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==PART II The Period of Fertiliziation==
=Part II - The Period of Fertilization=
The period of fertilization involves:
[[Book - Comparative Embryology of the Vertebrates 2|'''Part II - The Period of Fertilization''']]: [[Book - Comparative Embryology of the Vertebrates 2-4|4. Transportation of the Gametes (Sperm and Egg) from the Germ Glands to the Site where Fertilization Normally Occurs]] | [[Book - Comparative Embryology of the Vertebrates 2-5|5. Fertilization]]
 
( 1 ) The transportation of the f^amctcs to the site normal for the species where environmental conditions are suitable for gametic union (Chap. 4), and
 
(2) Fertilization or the union of the gametes (Chap. 5).
 
The union of the gametes may be divided into two phases, viz.:
 
( 1 ) The primary phase which is terminated when the sperm has made intimate contact with the egg’s surface, and
 
(2) The secondary phase or the fusion of the two gametes resulting in the initiation
of development.
 
 
175
 
 
 
 
4
 
 
Transportation of tlie Gametes (Sperm and from
tke Germ Glands to tke Site Wkere Fertiliziation
Normally Occurs
 
 
A. Introduction
 
1. Activities of the male and female gametes in their migration to the site of
fertilization
 
B. Transportation of the sperm within the male accessory reproductive structures
 
1. Transportation of sperm from the testis to the external orifice of the genital duct
in the mammal
 
a. Possible factors involved in the passage of the seminal fluid from the testis to
the main reproductive duct
 
1) Accumulated pressure within the seminiferous tubules
 
2) Activities within the efferent ductules of the testis
 
b. Movement of the semen along the epididymal duct
 
1) Probable immotility of the sperm
 
2) Importance of muscle contraction, particularly in the vas deferens
 
3) Summary of factors which propel the seminal fluid from the testis to the
external orifice of the reproductive duct in the mammal
 
2. Transportation of sperm in other vertebrates with a convoluted reproductive duct
 
3. Transportation of sperm from the testis in vertebrates possessing a relatively simple
reproductive duct
 
C. Transportation of sperm outside of the genital tract of the male
 
1. Transportation of sperm in the external watery medium
 
2. Transportation of sperm in forms where fertilization of the egg is internal
 
a. General features relative to internal fertilization
 
1) Comparative numbers of vertebrates practicing internal fertilization
 
2) Sites or areas where fertilization is effected
 
3) Means of sperm transfer from the male genital tract to that of the female
 
b. Methods of sperm transport within the female reproductive tract
 
1) When fertilization is in the lower or posterior portion of the genital tract
 
2) When fertilization occurs in the upper extremity of the oviduct
 
3) When fertilization occurs in the ovary
 
D. Sperm survival in the female genital tract
 
E. Sperm survival outside the male and female tracts
 
1, In watery solutions under spawning conditions
 
2. Sperm survival under various artificial conditions; practical application in animal
breeding
 
 
177
 
 
 
 
178
 
 
TRANSPORTATION OF THE GAMETES
 
 
F. Transportation of the egg from the ovary to the site of fertilization
 
1. Definitions
 
2. Transportation of the egg in those forms where fertilization occurs in the anterior
 
portion of the oviduct
 
a. Birds
 
b. Mammals
 
3. Transportation of the egg in those species where fertilization is effected in the
 
caudal portion of the oviduct or in the external medium
 
a. Frog
 
b. Other amphibia
 
c. Fishes
 
G. Summary of the characteristics of various mature chordate eggs together with the
 
site of fertilization and place of sperm entrance into the egg
 
A. Introduction
 
1. Activities of the Male and Female Gametes in Their
Migration to the Site of Fertilization
 
The first step in the actual process of fertilization and the reproduction of
a new individual is the transportation of the mature gametes from the place
of their development in the reproductive structures to the area or site where
conditions are optimum for their union (fig. 98). This transport is dependent
upon the development of the proper reproductive conditions in the male and
the female parent — a state governed by sex hormones. That is to say, the
sex hormones regulate the behavior of the parents and the reproductive ducts
in such a way that the reproductive act is possible.
 
The transport of the female gamete to the site of fertilization is a passive
one, effected by the behavior of the reproductive structures. Also, the transportation of the sperm within the confines of the male tract largely is a passive
affair. However, outside of the male reproductive tract, sperm motility is a
factor in effecting the contact of the sperm with the egg. Not only is sperm
motility a factor in the external watery medium of those species accustomed
to external fertilization, but also to some degree within the female genital
tract in those species utilizing internal fertilization. However, in the latter
case, sperm transport is aided greatly by the activities of the female genital tract.
 
B. Transportation of the Sperm Within the Male Accessory
Reproductive Structures
 
1. Transportation of Sperm from the Testis to the
External Orifice of the Genital Duct in
the Mammal
 
Sperm transport within the male genital tract of the mammal is a slow
process. It might be defined better by saying that it is efficiently slow, for the
ripening process of the sperm described in the previous chapter is dependent
 
 
 
WITHIN MALE REPRODUCTIVE STRUCTURES
 
 
179
 
 
 
(MOST FISH
MOST A N U R a)
 
 
Fig. 98. Sites of normal fertilization (x) in the vertebrate group. (A, C) Vertebrates
below mammals. (B) Mammalia.
 
upon a lingering passage of the sperm through the epididymal portion of the
male genital tract.
 
a. Possible Factors Involved in the Passage of the Seminal Fluid from
the Testis to the Main Reproductive Duct
 
1) Accumulated Pressure Within the Seminiferous Tubules. The oozing
of sperm and seminal fluid from the seminiferous tubules through the reU
tubules into the efferent ductules of the epididymis possibly may be the resul
of accumulated pressure within the seminiferous tubules themselves. Thi:
pressure may arise from secretions of the Sertoli cells, the infiltration of fluid!
from the interstitial areas between the seminiferous tubules, and by the addi
 
 
 
180
 
 
TRANSPORTATION OF THE GAMETES
 
 
tion of sperm to the contents of the tubules. As the seminiferous tubule is
blind at its distal end, increased pressure of this kind would serve efficiently
to push the contained substance forward toward the efferent ductules connecting the testis with the reproductive duct.
 
2) Activities Within the Efferent Ductules of the Testis. The time required
for sperm to traverse the epididymal duct in the guinea pig is about 14 to
16 days. However, when the efferent ductules between the testis and the
epididymal duct are ligated, the passage time is increased to 25 to 28 days
(Toothill and Young, ’31). The results produced by ligation of the ductuli
effe rentes in this experiment suggest: (a) That the force produced by the
accumulation of secretion within the seminiferous tubules and adjacent ducts
tends to push the sperm solution out of the seminiferous tubules into the
ductuli efferentes and thence along the epididymal duct, and/or (b) at least
a part of the propulsive force which moves the contents of the seminiferous
tubules through the rete tubules and efferent ductules and along the epididymal
duct arises from beating of cilia within the lumen of the efferent ducts. The
tall cells lining the latter ducts possess cilia which beat toward the epididymal
duct. As the sperm and surrounding fluid reach the efferent ductules, the
beating of these cilia would propel the seminal substances toward the epididymal duct.
 
b. Movement of the Semen Along the Epididymal Duct
 
1) Probable Immotility of the Sperm. The journey through the epididymal
duct as previously indicated is tedious, and secretion from the epididymal
cells is added to the seminal contents (fig. 99). Sperm motility evidently is
 
 
 
Fig. 99. Human epididymal cells. (Slightly modified from Maximow and Bloom: A
Textbook of Histology, Philadelphia, W. B. Saunders Co.) These cells discharge secretion
into the lumen of the epididymal duct. Observe large, non-motile stereocilia at distal
end of the cells.
 
 
 
WITHIN MALE REPRODUCTIVE STRUCTURES
 
 
181
 
 
not a major factor in sperm passage along the epididymal portion of the
reproductive duct, as conditions within the duct appear to suppress this motility. It has been shown, for example (Hartman, ’39, p. 681), that sperm
motility increases for trout sperm at a pH of 7.0 to 8.0, in the mammals a pH
of a little over 7.0 seems optimum for motility for most species, while in the
rooster a pH of 7.6 to 8.0 stimulates sperm movements. On the other hand,
an increase of the CO 2 concentration of the medium raises the hydrogen ion
concentration of the suspension. The latter condition suppresses sperm motility and increases the life of sea-urchin sperm (Cohn, ’17, ’18). These facts
relative to the influence of pH on the motility of sperm suggest that motility
during the slow and relatively long epididymal journey — a journey which may
take weeks — apparently is inhibited by the production of carbon dioxide by
the large aggregate of sperm within the lumen of the epididymal duct, a condition which serves to keep the spermatic fluid on the acid side. This suppressed activity of the sperm in turn increases their longevity. The matter of
sperm motility within the epididymal duct, however, needs more study before
definite conclusions can be reached relative to the actual presence or absence
of motility.
 
2) Importance of Muscle Contraction, Particularly of the Vas Deferens.
 
If sperm are relatively immobilized during their passage through the epididymal
duct by the accumulation of carbon dioxide, we must assume that their
transport through this area is due mainly to the activities of the accessory
structures together with some pressure from testicular secretion and efferentductule activity as mentioned above. Aside from the forward propulsion resulting from the accumulation of glandular secretion within the epididymal
duct, muscle contraction appears to be the main factor involved in effecting
this transport. The epididymal musculature is not well developed, and muscle
contraction in this area may be effective but not pronounced. However, added
to the contracture of the epididymal musculature is the contraction of the
well-developed musculature of the vas deferens (fig. 100). During sexual
stimulation this organ contracts vigorously, producing strong peristaltic waves
which move caudally along the duct. The activity of the vas deferens may
be regarded as a kind of “pump action” which produces suction sufficient to
move the seminal fluid from the caudal portions of the epididymis, i.e., from
the cauda epididymidis into the vas deferens where it is propelled toward the
external orifice. Furthermore, the removal of materials from the cauda epididymidis would tend to aid the movement of the entire contents of the
epididymal duct forward toward the cauda epididymidis. From this point of
view, the vas deferens is an efficient organ for sperm transport, while the
epididymal duct functions as a nursery and a “storage organ” for the sperm
(see Chap. 1). Some sperm also are stored in the ampullary portion of the
vas deferens (fig. 101 ), but this storage is of secondary importance inasmuch
as sperm do not retain their viability in this area over extended periods of time.
 
 
 
 
 
 
WITHIN MALE REPRODUCTIVE STRUCTURES
 
 
183
 
 
(4) the possibility of a weak sperm motility aiding the advance of the
sperm through the body of the epididymis must not be denied;
 
(5) the vigorous pumping action of the vas deferens, especially during
the stimulation attending ejaculation, serves to transport the sperm
from the “epididymal well” (the cauda epididymidis) through the vas
deferens to the external areas.
 
2. Transportation of Sperm in Other Vertebrates with a
Convoluted Reproductive Duct
 
The transportation of sperm in other vertebrates which possess an extended
and complicated reproductive duct similar to that of the mammal presumably
involves the same general principles observed above (fig. 105 A, B). However,
certain variations of sperm passage exist which are correlated with structural
modifications of the accessory reproductive organs. For example, the reproductive duct may be somewhat more tortuous and complicated in some instances, such as in the pigeon, turkey, and domestic cock (figs. 102, 105B).
That is, the entire deferent duct extending from the epididymis caudally to
the cloaca may be regarded as a sperm-storage organ, as sperm may be collected in large numbers all along the reproductive duct. As the cock is
capable of effecting repeated ejaculations over an extended period of time.
 
 
LUMEN
 
 
GLAND-LIKE
OUT POUCHING S
OF MAIN LUMEN
 
 
FOLDS OF
MUCOSA
 
 
Fig. 101. Portion of a cross section of the ampullary region of the ductus deferens in
man. Observe gland-like outpouchings of the main lumen and character of mucosal folds.
Surrounding the lumen may be seen the highly muscularized walls of the ampullary area.
 
 
 
 
184
 
 
TRANSPORTATION OF THE GAMETES
 
 
 
POSTERIOR
VENA CAVA
 
TESTES
M E SORC H i U M
ILIAC VEIN
 
EPI Dl DYM I S
 
KIDNEY
 
FEMORAL
VEIN
DOR S A L
AORTA
 
RENAL
PORTA L
VEIN
 
URETER
 
 
VA S
 
DEFERENS
 
 
C LO AC A
 
 
Fig. 102. Reproductive and urinary structures of the adult Leghorn cock. Observe that
the vas deferens is a much convoluted structure. (After Domm: In Sex and Internal Secretions, by Allen, et al., Baltimore, Williams & Wilkins, 1939.)
 
each contraction of the caudal portion of the deferential duct during sperm
discharge serves to move the general mass of seminal fluid posteriad in a
gradual manner. The reproductive conditions present in the cock fulfill the
requirements of a continuous breeder capable of serving many individual
 
 
 
 
Fig. 104. Modifications of the fins of male fishes with the resulting elaboration of an
intromittent organ. (A) Catnhusia affittix. (B) Ventral view of pelvic fins of Squalus
acanthias. (C) Dorsal view of left fin to show genital groove in intromittent structure.
 
