stained with haematoxylin, and acidophilic
'plasmatic' nucleoli, which failed to take this stain; in addition, some
nucleoli were found to exhibit a shell of strongly stained material
covering a non-staining centre. These various effects can readily be
obtained if egg sections are treated with haematoxylin under the
usual histological conditions, but when more refined methods for
demonstrating basophilia and acidophilia are employed the nucleoli,
as already noted, are found to be uniformly acidophilic and not
basophilic at all. Clearly, affinity for haematoxylin applied by
classical methods cannot be taken as denoting basophilia in nucleoli,
but there is no obvious explanation for the different forms of
staining, in particular the rather striking 'shell' form. It has been
mentioned (p. 32) that nucleolar inclusions may be so large as to
reduce the nucleolar material to a mere shell, but such nucleoli are
comparatively rare, whereas those showing the 'shell' type of
staining were to be found in almost every nucleus. Some of the
recent observations with electron microscopy suggest the possibility
of a structural reason for the 'shell' type of staining : rat pronuclear
Fig. 27
Electron micrograph of a golden hamster pronuclear egg. X 2,000.
34 THE MAMMALIAN EGG
nucleoli were reported to consist of a finely granular inner mass
surrounded by a thick zone of much denser material. The structure
was essentially the same as for oocyte nucleoli (p. 18) (Sotelo and
Porter, 1959). Hamster pronuclear nucleoli, on the other hand, did
not show the 'shell' when examined by electron microscopy (Fig.
27) although the method of fixation was similar. It may be that
nucleolar substance is prone to a physical change such as condensa-
tion under certain artificial conditions and in this state has a greater
affinity for osmium and some stains.
Anomalies of Pronuclei
Subnudei. In those eggs that are ovulated in the metaphase of the
second meiotic division, the chromosome group remains quiescent
until sperm penetration occurs or for 12 hr or more in the absence
of sperm penetration. In some unpenetrated eggs, the spindle
eventually regresses, however, and the chromosome group breaks
up or fragments, the chromosomes becoming scattered through the
egg cytoplasm (Fig. 28a). This course of events is well known in
the eggs of rats and mice and is commonly followed by the forma-
tion of a number, as many as twenty or thirty, of very small nuclei.
These are referred to as subnuclei; each is bounded by a nuclear
membrane and contains from one to several small nucleoli suspended
in a clear nucleoplasm (Fig. 28b, c). They can reasonably be re-
garded as being derived from isolated chromosomes, parts of
chromosomes or small groups of chromosomes.
Clearly, however, the term subnucleus is arbitrary, for the nuclei
vary greatly in size and there is no doubt that there exists a more or
less continuous series of nuclei extending from simple, diminutive
forms to those resembling pronuclei of normal size and complexity.
As the size of the nuclei increases, the number that can be formed
decreases, so that at one end of the series the egg contains a pro-
nucleus-like near-diploid nucleus together with a small subnucleus —
a nuclear state not far removed from that seen in the initial phase of
one form of parthenogenesis when a single diploid nucleus may be
present. These facts suggest that eggs have an innate tendency
towards parthenogenetic development and such a view has often
been advanced. The nuclear state as thus described docs not, how-
ever, represent the whole situation. Eggs with fragmented nuclei,
especially those with numerous subnuclei, commonly show a
cytoplasmic state that is clearly abnormal and marks them as
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS
35
degenerating. There is no evidence that these eggs can undergo any
kind of true embryonic development, though concomitant break-up
of the cytoplasm may have a superficial likeness to cleavage (p. 84).
Pw#,j
Jr "^x
Sl^i
1 W 1
*
%%
■Hhit^&*
P>« mM a
1 >*4 1,*<"ii *
- • #
S.N.
S,N
.'<?
Fig. 28
(<i) Vitelline chromosome group (Chr.) becoming scattered after emission of the second
polar body (2.Pb.) in a rat egg. X 1,000. (b) and (c) Subnuclei (S.N.) of various sizes near
apparently normal male pronuclei (£). X 800. (From Austin and Braden, 1954b.)
Since subnuclei are probably derived from scattered chromo-
somes, and chromosomes may go astray even under apparently
normal circumstances, it is not unexpected that subnuclei are
occasionally found in eggs undergoing otherwise normal fertiliza-
tion or cleavage. It seems very likely that the chromosomes involved
in subnuclei would not enter into syngamy in a normal manner and
may even fail to take part at all. If this is so, the resulting embryo
could come to carry chromosomal anomalies such as mosaicism or
36 THE MAMMALIAN EGG
hypodiploidy. The occurrence of subnuclei may be subject to
genetic influence: Braden (1957) found subnuclei far more com-
monly (7-2 per cent) in eggs undergoing fertilization in one colony
of mice (V stock) than in the others he investigated (o to 0-2 per cent).
The frequency of occurrence of subnuclei in rat and mouse eggs
undergoing fertilization may be greatly increased by experimental
conditions, such as artificial insemination late in oestrus (Blandau,
1952), treatment of the eggs in situ with heat shock or systemically
administered colchicine (Austin and Braden, 1954b; Edwards, 1958a;
Piko and Bomsel-Helmreich, i960), or treatment of the spermatozoa
with ultra-violet or X-irradiation or with radiomimetic drugs
(Edwards, 1957^, b, 1958b) (Fig. 28b, c).
Rudimentary parthenogenesis. The second-metaphase chromosome
group in unpenetrated eggs may not break up but instead give rise
directly to a single nucleus (Table 2) ; this would be diploid, unless
by a remote chance the first meiotic division has also failed, in
which case it would be tetraploid. In certain non-mammalian
animals, in which parthenogenesis occurs naturally or can be
induced artificially, a diploid nucleus is thus formed, the process
representing one of the mechanisms of 'regulation to diploidy' (see
also p. 76; and Tyler, 1941, and White, 1954). Alternatively,
unpenetrated eggs may show spontaneous resumption of the second
meiotic division and develop a single nucleus after the expulsion
of the second polar body (Table 2). This nucleus would be haploid
(or diploid if the first meiotic division had failed). Eggs of this kind
are rarely encountered in untreated subjects in mammals of most
species, but remarkably common in the golden hamster. In this
animal, about three-quarters of the eggs recovered some 20 hr after
ovulation were found to have undergone activation with expulsion
of the second polar body, and nearly one-third of them had devel-
oped single nuclei that resembled normal pronuclei (Austin, 1956a)
(Fig. 29). In this series of observations, only one normal-looking
2-cell egg was found at a later stage, so that the parthenogenesis of
the great majority of the hamster eggs must have been purely
rudimentary. Similar experiences were reported by Chang and
Fernandez-Cano (1958): among unpenetrated eggs recovered 13 to
40 hr after ovulation, about 40 per cent had formed single nuclei
with or without emission of the second polar body. Uninuclear eggs
have also been reported in untreated rats, mice and voles (Austin
and Braden, 1954c; Austin, 1957b), but it was not known whether
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS
37
mamm
Fig. 29
Stages in the development of a single nucleus in impenetrated golden-hamster eggs.
(From Austin, 1956a.)
38 THE MAMMALIAN EGG
these had developed with or without second-polar-body expulsion.
Cold-shock treatment (hypothermia) had no significant effect
upon the incidence with which unpenetrated hamster eggs under-
went activation or developed nuclei (Austin, 1956a). In rabbits,
sheep, rats and mice, however, the incidence is greatly increased by
cold shock as well as other forms of experimental stimuli. In most
rabbit eggs chilled in situ by the application of ice to the Fallopian
tube, single diploid nuclei were formed, the second meiotic divi-
sion being suppressed (Thibault, 1947, 1948, 1949; Chang, 1952a);
sheep eggs seemed to react in the same way (Thibault, 1949 ; Thibault
and Ortavant, 1949). In rabbits, other procedures were also effec-
tive: culture in vitro, or treatment with heat (47°C), with hypertonic
solutions or with suspensions of spermatozoa (Pincus, 1936b, 1939a),
hypothermia (Shapiro, 1942). (It has been claimed that partheno-
genesis in the rabbit can proceed to the birth of viable young:
Pincus, 1939a, c; Pincus and Shapiro, 1940a, b.) In rats, chilling
caused about 10 per cent of the eggs to show nucleus formation
and on the evidence available all these eggs could be held to have
completed the second meiotic division so that the nuclei were
probably haploid (Austin and Braden, i954d). In mice, the same
result, though at a higher incidence (about 40 per cent), required
a different treatment, namely heat shock (immersion of the Fallopian
tubes in water at 44 to 45 °C); a few eggs of the same kind were
recovered when the treatment had been merely ether anaesthesia
(Braden and Austin, 1954c).
There is no certain evidence that mammalian eggs developing
single nuclei, whether haploid or diploid, can give rise to embryos
capable of surviving to birth, but some embryonic development is
known to be possible — to 2- and 4-cell eggs in the sheep and rodents,
and to blastocysts in the rabbit (one of which implanted — Thibault,
1949). The nuclei themselves, however, have a definite interest. In
rats and mice, these nuclei were found to be capable of achieving
roughly twice the nuclear and nucleolar volumes of normal female
pronuclei, despite the fact that they derived from equivalent
chromosomal material; the possible significance of this observation
is discussed later (p. 47). Beatty (1954) has recorded the finding of
spontaneous haploid mouse embryos which had reached the blasto-
cyst stage of development (3 \ days) ; they may have arisen partheno-
genetically, but since they came from mated animals origins through
androgenesis or gynogenesis cannot be excluded.
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 39
Sometimes, when the second mciotic division proceeds spon-
taneously or after artificial activation, in impenetrated eggs, it is not
succeeded by the expulsion of the second polar body and, conse-
quently, two (haploid) nuclei are formed. This is a rarer event than
the formation of a single nucleus but has been reported in the rabbit
(Thibault, 1949), rat (Austin and Braden, i954d), mouse (Braden and
Austin, 1954c) and hamster (Austin, 1956a; Chang and Fernandez-
Cano, 1958). The two nuclei can look remarkably like normal
male and female pronuclei, but are considered incapable of under-
going normal syngamy, at least in the rabbit egg, and to be unlikely
therefore to lead to any further development (Thibault, 1949).
Gynogenesis and androgenesis. The presence of a single nucleus in
an egg that has been penetrated by a spermatozoon is generally
owing to failure of either the male pronucleus, as in gynogenetic
development, or of the female pronucleus, as in androgenetic
development. (It is just possible that the uninuclear state can
arise from fusion of male and female, or two male, pronuclei:
Pesonen, 1949; Austin and Braden, 1954b.) The nuclei are haploid
unless one or other of the meiotic divisions has been inhibited, but
they are nevertheless capable of growing in an apparently normal
way to a large size, sometimes becoming bigger than a normal
pronucleus. Instances of uninuclear eggs possibly representing
spontaneous early gynogenesis and androgenesis have been described
in rats (Austin and Braden, 1954c), mice (Austin and Bruce, 1956)
and hamsters (Austin, i956d), and after heat treatment in rats (Austin
and Braden, 1954b). Attempts to induce gynogenesis artificially in
the mouse by X-irradiation of the testes of the males yielded thirteen
uninuclear eggs that could have been undergoing this form of
development (Bruce and Austin, 1956), but evidence indicated that
normal cleavage was most unlikely to have ensued. X-irradiation
or ultra-violet irradiation of the spermatozoa, or injection of
colchicine solutions into the uterus through the cervix, resulted in
the production in mice of some instance of early gynogenesis and
androgenesis, and there were indications that, while neither form of
development was likely to be protracted, the androgenetic embryo
was a little the more viable (Edwards, 1954, 1957a, b, 1958b).
Intraperitoneal injections of colchicine in rats, given 2 J hr after
mating, have resulted in a high incidence of androgenetic eggs
(3 8 per cent of penetrated eggs) ; the time was highly critical : with
similar injections given at 2 hr after mating, the incidence was
40
THE MAMMALIAN EGG
only o*9 per cent (Piko and Bomsel-Hclmreich, i960). The mechan-
ism involved appeared to be the exclusion of the whole of the
female chromatin in a polar-body-like structure, formed amito-
tically.
Aiieuganty. Anomalies of pronuclei may involve, not the number
of male or female pronuclei present in an egg, but the ploidy of
one or both of the pronuclei (Table 2). Aneuploidy in pronuclei
constitutes the state of aneugamy. The condition can arise through
TABLE 2
The nine theoretically possible kinds of Ootid, with respect to
Number and Ploidy of Polar Bodies (pb) and Female Pronuclei (pn),
THAT COULD ARISE THROUGH SUPPRESSION OF ONE OR BOTH POLAR BODIES.
First polar body
Emitted
Suppressed; meiosis stopped at:
Metaphase
1 pb (2N)
1 pn (2N) (b)
Anaphase
->-
Emitted
2 pb (2N + N)
1 pn (N) (a)
2 pb (N + N)
2 pn (N + N) (c)
C.
~<3
Suppressed ; meiosis
stopped at:
S3
1
1 pb (2N)
1 pn (2N)
(J)
0 pb
1 pn (4N)
(e)
0 pb
2 pn (2N + 2N)
(0
1
1
"I
1 pb (2N)
2 pn (N + N)
(g)
0 pb
2 pn (2N + 2N)
00
0 pb
4 pn (N + N + N + N)
In the absence of sperm penetration, these classes describe forms of partheno-
genetic eggs. If fertilization is initiated, the corresponding ootids would display :
(a) normal fertilization; (b), (d) and (e) aneugamy ; (c), (f), (g), (h) and (i) polygyny.
fertilization by a normal spermatozoon of an egg deriving from a
uninuclear octaploid primary oocyte (8N in dna content and
chromatid count, 4N in chromosome number) or of an egg in
which one or both mciotic divisions have failed, or through fertiliza-
tion by a polyploid spermatozoon. Clearly, the number of com-
binations of these variables is large, so that a wide variety of forms
of aneugamy are possible. This group of anomalies is, however,
likely to remain largely hypothetical until studies are made on the
chromosome complements of pronuclei, which will probably be
STRUCTURE AND FUNCTION IN MAMMALIAN FGGS 41
most practicable during the prophase stages of the first cleavage
mitosis. A few possible examples of aneugamy have already been
recorded. Giant eggs undergoing fertilization and displaying a
single female pronucleus, which may well have been polyploid,
were recovered from rats (Austin and Braden, 1954c; see also p. 15).
Eggs from mated rats treated with colchicine had two normal-
looking pronuclei but no second polar body; the female pronuclei
seem likely to have been diploid (Austin and Braden, 1954b). Giant
spermatozoa are occasionally encountered (rat: R. Kinosita, i960,
personal communication; cat: M. W. H. Bishop and Austin, un-
published data); these are probably polyploid and could lead to
aneugamy if they are capable of fertilization. Dimegaly (two sizes)
and polymegaly (several sizes) of spermatozoa have long been known
in insects, nemertines, annelids, amphibians and birds; some forms
are considered to arise through suppression of one or both sperma-
tocyte divisions and would accordingly be polyploid (Wilson, 1928,
p. 303).
Polyandry mid polygyny. Eggs recovered from treated as well as
from untreated animals at the time of fertilization have occasionally
been found to possess three well-formed nuclei. In some instances,
these were named as one female and two male pronuclei (rat : Austin
and Braden, 1953a, b; Austin, 1956b; Odor and Blandau, 1956;
Braden, 1958a; Piko, 1958 — mouse: Braden, Austin and David,
1954; Edwards and Sirlin, 1956; Braden, 1957; Edwards, 1957a—
hamster: Austin and Braden, 1956 — field vole: Austin, 1957b — pig:
Pitkjanen, 1955; Hancock, 1959, 1961; Thibault, 1959). In other
instances, pronuclei were identified as one male and two female (rat :
Austin and Braden, 1953b; Austin and Braden, 1954b, c — mouse:
Pesonen, 1949; Braden, 1957; Edwards, 1957a, b — rabbit: Thibault,
1949; Austin, 1960b— hamster: Hamilton and Samuel, 1956; Chang
and Fernandez-Cano, 1958; Ohnuki, 1959 — pig: Thibault, 1959).
In others again, identification was not made (rat: Tafani, 1889;
Ludwig, 1954 — mouse: Kremer, 1924 — cat: R. Van der Stricht,
191 1 ; Hill and Tribe, 1924 — ferret: Mainland, 1930 — rabbit:
Amoroso and Parkes, 1947; Austin and Braden, 1953b — pig: Pit-
kjanen, 1955 — cow: Pitkjanen and I vankov, 1956 — sheep: Pitkjanen,
1958). The presence of one female and two male pronuclei con-
stitutes the state of polyandry and arises from polyspermy — the
participation of two spermatozoa in fertilization. The reported
D
42
THE MAMMALIAN EGG
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STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 43
normal incidence of the condition among penetrated eggs varies
somewhat in different species but has been found generally to be of
the order of i or 2 per cent (Table 3) but in the pig it may be as
high as 10 per cent (Pitkjanen, 1955). Polyandry may become much
more common with coitus late in oestrus, and following heat treat-
ment (Table 3). Piko and Bomsel-Helmreich (i960) found that
hyperthermia induced in rats produced 8 to 10 per cent polyspermic
(dispermic) eggs in the Sherman and Long-Evans strains, but only
3-5 per cent in the Wistar CF strain. Hancock (1959, 1961) reported
that the incidence of trinuclear eggs in pigs allowed coitus at the
start of oestrus or at 24, 30 and 40 to 48 hr later was o, 3, 13 and
41 per cent, respectively. His cytological evidence indicated that the
trinuclear state could be ascribed chiefly to polyandry. Thibault
(1959), on the other hand, maintained that the principle effect of
late mating or insemination in the pig is an increase in the incidence
of polygyny, the increase for polyandry being relatively small (from
1-8 to about 12 per cent).
The general uncommonness of polyandry under normal circum-
stances is attributable chiefly to the relatively small number of
spermatozoa reaching the site of fertilization (see Braden and Austin,
1954a) and to the fact that either the vitelline surface or the zona
pellucida, or both, tend to become impermeable to spermatozoa
after the entry of the first (see pp. 88 and 92).
Polyandry has been studied in some detail in the rat. It was
observed that the two male pronuclei develop in remarkably close
parallel with each other (Fig. 30a, b, c and e), a feature that may be
owing to the operation of a co-ordinating influence (see p. 47) or
to the necessarily closely synchronous entry of the spermatozoa.
The volumes achieved by the pronuclei at full development were
individually always less than those of the corresponding normal
pronuclei, and this was true too for nucleolar volumes (Fig. 3od, e
and f). Indeed, the sum of the nuclear volumes (about 7,300 tt3) and
of nucleolar volumes (about 800 /x3) in polyandric eggs did not
differ significantly from the corresponding figures for normal eggs
(about 8,000 /x3 and 800 /x3, respectively). At the approach of
syngamy, contact occurred just as often between the two male
pronuclei as between a male and the female, testifying to a lack of
specificity in the forces that draw the pronuclei together at this phase
of fertilization. By all appearances, the general course of syngamy
in polyandric eggs was the same as in normal eggs, except for the
44
THE MAMMALIAN EGG
presence initially of the extra male pronucleus and later of the extra
chromosome group. The three chromosome groups that eventually
become evident are similar in appearance and they move together
©
<§
®
®
Fig. 30
Pronuclei in rat eggs, (a), (b), (c) and (e) Stages in the development of polyandry arising
from dispermy, showing the close similarity throughout between the two male pronuclei.
(d), (e) and(/) Pronuclei at full development after monospermic, dispermic and trispermic
penetration, respectively. (Drawn from photographs.)
to form a single gathering in the centre of the egg. Almost in-
variably, a normal-looking bipolar spindle was found to have
formed (Fig. 31), despite the triploid number of chromosomes, and
the first cleavage division seemed to go through in the usual way.
Polyandric early embryos could be recognized by the possession of
two sperm tails in the cytoplasm, and such embryos, normal in
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS
45
appearance, were found up to the 8-cell stage (Austin and Braden,
1953b). Piko and Bomscl-Helmreich (i960) have recorded triploid
and mosaic (3N/2N) embryos at mid-gestation (11 days) in rats at
Fig. 31
Telophase first-cleavage spindle in a polyspermia rat egg. X 1,700.
(From Austin and Braden, 1953b.)
a frequency corresponding to that of polyandry, but were unable
to find any at later stages.
The other group of trinuclear eggs, namely those that have one
male and two female pronuclei, display the condition of polygyny
and can originate in three different ways : (a) The spermatozoon may
enter an egg deriving from a binuclear oocyte. Since binuclear
oocytes seem rarely to survive to maturation (p. 20), this source of
polygyny must be considered a most infrequent one. (b) The first
polar body may fail to form after the first meiotic division has gone
through to telophase; consequently, two second meiotic spindles
develop and lead to the presence of two female pronuclei in the
ootid. This also seems to be a most uncommon mechanism, but it
has been detected in untreated animals — in an outbred stock of
mice (V) at an incidence of about 2 per cent (Braden, 1957). (c) The
second polar body may fail to form after the second meiotic division
has gone through to telophase. This is probably the commonest of
the three processes responsible for the presence of two female
pronuclei and it has been induced under experimental conditions.
46 THE MAMMALIAN EGG
The application of heat to the Fallopian tubes of mice 3 hr after
mating increased the incidence of second-polar-body suppression
from 0-5 to 12-4 per cent (Braden and Austin, 1954b). Studies on
special groups of mice have revealed that, in the outbred stock just
mentioned (V), suppression of the second polar body occurs at
higher incidence than that of the first, namely, between 4 and 5 per
cent (Braden, 1957). Polar-body suppression is evidently a geneti-
cally controlled factor in these animals, and is the probable cause
of the triploidy recognized to be relatively common in this strain
of mice (Beatty and Fischberg, 1951). In contrast to the effect of
delayed mating in the rat, which often increases the frequency of
polyandry as already noted, delayed mating in the hamster has been
found to produce an even more striking increase in polygyny, thirty
out of eighty-eight penetrated eggs (34 per cent) showing this con-
dition (Chang and Fernandez-Cano, 1958). Polyandry was not
increased in incidence. Recent observations on pig eggs reveal that
the frequency with which polygynic eggs are found is greatly
increased, from o to 21 per cent, if coitus or artificial insemination
is effected more than 36 hr after the onset of oestrus (Thibault,
1959). Intraperitoneal injections of colchicine have been reported
to cause second-polar-body suppression at a high incidence (38 per
cent of penetrated eggs) in rats, if given 2 hr after mating; injection
at 2 \ hr resulted in suppression in only 11 per cent of eggs (Piko and
Bomsel-Helmreich, i960). (See also Fischberg and Beatty, I952-)
Suppression of the second polar body can accompany polyspermy
and so give a quadrinuclear egg containing two female and two
male pronuclei, and this has been reported in a pig egg (Thibault,
1959) and a rat egg (Austin and Walton, i960). Alternatively, an
egg may complete maturation normally but be entered by three
spermatozoa (trispermy) and so come to have one female and three
male pronuclei. The occurrence has been reported in untreated
rats (Austin, 1951b; Austin and Braden, 1953b), and in animals in
which hyperthermia had been induced (Austin, 1956b). Although
no measurements are recorded of the nuclei in trispermic eggs, it is
clear from the general appearance that the female and all the male
pronuclei each attain a smaller size than that of the corresponding
pronuclei in normal eggs (Fig. 3 of). One example of spontaneous
tetraspermy has been described in a rat egg — the five nuclei were all
well formed, the four male pronuclei being equally larger than the
female pronucleus (Piko, 1958). Tetra- and pentaspermic eggs have
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 47
been found in rats after induced hyperthermia, but their nuclear
state was too irregular to justify their description as truly quinque-
nuclear and sexinuclear eggs (Austin, 1955, 1956b).
NUCLEOCYTOPLASMIC RELATIONS IN FERTILIZATION
Certain aspects of nuclear development in eggs testify to the
closeness of the nucleocytoplasmic interdependence recognized as
a feature of cells in general. It is a common observation that the
chromosome groups emitted within polar bodies often do not give
rise to resting nuclei, and, on those rare occasions when the sperm
head becomes lodged in a polar body, or extruded from the vitellus
in a small mass of cytoplasm, it too fails to give rise to such a nucleus.
Presumably, the organelles that normally participate in nucleus
formation are often lacking from polar bodies; in addition, polar
bodies would probably be deficient in the necessary substrate. That
the availability of substrate material is a limiting factor in pronuclear
growth is strongly suggested by the subnormal size exhibited by
pronuclei in polyandric and polygynic eggs. This limitation in
growth stands in strong contrast to the supernormal size achieved
by female pronuclei in rudimentary parthenogenesis or gynogenesis.
Substrate availability is, however, evidently not the only condition
that determines the ultimate size of pronuclei. The volumes of
single nuclei developing in eggs were found to be less than the
combined volumes of normal male and female pronuclei, so it is
inferred that there must be yet another restricting influence, possibly
inherent in the nuclei themselves (Austin, 1952a; Austin and Braden,
1955). Such an influence, predominating in the female pronuclei of
eggs such as those of the rat and mouse, could underlie the large
difference in relative size of male and female pronuclei. On the
other hand, this pronuclear disparity could be ascribed equally well
to a greater affinity of the male pronucleus for cytoplasmic substrate.
Suppression of pronuclear development, apparently by influences
arising in or mediated by the cytoplasm, has been described in
urodele eggs : in polyspermic fertilization, the supernumerary male
pronuclei regress when syngamy is effected between the female
pronucleus and the successful male pronucleus (Fankhauser, 1948).
Evidence of a different nature was provided some years ago by
Brachet (1922) who noted that the development of the male pro-
nuclei and associated asters in polyspermic sea-urchin eggs proceeded
exactly synchronously with that of the female pronucleus and its
48 THE MAMMALIAN EGG
aster. There appeared to be a mechanism in the egg which, under
normal circumstances, could be held responsible for co-ordinating
the development of the pronuclei. Correlation of a similar kind has
been observed also in several phases of mammalian fertilization. In
the rat, the first nucleoli make their appearance at about the same
time in both pronuclei; the pronuclei reach their maximum size
together and, later, start simultaneously upon the process of syn-
gamy. Polyspermic (dispermic) rat eggs, too, provide evidence of
co-ordination in the striking similarity of form exhibited by the
two male pronuclei at the various stages of pronuclear development
(Austin, 1951c; Austin and Braden, 1953b, 1954b).
Attempts to disturb the synchrony of development of pronuclei,
by treatment with colchicine, cold shock or heat shock, yielded only
transient effect, the induced disturbance soon becoming corrected
(Austin and Braden, 1954b). In mouse eggs penetrated by X-irradi-
ated spermatozoa, the pronuclei often developed well but failed to
enter upon syngamy; it was surmised that irradiation had impaired
the male pronucleus, rendering it incapable of proceeding further,
and that the female pronucleus was unable to go forward alone
(Bruce and Austin, 1956). These observations add support for the
idea that, in eggs as in tissue cells, the cytoplasm exerts a controlling
influence over nuclear function, an idea for which a solid bio-
chemical foundation has already been laid through work on tissue
cells (see Brachet, 1957).
Nucleocytoplasmic relations in the synthesis of dna are discussed
in the next section.
Cleavage Nuclei
Fertilization may be said to end with the condensation of the
chromosomes in the male and female pronuclei and the coming
together of the two chromosome groups to form a single group.
These events can also be regarded as constituting the prophase of
the first cleavage mitosis, for the chromosomes proceed immediately
thereafter to become arranged as the metaphase plate of the first
cleavage spindle. There is now evidently a pause, since eggs re-
covered from rats at about the time of the first cleavage are more
often found in metaphase than in stages just preceding or succeeding.
The mitosis passes to telophase, cytoplasmic division occurs, and
interphase nuclei arc reconstituted from the chromosome groups
(Fig. 32). The mode of formation of the nuclei resembles that of
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 49
the female pronucleus after the second meiotic division, numerous
minute nucleoli appearing in the midst of the fading chromosome
group, while an encircling nuclear membrane becomes visible.
Some coalescence of nucleoli is associated with the subsequent
Fig. 32
Stages of cleavage in the rat egg. (From Austin, 1959c.)
growth of the nuclei. When fully grown, the nuclei of living 2-cell
rat eggs are similar in general structure to pronuclei, except that
fewer nucleoli develop and small elevations of material can often be
seen on the surface of some of the nucleoli. Nucleolar inclusions,
too, are occasionally met with, ranging from small spherical bodies
with a diameter a fraction of that of the nucleolus to others so large
that the nucleolar substance is reduced to a mere shell (Fig. 33). The
inclusions seen in 2-cell rat eggs are evidently composed of fluid
like the nuclear sap, for sometimes a nucleolus with a large inclusion
may be observed to 'break', releasing the contents which mingles
immediately with the nuclear sap. The nucleolar substance then
rapidly assumes a spherical form, now much smaller in diameter
than before. The nuclei of living 2-ccll rat eggs examined by phase-
contrast microscopy were often found to contain other structures
than nucleoli and their attached material. These were small,
50
THE MAMMALIAN EGG
eir
irregularly shaped masses, often with a complex structure; th
nature is conjectural.
After a time, changes occur in the 2-cell nucleus that presage the
next mitosis. The nucleus decreases in volume, the nucleoli diminish
Fig. 33
Nuclei from rat 2-cell eggs, showing nucleolar inclusions. X 1,200.
in size and number and disappear, and the chromosomes condense —
the course of events is similar to the first-cleavage prophase changes
of the pronuclei. Mitosis then advances through metaphase and
anaphase to telophase, the cytoplasm undergoes division, and nuclei
are reconstituted. Nuclear and nucleolar volumes are approximately
halved at each stage, and the number of nucleoli is reduced (Hert-
wig, 1939; Austin and Braden, 1953c) (Fig. 12). The overall size of
chromosome groups and the chromosomes themselves become
progressively smaller. By contrast, the nucleolus-associated material,
just discernible at the 2-cell stage, becomes increasingly prominent,
and, by the 16-cell stage in the rat, the perinucleolar elevations are
so large that they often conceal the nucleoli (Fig. 34a to d). Ultra-
violet microscopy at a wavelength of 2,600 A shows that the
material composing the elevations contains a high concentration of
nucleic acid, whereas the nuclear sap and the nucleoli have very
much less (Austin, 1953; Austin and Braden, 1953c) (Fig. 34c to j).
Observations by fluorescence microscopy, with acridine orange as
vital fluorochrome, reveal a similar distribution and indicate that
the nucleic acid in question is dna (Figs. 35 and 36). Histological
studies with Fculgen's reagent applied to fixed material provide
confirmation (Alfert, 1950; Braden and Austin, 1953), and it is
Fig. 35
Rat 2-cell egg. X 500.
Fig. 36
Rat 8-cell egg. X 500.
Facing page 50
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS
51
apparent that a system, resembling in certain respects the nucleolus-
associated-chromatin system of Caspersson (1950), becomes increas-
ingly more evident as cleavage proceeds. Not until implantation
231 ♦>
£
v:
■ w H
m \
i ft
•
1
1
m%
■F
Fig. 34
Nuclei in rat eggs: (a) 4-cell, (b), (c) and (e) to (j) 8-cell, (d) 16-cell. Photographs in (/),
(//) and(j) were taken by ultra-violet microscopy at 2,600 A, the remainder by phase-contrast.
(<*)-(</) X 2,000. (e)-(j') X 900. (From Austin, 1953.)
52 THE MAMMALIAN EGG
of the embryo occurs, however, is there evidence of the cyto-
plasmic basophilia and the high nucleolar rna concentration,
which form integral parts of the Caspersson system (Alfert, 1950),
and it therefore seems unlikely that protein synthesis is a quantita-
tively important feature of metabolism in the embryo during
cleavage. Consistently, Greenwald and Everett (1959) have reported
that evidence for active protein synthesis, as inferred from uptake
of [35S] methionine, was clearly shown by mouse ovarian eggs and
blastocysts, but not by embryos in the cleavage stages. Other
aspects of the nucleocytoplasmic relationship in processes of synthesis
are discussed later (p. 61).
On the other hand, there is no reason to doubt that dna syn-
thesis takes place during cleavage. The mammalian egg lacks the
large cytoplasmic stores of dna that have been demonstrated in
sea-urchin and frog eggs (Zeuthen, 195 1; HorT-Jorgensen, 1954)
and the total nuclear dna is doubled at each stage of cleavage
(Dalcq and Pasteels, 1955). The increasingly large size of the
perinucleolar masses can be attributed simply to the physical result
of the packing of the same amount of material into progressively
diminishing nuclei. Despite this effect, the characteristic concentra-
tion of DNA-protein designated the 'sex chromatin' (Barr, Bertram
and Lindsay, 1950; Graham, 1954) does not become discernible in
cat, monkey and human embryos until the end of cleavage, that is
to say, at the time of implantation or shortly beforehand (Austin and
Amoroso, 1957; Park, 1957; Austin, 1961b) — approximately when
the size of embryonic cells has reached the size of an average tissue
cell.
Cytoplasm
Physical Features
Yolk. Among the most characteristic features of the cytoplasm
of eggs is the presence of stored nutrient material (yolk or deuto-
plasm) and the manner in which it is distributed. On the basis of
the amount of yolk eggs contain, they can be classified into two
groups: those with much, the mcgalccithal eggs, and those with
little, the miolecithal eggs. This subdivision is somewhat arbitrary,
for there exists in the animal kingdom as a whole a continuous
series between the extreme forms. The mcgalccithal egg consists
essentially of a mass of yolk on the surface of which lies a small
cytoplasmic disc wherein the nuclear structures reside and which
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 53
alone undergoes cleavage. The relatively large size of these cg^s is
therefore attributable to the quantity of yolk that they carry. To
this group belong the eggs of birds, reptiles, fish and amphibians,
and also the oviparous mammals, the monotremes. The eggs of all
Fig. 37
Armadillo oocyte (Dasyptt* novemcinctus), showing segregation
of yolk. (Drawn from illustration by Newman, 1912.)
other mammals are typically miolecithal, the yolk being much
scantier and to varying degrees mingled with the cytoplasm; the
whole vitellus takes part in cleavage. Variations in the size of
miolecithal mammalian eggs are evidently due in no small measure
to variations in the mass of active cytoplasm, for larger eggs in this
series have larger nuclei. In the egg of the native cat Dasyurus (Fig.
io) and the armadillo Dasypus (Fig. 37), much of the yolk in the
oocyte and ootid is gathered at one pole and forms a separate body
during early cleavage. Suggestions of polarity in the arrangement
of the yolk components are seen also in other mammalian eggs,
such as those of the guinea-pig (Fig. 38), but here the yolk is disposed
as globules or droplets. In the eggs of some bats (Fig. 39), the cat
(Figs. 19, 20, 40 to 45), the ferret, the dog (Fig. 46), the fox and the
pig, very numerous globules are distributed more or less uniformly
throughout the vitellus. The eggs of man, monkey, the horse, the
cow, the sheep, the rabbit and the murine rodents mostly have a
granular yolk with a pattern of distribution characteristic for each
species. In certain inbred strains of mice, it has been shown that the
pattern is recognizably different with each strain (Braden, 1959)-
54
THE MAMMALIAN EGG
In some animals, distinct changes in the pattern of cytoplasmic
particulates follow sperm penetration ; these have been described in
the bat and guinea-pig (Van der Stricht, 1923), the rhesus monkey
Fig. 38
Guinea-pig egg. X 550.
**&Mm
*
Fig. 39
Egg of common pipistrelle bat, with late telophase
second-meiotic spindle. X 900.
(Lewis and Hartman, 1933, 1941), the mouse (Gresson, 1941) and
the rabbit (Nihoul, 1927; Austin and Bishop, 1957b).
Yolk material may become extruded from the cytoplasm and
accumulate in the perivitellinc space; the process is known as
deutoplasmolysis and is thought to represent either a disposal of
superfluous yolk that might otherwise interfere with cleavage, an
adjustment of the nucleocytoplasmic ratio or the provision of
nutrient materials for the developing embryo. The extruded yolk
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 55
differs in form and amount in different species; ejection occurs
chiefly about the time of fertilization and the first cleavage division.
Deutoplasmolysis has been described in the opossum Didclphys
Fig. 46
Dog morula. X 250. (E. C. Amoroso.)
(Hill, 1918; Hartman, 1919; McCrady, 1938), native cat Ddsyurus
(Hill, 1910), bat (Van der Stricht, 1909), guinea-pig (Lams, 1913),
cat (Van der Stricht, 1923; Hill and Tribe, 1924), pig (Heuser and
Streeter, 1929), ferret (Hamilton, 1934), horse (Hamilton and Day,
1945), field vole (Austin, 1957b) and rat (Odor, i960). In Didelphys,
the process takes an extreme form; the blastomeres of 2-cell and
4-cell eggs generally appear to have incomplete plasma-membrane
envelopes and the blastomere cytoplasm is in places continuous with
material that is eventually to be distinguished as discarded yolk.
Fine structure. Few investigations have yet been made on the fine
structure of mammalian egg cytoplasm, the most detailed being
those of Yamada, Muta, Motomura and Koga (1957) on the mouse,
and Sotelo and Porter (1959) and Odor (i960) on the rat. In oocytes
and ootids, the endoplasmic reticulum appeared to exist only in the
form of a few small vesicles deficient in rna particles (ribosomes),
although Odor noted the presence of many atypical membranous
elements before the preovulatory changes. In the 2-cell egg, there
were many more such vesicles and occasionally they showed
continuity with the outer layer of the nuclear membrane, in a
maimer that has often been described in tissue cells. Sotelo and
Porter suggest that this difference in the 2-cell egg marks the
56 THE MAMMALIAN EGG
beginning of a differentiation of the endoplasmic reticulum. Very
numerous small dense particles, identical with the rna particles
responsible for basophilia, were distributed throughout the cyto-
plasm and were more common in eggs undergoing fertilization and
early cleavage than in oocytes. The bulk of the vitellus was finely
granular in appearance and more or less uniform in texture; this
material was considered to be deutoplasmic in nature. Scattered
throughout, however, were numerous irregular masses of a more
densely granular nature, often connected by bridges or trabeculae
and containing many mitochondria and other small bodies; this
material probably represented the active cytoplasm. Among the
other small bodies just mentioned, there were many examples of
an unusual type of structure — a vesicle containing many small
vesicles. This was termed a 'multivesicular body' or 'vesicular
conglomerate' ; these bodies increased in number during maturation
and fertilization, and they were believed to break down in the later
stages, liberating their content of smaller vesicles. Odor (i960)
confirms the increased occurrence in the later stages of oocyte
growth. (Similar structures have been seen in glomerular epithelial
cells by Yamada, 1955, and in spider oocytes by Sotelo and Trujillo-
Cenoz, 1957.) In the ovarian oocyte, the surface of the vitellus was
found to be thrown up into microvilli which project a short distance
into the zona pellucida. Processes from the overlying follicle cells
also penetrate the zona and to a greater extent, often passing com-
pletely through, but no continuity appeared to be established
between the cytoplasm of follicle cells and oocyte (Fig. 47).
The ultrastructure of the cytoplasm in tubal eggs of the golden
hamster appears to be similar in general to that described by Sotelo
and Porter for the rat. Here, too, the finer more homogeneous
material making up the bulk of the vitellus is liberally interspersed
with irregular groups of a coarser substance containing bodies
resembling mitochondria (Fig. 27). Multivesicular bodies were not
seen.
Changes in size and form. Observations on the eggs of the rabbit
and the common laboratory rodents indicate that the size and shape
of the vitellus, in these eggs at least, is maintained dynamically and
not merely by physical conditions such as surface tension or cortical
rigidity. The vitellus can undergo a comparatively sudden reduction
in diameter, the contraction being associated with a release of fluid
into the perivitelline space. Contraction occurs most noticeably on
STRUCTURE AND FUNCTION IN MAMMAU \N EGGS 57
two occasions: at the time of expulsion of the first polar body and
shortly after the entry of the spermatozoon. In the former instance,
the change in volume is responsible for transforming the peri-
vitelline space from a potential state to a real one. The contraction
Fig. 47
Relations between follicle cells (stippled),
zona pellucida (horizontal lines) and vitellus
(black) in the late ovarian oocyte, as revealed
by published accounts based on electron
microscopy. (Semi-diagrammatic.)
following sperm entry is generally taken as a feature of activation,
and, indeed, it is also clickable by the various stimuli that are known
to be capable of initiating parthenogenetic development (p. 38).
Dauzier and Thibault (1956) maintain that contraction can be
induced in vitro by the mere presence of spermatozoa in the medium.
The decrease in volume after sperm entry has been observed in the
rabbit (Gregory, 1930; Pincus and Enzmann, 1932; Thibault, 1947-
1949), mouse (Sobotta, 1895), guinea-pig (Lams and Doormc,
1908), dog, cat and bat (Van der Stricht, 1923), rat (Gilchrist and
Pincus, 1932; Pincus and Enzmann, 1934; Pincus, 1936a; Austin
and Braden, 1954b), cow (Hamilton and Laing, 1946), hamster
(Austin, i956d) and pig (Pitkjanen and Sheglov, 1958). It has been
estimated to represent some 13 to 17 per cent of the vitelline volume
in the rat egg and about 9 per cent in the hamster egg, but was too
small for accurate assessment in the diminutive egg of the field vole
(Austin, 1957b). Krassovskaja (1935b) reports that the rabbit egg
increases in volume after the formation of the pronuclei and up to
the stage of the formation of the cleavage spindle.
58
THE MAMMALIAN EGG
The most obvious modifications in shape of the vitellus are those
occurring in polar-body emission and in cleavage, but others are
seen also. Prior to polar-body formation, the surface becomes
elevated in the region that overlies the second maturation spindle;
the elevation may persist for hours or even days in the absence of
fertilization, and eventually subsides when the spindle breaks up.
A similar elevation develops at the site of attachment of the sperma-
tozoon and lasts for a short while after entry of the spermatozoon
into the vitellus. This reaction is analogous in some respects to the
outgrowth of the fertilization cone of many invertebrate eggs.
Unfertilized eggs often undergo fragmentation and in these circum-
stances the cytoplasmic masses may take on bizarre shapes, presum-
ably under the influence of disorganized cleavage forces. Some eggs
penetrated by X-irradiated spermatozoa have been observed to
share the same fate (Bruce and Austin, 1956) (Figs. 48 and 49).
Figs. 48 and 49
Mouse eggs cleaved after fertilization with X-irradiated spermatozoa.
Bruce and Austin, 1956.)
420. (From
Another form of movement evinced by the egg cytoplasm is a
constant steady streaming or 'boiling' motion which can best be
demonstrated by time-lapse photography. This is evidently the
same phenomenon as cytoplasmic streaming or 'cyclosis' which is
well known in other mammalian cells under conditions of tissue
culture but especially in plant cells.
structure and function in mammalian eggs 59
Chemical Components
Much attention has been given to the distribution in eggs of
basophilia and of the nucleic acids, the presence of which basophilia
is generally held to denote. As the oocyte grows, a perinuclear
Fig. 50
Rat 8-cell egg as seen by dark-ground illumination,
showing distribution of granules. X 350.
band of rna develops in the cytoplasm (Vincent and Dornfeld,
1948). During fertilization, the cytoplasm in sections of fixed rat
eggs showed evenly distributed weak basophilia, and strong acido-
philia. In 4-cell and 8-cell eggs, the intensity of the basophilia was
strongly augmented but was restricted in distribution chiefly to the
regions about the nuclei; acidophilic material, too, had a perinuclear
arrangement (Braden and Austin, 1953). Observations based on the
ultra-violet absorption of living rat eggs showed that, during
fertilization, moderately strong absorption was associated with the
irregular masses of granular elements, while the hyaloplasm showed
a lower absorption evenly spread throughout. With successive
cleavage divisions, the granular elements gathered more and more
about the nuclei, leaving the peripheral cytoplasm free (Fig. 50);
the absorption in the peripheral hyaloplasm tended to diminish
(Austin and Braden, 1953c) (Fig. 51). Absorption in the hyaloplasm
is probably attributable to rna, while that associated with granular
elements seems more likely to be due to mononucleotides.
60
THE MAMMALIAN EGG
Living rat eggs have also been studied by fluorescence microscopy,
involving acridine-orangc staining and irradiation in the near ultra-
violet (Austin and Bishop, 1959a; Austin and Amoroso, 1959). In
the cytoplasm, only the granular elements fluoresced and these
wffH
Ultra-violet absorption by rat 8-cell egg showing distribution of
nucleic acids and nucleotides. X 500.
showed a brilliant red colour. The red fluorescent granules lay
chiefly in the neighbourhood of the germinal vesicle in the oocyte
(Fig. 15), but were irregularly distributed in numerous groups
throughout the cytoplasm in eggs undergoing fertilization and in
2-cell eggs (Figs. 25, 26 and 35). More distinct aggregation was
evident in 4-cell eggs, and at the 8-cell stage dense masses of red
granules were grouped about each nucleus (Fig. 36). It seemed
likely that, under the conditions of these experiments, the red
fluorescence was given by mitochondrial mononucleotides.
Sotelo and Porter (1959) point out that there is good reason to
believe that basophilia in tissue cells is located in the small dense
particles (Pallade's small granules, ribosomes) which, on isolation,
have been shown to contain high concentrations of rna. They
found particles of this kind (150 to 200 A in diameter) in the cyto-
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 61
plasm of rat oocytes and fertilized eggs, distributed throughout the
matrix of the cytoplasm and without any obvious association with
other cytoplasmic structures. There were more particles in fertilized
and developing eggs than in oocytes. It seems probable that the
rna in these particles is responsible for the ultra-violet absorption
reported by Austin and Braden (1953c) in the hyaloplasm of eggs;
the particles are certainly much too small to correspond to the red
fluorescent granules observed by fluorescence microscopy.
The extensive observations of A. M. Dalcq and his colleagues
(see Dalcq, 1955a, 1956, 1957; Borghese, 1957) have led to different
conclusions. In addition, their findings have been built into the
theory that cytoplasmic characteristics confer a bilateral symmetry
on the oocyte and the egg during fertilization, and later serve to
distinguish those regions of 2- and 4-cell eggs, and those blastomeres
of 8-cell eggs, that are to become either the inner cell mass or the
trophoblast of the blastocyst. Symmetry of the oocyte is held to be
due to the presence of a more 'condensed' form of cytoplasm,
containing more and larger mitochondrial granules, at the animal
pole and on one side of the animal-vegetal axis ; this region contains
more rna as indicated by the basophilia detectable by pyronine
staining, before but not after treatment with ribonuclease. On the
other side of the cell, the cytoplasm is somewhat vacuolated, con-
tains fewer granules, and, in animals such as the guinea-pig, is
distinguished by the presence of numerous fat globules. The planes
of cleavage are not clearly related to the plane of symmetry, but,
when the 8-cell stage is reached, four of the blastomeres are found
to be relatively larger than the others. The larger blastomeres
contain the more vacuolated cytoplasm and these are the ones
destined to constitute the trophoblast by increase in size with low
mitotic frequency. The smaller blastomeres, richer in rna, in-
crease further their content of nucleic acid as they rapidly multiply
to form the inner cell mass. Thus, both the form and distribution
of the rna bodies define the future development of parts of the
egg and early embryo. In both types of cell, the rna is described
as being associated with the larger mitochondrial granules which
are distributed in the outer regions of the cells. Some rna, how-
ever, accompanies the finer granules which gather near the nuclei.
No rna, apparently, is identified in the hyaloplasm.
For Dalcq, the distribution of rna is only part of the story.
As he and Pasteels (1955) have shown, doubling of the nuclear
62 THE MAMMALIAN EGG
dna occurs during the interphase before each cleavage of the egg,
and the extra dna must presumably be synthesized from cyto-
plasmic substrate. Dalcq maintains that mucopolysaccharide and
'plasmalogen' (possibly an acetalphosphatide), the concentrations of
which have been found to fall immediately after mitosis and build
up again during interphase, are precursors of the dna. Indeed, it
is felt that the accumulation of these precursor substances to a
threshold level might initiate the new division. The mucopoly-
saccharide is located in groups of mitochondria that occupy, in
4-cell eggs and onwards, the peripheral parts of the blastomeres
destined to form the trophoblast, and its concentration increases as
this structure develops. Plasmalogen, on the other hand, is found
in the hyaloplasm. Both mucopolysaccharide and plasmalogen are
believed to originate in the nucleoli (which were shown often to
have metachromatic inclusions) and to pass into the cytoplasm when
nucleoli press up against the nuclear membrane. It is suggested, too,
that smaller nucleoli sometimes escape in toto into the cytoplasm.
In these ways, the cytoplasm is thought to be activated by sub-
stances that have derived from the chromosomes through the inter-
mediation of the nucleoli.
Dalcq's theory is reminiscent in some respects of Kremer's (1924)
suggestion that substances originating in the cytoplasm pass into the
nucleus where they become specifically modified under the in-
fluence of genes, are stored in the nucleoli and eventually pass back
into the cytoplasm, within extruded nucleoli, as carriers of hereditary
characters. The idea, in general terms, seems reasonable enough,
though the transfer of nucleoli as such, or even of less organized
material, directly from the nucleus to the cytoplasm is inconsistent
with current views. It would be more acceptable to maintain that
the influence is indirect, a new substance being elaborated on the
cytoplasmic side of the nuclear membrane, but controlled in its
properties by gcnically determined agents within the nucleus.
There is a good deal of evidence that cytoplasmic rna is syn-
thesized under these conditions (see Brachet, 1957). In this connec-
tion, it is of special interest that in one species, the Chinese hamster,
there arc distinctive sacculations about the pronuclei (Fig. 52) and
cleavage nuclei which might well be associated with processes of
synthesis at the nuclear membrane.
Dalcq further postulates that, as the rna concentration increases
in the inner cell mass of the implanted blastocyst, small granules
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 63
charged with alkaline phosphatase are produced in progressively
larger quantities, first around the nuclei and then throughout the
cytoplasm. For both substances, increase in amount is considered
to indicate active protein synthesis. Consistently, with regard to the
Fig. 52
Pronucleus of the Chinese-hamster egg. X 1,200.
rna increase, Alfert (1950) in the mouse and Skreb (1957) in the
bat reported that the cytoplasmic basophilia of the embryo becomes
strongly augmented at the time of implantation, particularly in the
inner cell mass.
Organelles
Mitochondria. The high content of mitochondria in eggs is
indicated by Gresson's (1940a) finding that, in the centrifuged oocyte
of the mouse, mitochondria (identified by staining with Janus Green
B) occupy one of the broadest of the zones that become separated.
Early oocytes bear, near the germinal vesicle, a distinctive structure
known as the yolk nucleus (Balbiani's body, corps vitellin, etc.)
which consists of the centrosome surrounded by a zone of clear
cytoplasm and around this lies a dense array of mitochondria and
argentophilic components of the Golgi complex (p. 64). As the
oocyte grows, the mitochondria spread out in small groups through
the cytoplasm and come to occupy the regions immediately around
the germinal vesicle and in the periphery of the cell. It is during
these changes that yolk formation predominantly occurs (see Van
der Stricht, 1923). During the pronuclear phase, mitochondria tend
64 THE MAMMALIAN EGG
to be more numerous in the central than in the peripheral regions,
and later become closely gathered about the first cleavage spindle.
In the 2-cell stage, the distribution is similar to that of the pronuclear
egg (Gresson, 1941, 1948).
Mitochondria of a roughly spherical or oval form, but with the
characteristic internal cristae, have been described by Yamada,
Muta, Motomura and Koga (1957), Moricard (1958), Sotelo and
Porter (1959) and Odor (i960) in ultra-thin sections of mouse and
rat eggs, and their distribution corresponded broadly with that
observed by Gresson. Yamada et al. remarked on a feature that
seems peculiar to oocyte mitochondria, namely vacuolization. The
vacuoles vary in size, and are round or irregular in shape; they are
bounded by single membranes and appear less opaque than the
surrounding matrix. Bodies of the same size and shape as mito-
chondria and lying in the same pattern, are visible also in hamster
eggs, both penetrated and unpenetrated, but the absence of cristae
precludes their recognition as fully differentiated mitochondria
(Figs. 27 and 54). The arrangement of the red fluorescent granules
in rat eggs, evident after treatment with acridine orange, is also
similar to that of mitochondria (Figs. 15, 16, 25, 26, 35 and 36).
Golgi material. In early oocytes, a strongly argentophilic and
osmiophilic structure, identified as the Golgi apparatus, is readily
demonstrable associated with the yolk nucleus at one side of the
germinal vesicle. As the oocyte grows, the Golgi material breaks
up, becoming distributed around the nucleus and later throughout
the cell, often in association with the groups of mitochondria. In
centrifuged oocytes of the mouse, granules of Golgi material fill a
broad band separate from that occupied by mitochondria (Gresson,
1940a). During fertilization and the first cleavage division, the Golgi
granules tend to gather about the pronuclei, particularly just before
syngamy, and also about the 2-cell nuclei (Nihoul, 1927; Gresson,
1948).
By electron micrography of mouse and rat eggs, Yamada et a\.
(1957), Sotelo and Porter (1959) and Odor (i960) found a complex
structure containing paired membranes and a number of spherical
vacuoles which varied greatly in size; this was disposed close to one
pole of the nucleus in the early oocyte and was considered to consist
of Golgi material. In late oocytes, small groups of parallel mem-
branes were scattered chiefly through the peripheral cytoplasm. In
the cytoplasm near the arrays, there were numerous small vesicles
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 65
resembling elements identified as Golgi material. During fertiliza-
tion and cleavage, the distribution became more general. Odor
reported that the characteristic Golgi complex was never seen in
secondary oocytes and ootids.
Fig. 53
Loss of light refraction at the surface of a penetrated golden-hamster
egg (right). X 130. (From Austin, 1956c.)
Cortical granules. Low-power examination of hamster eggs with
dark-ground illumination shows that the vitelline cortex refracts
light much more before than after sperm penetration (Fig. 53). The
cause evidently resides in the possession by the cortex of numerous
small granules, in impenetrated but not in penetrated eggs. The
granules were estimated to be mostly between o-i and 0-5 {jl in
diameter and to number 50 to 100/ 100 /x3 of egg surface (Austin,
1956c). In their size, number and evident response to sperm penetra-
tion, the cortical granules in hamster eggs are similar to those in
sea-urchin eggs; when examined by high-power phase-contrast
microscopy, the resemblance in appearance between the cortical
granules of the two species is quite striking. Hamster cortical
granules, however, appear to be uniform in structure, except for
small variations in size, whereas the sea-urchin cortical granules
seemed, according to Endo (1952), to have light and dark halves.
The fine structure as determined by electron microscopy also
appears to differ, the hamster cortical granules presenting little
internal detail (Fig. 54), in contrast to the strikingly cristiate structure
of the sea-urchin cortical granules (see Afzelius, 1956-7). Hamster
cortical granules are thought to play a role in the zona reaction
(p. 92).
Division apparatus. The cytoplasmic organelles concerned with
the division of the nucleus are the centrosomes, asters and spindle.
The centrosome is best known in non-mammalian eggs; it is
66 THE MAMMALIAN EGG
generally seen as a small round body with a distinct core — the,
centriole. The centriole divides during the later phases of mitosis
and the centrosome soon afterwards, so that in the primary oocyte
there are first of all two centrioles within a single centrosome and
J
>
O
^ «
I «
% 6
£*&
•
Fig. 54
Electron micrograph of an impenetrated golden-hamster egg, showing the cortical
granules. X 14,000.
later two centrosomes each with a single centriole, and these
structures are disposed near the border of the nucleus. A similar
arrangement is found in embryos approaching the second and
subsequent cleavage divisions. At the start of the first meiotic
division, or of the second and subsequent cleavage mitoses, the
centrosomes take up positions at opposite poles of the nucleus, while
a characteristic radial or star-like structure, the aster, develops in the
cytoplasm immediately surrounding the centrosomes. When the
asters are fully grown, the nucleus appears to be supported between
them. With the condensation of the chromosomes and the dis-
appearance of the nuclear membrane, the achromatic spindle
develops between the asters and on this the chromosomes become
arranged. In the secondary oocyte, the centrosome at the vitelline
pole of the first maturation spindle divides to become the centres
of the asters and spindle of the second meiotic division. The cen-
trosome responsible for the origin of the first cleavage spindle has
been shown to arise in some species from the centriole introduced
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 67
by the fertilizing spermatozoon; in others, the egg and spermatozoon
are each thought to contribute a centriole. In eggs beginning
parthenogenetic development, the aster forms after division of a
centrosome that may have persisted from the second maturation
spindle or may have been generated de novo in the cytoplasm. The
nuclear sap evidently contributes something to the formation of the
spindle, so that the division apparatus is normally both cytoplasmic
and nuclear in origin, and predominantly the former. Under certain
experimental conditions, however, supernumerary asters (cytasters)
can be induced in invertebrate eggs (Wilson, 1928) and some
Fig. 55
Early telophase, first-meiotic spindle (rat). The intermediary
body is very distinct. X 2,000.
Fig. 56
Metaphase second-meiotic spindle
in a field- vole egg. x 1,500.
cleavage with cytasters has been seen in enucleated egg fragments
(Harvey, 1936), so that an active division apparatus can be formed
without direct nuclear contribution.
68 THE MAMMALIAN EGG
In mammalian eggs, only the spindle is easily detected, though
the likelihood is that the form and function of the division apparatus
resemble those in non-mammalian eggs. The spindle can be seen
in living eggs, with the aid of phase-contrast microscopy, as well as
Fig. 57
First polar body and metaphase second-meiotic spindle in an egt
of the golden hamster. X 1,200. (From Austin, 1956d.)
Figs. 58 and 59
First cleavage spindle of the field-vole egg at metaphase, seen in
equatorial and polar views, respectively. The X chromosome is clearly
recognizable. X ca. 900. (From Austin, 1957b.)
in fixed and stained preparations (Figs. 39, 40 and 55 to 58). In both
instances, the spindle presents itself as a transparent body, often with
faint longitudinal striations, and its existence is chiefly evident
through the absence of cytoplasmic particles. The refractility of the
component fibres is responsible for the stranded appearance; the
birefringence of the spindle in polarized light testifies to its con-
struction of longitudinally-orientated submicroscopic micelles. Late
anaphase and telophase spindles in eggs usually carry at the equator
a disc-shaped aggregation of granules constituting the intermediary
body (see also pp. 72 and 73). In ultrastructure, this body was found
to contain units made up of a pair of parallel membranes separated
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 69
by a lighter area averaging 360 A in width (Odor and Renninger,
i960). Dense, probably basophilic, material was associated with the
outer surface of the membranes. The units could be spindle fibres
with thickened walls or tubular structures through which the fibres
pass. In the various phases of division, the spindle with its attached
chromosomes behaves as a solid body when extruded by rupture
of a living egg. The shape of the spindle varies greatly: it is short
and fat at metaphase and early anaphase, and long and narrow at
telophase. Sometimes the metaphase spindle comes clearly to points
at each pole, at other times it appears barrel-shaped.
Centrosomes, centrioles and asters have been described in
mammalian eggs on several occasions : in the guinea-pig (Rubasch-
kin, 1905; Lams, 1913), bat (Van der Stricht, 1909), rat (Sobotta and
Burckhard, 19 10), cat (Van der Stricht, 191 1), dog (Van der Stricht
1923), rabbit (Amoroso and Parkes, 1948; Thibault, Dauzier and
Wintenberger, 1954; Dauzier and Thibault, 1956) and pig (Thibault,
i959)> but they are much less distinct than in non-mammalian eggs.
A suggestion of astral fibres can be seen in the rat egg shown in
Fig. 31.
Components of the spermatozoon. In those animals in which the
sperm tail follows the head into the vitellus at fertilization, the
components of the tail, in addition to those parts of the head that
are not incorporated into the male pronucleus, dissociate and
evidently become part of the cytoplasmic equipment of the embryo.
The sperm tail has been reported to enter the vitellus in the eggs of
the guinea-pig (Hensen, 1876; Rubaschkin, 1905; Lams and
Doorme, 1908; Lams, 191 3), bat (Van der Stricht, 1902; Levi,
1915), mouse (Lams and Doorme, 1908; Gresson, 1940b, 1941), rat
(Sobotta and Burckhard, 1910; Van der Stricht, 1923; Kremer,
1924; Gilchrist and Pincus, 1932; Macdonald and Long, 1934;
Austin and Smiles, 1948; Blandau and Odor, 1952), dog (Van der
Stricht, 1923), rabbit (Nihoul, 1927; Pincus, 1930; Austin and
Bishop, 1957b), ferret (Mainland, 1930), pig (Pitkjanen, 1955;
Hancock, 1958; Thibault, 1959), golden hamster (Austin, 19560";
Hamilton and Samuel, 1956; Ohnuki, 1959), field vole (Austin,
1957b), Chinese hamster, multimammate rat and Libyan jird (Austin
and Walton, i960). Nevertheless, entry of the tail cannot be
regarded as either universal or invariable in its occurrence : Rubasch-
kin, Sobotta and Burckhard, Nihoul and Pincus considered that it
did not always take place in the guinea-pig, rat and rabbit, Van der
70 THE MAMMALIAN EGG
Stricht (1923) maintained that it did not occur in the cat, and Austin
found that entry failed in about 45 per cent of field- vole eggs under-
going fertilization (Fig. 24) and in the great majority of Chinese
hamster eggs.
The tail of the spermatozoon may separate from the head soon
after entry into the vitellus and while the nucleus is taking on the
form of a male pronucleus, or it may remain attached to the pro-
nucleus for part or all of the pronuclear life span. In murine rodents,
separation appears to be the rule,
whereas in the bat (Van der Stricht,
1902) and guinea-pig (Lams, 191 3)
the tail generally retains its attach-
ment. In the rabbit, the attachment
certainly seems to persist on some
occasions (Fig. 60).
The components of the tail that
have been identified in the vitellus
are the centriole, mitochondria,
Golgi elements and the axial fila-
ments. The mitochondria and Golgi
Male pronucleus in rabbit egg with dements become detached during
sperm tail still attached, x 900. fertilization or shortly thereafter and
mingle with the particulates in the
egg cytoplasm (Gresson, 1940b, 1941) (Fig. 61). The tail filaments
are more persistent; they tend to become spread out as the outer
layers of the tail are lost (Fig. 62), and in the rat can be seen in 8-cell
eggs and even in the late blastocyst (Odor and Blandau, 1949). Sperm
centrioles have been reported in the eggs of the bat (Van der Stricht,
1909), rat (Sobotta and Burckhard, 1910), guinea-pig (Lams, 191 3),
dog (Van der Stricht, 1923), rabbit (Amoroso and Parkes, 1947) and
pig (Hancock, 1961).
Of the parts of the sperm head that are not involved in pronucleus
formation, only the perforatorium clearly persists and is readily
traced in the vitelline cytoplasm (Fig. 17). (This body was called the
acrosome when it was originally described in the rat spermatozoon
by Lenhossek, 1898, but the term used here is now the more gener-
ally accepted; 'acrosome' is best reserved for the extranuclear cap.)
The perforatorium is perhaps best seen in the rat egg where it takes
the form of a short curved bifurcated rod; it can generally be
discerned throughout the period of fertilization and sometimes in
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS
71
the 2-cell egg (Odor and Blandau, 1951b). In the earliest stages of
the transformation of the sperm head into the male pronucleus, the
perforatorium appears to have a third prong, originally lying along
w
■-
• *g
Fig. 61
Sperm tails in the eggs of (a) the Libyan jird, and (b) the golden hamster. The mid-piece
appears to be disintegrating in the manner of a thread becoming unwound. In (b), the
'smoke ring' is visible in the middle of the picture. X 1,800. ( (b) from Austin and Bishop,
1957b.)
part of the greater curvature of the sperm head (Austin and Sapsford,
1952; Austin and Bishop, 1958b); this conforms with its description
in the intact spermatozoon as a modified part of the nuclear mem-
brane (Leblond and Clermont, 1952a, b). The continuity of the
perforatorium with the rest of the nuclear membrane can be made
out a little more easily in the hamster egg (Austin and Bishop, 1958c).
72 THE MAMMALIAN EGG
The perforatorium probably plays a role in the penetration of the
spermatozoon through the zona pellucida and perhaps the vitelline
membrane.
Fig. 62
Rat sperm tails, (a) lying in the cytoplasm of a 2-cell egg,
(b) suspended in the surrounding medium after an egg has been
broken. The component fibrils are becoming separated. A
'smoke ring' is visible around the tail shown in (a). X 900.
Mechanism of Cell Division
Cytoplasmic division is an almost universal characteristic of cells
and as a general rule it immediately succeeds nuclear division. The
cell elongates and the surface around the lesser circumference dips
inwards towards the equator of the spindle. The equatorial plane is
often marked by the presence of the intermediary body (Fig. 55),
which consists of basophilic granules considered to be rna left
behind by the chromosomes after anaphase separation. The con-
striction continues until the cell is divided into two daughter cells
within each of which a resting nucleus is reconstituted. The plane
of cleavage passes to one side of the intermediary body and not
through it, and the residue of the spindle bearing this structure can
often be discerned shortly after cleavage (see, for example, Fig. 24
of De Robertis, Nowinski and Saez, 1954).
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 73
Several theories have been advanced to explain the mechanism of
cell division and these have been systematically reviewed by Swann
and Mitchison (1958) ; a detailed account is given also by Ris (1955).
Briefly, opinions are as follows. The initial elongation of the egg
could be attributable to extension of the spindle and the mechanical
effects exerted by the asters. These effects seem more likely to be
caused by traction by astral fibres attached to the surface of the cell,
pulling in the surface in the region between the spindle poles, rather
than by pressure against the surface external to the spindle poles.
A contributory factor leading to the dipping in of the cleavage
furrow may possibly be an alteration of surface properties in the
central region caused by some agent emanating from the breakdown
of the nucleus. Since cleavage necessarily involves considerable
increase in the area of the cell cortex, it is suggested that the motive
force for cell division may well be a passive extension of the cortex
brought about by addition of material in the regions external to the
spindle poles, the material possibly originating from the polar
groups of chromosomes. Associated with such a process, there is
almost certainly an active growth of the cortex in the depths of the
cleavage furrow, particularly during its terminal movements.
Polar-body Emission
Early views on the function of polar bodies included the sugges-
tions that they served as cushions to protect the vitellus (Rabl, 1876),
that they were a means of disposing of unwanted material (Semper,
1875 — 'a form of defaecation' ; Fol, 1875), and that they were
rudimentary cells having an atavistic significance (Giard, 1877)
(references cited by Blanchard, 1878). They were widely thought
to determine the direction of the cleavage furrow, which in many
non-mammalian eggs clearly begins at the animal pole near which
the polar bodies remain.
Emission of the polar body takes place after the meiotic division
has reached telophase, and follows much the same course with both
first and second polar bodies (Fig. 14). Initially, the telophase spindle
lies just below the surface of the egg and in a plane parallel to the
tangent. The first visible sign of polar-body formation is an indenta-
tion of the egg surface at a point immediately peripheral to the
equator of the spindle, which is marked by the presence of a very
distinct intermediary body. The spindle then moves inwards and
rotates about one pole until its long axis assumes approximately a
F
~4
THE MAMMALIAN EGG
radial orientation (Fig. 63) ; one chromosome group thus comes to
lie nearer the centre of the egg
■Kfcfr*
Fig. 63
Movements shown by the telophase
second-meiotic spindle of a recently
penetrated rat egg while under observa-
tion in vitro. X 1,400.
while the other remains close to the
surface. (Spindle rotation occurs in
rodents and some other animals, but
may not do so in all mammals. O.
Van der Stricht (1909), R. Van der
Stricht (191 1), Pearson and Enders
(1943) and J. L. Hancock (personal
communication, i960) maintain
that the spindle is always radially
orientated in the bat, cat, fox and
pig, respectively.) Concurrently,
the surface indentation deepens and
extends around the external pole of
the spindle so as to cut off the small
body of cytoplasm that contains
the more superficial chromosome
group. The cytoplasm composing
the polar body is generally charac-
terized by the presence of few
granular elements. For a while
after its formation the polar body
remains connected to the vitellus by
the spindle which can be shown by
manipulation to have appreciable
tensile strength (Odor and Blandau,
1951a). When the spindle is finally
transected, separation occurs just
medially to the intermediary body
(Blandau, 1945; Ward, 1948; Odor,
1955; Austin, I956d); the rna shed
by the chromosomes is thus jetti-
soned in the polar body.
In many non-mammalian ani-
mals, the first polar body divides
into two so that three polar bodies
are eventually formed; this is rare
in mammalian eggs, but has been
reported (Sobotta, 1895; Rubasch-
kin, 1905; Krassovskaja, 1934;
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 75
Odor, 1955). In mammals, the chromosomes in the first polar
body may remain clumped together, may undergo to varying
degrees a second meiotic division, or may become scattered in the
polar-body cytoplasm. Nucleus formation is most uncommon. On
the other hand, though chromosome scatter can also occur in the
second polar body, an interphase nucleus is frequently seen ; Braden
(1957) notes that in mice a nucleus is reconstituted in the second polar
body so often that its presence can serve to distinguish between
the two polar bodies. Consistently, Ward (1948) never saw nuclear
reconstitution in the first polar body in the hamster egg, though
it did occur in the second.
Mammalian tubal eggs are often recovered with no polar bodies
(before sperm penetration) or only one polar body (during fertiliza-
tion) owing to the break-up of the first polar body; the frequency
of this occurrence varies widely with strain and species. In the
hamster (Austin, I956d) and field vole (Austin, 1957b), the first polar
body persisted in all the freshly ovulated eggs examined; in rabbits,
the incidence of persistence was 88 per cent (Austin and Bishop,
!957b)» whereas in the mouse it was 10 per cent (Sobotta, 1895),
and, in rats, only 2 per cent (Sobotta and Burckhard, 1910), 1*3 per
cent (Austin and Braden, 1954b) or 6 per cent (Odor, 1955).
Emission of a polar body can suffer inhibition, either spontane-
ously or artificially, and this follows directly from failure of the
meiotic division to proceed beyond metaphase or anaphase, or to
failure of the telophase spindle to undergo rotation. Inhibition of
polar-body emission appears to be an inherited tendency (p. 45) and
to be favoured by delay in the time of fertilization (p. 46); emission
can be inhibited in rats by treatment with colchicine (p. 46). The
consequences of polar-body inhibition for pronuclear development
have already been discussed (p. 41 et seq. and Table 2) ; the genetic
consequences are dealt with systematically by Beatty (1957).
In general, the larger the egg, the relatively smaller the polar
body, but this is not a strict relationship — rodent eggs tend to have
disproportionately large polar bodies (see, for example, the guinea-
pig egg in Fig. 38). In any one species, the size of the polar body is
normally fairly constant, but under some circumstances it can vary
greatly. Presumably, the determining factor is the position taken
up by the meiotic spindle relative to the egg surface; experiments
on the eggs of the gastropods Crepidula (Conklin, 1917) and
Ilyamssa (Clement, 1935) showed that displacement of the meiotic
76 THE MAMMALIAN EGG
spindle by centrifugation resulted in the formation of giant polar
bodies, sometimes as large as the egg itself. Tyler (1932) found
that unfertilized Urechis eggs placed in hypotonic sea water for
an appropriate period underwent complete cleavage into two
blastomeres instead of emitting polar bodies, and subsequently these
eggs developed into embryos. Tyler was able to show that the first
cleavage division had been effected by the presumptive polar spindle
which had migrated to the centre of the egg ; this mechanism, by
maintaining diploidy in the embryo, had evidently made possible
the parthenogenetic development (see also Tyler, 1941). Observa-
tions indicate that, in mammalian eggs, cleavage by a presumptive
polar spindle can occur both spontaneously and in response to
experimental treatment. Spontaneous cleavage of the egg by a first
maturation spindle has been reported in the dog (Grosser, 1927) and
mouse (Pesonen, 1946a, b; Braden, 1957). Braden cites an un-
published observation by R. G. Edwards and himself on a mouse
egg, cleaved at the first meiosis, in which one 'blastomere' had been
penetrated by a spermatozoon so that there is certainly a possibility
that one or even both cells of such eggs can undergo fertilization
and proceed with development. This could give rise to mosaic or
gynandromorphic individuals.