 
 
186
 
 
TRANSPORTATION OF THE GAMETES
 
 
females. It is to be observed in this connection that Mann (’49) gives the
amount of ejaculate in the cock as 0.8 cc., highly concentrated with sperm.
 
Another variation found in certain birds is the presence of a seminal vesicle
located at the caudal end of the reproductive duct. This outgrowth is a spermstorage organ and is not comparable to the secretory seminal vesicle found
in mammals. Such seminal vesicles are found in the robin, ovenbird, wood
thrush, catbird, towhee, etc. These structures enlarge enormously during the
breeding season, but in the fall and winter months they shrink into insignificant
organs (Riddle, ’27). It is apparent that the seminal fluid is moved along
and stored at the distal (posterior) end of the reproductive duct in these
species. Other birds, such as the pigeon and mourning dove, lack extensively
developed seminal vesicles, but possess instead pouch-like enlargements of
the caudal end of the reproductive duct when the breeding season is at its
maximum.
 
In many lower vertebrates which practice internal fertilization, large seminal
vesicles or enlargements of the caudal end of the reproductive duct are present.
Such conditions are found in the elasmobranch fishes. These structures act as
sperm-storage organs during the breeding season.
 
3. Transportation of Sperm from the Testis in Vertebrates  
Possessing a Relatively Simple Reproductive Duct
 
In forms such as the frog, toad, and hellbender (figs. 9, 105C), the pressure
within the seminiferous tubules of the testis associated with contractions of
the reproductive duct serve to move the sperm along the reproductive duct.
At the time of spawning, a copious discharge of sperm is effected. In teleost
fishes, a general contraction of the testicular tissue and the muscles of the
abbreviated sperm duct propel the sperm outward during the spawning act
(fig. 105D). In teleosts, sperm are stored in the testis, or as in the perch,
large numbers may be accommodated within the reproductive duct (fig. 105D) .
Slight motility also may be a factor in effecting sperm transport down the
reproductive duct in the lower vertebrates.
 
C. Transportation of Sperm Outside of the Genital Tract of the Male
 
1. Transportation of Sperm in the External Watery Medium
 
In most teleost fishes and in amphibia, such as the frogs and toads, and
the urodeles of the families Hynobiidae and Cryptobranchidae (possibly also
the Sirenidae), fertilization is external and sperm are discharged in close
proximity to the eggs as they are spawned. Many are the ways by which this
relationship is established, some of which are most ingenious (fig. 103).
Sperm motility, once the watery medium near the egg is reached, brings the
sperm into contact with the egg in most instances. However, exceptional cases
are present where the sperm are “almost completely immobile,” such as in
 
 
 
TRANSPORTATION OF SPERM OUTSIDE GENITAL TRACT OF MALE
 
 
187
 
 
HEAD OF epididymis
I WEAK MUSCULAR CONTRACTION
 
I and accumulated pressure
 
/ /BODY OF EPIDIHymic STRONG MUSCULAR
EPIDIDYMIS / CONTRACTION
 
 
EFFERENT
 
DUCTULES
 
 
 
— 1
 
VAS DEFERENS
 
SPERM STORAGE IN
"EPIOIDYMAL WELL"
 
(THE CAUDA EPIDIDYMIDIS )
CILIARY ACT I 0 N
ACCUMULATED PRESSURE
SEMINIFEROUS TUBULES
 
 
EJ ADULATORY
DUCT
 
 
WEAK MUSCULAR CONTRACTION
 
 
 
 
SPERM DUCT
SPERM STORAGE
ACCUMULATED PRESSURE
 
SPERM STORAGE
 
 
D.
 
 
Fio. 105. Various types of reproductive ducts in male vertebrates. The possible activities which transport the sperm along the ducts are indicated. (A) Mammalian type
(B) Bird, urodele, elasmobranch fish type. (C) Frog type. (D) Teleost fish type.
 
the primitive frog, Discoglossus (see Hibbard, ’ 28 ). Here the sperm must
be deposited in close contact with the egg at the time of spawning. In fishes
which lay pelagic eggs (i.e., eggs that float in the water and do not sink to
the bottom), the male may swim about the female in an agitated manner
during the spawning act. This behavior serves to broadcast the sperm in relation to the eggs.
 
 
 
 
 
BROOD POUCH
OF MALE
 
 
Fig. 106. Brood pouch in the male pipefish. (A) Longitudinal view with left flap
pulled aside to show the developing eggs within the pouch. (B) Transverse section to
show relation of eggs to the pouch and dorsal region of the tail.
 
 
188
 
 
 
TRANSPORTATION OF SPERM OUTSIDE GENITAL TRACT OF MALE
 
 
189
 
 
2. Transportation of Sperm in Forms Where Fertilization
OF the Egg is Internal
 
a. General Features Relative to Internal Fertilization
 
1) Comparative Numbers of Vertebrates Practicing Internal Fertilization.
 
Of the 60,000 or more species of vertebrates which have been described, a
majority practice some form of internal fertilization of the egg. Internal fertilization, therefore, is a conspicuous characteristic of the reproductive phenomena of the vertebrate animal group.
 
2) Sites or Areas where Fertilization is Effected. The fertilization areas
(fig. 98) for those vertebrates which utilize internal fertilization are:
 
( 1 ) the lower portions of the oviduct near or at the external orifice,
 
(2) the oviduct, especially its upper extremity,
 
(3) possibly the peritoneal cavity,
 
(4) the follicles of the ovary, and
 
(5) the brood pouch of the male (figs. 98, 106).
 
Though the exact place where internal fertilization occurs may vary considerably throughout the vertebrate group as a whole, the specific site for each
species is fairly constant.
 
3) Means of Sperm Transfer from the Male Genital Tract to That of the
Female. In those fishes adapted to internal fertilization, sperm transport from
the male to the female is brought about by the use of the anal or pelvic fins
which are modified into intromittent organs (fig. 104). In the amphibia two
genera of Anura are known to impregnate the eggs within the oviduct of
the female. In the primitive frog, Ascaphus truei, the male possesses a cloacal
appendage or “tail,” used to transport the sperm from the male to the female,
and the oviducts become supplied with sperm which come to lie between the
mucous folds (Noble, ’31). (See fig. 107.) In East Africa, in the viviparous
toad, Nectophrynoides vivipara, fertilization is internal, and the young, a hundred or more, develop in each uterus. (See Noble, ’31, p. 74.) Just how the
sperm are transmitted to the oviduct and whether fertilization is in the lower
or upper parts of the oviduct in this species is not known.
 
In contrast to the conditions found in most Anura, the majority of urodele
amphibia employ internal fertilization. In many species the male deposits a
spermatophore or sperm mass (fig. 10). The jelly-like substance of the spermatophore of the salamanders is produced by certain cloacal or auxiliary
reproductive glands. The spermatophore may in some species be picked up
by the cloaca of the female or in other species it appears to be transmitted
directly to the cloaca of the female from the cloaca of the male. As the spermatophore is held between the lips of the cloaca of the female, it disintegrates
and the sperm migrate to and are retained within special dorsal diverticula of
the cloacal wall known as the spermatheca (Noble and Weber, ’29) (fig. 108).
 
 
 
 
Fig. 107. Intromittent organ of the tailed frog of America, Ascaphus iruei. (After
Noble, ’31.) (A) Cloacal appendage. (B) Ventral view of same. (C) Fully distended
 
appendage, showing spines on distal end. Opening of cloaca shown in the center.
 
 
SPERM ATHECA
 
 
 
Fig. 108. Diagrammatic sagittal sections of the cloacas of three salamanders, showing
types of spermatheca. (A) Necturus. (B) Amhystoma. (C) Desmognathus. (Redrawn from Noble, ’31.)
 
 
190
 
 
 
TRANSPORTATION OF SPERM OUTSIDE GENITAL TRACT OF MALE
 
 
191
 
 
In the male of the gymnophionan amphibia, a definite protrusiblc copulatory organ is present as a cloacal modification, and fertilization occurs within
the oviducts (fig. 109). Extensible copulatory organs are found generally in
reptiles and mammals, and are present also in some birds, such as the duck,
ostrich, cassowary, emu, etc. In most birds the eversion of the cloaca with
a slight protrusion of the dorsal cloacal wall functions very effectively as a
copulatory organ.
 
b. Methods of Sperm Transport Within the Female Reproductive Tract
 
1) When Fertilization Is in the Lower or Posterior Portion of the Genital
Tract. In many of the urodele amphibia, fertilization is effected apparently
in the caudal areas of the female genital tract or as the egg passes through the
cloacal region. It is probable in these cases that sperm motility is the means
of transporting the sperm to the egg from the ducts of the spermatheca or
from the recesses of the folds of the oviduct.
 
2) When Fertilization Occurs in the Upper Extremity of the Oviduct. In
several species of salamanders, fertilization of the egg and development of
the embryo occur within the oviduct. Examples are: Salamandra salamandra,
S. atra, Hydromantes genei and H. italicus, all in Europe, and the widely
spread neotropical urodele, Oedipus. The latter contains many species. The
exact region of the oviduct where fertilization occurs is not known, but presumably, in some cases, it is near the anterior end. Weber (’22) suggests that
fertilization may occur normally in the peritoneal cavity of Salamandra atra.
In these instances, the method by which the sperm reach the fertilization
area is not clear. It is probable that motility of the sperm themselves has
much to do with their transport, although muscular contraction and ciliary
action may contribute some aid.
 
On the other hand, studies of sperm transport in the female genital tract in
higher vertebrates have supplied some interesting data relative to the methods
and rate of transport. In the painted turtle, Chrysemys picta, sperm are deposited within the cloacal area of the female during copulation; from the
cloaca they pass into the vaginal portion of the oviduct and thence into the
uterus. It is possible that muscular contractions, antiperistaltic in nature, propel
the sperm from the cloaca through the vagina and into the uterus. It may be
that similar muscle contractions propel them through the uterus up into the
albumen-secreting portions of the oviduct, or it is possible that sperm motility
is the method of transport through these areas. However, once within the
albumen-secreting section of the oviduct, a band of pro-ovarian cilia (i.e.,
cilia which beat toward the ovary) (fig. IlOA, B) appears to transport the
sperm upward to the infundibulum of the oviduct (Parker, *31). Somewhat
similar mechanisms of muscular contraction, antiperistaltic in nature, and
beating of pro-ovarian cilia are probably the means of sperm transport in
the pigeon and hen (Parker, ’31). Antiperistaltic muscular contractions are
 
 
 
 
 
192
 
 
 
 
TRANSPORTATION OF SPERM OUTSIDE GENITAL TRACT OF MALE
 
 
193
 
 
known to be possible in the hen (Payne, ’14). Active muscular contractions
are suggested, as sperm travel upward to the infundibulum of the oviduct in
about one and one-half hours in the hen.
 
In the rabbit, antiperistaltic contractions of the cervix and body of the
uterus at the time of copulation pump or suck the sperm through the os uteri
from the vagina and transport them into the uterus at its cervical end (Parker,
’3 1 ) . This transportation occupies about one to three minutes. Passage through
the body of the uterus to the Fallopian tube occurs in one and one-half to
two hours after copulation. It is not clear whether sperm motility alone or
sperm motility plus uterine antiperistalsis effects this transportation. The transport of the sperm upward through the Fallopian tube to the infundibular
region takes about two hours more. The behavior of the uterine (Fallopian)
tube is somewhat peculiar at this time. Churning movements similar to that
of the normal activity of the intestine are produced. Also, temporary longitudinal constrictions of the wall of the tube produce longitudinal compartments along the length of the tube. Within these compartments cilia beat
vigorously in an abovarian direction (i.e., away from the ovary). The general
result of these activities is a thorough mixing and churning of the contents
of the tubes. At the same time these movements succeed in transporting the
sperm up the tube to the infundibular area. The entire journey through the
uterus and Fallopian tube consumes about four hours (Hartman, ’39, pp.
698-702; Parker, ’31).
 
Sperm transport through the female genital tract in the rabbit occupies a
relatively long period of time compared to that which obtains in certain other
mammalian species. The journey to the infundibular area of the Fallopian
tube takes only 20 minutes in the majority of cases in the ewe, following
normal service by the ram. The rate of sperm travel toward the ovaries is
approximately four cm. per minute (Schott and Phillips, ’41). The passage
time through the entire female duct may be considerably less than this in the
guinea pig, dog, mouse, etc. (Hartman, ’39, p. 698). It is probable that the
latter forms experience antiperistaltic muscular contractions of the uterine
cervix, uteri, and Fallopian tubes, which propel the sperm upward to the
infundibular region, the normal site of fertilization.
 