Cleavage of mouse eggs at the second meiosis was found by
Braden (1957) to be much more common than that at the first. The
incidence varied with the stock or strain: 0-9 per cent (in 910 eggs)
in A strain mice, 0-3 per cent (in 604 eggs) in V stock, 0*2 per cent
(in 1,335 eggs) in J stock, 0-9 per cent (in 456 eggs) in JS stock and
0-4 per cent (in 232 eggs) inJNS stock; no examples were found
among 1,073 eggs of CBA strain mice, among 749 eggs of C57BL
strain or among 645 eggs of RIII strain. When the cleavage took
place in an egg that had been penetrated by a spermatozoon, one
of the cells contained a male and a female pronucleus, and usually
the sperm tail as well, while the other cell contained only a single
nucleus similar in size to the female pronucleus of the first cell ;
sometimes the sperm tail lay partly in one cell and partly in the
other. Two-celled eggs with two nuclei in one blastomere and one
in the other, which may well have arisen in this way, have also been
described by Van der Stricht (1923) in the bat (Fig. 64), Austin and
Braden (1953b) in the rat, Austin and Braden (1954c) and Edwards
(1957a, b, 1958b) in the mouse and Hancock (1961) in the pig.
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS
77
Fig. 64
A 2-cell bat egg showing two
nuclei in one blastomere. (Drawn
from an illustration by Van der
Stricht, 1923.)
The artificial induction of cleavage of mouse eggs at the second
meiosis was reported by Braden and Austin (1954c) who termed
the phenomenon 'immediate cleavage'. The effective agent was the
application of heat (44 to 45 CC) to the eggs in situ for 5 to 10 min
at 8 to 12 hr after ovulation. Five such
eggs were seen, representing 7-5 per cent
of the eggs recovered. Nine eggs out of
a total of 98 recovered from mice sub-
jected to deep ether anaesthesia were also
judged to have developed through 'im-
mediate cleavage', eight of these eggs
were 2-cell and one had advanced to
the 4-cell stage. When heat treatment
was applied to mice 3 to 4 hr after
mating, four out of 132 eggs recovered
were 2-cell and were considered to have
arisen by 'immediate cleavage'; all four
contained a spermatozoon and two of
them had two nuclei in one 'blastomere' and one in the other
(Braden and Austin, 1954b). As with cleavage at the first meiosis,
the development of mosaic individuals after 'immediate cleavage' is
a possibility. Edwards (1958b) has reported twelve instances of
penetrated mouse eggs cleaved at the second meiosis, each with
two nuclei (pronuclei) in one blastomere and one in the other; the
mice had received intrauterine injections of nitrogen mustard just
before ovulation and mating. Similar eggs were recovered from
mice mated to males that had been injected with triethylenemelamine
(Cattanach and Edwards, 1958).
The penetration of spermatozoa into apparently normal polar
bodies has been reported: invertebrates (Wilson, 1928), guinea-pig
(Hensen, 1876). Edwards and Sirlin (1959) observed a spermatozoon
within a small mass of cytoplasm which resembled a polar body,
but they pointed out that in reality the spermatozoon may have
entered the vitellus and subsequently been extruded with some of
the cytoplasm. The same explanation was put forward by Austin
and Braden (1954c) for two rat eggs observed in a similar state.
In most mammals, the first polar body is emitted shortly before
ovulation and the second after the egg has reached the Fallopian
tube and as a consequence of sperm penetration, but there are some
exceptions to this rule. In the tenrecs (Madagascan insectivores), the
78 THE MAMMALIAN EGG
spermatozoon is said to enter the ovarian follicle and initiate fertiliza-
tion there, and so the eggs emit both the polar bodies before leaving
the follicle (Bluntschli, 1938; Strauss, 1938, 1950). The same rela-
tions may hold also for the shrew Blarina hrevicorda (Pearson, 1944).
The eggs of the dog, fox and possibly the horse are ovulated as
primary oocytes and must produce both polar bodies after reaching
the Fallopian tube (Van der Stricht, 1923 ; Pearson and Enders,
1943; Hamilton and Day, 1945). In the dog, sperm penetration
occurs early, sometimes whilst the egg still has a germinal vesicle,
but the formation of the male pronucleus does not begin until the
second meiotic division is in progress. In the fox, on the other hand,
sperm penetration is delayed until after the formation of the first
polar body. Some details of time relations are given by Austin and
Walton (i960).
Cleavage of the Fertilized Egg
As the first cleavage mitosis reaches telophase, the vitellus of the
egg elongates, the surface dips in around the lesser circumference
and the constriction continues until the egg is divided into two
blastomeres, within each of which a resting nucleus becomes con-
stituted. The plane of cleavage is said to follow a line passing
through the positions formerly occupied by the centres of the two
pronuclei as they lay at syngamy (Van der Stricht, 1923). Division
of the blastomeres of the 2-cell egg is seldom synchronous, so that
a 3 -cell stage is normally interposed between the 2-cell and 4-cell
stages. Similarly, though the stages of eight cells, sixteen cells,
thirty-two cells and so on are customarily mentioned as representa-
tive of steps in embryonic development, and are in fact most often
met with, all the intermediate cell numbers are also seen. With each
successive stage of cleavage, the size of the blastomeres is roughly
halved, until it reaches about that of most of the tissue cells in the
organism concerned. During cleavage, the total mass of cytoplasm
actually decreases, presumably because yolk materials are used up
to provide energy for the maintenance and division of the cells.
The diminution in cytoplasmic volume from the i-cell stage to the
8-cell stage has been found to be about 20 per cent in the cow,
40 per cent in the sheep, 30 per cent in the ferret and 25 per cent in
the mouse (see Hamilton and Laing, 1946). Cell divisions subsequent
to the cleavage phase are associated with increase in size (growth) of
the embryo and with intake of nutrients by the embryo.
Fig. 40
Cat 2-cell egg with a second-cleavage spindle at telophase. X 700.
(Zenker; Heidenhain H and E). (E. C. Amoroso.)
Fig. 41
Cat 4-cell egg. X 700. (Zenker formol; Weigert H and E.)
(E. C. Amoroso.)
Facing page 78
Fig. 42
Cat 8-cell egg; only six blastomeres are visible in this section.
X 700. (Zenker formol with acetic acid; Masson trichrome.)
(E. C. Amoroso.)
Fig. 43
Cat morula. X 700. (Zenker formol with acetic acid and post-
osmication; Weigert haematoxylin. Fat droplets stained.)
(E. C. Amoroso.)
Fig. 69
A well-expanded cat blastocyst. X 270. (Bouin; Weigert H and E.)
(E. C. Amoroso.)
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 79
Cytoplasmic cleavage can be inhibited or even, if it has not
advanced too far, reversed, whereupon a single cell is reformed with
a resting nucleus. The process is best known at the first division of
the egg and can be followed later by normal cleavage, the resulting
embryo now having twice the previous chromosome number.
Some forms of invertebrate parthenogenesis involve first-cleavage
inhibition as a means of regulation to diploidy. Inhibition after
fertilization results in tetraploidy. The few relevant observations
that have been made in mammals are set out clearly by Beatty (1957)
(see also Edwards, 1958a).
The sizes of the blastomeres produced by the early cleavage
divisions are generally unequal, so that the morula in many animals
comes to be made up of larger and smaller cells which tend to
aggregate towards opposite poles (Fig. 44). The smaller cells are
destined to form the inner cell mass of the blastocyst and the larger
cells the trophoblast. Views concerning other distinguishing charac-
teristics of these two cell types have already been discussed (p. 61).
In those animals in which the sperm tail enters the vitcllus at
fertilization (p. 69), the residue of this structure may, to judge from
studies on the rat, mouse and hamster, come to lie wholly within one
blastomere at the 2-cell stage, or be 'shared' by the two cells, passing
across from one to the other in the region of contact between them.
Similar distributions may be seen at later cleavage stages, though
the fate of the sperm tail becomes progressively more difficult to
determine, even in the rodent eggs, owing to its gradual dissolution.
A small distinct dark circle of material seems to be accumulated
by the cleavage furrow in its inward movement and to persist for
a while after cleavage is completed. It rather resembles a smoke
ring, and may lie free in the cytoplasm of one of the blastomeres or
come to surround a sperm tail (Austin and Braden, 1953b) (Figs. 61b
and 62a). If, during microscopical examination, the sperm tail is
extruded from the egg by pressure on the coverglass, the 'smoke
ring' can still be seen surrounding the tail; it appears to have some
solidity. In those polyspermic 2-cell eggs in which both sperm tails
are shared between the two blastomeres, the 'smoke ring' may be
deposited around the tails and give the appearance of binding them
together (Fig. 65).
The characteristic feature of the blastocyst is its thin-walled
bladder-like form, but wide variations on this basic pattern occur
among animals. The overall dimensions of the rodent embryo, as
80 THE MAMMALIAN EGG
typified in the rat, mouse, hamster and guinea-pig, do not alter
appreciably during the development of the blastocyst and up to the
time of implantation. Generally, the zona pellucida remains un-
changed until shortly before implantation, though it was often
Fig. 65
'Smoke rings' apparently binding together the two
sperm tails in 2-cell polyspermic rat eggs, (a) X 800;
(b) x 3,000. (From Austin and Bradem 1953b.)
found to undergo some expansion in the hamster, with concomitant
increase in the size of the perivitelline space (Austin, iQ56d). On the
other hand, in the rabbit, ferret, dog (Fig. 66) and cat (Figs. 67 to
69), and in man and ape, the embryo expands some fifty- or hun-
dredfold in diameter, becoming strongly distended by the fluid that
accumulates in the blastocoele. Extreme forms of blastocyst are
found in the ungulates wherein it is a relatively enormous flaccid
spindle-shaped structure, containing little fluid. Form of blastocyst
is related to mode of implantation, which tends to be superficial
with the larger ones and interstitial with the smaller (see Amoroso,
1952).
Studies have been made on the nature of the fluid in the rabbit
blastocyst, and these have shown that its composition differs in
Fig. 44
Cat morula. X 700. (Zenker formol with acetic acid; Masson
trichromc.) (E. C. Amoroso.)
Fig. 45
Cat morula. X 700. (Bouin; Weigert H andE.)
(E. C. Amoroso.)
Facing page 80
Fig. 67
A cat early blastocyst. X 550. (Bouin; Wcigcrt H and E.)
(E. C. Amoroso.)
Fig. 68
A cat blastocyst at a later stage, after differentiation of the endoderm.
X 450. (Bouin; Weigcrt H andE.) (E. C. Amoroso.)
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS SI
several particulars from that of blood serum. One day before
implantation (Day 6), the fluid contains very little protein or glucose,
but the concentration of both substances approaches that in serum
by Day 8 ; data showed that the increase was due to passage of the
Fig. 66
Dog blastocysts as seen by dark-ground illumination.
(E. C. Amoroso.)
65.
substances to the blastocyst from the maternal blood stream. During
the same period, the phosphorus content doubled and the chlorides
increased about threefold. On the other hand, the concentrations of
potassium and bicarbonate were higher on Day 6 than later and
declined to maternal serum levels as implantation proceeded.
Thiamin, riboflavin, nicotinic acid and vitamin B12 were all present
in assayable amounts in the blastocyst fluid (Brambell and Hem-
mings, 1949; Jacobsen and Lutwak-Mann, 1956; Kodicek and
Lutwak-Mann, 1957; Lutwak-Mann, 1954, 1959, i960).
Shortly before implantation, the guinea-pig egg displays a num-
ber of slender protoplasmic processes which extend out through the
zona pellucida from the abembryonal cells of the blastocyst (Spee,
1893, 1901; Blandau, 1949a, b; Amoroso, 1959). These processes
move about actively, rather in the manner of pseudopodia, and are
considered to play an important role in the initiation of implanta-
82 THE MAMMALIAN EGG
tion. When attachment occurs, processes from the abembryonal
cells can be seen passing between the cells of the uterine epithelium.
The zona pellucida is generally shed soon after attachment has been
effected. Similar protoplasmic processes are reported to develop in
mouse blastocysts cultured in vitro (Whitten, 1957). In the rat, it
has been found that eggs recovered just before implantation fre-
quently lack the zona pellucida and in many of those that are still
entire the embryo is found protruding in part through a hole in
the membrane, as if in the act of escape (Z. Dickmann, personal
communication, i960). Possibly, pre-implantation escape of the
rat embryo from the zona pellucida is effected by the same means
as post-implantation escape in the guinea-pig. It is also tempting to
suppose that the mechanism by which the protoplasmic processes
traverse the zona pellucida may be the same as that employed by
the spermatozoon in its penetration into the egg.
During their free existence, from ovulation to implantation, eggs
and embryos have a measure of independence from the maternal
organism and enjoy some protection from many of the environ-
mental influences that exert effect upon the mother. They are not,
however, completely immune to interference. Disturbance in the
rate of their transport to the uterus and alteration in the properties
of the tubal and uterine secretions can result in death of pre-implan-
tation embryos — both effects can be produced by injections of agents
such as oestradiol, ethinyl-oestradiol, diethylstilboestrol, oestriol
and testosterone (Burdick, Emmerson and Whitney, 1940; Burdick
and Pincus, 1935; Burdick and Whitney, 1937; Burdick, Whitney
and Pincus, 1937; Parkes, Dodds and Noble, 1938; Pincus and
Kirsch, 1936; Velardo, Raney, Smith and Sturgis, 1956; Whitney
and Burdick, 1936, 1937). In addition, several antimitotic agents,
such as D-usnic acid and more especially podophyllotoxin, have
been found on injection into rats to be lethal to the free embryos in
doses well tolerated by the mother (Wiesner and Yudkin, 1955).
Similar results were reported for the triphenyl ethanol derivative
known as MER-25, when given by oral administration to rats and
rabbits (Segal and Nelson, 1958 ; Chang, 1959b), and for 6-mercapto-
purine, 8-azaguanine, tricthylene-thiophosphoramide (Thiotepa),
/y-bis-i, 6-chloroethylamino-D-mannitol (Degranol), triethyleneme-
lamine (TEM), N-desacctylmethyl-colchicine (Colcemide) and
N-desacetylthiol-colchicine (Thiolcolceran) when injected into
rabbits (Hay, Adams and Lutwak-Mann, i960).
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 83
There is little really precise information on the cleavage rates of
mammalian eggs in vivo; this is owing to the difficulty of knowing
the exact time of ovulation, to the fact that fertilization may be
initiated at any time over a period of 12 hr after ovulation or even
longer, and, in polytocous animals, to the scatter in the time of
penetration of the eggs. In addition, it is reasonable to suppose that
eggs in any one species do not all develop at the same rate. Finally,
since the actual process of cleavage occurs relatively rapidly, direct
observation is rare, the time of cleavage has generally to be inferred
from the condition of eggs on recovery from the animal and a large
number of observations are necessary for even approximate esti-
mates. As a result, for animals of many species the figures so far
available from published reports show an extremely wide range of
variation and are almost valueless. Perhaps the most useful con-
clusions that can be drawn from this material are as follows : (a) The
best estimates are those for the rabbit ; this is largely because ovula-
tion is induced by coitus and is known to occur about 10 hr after
the stimulus. The most advanced eggs undergo the first cleavage at
about 12 hr after ovulation, the second at 16 hr, the third at 22 hr
and the fourth (becoming 16-celled) at 30 hr. The blastocoele is
first evident at about 60 hr and the main expansion of the blastocyst
takes place in the region of 90 hr (Lewis and Gregory, 1929a, b).
(b) The next most accurate estimates are those for some of the
laboratory rodents, owing to the large number of observations made
on them. Mouse eggs seem to develop quickest, the earliest be-
coming 2-celled at about 17 hr after ovulation, 4-celled at 38 hr,
and 8-celled at 47 hr. The blastocyst is recognizable at about 63 hr.
Clearly, the cleavage rate in the mouse is much slower than in the
rabbit; the impression that the mouse embryo 'catches up' at the
blastocyst stage is attributable to the fact that rodent blastocysts are
formed of many fewer cells than are rabbit blastocysts. Rat and
golden-hamster eggs cleave even more slowly, the earliest entering
the 2-cell stage at about 15 hr after ovulation, the 4-cell at 40 hr,
the 8-cell at 60 hr and the blastocyst at 80 hr. hi these three rodents,
sperm penetration commonly occurs 2 to 5 hr after ovulation, so
that fertilization can be said to require about 12 hr. (These figures
are based on the reports of Beatty, 1956a, who summarizes earlier
data on cleavage rates; Austin and Braden, 1954a; Braden and Austin,
1954b; Austin, I956d; Chang and Fernandez-Cano, 1958; and the
author's unpublished observations.) (c) From the data summar-
84 THE MAMMALIAN EGG
ized by Amoroso, Griffiths and Hamilton (1942), it can be inferred
that the eggs of the goat, cow, sheep and pig pass from the 2-cell
stage to the 128-cell stage (six cleavages) in a mean time of about
112 hr (arriving at this point between 140 and 170 hr after coitus).
This represents a cleavage rate of about 19 hr per stage, an interval
of about the same duration as with rodent eggs. The blastocoele is
reported to be formed at about 5 days in the goat, 8 to 9 days in
the cow, 6 to 7 days in the sheep and 5 to 6 days in the pig (Beatty,
1956a). (Data on some other animals are given by Boyd and Hamil-
ton, 1952, and Beatty, 1956a.)
The process of cleavage as thus far considered pertains- to meta-
therian and eutherian eggs. Cleavage in the prototherian (mono-
treme) egg is similar to that in other megalecithal eggs in that the
large mass of yolk is unaffected and even the cytoplasm does not
become divided into separate cells in the early stages. Cleavage
furrows divide the germinal disc into progressively smaller areas,
the cytoplasm in the deeper regions of each cell retaining continuity
with that of the other cells and with the yolk mass. Later, as the
number of cells increases, they do become separate units and form
a flattened blastodisc. With further cellular divisions, the blastodisc
comes to consist of several layers and a single layer of cells extends
out over the surface of the yolk. When the yolk is entirely covered,
the embryo is held to have reached the blastocyst state, though a
true blastocoele is apparently not represented. (For further details,
see Boyd and Hamilton, 1952.)
Fragmentation of Eggs
It has long been known that both ovarian oocytes and tubal eggs
are prone to undergo cytoplasmic division, apparently spontaneously
and often in a manner that superficially resembles normal cleavage.
The phenomenon has been described in a number of species : bat
(Van der Stricht, 1901), guinea-pig (Rubaschkin, 1906), armadillo
(Newman, 191 3), mouse (Kingery, 19 14), opossum (Hartman,
1919), water vole (Sansom, 1920), rabbit (Champy, 1923), rat (Mann,
1924), man (Krafka, 1939), ferret (Chang, 1950c, 1957b), hamster
(Skowron, 1956) and pig (Dziuk, i960). Though several authors
were attracted by the idea that parthenogenesis might on occasion
be displayed by mammalian eggs, the general conclusion was that
most if not all the instances of apparent cleavage were in fact caused
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 85
by a disorganization and degenerative fragmentation of the egg (see
also Thibault, 1949, I952)- Rarely if ever does the nuclear state of
such eggs resemble that seen in normal cleavage; the 'blastomercs'
contain one or more subnuclei, or apparently no nuclear material
at all. Absence of nuclear material from egg fragments suggests that
the egg cytoplasm can undergo amitotic division, possibly through
the activity of cy tasters.
Fragmentation of ovarian eggs was found to be more likely to
occur in immature animals (Bacsich and Wyburn, 1945), and the
frequency increased when the eggs were released from the ovary
by artificially-induced ovulation (Austin, 1949b; Chang, 1950c).
This might be interpreted as an augmentation of an innate tendency
to development, but it seems more reasonable to infer that condi-
tions within the immature animal, perhaps more especially within its
genital tract, constitute a somewhat unfavourable environment for
the egg and conduce to its disorganization. Consistently, it has been
found that about one-third of the eggs fertilized in hypophy-
sectomized rats (Rowlands and Williams, 1946) and more than half
the eggs fertilized in immature rats (Austin, 1950b), after induced
ovulation, underwent fragmentation instead of normal cleavage.
Degeneration, involving fragmentation, may also be attributable to
defects inherent in the eggs (Hartman, 1953).
Examination of unpenetrated rat eggs reveals that the second
meiotic chromosomes become scattered some hours after the normal
time of sperm penetration (Fig. 28a), and this occurrence no doubt
underlies the subsequent cytoplasmic fragmentation. Delay in the
time of fertilization or the application of agents that interfere with
the normal organization of chromosomes during cleavage of the
fertilized egg may therefore be expected to favour or even promote
fragmentation. Increase in the frequency of fragmentation has
indeed been found to follow artificial insemination in rats when
this is done after the time of ovulation (Odor and Blandau, 1956),
and has also been seen as a result of the application of irradiations or
radiomimetic agents to spermatozoa before fertilization, although
with these treatments the chief effect appeared to be delay of
cleavage or even complete arrest of cell division (Brenneke, 1937;
Amoroso and Parkes, 1947; Parkes, 1947; Bruce and Austin, 1956;
Chang, Hunt and Romanoff, 1958; Edwards, 1957a, b, 1958b).
86
THE MAMMALIAN EGG
Membranes and Investments
Vitelline Membrane
The egg cytoplasm, like that of other cells, is limited by a plasma
or permeability membrane. In mammalian eggs, the plasma mem-
brane is generally called the vitelline membrane, but it is not as well
developed as the vitelline membrane in the eggs of Sauropsida, nor
is it to be identified with the vitelline membrane of invertebrate
eggs, a structure that becomes modified after sperm entry and rises
from the egg surface as the fertilization membrane. Alone among
the eggs of placental mammals,
the hamster egg has been said to
develop a fertilization membrane
(Graves, 1945; Venable, 1946),
but this could not be seen in
living eggs (Samuel and Hamil-
ton, 1942; Austin, i956d) and
there seems to be no evidence for
its existence in sections examined
by the electron microscope (Fig.
70).
The vitelline membrane may
be considered to have essentially
the same structure and the same
properties of diffusion and active
transport as the plasma mem-
brane of tissue cells. (The struc-
ture and properties of the cell membrane have recently been
discussed by Fitton Jackson, 1961, and Weiss, 1961.) Osmotic
regulation in the vitellus is considered later as a feature of metabolism
(p. in). Active transport is probably involved in the fluid extrusion
associated with first-polar-body emission and with activation of the
egg (P- 56).
As revealed by means of the electron microscope, the vitelline
membrane of the early oocyte is a smooth uncomplicated layer
against which the plasma membranes of the surrounding follicle
cells are closely applied. As the follicle develops, the vitelline mem-
brane becomes thrown up into numerous microvilli some of which
form interdigitations with the surface of the follicle cells or of
processes arising from them. With the formation and growth of
Fig. 70
Electron micrograph of a penetrated
golden-hamster egg, showing part of the
sperm tail apparently enclosed within a
vesicle. X 14,000.
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS «S7
the zona pellucida, egg and follicle cells become separated, but the
microvilli continue to project up to about half-way through the
membrane, and many of the follicle-cell processes, passing com-
pletely through, retain contact with the vitelline surface (Fig. 47).
The microvilli diminish and disappear shortly before ovulation
(Yamada, Muta, Motomura and Koga, 1957; Moricard, 1958;
Chiquoine, 1959, i960; Sotelo and Porter, 1959; Anderson and
Beams, i960; Odor, i960).
The vitelline membrane must be intimately involved in the
attachment of the spermatozoon to the vitelline surface, and in at
least the initial phases of sperm engulfment. Observations in rat
eggs show that the sperm head usually comes to lie flat upon the
vitelline surface and to remain thus for an appreciable time before
it is engulfed (see Austin and Braden, 1956); a similar relationship
has also been reported in the rabbit (Dauzier and Thibault, 1956).
Particles taken into phagocytic cells apparently continue to be
surrounded by plasma membrane, and thus in a sense remain 'out-
side' the cell. Sperm penetration has points of resemblance with
phagocytosis (Loeb, 19 17) and spermatozoa seem prone to engulf-
ment by various cells: they are known to be taken up readily by
macrophages (Hoehne, 1914; Hoehne and Behnc, 19 14) and poly-
morphonuclear leucocytes (Yochem, 1929; Merton, 1939; Austin,
1957c), and apparently even by epithelial cells (Austin and Bishop,
1959b; Austin, 1959a, 1960a). In addition, the appearances presented
by the ultra-thin section of the hamster egg shown in Fig. 70 are
consistent with the idea of phagocytosis — the sperm tail is apparently
contained within a vesicle in much the same way as a phagocytosed
particle, and the vesicle is presumably limited by an invaginated
portion of the vitelline membrane. Nevertheless, recent observa-
tions of Szollosi and Ris (1961), based on electron micrographs of
rat spermatozoa in the act of entering the vitellus, make it clear that
the mechanism involved is essentially different from phagocytosis
(see Frontispiece). These authors postulate that, when the fertilizing
spermatozoon comes into contact with the vitellus, the cell mem-
branes of both the spermatozoon and the egg rupture in the area of
contact and unite with each other. The sperm cell membrane thus
becomes continuous with the vitelline membrane and is left behind
on the surface of the vitellus as the spermatozoon passes into the
cytoplasm. Membrane fusion is held to entail the force responsible
for the movement of the spermatozoon into the vitellus. Similar
88 THE MAMMALIAN EGG
findings have been made on sperm penetration in Hydroides (A. L.
Colwin and L. H. Colwin, personal communication, i960).
The properties of the sperm head and vitelline membrane that
permit attachment can evidently be abolished — many spermatozoa
treated with hyaluronidase inhibitor seem unable to stick on the
vitelline surface (Parkes, Rogers and Spensley, 1954) and eggs sub-
jected to heat treatment often appear to have an impermeable
vitcllus (Austin and Braden, 1956). There is evidence too that these
properties of sperm head and vitelline membrane are subject to
genetic influence; Krzanowska (i960) reports that the low fertility
of an inbred strain of mice (E strain) could be attributed to a low
fertilization rate, and that a remarkably high proportion of the un-
fertilized eggs (varying from 13*1 to 18-7 per cent) contained
spermatozoa in the perivitelline space. The eggs were not activated
either, which certainly implies that no attachment to the vitelline
surface had occurred. The proportion of such eggs was greatly
reduced by outcrossing in either direction.
Attachment of the spermatozoon to the vitelline membrane is
generally effected only by the first one to make contact with it, and
subsequent spermatozoa are thus unable to pass into the vitellus and
take part in fertilization. The change in reactivity of the vitelline
surface reflects the operation of the block to polyspermy, a defence
mechanism protecting the egg against the occurrence of polyandry
(p. 41). The efficiency of the block to polyspermy has been found
to vary in different stocks and strains of rats and mice (Table 3). In
the sea-urchin egg, the block to polyspermy is considered to be a
change propagated over the egg cortex in two phases: a fast partial
block affects the whole surface in one or two seconds and a complete
block is established in about 60 sec (Rothschild, 1954, 1956; Roths-
child and Swann, 1949, 195 1, 1952). Whether the mammalian block
to polyspermy is biphasic and how long it takes to pass over the
vitelline surface are, as yet, unanswered questions. Some similarities,
however, have been demonstrated — in both groups of animals the
block loses efficiency, presumably by slowing down, as the egg
becomes stale or ages, and this change is hastened by heat treatment.
The aging effect in mammalian eggs is shown by the greater fre-
quency with which polyspermy is encountered in animals that have
copulated or been inseminated fiear the end of oestrus (p. 43), and
the effect of the local application of heat or of the induction of
hyperthermia is summarized in Table 3.
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 89
The block to polyspermy is only one of several mechanisms that
help to preserve monospermic fertilization, others being the zona
reaction (to be discussed shortly), the limitation of the numbers of
spermatozoa reaching the site of fertilization (see Austin and Bishop,
1957a; Austin and Walton, i960) and possibly also the impedance
offered by the cumulus oophorus (also to be discussed shortly). The
relative importance of these mechanisms differs in different species
but all species appear to possess a block to polyspermy.
Zona Pellucida
The zona pellucida is a relatively thick transparent membrane
which is best developed in the eggs of placental mammals but is
recognizable also in those of marsupials and monotremes (Fig. 10)
and even of reptiles, though here the corresponding membrane is
perhaps better termed the zona radiata. The zona pellucida is
deposited first as an interrupted intercellular structure related to
single follicle cells ; in addition, the processes and regions of follicle
cells near the egg appear to contain an amorphous substance resem-
bling the material of the zona (Chiquoine, 1959, i960; Trujillo-
Cenoz and Sotelo, 1959). These two observations support the idea
that the zona pellucida is a product of the follicle cells rather than
of the egg. As the follicle grows, the layer of new material becomes
continuous around the oocyte and increasingly separates the follicle
cells from the egg surface. As a result, the follicle-cell processes that
maintain contact with the egg surface become extremely attenuated.
Initially, the zona pellucida lies in close apposition to the vitellus
but becomes separated by the fluid extruded from the vitellus at the
time of first-polar-body emission. In the cat, the zona pellucida
appears to show further accretion after ovulation, whilst it is passing
through the Fallopian tube (Austin and Amoroso, 1959) (compare
Figs. 19, 20, 40, 41, 44, 45). The matrix of the zona pellucida is
essentially homogeneous, even by electron microscopy.
The zona pellucida of rat and rabbit eggs has been shown to
consist of neutral or weakly acidic mucoprotein ; it is dissolved by
strong reducing or oxidizing substances, the rat zona more easily
than that of the rabbit, the most effective agent being a mixture of
hydrogen peroxide and ascorbic acid ; 2 and 4 per cent urea solutions
dissolved only the rat zona (Braden, 1952). Deane (1952) found that
in tests on histological sections silver is precipitated in the rat zona
pellucida from acid solution and she concluded that the membrane
90 THE MAMMALIAN EGG
contains ascorbic acid. Koneckny (1959) reported that the nieta-
chromasia exhibited by the external and especially by the most
internal layers of the zona pellucida of cat follicular oocytes is
removed by treatment with hyaluronidase ; from this, it was inferred
that hyaluronic acid is a normal component of the zona. Strong
staining of the membrane with Sudan B was interpreted to indicate
the presence of lipoprotein. Solution of the zona pellucida is
obtained with acid media: pH 4-5 to 5 for the rat zona pellucida,
pH 3 for the rabbit, pH 2-8 for the hamster, pH 2-4 for the field
vole (Hall, 1935; Harter, 1948; Braden, 1952; Austin, i956d, 1957b).
The zona pellucida is digested by some enzymes and not by others,
distinct species differences being displayed (Table 4). It appears to
be morphologically unaffected by hyaluronidase. The mouse, rat
and rabbit zona pellucida is digested by trypsin more readily before
sperm penetration than after (Smithberg, 1953; Chang and Hunt,
1956), a change that presumably reflects the occurrence of a zona
reaction. The zona pellucida of rabbit, rat and hamster eggs is
permeable to substances of a molecular weight of 1,200 or less, but
not to those of m.w. 16,000 (Austin and Lovelock, 1958). This
means that the vitellus can be considered directly accessible to all
the known essential food components, including vitamins, to the
great majority of pharmacologically active compounds, and to all
natural steroid hormones. It would be inaccessible to most enzymes,
antigens, antibodies, protein hormones, and substances of the nature
of the invertebrate fertilizins and antifertilizins.
Passage of spermatozoa through the rodent zona pellucida is a
very rapid process, judging from the infrequency with which eggs
are recovered with spermatozoa in the act of penetrating this
membrane. It has been remarked by some of the investigators who
have recorded mouse and rat eggs in this condition that the sper-
matozoa appeared to be in the act of passing obliquely through the
zona pellucida (Sobotta, 1895; Sobotta and Burckhard, 1910); more
recent observations certainly support this idea, for not only have
sperm heads regularly been found to lie obliquely in the thickness
of the zona pellucida in hamster and guinea-pig eggs, but the slits
left in the zona by penetrating spermatozoa, as observed in guinea-
pig and Libyan-jird eggs, were found to follow a curved, oblique
path (Austin and Bishop, 1958c). No adequate reason has yet been
advanced to account for this direction of penetration.
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS
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92 THE MAMMALIAN EGG
The act of sperm penetration is thought to depend on an enzyme
or similar agent associated with the perforatorium in the sperm head
which is exposed when the acrosome is detached (Austin and Bishop,
1958a, b, c). Although a number of points of indirect evidence
favour the involvement of a lytic agent in sperm passage through
the zona pellucida, and analogous mechanisms are known in
invertebrates, no success has yet been obtained in attempts to extract
such an agent from mammalian spermatozoa. It is possible that the
hypothetical zona lysin is active only while attached to the per-
foratorium. Dauzier and Thibault (1956) report that uterine
polymorphonuclear leucocytes enter eggs in culture; since it is
conceivable that the mechanism of penetration is similar, investiga-
tions on this problem might profitably include study of these cells.
Study of the numbers of spermatozoa entering the eggs of rats
and mice showed that the zona pellucida could reasonably be held
to undergo a change after the entry of the first spermatozoon which
tended to exclude other spermatozoa, and this change was termed
the zona reaction (Braden, Austin and David, 1954). The zona
reaction is thus a mechanism, like the block to polyspermy, that
helps to prevent the occurrence of polyspermic fertilization. In the
rat, the mean time the reaction takes to reach completion was
estimated to be not less than 10 min nor more than ij to 2 hr. In
the rat, mouse, guinea-pig, cat and ferret, the reaction may be
classed as moderately efficient — though the number of spermatozoa
that pass through the zona is limited, it is not merely the fertilizing
spermatozoon that is successful, and eggs are often seen in which
one or, less commonly, a few supplementary spermatozoa are
present in the perivitelline space, excluded from the vitellus by the
block to polyspermy. By contrast, supplementary spermatozoa are
rarely if ever to be found in the perivitelline space of the eggs of
the hamster, field vole, dog and sheep, and in these animals the
reaction may be classed as highly efficient. At the other extreme,
the eggs of the rabbit (see Adams, 1955), pika (Harvey, 1958) and
mole (Heape, 1886) appear to lack a zona reaction for they regularly
have quite large numbers of supplementary spermatozoa, the rabbit
egg often as many as 200 or 300. The eggs of the pocket gopher,
with 'several' to 'numerous' spermatozoa in the perivitelline space
(Mossman and Hisaw, 1940), presumably have a very slow reaction.
The best explanation of the mechanism of the zona reaction
seems to be that attachment of the fertilizing spermatozoon to the
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 93
vitelline surface causes the release of a substance which diffuses
through the perivitelline fluid and renders the zona pellucida im-
permeable to spermatozoa (Fig. 71) (Austin and Braden, 1956).
This theory invokes a system that, as Rothschild (1956) points out,
Fig. 71
Diagrams of rat eggs to show how the zona reaction is believed to spread out in relation
to the point of sperm attachment on the surface of the vitellus. (From Austin and Bishop,
1957b.)
is widespread in the animal kingdom : the arousal by sperm penetra-
tion of a reaction that is propagated over the egg surface and is
associated with the release of an agent that has the function of
rendering a membrane impermeable to spermatozoa. In sea-urchins,
the response to contact by the fertilizing spermatozoon involves the
sudden expansion ('explosion') of cortical granules, the contents of
which apparently unite with the vitelline membrane converting it
into the sperm-impermeable fertilization membrane (Fig. 72).
Elevation of the fertilization membrane is thought to be due to the
osmotic effect of colloids released in the reaction. Cortical granules
of a different kind have been described in Nereis and these are packed
in regularly arranged alveoli; the reaction to sperm penetration is
also different in detail but presents the common features of cortical
propagation, release of specific substances (which produce a volum-
inous jelly coat in this instance), and alteration of the vitelline
membrane (Costello, 1949). Fish-egg alveoli do not resemble those
of Nereis in appearance, nor the cortical granules of sea-urchin eggs,
but here again there is a propagated change and the alteration of a
membrane ('hardening' of the chorion) evidently under the action
of substances released from the alveoli (see Rothschild, 1958; Zotin,
1958).
94
THE MAMMALIAN EGG
The following observations support the suggestion that the
mammalian zona reaction belongs to this general series of reactions:
(a) In rat eggs penetrated by two spermatozoa, the slits left in the
zona pellucida were more often in opposite hemispheres than in the
Fig. 72
Diagrams of a sea-urchin egg to show how the cortical granules are
considered to react to sperm contact with the vitellus and take part in
the elevation of the fertilization membrane.
same one, a distribution that points to a propagated reaction (Braden,
Austin and David, 1954). (b) Unfertilized mouse eggs with
perivitelline spermatozoa well past the time of fertilization have
been observed after heat treatment of eggs (Austin and Braden,
1956) and in a certain inbred strain of mice (Krzanowska, i960); in
both instances, attachment of the sperm head to the vitelline surface
had evidently failed and in both instances the zona reaction had
failed also, for the number of perivitelline spermatozoa was much
higher than is seen in normally fertilized eggs, (c) In one mammal
at least, the golden hamster, cortical granules exist which disappear
following sperm contact with the vitellus (p. 65). The inferred
relationship between the zona reaction and the cortical-granule
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS
95
response in the hamster egg is illustrated in Fig. 73. Possibly, in
mammalian eggs that exhibit the zona reaction, but lack distinct
cortical granules, the active agent is carried in a more dispersed form
in the vitelline cortex.