In the marsupial group the lateral vaginal canals complicate the sperm
transport problem. In the opossum, the bifid terminal portion of the penial
organ (fig. 1 14A) probably transmits the sperm to both lateral vaginal canals
simultaneously, where they are churned and mixed with the taginal contents.
From the lateral vaginal canals the sperm are passed on to the median vaginal
cul-de-sac. From this compartment they travel by their own motive power
or are propelled upward through the uterus and Fallopian tubes to the infundibular area of the latter (figs. 34, 35, 114).
 
The foregoing instances regarding sperm transport in the female mammal
involve active muscle contractions presumably mediated through nerve im
 
 
 
Fig. 111. Dorsal view of anterior end of uterine horn of the common opossum, Didelphys
virginiana, showing relation of ovary to infundibulum.
 
 
 
194
 
 
 
TRANSPORTATION OF SPERM OUTSIDE GENITAL TRACT OF MALE
 
 
195
 
 
 
^IG. 113. Open body cavity of adult female of Rana pipicns, showing distribution of
cilia and ostium of oviduct. (Slightly modified from Rugh, ’35.)
 
5ulses aroused during the reproductive act or orgasm together with the actual
Dresence within the reproductive tract of seminal fluid. However, this nervenuscular activity is assuredly not the only means of sperm transport although
t may be the more normal and common method. A slower means of trans)ort, that of sperm motility, plays an important role in many instances. This
s suggested by such facts as fertility being equal in women who experience
10 orgasm during coitus compared to those who do; proven fertility in rabbits
ind dogs whose genital tracts are completely de-afferented by spinal section;
ind conception by females artificially inseminated intra vaginum. (See Hartnan, ’39, p. 699.) Moreover, Phillips and Andrews (’37) have shown that
*at sperm injected into the vagina of the ewe along with ram sperm lag behind
he ram sperm in their migration upward in the genital tract. That is, the
ibnormal environment of the genital tract of the ewe in which the rat sperm
 
 
 
196
 
 
TRANSPORTATION OF THE GAMETES
 
 
were placed may have affected their motility, as well as their ability to survive.
(See Yochem, ’29.)
 
The above data suggest relationships in many of the vertebrates which
doubly assure that sperm will reach the proper site for fertilization in the
oviduct. One aspect of this assurance is the physiological behavior of the
anatomical structures of the oviduct, which may express itself by ciliary beating
in some instances or, in other cases, by muscle contraction. On the other hand.
 
 
 
Fig. 114. Bifid penis of the male opossum; diagram of female reproductive tract. (A)
Extended penis. (After McCrady, Am. Anat. Memoirs, 16, The Wistar Institute of
Anatomy and Biology, Philadelphia.) (B) Female reproductive tract.
 
 
 
SPERM SURVIVAL IN FEMALE GENITAL TRACT
 
 
197
 
 
if this method fails or is weakened, sperm motility itself comes to the rescue,
and sperm are transported under their own power.
 
In view of the above-mentioned behavior of the oviduct in transporting
sperm, it is important to observe that the estrogenic hormone is in a large
way responsible for the activities of the oviduct during the early phases of
the reproductive period and, consequently, influences the conditions necessary
for sperm transport. It enhances this process by arousing a state of irritability
and reactivity within the musculature of the uterus and the Fallopian tubes.
It also induces environmental conditions which are favorable for sperm survival within the female genital tract.
 
3) When Fertilization Occurs in the Ovary. In certain viviparous fishes
the egg is fertilized in the ovary (e.g., Gambusia affinis; Heterandria jormosa).
(See Turner, ’37, ’40; Scrimshaw, ’44.) As the sperm survive for months in
the female tract, sperm transport is due probably to the movements of the
sperm themselves. Motility evidently is a factor in the case of the eutherian
mammal, Ericulus, where ovarian fertilization presumably occurs according to
Strauss, ’39.
 
 
D. Sperm Survival in the Female Genital Tract
 
The length of life of sperm in the female genital tract varies considerably
in different vertebrates. In the common dogfish, Squalus acanthias, and also
in other elasmobranch fishes, sperm evidently live within the female genital
tract for several months, and retain, meanwhile, their ability to fertilize. In
the ordinary aquarium fish, the guppy (Lebistes), sperm may live for about
one year in the female tract (Purser, ’37). A long sperm survival is true also
of the “mosquito fish,” Gambusia. Within the cloacal spermatheca of certain
urodele amphibia, sperm survive for several months. Within the uterus of the
garter snake they may live for three or more months (Rahn, ’40), while in
the turtle, Malaclemys centrata, a small percentage of fertile eggs (3.7 per
cent) were obtained from females after four years of isolation from the male
(Hildebrand, ’29). Sperm, within the female tract of the hen, are known to
live and retain their fertility for two or three weeks or even longer (Dunn, ’27) .
In the duck the duration of sperm survival is much shorter (Hammond and
Asdell, ’26).
 
Among mammals, the female bat probably has the honor of retaining
viable sperm in the genital tract for the longest period of time, for, while the
female is in hibernation, sperm continue to live and retain their fertilizing
power from the middle of autumn to early spring (Hartman, ’33; Wimsatt,
’44). According to Hill and O’Donoghue (T3) sperm can remain alive within
the Fallopian tubes of the Australian native cat, Dasyurus viverrinus, for “at
least two weeks.” However, it is problematical whether such sperm are capable
of fertilizing the egg, for motility is not the only faculty necessary in the
fertilization process. In most mammals, including the human female, sperm
 
 
 
198
 
 
TRANSPORTATION OF THE GAMETES
 
 
survival is probably not longer than 1 to 3 days. In the rabbit, sperm are in
the female genital tract about 10 to 14 hours before fertilization normally
occurs; they lose their ability to fertilize during the early part of the second
day (Hammond and Asdell, ’26). In the genital tract of the female rat, sperm
retain their motility during the first 17 hours but, when injected into the
guinea pig uterus, they remain motile for only four and one-half hours. However, guinea-pig sperm will remain alive for at least 41 hours in the guineapig uterine horns and Fallopian tubes (Yochem, ’29).
 
£. Sperm Survival Outside the Male and Female Tracts
 
1. In Watery Solutions Under Spawning Conditions
In watery solutions in which the natural spawning phenomena occur, the
life of the sperm is of short duration. The sperm of the frog, Rana pipiem,
may live for an hour or two, while the sperm of Fundulus heterocUtus probably
live 10 minutes or a little longer. In some other teleost fishes, the fertilizing
ability is retained only for a few seconds.
 
 
2. Sperm Survival Under Various Artificial Conditions;
Practical Application in Animal Breeding
 
 
One of the main requisites for the survival of mammalian and bird sperm
outside the male or female tract is a lowered temperature. The relatively high
temperature of 45 to 50"^ C. injures and kills mammalian sperm while body
temperatures are most favorable for motility of mammalian and bird sperm;
lower temperatures reduce motility and prolong their life. Several workers
have used temperatures of 0 to 2° C. to preserve the life of mammalian and
fowl sperm, but a temperature of about 8 to 12*^ C. is now commonly used
in keeping mammalian and fowl sperm for purposes of artificial insemination.
Slow freezing is detrimental to sperm, but quick freezing in liquid nitrogen
permits sperm survival even at a temperature of -—195° C. (See Shettles, ’40;
Hoaglund and Pincus, ’42.)
 
Another requirement for sperm survival outside the genital tract of the
male is an appropriate nutritive medium. Sperm ejaculates used in artificial
insemination generally are diluted in a nutritive diluent. The following diluent
(Perry and Bartlett, ’39) has been used extensively in inseminating dairy
cattle:
 
 
Na 2 S 04 1.36 gr. )
 
Dextrose 1.20 gr. > per 100 ml. H 2 O.
 
Peptone 0.50 gr. )
 
 
Also, a glucose-saline diluent has been used with success (Hartman, ’39,
p. 685). Its composition is as follows:
 
 
Glucose
 
Na2HP0ol2H.,0
 
NaCl
 
KH2PO,
 
 
30.9 gr. \
 
t n /• Pe'' 1000 ml. HjO.
2.0 gr. ( ^
 
0.1 gr. )
 
 
 
TRANSPORTATION OF EGG FROM OVARY TO SITE OF FERTILIZATION
 
 
199
 
 
Some workers in artificial insemination use one type of diluent for ram
sperm, another for stallion sperm, and still another for bull sperm, etc.
 
Artificial insemination of domestic animals and of the human female is
extensively used at present. It is both an art and a science. In the hands of
adequately prepared and understanding practitioners, it is highly successful.
The best results have been obtained from semen used within the first 24
hours after collection, although cows in the Argentine have been inseminated
with sperm sent from the United States seven days previously (Hartman, ’39,
p. 685).
 
F. Transportation of the Egg from the Ovary to the Site of Fertilization
 
1. Definitions
 
The transportation of the egg from the ovary to the oviduct is described
as external (peritoneal) migration of the egg, whereas transportation within
the confines of the female reproductive tract constitutes Internal (oviducal)
migration. It follows from the information given above that the site of fertilization determines the extent of egg migration. In those species where external fertilization of the egg is the habit, the egg must travel relatively long
distances from the ovary to the watery medium outside the female body. On
the other hand, in most species accustomed to internal fertilization, the latter
occurs generally in the upper region of the oviduct. Of course, in special
cases as in certain viviparous fishes, such as Gambusia affinis and Heterandria
formosa, fertilization occurs within the follicle of the ovary and migration of
the egg is not necessary. The other extreme of the latter condition is present
in such forms as the pipefishes. In the latter instance the female transfers the
eggs into the brood pouch of the male; here they are fertilized and the embryos
undergo development (fig. 106).
 
2. Transportation of the Egg in Those Forms Where
Fertilization Occurs in the Anterior Portion
OF the Oviduct
 
a. Birds
 
A classical example of the activities involved in transportation of the egg
from the ovary to the anterior part of the oviduct is to be found in the birds.
In the hen the enlarged funnel-shaped mouth of the oviduct or infundibulum
actually wraps itself around the discharged egg and engulfs it (fig. 31). Peristalsis of the oviduct definitely aids this engulfing process. Two quotations
relative to the activities of the mouth of the oviduct during egg engulfment
are presented below. The first is from Patterson, TO, p. 107:
 
Coste describes the infundibulum as actually embracing the ovum in its follicle
at the time of ovulation, and the writer [i.e., Patterson] has been able to confirm
his statement by several observations. If we examine the oviduct of a hen that is
laying daily, some time before the deposition of the egg, it will be found to be
 
 
 
200
 
 
TRANSPORTATION OF THE GAMETES
 
 
inactive; but an examination shortly after laying reveals the fact that the oviduct
is in a state of high excitability, with the infundibulum usually clasping an ovum
in the follicle. In one case it was embracing a follicle containing a half-developed
ovum, and with such tenacity that a considerable pull was necessary to disengage
it. It seems certain, therefore, that the stimulus which sets off the mechanism for
ovulation is not received until the time of laying, or shortly after.
 
If the egg falls into the ovarian pocket (i.e., the space formed around the
ovary by the contiguous body organs ) , the infundibulum still is able to engulf
the egg. Relative to the engulfment of an egg lying within the ovarian pocket,
Romanoff and Romanoff, ’49, p. 215, states:
 
The infundibulum continues to advance, swallow, and retreat, partially engulfing
the ovum, then releasing it. This activity may continue for half an hour before the
ovum is entirely within the oviduct.
 
b. Mammals
 
In those mammals in which the ovary lies free and separated from the
mouth of the oviduct (figs. 29, 111) it is probable that the infundibulum
moves over and around the ovary intermittently during the ovulatory period.
Also, the ovary itself changes position at the time when ovulation occurs,
with the result that the ovary moves in and out of the infundibular opening
of the uterine tube (Hartman, ’39, p. 664). In the Monotremata (prototherian
mammals) during the breeding season, the enlarged membranous funnel
(infundibulum) of the oviduct engulfs the ovary, and a thick mucous-like
fluid lies in the area between the ovary and the funnel (Flynn and Hill, ’39).
At ovulation the relatively large egg passes into this fluid and then into the
Fallopian tube. In the rat and the mouse which have a relatively closed ovarian
sac, the bursa ovarica, around the ovary (figs. 37, 112) contractions of the
Fallopian tube similar to those of other mammals tend to move the fluid and
contained eggs away from the ovary and into the tube. Thus it appears that
the activities of the mouth and upper portions of the oviduct serve to move
the egg from the ovarian surface into the reproductive duct at the time of
ovulation in the mammal and bird. This method of transport probably is present
also in reptiles and elasmobranch fishes. In the mammal this activity has been
shown to be the greatest at the time of estrus. The estrogenic hormone, therefore, is directly involved in those processes which transport the egg from the
ovary into the uterine tube.
 
In women, and as shown experimentally in other mammals, the removal
of the ovary of one side and the ligation or removal of the Fallopian tube on
the other side does not exclude pregnancy. In these cases, there is a transmigration of the egg from the ovary on one side across the peritoneal cavity to
the opening of the Fallopian tube on the other where fertilization occurs.
This transmigration is effected, presumably, by the activities of the intact
infundibulum and Fallopian tube of the contralateral side.
 
Another aspect of egg transport in the mammal is the activity of the cilia
 
 
 
TRANSPORTATION OF EGG FROM OVARY TO SITE OF FERTILIZATION
 
 
201
 
 
lining the fimbriae, mouth, and to a great extent, the ampullary portions of
the uterine (Fallopian) tube itself. The beating of these cilia tend to sweep
small objects downward into the Fallopian tube. However, these activities
are relatively uninfluential in comparison to the muscular activities of the
infundibulum and other portions of the Fallopian tube.
 
Egg transport between the ovary and the oviduct is not always as efficient
as the above descriptions may imply. For, under abnormal conditions the
egg “may lose its way” and if fertilized, may begin its development withiir
the spacious area of the peritoneal cavity. This sort of occurrence is called
an ectopic pregnancy. In the hen, also, some eggs never reach the oviduct
and are resorbed in the peritoneal cavity.
 
3. Transportation of the Egg in Those Species Where
Fertilization is Effected in the Caudal Portion
OF the Oviduct or in the External Medium
 
a. Frog
 
In the adult female of the frog (but not in the immature female or in the
male) cilia are found upon the peritoneal lining cells of the body wall, the
lateral aspect of the ovarian ligaments, the peritoneal wall of the pericardial
cavity and upon the visceral peritoneum of the liver. Cilia are not found on
the coelomic epithelium supporting and surrounding the digestive tract, nor
are they found upon the epithelial covering of the ovary, kidney, lung, bladder,
etc. (fig. 113). (See Rugh, ’35.) This ciliated area has been shown to be
capable of transporting the eggs from the ovary anteriad to the opening of
the oviduct on either side of the heart (fig. 113) (Rugh, ’35). In this form,
therefore, ciliary action is the main propagating force which transports the
egg (external migration) from the ovary to the oviduct. Internal migration
of the egg (transportation of the egg within the oviduct) also is effected mainly
by cilia in the common frog, although the lower third of the oviduct “is abundantly supplied with smooth muscle fibers,” and “shows some signs of peristalsis” (Rugh, ’35). The passage downward through the oviduct to the uterus
consumes about two hours at 22 C. and, during this transit, the jelly coats
are deposited around the vitelline membrane. The jelly forming “the innermost
layer” is deposited “in the upper third of the oviduct, and the outermost layer
just above the region of the uterus.” The ciliated epithelium, due to the spiral
arrangement of the glandular cells along the oviduct, rotates the egg in a spiral
manner as it is propelled posteriad (Rugh, ’35). Once within the uterus, the
eggs are stored for various periods of time, depending upon the temperature.
During amplexus, contractions of the uterine wall together, possibly, with
contractions of the musculature of the abdominal wall, expel the eggs to the
outside. At the same time, the male frog, as in the toad, discharges sperm
into the water over the eggs (fig. 103). In the toad, the eggs pass continu
 
 
202
 
 
TRANSPORTATION OF THE GAMETES
 
 
ously through the oviduct and are not retained in the uterus as in the frog
(Noble, ’31, p. 282).
 
 
b. Other Amphibia
 
The transport of the eggs to the site of fertilization in other anuran amphibia
presumably is much the same as in the frog, although variations in detail
may occur. In the urodeles, however, conditions appear to diverge from the
frog pattern considerably. As mentioned previously, fertilization of the eggs
of Salamandra atra may occur within the peritoneal cavity before the egg
reaches the oviduct, while fertilization in most urodeles occurs internally in
the oviduct, either posteriorly or in some cases more anteriorly. In this amphibian group, the ostium of the oviduct is funnel-shaped and is open, whereas
in the frog it is maintained in a constricted condition and opens momentarily
as the egg passes through it into the oviduct. (Compare figs. 34, 113.) The
open condition of the oviducal ostium in the urodeles suggests that the ostium
and anterior part of the oviduct may function as a muscular organ in a manner
similar to that of birds and mammals.
 
c. Fishes
 
Egg transport in the fishes presents a heterogeneous group of procedures.
In the cyclostomes the eggs are shed into the peritoneal cavity and are transported caudally on either side of the cloaca to lateral openings of the urogenital sinus. The eggs pass through these openings into the sinus and through
the urogenital papilla to the outside. Contractions of the musculature of the
abdominal wall may aid egg transport.
 
In most teleost fishes, the contraction of ovarian tissue together with probable contractions of the short oviduct is sufficient to expel the eggs to the
outside (fig. 28). A somewhat similar condition is found in the bony ganoid
fish, Lepisosteus, where the ovary and oviduct are continuous. However, in
the closely related bony ganoid, Amia, the eggs are shed into the peritoneal
cavity and make their way into an elongated oviduct with a wide funnelshaped anterior opening and from thence to the outside. A similar condition
is found in the cartilaginous ganoid, Acipenser. In the latter two forms, the
anatomy of the reproductive ducts in relation to the ovaries suggests that the
egg-transport method from the ovary to the ostium of the duct is similar to
that found in birds and mammals. Muscular contractions of the oviduct
probably propel the egg to the outside where fertilization occurs. This may
be true also of the salmon group of fishes, including the trout, where a short,
open-mouthed oviduct is present. In the lungfishes (Dipnoi) the anatomy of
the female reproductive organs closely simulates that of urodele amphibia.
It is probable that egg transport in this group is similar to that of the urodeles,
although fertilization in the Dipnoi is external.
 
 
 
G. Siunmary of the Characteristics of Various Mature Chordate Eggs Together with the Site of Fertiluatioii and Place
 
of Sperm Entrance into the Egg
 
 
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Bibliography
 
 
Cohn, E. J. 1917. The relation between
the hydrogen ion concentration of sperm
suspensions and their fertilizing power.
Anat. Rec. 11:530.
 
. 1918. Studies in the physiology
 
of spermatozoa. Biol. Bull. 34:167.
 
Dunn, L. C. 1927. Selective fertilization
in fowls. Poul. Sc. 6:201.
 
Flynn, T. T. and Hill, J. P. 1939. The development of the monotremata: Part IV.
Growth of the ovarian ovum. Maturation, fertilisation and early cleavage.
Trans. Zool. Soc. London 24: Part
6:445.
 
Hammond, J, and Asdell, S. A. 1926. The
vitality of spermatozoa in the male and
female reproductive tracts. Brit. J. Exper.
Biol. 4:155.
 
Hartman, C. G. 1933. On the .survival of
spermatozoa in the female genital tract
of the bat. Quart. Rev. Biol. 8:185.
 
. 1939. Chap. 9. Physiology of eggs
 
and spermatozoa in Allen, et al., Sex
and Internal Secretions. 2d ed., The Williams & Wilkins Co., Baltimore.
 
Hibbard, H. 1928. Contribution a I’etude
de I’ovogenese de la fecondation et de
I’histogenese chez, Discoglossus Pictus
Otth. Arch, biol., Paris. .38:251.
 
Hildebrand, S. F. 1929. Review of experiments on artificial culture of diamondback terrapin. Bull. U. S. Bur. Fisheries.
45:25.
 
Hill, J. P. and O’Donoghue, C. H. 1913.
The reproductive cycle in the marsupial
Dasyurus viverrinus. Quart. J. Micr. Sc.
59:133.
 
Hoaglund, H. and Pincus, G. 1942. Revival of mammalian sperm after immersion in liquid nitrogen. J. Gen. Physiol.
25:337.
 
Mann, T. 1949. Metabolism of semen.
Adv. in Enzymology. 9:329.
 
Noble, G. K. 1931. Biology of the Amphibia. McGraw-Hill Book Co., Inc.,
New York.
 
 
and Weber, J. A. 1929. The sper
matophores of Desmognathus and other
plethodontid salamanders. Am. Mus.
Novit. No. 351.
 
Parker, G. H. 1931. The passage of sperms
and of eggs through the oviducts in terrestrial vertebrates. Philos. Tr. Roy. Soc.,
London, s.B. 219:381.
 
Patterson, J. T. 1910. Studies on the early
development of the hen’s egg. 1. History of the early cleavage and of the
accessory cleavage. J. Morphol. 21:101.
 
Payne, L. F. 1914. Vitality and activity
of sperm cells and artificial insemination
of the chicken. Circular No. 30, Oklahoma Agricultural Experimental Station,
Stillwater, Oklahoma.
 
Perry, E. J. and Bartlett, J. W. 1939. Artificial insemination of dairy cows. Extension Bulletin 200 from New Jersey
State College of Agriculture and Agriculture Experimental Station, New Brunswick, New Jersey.
 
Phillips, R. W. and Andrews, F. N. 1937.
The speed of travel of ram spermatozoa.
Anat. Rec. 68:127.
 
Purser, G. L. 1937. Succession of broods
of Lebistes. Nature, London. 140:155.
 
Rahn, H. 1940. Sperm viability in the
uterus of the garter snake Thamnophis.
Copeia. 2:109-115.
 
Riddle, O. 1927. The cyclical growth of
the vesicula seminalis in birds is hormone controlled. Anat. Rec. 37:1.
 
Romanoff, A. L. and Romanoff, A. J.
1949. The Avian Egg. John Wiley &
Sons, Inc., New York.
 
Rugh, R. 1935. Ovulation in the frog.
II. Follicular rupture to fertilization. J.
Exper. Zool. 71:163.
 
Schott, R. G. and Phillips, R. W. 1941.
Rate of sperm travel and time of ovulation in sheep. Anat. Rec. 79:531.
 
Scrimshaw, N. S. 1944. Embryonic growth
in the viviparous poeciliid, Heterandria
formosa. Biol. Bull. 87:37.
 
 
208
 
 
 
BIBLIOGRAPHY
 
 
209
 
 
Shettles, L. B. 1940. The respiration of
human spermatozoa and their response
to various gases and low temperatures.
Am. J. Physiol. 128:408.
 
Toothill, M. C. and Young, W. C. 1931.
The time consumed by spermatozoa in
passing through the epididymis in the
guinea pig. Anat. Rec. 50:95.
 
Turner, C. L. 1937. Reproductive cycles
and superfetation in poeciliid fishes. Biol.
Bull. 72:145.
 
 
. 1940. Adaptations for viviparity
 
in jenynsiid fishes. J. Morphol. 67:291.
 
Weber, A. 1922. Le fecondation chez la
salamandre alpestre. Compt. rend, de
I’Assoc. d. anat. 17:322.
 
Wimsatt, W. A. 1944. Further studies on
the survival of spermatozoa in the female reproductive tract of the bat. Anat.
Rec. 88:193.
 
Yochem, D. E. 1929. Spermatozoon life
in the female reproductive tract of the
guinea pig and rat. Biol. Bull. 56:274.
 