Fig. 73
Diagrams of golden-hamster eggs to show the possible rela-
tion between sperm attachment, disappearance of cortical
grannies and spread of the zona reaction.
The zona pellucida may be responsible in some instances for the
failure of heterologous fertilization. Viable hybrids are known in
a wide range of animals (Gray, 1954) and cross-insemination between
S Sylvilagus and + Oryctolagus (Chang and MacDonough, 1955;
Chang, i960), and S Lepus and £ Oryctolagus (Adams, 1957; R. G.
Edwards, personal communication, i960) was shown to result
in early embryos that pass through apparently normal cleavage,
though they degenerate soon afterwards. On the other hand,
persistent failure of sperm penetration has been reported after
artificial insemination of rats with bull, mouse, guinea-pig, rabbit
and Mastomys spermatozoa, of mice with rat, Apodemus, Microtus
and Mastomys spermatozoa, and of Mastomys with mouse and rat
spermatozoa — with the single exception of a Mastomys egg that was
96 THE MAMMALIAN EGG
found to contain two rat spermatozoa in the peri vitelline space
(Leonard and Perlman, 1949; A. K. Tarkowski, A. W. H. Braden,
R. G. Edwards and C. R. Austin, unpublished data). In the great
majority of these experiments, the foreign spermatozoa achieved the
site of fertilization, often in numbers that were well within the
normal range. Provisionally, it is suggested that the zona pellucida
is resistant to penetration by spermatozoa of other than closely
related species, though the possibility cannot yet be excluded that
it is primarily the process of capacitation that is involved in this
distinction.
Another phenomenon in which the zona pellucida possibly plays
a role is that of selective fertilization. Braden (1958b) showed that
the fertilization efficiency of spermatozoa is influenced by the genetic
constitution of the male, and later (Braden, 1958c) concluded that
the chances of egg penetration by spermatozoa could be influenced
by a single genetic locus (the T locus). Evidence showed that
spermatozoa carrying a t allele were in some way handicapped for
the task of traversing the utero-tubal junction (Braden and Glueck-
sohn-Waelsch, 1958), but more recent information indicates that the
transmission ratio of t and T is also influenced by the genotype of
the egg, and tins appears to mean that the ease of penetration of
eggs differs under genetic control (Braden, i960; Bateman, i960).
The mechanism is as yet unknown but may well involve properties
of the zona pellucida.
Cumulus Oophorus
The cumulus oophorus or membrana granulosa is the mass of
cells that comes to surround the oocyte as the follicle grows. At
ovulation, the egg passes to the Fallopian tube still surrounded, in
most animals, by the cumulus; in the opossum, the egg is said to
reach the Fallopian tube already freed of the follicle cells. In other
animals, the investment persists for very variable periods of time.
The cumulus in the sheep, cow, horse and man breaks up readily
and sperm penetration is considered normally to be into eggs free
of cells (denuded eggs). In the rodents, the rabbit and the pig,
denudation occurs during the period of sperm penetration or shortly
thereafter. Cat and dog eggs retain a coating of follicle cells even
after the first cleavage division.
The cumulus oophorus is made up of large numbers of follicle
cells embedded in a transparent jelly-like matrix (Fig. 74). The
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS
97
immediately surrounding cells are anchored to the egg by processes
that ramify, forming a network on the surface of the zona, and, as
already noted, extend through the zona to make contact with the
vitellus (early descriptions were given by Heape, 1886, and Fischer,
Fig. 74
Rat egg surrounded by cumulus oophorus; follicle cells embedded
in a hyaluronic-acid matrix. X 125.
1905). While there is no doubt that the contact is real, it is empha-
sized that there is no evidence of cytoplasmic continuity between
follicle-cell process and vitellus (Chiquoine, 1959, i960; Sotelo and
Porter, 1959). It has long been maintained that the follicle cells
have a nutritive function in relation to the oocyte; direct evidence
for the transfer of lipid material has been obtained by Wotton and
Village (195 1) in the ovary of the kitten. The cells are held together
partly by intercellular attachment and partly, especially in the
periphery of the cumulus, by the matrix. The layers of follicle cells
nearest the egg are much more densely packed and present a distinc-
tive radial pattern, forming a structure known as the corona radiata
(Figs. 74 and 75). During pre-ovulatory maturation and as time
passes after ovulation, the follicle cells show degenerative changes
and tend to disperse: the processes are withdrawn from the zona
pellucida and the cells migrate out of the matrix. Thus, in rats and
mice that have not been mated, it is possible to find, on the second
day after ovulation, eggs bearing a mass of matrix about them
which is almost or completely free of follicle cells. Generally,
however, in unmated animals, the entire cumulus breaks down
.
***
98 THE MAMMALIAN EGG
liberating denuded eggs. The mechanism responsible for this dis-
integration is unknown, though evidence shows that enzymic acti-
vity or mechanical movement within the Fallopian tube is partly
responsible, at least in the rabbit (Swyer, 1947). In the rat, mouse
and hamster, it seems possible
that the cells in the cumulus
surrounding freshly-ovulated
eggs are still too tightly packed
to permit sperm penetration
into the eggs : penetration was
found to begin 3 to 4 hr after
ovulation, whereas in the rab-
bit it appears to start imme-
diately after ovulation (Austin
and Braden, 1954a; Austin,
I956d; Strauss, 1956). Braden
(1958b) showed that in two
inbred strains of mice the
delay in sperm penetration
FlG- 75 differed in duration and so also
radiata^xIsO. Ulbal ^^ ^ ^ C°r°Ua did the dellsity °f the CUmU"
lus and the rate at which the
investment ultimately broke up. Study of the heritability of these
features confirmed the belief that they are determined by the geno-
type of the female. It has also been shown that the density of the
cumulus can be reduced, and the delay in sperm penetration short-
ened, by treating the females with injections of gonadotrophs
which provoke ovulation (Braden, i960).
The matrix of the cumulus contains protein but is largely com-
posed of the acid mucopolysaccharide known as hyaluronic acid,
which is also a constituent of several tissues, notably synovial fluid,
umbilical cord, vitreous humor, aqueous humor and the ground
substance of connective tissue. It is readily liquefied by proteolytic
enzymes, such as trypsin, chymotrypsin, pepsin and mould protease
(Braden, 1952, 1955), and by the specific enzyme hyaluronidase,
which spermatozoa carry. The permeability of the matrix to
solutes is perhaps slightly less than that of the zona pellucida, but
still sufficient to allow passage of substances of m.w. 1,200 (Austin
and Lovelock, 1958). The various properties of the cumulus matrix
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 99
are much more constant among different species than are those of
the zona pellucida.
The cumulus masses surrounding freshly ovulated rodent eggs arc
quickly broken up, and the eggs thus denuded, by treatment with
sperm suspension (as noted by Schenk, 1878) or with solutions of
hyaluronidase. This is not true, however, for oocytes recovered
from large ovarian follicles, and the difference is probably to be
attributed to the firmer attachment between the follicle cells before
ovulation. Hyaluronidase solutions also fail fully to denude ovu-
lated rabbit, dog and cat eggs; the more densely-packed cells in the
immediate vicinity of the egg, the corona radiata, evidently retain
sufficient direct attachment to the egg and to each other to maintain
their positions in the absence of matrix.
In studies with the rodents and the rabbit, it has frequently been
remarked that the cumulus disintegrates more rapidly in mated
animals than in those that have not mated. It is reasonable to hold
that disintegration is owing to the action of hyaluronidase liberated
from spermatozoa that reach the site of fertilization. The hyaluroni-
dase carried by spermatozoa is probably associated with the acrosome
(Leuchtenberger and Schrader, 1950; Schrader and Leuchtenbergcr,
1951; Bishop and Austin, 1957), and in ejaculated and epididymal
spermatozoa appears to be released only by the moribund cells (see
Mann, 1954), in which the acrosome becomes visibly changed or
detached (Austin and Bishop, 1958b). Before spermatozoa can take
part in fertilization, they need to undergo a form of physiological
preparation called 'capacitation' in the female genital tract (Chang,
1951a, 1955b, 1958; Austin, 1951a, 1952b; Noyes, 1953; Austin and
Braden, 1954a; Noyes, Walton and Adams, 1958); this evidently
involves a change in the acrosome of the living spermatozoon
resembling in appearance that shown by the acrosome of the
moribund spermatozoon (Austin and Bishop, 1958c). When tested
under specific conditions /'// vitro, epididymal and ejaculated sperma-
tozoa are unable to pass into the cumulus matrix, whereas cumulus
masses recovered from mated animals are often found to contain
spermatozoa that move freely through the cumulus — these sperma-
tozoa exhibit the acrosome change. It is therefore inferred that the
acrosome alteration involved in capacitation permits the release of
hyaluronidase, which enables the spermatozoon to penetrate the
cumulus by liquifying the matrix in the vicinity of its head (Austin,
1948, 1960c, i96id). The altered acrosome is easily detached and
100 THE MAMMALIAN EGG
it is suggested that when the spermatozoon reaches the egg the
acrosome is removed, laying bare the perforatorium (Austin and
Bishop, 1958b, c), the probable function of which has already been
discussed (p. 92).
It is tempting to argue that the capacitation change of the mam-
malian acrosome is analogous to the 'acrosome reaction' exhibited
by spermatozoa of several invertebrate species (see Dan, 1956;
Col win and Col win, 1957; Franzen, 1958). The invertebrate
acrosome reaction is provoked by substances in the jelly coats
covering eggs or diffusing from the eggs into the medium; it finds
expression in the protrusion of an acrosome filament and the release
of lytic agents, both processes evidently making possible the entry
of the spermatozoon into the egg. The reason for drawing this
parallel is to support the suggestion that the normal capacitation
process may turn out to be a reaction of the spermatozoon, not to
tubal or uterine secretions, but to substances in or emanating from
the cumulus masses as they lie in the Fallopian tube. As yet, how-
ever, it has not been found possible to obtain the mammalian
acrosome reaction by merely placing spermatozoa and cumulus
masses together in vitro, and so it is necessary to suppose that capacita-
tion within the female tract involves also a preliminary phase in
which substances present in the ejaculate and exerting an inhibitory
effect are removed from the spermatozoa.
If the ideas just set out on the passage of spermatozoa through
the cumulus are substantially true, the cumulus, in those animals in
which it persists, could be regarded as constituting another line of
defence against the danger of polyspermic fertilization, by providing
a hindrance to sperm passage which individual spermatozoa may
well vary in their ability to overcome. On the other hand, it can
also be argued that the cumulus improves the chances of fertilization
by providing a larger target for spermatozoa to encounter and by
orientating the spermatozoa towards the egg, through the radial
arrangement of the follicle cells. Perhaps, these two functions
would not necessarily be conflicting.
Mucin Coat of the Rabbit Egg
The mucin coat, originally designated the 'albumen' coat and
formed of material secreted by the epithelium of the rabbit Fallopian
tube, becomes deposited in the final stages of disintegration of the
cumulus, and often imprisons a few coronal cells. It shows distinct
STRUCTURE AND FUNCTION IN MAMMALIAN EGGS 101
concentric layers between which debris and occasional cells, includ-
ing spermatozoa, may be trapped. Not only eggs but other objects
also, such as fragments of sloughed epithelium and experimentally
introduced foreign bodies, similarly receive a mucin coat as they
pass along the tube. Deposition is evidently continuous, so that on
entry into the uterus eggs often carry a mucin layer the thickness
of which is equal to or greater than the original diameter of the
egg, including the zona pellucida; in other words, the overall
diameter of the egg undergoes at least a threefold increase (Fig. 10).
The thickness of the mucin coat was reported to be diminished by
the injection of oestradiol into the rabbit (Green wald, 1957) and
increased by the injection of progesterone (Greenwald, 1958);
Noyes, Adams and Walton (1959), on the other hand, found that
mucin deposition was not prevented by ovariectomy and might, in
fact, be increased by the administration of small doses of oestrogen
to ovariectomized rabbits. The last-named authors consider that the
thickness of the mucin coat depends more upon the time spent by
the egg in the mucin-depositing regions of the tube than upon
variations in the secretory activity of the tubal epithelium.
The material constituting the mucin layer has been characterized
as a strongly acidic mucoprotein (Braden, 1952; Bacsich and
Hamilton, 1954). It was found to be digestible by trypsin, chymo-
trypsin and pepsin, but not by mould protease; it was insoluble
through the pH range of 2-0 to 9-0 and soluble in more alkaline
media than this; it was dissolved by hydrogen peroxide, with or
without ascorbic acid, but not by urea solutions or a variety of
oxidizing and reducing agents (Braden, 1952). Permeability studies
have shown that the mucin coat, like the zona pellucida, permits
the passage of dissolved substances of m.w. 1,200 or less (Austin and
Lovelock, 1958).
The mucin coat is impenetrable to spermatozoa and its deposition
has therefore been said to limit the fertilizable life of the rabbit egg
(Pincus, 1930; Hammond, 1934). The time that deposition begins
has been variously put at 5 hr after ovulation (Pincus, 1930), 6 hr
(Hammond, 1934), not more than 8 hr (Braden, 1952) and 10 to
14 hr (Chang, 195 id, 1955c). The range in estimates may be owing
to the fact that they are based on observations on mated animals,
in which cumulus dispersal would have been expedited to varying
degrees by the hyaluronidase released from spermatozoa at the site
of fertilization. In unmated animals, cumulus dispersal is much
102 THE MAMMALIAN EGG
slower and may take as long as 17 hr (Pincus, 1930); mucin deposi-
tion would be similarly delayed. These considerations give force to
Chang's (195 id) contention that the demonstrably short fertilizable
life of the rabbit egg should not be ascribed to its acquisition of a
mucin coat.
When the rabbit blastocyst expands in the uterus, the mucin coat,
as Boving (1954) points out, is reduced to a thickness of only a few
microns, while the zona pellucida must become vanishingly thin.
Boving found, nevertheless, that the rabbit blastocyst is surrounded
by two distinct membranes and he suggests that the outer mem-
brane, which he calls the 'gloiolemma', is secreted by the uterus and,
by virtue of its adhesive property, is intimately involved in the
implantation reaction.
Outer Coats of Marsupial and Monotreme Eggs
The eggs of the opossum Didelphis (Hartman, 1916, 1919; Hill,
191 8), the native cat Dasyurus (Hill, 1910) and the wallaby Setonix
(Sharman, 1955a) acquire a coating of jelly-like material in their
passage through the Fallopian tube (Fig. 10); this is referred to as
albumen although its chemical nature does not seem to have been
investigated. In the opossum, more albumen is added in the uterus,
the final thickness of the coat amounting to rather more than the
original diameter of the egg. Both opossum and native-cat eggs
receive in addition a shell membrane, which becomes thicker with
time. The opossum egg is also described as having a shell, but this
is non-calcareous and leathery in texture.
The eggs of the monotremes, the duck-billed platypus Ornitho-
rhynchus and the spiny anteater Tachyglossus (= Echidna), resemble
bird and reptile eggs rather than those of marsupials and placental
mammals (Caldwell, 1887; Gatenby and Hill, 1924; Flynn, 1930;
Hill, 1933 ; Flynn and Hill, 1939) ; they become covered with a broad
layer of albumen, a shell membrane and a leathery shell (Fig. 10).
MANIPULATION OF EGGS
Microscopy
Suitable fluid media for the recovery and handling ofliving eggs
for microscopical examination are blood serum, 0-9 per cent sodium-
chloride solution and a number of buffered isosmotic saline solutions,
such as Tyrode's, Locke's, Simm's, Gey's and Hank's solutions.
Eggs deteriorate less rapidly in vitro when suspended in media
containing substances of high molecular weight, and accordingly the
saline solutions mentioned are improved by the addition of materials
such as hen-egg albumen and crystalline bovine serum albumen.
Follicular oocytes can be obtained by placing the ovary in a fluid
medium in a suitable container, incising the follicle wall and teasing
out the contents (small ovaries) or flushing out the contents with
the fluid (large ovaries). Ovulated oocytes and eggs undergoing
fertilization or cleavage are recovered by somewhat different
methods according to the animal involved. From the rabbit
Fallopian tube, eggs are best obtained by flushing. The tube is
removed from the abdomen by transecting the uterus about half an
inch from the utero-tubal junction and cutting through the fat and
other tubal adnexae, with care to avoid nicking the tube. The
specimen is placed on a cork pad and the tube trimmed of most of
the adherent tissue so that it can be straightened out. The attached
portion of the uterus is cut away to reveal the uterine opening of
the tube. A finely-drawn Pasteur pipette with a capillary having an
external diameter of about 0-5 mm is charged with the flushing
solution and inserted into the isthmus of the tube through the
uterine opening. The Fallopian tube is held vertically above a
suitable receptacle such as a glass cavity-block or watch-glass and
the solution propelled through it so as to wash the eggs into the
receptacle. Essentially the same method can be used for the Fallopian
tubes of the domestic animals and man.
In murine rodents, recovery of eggs from the Fallopian tube
involves first the removal of the tube from the abdominal cavity by
cutting through the utero-tubal junction on the one hand and the
mesosalpinx and ovarian capsule on the other with the aid of fine-
pointed scissors. It is advantageous to leave the ovary behind,
103
104 THE MAMMALIAN EGG
though to do this without damaging the Fallopian tube requires
care, especially in small animals. Eggs that are still surrounded by
cumulus oophorus and grouped together in the extended part of the
ampulla are released simply by slitting the ampulla with an instru-
ment such as a Graefe knife, whereupon the cumulus masses gener-
ally emerge without further aid. Denuded eggs may be recovered
either by flushing or by manipulation. The flushing method is the
same in principle as that described for the rabbit Fallopian tube
except that the pipette used is necessarily of smaller dimensions. It
may be found helpful to make a small bulbous enlargement at the
tip of the pipette as this tends to retain it after it has been inserted
into the tube. The Fallopian tube can be flushed in either direction —
some authors prefer to insert the pipette into the lumen of the
isthmus, others pass it through the infundibulum. Recovery by
manipulation, on the other hand, involves the application of pressure
to the Fallopian tube in such a way as to drive the contents along
the tube and finally through the infundibulum, or, if preferred,
through an opening made in the wall of the tube. Pressure is applied
with a pair of dissecting needles. As a possible refinement, the
Fallopian tube may, on removal from the animal, be placed in
liquid paraffin in a Petri dish; this permits the eggs to be dissected
from the tube, and transferred to a microscope slide, while still
surrounded by their natural fluid medium. The method is perhaps
appropriate only in the murine rodents and when there is an appreci-
able accumulation of fluid in the tube, as is the case for a limited
period after ovulation. Phases in the fertilization of rat eggs were
found to continue in vitro more surely when the eggs had been
recovered in this way than when they were surrounded by artificial
medium (Austin, 1950a, 1951a). The method has also been applied
to hamsters (Ohnuki, 1959).
If large numbers of follicle cells are still attached to the zona
pellucida they tend to obscure the finer details within the eggs when
these are examined with the higher powers of the microscope;
accordingly, the cumulus should first be removed by treatment
with solutions of hyaluronidase or trypsin. This procedure is
ineffective, however, with follicular oocytes, from which the
adherent cells must be removed by dissection. The corona radiata
of the rabbit egg is also resistant to removal by enzymes but can be
dislodged if the eggs are vigorously propelled into and out of a fine
pipette.
MANIPULATION OF EGGS 105
Recovery of eggs from the guinea-pig Fallopian tube may be
troublesome owing to the large amount of fat that often surrounds
the tube; both flushing and manipulative techniques, however, have
been successfully employed.
Eggs have been obtained by several investigators from the
Fallopian tubes of living animals (domestic animals, rabbit and man)
under anaesthesia (Appendix No. i; also Krassovskaja, 1934, from
the rabbit). This can be done by placing a clamp near the tubal end
of the uterus and injecting fluid into the isolated part of the uterine
lumen; the fluid flows along the Fallopian tube, carrying the eggs
with it, and can be collected as it escapes from the abdominal ostium.
When resistance is offered by the utero-tubal junction, as in the
rabbit, the fluid may be injected instead into the ampulla, by means
of a syringe inserted into the infundibulum; an opening is made in
the tubal end of the uterus and a short length of glass tubing inserted
into the isthmus through which the flushing solution runs (see Avis
and Sawin, 195 1).
Recovery of cleaving eggs and blastocysts from the uterus is also
effected by flushing, though manipulation can be used with the
smaller rodent uteri. To extract large blastocysts without damage, it
may be necessary to make a large incision in the uterine wall, and
ungulate blastocysts are generally obtained in this way. Neverthe-
less, early bovine blastocysts have been removed from the living
animal without operative interference — this was done with the aid
of a special flushing tube or catheter which had separate lumina, one
for admitting the fluid to the uterine cavity and the other for
draining off fluid together with the suspended eggs (Rowson and
Dowling, 1949; Dracy and Petersen, 1951; Donker, 1955).
For detailed study, eggs are taken up with a little of the surround-
ing medium into a finely-drawn Pasteur pipette and transferred to
a microscope slide. The capillary of the pipette should be about
2" to 3" long with an internal diameter a little larger than that of
the oocyte, namely of the order of 0-2 to 0-4 mm — it has been found
that fluid movements are most easily controlled with pipettes of
these dimensions. The egg should not be drawn more than half an
inch or so into the capillary, and certainly not into the wider portion
of the pipette, because there is then a risk that it will be left behind
in the pipette when the fluid is expelled. The same pipettes can be
used for transferring larger objects, such as an entire granulosa-cell
mass, by drawing the mass onto the tip of the pipette and holding
H
106 THE MAMMALIAN EGG
it there by maintaining slight negative pressure within the pipette.
Alternatively, larger-bore pipettes may be preferred for the larger
objects.
After the egg has been placed on the slide, it is covered by a
coverglass to the edges of which a little vaseline has been applied.
The purpose of the vaseline is to prevent the coverglass from being
drawn down close to the slide by the surface tension of the fluid —
which would be very likely to crush the egg — and to permit some
control of the compression applied to the egg. Spaces should be
left in the vaseline edging to allow the escape of air and medium.
It is recommended that the volume of medium deposited with the
egg on the slide should be as small as practicable — if the volume is
too large the fluid may run to the edge of the coverglass, carrying
the egg with it. This consideration is especially important with
denuded eggs and when several have been placed on the one slide;
in studies with a high-powered microscope, it is specially convenient
to have all the eggs close together, thus avoiding the need to hunt
for each one over a wide area.
Once the coverglass is in position, and contact has been made
with the fluid droplet, pressure is applied with the fingers to opposite
edges of the coverglass while progress is watched through a dissect-
ing microscope. The coverglass is depressed until it just makes
contact with the surface of the egg or with cells closely investing it.
The slide is then transferred to the stage of a high-powered micro-
scope and compression continued in the same way while the results
are observed at low magnification (16-mm objective). Within
limits, the more the egg is flattened the clearer will the internal
details be at high magnification (2-mm objective), but some experi-
ence is needed to know just how much an egg can be compressed;
excess pressure will either rupture the egg or cause it to degenerate
rapidly. When suitable flattening has been achieved, more medium
may be run under the coverglass to prevent the preparation from
drying out. If flattening has not been excessive, it is generally
possible to change the orientation of structures within the egg, and
so obtain optimal presentation of a selected detail, by gently sliding
the coverglass and so rolling the egg. Sometimes, however, the egg
becomes adherent to one of the glass surfaces and will not roll.
Eggs set up on a slide in this way may be fixed and stained by
drawing the appropriate solutions under the coverglass: a drop of
the solution is deposited on the slide in contact with one edge of
MANIPULATION OF EGGS 107
the coverglass and a piece of filter paper is held against the opposing
edge to absorb the fluid from that side. A convenient fixative is a
mixture of 5 ml glacial acetic acid and 95 ml absolute ethyl alcohol;
nuclear structures can then be satisfactorily stained with a o-i per
cent aqueous solution of toluidine blue. After such treatment, the
edges of the coverglass can be sealed with paraffin or beeswax so as
to make a semi-permanent preparation. Some authors prefer to fix
and stain the eggs with the use of a single solution, and good results
have been obtained with aceto-carmine (0-5 per cent carmine dis-
solved in 45 per cent acetic acid) (Chang, 1952a; Spalding, Berry
and Momt, 1955; Berry and Savcry, 1958; Hancock, 1958).
The optical equipment most generally preferred for the high-
power study of living eggs is the phase-contrast microscope fitted
with negative contrast objectives. Illumination for viewing is best
obtained from a very bright point source, the light passing through
a monochromatic green filter; for photomicrography, the filter
should be appropriate to the type of emulsion used. An alternative
optical system is the anoptral phase contrast, which is said to have
some advantages, notably the avoidance of flare around highly
refractile structures (Wilska, 1954). The interference microscope,
invaluable for the study of tissue-culture cells and the like — since it
permits the determination of dry-matter content and presents a very
satisfactory colour-contrast picture at low magnifications (Hale,
1958) — is not appropriate for detailed observations on eggs owing to
their large size and manifold inclusions, and because resolution is
poor at high power. Another recent development is fluorescence
microscopy, in which eggs treated with vital fluorochromes such
as acridine orange are subjected to ultra-violet radiation of relatively
long wavelength and examined with a conventional bright-field
microscope fitted with a dark-ground condenser. With acridine-
orange staining, information can be obtained on the distribution in
living eggs of dna which gives a bright green fluorescence; the
striking red fluorescence which granular bodies display seems likely
to be due to mononucleotides (Austin and Bishop, 1959a). Finally,
there is the ultra-violet microscope, the use of which offers two
advantages: with radiations of the shorter wavelengths available,
higher degrees of resolution can be obtained than with light micro-
scopy, and the distribution of substances having sharp absorption
maxima, such as the nucleic acids, can be studied. The characteristic
strong absorption of nucleic acids at a wavelength of 2,600 A is
108 THE MAMMALIAN EGG
attributable to their purine and. pyrimidine bases. For work with
ultra-violet microscopy, it is necessary to have a powerful source of
radiation and an optical system composed of quartz. It is possible
to incorporate the phase-contrast principle in ultra-violet micro-
scopy, thus obtaining extremely good resolution of details in living
cells (Taylor, 1950; Smiles and Dobson, 1955). A difficulty inherent
in ultra-violet microscopy is that critical focusing cannot be done
by eye, except with expensive electronic scanning and cathode-ray
equipment, and so the common practice is to take a succession of
photographs passing through the estimated focal plane of the
selected detail.
Eggs may be prepared for histological study in situ by placing the
ovary or Fallopian tube, or parts thereof, in a selected fixative, and
dehydrating and embedding in the usual way. This general pro-
cedure is the classical one, followed by Sobotta (1895), Van der
Stricht (1902), Rubaschkin (1905) and many others since. It is con-
venient and provides good permanent records, but it has disadvan-
tages : the state of the eggs cannot be examined before fixation, the
plane of the sections relative to internal structures of the egg is
entirely fortuitous, and it is often necessary to prepare rather a large
number of sections to be sure of including all the eggs in the
specimen.
These disadvantages are overcome in the following ways : (a) The
eggs are recovered in the fresh state, by the means described earlier,
and examined and photographed under the low powers of the
microscope. They are then transferred to fixative solution in a
cavity-block. If required for electron microscopy, they are fixed
in a buffered solution of osmium tetroxide, passed through a series
of alcohol solutions, and finally into the monomer mixture, all
solutions being contained in cavity-blocks. Finally, the eggs are
deposited in some partially polymerized monomer mixture in the
lower half of a gelatin capsule (No. 00), and moved with a fine wire
into a close group at the bottom. The capsule is then placed in an
oven at 6o°C until polymerization is complete (1 or 2 days). Eggs
required for conventional microscopy are more easily handled by
a method such as that described by Dalcq (195 1). In a small Petri
dish, a mound of agar is built up; a cavity is produced in the top by
blowing a small bubble with a pipette while the agar is still fluid
and opening this later with a hot needle. The agar is covered with
fixative solution (Dalcq recommends alcohol : formalin : acetic acid,
MANIPULATION OF ECKiS 109
6:3:1) and the eggs are placed in the cavity. After fixation, which
requires about 2 hr, the fixative is drawn off with a pipette, first
from about the agar and then from the cavity, care being taken not
to remove any eggs. The eggs are gathered together with a fine
needle and a drop of albumen solution, such as Meyer's egg albumen,
placed on them. This is followed by a drop of 90 per cent alcohol
which coagulates the albumen and immobilizes the eggs. The agar
mound is then taken through the alcohols to water, the cavity is
filled with melted agar and the mound returned through the
alcohols for embedding in paraffin. (/;) After fixation, the eggs can
be stained with carmine, which brings up the nuclear structures,
and cleared in glycerol — whole eggs thus treated were often pre-
ferred to sections, in the days before sufficiently good microtomes
were available, and the observations of Van Bcneden and Julin
(1880) were made in this way. The procedure allows of the orienta-
tion of eggs before embedding, a technique that was developed
particularly skilfully by Samuel (1944) and Amoroso and Parkes
(i947).
A technique described recently by Moog and Lutwak-Mann
(1958) is a convenient one for making permanent flat mounts of
rabbit blastocysts. On recovery from the uterus, the blastocyst is
rinsed in saline solution and fixed for 1 hr or more in absolute
methanol. The blastocyst is then placed, embryonic shield down-
wards, on a coverslip immersed in methanol deep enough to cover
the blastocyst, the abembryonal pole is punctured with dissecting
needles and the wall is torn into strips extending to the edge of the
embryonic shield. The strips are laid out radially so that the prepara-
tion is star-shaped, and generally it is possible to avoid serious
wrinkling. The preparation is allowed to dry and can then be
stained, dehydrated and mounted like a tissue section. A suitable
stain is Mayer's acid haemalum applied for 20 to 40 min.
Transfer
A considerable amount of work has now been done on the
transfer of eggs from one individual to another; the methods
employed and the results obtained have been reviewed and discussed
by Pincus (1936a), Nicholas (1947), Pincher (1948), Chang (1949b,
i95od, 1951b), Dowling (1949), Hervcy (1949), Kyle (i949)> Ham-
mond (1950a, b), Giuliani (195 1), Davidov (1952), Lamming and
Rowson (1952), Dracy (1953a, b, 1955), Willett (1952, 1953),
110 THE MAMMALIAN EGG
Donker (1955), Henriet (1955), Dziuk, Donker, Nichols and
Peterson (1958), Hafez (1958) and Noyes and Dickmann (i960).
The original reports are summarized in Appendix No. 1.
It has been demonstrated that :
(a) Normal young animals can be born from embryos transferred
during the early cleavage stages ; this has been shown in the rabbit,
mouse, rat, sheep, cow and pig.
(b) Follicular or tubal oocytes can undergo fertilization after
transfer to a mated recipient animal and develop to normal birth;
this has been shown in the rabbit, mouse, rat and sheep.
(c) Eggs and cleavage embryos can tolerate wide variations in
environmental conditions between recovery from the donor and
lodgement in the host. Rabbit oocytes have survived storage at
o°C for 72 hr and at io°C for 96 hr, and rabbit embryos storage at
o°C for 78 to 102 hr or at io°C for 80 to 101 hr (Chang, 1947,
1948a, b, c, 1952a). Unfertilized rabbit eggs and fertilized eggs in
various stages of cleavage have been subjected in vitro to irradiation
from radiocobalt, and then transferred to suitable recipients (Chang,
Hunt and Romanoff, 1958; Chang and Hunt, i960). Even 65,000 r
did not prevent unfertilized eggs undergoing fertilization after
transfer, though subsequent development failed; most eggs, how-
ever, whether unfertilized or cleaving, were prevented by treatment
with 100 or 200 r from advancing far in embryonic development.
Apparently normal young rabbits and mice have been born from
2-cell eggs in which one blastomere was destroyed (Seidel, 1952;
Tarkowski, 1959a, b), and some embryonic development was
possible even from 4-cell eggs in which three blastomeres were
destroyed (Seidel, 1956, i960; Tarkowski, 1959a, b). Rabbit and
mouse embryos have been grown in culture for 1 or 2 days and
then, on being transferred to recipients, have developed to birth
(Chang, 1948c, 1950b; Biggers and McLaren, 1958; McLaren and
Biggers, 1958). Mouse oocytes have retained their capacity for
fertilization and extensive development after being frozen for \ to
31 hr (Sherman and Lin, 1958, 1959). Sheep embryos have with-
stood transfer to the rabbit genital tract for a week and then, after
retransfer to the uterus of a sheep, have developed for a further 10
to 12 days (Averill, Adams and Rowson, 1955; Averill, 1956).
(d) The chances of implantation and survival of transferred
embryos depends upon a fairly close synchronization between the
post-ovulatory age of the uterine environment and the age of the
MANIPULATION OF EGGS 111
embryo, embryos a little in advance of the uterine changes having
the best chances. This has been shown in the rabbit (Chang, 1950a,
I95id), mouse (Fekete and Little, 1942; Runner and Palm, 1953;
McLaren and Michic, 1956), rat (Nicholas, 1933b; Dickmann and
Noyes, i960; Noyes and Dickmann, i960) and sheep (Avcrill and
Rowson, 1958). Only limited development seems possible in
interspecific and intergencric transfers. The transfers tested have
been: reciprocally between sheep and goat (Warwick and Berry,
1949, 1951; Warwick, Berry and Horlacher, 1934), between sheep
and rabbit (Averill, 1956; Averill, Adams and Rowson, 1955), and
reciprocally between rabbit, mouse, rat and guinea-pig (Briones and
Beatty, 1954).
Other problems that have been attacked by the egg-transfer
technique include : the developmental capacity of eggs from imma-
ture rabbits (Adams, 1953, 1954) and mice (Runner and Palm, 1953 ;
Gates, 1956; Edwards and Gates, 1959), and of eggs from pseudo-
pregnant rabbits (Black, Otto and Casida, 195 1), and the specific
effect of the maternal environment upon the characters of the young
animal (Fekete, 1947; Fekete and Little, 1942; Venge, 1950; McLaren
and Michie, 1958; Green and Green, 1959). Brochart (1954) re-
ported that he was able to demonstrate, both with transfer and
culture techniques, the survival of some rabbit 2-cell eggs in which
the blastomeres had been mechanically separated. There are also
problems of a technical nature that have drawn attention, the one
of greatest practical importance probably being that of the transfer
of early uterine blastocysts between cows without recourse to
surgery; a successful procedure has yet to be developed.
Studies on Eggs maintained in vitro
Metabolism. Observations on the metabolism of invertebrate eggs,
especially of sea-urchin eggs, are numerous and extensive, and
consideration of this subject is apt to account for a major part of
treatises on invertebrate fertilization and early development (see, for
example, Runnstrom, 1949; Brachet, i960). By contrast, very little
information is available on the metabolism of mammalian eggs and
early embryos, chiefly because they are difficult to obtain in even
moderate numbers. A few attempts have been made to determine
the oxygen uptake of eggs. Dragoiu, Benetato and Opreanu (1937)
made observations on cow eggs with the Warburg apparatus, but
their results are of doubtful significance because the eggs they used
112 THE MAMMALIAN EGG
were still surrounded by follicle cells. Subsequent investigations
were more critical and in each of these the method involved the use
of the Cartesian-diver technique. Boell and Nicholas (1939a, b, c,
1948) studied various cleavage stages in the rat and recorded figures
for oxygen uptake which lay mostly within the range of 0-5 to
i-o m/xl 02/egg/hr (i-o m/xl = io-6 ml). Rabbit eggs were studied
by Smith and Kleiber (1950) and Fridhandler, Hafez and Pincus
(1956a, b, 1957). Smith and Kleiber reported that the oxygen
uptake increased from about 26 m/xl/egg/hr for the i-cell egg to
about 60 mtJ/egg/hr for the morula and they pointed out that the
early embryo has a very much higher uptake, weight for weight,
than the adult organism. Fridhandler et ah found little difference
in oxygen consumption during the cleavage stages and the figure
they recorded was o-6i m/xl/egg/hr — remarkably at variance with
Smith and Kleiber's results. Early blastocysts displayed a sudden
increase in oxygen requirements with an uptake of 2-56 m^l/egg/hr.
According to Fridhandler and his associates, the addition of fluoride,
phlorizin, malonate, malonate-fumarate combinations, pyruvate or
glucose had little effect on oxygen uptake, and cyanide produced
only mild depression except when used at the high concentration of
i-o M. Eggs at the 1- to 16-cell stages showed no sign of glycolytic
activity, but late morulae and blastocysts did, at least in the presence
of exogenous glucose. It was inferred that the data showed evidence
of the emergence of an enzyme complex in the early developing
embryo.
Since rabbit eggs fail to enter the blastocyst stage when cultured
in serum under anaerobic conditions, this phase of development was
considered by Pincus (1941) to be a period in which the metabolism
of the embryo is delicately poised and therefore appropriate for
metabolic studies. He found that the addition of potassium cyanide
also inhibited blastocyst formation; glucose did not stimulate the
process nor was it taken up. Pyruvate (io_3m to io~2m), cysteine and
glutathione, on the other hand, did stimulate blastocyst growth.
Pincus concluded that energy for growth is derived chiefly from the
Meyerhof system, sulphydril compounds maintaining the enzymes.
The osmotic regulation of eggs has also received little attention.
It has often been observed that eggs kept in 0-9 per cent (isosmotic)
sodium-chloride solution soon show shrinkage of the vitellus.
Presumably the effect is to be attributed to the absence of colloids,
for eggs maintain their volume much better in saline solution if it
MANIPULATION OF EGGS
113
contains also some egg albumen or serum albumen. Since proteins
evidently cannot pass through the zona pellucida (p. 90) the influence
must reside in their osmotic effect at the surface of this membrane.
Active transport of potassium ions seems to be demonstrable in
eggs. Rat 2-cell eggs maintained for 18 hr in isosmotic solutions
of differing Na:K ratio displayed distinct differences in volume —
those in the higher concentrations of the potassium ion expanding
to the limits of the zona pellucida (Fig. 76).
Fig. 76
Rat 2-cell eggs after being held for 18 hr in media consisting of different proportions of
isosmotic sodium-chloride and potassium-chloride solutions, (a) NaCl alone; (b) 9.5 ml NaCl,
0-5 ml KC1; (r) 8 ml NaCl, 2 ml KC1; (d) 5 ml NaCl, 5 ml KC1. X 330.
Fertilized i-cell rabbit eggs placed in homologous serum at 20 C
containing 2-5, 3-75, 5 and 7-5 per cent glycerol were observed to
contract and re-expand during the hour they were left at each stage.
114 THE MAMMALIAN EGG
In the course of subsequent passage through 10 and 15 per cent
glycerol, however, the eggs shrank irreversibly, and from the
results of attempts to culture these eggs it was considered that they
had been killed. On the other hand, eggs treated with the same
concentrations of glycerol, but at 37°C and for 10 min at each
stage, contracted only slightly and soon re-expanded. The data
suggest that eggs are more permeable to glycerol at 37°C than at
20°C. When the eggs were freed of glycerol and placed in culture
in serum, most of them developed to morulae, showing that rabbit
eggs can tolerate exposure to relatively high concentrations of
glycerol at body temperature (Smith, 1952).
Mouse eggs (unfertilized tubal oocytes) exposed to a medium
composed of a suspension of egg yolk in Locke's solution, to which
sodium citrate had been added, showed only slight shrinkage when
held at 5°C for up to 2 hr. If the medium contained in addition
glycerol at a concentration of 5 per cent, however, the eggs shrank
considerably and became crenated. The effect took place within
15 min; no re-expansion occurred in the subsequent 1 to ij hr,
suggesting that glycerol had not entered the vitellus during the
period of observation. Vitelline shrinkage evidently had little effect
on the viability of the eggs, for when they were transferred to
recipient mated mice 22-8 per cent developed normally, a propor-
tion that was comparable to that found with untreated eggs (Lin,
Sherman and Willett, 1957).
Influence on spermatozoa. The spermatozoa of some primitive
plants are attracted towards the eggs by substances emanating from
the eggs ; this is probably best established for the ferns, in which the
attracting substance is L-malic acid (see Rothschild, 1956). The fern
spermatozoa are said to become orientated by chemotaxis, swim-
ming persistently towards higher concentrations of malic acid and
so reaching the eggs more surely than they would have otherwise.
Several claims have been made that a similar mechanism exists in
the animal kingdom, but so far they have not received general
acceptance. The main reason for this is the difficulty of distinguish-
ing between an attractive effect and a trapping action, these two
influences being likely to have very similar consequences in the
distribution and behaviour of the spermatozoa. Thus, in one in-
vestigation, the concentration of mouse spermatozoa was found to
be much higher in the region of cumulus oophorus immediately
surrounding the eggs than in peripheral parts of the cumulus ; but a
MANIPULATION OF EGGS 115
more acceptable explanation than the operation of chemotaxis is
simply that the resistance ofFered by the densely-packed, radially-
arranged follicle cells around the eggs tends to detain spermatozoa
there (Braden, 196 1). Another recent inquiry into the problem was
that of Schwartz, Brooks and Zinsser (1958), who noted that human
spermatozoa suspended in a neutral medium on a slide tended to
congregate in regions in which had been deposited fluids from
follicles or ovarian cysts, or hen egg-white ; they concluded that the
effect was caused by chemotaxis since the motility of the sperma-
tozoa in these regions was increased and this would tend to coun-
teract any trapping action.
An influence of a different kind exerted by eggs on spermatozoa
is that described by Bishop and Tyler (1956) ; they maintained that
a substance akin to the fertilizin of sea-urchin and other invertebrate
eggs diffuses from the zona pellucida and reacts with spermatozoa
in such a way as to increase their tendency to become attached to
surfaces by their heads. In slide preparations, the effect is seen in the
greater frequency of head-to-head agglutination of spermatozoa
nearer the eggs than of those further away. In nature, the action
of this 'fertilizin' could be responsible for attachment of spermatozoa
to the surface of the zona pellucida, preparatory to their penetration
of this membrane. The agent was detected in association with the
oocytes and freshly ovulated eggs of rabbit, mouse and cow, and
the reaction with spermatozoa was largely species specific. The
agent did not appear to be released by rabbit eggs that had acquired
mucin coats — presumably, it could not diffuse through the mucin
layer and this conforms with Bishop and Tyler's suggestion that it
may be a glycoprotein.
The term 'fertilizin' is used also by Thibault and Dauzier (i960)
for an agent with a somewhat different action. In the course of
experiments on the fertilization of rabbit eggs in vitro (p. 122), these
authors noted that both the proportion of eggs developing pro-
nuclei and the number of spermatozoa entering eggs were increased
if the eggs were held in vitro for 2 to 4 hr before the addition of
spermatozoa. An even greater improvement was achieved by
washing the eggs before semination. On the other hand, good
results could be had with freshly recovered eggs if the spermatozoa
used were obtained by removing the undiluted uterine fluid of a
mated animal instead of flushing the uterus with an artificial
medium, which was the procedure normally followed. Thibault
116 THE MAMMALIAN EGG
and Dauzier infer that the egg emits an agent resembling fertilizin,
which, however, does not agglutinate spermatozoa but instead
repells or immobilizes them. Further, they consider that the female
genital tract contains a substance that normally neutralizes the
'fertilizin'.
The relations between Bishop and Tyler's 'fertilizin' and Thibault
and Dauzier's 'fertilizin' have still to be elucidated. The former has
the characteristic effect that invertebrate fertilizin has, that of
agglutinating spermatozoa, but whether it can render spermatozoa
incapable of fertilization, as invertebrate fertilizin can, is not known.
Thibault and Dauzier's agent, though it does not agglutinate
spermatozoa, still has a right to be called 'fertilizin' for it renders
spermatozoa infertile, and invertebrate fertilizins are known that
have this effect on spermatozoa without agglutinating them (see
Metz, 1957). Another relation that needs to be investigated is that
between the strong agglutination inhibitor in vaginal washings
(Smith, 1949b), the female 'sperm antagglutin' (see Lindahl, i960,
for outline and references) and the factor in uterine secretions that
Thibault and Dauzier maintain opposes their 'fertilizin'. It is also
tempting to speculate that the acrosome reaction of mammalian
spermatozoa, as a feature of capacitation, may be evoked by sub-
stances emanating from the freshly ovulated eggs or their cumulus
investments (p. 96) and related in some way to the 'fertilizins' just
described.
Resistance to low temperatures. When fertilized (2-cell) rabbit eggs
in serum were cooled slowly to o, 5 or io°C, most of those stored
for 24 hr, and about half of those stored for 72 hr, were able
to undergo apparently normal cleavage on subsequent culture.
Nearly 25 per cent of eggs kept at io°C for 144 hr survived, but none
of those kept for 168 hr. Eggs were also transferred after storage
to recipient animals and litters were born from eggs that had been
held at o°C for up to 102 hr (Chang, 1947, 1948a, b, c). Blastocysts
proved to be less resistant — they could grow after 1 day at o°C or
2 days at io°C, but the birth of young was recorded only from
blastocysts stored for 1 day at io°C (Chang, 1950b). Unfertilized
eggs recovered 2 hr after ovulation could be kept at o°C for 48 to
72 hr, or at io°C for up to 96 hr, and still undergo fertilization after
transfer, but though fertilization seemed normal, most of the
embryos degenerated before birth (Chang, 1952a, 1953, 1955^, d).
MANIPULATION OF EGGS 117
Better prospects are offered when eggs receive some protection
from the ill-effects of low temperatures by treatment with glycerol.
Fertilized (i-cell) rabbit eggs treated at 37°C with glycerol at final
concentrations of 10 to 20 per cent were subjected to various low
temperatures and then thawed, freed of glycerol and placed in
culture. More than half the eggs kept at — I5°C for 2 or 3 days,
and 10 to 30 per cent of those kept for 4 to 7 days, developed well
in culture. Out of about 600 eggs left for up to 2 days at — 79°C,
— i6o°C, or — ioo°C, however, only six passed through a few cleav-
age divisions in culture (Smith, 1952, 1953a). Mouse eggs (unferti-
lized) have so far proved to have little resistance to low temperatures
even with protection from glycerol, The eggs were handled in
a medium composed of Locke's solution, to which was added
some sodium citrate, together with glycerol at a concentration of
5 per cent. After chilling, they were transferred to mated recipient
mice. Of eggs kept at 5°C for ij to 2 hr, 22-8 per cent developed
to embryos that seemed normal at autopsy on the 19th day of
pregnancy, but only two eggs out of 276 survived storage for 24 hr,
and none storage for 3 days. Rapid cooling to — 21 °C, followed by
immediate rewarming, had no apparent effect on viability, but only
seven out of sixty eggs developed after being kept at — io°C for
3 \ hr, and four out of sixty-six at o°C for 6 hr (Lin, Sherman and
Willett, 1957; Sherman and Lin, 1958, 1959)-
Most impressive are the results obtained by freezing follicular
oocytes within pieces of ovarian tissue, though these eggs cannot
be said to have been treated in vitro, in the strict sense of the term.
Observations based on the development of oocytes within sub-
cutaneous grafts of rat ovarian tissue have suggested that a few
oocytes (less than 10 per cent) are still viable after treatment with
15 per cent glycerol and freezing to — 79°C (Deanesly, i954> x957;
Green, Smith and Zuckerman, 1956). Proof of viability was
supplied by results obtained with the technique of orthotopic
grafting in mice. Oocytes from ovaries frozen at — 79°C for as
long as 6 weeks have been found capable of subsequent development
into normal young (Parrott, 1958, i960; Parrott and Parkes, i960).
Development in culture. Oocytes have been kept in vitro, under
tissue-culture conditions, to obtain their maturation prior to transfer
to recipient mated animals (Chang, 1955a, d) or prior to the
attempted induction of fertilization /'// vitro (Rock and Menkin,
1944; Menkin and Rock, 1948). In the great majority of investiga-
118 THE MAMMALIAN EGG
tions, however, penetrated or fertilized eggs have been placed in
culture so as to permit further development under artificial condi-
tions (Appendix No. 2). Some authors combined storage or culture
with subsequent transfer to suitable recipient animals in order to
demonstrate that the treatment in vitro had no permanent ill-effect
upon the embryo (Chang, 1948a, b, c, 1950b; Adams, 1956; Biggers
and McLaren, 1958; McLaren and Biggers, 1958).
Most success in culture has been had with rabbit eggs, which
undergo apparently normal cleavage from the i-ccll to the early
blastocyst stage, provided the medium contains about 50 per cent
or more of serum. Blastocyst expansion fails, however, and the
embryos collapse and become disorganized. The eggs of other
mammals have been found even more refractory to culture ; so far,
they have not been found to undergo more than one or two divisions
when placed in culture at the i-cell stage, but 4- to 8-cell mouse
eggs have often been shown capable of developing to blastocysts.
Here again, proteins, such as egg-white or serum, are evidently
essential components of the medium.
Fertilization in vitro. It is evident that the ease with which the
fertilization of many non-mammalian eggs can be obtained under
artificial conditions fostered the belief that mammalian eggs should
readily undergo fertilization in vitro. As a result, the consequences
of placing eggs and spermatozoa together /'// vitro were often inter-
preted on the assumption that fertilization must inevitably be
occurring or have taken place and that the provision of proof would
be an act of supererogation. The need for a more critical evaluation
of observations became apparent as the appreciation grew that eggs
could be activated to a degree of parthenogenetic development by
conditions they encountered under experiment, that ejaculated
spermatozoa were accompanied by substances detrimental to eggs,
that the sperm concentrations that seemed appropriate in tests were
in fact vastly greater than those normally occurring in vivo, and that
spermatozoa require to undergo capacitation before they become
capable of fertilization. In addition, the pitfalls inherent in some of
the experimental procedures have not always been clearly recog-
nized. Undoubtedly, the best criterion of the occurrence of fertiliza-
tion in vitro is the development of foetuses or the birth of young
from eggs transferred to recipient animals after treatment with
spermatozoa. Preferably, the progeny should in addition be of both
sexes and genetically distinguishable as deriving from the transferred
MANIPULATION OF EGGS 119
eggs. But if the recipient has been brought into a suitable state by
prior mating with a vasectomized male, there is the obvious danger
that the vasectomy was incompletely effective and that the male
was still ejaculating spermatozoa. Clearly, a better procedure is to
prepare the recipient by appropriate hormone treatment. Again,
eggs transferred after treatment with spermatozoa may be accom-
panied by free spermatozoa which later effect fertilization within
the recipient female tract — fertilization either of the transferred eggs
or of the recipient's eggs. This could happen even if the eggs under
test are carefully washed immediately before transfer, for it is
extremely difficult to remove adherent or accompanying sperma-
tozoa altogether. The danger that the recipient's eggs may be
fertilized can be taken into account by the use of genetic markers.
Probably the best way to circumvent the risk that the transferred
eggs are fertilized in this way is to transfer them only after they have
been kept in culture until the occurrence of cleavage (or fragmenta-
tion) indicates that the stage of fertilization is past. (There are several
other possible sources of error, in addition to those just described,
and these arise chiefly from the production of artefacts during
preparation of the eggs for histological study and from the mis-
interpretation of objects seen in histological sections. These points
have been discussed on several occasions: Chang and Pincus, 195 1;
Smith, 195 1 ; Austin and Bishop, 1957b; Chang, 1957a; Austin and
Walton, i960; Austin, 1961c.)
In view of the difficulties of establishing conclusively the occur-
rence of fertilization /'// vitro, it is not surprising that the great
majority of the claims for success, the more detailed of which are
shown in Table 5, are far from convincing. For various reasons, the
claims that seem to merit the most serious consideration are those
of Dauzier and his colleagues (Dauzier, Thibault and Wintenberger,
1954; Thibault, Dauzier and Wintenberger, 1954; Dauzier and
Thibault, 1956, 1959; Thibault and Dauzier, i960), of Moricard
(1954a, b) and of Chang (1959a).
Dauzier and his associates recovered eggs from rabbits soon after
artificially induced ovulation and held them under conditions that
were considered unlikely to provoke parthenogenetic development,
in the light of Thibault's (1949) earlier experience with this pheno-
menon. The eggs were maintained in Locke's solution in short
lengths of glass tubing. Spermatozoa in suspension were obtained
by flushing the tubal, uterine or vaginal lumina of rabbits mated
120
THE MAMMALIAN EGG
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MANIPULATION OF EGGS
121
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122 THE MAMMALIAN EGG
12 hr previously, and a small volume of this suspension was added
to the eggs. The glass tubes were sealed at both ends with liquid
paraffin. The preparation was incubated for 2 to 6 hr and the eggs
then fixed and prepared for histological examination. Some eggs
were transferred to homologous serum and kept in culture (in glass
tubes) to permit cleavage before being removed for histological
study. In sections of eggs, various stages were seen in the develop-
ment of two nuclei, which resembled in general appearance and
staining reactions normal male and female pronuclei. Sperm tails
could sometimes be identified in the vitellus and, in some of the
experiments, some of the eggs contained supplementary spermatozoa
in the perivitelline space. With the longer periods of incubation,
some eggs underwent cleavage and this appeared to have occurred
in a normal manner. Very few eggs that were subjected to the same
treatments, but without the addition of spermatozoa, showed any
sign of activation.
Moricard's work was of a somewhat similar nature: he placed the
freshly recovered rabbit eggs in a watch-glass under liquid paraffin
and added to them a suspension of spermatozoa recovered from the
uterus of an animal that had been mated 10 hr previously. After
incubating the eggs, he found that spermatozoa could be seen in
the perivitelline space of whole unfixed eggs (examined by phase-
contrast microscopy) and noted the development of pronuclei.
In addition to the cytological data, Dauzier and his associates
reported that they obtained only negative results when suspensions
of freshly ejaculated spermatozoa were used. No penetration was
recorded when the female rabbit, from whose genital tract the
sperm suspension was prepared, had been mated only 4 or 6 hr
previously, and the frequency increased with longer intervals from
mating, from some penetration at 8 hr up to a maximum of about
25 per cent at 12 hr. At 16 hr, the penetration frequency was low
again. In the most recent report of the series, evidence is adduced
in support of the idea that rabbit eggs emit a form of 'fertilizin'
which tends to inhibit sperm penetration and which is normally
neutralized by a substance in the secretions of the female genital
tract (see p. 115). Consistently, eggs washed several times after
recovery were found to have been penetrated much more frequently
(about 70 per cent) and to contain more supplementary spermatozoa
than eggs seminated without this treatment.
MANIPULATION OF EGGS 123
All these data constitute strong support for the claim that the eggs
investigated had indeed been fertilized in vitro, but it would have
been a much more convincing case had the authors transferred eggs
to recipients and recorded the birth of young. Curiously enough,
they do not appear to have tried transfer, and so it was left to Chang
(1959a) to take this important step and so provide what can reasonably
be regarded as proof. Having previously made several unsuccessful
attempts (see Chang, 1957a), he now followed the method used by
Dauzier and his associates, with minor modifications. Sperm
suspensions were made by flushing the uterine horns of rabbits
mated 12 hr beforehand with Krebs-Ringer bicarbonate solution
and placed in i-5-ml capacity Carrel flasks. Eggs were recovered
2 to 3 hr after ovulation with the same physiological solution and
placed in the sperm suspensions. The flasks were attached to a
rocking device within an incubator at 38°C and left for 3 to 4 hr.
After this time, the eggs were taken out and transferred to 8-ml
capacity Carrel flasks containing fresh homologous serum which
had earlier been heated to 55°C for 20 min. After incubation for
a further 18 hr, the eggs were removed and examined in the fresh
state. They were then transferred to recipient rabbits in which
ovulation had been artificially induced about 8 hr previously.
Chang reported that, when the eggs were examined in the fresh
state, 55 out of 266 (21 per cent) appeared to have undergone
normal cleavage into four cells. Of the fifty-five eggs, thirty-six
were transferred to six recipients. Two of the recipients did not
become pregnant, but the other four yielded fifteen living young.
From the observations of these investigators, it is reasonable to
conclude that the fertilization of rabbit eggs in vitro can in fact be
procured, provided that the spermatozoa used have been recovered
from the female genital tract some hours after mating or artificial
insemination. Within limits, other conditions, such as the chemical
nature of a suspending medium, the oxygen partial pressure and the
redox potential, are evidently of minor significance compared to
the need for employing spermatozoa that have undergone capacita-
tion. This does not necessarily mean, however, that all reports
relating to the use of epididymal or ejaculated spermatozoa should
be doubted, for the experiments of Noyes, Walton and Adams
(1958) suggest that it is possible for capacitation to take place in vitro
under certain conditions. Of special interest in this connection is the
work of Smith (195 1) who maintained that sperm penetration took
124
THE MAMMALIAN EGG
place when she incorporated scrapings of Fallopian-tube mucosa in
the medium but not otherwise. Establishment of the conditions
required for capacitation in vitro is certainly the next important step
to be taken in this field of research. (Other problems relating to the
fertilization of mammalian eggs in vitro have recently been discussed:
Austin, 1961c.)
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REFERENCES AND AUTHOR INDEX
(Numbers in square brackets refer to the pages on which the work is cited iu this book)
Adams, C. E.:
(1953) 'Some aspects of ovulation, recovery and transplantation of ova in the immature
rabbit.' Mammalian Germ Cells, p. 198. Ed. G. E. W. Wolstenholme, M. P. Cameron
and J. S. Freeman. Churchill, London. [Ill, 130]
(1954) 'The experimental shortening of the generation interval.' Proc. Brit. Soc. Anim.
Prod. p. 97. [Ill, 130]
(1955) 'The frequency of occurrence of supernumerary spermatozoa in rabbit ova.' Studies
on Fertility, 1, 130. [92]
(1956) 'Egg transfer and fertility in the rabbit.' Proc. IHrd int. Congr. Anim. Reprod.,
Cambridge, Section 3, p. 5. [118, 132, 146]
(1957) 'An attempt to cross the domestic rabbit (Oryctolagus cuniculus) and hare (Lepus
europaeus). Nature, Lond. 180, 853. [95]
Afzelius, B. A.:
(1956) 'The ultrastructure of the cortical granules and their products in the sea urchin
egg as studied with the electron microscope.' Exp. Cell Res. 10, 257. [65]
(1957) Electron microscopy of sea urchin gametes. Almquist & Wiksell, Uppsala. [65]
Alfert, M. (1950) 'A cytochemical study of oogenesis and cleavage in the mouse.' J. cell.
comp. Physiol. 36, 381. [18, 30, 31, 50, 52, 63]
Allen, P., Brambell, F. W. R., & Mills, I. H. (1947) 'Studies on sterility and prenatal
mortality in wild rabbits. I. The reliability of estimates of prenatal mortality based on
counts of corpora lutea, implantation sites and embryos.' J. exp. Biol. 23, 312. [20]
Amoroso, E. C:
(1952) 'Placentation.' Marshall's Physiology of Reproduction, 3rd edn., vol. 2, p. 127. Ed.
A. S. Parkes. Longmans, Green & Co., London. [14, 80]
(1959) 'The attachment cone of the guinea-pig blastocyst as observed under time-lapse
phase-contrast cinematography.' Implantation of Ova. Mem. Soc. Endocrin., No. 6, p. 50.
Ed. P. Eckstein. Cambridge University Press. [81]
Amoroso, E. C, Griffiths, W. F. B., & Hamilton, W. J. (1942) 'The early development
of the goat (Capra hircus). J. Anat., Lond. 76, 377. [14, 84]
Amoroso, E. C, & Parkes, A. S. (1947) 'Effects on embryonic development of X-irradiation
of rabbit spermatozoa in vitro. Proc. roy. Soc. B, 134, 57. [41, 69, 70, 85, 109]
Anderson, E., & Beams, H. W. :
(1956) 'Evidence from electron micrographs for the passage of material through pores of
the nuclear membrane. J. hiophys. biochem. Cytol. 2, Suppl. p. 439. [20]
(1960) 'Cytological observations on the fine structure of the guinea-pig ovary with special
reference to the oogonium, primary oocyte and associated follicle cells.' J. Ultrastructure
Res. 3, 432. [87]
Anderson, N. G. (1953) 'On the nuclear envelope.' Science, 117, 517. [20]
Austin, C. R.:
(1948) 'Function of hyaluronidase in fertilization.' Nature, Lond. 162, 63. [99]
(1949a) 'Fertilization and the transport of gametes in the pseudopregnant rabbit.' J.
Endocrin. 6, 63. [127]
(1949b) 'The fragmentation of eggs following induced ovulation in immature rats.'
J. Endocrin. 6, 104. [85]
(1950a) 'Fertilization of the rat egg.' Nature, Lond. 166, 407. [104]
149
150 THE MAMMALIAN EGG
(1950b) 'The fecundity of the immature rat following induced superovulation.' J. Endocrin.
6,293. [85]
(1951a) 'Observations on the penetration of the sperm into the mammalian egg.' Atist.J.
sci. Res. B, 4, 581. [99, 104]
(1951b) 'The formation, growth and conjugation of the pronuclei in the rat egg.' J. R.
tnicr. Soc. 71, 295. [46]
(1951c) 'Activation and the correlation between male and female elements in fertilization.'
Nature, Land. 168, 558. [48]
(1952a) 'The development of pronuclei in the rat egg, with particular reference to quanti-
tative relations.' Aust. J. sci. Res. B, 5, 354. [25, 27, 47]
(1952b) 'The "capacitation" of the mammalian sperm.' Nature, Lond. 170, 326. [99]
(1953) 'Nucleic acids associated with the nucleoli of living segmented rat eggs.' Exp. Cell
Res. 4, 249. [50, 51]
(1955) 'Polyspermy after induced hyperthermia in rats.' Nature, Land. 175, 1038. [47]
(1956a) 'Activation of eggs by hypothermia in rats and hamsters.' J. exp. Biol. 33, 338. [24,
36, 37, 38, 39]
(1956b) 'Effects of hypothermia and hyperthermia on fertilization in rat eggs.' J. exp.
Biol. 33,348. [41,42,46,47]
(1956c) 'Cortical granules in hamster eggs.' Exp. Cell Res. 10, 533. [65]
(1956d) 'Ovulation, fertilization and early cleavage in the hamster (Mesocricetus auratus):
J. R. tnicr. Soc. 75, 141. [39, 57, 68, 69, 74, 75, 80, 83, 86, 90, 91, 98]
(1957a) 'Oestrus and ovulation in the field vole (Microtus agrestis).' J. Endocrin. 15, iv. [11]
(1957b) 'Fertilization, early cleavage and associated phenomena in the field vole (Microtus
agrestis): J. Anat., Lond. 91, 1. [15, 30, 36, 41, 42, 55, 57, 68, 69, 75, 90]
(1957c) 'Fate of spermatozoa in the uterus of the mouse and rat.' J. Endocrin. 14, 335. [87]
(1959a) 'Entry of spermatozoa into the Fallopian tube mucosa.' Nature, Lond. 183, 908.
[87]
(1959b) 'The role of fertilization.' Perspectives Biol. Med. 3, 44. [8]
(1959c) 'Fertilization and development of the egg.' Reproduction in Domestic Animals, vol. 1,
chap. 12. Ed. H. H. Cole & P. T. Cupps. ^Academic Press, New York. [22, 49]
(1960a) 'Fate of spermatozoa in the female genital tract.' J. Reprod. Fertil. 1, 151. [87]
(1960b) 'Anomalies of fertilization leading to triploidy.' J. cell. comp. Physiol. 56, Suppl 1,
p. 1. [24, 41]
(1960c) 'Capacitation and the release of hyaluronidase from spermatozoa.' J. Reprod.
Fertil. 1, 310. [99]
(1961a) 'Egg.' Encyclopedia of Biological Sciences. Ed. P. Gray. Reinhold, New York. [15]
(1961b) 'Sex chromatin in embryonic and fetal tissue.' Acta cytol. 5 (in press). [52]
(1961c) 'Fertilization of mammalian eggs in vitro.' Int. Rev. Cytol. (in press). [119, 124]
(1961d) 'Significance of sperm capacitation.' Proc. IVth int. Congr. Anim. Reprod., Hague
(in press). [99]
Austin, C. R., & Amoroso, E. C:
(1957) 'Sex chromatin in early cat embryos.' Exp. Cell Res. 13, 419. [52]
(1959) 'The mammalian egg.' Endeavour, 18, 130. [15, 30, 32, 60, 89]
Austin, C. R., & Bishop, M. W. H.:
(1957a) 'Preliminaries to fertilization in mammals.' The Beginnings of Embryonic Develop-
ment, p. 71. Ed. A. Tyler, R. C. von Borstel and C. B. Metz. American Association
for the Advancement of Science, Washington. [89]
(1957b) 'Fertilization in mammals.' Biol. Rev.il, 296. [23, 54, 69, 71, 75, 119]
(1958a) 'Capacitation of mammalian spermatozoa.' Nature, Lond. 181, 851. [92]
(1958b) 'Some features of the acrosome and perforatorium in mammalian spermatozoa.'
Proc. roy. Soc. B, 149, 234. [71, 92, 99, 100]
(1958c) 'Role of the rodent acrosome and perforatorium in fertilization.' Proc. roy. Soc. B,
149, 241. [71, 90, 92, 99, 100]
REFERENCES AND AUTHOR INDEX 151
(1959a) 'Differential fluorescence in living rat eggs treated with acridine orange.' Exp. Cell
Res. 17, 35. [17, 30, 32, 60, 107]
(1959b) 'Presence of spermatozoa in the uterine-tube mucosa of bats.' J. Endocrin. 18, viii.
[87]
Austin, C. R., & Braden, A. W. H. :
(1953a) 'Polyspermy in mammals.' Nature, Land. 172, 82. [41]
(1953b) 'An investigation of polyspermy in the rat and rabbit.' Aust. J. biol. Sci. 6, 674.
[41, 42, 45, 46, 48, 76, 79, 80]
(1953c) 'The distribution of nucleic acids in rat eggs in fertilization and early segmentation.
I: Studies on living eggs by ultraviolet microscopy.' Aust. J. biol. Sci. 6, 324. [17, 32,
50, 59, 61]
(1954a) 'Time relations and their significance in the ovulation and penetration of eggs in
rats and rabbits.' Aust. J. biol. Sci. 7, 179. [83, 98, 99]
(1954b) 'Induction and inhibition of the second polar division in the rat egg and subsequent
fertilization.' Aust. J. biol. Sci. 7, 195. [24, 35, 36, 39, 41, 42, 48, 57, 75]
(1954c) 'Anomalies in rat, mouse and rabbit eggs.' Aust. J. biol. Sci. 7, 537. [12, 15, 36, 39,
41, 76, 77]
(1954d) 'Nucleus formation and cleavage induced in unfertilized rat eggs.' Nature, Loud.
173, 999. [38, 39]
(1955) 'Observations on nuclear size and form in living rat and mouse eggs.' Exp. Cell
Res. 8, 163. [47]
(1956) 'Early reactions of the rodent egg to spermatozoon penetration.' J. exp. Biol. 33,
358. [41, 42, 87, 88, 93, 94]
Austin, C. R., & Bruce, H. M. (1956) 'Effect of continuous oestrogen administration on
oestrus, ovulation and fertilization in rats and mice.' J. Endocrin. 13, 376. [39]
Austin, C. R., & Lovelock, J. E. (1958) 'Permeability of rabbit, rat and hamster egg mem-
branes.' Exp. Cell Res. 15, 260. [90, 98, 101]
Austin, C. R., & Sapsford, C. S. (1952) 'The development of the rat spermatid.' J. R.
micr. Soc. 71, 397. [71]
Austin, C. R., 3c Smiles, J. (1948) 'Phase-contrast microscopy in the study of fertilization
and early development of the rat egg.' J. R. micr. Soc. 68, 13. [69]
Austin, C. R., & Walton, A. (1960) 'Fertilization.' Marshall's Physiology of Reproduction,
3rd edn., vol. 1, pt. 2. Ed. A. S. Parkes. Longmans, London. [15, 46, 69, 78, 89, 119]
Averill, R. L. W. (1956) 'The transfer and storage of sheep ova.' Proc. IHrd int. Congr.
Anint. Reprod., Cambridge, Section 3, p. 7. [110, 111, 142]
Averill, R. L. W., Adams, C. E., & Rowson, L. E. A. (1955) 'Transfer of mammalian ova
between species.' Nature, Loud. 176, 167. [110, 111, 141]
Averill, R. L. W., & Rowson, L. E. A. (1958) 'Ovum transfer in sheep.' J. Endocrin. 16,
326. [Ill, 142]
Avis, F. R., & Sawin, P. S. (1951) 'A surgical technique for the reciprocal transplantation
of fertilized eggs in the rabbit.' J. Hered. 42, 259. [105, 128]
Bacsich, P. (1949) 'Multinuclear ova and multiovular follicles in the young human ovary
and their probable endocrinological significance.' J. Endocrin. 6, i. [20]
Bacsich, P., 8c Hamilton, W. J. (1954) 'Some observations on vitally stained rabbit ova
with special reference to their albuminous coat.' J. Embryol. exp. Morph. 2, 81. [101]
Bacsich, P., & Wyburn, G. M. (1945) 'Parthenogenesis of atretic ova in the rodent ovary.'
J. Anat., Lond. 79, 177. [85]
Baer, K. E. von (1827) 'De ovi mammalium et hominis genesi.' Lipsiae. [2, 3]
Barr, M. L., Bertram, L. F., & Lindsay, H. A. (1950) 'The morphology of the nerve cell
nucleus, according to sex.' Anat. Rec. 107, 283. [52]
Barry, M.:
(1838) 'Researches in embryology — first series.' Phil. Trans, pt. 1, 301. [4]
152 THE MAMMALIAN EGG
(1839) 'Researches in embryology — second series.' Phil. Trans, pt. 2, 307. [4, 11, 13]
(1843) 'Spermatozoa observed within the mammiferous ovum.' Phil. Trans. 133, 33. [5]
Bateman, N. (1960) 'Selective fertilization at the T-locus of the mouse.' Genet. Res., Camb. 1,
226. [96]
Beatty, R. A.:
(1951a) 'Heteroploidy in mammals.' Anim. Breed. Abstr. 18, 283. [24]
(1951b) 'Transplantation of mouse eggs.' Nature, Lond. 168, 995. [134]
(1954) 'Haploid rodent eggs.' Caryologia 6 (Suppl. Pt. 2), 784. [38]
(1956a) 'Ovum characteristics: mammals.' Handbook of Biological Data, p. 124. Ed. W. S.
Spector. W. B. Saunders Co., Philadelphia. [14, 15, 83, 84]
(1956b) 'Melanizing activity of semen from rabbit males of different genotvpe.' Proc. roy.
phys. Soc, Edinb. 25, 39. [23]
(1957) Parthenogenesis and polyploidy in mammalian development. Cambridge University
Press. [23, 24, 75, 79]
Beatty, R. A., & Fischberg, M. (1951) 'Heteroploidy in mammals. 1. Spontaneous hetero-
ploidy in pre-implantation mouse embryos.' J. Genet. 50, 345. [46]
Beatty, R. A., & Napier, R. A. N. (1960) 'Genetics of gametes. II. Strain differences in
characteristics of rabbit spermatozoa.' Proc. roy. Soc. Edinb., B, 68, 17. [23]
Beatty, R. A., & Sharma, K. N. (1960) 'Genetics of gametes. III. Strain differences in
spermatozoa from eight inbred strains of mice.' Proc. roy. Soc. Edinb., B, 68, 25. [23]
Berry, R. O., & Savery, A. P. (1958) 'A cytological study of the maturation process of the
ovum of the ewe during normal and induced ovulation.' Reproduction and Infertility,
p. 75. III. Symposium. Ed. F. X. Gassner. Pergamon Press, London. [107]
Biedl, L., Peters, H, & Hofstatler, R. (1922) 'Experimented Studien uber die Einnistung
und Weiterentwicklung des Eies im Uterus.' Z. Geburtsh. Gyndk. 84, 59. [125]
Biggers, J. D., & McLaren, A. (1958) ' "Test-tube" animals — the culture and transfer of
early mammalian embryos.' Discovery, Oct. 1958, p. 423. [110, 118, 136, 147]
Bischoff, T. L. W. :
(1842a) Entwicklungsgeschichte des Kanincheneies. Braunschweig. [5, 11, 13]
(1842b) Entwickelungsgeschichte des Menschen und der Sdugethiere. Leipzig. [5]
(1845) Entwickelungsgeschichte des Hundeeies. Braunschweig. [5]
(1852) Entwickelungsgeschichte des Meerschweinchens. Giessen. [5]
(1854a) Bestdtigung des von Dr. Newport bei den Batrachiern und Dr. Barry bei den Kaninchen
behaupteten Eindringens der Spermatozoiden in das Ei. Giessen. [5]
(1854b) Entwickelungsgeschichte des Relies. Giessen. [5]
(1863) 'Ueber die Ranzzeit des Fuchses und die erste Entwickelung seines Eies.' Sitzungsber.
meth. phys. CI., 13juni. [5]
Bishop, D. W., & Tyler, A. (1956) 'Fertilizes of mammalian eggs.' J. exp. Zool. 132, 575.