 
 
5
 
Fertili!z;atioii
 
 
A. Definition of fertilization
 
B. Historical considerations concerning gametic fusion and its significance.
 
C. Types of egg activation
 
1. Natural activation of the egg
 
2. Artificial activation of the egg
 
a. Object of studies in artificial parthenogenesis
 
b. Some of the procedures used in artificial activation of the egg
 
c. Results obtained by the work on artificial parthenogenesis
 
D. Behavior of the gametes during the fertilization process
 
1. General condition of the gametes when deposited within the area where fertilization is to occur
 
a. Characteristics of the female gamete
 
1) Oocyte stage of development
 
2) Inhibited or blocked condition
 
3) Low level of respiration
 
4) Loss of permeability
 
b. Characteristics of the male gamete
 
2. Specific activities of the gametes in effecting physical contact of the egg with the
sperm
 
a. Activities of the female gamete in aiding sperm and egg contact
 
1) Formation of egg secretions which influence the sperm
 
a) Fertilizin complex
 
b) Spawning-inducing substances
 
b. Activities of the male gamete in aiding the actual contact of the two gametes
 
1) Sperm secretions
 
a) Secretions producing lysis
 
b) Secretions related specifically to the fertilization reactions
 
c) Secretions which induce the spawning reaction in the female
 
2) Relation and function of sperm number in effecting the contact of the sperm
with the egg
 
3) Influences of the seminal plasma in effecting sperm contact with the egg
 
4) Roles played by specific structural parts of the sperm in effecting contact
with the egg
 
a) Role of the flagellum
 
b) Role of the acrosome in the egg-sperm contact
 
5) Summary of the activities of the egg and sperm in bringing about the primary or initial stage of the fertilization process, namely, that of egg and
sperm contact
 
 
210
 
 
 
 
DEFINITION
 
 
211
 
 
3. Fusion of the gametes or the second stage of the process of fertilization
 
4. Detailed description of the processes involved in gametic union as outlined above
 
a. Separation and importance of a protective egg membrane, exudates, etc.
 
b. Fertilization cone or attraction cone
 
c. Some changes in the physiological activities of the egg at fertilization
 
d. Completion of maturation divisions, ooplasmic movements, and copulatory
paths of the male and female pronuclei in eggs of various chordate species
 
1) Fertilization in Styela (Cynthia) partita
 
a) Characteristics of the egg before fertilization
 
b) Entrance of the sperm
 
c) Cytoplasmic segregation
 
d) Copulatory paths and fusion of the gametic pronuclei
 
2) Fertilization of Amphioxus
 
3) Fertilization of the frog’s egg
 
4) Fertilization of the teleost fish egg
 
5) Fertilization in the egg of the hen and the pigeon
 
6) Fertilization in the rabbit
 
7) Fertilization in the Echidna, a prototherian mammal
 
E. Significance of the maturation divisions of the oocyte in relation to sperm entrance
and egg activation
 
F. Micropyles and other physiologically determined areas for sperm entrance
 
G. Monospermic and polyspermic eggs
 
H. Importance of the sperm aster and the origin of the first cleavage amphiaster
 
I. Some related conditions of development associated with the fertilization process
 
1. Gynogenesis
 
2. Androgenesis
 
3. Merogony
 
J. Theories of fertilization and egg activation
 
 
A. Definition of Fertilization
 
'][he union or fusion (syngamy) of the oocyte or egg (female gamete) with
the sperm (male gamete) to form a zygote is known as fertilization. From
this zvgotic fusion the new individual arises^ Strictly speaking, the word fertilization denotes the process of making the egg fruitful (i.e., develop) by
means of the sperm’s contact with the egg, and as such may not always imply
a fusion of the sperm with the egg. In certain types of hybrid crosses, such
as in the toad egg (Bufo) inseminated with urodele sperm (Triton), egg activation may occur without fusion of the sperm nucleus with the egg nucleus.
Ordinarily, however, the word fertilization denotes a fusion of the two gametes
(see Wilson, ’25, pp. 460-461).
 
The word zygote is derived from a basic Greek word which means to join
or yoke together) The word is particularly appropriate in reference to the
behavior of the nuclei of the two gametes during fertilization. (For, during
gametic union, the haploid group of chromosomes from one gamete is added
to the haploid group from the other, restoring the diploid or normal number
of chromosomes^^ln most instances, each chromosome from one gamete has
a mate or homologue composed of similar genes in the other gamete. There
 
 
212
 
 
FERTILIZATION
 
 
fore, the union of the two haploid groups of chromosomes forms an integrated
association in which pairs of similar genes are yoked together to perform their
functions in the development of the new individual.
 
In most animal species aside from the union of the chromosome groups,
there is a coalescence of most of the cytoplasm of the male gamete with that
of the female gamete as the entire sperm generally enters the egg (figs. 115,
118), However, in some species the tail of the sperm may be left out, e.g.,
rabbit, starfish, and sea urchin, while in the marine annelid. Nereis, the head
of the sperm alone enters, the middle piece and tail being left behind.
 
/^he morphological fusion of the two sets of nucleoplasms and cytoplasms
of the gametes during fertilization is made possible by certain physiological
changes which accompany the fusion process. These changes begin the instant
that a sperm makes intimate contact with the surface of the oocyte (or egg).
As a result, important ooplasmic activities are aroused within the egg which
not only draw the sperm into the ooplasm but also set in motion the physicochemical machinery which starts normal development. The initiation of normal
development results from the complete activation of the egg. Partial activation
of the egg is possible, and in these instances, various degrees of development
occur which are more or less abnormal. Partial activation of the egg happens
in most instances when the various methods of artificial activation (see p. 217)
are employe^
 
While the main processes of activation leading to development are concerned with the organization and substances of the egg, one should not overlook the fact thatTr/ie sperm also is activated (and in a sense, is fertilized)
during the fusion process. Sperm activation is composed of two distinct phases:
 
 
( 1 )
 
( 2 )
 
 
Before the sperm makes contact with the oocyte or egg, it is aroused
by environmental factors to swim and move in a directed manner and
is attracted to the oocyte or egg by certain chemical substances secreted
by the latter; and
 
after its entrance into the egg’s substance, the sperm nucleus begins
to enlarge and its chromosomes undergo changes which make it possible for them to associate with the egg chromosomes in the first
cleavage spindle. Also, the first cleavage amphiaster in the majority
of animal species appears to arise within the substance of the middle
piece of the sperm as a result of ooplasmic stimulation^
 
 
In the process of normal fertilization it is clear, therefore, that two main
conditions are satisfied:
 
 
(1) There is a union of two haploid chromosome groups, one male and
the other female, bringing about the restoration of a proper diploid
genic balance; and
 
(2) an activation of the substances of the fused gametes, both cyto
 
 
HISTORICAL CONSIDERATIONS
 
 
213
 
 
plasmic and nuclear, is effected, resulting in the initiation of normal
development.
 
The biochemical and physiological factors which accomplish the union of
the haploid chromosome groups and the activation of the gametes are the
objectives of one of the main facets of embryological investigation today.
 
B. Historical Considerations Concerning Gametic Fusion and Its
 
Significance
 
The use of the word “fertilization” in the sense of initiating development
and the idea of making fertile or fruitful, which the w<^ arouses in one’s
mind, reaches back to the dawn of recorded history. (U he concept of this
fruitfulness as being dependent upon the union of one sex cell with another
sex cell and of the fusion of the two to initiate the development of a new
individual originated in the nineteenth century. However, Leeuwenhoek, in
1683, appears to have been the first to advance the thesis that the egg must
be impregnated by a seminal animalcule (i.e., the sperm) in order to become fruitfi^ but the real significance of this statement certainly was not
appreciated by him.
 
Moreover, to Leeuwenhoek, the idea behind the penetration of the egg by
the seminal animalcula was to supply nourishment for the latter, which he
believed was the essential element in that it contained the preformed embryo
in an intangible way. That is, the sperm animalcule of the ram contains a
lamb, which does not assume the external appearance of one until it has
been nourished and grown in the uterus of the female (Cole, ’30, pp. 57, 165).
It should be added parenthetically that actual presence of the little animalcules
as living entities had previously been called to Leeuwenhoek’s attention in
1677 by a Mr. Ham (Cole, ’30, p. 10).
 
In the years that followed Leeuwenhoek the exact interpretation to be
applied to the seminal animalcules (sperm) was a matter of much debate.
Many maintained that they were parasites in the seminal fluid, the latter being
regarded as the essential fertilizing substance in the male semen.An 1827,
von Baer, who regarded the sperm as parasites, named them spermatozoa,
that is, parasitic animals in the spermatic flui^ (Cole, ’30, p. 28). Finally,
in the years from 1835-1841, Peltier, Wagner, Lallemand, and Kolliker, established the non-parasitic nature of the sperm.(^Kolliker in 1841 traced their
origin from testicular tissue^) and thus settled the argument once and for all
as to the true nature of the seminal animals or sperm.
 
Various individuals have laid claim to the honor of being the first to describe the sperm’s entry into the egg at fertilization, but the studies of Newport
and Bischoff (1853, 1854) resulted in the first exact descriptions of the
process. (See Cole, ’30, pp. 191-195.) Thus the general proposition set forth
by Leeuwenhoek 170 years earlier became an accepted fact, although the
 
 
 
214
 
 
FERTILIZATION
 
 
illumination of the details of sperm and egg behavior during fertilization really
began with the studies of O. Hertwig in 1875. The more important studies
which have shed light upon the problems involved in gametic fusion are presented below:
 
(1) O. Hertwig, 1875, 1877, in the former paper, described the fusion of
the egg and sperm pronuclei in the Mediterranean sea urchin, Toxopneustes lividus. One aspect of the work published in 1877 was concerned with the formation of the polar bodies in Haemopis and
Nephelis. In a part of the latter publication O. Hertwig presented
descriptions of sperm migration from the periphery of the egg and
the ultimate association of the sperm and egg pronuclei during the
fertilization in the frog, Rana temporaria (fig. 1191, J).
 
(2) Fol, 1879, contributed detailed information relative to the actual entrance of the sperm into the sea-urchin egg and showed that in the
eggs of various animal species only one sperm normally enters. He
also described the formation of the fertilization membrane in the egg
of the sea urchin, Toxopneustes lividus.
 
(3) Mark, 1881, made important contributions relative to the formation
of the polar bodies in the slug, Deroceras laeve (Umax campestris).
He also presented information which showed that the egg and sperm
pronuclei, although associated near the center of the egg during fertilization, do not actually form a fusion nucleus in this species as described for the sea urchin by O. Hertwig. This is an important contribution to the fertilization problem, as fusion nuclei are not formed
in all animal species.
 
(4) Van Beneden, 1883, in his studies on maturation of the egg and fertilization in A scans megalocephala, demonstrated that half of the
chromatin material of the egg nucleus was discharged in the maturation divisions. (He erroneously thought, however, that the female
ejected the male chromosomes at this time, and in the male, the reverse
process occurred.) (See fig. 133C, D.) He demonstrated also that the
two pronuclei in Ascaris do not join to form a fusion nucleus at fertilization. His work revealed further that the male and female pronuclei
each contributes the haploid or half the normal number of chromosomes at fertilization and that each haploid group of chromosomes
enters the equatorial plate of the first cleavage spindle as an independent unit (fig. 133F-1). Upon the equatorial plate each chromosome divides and contributes one chromosome to each of the two
daughter nuclei resulting from the first cleavage division. This contribution of the haploid number (half the typical, somatic number) of
chromosomes from each parent Van Beneden assumed to be a fundamental principle of the fertilization process. This principle was defi
 
 
HISTORICAL CONSIDERATIONS
 
 
215
 
 
nitely established by later workers and it has become known as Van
Beneden’s Law.
 
(5) Boveri, 1887 and the following years, further established the fact of
Van Beneden’s Law and also demonstrated that half of the chromosomes of the cells derived from the zygote are maternal and half are
paternal in origin. (Fig. 133 is derived from Boveri’s study of Ascaris.)
In ’00 and ’05 he emphasized the importance of the centrosome and
centrioles and presented the theory that the centrosome contributed
by the sperm to the egg at the time of fertilization constituted the
dynamic center of division which the egg lacked; hence, it was a causal
factor in development. This latter concept added new thinking to the
fertilization problem, for O. Hertwig, 1875, had suggested that the
activation of the egg was due to the fusion of the egg and sperm
nuclei. The centrosome theory of Boveri eventually became one of
the foremost theories of egg activation (see end of chapter).
 
(6) During the last five years of the nineteenth century, intensive studies
on artificial activation of the egg (artificial parthenogenesis) were
initiated. This matter is discussed on page 217 in the section dealing
with artificial activation.
 
(7) Another attack on the problem of fertilization and its meaning had
its origin in the “idioplasm theory” of Nageli. This theory (1884)
postulated an “idioplasm” carried by the germ cells which formed
the essential physical basis of heredity. A little later O. Hertwig,
Kolliker, and especially Weismann, identified the idioplasm of Nageli
with the chromatin of the nucleus. In the meantime, Roux emphasized
the importance of the chromatin threads of the nucleus and stated
that the division of these threads by longitudinal splitting (separation)
during mitosis implied that different longitudinal areas of these threads
embodied different qualities, (See Wilson, ’25, p. 500.) In harmony
with the foregoing ideology and as a result of his own intensive work
on maturation and fertilization in Ascaris and also upon other forms,
Boveri came to the conclusion in 1902 and 1907 (Wilson, ’25, p. 916)
that development was dependent upon the chromosomes and further
that the individual chromosomes possessed different qualities.
 
(8) As a result, the field of biological ideas was at this time well plowed
and ready for another important suggestion. This came in ’01 and ’02
when McClung offered the view that the accessory chromosome described by Henking (1891) as the x-chromatin-element or nucleolus
was, in the germ cell of the male grasshopper, a sex chromosome
which carried factors involved in the determination of sex. McClung
first made this suggestion and stated a definite hypothesis, immediately
stimulating work by others; in a few years McClung’s original suggestion was well rounded out and the complete cycle of sex chromo
 
 
216
 
 
FERTILIZATION
 
 
somes in the life history was formulated. E. B. Wilson led this work,
and the theory that he formulated became the assured basis of cytological and genetical sex studies constituting one of the greatest present
day advances in zoology. “McClung’s anticipation of this theory is a
striking example of scientific imagination applied to painstaking observation” (Lillie, F. R., ’40).
 