[115]
Bishop, M. W. H. (1960) 'The possibility of controlling sex ratio at conception. I. Spermato-
genesis and the individuality of the spermatozoon.' Sex Differentiation and Develop-
ment. Mem. Soc. Endocrin., No. 7, p. 81. Ed. C. R. Austin. Cambridge University
Press. [23]
Bishop, M. W. H, & Austin, C. R. (1957) 'Mammalian spermatozoa.' Endeavour, 16, 137.
[99]
Bishop, M. W. H, & Walton, A. (1960) 'Spermatogenesis and the structure of mammalian
spermatozoa.' Marshall's Physiology of Reproduction, 3rd edn., vol. 1, pt. 2, p. 1. Ed.
A. S. Parkes. Longmans, Green & Co., London. [24]
Black, W. G., Otto, G., & Casida, L. E. (1951) 'Embryonic mortality in pregnancies in-
duced in rabbits of different reproductive stages.' Endocrinology, 49, 237. [Ill, 128]
Blanchard, R. (1878) 'La fecondation dans la serie animale, d'apres les publications les plus
recentes. Revue bibliographique.' J. Anat. Physiol. 14, 551, 701. [73]
REFERENCES AND AUTHOR INDEX 153
Blandau, R. J. :
(1945) 'The first maturation division of the rat ovum.' Anat. Rec. 92, 449. [74]
(1949a) 'Observations on implantation of the guinea-pig ovum.' Anat. Rec. 103, 19. [81]
(1949b) 'Embryo-endometrial interrelationship in the rat and guinea-pig.' Anat. Rec. 104,
331. [81]
(1952) 'The female factor in fertility and infertility. I: Effects of delayed fertilization on
the development of the pronuclei in rat ova.' Fertil. & Steril. 3, 349. [36]
(1954) 'The effects on development when eggs and sperm are aged before fertilization.'
Ann. N. V. Acad. Sci. 57, 526. [13]
Blandau, R. J., & Odor, D. L.:
(1950) 'Observations on fertilization of rat ova.' Anat. Rec. 106, 177. [28]
(1952) 'Observations on sperm penetration into the ooplasm and changes in the cyto-
plasmic components of the fertilizing spermatozoon in rat ova.' Fertil. & Steril. 3, 13
[69]
Blandau, R. J., & Young, W. C. (1939) 'The effects of delayed fertilization on the develop-
ment of the guinea-pig ovum.' Amer. J. Anat. 64, 303. [13]
Block, E. (1953) 'Quantitative morphological investigation of follicular system in newborn
female infants.' Acta Anat. 17, 201. [8]
Bluntschli, H. (1938) 'Le developpement primaire et l'implantation chez un centetine
(Hemicentetes).' C. R. Ass. Anat. Bale 1, 39. [13, 78]
Bodenhelmer, F. S., & Lasch, W. (1957) 'The primordial egg in the ovary of the adult
female of the Levant vole (Microtns giintheri D.a.A.).' Stud. Biol. Hist. {Jems.) 1, 9. [8]
Bodenheimer, F. S., & Sulman, F. (1946) 'The oestrous cycle of Microtns giientheri D. and A.
and its ecological implications.' Ecology, 27, 255. [11]
Boell, E. J., & Nicholas, J. S. :
(1939a) 'Respiratory metabolism of mammalian eggs and embryos.' Science, 90, 411. [112]
(1939b) 'Respiratory metabolism of mammalian eggs and embryos.' Anat. Rec. 73 (Suppl.),
9. [112]
(1939c) 'Respiratory metabolism of mammalian eggs and embryos.' Anat. Rec. 75 (Suppl.),
66. [112]
(1948) 'Respiratory metabolism of the mammalian egg.' J. exp. Zool. 109, 267. [112]
Boot, L. M., & Muhlbock, O. (1953) 'Transplantation of ova in mice.' Acta physiol. pharm.
need. 3, 133. [135]
Borghese, E. (1957) 'Recent histochemical results of studies on embryos of some birds and
mammals.' Int. Rev. Cytol. 6, 289. [61]
Boveri, T. (1891) 'Befruchtung.' Ergebn. Anat. EntwGesch. 1, 386. [6]
Boving, B. G. (1954) 'Blastocyst-uterine relationships.' Cold Spring Harbor Synip. quant.
Biol. 19, 9. [102]
Boyd, J. D., &: Hamilton, W. J. (1952) 'Cleavage, early development and implantation of
the egg.' Marshall's Physiology of Reproduction, 3rd edn., vol. 2, chap. 14. Ed. A. S.
Parkes. Longmans, Green & Co., London. [15, 84]
Brachet, A.:
(1912) 'Developpement in vitro de blastodermes et de jeunes embryons de mammiferes.'
C. R. Acad. Sci., Paris, 155, 1191, 1912. [144]
(1913) 'Recherches sur le determinisme hereditaire de l'oeuf des mammiferes. Developpe-
ment in vitro de jeunes vesicules blastodermiques du lapin.' Arch. Biol., Paris, 28, 447,
1913. [144]
(1922) 'Recherches sur la fecondation prematuree de l'oeuf d'oursin (Paracentrotus lividus).'
Arch. Biol, Liege 32, 205. [47]
Brachet, J. :
(1957) Biochemical cytology. Academic Press Inc., New York. [19, 48, 62]
(1960) The biochemistry of development. Pergamon Press, London. [Ill]
L
154 THE MAMMALIAN EGG
liRADEN, A. W. H. :
(1952) 'Properties of the membranes of rat and rabbit eggs.' Aust. J. sci. Res. B, 5, 460.
[89, 90, 91, 98, 101]
(1955) 'The reactions of isolated mucopolysaccharides to several histochemical tests.'
Stain Tech. 30, 19. [98]
(1957) 'Variation between strains in the incidence of various abnormalities of egg matura-
tion and fertilization in the mouse.' J. Genet. 55, 476. [23, 36, 41, 42, 45, 46, 75, 76]
(1958a) 'Strain differences in the incidence of polyspermia in rats after delayed mating.'
Fertil. & Steril. 9, 243. [41, 42]
(1958b) 'Variation between strains of mice in phenomena associated with sperm penetration
and fertilization.' J. Genet. 56, 37. [96, 98]
(1958c) 'Influence of time of mating on the segregation ratio of alleles at the T-locus in
the house mouse.' Nature, Lond. 181, 786. [96]
(1959) 'Strain differences in the morphology of the gametes of the mouse.' Aust. J. biol.
Sci. 12, 65. [23, 53]
(1960) 'Genetic influences on the morphology and function of the gametes.' J. cell. comp.
Physiol. 56, Suppl. 1, p. 17. [96, 98]
(1961) 'Spermatozoon penetration and fertilization in the mouse.' Int. Symp. exp. Biol.
(in press). [23, 115]
Braden, A. W. H., & Austin, C. R.:
(1953) 'The distribution of nucleic acids in rat eggs in fertilization and early segmentation.
II: Histochemical studies.' Aust. J. biol. Sci. 6, 665. [30, 32, 50, 59]
(1954a) 'The number of sperms about the eggs in mammals and its significance for normal
fertilization.' Aust. J. biol. Sci. 7, 543. [43]
(1954b) 'Fertilization of the mouse egg and the effect of delayed coitus and of hot-shock
treatment.' Aust. J. biol. Sci. 7, 552. [23, 42, 46, 77, 83]
(1954c) 'Reactions of unfertilized mouse eggs to some experimental stimuli.' Exp. Cell
Res. 7, 277. [38,39,77]
(1954d) 'The fertile life of mouse and rat eggs.' Science, 120, 361. [13]
Bkaden, A. W. H., Austin, C. R., & David, H. A. (1954) 'The reaction of the zona pellucida
to sperm penetration.' Aust. J. biol. Sci. 7, 391. [41, 92, 94]
Braden, A. W. H., & Gluecksohn-Waelsch, S. (1958) 'Further studies of the effects of
the T-locus in the house mouse on male fertility.' J. exp. Zool. 138, 431. [96]
Brambell, F. W. R. :
(1935) 'Reproduction in the common shrew (Sorex araneus Linnaeus). I. The oestrous cycle
of the female.' Phil. Trans. B, 225, 1. [11]
(1956) 'Ovarian changes.' Marshall's Physiology of Reproduction, 3rd edn., vol. 1, pt. 1,
chap. 5. Ed. A. S. Parkes. Longmans, Green & Co., London. [8]
Brambell, F. W. R., Fielding, U., & Parkes, A. S. (1928) 'Changes in the ovary of the
mouse following exposure to X-rays. 4. The corpus luteum in the sterilized ovary, and
some concluding experiments.' Proc. roy. Soc. B, 102, 385. [8]
Brambell, F. W. R., & Hall, K. (1937) 'Reproduction of the lesser shrew (Sorex minutus
Linnaeus).' Proc. zool. Soc, Lond., p. 957. [11]
Brambell, F. W. R., &: Hemmings, W. A. (1949) 'The passage into the embryonic yolk-sac
cavity of maternal plasma proteins in rabbits.' J. Physiol. 108, 177. [81]
Brambell, F. W. R., & Parkes, A. S. (1927) 'Changes in the ovary of the mouse following
exposure to X-rays. 3. Irradiation of the non-parous adult.' Proc. roy. Soc. B, 101,
316. [8]
Brambell, F. W. R., Parkes, A. S., &: Fielding, U. :
(1927a) 'Changes in the ovary of the mouse following exposure to X-rays. 1. Irradiation
at three weeks old.' Proc. roy. Sec. B, 101, 29. [8]
(1927b) 'Changes in the ovary of the mouse following exposure to X-rays. 2. Irradiation
at or before birth.' Proc. roy. Soc. B, 101, 95. [8]
REFERENCES AND AUTHOR INDEX 155
Brenneke, H. (1937) 'Strahlenschadigung von Mause- und Rattensperme, beobachtet an
der Fruhentwicklung der Eicr.' Strdhlentherapie, 60, 214. [85]
Briones, H., & Beatty, R. A. (1954) 'Interspecific transfers of rodent eggs.' J. exp. Zool.
125,99. [Ill, 131, 135]
Brochart, M. (1954) 'Attempted experimental production of identical twins in rabbits.'
Nature, Lond. 173, 160. [Ill]
Bruce, H. M., &: Austin, C. R. (1956) 'An attempt to produce the Hertwig effect by
X-irradiation of male mice.' Studies on Fertility, 8, 121. [39, 48, 58, 85]
Burdick, H. O., Emmerson, B. B., & Whitney, R. (1940) 'Effects of testosterone propionate
on pregnancy and on passage of ova through the oviducts of mice.' Endocrinology, 26,
1081. [82]
Burdick, H. O., & Pincus, G. (1935) 'The effect of oestrin injections upon the developing
ova of mice and rabbits.' Amer.J. Physiol. Ill, 201. [82]
Burdick, H. O., & Whitney, R. (1937) 'Acceleration of the rate of passage of fertilized ova
through the Fallopian tubes of mice by massive injections of an estrogenic substance.
Endocrinology 21, 637. [82]
Burdick, H. O., Whitney, R., & Pincus, G. (1937) 'The fate of mouse ova tube-locked by
injections of oestrogenic substances.' Anat. Rec. 67, 513. [82]
Caldwell, W. H. (1887) 'The embryology of Monotremata and Marsupialia. I.' Phil. Trans.
B, 178, 463. [102]
Casida, L. E., Warwick, E. J., & Meyer, R. K. (1944) 'Survival of multiple pregnancies
induced in the ewe following treatment with pituitary gonadotropins.' J. Anim. Sci. 3,
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Caspersson, T. O. (1950) Cell growth and cell function. Norton & Co., New York. [51]
Cattanach, B. M., & Edwards, R. G. (1958) 'The effects of triethylenemelamine on the
fertility of male mice.' Proc. roy. Soc. Edinb. 67, 54. [77]
Champy, C. (1923) 'Parthenogenesc experimentale chez le lapin.' C. R. Soc. Biol., Paris, 96,
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Chang, M. C:
(1947) 'Normal development of fertilized rabbit ova stored at low temperature for several
days.' Nature, Lond. 159, 602. [110, 116, 126]
(1948a) 'The effects of low temperature on fertilized rabbit ova in vitro, and the normal
development of ova kept at low temperature for several days.' J. gen. Physiol. 31, 385.
[110, 116, 118, 126]
(1948b) 'Probability of normal development after transplantation of fertilized rabbit ova
stored at different temperatures.' Proc. Soc. exp. Biol., N.Y., 68, 680. [110, 116, 118, 126]
(1948c) 'Transplantation of fertilized rabbit ova — the effect on viability of age, in vitro
storage period, and storage temperature.' Nature, Lond. 161, 978. [110, 116, 118, 126]
(1949a) 'Effects of heterologous sera on fertilized rabbit ova.' J. gen. Physiol. 32, 291. [145]
(1949b) 'Artificial insemination of rabbits and transplantation of rabbit eggs. (Motion
picture.)' Anat. Rec. 105, 550. [109]
(1950a) 'Development and fate of transferred rabbit ova or blastocysts in relation to the
ovulation time of recipients.' J. exp. Zool. 114, 197. [Ill, 127]
(1950b) 'Transplantation of rabbit blastocysts at late stage; probability of normal develop-
ment and viability at low temperature.' Science, 111, 544. [110, 116, 118, 127]
(1950c) 'The effect of seminal plasma on fertilized rabbit ova.' Proc. nat. Acad. Sci., Wash.
36, 188. [145]
(1950d) 'Der gegenwartige Stand der Saugetierei-transplantation.' Wien. tierdrztl. Mschr.
12,913. [109]
(1950e) 'Cleavage of unfertilized ova in immature ferrets.' Anat. Rec. 108, 31. [84, 85]
(1951a) 'Fertilizing capacity of spermatozoa deposited into the Fallopian tubes.' Nature,
Lond. 168, 697. [99]
156 THE MAMMALIAN EGG
(1951b) 'The problems of superovulation and egg transfer in cattle.' Proc. 1st nat. Egg-,
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(1951c) 'Maintenance of pregnancy in intact rabbits in the absence of corpora lutea.' Endo-
crinology, 48, 17. [129]
(1951d) 'Fertility and sterility as revealed in the study of fertilization and development of
rabbit eggs.' Fertil. & Steril. 2, 205. [101, 102, 129]
(1952a) 'Fertilizability of rabbit ova and the effects of temperature in vitro on their subsequent
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(1952b) 'Effects of delayed fertilization on segmenting ova, blastocysts and fetuses in
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(1953) 'Fertilizability of rabbit germ cells.' Mammalian Germ Cells, p. 226. Ed. G. E. W.
Wolstenholme, M. P. Cameron and J. S. Freeman. Churchill, London. [116, 130]
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(1955a) 'Fertilization and normal development of follicular oocytes in the rabbit.' Science
121, 867. [116, 117, 131, 146]
(1955b) 'Development of fertilizing capacity of rabbit spermatozoa in the uterus.' Nature,
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(1955c) 'Vital stain of rabbit eggs in vitro during fertilization.' Anat. Rec. 121, 427. [101]
(1955d) 'The maturation of rabbit oocytes in culture and their maturation, activation,
fertilization and subsequent development in the Fallopian tubes.' /. exp. Zool. 128, 379.
[116, 117, 131]
(1957a) 'Some aspects of mammalian fertilization.' The Beginnings of Embryonic Develop-
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(1957b) 'Natural occurrence and artificial induction of parthenogenetic cleavage of ferret
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(1958) 'Capacitation of rabbit spermatozoa in the uterus with special reference to the
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(1959a) 'Fertilization of rabbit ova in vitro.' Nature, Eond. 184, 466. [119, 121, 123]
(1959b) 'Degeneration of ova in the rat and rabbit following oral administration of
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[82]
(1960) 'Fertilization of domestic rabbit (Oryctolagus cuniculus) ova by cottontail rabbit
(Sylvilagtis transitionalis) sperm.' J. exp. Zool. 144, 1. [95]
Chang, M. C, & Fernandez-Cano (1958) 'Effects of delayed fertilization on the develop-
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36, 39, 41, 42, 46, 83]
Chang, M. C, & Hunt, D. M.:
(1956) 'Effects of proteolytic enzymes on the zona pellucida of fertilized and unfertilized
mammalian eggs.' Exp. Cell Res. 11, 497. [90, 91]
(1960) 'Effects of in vitro radiocobalt irradiation of rabbit ova on subsequent development
in vivo with special reference to the irradiation of maternal organism.' Anat. Rec. 137,
511. [110, 132]
Chang, M. C, Hunt, D. M., & Romanoff, E. B. (1958) 'Effects of radiocobalt irradiation
of unfertilized or fertilized rabbit ova in vitro on subsequent fertilization and development
in vivo: Anat. Rec. 132, 161. [85, 110, 132]
Chang, M. C, & McDonough, J. J. (1955) 'An experiment to cross the cottontail and the
domestic rabbit.' J. Hered. 46, 41. [95]
Chang, M. C, & Marden, W. G. R. (1954) 'The aerial transport of fertilized mammalian
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Chang, M. C, & Pincus, G. (1951) 'Physiology of fertilization in mammals.' Physiol. Rev.
31, 1. [119]
REFERENCES AND AUTHOR INDEX 157
Chiquoine, A. D. :
(1959) 'Electron microscopic observations on the developmental cytology of the mam-
malian ovum.' Anat. Rec. 133, 258. [87, 89, 97]
(1960) 'The development of the zona pellucida of the mammalian ovum.' Atner.J. Anat.
106, 149. [87, 89, 97]
Chitty, H., & Austin, C. R. (1957) 'Environmental modification of oestrus in the vole.'
Nature, Lond. 179,592. [11]
Clement, A. C. (1935) 'The formation of giant polar bodies in centrifuged eggs of Ilyanassa.'
Biol. Bull. Woods Hole, 69, 403. [75]
Cleveland, L. R. :
(1958a) 'Photographs of fertilization in the smaller species of Trichonympha* J. Protozool.
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Clewe, T. H., Yamate, R. M., & Noyes, R. W. (1958) 'Maturation of ova in mammalian
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Colwin, A. L., & Colwin, L. H. (1957) 'Morphology of fertilization : acrosome filament
formation and sperm entry.' The Beginnings of Embryonic Development , p. 135. Ed. A.
Tyler, R. C. von Borstel and C. B. Metz. [100]
Conklin, E. G. (1917) 'Effects of centrifugal force on the structure and development of the
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Corner, G. W. (1933) 'The discovery of the mammalian ovum.' Lectures on the History of
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Coste, J. (1834) 'Recherches sur la generation des mammiferes.' Ann. Sci. nat. 2, 1. [4]
Costello, D. P. (1949) 'The relations of the plasma membrane, vitelline membrane, and
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Costello, D. P., Davidson, M. E., Eggers, A., Fox, M. H., & Henley, C. (1957) 'Methods
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Cruickshank, W. (1797) 'Experiments in which, on the third day after impregnation, the
ova of rabbits were found in the Fallopian tubes; and on the fourth day after impregna-
tion in the uterus itself; with the first appearance of the foetus.' Phil. Trans, pt. 1, 197.
[2,3]
Dalcq, A. M.:
(1951) 'New descriptive and experimental data concerning the mammalian egg, principally
of the rat. I, Ha, b.' Proc. Acad. Sci. Amst. C, 54, 351. [32, 108]
(1952) 'Effets de la centrifugation sur l'oocyte de 2e ordre et l'oeuf fecondc indivis du rat.'
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Dalcq, A. M., & Pasteels, J. (1955) 'Determination photometrique de la teneur relative en
DNA des noyaux dans les oeufs en segmentation du rat et de la souris.' Exp. Cell Res.
Suppl. 3, p. 72. [52, 61]
158 THE MAMMALIAN EGG
Dalcq, A., & Van Egmond, M. (1953) 'Effets de la centrifugation sur l'oocyte de trois
mammiferes (rat, hamster, taupe).' Arch. Biol., Paris, 64, 311. [18]
Dan, J. C. (1956) 'The acrosome reaction.' Int. Rev. Cytol. 5, 365. [100]
Dauzier, L., & Thibault, C.:
(1956) 'Recherche experimentale sur la maturation des gametes males chez les mammiferes,
par l'etude de la fecondation in vitro de l'oeuf de lapine.' Proc. IHrd int. Congr. Atiim.
Reprod., Cambridge, Section I, p. 58. [31, 57, 69, 87, 92, 119, 121]
(1959) 'Donnees nouvelles sur la fecondation in vitro de l'oeuf de la lapine et de la brebis.'
C. R. Acad. Sci. 248, 2655. [119, 121]
Dauzier, L., Thibault, C., & Wintenberger, S. (1954) 'La fecondation in vitro de l'oeuf
de la lapine.' C. R. Acad. Sci., Paris, 238, 844. [119, 121]
Davis, D. E., & Hall, O. (1950) 'Polyovuly and anovular follicles in the wild Norway rat.'
Anat. Rec. 107, 187. [20]
Davidov, S. G. (1952) 'The wider use of the achievements of Micurin agrobiology in animal
breeding (trans, title).' Anint. Breed. Abstr. 20, 9. [109]
Dawson, A. B. (1951) 'Histogenic interrelationships of oocytes and follicle cells. A possible
explanation of the mode of origin of certain polyocular follicles in the immature rat.'
Anat. Rec. 110, 181. [20]
Dawson, A. B., & Friedgood, H. B. (1940) 'The time and sequence of preovulatory changes
in the cat ovary after mating or mechanical stimulation of the cervix uteri.' Anat. Rec.
76,411. [10]
Deane, H. W. (1952) 'Histochemical observations on the ovary and oviduct of the albino
rat during the estrous cycle.' Amer. J. Anat. 91, 363. [89]
Deanesly, R. :
(1944) 'The reproductive cycle of the female weasel (Mustela nivalis).' Proc. zool. Soc,
Lond. 114, 339. [10]
(1954) 'Immature rat ovaries grafted after freezing and thawing.' J. Endocrin. 11, 197. [117]
(1957) 'Egg survival in immature rat ovaries grafted after freezing and thawing.' Proc.
roy. Soc. B, 147, 412. [117]
Dederer, P. H. (1934) 'Polyovular follicles in the cat.' Anat. Rec. 60, 391. [20]
Defrise, A. (1933) 'Some observations on living eggs and blastulae of the albino rat.' Anat.
Rec. 57, 239. [147]
Dempsey, E. W. (1939) 'Maturation and cleavage figures in ovarian ova.' Anat. Rec. 75, 223.
[21]
De Robertis, E. D. P., Nowinski, W. W., & Saez, F. A. (1954) General cytology, 2nd edn.
W. B. Saunders Co., Philadelphia. [19, 72]
Desaive, P.:
(1940) 'Contribution radio-biologique a l'etude de l'ovaire.' Arch. Biol., Paris, 51, 5. [8]
(1941) 'Contribution radio-biologique a la demonstration de la fixite, dans l'ovaire de
lapine adulte, des sources du dcveloppement folliculaire.' Acta, neerl. morph. 4, 10. [8]
Dickmann, Z., & Noyes, R. W. (1960) 'The fate of ova transferred into the uterus of the
rat.' J. Reprod. Fertil. 1, 197. [Ill, 140]
Diomidova, H. A., & Kusnetzova, N. A. (1935) 'Semination of rabbit eggs in vitro' (trans.
title). Zh. Biol. 4, 250. [120]
Donker, F. D. (1955) 'Recovery and transplantation of ova.' Mich. St. Univ. Centennial
Symposium. Rep. Reprod. Infertility. [105, 110]
Dowling, D. F. (1949) 'Problems of the transplantation of fertilized ova.' J. agric. Sci. 39,
374. [109, 127, 143]
Dracy, A. E. :
(1953a) 'The future of ova transfer.' Iowa St. Coll. J. Sci. 28, 101. [109]
(1953b) 'Progesterone and relaxin as aids in ova transfer.' Bull. S. Dak. agric. Exp. Sta.
No. 66, p. 130. [109]
REFERENCES AND AUTHOR INDEX 159
(1955) 'The transplantation of ova from mammals.' Mich. St. Univ. Centennial Symposium.
Rep. Reprod. Infertility. [109]
Dracy, A. E., & Petersen, W. E. (1951) 'Technique for isolating fertilized bovine ova.'
Proc. \st nat. Egg-Transfer Breed. Cottf., Texas, p. 13. [105]
Dragoiu, I., Benetato, G., & Opreanu, R. (1937) 'Recherches sur la respiration des ovo-
cytes des mammifercs.' C. R. Soc. Biol., Paris, 126, 1044. [Ill]
Drips, D. (1919) 'Studies on the ovary of the spermophile (Spermophiltis citellus tridecemlineatus)
with special reference to the corpus luteum.' Amer.J. Anat. 25, 117. [11]
Duke, K. L. (1949) 'Some notes on the histology of the ovary of the bobcat (lynx) with
special reference to the corpora lutea.' Anat. Rec. 103, 111. [10]
Dziuk, P. (1960) 'Frequency of spontaneous fragmentation of ova in unbred gilts.' Proc. Soc.
exp. Biol., N.Y., 103,91. [84]
Dziuk, P. J., Donker, J. D., Nichols, J. R., & Peterson, W. E. (1958) 'Problems associated
with the transfer of ova between cattle.' Tech. Bull. Minn, agric. Exp. Sta. No. 222. [110]
Dziuk, P. J., & Peterson, W. E. (1954) 'Attempts at non-surgical transfer of bovine ova.'
J. Anim. Sci. 13, 1019. [143]
Eckstein, P. (1959) 'Implantation of ova.' Mem. Soc. Endocrin. No. 6. Cambridge University
Press. [14]
Edwards, R. G. :
(1954) 'The experimental induction of pseudogamy in early mouse embryos.' Experientia,
10,499. [39]
(1957a) 'The experimental induction of gynogenesis in the mouse. I: Irradiation of the
sperm by X-rays.' Proc. roy. Soc. B, 146, 469. [36, 39, 41, 76, 85]
(1957b) 'The experimental induction of gynogenesis in the mouse. II: Ultra-violet irradia-
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(1958a) 'Colchicine-induced heteroploidy in the mouse. II: The induction of tetraploidy
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(1958b) 'The experimental induction of gynogenesis in the mouse. Ill: Treatment of
sperm with trypaflavine, toluidine blue, or nitrogen mustard.' Proc. roy. Soc. B, 149,
117. [36, 39, 76, 77, 85]
Edwards, R. G., & Gates, A. H. (1959) 'Embryonic development in superovulated mice
not receiving the coital stimulus.' Anat. Rec. 135, 291. [Ill, 137]
Edwards, R. G., & Sirlin, J. L. :
(1956) 'Labelled pronuclei in mouse eggs fertilized by labelled sperm.' Nature, Lond. 177,
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(1957) 'Studies in gamctogencsis, fertilization and early development in the mouse, using
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(1958) 'Radioactive tracers and fertilization in mammals.' Endeavour, 17, 42. [18]
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Endo, Y. (1952) 'The role of the cortical granules in the formation of the fertilization mem-
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Engle, E. T. (1927) 'Polyovular follicles and polynuclear ova in the mouse.' Anat. Res. 35,
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Fankhauser, G. (1948) 'The organization of the amphibian egg during fertilization and
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160 THE MAMMALIAN EGG
Fawcett, D. W. (1950) 'The development of mouse ova under the capsule of the kidney.'
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Fawcett, D. W., Wislocki, G. B., & Waldo, C. M. (1947) 'The development of mouse
ova in the anterior chamber of the eve and in the abdominal cavity.' Amer.J. Anat. 81,
413. [133]
Fekete, E. :
(1947) 'Differences in the effects of uterine environment upon development in the DBA
& C57 Black strains of mice.' Anat. Rec. 98, 409. [Ill, 133]
(1950) 'Polyovular follicles in the C58 strain of mice.' Anat. Rec. 108, 699. [20]
Fekete, E., & Little, C. C. (1942) 'Observations on the mammary tumor incidence in mice
born from transferred ova.' Cancer Res. 2, 525. [Ill, 132]
Fischberg, M., & Beatty, R. A. (1952) 'Heteroploidy in mammals. II: Induction of
triploidy in pre-implantation mouse eggs.' J. Genet. 50, 455. [46]
Fischer, A. (1905) 'Zur Kenntnis der Struktur des Oolemmas der Saugethiereizellen.' Anat.
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Flynn, T. T. (1930) 'On the unsegmented ovum of Echidna (Tachyglossus).' Quart. J. micr.
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Flynn, T. T., & Hill, J. P. (1939) 'The development of the Monotremata. IV: Growth of
the ovarian ovum, maturation, fertilization and early cleavage.' Tram. zool. Soc. Lend.
24, 445. [13, 15, 26, 102]
Fol, H. :
(1877a) 'Sur les phenomenes intimes de la fecondation.' C. R. Acad. Set., Paris, 84, 268. [5]
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357. [5]
(1879) 'Recherches sur la fecondation et la commencement de l'henogenie chez divers
animaux.' Mem. Soc. Phys., Geneve, 26, 89. [5]
Foster, M. A. (1934) 'The reproductive cycle of the female ground squirrel, Citellus tridecem-
lineatus (Mitchill). Amer.J. Anat. 54, 487. [11]
Franzen, A. (1958) On sperm morphology and acrosome filament formation in some Annelida,
Echiuroidea, and Tunicata. Almquist & Wiksells, Uppsala. [100]
Fridhandler, L., Hafez, E. S. E., & Pincus, G.:
(1956a) '02 uptake of rabbit ova.' Proc. Hlrd int. Congr. Anim. Reprod., Cambridge, Section
1, p. 48. [112]
(1956b) 'Respiratory metabolism of mammalian eggs.' Proc. Soc. exp. Biol., N.Y. 92, 127.
[112]
(1957) 'Developmental changes in the respiratory activity of rabbit ova.' Exp. Cell Res. 13,
132. [112]
Gatenby, J. B., & Hill, J. P. (1924) 'On an ovum of Omithorhytichus exhibiting polar bodies
and polyspermy.' Quart. J. micr. Sci. 68, 229. [102]
Gates, A. (1956) 'Viability and developmental capacity of eggs from immature mice treated
with gonadotrophins.' Nature, Lond. 177, 754. [Ill, 136]
Gates, A., & Runner, M. (1952) 'Factors affecting survival of transplanted ova of the
mouse.' Anat. Rec. 113, 555 (Abstr.). [134]
Gay, H. (1956) 'Chromosome-nuclear membrane-cytoplasmic interrelations in Drosophila.'
J. biophys. biochem. Cytol. 2, Suppl. p. 407. [20]
Geller, F. C. (1930) 'Zellveranderungcn im Eierstock der geschlcchtsreifen weissen Maus
nach Rbntgenbestrahlung.' Arch. Gynaek. 141, 61. [8]
Genther, I. T. (1931) 'Irradiation of the ovaries of guinea-pigs and its effect on the oestrous
cycle.' Amer.J. Anat. 48, 99. [8]
Gilchrist, F., & Pincus, G. (1932) 'Living rat eggs.' Anat. Rec. 54, 275. [57, 69]
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Batav. |1]
REFERENCES AND AUTHOR INDEX 161
Graham, M. A. (1954) 'Sex chromatin in cell nuclei of the cat from the early embryo to
maturity.' Anat. Rec. 119, 469. [52]
Graves, A. P. (1945) 'Development of the golden hamster, Cricetus auratus Waterhouse,
during the first nine days.' Amer.J. Anat. 77, 219. [86]
Gray, A. P. (1954) Mammalian hybrids. Commonwealth Agricultural Bureaux, Farnham
Royal. [95]
Green, E. L., & Green, M. C:
(1953) 'Modification of difference in skeletal types between reciprocal hybrids by trans-
plantation of ova in mice.' Genetics, 38, 666 (Abstr.). [135]
(1959) 'Transplantation of ova in mice. (An attempt to modify the number of presacral
vertebrae.)' J. Hered. 50, 109. [Ill, 137]
Green, S. H., Smith, A. U., & Zuckerman, S. (1956) 'The number of oocytes in ovarian
autografts after freezing and thawing.' J. Endocrin. 13, 330. [117]
Greenwald, G. S.:
(1956) 'The reproductive cycle of the field mouse, Microtus calif or nicus.' J. Mam. 37,
213. [11]
(1957) 'Interruption of pregnancy in the rabbit by the administration of estrogen.' J. exp.
Zool. 135, 461. [101]
(1958) 'Endocrine regulation of the secretion of mucin in the tubal epithelium of the
rabbit.' Anat. Rec. 130, 477. [101]
Greenwald, G. S., & Everett, N. B. (1959) 'The incorporation of S35 methionine by the
uterus and ova of the mouse.' Anat. Rec. 134, 171. [52]
Gregory, P. W. (1930) 'The early embryology of the rabbit.' Contr. Embryol. Cameg. Instn.
21, 141. [57, 144]
Gresson, R. A. R.:
(1940a) 'A cytological study of the centrifuged oocyte of the mouse.' Quart. J. micr. Sci.
81, 569. [63, 64]
(1940b) 'Presence of the sperm middle-piece in the fertilized egg of the mouse (Mus
musculus): Nature, Lond. 145, 425. [69,70]
(1941) 'A study of the cytoplasmic inclusions during the maturation, fertilization and the
first cleavage division of the egg of the mouse.' Quart. J. micr. Sci. 83, 35. [54, 64, 69, 70]
(1948) Essentials of general cytology. Edinburgh University Press. [64]
Grobstein, C. (1949) 'Behaviour of components of the early embryo of the mouse in culture
and in the anterior chamber of the eye.' Anat. Rec. 105, 490. [133]
Grosser, O. (1927) 'Friihentwicklung, Eihautbildung und Placentation des Menschen und
der Saugetiere.' Dtsch. Frauenheilkunde, 5, 1. [76]
Grusdew, W. S. (1896) 'Versuche iibcr die kiinstlichc Befruchtung von Kanincheneiern.
Arch. Anat. Entw. 269, 304. [125]
Guiliani, R. (1951) 'Superovulazionc c trapianto degli ovuli nelle vacche. (Superovulation
and ovum transfer in cattle).' Riv. Zootec, Firenze, 24, 269. [109]
Hafez, E. S. E. (1958) 'Techniques of collection and transplantation of ova in farm animals.'
J. Amer. vet. med. Ass. 133, 506. [110]
Hale, A. J. (1958) The interference microscope in biological research. E. & S. Livingstone, Edin-
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Hall, B. V. (1935) 'The reactions of rat and mouse eggs to hydrogen ions.' Proc. Soc. exp.
Biol., N.Y. 32, 747. [90]
Ham, A. W. (1957) Histology, 3rd edit. J. B. Lippincott Co., Philadelphia. [9]
Hamilton, W. J. (1934) 'The early stages in the development of the ferret. Fertilization to
the formation of the prochordal plate.' Trans, roy. Soc. Ediub. 58, 251. [55]
Hamilton, W. J., & Day, F. T. (1945) 'Cleavage stages of the ova of the horse, with notes
on ovulation.' J. Anat., Lond. 79, 127. [12, 55, 78]
162 THE MAMMALIAN EGG
Hamilton, W. J., & Laing, J. A. (1946) 'Development of the egg of the cow up to the
stage of blastocyst formation.' J. Anat., Land. 80, 194. [57, 78]
Hamilton, W. J., & Samuel, D. M. (1956) 'The early development of the golden hamster
(Cricetus auratus).' J. Anat., Lond. 90, 395. [31, 41, 69]
Hammond, J. :
(1934) 'The fertilization of rabbit ova in relation to time. A method of controlling the
litter size, the duration of pregnancy and the weight of the young at birth.' J. exp.
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An. Fac. Med. Montevideo, 35, 810. [109]
(1950b) 'The possibility of artificial pregnancy in cattle.' J. Minist. Agric. 57, 67. [109]
Hammond, J., & Walton, A. (1934) 'Notes on ovulation and fertilization in the ferret.'
J. exp. Biol. 11, 307. [10]
Hammond, J., Jr.:
(1949a) 'Recovery and culture of tubal mouse ova.' Nature, Lond. 163, 28.' [146]
(1949b) 'Survival of mouse ova in vitro: and induced multiple pregnancies in cattle.' Proc.
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Hancock, J. L. :
(1958) 'The examination of pig ova.' Vet. Rec. 70, 1200. [69, 107]
(1959) 'Polyspermy of pig ova.' Aniw. Prod. 1, 103. [41, 43]
(1961) 'Fertilization in the pig.' J. Reprod. Fertil. 2. (In press.) [32, 41, 43, 70]
Hansson, A. (1947) 'The physiology of reproduction in mink (Mustela vison Skreb) with
special reference to delayed implantation.' Acta zool. 28, 1. [10]
Harrison, R. J. (1948) 'The changes occurring in the ovary of the goat during the estrous
cycle and in early pregnancy.' J. Anat., Lond. 82, 21. [20]
Harter, B. T. (1948) 'Glycogen and carbohydrate-protein complexes in the ovary of the
white rat during the oestrous cycle.' Anat. Rec. 102, 349. [90]
Hartman, C. G. :
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Morph. 32, 1. [55, 84, 102]
(1924) 'Observations on the viability of the mammalian ovum.' Anwr.J. Ohstet. Gynec. 7.
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(1926) 'Polynuclear ova and polyovular follicles in the opossum and other mammals, with
special reference to the problem of fecundity.' Amer.J. Anat. 37, 1. [20]
(1928) 'The breeding season of the opossum, Didelphis virginiana, and the rate of intra-
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(1929) 'How large is the mammalian egg?' Quart. Rev. Biol. 4, 373. [15]
(1953) 'Early death of the mammalian ovum with special reference to the aplacental
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Harvey, Elmer B. (1958) 'Tubal ovum in Ochotonidae (Lagomorpha).' Anat. Rec. 132,
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Harvey, Ethel B. :
(1936) 'Parthenogenetic mcrogony or cleavage without nuclei in Arhacia punetulataJ1 Biol.
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Harvey, W. (1651) Exercitationes de generatione animalium. Amstelodami, and Londini. [1]
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REFERENCES AND AUTHOR INDEX 163
Heape, W.:
(1886) 'The development of the mole (Talpa europea), the ovarian ovum, and segmenta-
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(1890) 'Preliminary note on the transplantation and growth of mammalian ova within a
uterine foster-mother.' Proc. roy. Soc. 48, 457. [6, 125]
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Henriet, L. (1955) 'La transplantation ovulaire.' Ann. Med. vet. 5, 343. [110]
FIensen, V. (1876) 'Beobachtungen iiber die Befruchtung und Entwicklung des Kaninchens
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Hertwig, O. (1876) 'Beitrage zur Kenntniss der Bildung, Befruchtung und Theilung des
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Hertwig, G. (1939) 'Der Furchungsprozess des Miuseeies, ein Beispiel fiir die wiederholtc
Volumenhalbierung polymerer Kerne und Chromosomen durch multiple succedan-
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Hervey, C. (1949) 'Thirty calves a year from your best cow!' Fm.J. 73, 46. [109]
Heuser, C. H, & Streeter, G. L. (1929) 'Early stages in the development of pig embryos,
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Hill, J. P.:
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Hill, J. P., & Tribe, M. (1924) 'The early development of the cat (Felis domestica).' Quart.
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Hoehne, O. (1914) 'Experimentelle Untersuchungen liber des Schiksal arteigener and
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Hoehne, O., & Behne, K. (1914) 'Uber die Lebensdauer homologer und heterologcr
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Hunter, G. L. (1956) 'The maternal influence on size in sheep.' J. agric. Sci. 48, 36. [142]
Hunter, G. L., Adams, C. E., & Rowson, L. E.:
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[29]
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Jacobson, W., & Lutwak-Mann, C. (1956) 'The vitamin B12 content of the early rabbit
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164 THE MAMMALIAN EGG
Kent, H. A.:
(1959) 'Reduction of polyovular follicles and polynuclear ova by estradiol monobenzoate.'
Anat. Rec. 134, 455. [20]
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Kerckring, T. (1672) 'An account of what hath been of late observed by Dr. Kerckringius
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Kingery, H. M. (1914) 'So-called parthenogenesis in the white mouse.' Biol. Bull., Woods
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Kodicek, E., & Lutwak-Mann, C. (1957) 'The pattern of distribution of thiamine, ribo-
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Konecny, M. (1959) 'Etude histochimique de la zone pellucide des ovules de chatte.' C. R.
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Krafka, J. (1939) 'Parthenogentic cleavage in the human ovary.' Anat. Rec. 75, 19. [84]
Krassovskaja, O. V.:
(1934) 'Fertilization of the rabbit egg outside the organism. II. Early stages of rabbit egg
development outside the organism. Russk. Arkh. Anat. 13, 415. [74, 105, 120, 144]
(1935a) 'Cytological study of the heterogeneous fertilization of the egg of the rabbit outside
the organism.' Acta Zool., Stockh. 16, 449. [120]
(1935b) 'Fertilization of the rabbit egg outside the organism. III. Variations in size of rabbit
eggs before and after fertilization (trans, title).' Biol. Zh. 4, 251. [57, 120]
Krassovskaja, O. V., & Diomidova, H. A. (1934) 'Fertilization of the egg of the rabbit in
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Kremer, J. (1924) 'Das Verhalten der Vorkerne im befruchteten Ei der Ratte und der Maus
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41, 62, 69]
Krzanowska, H. (1960) 'Studies on heterosis. II. Fertilization rate in inbred lines of mice,
and their crosses.' Folia biol. 8, 269. [88, 94]
Kvasnickii, A. V. :
(1950) 'Homoplastic transplantation of ova.' Vestu. Ed. Akad. Zemed. 24, 529. In Anim.
Breed. Abstr. 19, 233 (1951). [128]
(1951) 'Interbreed transplantation of ova.' Sovetsk. Zootek. 1, 36. In Anim. Breed. Abstr. 19,
224(1951). [143]
Kvasnickii, A. V., & Mankovskaja, M. N. (1949) ' "Vegetative hybridization" in animal
breeding.' Priroda, Mosk. 11, 39. In Anim. Breed. Abstr. 18, 314 (1950). [127]
Kvasnickii, A. V., & Martynenko, N. A. (1951) 'The effects of the maternal organism on
progeny.' Sovetsk. Zootek. 7, 63. In Anim. Breed. Abstr. 20, 69 (1952). [129]
Kyle, W. H. (1949) 'The effect of successful embryo transplantations on the progress expected
from selection.' J. Anim. Sci. 8, 607. [109]
Laing, J. A. (1957) 'Female fertility.' Progress in the Physiology of Farm Animals, vol. 3, chap.
17. Ed. J. Hammond. Butterworths, London. [13]
Lamming, G. E., & Rowson, L. E. A. (1952) 'Superovulation and ovum transplantation in
cattle.' Proc. Hnd int. Congr. Anim. Reprod., Copenhagen, 1, 144. [109]
Lams, H. (1913) 'Etude de l'oeuf de cobaye aux premiers stades de l'embrvogencse.' Arch.
Biol., Paris, 28, 229. [55, 69, 70]
Lams, H., & Doorme, J. (1908) 'Nouvelles recherches sur la maturation et la fecondation
de l'oeuf des mammifcres.' Arch. Biol, Paris, 23, 259. [57, 69]
Lane, C. E. (1938) 'Aberrant ovarian follicles in the immature rat.' Anat. Rec. 71, 243. [20]
Leblond, C. P., & Clermont, Y. :
(1952a) 'Spermiogenesis of rat, mouse, hamster and guinea-pig as revealed by the "periodic
acid-fuchsin sulfurous acid" technique.' Amer.J. Anat. 90, 167. [71]
REFERENCES AND AUTHOR INDEX 165
(1952b) 'Spermatogenesis and sperm maturation. Definition of the stages of the cycle of
the seminiferous epithelium in the rat.' Ann. N.Y. Acad. Sci. 55, 548. [71]
Leonard, S. L., & Perlman, P. L. (1949) 'Conditions affecting the passage of spermatozoa
through the utero-tubal junction of the rat.' Anat. Rec. 104, 89. [96]
Lenhossek, M. v. (1898) 'Untersuchungen iiber Spermatogenese.' Arch. mikr. Anat. 51, 215.
[70]
Leuchtenberger, C, &: Schrader, F. (1950) 'The chemical nature of the acrosome in the
male germ cells.' Proc. nat. Acad. Sci., Wash. 36, 677. [99]
Levi, G. (1915) 'II comportamento dei condriosomi durante i pui precoci periodi dello
svillupo dei mammiferi.' Arch. Zellforsch. 13, 471. [69]
Lewis, W. H., & Gregory, P. M. :
(1929a) 'Moving pictures of developing living rabbit eggs (Abstr.).' Anat. Rec. 42, Suppl.,
p. 27. [7, 83, 144]
(1929b) 'Cinematographs of living developing rabbit eggs.' Science, 69, 226. [7, 83, 144]
Lewis, W. H., & Hartman, C. G.:
(1933) 'Early cleavage stages of the eggs of the monkey (Macacus rhesus).' Contr. Embryol.
Cameg. Instn. 24, 187. [54]
(1941) 'Tubal ova of the rhesus monkey.' Contr. Embryol. Cameg. Instn. 29, 7. [54]
Liche, H. (1939) 'Oestrous cycle in the cat.' Nature, Loud. 143, 900. [10]
Lin, T. P., Sherman, J. K., & Willett, E. L. (1957) 'Survival of unfertilized mouse eggs in
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Lindahl, P. E. (1960) 'Some factors influencing the biological activity of sperm antagglutins.'
J. Reprod. Fertil. 1, 3. [116]
Loeb, J. (1917) 'Fecondation et phagocytose.' Ann. Inst. Pasteur, 31, 437. [87]
Long, J. A. (1912) ^Studies on early stages of development in rats and mice.' Univ. Calif.
Publ. Zool. 9, 105. [6, 146]
Lopyrin, A. I., Loginova, N. V., & Karpov, P. L. :
(1950a) 'Experiment in interbreed transference of ova in sheep.' Sovetsk. Zooteh. 8, 50,
Anim. Breed. Abstr. 18, 415 (1950). [141]
(1950b) 'Changes in the exterior of lambs as a result of interbreed embryonic transfer.'
Dokl. Akad. Nank SSSR. 74, 1019. In Anim. Breed. Abstr. 19, 355 (1951). [141]
(1951) 'The effects of changed conditions during embryogenesis on growth and develop-
ment of lambs.' Sovetsk. Zooteh. 11, 83. In Anim. Breed. Abstr. 20, 153 (1952). [141]
Ludwig, K. S.:
(1953) 'Sur quelques aspects cytologique et cytochimique de la fecondation chez les
Rongeurs.' C. R. Acad. Sci., Paris, 237, 496. [30]
(1954) 'Das Verhalten der Thymonukleinsaure (DNA) wahrend der Befruchtung und den
ersten segmentationsstadien bei der Ratte und dem Goldhamster.' Arch. Biol., Paris, 65,
135. [30,41]
Lutwak-Mann, C:
(1954) 'Some properties of the rabbit blastocyst.' J. Embryol. exp. Morph. 2, 1. [81]
(1959) 'Biochemical approach to the study of ovum implantation in the rabbit.' Implanta-
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[81]
(1960) 'Some properties of the early embryonic fluids in the rabbit.' J. Reprod. Fertil. 1,
316. [81]
McCrady, E. (1938) 'The embryology of the opossum.' Amer. anat. Mem. No. 16. [55]
Macdonald, E., & Long, J. A. (1934) 'Some features of cleavage in the living egg of the
rat.' Amer. J. Anat. 55, 343. [69]
McLaren, A., & Biggers, J. D. (1958) 'Successful development and birth of mice cultivated
in vitro as early embryos.' Nature, Lond. 182, 877. [110, 118, 136, 147]
166 THE MAMMALIAN EGG
McLaren, A., & Michie, D.:
(1954) 'Transmigration of unborn mice.' Nature, Land. 174, 844. [135]
(1956) 'Studies on the transfer of fertilized mouse eggs to uterine foster-mothers. I. Factors
affecting the implantation and survival of native and transferred eggs.' J. exp. Biol. 33,
394. [Ill, 136]
(1958) 'An effect of the uterine environment upon skeletal morphology in the mouse.'
Nature, Loud. 181, 1147. [Ill]
(1959a) 'The spacing of implantations in the mouse uterus.' Implantation of Ova. Mem.
Soc. Endocrin. No. 6, p. 65. Ed. P. Eckstein. Cambridge University Press. [137]
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Mainland, D.:
(1928) 'The pluriovular follicle, with reference to its occurrence in the ferret.' J. Anat.
Lond. 62, 139. [20]
(1930) 'The early development of the ferret: the pronuclei.' J. Anat., Lond. 64, 262. T41,
69]
Making, S. (1941) 'Studies on the murine chromosomes. 1. Cytological investigations of
mice, included in the genus Mns.' J. Fac. Sci., Hokkaido Univ. 7, 305. [19, 21]
Mandl, A. M. (1959) 'A quantitative study of the sensitivity of oocytes to X-irradiation.'
Proc. roy. Soc. B, 150, 53. [8]
Mann, M. C. (1924) 'Cytological changes in the unfertilized tubal eggs of the rat.' Biol.
Bull, Woods Hole, 46, 316. [84]
Mann, T. (1954) The biochemistry of semen. Methuen, London. [99]
Marden, W. G. R., & Chang, M. C:
(1952a) 'The aerial transport of fertilized mammalian ova.' Proc. Ibid int. Congr. Anim.
Reprod., Copenhagen, 1, 140. [129]
(1952b) 'The aerial transport of mammalian ova for transplantation.' Science, 115, 705.
[129]
Marshall, A. J. (1949) 'Pre-gestational changes in the giant fruit bat (Pteropus giganteus),
with special reference to an asymmetrical endometrial reaction.' Proc. Linn. Soc. Lond.
161, 26. [11]
Mather, W. B. (1950) 'The technique of rabbit blastoderm culture.' Pap. Dep. Biol. Unit'.
Qd. 2, No. 15. [145]
Matthews, L. H. (1947) 'A note on the female reproductive tract in the tree kangaroo
(Dendrolagus): Proc. zool. Soc. 117, 313. [10]
Meissner, G. (1855) 'Beobachtungen iiber des Eindringen der Samenelemente in den Dotter.'
Z. wiss. Zool. 6, 208. [5]
Menkin, M. F., & Rock, J. (1948) 'In vitro fertilization and cleavage of human ovarian eggs.'
Amer.J. Obstet. Gynec. 55, 440. [117, 120]
Merton, H. (1939) 'Reproduction in the albino mouse. III. Duration of life of sperm in the
female reproductive tract.' Proc. roy. Soc, Edinb. 59, 207. [87]
Metz, C. B. (1957) 'Specific egg and sperm substances and activation of the egg.' The
Beginnings of Embryonic Development. Ed. Albert Tyler, R. C. von Borstel & Charles B.
Metz. American Association for the Advancement of Science, Washington. [116]
Moog, F., & Lutwak-Mann, C. (1958) 'Observations on rabbit blastocysts prepared as flat
mounts.' J. Embryol. exp. Morph. 6, 57. [109]
Moore, N. W., Rowson, L. E. A., & Short, R. V. (1960) 'Egg transfer in sheep. Factors
affecting the survival and development of transferred eggs.' J. Reprod. Fertil. 1, 332.
[142]
Moricard, R.:
(1949) 'Penetration in vitro du spermatozoi'de dans l'ovule des mammiferes et niveau du
potentiel d'oxydo-reduction tubaire.' C. R. Soc. (rang. Gynec. 19, 226. [120]
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level.' Nature, Lond. 165, 763. [120]
REFERENCES AND AUTHOR INDEX 167
(1950b) 'Premieres observations de la penetration du spermatozoi'de dans la membrane
pellucide d'ovocytes de lapine fccondes in vitro niveau de potential d'oxydo reduction
de la secretion tubaire.' C. R. Ass. Anat. Louvain, No. 63, p. 337. [120]
(1954a) 'Observation of in vitro fertilization in the rabbit.' Nature, Loud. 173, 1140. [119,
121]
(1954b) 'Penetration spermatique obtenue in vitro au travers de la membrane pellucide
d'ovocytes de lapine cultives dans les liquides de secretion utero-tubaire.' C. R. Soc.
Biol, Paris, 148, 423. [119, 121]
(1958) 'Fonction meiogene et fonction oestrogene du follicule ovarien des mammiferes
(cytologie golgienne, traceurs, microscopie electronique).' Ann. Endocr., Paris, 19, 943.
[64, 87]
Moricard, R., & Bossu, J. (1949) 'Premieres etudes du passage du spermatozoi'de au travers
de la membrane pellucide d'ovocytes de lapine fecondes in vitro.' Bull. Acad. nat. Med.
133,659. [120]
Mossman, H. W., & Hisaw, F. L. (1940) 'The fetal membranes of the pocket gopher illus-
trating an intermediate type of rodent membrane formation. I. From the unfertilized
tubal egg to the beginning of the allantois.' Atner.J. Anat. 66, 367. [92]
Mulnard, J. (1955) 'Contribution a la connaissance des enzymes dans l'ontogenese. Les
phosphomonoesterases acide et alcaline dans la developpement du rat et de la souris.'
Arch. Biol, Paris, 66, 525. [32]
Nelson, H. (1851) 'On the reproduction of Ascaris mystax.1 Proc. roy. Soc. B, 6, 86. [5]
Newman, H. H. :
(1912) 'The ovum of the nine-banded armadillo. Growth of the ovocytes, maturation and
fertilization.' Biol. Bull., Woods Hole, 23, 100. [29, 53]
(1913) 'Parthenogenetic cleavage of the armadillo ovum.' Biol. Bull., Woods Hole, 25, 59.
[84]
Newport, G. (1853) 'On the impregnation of the ovum in the Amphibia (2nd ser. rev.) and
on the direct agency of the spermatozoon.' Phil. Trans. 143, 233. [5]
Nicholas, J. S.:
(1933a) 'The development of rat embryonic tissues after transplantation of the egg to the
kidney.' Anat. Rec. 55, 31 (Abstr.). [138]
(1933b) 'Development of transplanted rat eggs.' Proc. Soc. exp. Biol., N.Y. 30, 1111. [Ill,
138]
(1934) 'The induction of artificial pregnancy in virgin rats.' Anat. Rec. 58, 31. (Abstr.).
[138]
(1942) 'Experiments on developing rats. IV. The growth and differentiation of eggs and
egg-cylinders when transplanted under the kidney capsule.' J. exp. Zool. 90, 41. [138]
(1947) 'Experimental approaches to problems of early development in the rat.' Quart.
Rev. Biol. 22, 179. [109]
Nicholas, J. S., & Hall, B. V. :
(1934) 'The development of isolated blastomeres of the rat.' Anat. Rec. 58, 83. (Abstr.).
[139]
(1942) 'Experiments on developing rats. II. The development of isolated blastomeres and
fused eggs.' J. exp. Zool. 90, 441. [139]
Nihoul, J. (1927) 'Recherches sur l'appareil endocellulaire de Golgi dans les premiers stades
du developpement des mammiferes.' Cellule, 37, 23. [54, 64, 69]
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New York. [5]
Noyes, R. W. :
(1952) 'Fertilization of follicular ova.' Fertil. & Steril. 3, 1. [139]
(1953) 'The fertilizing capacity of spermatozoa.' West. J. Surg. 61, 342. [99]
168 THE MAMMALIAN EGG
Noyes, R. W., & Dickmann, Z. (1960) 'Relationship of ovular age to endometrial develop-
ment.' J. Reprod. Fertil. 1, 186. [110, 111, 140]
Noyes, R. W., Adams, C. E., & Walton, A. (1959) 'The transport of ova in relation to
the dosage of oestrogen in ovariectomized rabbits.' J. Endocrin. 18, 108. [101]
Noyes, R. W., Walton, A., &: Adams, C. E. (1958) 'Capacitation of rabbit spermatozoa.'
Nature, Lond. 181, 1209. [99, 123]
Noyes, R. W., Yamate, A. M., & Clewe, T. H. (1958) 'Ovarian transplants to the anterior
chamber of the eye.' Fertil. & Steril. 9, 99. [139]
Oakberg, E. F.:
(1958) 'The effect of X-rays on the mouse ovary.' Proc. Xth int. Congr. Genetics, 2, 207. [8]
(1960) 'Irradiation damage to animals and its effect on their reproductive capacity.' J. Dairy
Sci. 43, Suppl., p. 54. [8]
O'Donoghue, C. H. (1912) 'The corpus luteum in the non-pregnant Dasyurus and polyovular
follicles in Dasyurus: Anat. Auz. 41, 353. [20]
Odor, D. L.:
(1955) 'The temporal relationship of the first maturation division of rat ova to the onset
of heat.' Amer.J. Anat. 97, 461. [21, 74, 75]
(1960) 'Electron microscopic studies on ovarian oocytes and unfertilized tubal ova in the
rat.' J. biophys. biochem. Cytol. 7, 567. [19, 55, 56, 64, 87]
Odor, D. L., & Blandau, R. J.:
(1949) 'The frequency of occurrence of supernumerary sperm in rat ova.' Anat. Rec. 104,
1. [70]
(1951a) 'Observations on the formation of the second polar body in the rat ovum.' Anat.
Rec. 110, 329. [74]
(1951b) 'Observations on fertilization and the first segmentation division in rat ova.'
Amer.J. Anat. 89, 29. [28, 32, 71]
(1956) 'Incidence of polyspermy in normal and delayed matings in rats of the Wistar
strain.' Fertil. & Steril. 7, 456. [41, 42, 85]
Odor, D. L., & Renninger, D. F. (1960) 'Polar body formation in the rat oocyte as observed
with the electron microscope.' Anat. Rec. 137, 13. [69]
Ohno, S., Kaplan, W. D., & Kinosita, R.:
(1957) 'Conjugation of the heteropyknotic X and Y chromosomes of the rat spermatocyte.'
Exp. Cell Res. 12, 395. [16]
(1958) 'A photographic representation of mitosis and meiosis in the male of Rattus norve-
gicus.' Cytologia, 23, 422. [16]
(1960) 'On isopyknotic behavior of the XX-bivalent in oocytes of Rattus norvegicus.'
Exp. Cell Res. 19, 637. [16]
Ohnuki, Y. (1959) 'A phase microscopy study on the morphological and structural changes
in living hamster eggs during ovulation, fertilization and early cleavage.' Cytologia,
Tokyo, 24, 348. [41, 69, 104]
Oppenheimer, J. M. (1957) 'Embryological concepts in the twentieth century.' Survey biol.
Progr. 3, 1. [6]
Ota, T. (1934) 'Polyovular follicles in dogs.' Jap. J. Obstet. Gynec. 17, 207. [20]
Pankratz, D. S. (1938) 'Some observations on the Graafian follicles in an adult human ovary.'
Anat. Rec. 71, 211. [20]
Park, W. W. (1957) 'The occurrence of sex chromatin in early human and macaque em-
bryos.' J. Anat., Lond. 91, 369. [52]
Parkes, A. S. (1947) 'Effects on early embryonic development of irradiation of spermatozoa.'
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Parkes, A. S., Dodds, E. C, & Noble, R. L. (1938) 'Interruption of early pregnancy by
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REFERENCES AND AUTHOR INDEX 169
Parkes, A. Sm Rogers, H. J., & Spensley, P. C. (1954) 'Biological and biochemical aspects
of the prevention of fertilization by enzyme inhibitors.' Studies on Fertility, 6, 65. [88]
Parrott, D. M. V.:
(1958) 'Fertility of orthotopic ovarian grafts.' Studies on Fertility, 9, 137. [117]
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J. Reprod. Fertil. 1, 230. [117]
Parrott, D. M. V., & Parkes, A. S. (1960) 'Dynamics of the orthotopic ovarian graft.'
Sex Differentiation and Development. Mem. Soc. Endocrin., No. 7, p. 71. Ed. C. R. Austin.
Cambridge University Press. [117]
Pearson, O. P. (1944) 'Reproduction in the shrew (Blarina brevicorda Say).' Amer. J. Anat.
75, 39. [11, 13, 78]
Pearson, O. P., &Enders, R. K. (1943) 'Ovulation, maturation and fertilization in the fox.'
Anat. Rec. 85, 69. [12, 74, 78]
Pesonen, S.:
(1946a) 'Abortive egg cells in the mouse.' Hereditas, 32, 93. [23, 76]
(1946b) 'Uber Abortiveier. 1.' Acta obstet. gynec. scand. Suppl. 2, p. 152. [23, 76]
(1949) 'On abortive eggs. III. On the cytology of fertilized ova in the mouse.' Ann. Chir.
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Piko, L. (1958) 'Etude de la polyspermie chez le rat.' C. R. Soc. Biol., Paris, 10, 1356. [41,
42, 46]
Piko, L., & Bomsel-Helmreich, O. (1960) 'Triploid rat embryos and other chromosomal
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42, 43, 45, 46]
Pincher, C. (1948) 'Transplanting mammal's eggs.' Discovery, 9, 52. [109]
Pincus, G.:
(1930) 'Observations on the living eggs of the rabbit.' Proc. toy. Soc, B, 107, 132. [11,
69, 101, 102, 120]
(1936a) The eggs of mammals. Macmillan, New York. [14, 57, 109, 120]
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[38, 120]
(1939b) 'The maturation of explanted human ovarian ova.' Amer. J. Physiol. 126, 600.
[148]
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Proc. nat. Acad. Sci., Wash. 25, 557. [126]
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[112, 145]
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Egg-Transfer Breed. Conf, Texas, p. 18. [148]
Pincus, G., &Enzmann, E. V.:
(1932) 'Fertilization in the rabbit.' J. exp. Biol. 9, 403. [57]
(1934) 'Can mammalian eggs undergo normal development in vitro?' Proc. nat. Acad.
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Pincus, G., & Kirsch, R. E. (1936) 'The sterility in rabbits produced by injections of oestrone
and related compounds.' Amer. J. Physiol. 115, 219. [82]
Pincus, G., & Saunders, B. (1939) 'The comparative behaviour of mammalian eggs in
vivo and in vitro. VI. The maturation of human ovarian ova.' Anat. Rec. 75, 537. [148]
M
170 THE MAMMALIAN EGG
Pincus, G., & Shapiro, H. :
(1940a) 'The comparative behaviour of mammalian eggs in vivo and in vitro. VII. Further
studies on the activation of rabbit eggs.' Proc. Atner. phil. Soc. 83, 631. [38]
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Pincus, G., & Werthessen, N. T. (1938) 'The comparative behaviour of mammalian eggs
in vivo and in vitro. III. Factors controlling the growth of the rabbit blastocyst.' J. exp.
Zool. 78, 1. [144]
PlTKJANEN, I. G.:
(1955) 'Ovulation, fertilization and early embryonic development in the pig' (trans, title).
Izv. Acad. Nauk S.S.S.R. Ser. Biol., No. 3, p. 120. [31, 41, 43, 69]
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Izv. Acad. Nauk S.S.S.R.. Ser. Biol., No. 3, p. 291. [41]
Pitkjanen, I. G., & Ivankov, M. F. (1956) 'Fertilization and early stages of embryonic
development in the cow' (trans, title). Izv. Acad. Nauk S.S.S.R. Ser. Brol., No. 3, p. 77.
[41]
Pitkjanen, I. G., & Sheglov, O. V. (1958) 'Dimensions of pig eggs' (trans, title). Works
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& V. Hamburger. W. B. Saunders, Philadelphia. [73]
Robinson, A. (1918) 'The formation, rupture and closure of ovarian follicles in ferrets and
ferret-polecat hybrids, and some associated phenomena.' Trans, roy. Soc, Edinb. 52,
303. [10]
Rock, J., & Menkin, M. F. (1944) 'In vitro fertilization and cleavage of human ovarian eggs.'
Science, 100, 105. [117, 120]
Rothschild, Lord:
(1954) 'Polyspermy.' Quart. Rev. Biol. 29, 332. [88]
(1956) Fertilization. Methuen, London. [88, 93, 114]
(1958) 'Fertilization of fish and lamprey eggs.' Biol. Rev. 33, 372. [93]
Rothschild, Lord, & Swann, M. M. :
(1949) 'The fertilization reaction in the sea-urchin egg. A propagated response to sperm
attachment.' J. exp. Biol. 26, 164. [88]
(1951) 'The conduction time of the block to polyspermy in the sea-urchin egg.' Exp.
Cell Res. 2, 137. [88]
(1952) 'The fertilization reaction in the sea-urchin. The block to polyspermy.' J. exp.
Biol. 29, 469. [88]
Rowlands, I. W., & Williams, P. C. (1946) 'Fertilization of eggs in hypophysectomized
rats.' J. Endocrin. 4, 417. [85]
Rowson, L. E., & Dowling, D. F. (1949) 'An apparatus for the extraction of fertilized eggs
from the living cow.' Vet. Rec. 61, 191. [105]
Rubaschkin, W. :
(1905) 'Uber die Reifungs- und Befruchtungsprozesse dcs Mcerschweincheneies.' Anat.
Hefte, 29, 509. [69, 74, 108]
(1906) 'Uber die Veranderungen den Eier in den zugrunde gehenden Graafschen Follikeln.'
Anat. Hefte, 32, 255. [84]
Runner, M. N.:
(1947a) 'Development of mouse eggs in the anterior chamber of the eye.' Anat. Rec. 98, 1.
[133]
(1947b) 'Attempts at in vitrc semination of mouse eggs.' Anat. Rec. 99, 564. [133]
(1949) 'Limitation of litter size in the mouse following transfer of ova from artificially
induced ovulations.' Anat. Rec. 103, 585. [134]
REFERENCES AND AUTHOR INDEX 171
(1951) 'Differentiation of intrinsic and maternal factors governing intrauterine survival of
mammalian young.' J. exp. Zool. 116, 1. [134]
Runner, M. N., & Gates, A. (1954) 'Sterile, obese mothers.' J. Hertci. 45, 51. [135]
Runner, M. N., & Palm, J. (1953) 'Transplantation and survival of unfertilized ova of the
mouse in relation to postovulatory age.' J. exp. Zool. 124, 303. Till, 135]
Runnstrom, J. (1949) 'The mechanism of fertilization in metazoa.' Advanc. Enzymol. 9,
241. [Ill]
Russell, L. B., & Freeman, M. K. (1958) 'The influence of dose-rate on the sterilizing effect
of radiation in female mice.' Radiation Res. 9, 174. [8]
Russell, W. L., Russell, L. B., Steele, M. H., & Phipps, E. L. (1959) 'Extreme sensitivity
of an immature stage of the mouse ovary to sterilization by irradiation.' Science, 129,
1288. [8]
Russell, L. B., Stelzner, K. F., & Russell, W. L. (1959) 'Influence of dose rate on radiation
effect on fertility of female mice.' Proc. Soc. exp. Biol., N.Y. 102, 471. [8]
Rowson, L. E., & Dowling, D. F. (1949) 'An apparatus for the extraction of fertilized
eggs from the living cow.' Vet. Rec. 61, 191. [105]
Samuel, D. M. (1944) 'The use of an agar gel in the sectioning of mammalian eggs.' J. Anat.,
Lond. 78, 173. [109]
Samuel, D. M., & Hamilton, W. J. (1942) 'Living eggs of the golden hamster (Cricetus
auratus): J. Anat., Lond. 76, 204. [86]
Sansom, G. S. (1920) 'Parthenogenesis in the water vole, Microtus amphibius.' J. Anat.,
Lond. 55, 68. [84]
Schenk, S. L. (1878) 'Das Saugetierei kunstlich befruchter ausserhalb des Muttertieres.' Mitt.
Etnhr. Inst. K. K. Univ. Wien. 1, 107. [6, 99, 120]
Schotterer, A. (1928) 'Beitrag zur Feststellung derEianzahl in verschiedenen Altersperioden
bei der Hiindin.' Anat. Anz. 65, 177. [8]
Schrader, F., &: Leuchtenberger, D. (1951) 'The cytology and chemical nature of some
constituents of the developing sperm.' Chroniosonia, 4, 404. [99]
Schwann, Th. (1839) 'Mikroscopische Untersuchungen iiber die Uebereinstimmung in der
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Schwartz, R., Brooks, W., & Zinsser, H. H. (1958) 'Evidence of chemotaxis as a factor
in sperm motility.' Fertil. & Steril. 9, 300. [115]
Segal, S. J., & Nelson, W. O. (1958) 'An orally active compound with antifertility effect
in rats.' Proc. Soc. exp. Biol., N.Y. 98, 431. [82]
Seidel, F. :
(1952) 'Die Entwicklungspontenzen einer isolierten Blastomere des Zweizellenstadiums
in Saugetierei.' Naturwissenschaft. 39, 355. [110, 130]
(1956) 'Nachweis eines Zentrums zur Bildung der Keimscheiber im Saugethierei.' Natiir-
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(1960) 'DieEntwicklungsfahigkeiten isolierter Furchungszellen aus demEi des Kaninchens.'
Arch. EntwMech. 152, 43. [110]
Serebrjakov, P. N., & Kraseninnikova, A. I. (1951) 'Interbreed transplantation of fertilized
rabbit ova.' Sovetsk. Zootek. 11, 43. In Anim. Breed. Abs. 19, 234 (1951). [129]
Shah, M. K. (1956) 'Reciprocal egg transplantations to study the embryo-uterine relationship
in heat-induced failure of pregnancy in rabbits.' Nature, Lond. 177, 1134. [132]
Shapiro, H. (1942) 'Parthenogenetic activation of rabbit eggs.' Nature, Lond. 149, 304. [38]
Sharma, K. N. (1960) 'Genetics of gametes. IV. The phenotype of mouse spermatozoa in
four inbred strains and then Fx crosses.' Proc. roy. Soc, Edinb. B, 68, 54. [23]
Sharman, G. B.: •
(1955a) 'Studies on marsupial reproduction. II. The oestrous cycle of Setonix brachyurus.'
Aust.J. Zool. 3, 44. [13, 102.]
172 THE MAMMALIAN EGG
(1955b) 'Studies on marsupial reproduction. III. Normal and delayed pregnancy in
Setonix brachyurus.' Aust.J. Zool. 3, 45. [13]
Sherman, J. K., & Lin, T. P.:
(1958) 'Survival of unfertilized mouse eggs during freezing and thawing.' Proc. Soc. exp.
Biol., NY. 98, 902. [110, 117, 137]
(1959) 'Temperature shock and cold-storage of unfertilized mouse eggs.' Fertil. & Steril.
10,384. [110, 117]
Shettles, L. B. (1953) 'Observations on human follicular and tubal ova.' Amer. J. Obstet.
Gynec. 66, 235. [121]
Shettles, L. B. A. (1955) 'A morula stage of human ova developed in vitro.' Fertil. & Steril.
6, 287. [121, 148]
Sirlin, J. L., & Edwards, R. G. (1959) 'Timing of DNA synthesis in ovarian oocyte nuclei
and pronuclei of the mouse.' Exp. Cell Res. 18, 190. [31]
Skowron, S. (1956) 'The development of the oocytes in Graafian follicles of the golden
hamster Mesocricetus auratus' (trans, title). Folia Biol. 4, 23. [20, 84]
Skreb, N. (1957) 'Etudes cytologiques sur l'oeuf de quelques cheiropteres.' Arch. Biol.,
Paris, 68, 381. [63]
Slater, D. W., & Dornfeld, E.J. (1945) 'Quantitative aspects of growth and oocyte produc-
tion in the early prepubertal rat ovary.' Amer. J. Anat. 76, 253. [8]
Smiles, J., & Dobson, M. J. (1955) 'Direct ultra-violet and ultra-violet phase-contrast
micrography of bacteria from the stomachs of the sheep.' J. roy. micr. Soc. 75, 244. [108]
Smith, A. H., & Kleiber, M. (1950) 'Size and oxygen consumption in fertilized eggs.' J. cell.
comp. Physiol. 35, 131. [112]
Smith, A. U. :
(1949a) 'Cultivation of rabbit eggs and cumuli for phase-contrast microscopy.' Nature,
Lond. 164, 1136. [145]
(1949b) 'Some antigenic properties of mammalian spermatozoa.' Proc. roy. Soc. B, 136,
46. [116]
(1951) 'Fertilization in vitro of the mammalian egg.' The Biochemistry of Fertilization
and the Gametes. Biochem. Soc. Symp. 7, 3. [119, 121, 123]
(1952) 'Behaviour of fertilized rabbit eggs exposed to glycerol and to low temperatures.'