Not only were the sex chromosomes studied, but other chromosomes as well, and an intense series of genetical investigations were
initiated by Morgan and his students and others which succeeded in
tying a large number of hereditary traits to individual chromosomes
and also to definite areas or parts of the chromosomes. Thus the
assumptions of Roux and Boveri were amply demonstrated. Moreover, these observations established experimental proof for the concept that in the gametic fusion which occurs during fertilization, the
chromosomes pass from one generation to the next as individual
entities, carrying the hereditary substances from the parents to the
offspring. The heredity of the individual was in this way demonstrated
to be intimately associated with the reunion of the haploid groups of
chromosomes in the fertilization process.
 
")Cc. Types of Egg Activation
 
 
1. Natural Activation of the Egg
 
(^Natural parthenogenesis, i.e., the development of the egg spontaneously
without fertilization was suggested by Goedart, in 1667, for the moth, Orgyia
gnastigma, and by Bonnet, in 1745, in his study of reproduction in the aphid.’
(See Morgan, ’27, p. 538.) Since this discovery by Goedart and Bonnet, many
observations and cytological studies have shown thatflhere are two kinds of
eggs which are capable of natural parthenogenesis:
 
 
(1 ) That which occurs in the so-called, non-sexual egg, i.e., the egg which
has not undergone the maturation divisions and, hence, has the diploid
number of chromosomes; and
 
(2) that which results in the sexual egg, i.e., the egg which has experienced meiosis (Chap. 3 ) and thus has the reduced or haploid number
of chromosome (Sharp, ’34, pp. 409, 410).
 
 
Parthenogenesis from a non-sexual egg is found in daphnids, aphids, flatworms, and certain orthopterans. In the case of the sexual egg, parthenogenesis
normally occurs in bees, wasps, ants, some true bugs, grasshoppers, and
arachnids. Jn this type of egg, development may result with or without fertilization i^For example, in the honeybee. Apis mellifica, haploid males arise
from eggs which are not fertilized, workers and queens from fertilized eggs.
 
Extensive studies of the animal kingdom as a whole have demonstrated,
however(^that the majority of oocytes or eggs depend upon the fertilization
 
 
 
EGG ACTIVATION
 
 
217
 
 
process for activation. Consequently, eggs may be regarded in the following
light :fSome eggs are self-activating and develop spontaneously, while others
require sperm activation before development is initiat^ However, the differences between these two types of eggs may not be as real as it appears, for
it is probable that subtle changes in the environment of the so-called selfactivating or parthenogenetic eggs are sufficient to activate them, whereas in
those eggs which require fertilization a strong, abrupt, stimulus is requisite
to extricate them from their blocked condition and to start development. This
idea is enhanced by the information obtained from the methods employed
in the studies on artificial activation of the egg of different animal species.
 
2. Artificial Activation of the Egg
a. Object of Studies in Artificial Parthenogenesis
 
“The ultimate goal in the study of artificial parthenogenesis is the discovery
of the chemical and physical forces which are assumed to cause the initiation
of development” (Heilbrunn, T3). A brief resume of some of the results obtained in the studies on artificial activation of the egg is considered in the
following paragraphs.
 
b. Some of the Procedures Used in Artificial Activation of the Egg
 
^^^Ichomiroff, 1885 (Morgan, ’27, p. 538), stated that eggs from virgin
silkworm moths could be activated by rubbing or by treatment with sulfuric
acid. Somewhat later Mead, 1896-1897, published results of studies on artificial parthenogenesis in the annelid worm, Chaetopterus. The egg of this
worm has the germinal vesicle intact when it is deposited in sea water. Almost
immediately after entrance into sea water, the germinal vesicle breaks down,
and the chromatin proceeds to form the spindle for the first maturation
division. At this point, however, it stops and awaits the entrance of the sperm
for further activation. Thus, the immersion of the Chaetopterus egg in sea
water under normal spawning conditions partially activates the egg' (Mead,
1897). Mead attempted by artificial means to complete this activation initiated by the sea water. In doing so, he took eggs from normal sea water,
which thus had the first polar spindle, and placed them in sea water to which
Va to Vi per cent of potassium chloride had been added. Eggs thus treated
proceeded to form normal polar bodies and the beginnings of the first cleavage
occurred. Development ceased, however, at this point. These eggs were further
activated, but not completely activated. Two steps in partial activation are
here demonstrated:
 
( 1 ) When the egg is spawned into sea water, the nuclear membrane breaks
down and the first polar spindle is formed; and
 
(2) by the immersion in hypertonic sea water the first and second maturation divisions occur, and the first cleavage begins.
 
 
 
218
 
 
FERTILIZATION
 
 
This experiment by Mead was one of a number of pioneering studies made
during this period in an endeavor to activate artificially the egg. Another such
experiment was reported by R. Hertwig (1896), using eggs of the sea urchin.
In a strychnine solution the nucleus underwent changes preparatory to the
first division. Also, Morgan (1896) found it possible to produce cleavage,
normal and abnormal, if unfertilized eggs of the sea urchin, Arbacia, were
placed in sea water to which certain amounts of sodium chloride had been
added and then were returned to normal sea water. Morgan (’00) later
found that magnesium chloride added to sea water induced cleavage in eggs
treated for various intervals. Loeb, in 1899 (see Loeb, ’06), initiated a
series of experiments on activation of the sea-urchin egg. As a result of many
similar experiments, Loeb reached the conclusion that membrane formation
was the essential part of the activation process in that it stimulates the formation of the membrane by initiating cytolysis of the egg (see Loeb’s theory at
end of chapter). Consequently, he sought substances which would elicit
membrane formation and found that monobasic fatty acids, such as butyric,
acetic, formic, or valerianic, would produce membrane formation, and, also,
that ether, bile salts, saponin, etc., would do the same. However, although
these substances aroused certain initial activities of the egg, Loeb found it
necessary to apply a so-called corrective treatment of hypertonic sea water
to arrest the cytolytic effect of the first treatment, which, according to his
belief, restored respiration to its normal level. As a result, Loeb perfected a
treatment as follows which produced development in a considerable number
of sea-urchin eggs so exposed (Loeb, ’06):
 
(1) Unfertilized eggs were placed for Vi to IVi min. in 50 cc. of sea water
to which 3 cc. of N/10 solution of butyric or other monobasic fatty
acid had been added. This treatment effected membrane formation
when the eggs were returned to normal sea water, provided the eggs
had been exposed long enough to the acid.
 
(2) The eggs were allowed to remain in normal sea water for 5 to 10
min. and then were subjected to the corrective treatment in hypertonic
sea water, made by adding 15 cc. of 2 Vi N. NaCl solution to 100 cc.
normal sea water, and allowed to remain for 20 to 50 min. Lesser
times of exposures also were used successfully.
 
( 3 ) Following this treatment, the eggs were returned to normal sea water.
 
An example of an entirely different method from the solution technics
employed above on the sea-urchin egg is that of Guyer (’07) and especially
Bataillon (’10, ’ll, ’13) on the egg of the frog. The method employed by
the latter with success was as follows: Eggs were punctured with a fine glass
or platinum needle and then covered for a time with frog blood. Puncturing
alone is not sufficient; a second factor present in the blood is necessary for
successful parthenogenetic development. The number of actual developments
 
 
 
EGG ACTIVATION
 
 
219
 
 
procured by this method is small, however. In many cases an early cleavage
or larval stage is reached, but the advanced tadpole state or that of the fully
developed frog is quite rare.
 
The method introduced by Bataillon is still used extensively in studies on
artificial parthenogenesis in the frog. Recently, Shaver (’49) finds that the
“second factor” is present on certain cytoplasmic granules obtained by centrifugal fractionation. Heat at 60° C. and the enzyme, ribonuclease, destroy
the second-factor activity. Successful second-factor granules were obtained
from blood, early frog embryos, and “extracts of testis, brain, lung, muscle
and liver.” This author also reports that heparin suppresses parthenogenetic
cleavage.
 
In some of these parthenogenetically stimulated eggs of the frog, the diploid
chromosome relationships appear to be restored during early cleavage; in
others both diploid and triploid cells may be present. Some of these tadpoles
may be completely triploid (Parmenter, ’33, ’40). However, a large percentage
remain in the haploid condition (Parmenter, ’33).
 
A third method of approach in stimulating parthenogenetic development
was used by Pincus (’39) and Pincus and Shapiro (’40) on the rabbit. In
the former work, Pincus reports the successful birth of young from tubal
eggs activated by exposure to a temperature of 47° C. for three minutes. The
treated eggs were transplanted into the oviducts of pseudopregnant females.
In the latter work, eggs were exposed to a cooling temperature in vivo, that
is, the eggs were allowed to remain in the Fallopian tube during exposure to
cold. The birth of one living female was reported from such parthenogenetic
stimulation.
 
The foregoing experiments illustrate three different procedures used on
three widely separated animal species, namely, changing the external chemical
environment of the egg, a tearing or injuring of the egg’s surface followed
by the application of substances obtained from living tissues, and, finally,
changing the physical environment of the egg. To these three general approaches may be added that of mechanical shaking. For example, Mathews
(’01 ) states that mechanical shaking of the eggs of the starfish, Asterias jorbesi,
results in the development of a small percentage of eggs to the free-swimming
blastula stage.
 
Some of the recent work on the initiation of development and in stimulating
cells to divide has emphasized the importance of cellular injury as a factor.
Little is known concerning the mode of action of the injuring substances.
Harding (’51) concludes that an acid substance is released as the result of
“injury” and that this acid substance causes “an increase in protoplasmic
viscosity and initiates cell division” in the sea-urchin egg. (Cf. theory of R. S.
Lillie at end of chapter.)
 
That no single method has been found which activates eggs in general is
not surprising. The eggs of different species are not only in different states of
 
 
 
220
 
 
FERTILIZATION
 
 
maturation (i.e., development) when normally fertilized (fig. 137), but they
behave differently during the normal fertilization process. In some eggs, such
as the egg of Chaetopterus, there is only a slight change within the egg cortex
during fertilization, whereas in the egg of the teleost fish, the egg of the frog,
or in the egg of the urochordate, Styela, marked cortical changes involving
mass movements of protoplasmic materials can be demonstrated.
 
c. Results Obtained by the Work on Artificial Parthenogenesis
 
The question naturally arises: What has the work on artificial activation
of the egg contributed to the solution of the problems involved in egg activation? It has not, of course, solved the problem, but it has contributed much
toward a better understanding of the processes concerned with egg activation
and of the general problem of growth stimulation including cell division. We
may summarize the contributions of this work as follows:
 
( 1 ) It has demonstrated that the egg in its normal development reaches
a condition when a factor (or factors) inhibits further development.
That is, it becomes blocked in a developmental sense.
 
(2) It has shown that this inhibited state may be overcome and development initiated by appropriate types of treatment.
 
(3) It has revealed that activation of the egg is possible only at the time
when normal fertilization occurs in the particular species. In other
words, activation is possible only when favorable conditions are developed in the egg — conditions which enable it to respond to the activating stimulus.
 
(4) It has demonstrated that one of the primary conditions necessary for
the initiation of division or cleavage of the egg is an initial increase
in the viscosity of the egg’s cytoplasm.
 
(5) Certain experiments suggest that chemical compounds, such as heparin
or heparin-like substances, may suppress cleavage and cell division,
presumably due to their ability to decrease the viscosity of the egg.
 
(6) It therefore follows that substances and conditions which tend to increase the egg’s viscosity tend to overcome the inhibited state referred
to in ( 1 ) above and thus initiate development.
 
(7) Recent evidence suggests that substances which produce cell injury
tend to initiate cell division in the egg. As states of injury have been
shown to produce growths of various kinds during embryonic development and also during the post-embryonic period, it is probable that
the principles involved in egg activation are similar to those which
cause differentiation and growth in general.
 
(8) A common factor, therefore, involved in egg stimulation and other
types of growths, including tumor-like growths, is the liberation of
some substance in the egg or in a cell which overcomes an inhibiting
 
 
 
BEHAVIOR OF THE GAMETES
 
 
221
 
 
factor (or factors) and thus frees certain morphogenetic or developmental conditions within the egg or within a cell. Once the inhibiting
or blocking condition is overcome, differentiation and growth begin.
 
(9) Finally, the work on artificial parthenogenesis has demonstrated that
the egg is an organized system which, when properly stimulated, is
able to produce a new individual without the aid of the sperm cell.
This does not mean that the sperm is not an important factor in normal
fertilization, but rather, that the egg has the power to regulate its
internal conditions in such a way as to compensate for the absence
of the sperm.
 
D. Behavior of the Gametes During the Fertilization Process
 
The activities of the gametes during the fertilization process may be divided
for convenience into two major steps:
 
( 1 ) activities of the gametes which bring about their physical contact with
each other, and
 
(2) activities which result in the actual fusion of the gametes after this
contact is made.
 
Before considering these two major steps, we shall first observe certain of
the characteristics of the two gametes when they are about to take part in
the fertilization process.
 
1. General Condition of the Gametes when Deposited
Within the Area Where Fertilization Is to Occur
 
a. Characteristics of the Female Gamete
 
1) Oocyte Stage of Development. In the case of most animal species, the
female gamete is in the oocyte stage when it enters into the fertilization
process. (See Chap. 3; also fig. 137.)) In the dog and fox the female gamete
is in the primary oocyte stage, and'1>oth maturation processes happen after
sperm entrance (fig. 115). In the protochordate, Styela, the first maturation
spindle already is formed when the sperm enters (fig. 116A-D), and(^n
Amphioxus the first polar body has been given off, and the second maturation spindle is developed when the sperm enters (fig. il7A-D). The last
condition probably holds true for most vertebrate species (figs. 1 18B; 1 19D).
However, in the invertebrates, the sea-urchin egg experiences both maturation
divisions normally before sperm entryj
 
2) Inhibited or Blocked Condifion(^hen the female gamete thus reaches
a state of development determined for the species, its further development
is blocked or inhibited, and its future development depends on the circumvention of this state of inhibition^ If not fertilized or artificially aroused when
this inhibited state is reached, the oocyte or egg begins cytolysis. Eggs ferti
 
 
222
 
 
FERTILIZATION
 
 
lized, when these degenerative (cytolytic) conditions are initiated, fail to
develop normally^ If allowed to continue, cytolysis soon produces a condition
in which development is impossible, and dissolution of the egg results.
 
3) (Ebw Level of Respiration. While the egg is in this inhibited or arrested
state awaiting the event of fertilization, respiration is carried on at a steady
but low levels This respiratory level varies in different animal species (fig.
120). That this respiration rate may not be the direct cause of egg inhibition,
is shown by the fact that the rate of respiration does not always increase imme
 
 
Fig. 115. (A) Early fertilized egg in upper Fallopian tube of the bitch (dog). Observe
the female nucleus before the first maturation division together with the sperm head
and tail. Note that the sperm, as in other mammals, enters the nuclear pole of the egg.
Observe further that the zona pellucida and the ooplasm are contiguous. (B) Section
of the egg of the dog, taken from the upper part of the Fallopian tube. Observe the
following features: (1) The sperm pronucleus is forming; (2) the egg nucleus has now
entered the meta^hase of the first maturation division; (3) the ooplasm of the egg has
shrunk away from the zona pellucida and a space is present between the egg and the
zona. This space is the perivitelline space, containing an ooplasmic exudate. (C) Section of the egg in the Fallopian tube of the bitch, showing the formation of the first
polar body.
 
diately following fertilization in all species (fig. 120). (Consult Brachet, J.,
’50, p. 105.) Among the vertebrates, the low rate of oxygen consumption
of the unfertilized egg has been shown to continue for some time after fertilization in the toad and frog egg and also in the egg of the teleost fish, Fundulus
heteroclitus. However, in the case of the egg of the lamprey the respiration
rate rises after fertilization (Brachet, J., ’50, p. 108).
 
.Loss of Permeability. A final characteristic of the female gamete immediately before fertilization is the loss of permeability of the egg surface
to various substanceD Correlated with this fact is the presence of definite
ooplasmic or other egg membranes associated with the egg surface. The relationship between the ooplasmic surface of the egg and these membranes is
altered greatly after fertilization when the egg and the membranes tend to
separate. To what extent the loss of permeability of the egg surface is caused
by the intimate association of these membranes with the egg surface is problematical. The evidence to date suggests that under normal circumstances
 
 
 
BEHAVIOR OF THE GAMETES
 
 
223
 
 
they are integrated with the conditions which restrict permeability and egg
activation.
 
h. Characteristics of the Male Gamete
 
LJn contrast to the inertia and metabolic quietude experienced by the ^male
gamete, the gamete of the male experiences quite opposite conditions. ^When
the sperm, for instance, is brought into an environment which favors motility,
such as the posterior region of the vas deferens of the mammal, it becomes
highly motile and continues this motility in the female genital trac^ Similarly,
the normal sperm of other vertebrate species is a very active cell when placed
in the normal fertilization area (fig. 121, primary phase of fertilization). To
quote from J. Brachet (’50), page 91: “This very active cell has an intense
metabolism and the maintenance of this latter (condition) is indispensable
to the continuance of motility.” As mentioned previously (Chap. 1 ),^his high
respiratory metabolism at least in some species is supported partially by the
utilization of a simple sugar in the seminal fluid as the sperm ‘(fs rich in oxidative enzymes and in hydrogen transporters’^ (J. Brachet, ’50).
 
2. Specific Activities of the Gametes in Effecting Physical
Contact of the Egg with the Sperm
 
(Consult fig. 121, primary phase of fertilization.)
 
While the gametes are in the condition mentioned, they are physiologically
adapted to fulfill certain definite activities which enhance their contact with
each other and bring about actual union in the fertilization process.
 
a. Activities of the Female Gamete in Aiding Sperm and Egg Contact
 
l)CFormation of Egg Secretions Which Influence the Sperm. The activities
of the female gamete at this time are concerned mainly with the effusion of
certain egg secretions. These secretions are known as gynogamic substances,
or gynogamones. They are elaborated by the egg when the latter becomes
physiologically mature, i.e., when it becomes fertilizablev(fig. 137).
 
A study of the natural secretions of the egg in relation to the fertilization
process has occupied the attention of numerous investigators. These studies
began during the early part of the twentieth century. (in reference to the egg,
two main groups of substances have been recognized:
 
( 1 ) the fertilizin complex, and
 
(2) substances which induce the spawning reactions in the male.
 
a) Fertilizin Comple?^ Some of the earliest studies upon fertilizin substances were made by von Dungern in ’02, Schiicking in ’03, and De Meyer
in Tl. In these experiments an egg-water solution was obtained by allowing
ripe eggs of the sea urchin to stand in sea water for a period of time or by
disintegrating the eggs. All of these observers found tha^some substance from
 
 
 
 
 
 
 
 
 
Fig. 116. Fertilization and maturation of the egg in the urochordate, Styela (Cynthia)
partita, (After Conklin, ’05.) (A) Egg shortly after spawning but before sperm entrance.
 
The spindle fibers of the first maturation division are forming, and the nucleoplasm is
located toward the animal pole. (B) Egg showing the spindle for first maturation division. Observe the sperm nucleus just inside the ooplasmic membrane near the midvegetal
pole of the egg. The nucleoplasm (karyoplasm) of the female nucleus has spread into a
thin cap at the animal pole. (C) Metaphase of first division spindle (1, D.S.) nearly
parallel to the surface of the egg; no centrosomes are present. (D) Higher powered
representation of sperm a little later than that shown in (B). The aster for the first
cleavage spindle is forming in the middle piece of the sperm. (E) Slightly more advanced stage than that shown in (B). Collection of yellow-pigmented, peripheral protoplasm (PL.) is shown at bottom of the egg. (F) Anaphase of second polar spindle.
Sperm aster enlarging. (See (G) and (H).) (G) Separation of first polar body, (H)
Metaphase of second polar spindle, paratangential in position. (1) First polar body
(1 P.B.) formed; chromatin of second spindle (CHR.j. Sperm has revolved 180®; sperm
aster enlarging. (J) Telophase of second polar spindle. Sperm aster enlarges, and sperm
nucleus assumes vesicular condition. (K) Separation of second polar body. (L) Two
 
(Continued on facing page.)
 
 
224
 
 
 
BEHAVIOR OF THE GAMETES
 
 
225
 
 
the egg, when present in dilute solution, caused the sperm of the sea urchin to
loose their motility and to become clumped together or agglutinate^A little
later, F. R. Lillie, ’13, ’14, ’15, studied the activity of the egg water of the
sea urchin, Arbacia, extensively. Lillie associated the egg secretion found in
the egg water with a definite theory concerning the mechanism of fertilization.
He called ^e substance given off when the sea-urchin egg is allowed to stand
in sea water, “fertilizin’’; for, according to his results, washed eggs deprived
of this egg secretion fail to fertilize. Only ripe eggs give off fertilizin according
to his observations. Lillie found further that the activities of the sperm, introduced by means of a pipette into the egg-water solution are changed greatly.
At first they are activated, to be followed by an agglutination. Moreover, a
drop of egg water introduced into a sperm suspension activates the sperm
and appears to influence them chemically, causing them to be attracted to
the drop. (Lillie therefore concluded that fertilizin has a threefold action upon
the sperm:
 
(1) that it activates the sperm (that is, stimulates their movement),
 
(2) attracts the sperm by a positive chemotaxis, and
 
(3) agglutinates the sperm, that is, causes the sperm to associate in clumps.)
 
The agglutination effect F. R. Lillie found is reversible in most sea-urchin
sperm, providing the egg water containing fertilizin is not allowed to act too
long. On the other hand, in the annelid, Nereis, agglutination of the sperm is
“essentially permanent” (Lillie, F. R., ’13). Lillie placed most emphasis upon
the “agglutinin” factor in the egg water. He further postulated that fertilizin
not only affected the sperm, but dso activates the egg to cause its development
(see theory at end of chapter ).(j^illie also obtained another substance from
crushed or laked eggs which combines “with the agglutinating group of fertilizin, but which is separate from it as long as the egg is inactive.” This
substance present within the egg he called “antifertilizin^
 
Since the time of F. R. Lillie’s original contribution, the subject of fertilizin
and antifertilizin has been actively investigated by various students of the
problem. Some investigators criticized the conclusipns drawn by Lillie, but
more recent work substantiates them. For examplei^M. Hartmann, et al. (’40),
working on the sea urchin, Arbacia pustulosa, an^Tyler and Fox (’40) and
Tyler (’41), using eggs from Strongylocentrotus purpuratus, find that fertilizin
 
Fig. 116 — (Continued)
 
polar bodies (P.B.); fusion of chromosomal vesicles to form egg pronucleus (E.N.).
(M) Movement of sperm nucleus, aster, and of surrounding yellow-pigmented and clear
protoplasm to the posterior pole of the egg. The copulation path of egg pronucleus
(E.N.) to meet the sperm nucleus is in progress. (N) Sperm aster has divided; egg
pronucleus progresses along its copulation path toward posterior pole of egg to meet the
male pronucleus. (O) Egg and sperm pronuclei are making contact with each other.
 
(P) Pronuclei associate and begin to form early prophase conditions of the first cleavage.
 
(Q) Metaphase of first cleavage. (R) Anaphase of first cleavage. (S) Late anaphase
of first cleavage.
 
 
 
 
 
Fig. 1 17, (See facing page for legend.)
226
 
 
 
 
BEHAVIOR OF THE GAMETES
 
 
227
 
 
is present and that it is associated with the jelly layer around the egg. Tyler
(’41) concludes:
 
( 1 ) When fertilizin is present in the form of a gelatinous coat around the
egg, it enhances fertilization;
 
(2) when present only in solution around the egg after the gelatinous coat
is removed, it hinders fertilization by agglutinating the sperm; and
 
(3) that fertilizin is not entirely essential since eggs can be fertilized when
the jelly coat is removed, but a greater number of sperm are needed
under these circumstances^
 
Tyler also has detected antifertilizin below the surface of the egg and by
crushing the eggs was able to show thal(^ntifertilizin from the interior of the
egg is able to neutralize the fertilizin of the jelly coat surrounding the eg^
(Tyler, ’40, ’42). In Germany, Hartmann and his associates (see Hartmann,
M., et al., ’39, a and b, ’40) have demonstrated that by exposing fertilizin
to heat or light one may separate the “agglutinating factor” from the “activating factor.” Heat at 95° C. destroys the “agglutinating factor,” while exposure to bright light causes the “chemotactic” and “activating” factors to
disappear. The factual presence of the egg products, fertilizin and antifertilizin,
postulated by Lillie thus is well established.
 
Fertilizin appears to be widely distributed as an egg secretion among animals, invertebrate and vertebrate. Among the latter it has been identified in
cyclostomes, certain teleost fishes, and in the frog, Rana pipiens (Tyler, ’48).
Moreover, it is becoming increasingly clear that the term, fertilizin, as employed originally by F. R. Lillie, includes more than one secretion. How many
separate enzymes or other substances may be included under the general terms
of fertilizin and antifertilizin remains for the future to determine. Moreover,
the exact presence of particular gynogamic substances in the egg secretions
of different animal species may vary considerably. For example, the spermactivating principle may not be present in all animal species. In fact, there
is good evidence to show that it is not present, for example, in all species
of sea urchins.
 