Nature, Lond. 170, 374. [114, 117, 145]
(1953a) 'In vitro experiment with rabbit eggs.' Mammalian Germ Cells, p. 217. Ed. G. E. W.
Wolstenholme, M. P. Cameron and J. S. Freeman. Churchill, London. [117, 145]
(1953b) In discussion after paper by Venge (1953). [121]
Smithberg, M. (1953) 'The effect of different proteolytic enzymes on the zona pellucida of
mouse ova.' Anat. Rec. 117, 554. [90, 91]
Sobotta, J. (1895) 'Die Befruchtung und Furchung der Eies der Maus.' Arch. mikr. Anat. 45,
15. [5, 57, 74, 75, 90, 108]
Sobotta, J., & Burckhard, G. (1910) 'Reifung und Befruchtung des Eies der weissen
Ratte.' Anat. Hefte, 42, 433. [69, 70, 75, 90]
Sotelo, J. R. (1959) 'An electron microscope study of the cytoplasmic and nuclear com-
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Sotelo, J. R., & Porter, K. R. (1959) 'An electron microscope study of the rat ovum.'
J. biophys. biochem. Cytol. 5, 327. [19, 26, 34, 55, 60, 64, 87, 97]
Sotelo, J. R., & Trujillo-Cenoz, O. (1957) 'Electron microscope study of the vitelline
body of some spider oocytes.' J. biophys. biochem. Cytol. 3, 301. [56]
Spalding, J. F., Berry, R. O., & Moffit, J. G. (1955) 'The maturation process of the ovum
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Spee, F. Graf:
(1893) 'Beitrag zur Entwickelungsgeschichte der friiheren Stadien des Meerschweinchens
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REFERENCES AND AUTHOR INDEX 173
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3, 130. [81]
Squier, R. R. (1932) 'The living egg and early stages of its development in the guinea-pig.'
Contr. Embryol. Carneg. Instn. 32, 223. [147]
Stockard, A. H. (1937) 'Studies on the female reproductive system of the prairie dog,
Cynomys leucurus. 2. Normal cyclic phenomena of the ovarian follicles.' Pap. Mich.
Acad. Sci. 22, 671. [20]
Strauss, F. :
(1938) 'Die Befruchtung und der Vorgang der Ovulation bei Ericulus aus der Familie der
Centetiden.' Biomorphosis, 1, 281. [13, 78]
(1950) 'Ripe follicles without antra and fertilization within the follicle: a normal situation
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(1954) 'Das Problem des Befruchtungsortes des Saugetiereies.' Bull, schweiz. Akad. med.
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(1956) 'The time and place of fertilization of the golden hamster egg.' J. Embryol. cxp.
Morph. 4, 42. [98]
Swann, M. M., &' Mitchison, J. M. (1958) 'The mechanism of cleavage in animal cells.'
Biol Rev. 33, 103. [73]
Swyer, G. I. M. (1947) 'A tubal factor concerned in the denudation of rabbit ova.' Nature,
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Szoliosi, D. G., & Ris, H. (1961) 'Observations on sperm penetration in the rat.' J. biophys.
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Tafani, A. (1889) 'La fecondation et la segmentation etudiees dans les oeufs des rats.' Arch.
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Tarkowski, A. K. :
(1959a) 'Experiments on the development of isolated blastomeres of mouse eggs.' Nature,
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(1959b) 'Experimental studies on regulation in the development of isolated blastomeres of
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(1959c) 'Experiments on the transplantation of ova in mice.' Acta Theriologica, 2, 251. [138]
Taylor, E. W. (1950) 'The application of phase-contrast to the ultra-violet microscope.'
Proc. roy. Soc. B, 137, 332. [108]
Thibault, C.:
(1947) 'La parthenogenese experimentale chez le lapin.' C. R. Acad. Set., Paris, 224, 297.
[38, 57]
(1948) 'L'activation et la regulation de l'ovocyte parthenogenetique de lapine.' C. R. Soc.
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(1952) 'La fecondation chez les mammiferes et les premiers stades de developpement.'
Proc. Ilnd int. Congr. Physiol Path. Anitn. Reprod. artif. Insem., Copenhagen, Section 1, p. 7.
[85]
(1959) 'Analyse de la fecondation de l'oeuf de la truie apres accouplement ou insemination
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Thibault, C., & Dauzier, L. (1960) ' "Fertilisines" et fecondation in vitro de l'oeuf de lapine.'
C. R. Acad. Sci., Paris, 250, 1358. [115, 119, 121]
Thibault, C., Dauzier, L., & Wintenberger, S. (1954) 'Etude cytologique de la feconda-
tion in vitro de l'oeuf de la lapine.' C. R. Soc. Biol, Paris, 148, 789^ [69, 119, 121]
Thibault, C., & Ortavant, R. (1949) 'Parthenogenese experimentale chez le brebis.'
C. R. Acad. Sci., Paris, 228, 510. [38]
174 THE MAMMALIAN EGG
Trujillo-Cenoz, O., & Sotelo, J. R. (1959) 'Relationships of the ovular surface with
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Tyler, A. :
(1932) 'Changes in volume and surface of Urechis eggs upon fertilization.' J. exp. Zool. 63,
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(1941) 'Artificial parthenogenesis.' Biol. Rev. 16, 291. [36, 76]
Umbaugh, R. E. :
(1949) 'Superovulation and ovum transfer in cattle.' Atner.J. vet. Res. 10, 295. [143]
(1951a) 'Superovulation and ovum transfer in cattle.' Fertil. & Steril. 2, 243. [143]
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Van Beneden, E. (1875) 'Le maturation de l'oeuf, la fecondation et les premieres phases du
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Van Beneden, E., & Julin, C. (1880) 'Observations sur la maturation, la fecondation et la
segmentation de l'oeuf chez les chiroteres.' Arch. Biol., Paris, 1, 551. [5, 109]
Van de Kerckhove, D. (1959) 'Content of deoxyribonucleic acid of the germinal vesicle of
the primary oocyte in the rabbit.' Nature, Lend. 183, 329. [18]
Van der Stricht, O. :
(1901) 'L'atresie ovulaire et l'atresie folliculaire du follicule de De Graaf dans l'ovaire de
chauve-souris.' Verb. Anat. Ges. Jena, 15. [84]
(1902) 'Le spermatozoide dans l'oeuf de chauve-souris (V. noctula).' Verh. anat. Ges. 16
Versamml., Halle, p. 163. [69, 70, 108]
(1909) 'La structure de l'oeuf des mammiferes (Chauve-souris, Vesperugo noctula) Troisieme
partie. L'oocyte a la fin du stade d'accroissement, au stade de la maturation, au stade de
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2, 1. [13, 55, 69, 70, 74]
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d'apres les travaux du Laboratoire d'Histologie et d'Embryologie de l'Universite de
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Van der Stricht, R. (1911) 'Vitellogenese dans l'ovule de chatte.' Arch. Biol., Paris, 26, 365.
[41, 69, 74]
Vara, P., & Pesonen, S. (1947) 'Uber Abortiveier. II: Untersuchungen iiber die im
Chromosomensatz der Saugetiereizelle wahrend der Reifeteilungen sich abspielcnden
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Velardo, J., Raney, N. M., Smith, B. G., & Sturgis, S. H. (1956) 'Effect of various steroids
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Venable, J. H. (1946) 'Pre-implantation stages in the golden hamster (Cricetus auratus).'
Anat. Rec. 94, 105. [86]
Venge, O.:
(1950) 'Studies of the maternal influence on the birth weight in rabbits.' Aita zool., Stockh.
31, 1. In Anivt. Breed. Abstr. 18, 194 (1950). [Ill, 128]
(1953) 'Experiments on fertilization of rabbit ova in vitro with subsequent transfer to alien
does.' Mammalian Germ Cells, p. 243. Ed. G. E. W. Wolstenholme, M. P. Cameron
and J. S. Freeman. Churchill, London. [121, 130]
Vincent, W. S. (1955) 'Structure and chemistry of nucleoli.' Int. Rev. Cytol. 4, 269. [19, 28]
Vincent, W. S., & Dornfeld, E. J. (1948) 'Localization and role of nucleic acids in the
developing rat ovary.' Amer. J. Anat. 83, 437. [18,59]
REFERENCES AND AUTHOR INDEX 175
Ward, M. C. (1948) 'The maturation division of the ova of the golden hamster Cricetus
auratus: Anat. Rec. 101, 663. [74, 75]
Warwick, B. L., & Berry, R. O.:
(1949) 'Inter-generic and intra-specific embryo transfers in sheep and goats.' J. Hered. 40,
297. [Ill, 141]
(1951) 'Inter-generic and intra-specinc embryo transfers in sheep and goats.' Proc. 1st nat.
Egg-Transfer Breed. Conf., Texas, p. 5. [Ill, 141]
Warwick, B. L., Berry, R. O., & Horlacher, W. R. (1934) 'Results of mating rams to
angora female goats.' Proc. Amer. Soc. Anitn. Prod. p. 225. [Ill, 140]
Washburn, W. W., Jr. (1951) 'A study of the modifications in rat eggs observed in vitro and
following tubal retention.' Arch. Biol, Paris, 62, 439. [147]
Waterman, A. J. (1943) 'Studies of normal development of the New Zealand White strain
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Weiss, L. (1961) 'The cell surface in relation to hormone action.' Cell Mechanisms in Hormone
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White, M. J. D. (1954) Animal cytology and evolution, 2nd edn. Cambridge University Press.
[7, 23, 36]
Whitney, L. F., & Underwood, A. B. (1952) The raccoon. Practical Science Publishing Co.,
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Whitney, R., & Burdick, H. O. :
(1936) 'Tube-locking of ova by oestrogenic substances.' Endocrinology, 20, 643. [82]
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Whitten, W. K. :
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Wiesner, B. P., & Yudkin, J. (1955) 'Control of fertility by antimitotic agents.' Nature,
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Willett, E. L.:
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176 THE MAMMALIAN EGG
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ADDENDUM
The important observations of A. L. Colwin and L. H. Colwin, referred to on p. 88,
have now been published:
Colwin, A. L., & Colwin, L. H.:
(1961a) 'Fine structure of the spermatozoon of Hydroides hexagonus (Annelida), with special
reference to the acrosomal region.' J. biophys. biochem. Cytol. 10, 211.
(1961b) 'Changes in the spermatozoon during fertilization in Hydroides hexagonus (Anne-
lida). II. Incorporation with the egg.' J. biophys. biochem. Cytol. 10, 255.
Colwin, L. H., & Colwin, A. L. (1961) 'Changes in the spermatozoon during fertilization
in Hydroides hexagonus (Annelida). I. Passage of the acrosomal region through the
vitelline membrane.' J. biophys. biochem. Cytol. 10, 231.
SUBJECT INDEX
Acrosome, hyaluronidase in 99, 100; re-
action of invertebrate spermatozoa 100
Activation of egg, by : sperm entry 22, 24 ;
other stimuli 36-39
Adenine-8-14C 31
Ageing of eggs, effects of 36, 43, 46, 85, 88
Albumen coat in monotreme and marsupial
eggs 14, 102
Alveoli in fish eggs 93
Amitotic division 85
Anaesthesia, ether, as: activating stimulus
38; stimulus to 'immediate cleavage' 77
Androgenesis 38, 39
Aneugamy 40, 41
Artificial insemination late in oestrus 36, 43,
46, 85, 88
Aster, in meiosis and mitosis 66; visible in
mammalian egg 45 (Fig. 31)
Attachment of embryo in uterus (see Im-
plantation)
Blastocyst, agents lethal to 82; composition
of fluid of 80, 81; development of
cytoplasmic processes from 81, 82;
histochemistry of 52, 61; morphology
of 4, 79-81, 83; outline of development
of 9, 12; parthenogenetic, in rabbit 38;
preparation of flat mounts of 109
Block to polyspermy 22, 42 (Table 3), 43,
88, 89
Capacitation 96, 99, 100; in vitro 123, 124
Cell division, mechanism of 72, 73
Centriole 66-69
Centrosome, in oocyte 63; as part of divi-
sion apparatus 65-69; structure of 66
Chemotaxis 114, 115
Chorion of fish egg, change after sperm
entry 93
Chromosomes (see also Genes), chiasmata
and 'crossing-over' in 16, 21, 23; divi-
sion of centromeres of, in meiosis 22 ;
lampbrush 16; nuclcolus-organizing
loci of 27, 28 ; reduction in number of,
during meiosis 21-23; scatter of, from
second meiotic spindle 24, 34, 35, 85;
X and Y 16, 68 (Fig. 59)
in: cleavage nuclei 48-50; oocyte nuclei
16, 17, 19, 21; polar body 75; pronuclei
12, 25-27, 31, 44; sperm head 24
uniqueness of genotype of ootid 23
Cinematography of eggs 6
Cleavage (see also Blastocyst, Mitosis,
Morula, Vitellus), in monotreme eggs
84; inhibition of 79; mechanism of 72,
73, 78, 79; outline of 9, 12; rates of 83,
84
2-cell egg, development after destruction
of one blastomere 110, 130, 138;
'giant' 21; histochemical properties of
59-61; parthenogenetic 36, 38; resis-
tance to low temperatures 116, 117;
'smoke-ring' in 79; with two nuclei in
one blastomere 76, 77
4-cell egg, development after destruction
of one to three blastomeres 110, 132,
138; histochemistry of 59-61; par-
thenogenetic 38
8-cell egg, histochemistry of 59-61 ;
polyspcrmic 45
Coats of marsupial and monotreme eggs 14
(Fig. 10), 102
Colchicine 24, 36, 39, 41, 42, 46
Cold shock 38
Cortical granules 65, 93-95
Culture and maintenance of eggs in vitro 6,
7, 117, 118, Appendix No. 2; as acti-
vating stimulus 38; at low tempera-
tures 116, 117
Cumulus oophorus, appearance of 3, 97
(Fig. 74); as aid to sperm penetration
100; as check to early sperm penetra-
tion 98; as check to polyspermy 89,
100; break-up of 98, 99; chemical
properties of 98, 99; construction of
96, 97; effect of enzymes on 98, 99;
migration of follicle cells from 97, 98 ;
penetration of, by spermatozoon 99,
100; permeability of 98; state at ovula-
tion, and persistence of 96
relation of follicle cells to vitellus 97
Cyclosis 58
Cytaster 67, 85
177
178
SUBJECT INDEX
Cytoplasm of egg (see also names of organ-
elles), basophilia of 52, 56, 59-61, 63;
endoplasmic reticulum in 55, 56; fine
structure of 55, 56; presence of dna
in 52; rna in hyaloplasm of 61
changes in cytoplasm with : activation 24 ;
degeneration 34, 35 ; sperm penetration
54
Delayed mating as cause of increase in
polyandry and polygyny 42, 43, 46, 88
Deoxyribonucleic acid (dna), demonstration
of, in eggs by: fluorescence microscopy
107; U.V. microscopy 107, 108;
synthesis in embryo 61, 62
in: cleavage nuclei 50-52; cytoplasm of
sea-urchin and frog eggs 52; oocyte
nuclei 17, 18; pronuclei 30-32; sperm
head 24
Discovery of eggs 1-7
Division apparatus (see also Spindle) 65-69
Egg cells in simple animals 7
Electron microscopy (see Microscopy)
Embryo (see also Blastocyst and Cleavage),
early development, details of 61, 62;
outline of 9, 12
parthenogenetic 38, 39; polyspermic 44, 45
pre-implantation, agents lethal to 82
Fallopian tube, passage of eggs through 9,
12, 13; recovery of eggs from 103-105
Fertile life of eggs 13, 102
Fertilization cone 58
Fertilization in vitro 6, 104, 115, 116, 118-
124
Fertilization membrane 86, 93
'Fertilizin' of mammalian eggs 115, 116, 122
Follicle cells, processes penetrating zona
pellucida 56, 57, 87, 89, 97
Follicle, Graafian (or ovarian), formation of
8, 9, 12; penetration of eggs in 13, 78;
pluriovular 20; recovery of eggs from
103
Fragmentation of eggs 35, 58, 84, 85
Freezing of eggs 1 1 7
Genes, influence on: density of cumulus 98;
egg penetration 96; frequency of sub-
nuclei 36; 'immediate cleavage' 76;
incidence of polyandry 42, 43; inci-
dence of polygyny 45, 46; sperm
attachment to vitellus 88, 94
reassortment of, as feature of sexual
reproduction 8; recombination of, in
meiosis 23; relations of, in sperm head
24
Germ cell, primordial 7, 8
Germinal vesicle (see Nucleus of primary
oocyte)
'Giant' eggs 20, 21, 41
Golgi apparatus 63-65
Gloiolemma 102
Glycerol, treatment of eggs with 113, 114,
117, 137, 145
Glycine-2-14C 18
Gynandromorph 76
Gynogenesis 38-40
Haploidy 38
Heat shock, effects on eggs 36, 38, 39, 42,
43, 46, 77
Heterologous fertilization 95, 96
Histology and histochemistry of eggs 18,
31-34, 50, 108
History of ideas on eggs and fertilization 1-7
Hyaluronic acid 90
Hyaluronidase 90, 99; release from acro-
some by capacitation 99, 100
Hypertonic solutions as activating agents 38
Hypodiploidy 36
Hypothermia, as activating stimulus 38
'Immediate cleavage' 76, 77
Implantation, and increase in cytoplasmic
basophilia of egg 51, 52, 63; and pro-
perties of blastocyst 80-82,102
inhibition of, by steroid hormones and
other agents 82
of parthenogenetic blastocysts 38
Intermediary body of spindle 67-69, 72-74
Irradiation of: spermatozoa (U.V.) 36, 39,
85; (X) 36, 39, 58, 85
testis (X) 39
Life history of egg, outline 8-14
Manipulation of eggs 103-124
Maturation (see also Meiosis and Polar body),
details of 21-24; outline of 8, 9, 12
stage of, at ovulation and sperm penetra-
tion 12, 16, 77, 78
Media for maintaining eggs in vitro 103
Megalecithal egg, classification 52
SUBJECT INDEX
179
Mciosis, details of 21-24; outline of 8, 9, 12
first meiotic division, inhibition of 23, 24,
36, 40 (Table 2)
role of division apparatus in 66
second meiotic division, induction of, by
sperm penetration 24; by artificial
stimuli 24, 36-39; spontaneously 24,
34, 36, 37, 39; inhibition of 23, 24, 36,
38, 40 (Table 2); regression of 34, 35
Membrana granulosa (see Cumulus
oophorus)
Membrane fusion, as mechanism of sperm
entry into vitellus Frontispiece, 87, 88
Metabolism of eggs 111, 114
[35S]methionine 52
Microscopy, dark-ground 59, 65; electron
Frontispiece, 18-20, 26, 33, 34, 55, 56,
60, 61, 64-66, 68, 69, 86, 87, 89, 97;
fluorescence 17, 31, 32, 50, 60, 61, 64,
107; interference 107; phase-contrast
17, 20, 25, 26, 49-51, 68, 107; U.V. 17,
31, 32, 50, 51, 59-61
fixation and staining of eggs under micro-
scope 106, 107
preparation of eggs for: electron micro-
scopy 108; histology 108, 109; phase-
contrast microscopy 103-106
Miolecithal egg, classification 52
Mitochondria, structure of 64; number and
distribution of 56, 63, 64
Mitosis, course of first division 48, 49;
course of subsequent divisions 50;
prophase of first cleavage 26, 27, 48
Morula 9, 12, 61
Mosaicism 35, 45, 76, 77
Mucin coat of rabbit egg, chemical and
physical properties of 101; effect of
hormones on 101 ; impermeability of,
to spermatozoa 101 ; morphology of
14 (Fig. 10), 100, 101 ; time of deposition
of 101, 102
Multivesicular body 56
Nitrogen mustard, and 'immediate cleavage'
77
Nuclcocytoplasmic relations in fertilization
47, 48, 50-52, 61-63
Nucleic acids (see also Deoxyribonucleic acid
and Ribonucleic acid), synthesis of 18,
31, 61, 62
Nucleolus, chemical and physical properties
of 17-19, 32-34; inclusions in 17, 32,
49; passage of, into cytoplasm 19, 20
in: cleavage nuclei 49; oocyte nuclei 17,
18, 21; pronuclei 24-34; subnuclei 34,
35
nucleoloneme 19
perinucleolar material or nucleolus-associ-
ated chromatin 17, 19, 49
Nucleus, sizes of 16-18
cleavage nucleus, chemical properties of
50-52; formation of 48, 49; reduction
in volume of, during cleavage 50;
structure of 49, 50
ootid nucleus (see Pronucleus)
polar-body nucleus 47, 75
primary-oocyte nucleus, chemical proper-
ties of 17, 18; form of chromosomes in
16; migration of nucleoli from 19, 20;
multinuclear 20, 21 ; structure of 16-19
sub-nucleus 24, 34-36
zygote nucleus of invertebrates 26
Oocyte, primary (follicular or ovarian),
fragmentation of 84, 85; freezing of
117; 'giant' 20, 21; mitochondria in
63, 64; multinuclear 20, 21, 23, 24, 45;
nucleus of 16-21; number in ovary 8;
octaploid 40; outline of development
of 8, 9, 12; ovulated 12; protein syn-
thesis in 52; resistance to low tempera-
tures 110, 117; rna in 59, 61
secondary 9, 12, 23, 45, 84, 85, 110, 116,
117
Oogenesis 8
Oogonia 8, 16, 21; fusion of 21
Ootid, definition of 12; uniqueness of
genotype 23
Ovulation, induced by coitus 4, 10-11
(Table 1), 12
Parthenogenesis 24, 34, 36-39, 57, 67, 84
Passage of eggs through Fallopian tube 13
Perforatorium 92, 100
Perivitelline space, formation of 57; sperma-
tozoa in 88, 92, 94
Phenotype of eggs, influenced by genotype
23
Plasma membrane (see Vitelline membrane)
Plasmalogen 62
Polar body, details of formation of 21-24,
73, 74; nucleus formation in 47; outline
of formation of 12; sizes of 75, 76;
time of emission of, in relation to
ovulation and sperm penetration 77, 78
180
SUBJECT INDEX
Polar body — continued
first polar body, disappearance of 22, 75 ;
inhibition of 20, 21, 40 (Table 2), 45, 75
second polar body, induction of 36; in-
hibition of 39, 40 (Table 2), 45, 46, 75
polar-body-like structure containing egg
chromatin 40; containing sperm head,
47,77
Polyandry, mechanism of 43, 47; incidence
of 41-43 (Table 3)
Polygyny, mechanism of 40 (Table 2), 45-
47; incidence of 41, 43
Polymorphonuclear leucocytes penetrating
into eggs 92
Polyspermy (see Block to polyspermy, Pro-
nucleus, Polyandry)
Pronucleus, augmentation of dna in 31;
growth and development of 24-31, 43,
46-48; numbers of, in one egg 23, 24;
sizes of 17, 25, 26, 43, 46-48
androgenesis 38, 39
differences between male and female pro-
nuclei: volume 28, 29; staining proper-
ties 31, 32
female, diploid 23, 24, 36, 38, 40 (Table
2), 41; origin of 24,25, 30
fusion of male and female or of two male
pronuclei 26, 39
gynogenesis 38, 39, 47
male, origin of 24, 25, 30; polyploid 41
nucleocytoplasmic ratio 29
polyandry 41—47
polygyny 23, 24, 40 (Table 2), 41, 45, 46
rudimentary parthenogenesis, with one
nucleus 36-38, 40 (Table 2), 41, 47;
with two nuclei 39, 40 (Table 2)
synchronization between male and female
pronuclei 47, 48
syngamy 9, 26, 27, 31, 35, 43, 47
Protein synthesis 18, 52, 63
Radiomimetic drugs, treatment of sperma-
tozoa with 36
Recovery of eggs, from: Fallopian tube in
living animal 105; ovary and Fallopian
tube ^103-105
Regulation to diploidy 36, 76, 79
Reproduction, asexual 7
Ribonucleic acid (rna), absence from pro-
nuclear nucleoli 32, 33; in: egg cyto-
plasm 55, 56, 59-63; oocyte nucleoli
17, 18
Ribosomcs 55, 56, 60
Selective fertilization 96
Sex chromatin 52
Shell and shell membrane in monotreme and
marsupial eggs 14, 102
Sizes of eggs 13-15, 52, 53, 56, 57
'Smoke ring' 71 (Fig. 61), 72 (Fig. 62), 79,
80 (Fig. 65)
Sperm, spermatozoon, acrosome in egg
penetration 99, 100; antagglutin 116;
dimegaly of 41; dna in 24; 'giant' 41;
head changes in egg cytoplasm Frontis-
piece, 12, 24, 25, 30, 70-72; influence of
egg on 114-116; mid-piece in egg cyto-
plasm Frontispiece, 69-72; number at
site of fertilization 43, 89, 96; poly-
megaly of 41; polyploid 40, 41; size
of nucleus of 15; supplementary 92;
suspensions of, as activating stimulus
38, 57; tail in egg cytoplasm 44, 69-72,
79
penetration, effect on hamster cortical
granules 65; impermeability to, of
rabbit-egg mucin coat 101; into polar
body 77; site of 13; through cumulus
oophorus 99, 100; through vitelline
membrane 87, 88; through zona
pellucida 90, 92
Spindle (see also Intermediary body), function
of, in cell division 65-69, 12-1 A;
structure of 68, 69; two first meiotic
spindles in one egg 21 ; two second
meiotic spindles in one egg 23, 45
Syngamy of pronuclei 9, 26, 27, 31, 35, 43,
^47
T locus, effect of, on sperm penetration
96
Temperatures, low, resistance of eggs to 113,
114, 116, 117, 137, 145
Tetraploidy 21, 79
Transfer of eggs between animals 6, 109-
111, Appendix No. 1
Transport of eggs through Fallopian tube
(see Passage of . . .)
Triethylcnemelamine and 'immediate cleav-
age' 77
Triploidy 21, 23, 24, 45, 46
Uterus, recovery of blastocysts from 105;
implantation in (see Implantation)
Vesicular conglomerate 56
SUBJECT INDEX
181
Vitelline membrane, inhibition of sperm
attachment to 88; permeability of 86;
sperm penetration through 72, 87-89,
92, 93; structure of 56, 86
block to polyspermy 22, 43, 88, 89
Vitellus {see also Cytoplasm), diminution of,
dining cleavage 78, 79; sizes of, in
different animals 13-15
contraction of, with: maturation 22, 56,
57; non-specific activation 57; sperm
penetration 22, 24, 56, 57
elevation of vitelline surface over matura-
tion spindle and sperm head 58
Yolk, amount and distribution of 15, 52, 53,
56, 84; assimilation of, during cleavage
78; deutoplasmolysis of 54, 55; syn-
thesis of 16, 63
yolk nucleus 63
Zona pellucida, chemical and physical pro-
perties of 89-91 ; effect of enzymes on
90, 91; formation of 89; 'lysin' 92;
penetration of, by spermatozoon 72,
90, 92; perforation and shedding of, by
blastocyst 81, 82; structure of 14 (Fig.
10), 56, 80, 89
zona reaction 22, 43, 92-95
Zona radiata 89
Zygote (see Embryo)
INDEX OF ORGANISMS
Amphibia 16, 19, 41, 53
Annelida 41
Ape 80
Armadillo, Dasypus novemcinctus 29 (Fig.
23), 53 (Fig. 37), 84
Ascaris lumbricoides 5, 15
Bats 5, 63; Pipistrellus pipistrellus 53, 54
(Fig. 39); P. (=Vesperugo) dasycnemus
29; P. (=Vesperugo) mystacinus 29;
P. (=Vesperugo) noctula 13, 54, 55, 57,
69, 70, 74, 76, 77 (Fig. 64), 84; Pteropus
giganteus 11
Birds 1, 16, 41, 53, 102
Bobcat, Lynx rufus 10
Bull, Bos taurus 95
Cat, Felis catus 10, 13 (Fig. 9), 14, 41, 52, 53,
55, 57, 69, 70, 74, 80, 89, 90, 92, 96, 97,
99; Colour Figs. 19, 20, 40-45, 67-69
Clam, Spisula spp. 13 (Fig. 9)
Coelenterata 13 (Fig. 9)
Cotton-rat, Sigmodon hispidus 15 (Fig. 11)
Cow, Bos taurus 2 (Fig. 1), 13 (Fig. 9), 14,
41, 53, 57, 78, 84, 96, 110, 111, 115, 143,
148
Crab, Libinia spp. 13 (Fig. 9)
Crisia spp. 13 (Fig. 9), 15
Deer 4
Dog, Canis familiar is 3 (Fig. 3), 4, 5 (Fig. 5),
8, 12 (Fig. 8), 13 (Fig. 9), 14, 20, 53, 55
(Fig. 46), 57, 69, 70, 76, 78, 80, 81 (Fig.
66), 92, 96, 99
Duck-billed platypus, Ornitlwrhynchus para-
doxus 15, 102
Echinodermata 13 (Fig. 9)
Fern, Pteridium aquilinum 114
Ferret, Mustek furo 10, 14, 29, 41, 53, 55, 69,
78, 80, 84," 92
Fish 13 (Fig. 9), 15, 16, 53, 93
Fluke, Chinese liver, Clonorchis sinensis 15
Fox, Vulpes fulva 12, 53, 74, 78
Frog 5, 13 "(Fig. 9), 15, 52
Goat, Capra hircus 84, 111, 140, 141, 148
Guinea-pig, Cavia porcellus 14, 21, 28, 53,
54 (Fig. 38), 55, 57, 61, 69, 70, 75, 77,
80-82, 84, 90, 92, 95, 105, 111, 120, 135,
147
Hamster, Chinese, Cricetulus griseus 28, 29,
62, 63 (Fig. 52), 69, 70
Hamster, golden, Mesocricetus auratus 14
(Fig. 10), 24, 29 (Fig. 22), 31, 33 (Fig.
27), 34, 36, 37 (Fig. 29), 38, 39, 41, 42,
46, 56, 57, 64, 65 (Fig. 53), 66 (Fig. 54),
68 (Fig. 57), 69, 71 (Fig. 61), 75, 79, 80,
83, 84, 86 (Fig. 70), 87, 90-92, 94, 95
(Fig. 73), 98, 104
Hare, Lepus europaeus 95
Hedgehog, Erinaceus europaeus 98 (Fig. 75)
Horse, Equus cahallus 12, 13 (Fig. 9), 14, 53,
55, 78, 96
Hydroides hexagonus 88
Ilyanassa obsoleta 75
Insects 20, 41
Jird, Libyan, Meriones libycus 28, 29 (Fig.
22), 69, 71 (Fig. 61), 90
Kangaroo, tree, Dendrolagus matschiei 10
Limpet, Crepidula spp. 75
Malarial parasite, Plasmodium spp. 7
Man 2 (Fig. 1), 8, 9 (Fig. 7), 13 (Fig. 9), 14
(Fig. 10), 29, 52, 53, 80, 84, 96, 103, 105,
115, 120, 121, 148
Marsupialia 13 (Fig. 9), 89, 102
Mink, Mustela vison 10
Mole, Talpa europaea 92
Mollusca 13 (Fig. 9)
Monkey 14, 53; Macacus rhesus 52, 54
Monotremata 13 (Fig. 9), 26, 53, 89, 102
Mouse, Mus musculus 5, 6 (Fig. 6), 12, 14, 15,
18-20, 23, 24, 28, 31, 34, 36, 38, 39, 41,
42, 45-48, 52-55, 57, 58 (Figs. 48 and
49), 63, 64, 69, 75-80, 82-84, 88, 90-92,
94, 95, 97, 98, 110, 111, 114, 115, 117,
131-138, 146, 147
182
INDEX OF ORGANISMS
183
Mouse, field, Microtus californicus 11
Mouse, wood, Apodemus sylvaticus 95
Native cat, Dasyurus viverrinus 13 (Fig. 9),
14 (Fig. 10), 15, 29, 53, 55, 102
Nemertea 13 (Fig. 9), 41
Nereis limbata 93
Opossum, Didelphis aurata 55, 102; D.
virginiana 13, 14 (Fig. 10), 20, 29, 84
Pig, Sus scrofa 14, 29, 31, 32, 41, 43, 46, 53,
55, 57, 69, 70, 74, 76, 84, 96, 110, 143
Pika, Ochotona princeps 92
Platyhelmia 13 (Fig. 9)
Pocket gopher, Geotnys bursarius 92
Polychaeta 13 (Fig. 9)
Rabbit, Oryctolagus cuniculus 1, 2, 3 (Fig. 2),
4 (Fig. 4), 5, 7, 11, 13 (Fig. 9), 14 (Fig.
10), 15, 24, 28 (Fig. 21), 29, 31, 38, 39,
41, 53, 54, 56, 57, 69, 70 (Fig. 60), 75,
80, 82-84, 87, 89-92, 95, 96, 98-105,
109-123, 125-132, 135, 141-146
Rabbit, cotton-tail, Sylvilagus transitionalis
95
Raccoon, Procyon lotor 10
Rat, Rattus norvegicus, Frontispiece, 8, 12,
14-16, 17 (Fig. 12), 18 (Fig. 13), 19, 22
(Fig. 14), 24, 25 (Fig. 17), 26, 27 (Fig.
18), 28, 33, 34, 35 (Fig. 28), 36, 38, 39,
41-43, 44 (Fig. 30), 45 (Fig. 31), 46-48,
49 (Fig. 32), 50 (Fig. 33), 51 (Fig. 34),
55-57, 59 (Fig. 50), 60 (Fig. 51), 61, 64,
67 (Fig. 55), 69, 70, 72 (Fig. 62), 74
(Fig. 63), 75-77, 79, 80 (Fig. 65), 82-85,
87-92, 93 (Fig. 71), 94-96, 97 (Fig. 74),
98, 104, 110-112, 113 (Fig. 76), 117, 135,
138-140, 147; Colour Figs. 15, 16, 25,
26, 35, 36
Rat, multimammate, Rattus ( = Mastomys)
natalensis 28, 69, 95
Reptiles 16, 53, 89, 102
Rodents 13 (Fig. 9), 15, 56, 75, 79, 83, 84,
99, 105; murine 53, 70, 103, 104
Sauropsida 86
Sea-squirt, Amaroucium constellatum 13 (Fig.
9)
Sea urchins 52, 65, 93, 94 (Fig. 72), 111, 115;
Arbacia pnnctulata 15; Paracentrotus Hin-
dus 47; Psammechinus miliar is 88;
Toxopneustes lividus 5
Sheep, Oi'is aries 13 (Fig. 9), 14 (Fig. 10),
38, 41, 53, 78, 84, 92, 96, 110, 111, 121,
140-142, 148
Shrew, common, Sorex araneus 1 1
Shrew, lesser, S. minutus 11
Shrew, mole, Blarina brcvicorda 11, 13, 78
Spider 56
Spiny anteater, Tachyglossus ( = Echidna) 13,
14 (Fig. 10), 15, 26, 29, 62
Sporozoa 7
Squid, Loligo pealii 13 (Fig. 9)
Squirrel, ground, Citellus tridecemlineatus 11
Starfish, Asterias glacialis 5 ; Henricia sanguino-
lenta 13 (Fig. 9)
Tenrecs, Madagascan, Centetes, Ericulus
Hemicentetes spp. 13, 77
Trichonympha spp. 7
Tunicata 13 (Fig. 9)
Ungulates 80
Urechis caupo 76
Vole, field, Microtus agrestis 11, 13 (Fig. 9),
14 (Fig. 10), 15, 29, 30 (Fig. 24), 32, 36,
41, 42, 55, 57, 67 (Fig. 56), 68 (Figs. 58
and 59), 69, 70, 75, 90, 92, 95
Vole, Levant or Asiatic, Microtus giintheri 8,
11
Vole, water, M. amphibius 84
Wallaby, Setonix brachyurus 13, 29, 102
Weasel, Mustek frenata and M. nivalis 10
Whelk, Busycon spp. 13 (Fig. 9)
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