 
Fig. 117. Fertilization and maturation of the egg in Amphioxus. (A, B, FI after Cerfontaine, ’06; C-I after Sobotta, 1897.) (A) Metaphase of first maturation division
 
before sperm entrance. (B) Anaphase of first maturation division before sperm entrance.
(C) First polar body and metaphase of second maturation division before sperm entrance.
Observe the first or primary fertilization membrane. (D) Sperm has entered near vegetal
pole of egg. (F) Outer egg membrane has enlarged and is now much thinner; the
second egg membrane is separating from the egg, and the second polar body is forming.
 
(F) Outer and inner egg membranes have fused and expanded; pronuclei of sperm and
egg are evident; the sperm aster is to be observed in connection with the sperm nucleus.
 
(G) Meeting of the two pronuclei between the developing amphiaster. (H) Fusion
nucleus complete. (1) Diploid chromosomes now evident preparatory to the first
cleavage of the egg.
 
 
 
228
 
 
FERTILIZATION
 
 
(The general term “gamones” (Hartmann, M., ’40) has been applied to the
substances produced by the gametes at the time of fertilization. The Hartmann
school further has identified the factor responsible for chemotaxis and activation as “echinochrome A,” tl^t is, the bluish-red pigment of the egg, and
have called it “Gynogamone IJ^This factor will attract sperm and stimulate
their movements “at the enormous dilution^of 1 part in 2,500,000,000 parts
of water” (Brachet, J., ’50, p. 96). However, Tyler has not been able to
detect echinochrome in the egg of the Pacific coast sea urchin, Strongylocentrotus. But the egg water of this species does activate the sperm of this species,
which suggests that the activating factor may be something else than echinochrome. (Xo the agglutinating factor, M. Hartmann and his associates have
given the name “Gynogamone Il.j^
 
The exact identity of these gamones with particular chemical substances
present in the egg water at the present time is impossible. To quote from
J. Brachet, ’50, p. 99:
 
It is clear that research in this field is complicated by the fact that a number of
agents activate the movements of sperm (alkalinity, glutathione, echinochrome (?),
etc.) . . . There is strong evidence in favor of the protein nature of agglutinin,
while the sperm-activating principle is probably a substance with a small molecule,
its identity with echinochrome being doubtful at the present time.
 
bl Spawning-inducing Substances. In addition to the fertilizin substances which act in effecting the actual contact of the sperm with the egg,
a spawning-inducing agent is present in the egg water of certain species. In
the annelid, Nereis, for example, there is something present in the egg water
which induces spawning in the male^^^ownsend (’39) suggested that this
substance may be glutathione, but Tyler (’48) does not readily concur with
this conclusion. A spawning-inducing agent is found also in the egg water of
oysters (Galtsoff, ’40). Among the vertebrates, the spawning behavior of the
female appears to be the important factor in inducing the male reactiofQ
 
b. Activities of the Male Gamete in Aiding the Actual Contact
of the Two Gametes
 
^^The activities of the male gamete, including those of seminal fluid, are
much more complicated and devious than those of the female gamete. These
activities entail:
 
( 1 )(^production of certain sperm secretions,
 
(2) activities of large sperm numbers,
 
(3) presence of a healthy seminal plasma or protective substance for the
sperm, and
 
(4) physical movements and functioning of specific parts of the sperm cell
itself.
 
(1) Sperm Secretions. The sperm secretions are known as androgamic substances or androgamone£) These substances have been the object of much
 
 
 
BEHAVIOR OF THE GAMETES
 
 
229
 
 
Study since the initial endeavors of Fieri in 1899. (in recent years, three general types of substances have come to be recogniz^ in relation to the sperm
of different species. These three groups of substances are:
 
(1) secretions which cause lysis,
 
(2) a substance or substances related specifically to the fertilization reaction (i.e., egg and sperm contact), and
 
(3) substances which bring about the spawning reaction in the female.
 
a) Secretions PRODUcjiNG Lysis. To cite the importance of lytic substances produced by the sperm, reference is made first to the situation in the
amphibian, Discoglossus pictus. In this primitive anuran, the sperm, although
about 2 mm. long, are almost incapable of motility. However, they do accumulate in the region of a thickened portion of the egg capsule which overlies
a depressed area of the egg. They are capable of passing through this thickened
area of jelly by the aid of a digestive enzyme probably associated with the
acrosome (Hibbard, ’28j^Hibbard also suggests that “nuclear fluids” accumulate in the bottom of the egg depression and these fluids attract the sperm
to the thickened area of the capsule. If so, here is an example of two chemical
substances, one elaborated by the egg and the other by the sperm, both working together to bring about fertilization. In substantiation of Hibbard’s views
of the presence of a lytic enzyme associated with the sperm of this species,
Wintrebert (’29) found that extracts from the sperm contained an enzyme
which is capable of digesting the inner jelly coat of the egg.
 
More recently, Tyler (’39) has found that sea-water extracts of frozen
and thawed sperm of two mollusks (Megathura crenulata and Haliotis cracherodii) were able to dissolve the egg membranes of the respective species.
Cross-species reactions were not obtained, however. Strong extracts of concentrated sperm suspensions bring about egg-membrane disappearance in less
than one-half minute, but with the jelly coat present around the egg it takes
about three minutes. Also, Runnstrom and his collaborators (’44, ’45, a and
b, ’46) made methanol extracts of sea-urchin sperm which were able to liquefy
the superficial cortical area of the egg.
 
most interesting enzyme, known as hyaluronidase, has been extracted
from mammalian testes and from mammalian sperm. This substance is capable
of dispersing the follicle cells of the corona radiata present around most mammalian eggs when discharged from the ovary^^ ( Sheep and opossum eggs as
well as those of the monotremes do not possess a layer of follicle cells around
the newly ovulated egg.) (This dispersing effect aids fertilization, for it enables
sperm to reach the egg surface before degeneration processes occur in the
egg^ Rowlands (’44) effected artificial insemination in the rabbit with dilute
sperm solutions by adding the enzyme hyaluronidase from other sperm to the
dilute suspensions. Without the addition of hyaluronidase, fertilization did not
result. In certain cases in women where artificial insemination was tried but
 
 
 
230
 
 
FERTILIZATION
 
 
failed when semen alone was used, the addition of hyaluronidase from bull testis
to the semen produced successful fertilization (Leonard and Kurzrok, ’46).
 
b)''/SECRETiONS Related Specifically to the Fertilization Reactions. The substances mentioned in the preceding paragraphs are related to
the general fertilization process, but they may not be related specifically to
the reactions which bring the sperm in direct contact with the egg(ln the egg
we have observed the presence of fertilizin which stimulates a series of sperm
activities directed to this end. Similarly, in the male gamete, sperm of various
species seem capable of producing “androgamic substances which neutralize,
in part, the action of the gynogamic substances and thus assure the precise
mechanism necessary for precise fusion of the gamete$i^ (J. Brachet, ’50).
 
An introductory study by Frank (’39) suggests the presence of a sperm
substance which reacts directly with the fertilizin complex of the egg. It was
shown by this investigator that an extract from the sperm of the sea urchin,
Arbacia, is:
 
( 1 ) (able to destroy the sperm agglutinating factor when added to a solution
 
of fertilizin derived from the sea-urchin egg, and
 
(2) possesses the power to agglutinate eggs of the same specie^
 
Other students of the problem have found a similar substance associated
with the sperm. (See Hartmann, Schartau, and Wallenfels, ’40; Southwick, ’39;
Tyler, ’40.) (The general term “sperm antifertilizin” has been given to this
substanc^(or substances) by Tyler and O’Mel veney (’41) (Sperm antifertilizin unites with fertilizin produced by the egg, with the resulf that the sperm
is entrapped at the egg’s surface. Tyler and O’Melveney (’41) regard the
reaction between antifertilizin of the sperm and fertilizin of the egg to be the
“initial (perhaps essential) step in the union of the gametes whereby the
spermatozoon is entrapped by the . . . fertilizin, on the egg.’’/
 
C)ySECRETIONS WHICH INDUCE THE SPAWNING REACTION IN THE FEMALE.
Galtsoff (’38) has shown that the presence of sperm of the oyster, Ostrea
virginica, “easily induces spawning in oysters.’’ He also found that the spawning reaction is specific in that sperm of different species cannot provoke it.
The active principle of the sperm suspension is thermolabile and insoluble
in water. However, it may be readily extracted in 95 per cent ethyl alcohol
and benzene.
 
To what extent spawning-inducing substances may be present in other animal
species is questionable, but it may not be an uncommon phenomenon, especially in sedentary species, such as the oyster and other mollusks. In the vertebrate group, surface contact of the male and female bodies is an important
factor in many cases.
 
2) Relation and Function of Sperm Number in Effecting the Contact of
the Sperm with the Egg. In the preceding chapter, sperm transport is considered. This transportation journey is an efficient one with regard to the end
 
 
 
BEHAVIOR OF THE GAMETES
 
 
231
 
 
achieved, namely, contact of a single sperm with an egg, but from the viewpoint of sperm survival it may appear as waste and caprice. This fact is especially true in those forms utilizing fertilization where only one or a very few
eggs are fertilized. It has been shown by Walton (’27) in experiments dealing
with artificial insemination in the rabbit, when dilution of the sperm is such
that the number falls below 3,000 to 4,000 per cc., fertilization does not take
place. Recent observations by Farris (’49) on the human suggest that numbers of sperm below 80,000,000 per cc. are precarious when conception is
the end to be achieved. (For the total number of sperm ejaculated by certain
males during a single copulation, see Chap. l.)(j\lthough exceedingly large
numbers of sperm are deposited in the posterior area of the female reproductive tract, the number becomes less and less as the ovarian end of the
duct is reached. The ability of effective sperm transport within the female
tract probably varies considerably in different speeies and with different females in the same species^j^he rat and the dog appear to be more efficient
in this respect than the rabbit.
 
The relation of sperm numbers to the efficiency of the fertilization process
is not to be considered merely as a mechanical hit and miss device, whereby
the presence of a greater number of sperm may assure an accurate “hit” or
sperm-egg collision (Rothschild, Lord, and Swann, ’51). Hammond (’34)
has shown in the rabbit that fertilization is not effected by the few sperm
which reach the region of the egg first, but by the later aggregations of numbers of sperm. The work on hyaluronidase mentioned on page 229 suggests
strongly that one object of the excess sperm is to transport hyaluronidase to
the vicinity of the egg. The presence of this enzyme close to the egg possibly
facilitates the passage through the cells of the corona radiata and also through
the zona pellucida of the single sperm which makes contact with the egg in
the process of fertilization (Tyler, ’48).(The general result should be regarded
as the working of a cooperative enterprise, where many sperm aid in the dissolution of the interference in order that one sperm may reach the egg’s surface^
 
3) Influences of the Seminal Plasma in Effecting Sperm Contact with the
Egg. The importance of the seminal plasma (i.e., the fluid part of the semen;
see Chap. 1) cannot be overestimated (Mann, ’49). It is, to a great extent,
^he natural environment and at the same time the nutritive medium for the
sj^rm during the transport from the male ducts through the external medium
or within the lower region of the female genital tract. Its functions may be
stated as follows:
 
(1) It increases the motility of the sperm;
 
(2) it has a high buffering capacity, which protects the sperm from injurious acids or other injurious substances; and
 
(3) it is a vehicle for nutritive substances, such as fructose, vitamin C,
and the B complex which provide nourishment for the spenn^
 
 
 
232
 
 
FERTILIZATION
 
 
The B group of vitamins may be directly related to sperm motility. Other
substances, such as iron, copper, etc., are present. One should consider the
seminal plasma, therefore, as a most important association of substances which
aids in producing a protective environment for the sperm while the latter is
in migration to the egg.
 
The importance of the environment of the sperm and also that of the egg
cannot be overemphasized. If normal fertilization is to be effected, optimum
conditions for both sperm and egg must be present. An example of this fact
is shown by the observations of Reighard on fertilization of the walleyed pike.
(See Morgan, *27, p. 18.) The best results with the eggs of this teleost fish
were obtained when the eggs were fertilized as soon as they entered the water
from the female genital tract. After two minutes only 40 per cent of the eggs
segment, and after ten minutes no eggs segment. For many fish, “dry fertilization” gives the best results. Dry fertilization consists in stripping the female
to force out the eggs into a dry container and then stripping the milt (seminal
fluid) from the male directly over the eggs. The eggs are then placed in water
after a few minutes. This work suggests strongly that a deleterious environment for either the egg or the sperm is disturbing to the fertilization process.
 
4) Roles Played by Specific Structural Parts of the Sperm in Effecting
Contact with the Egg: a) Role of the Flagellum. As indicated in the
foregoing paragraphs, when the sperm cells have reached the normal fertilization site, the activities which bring about actual contact of the sperm with
the egg largely is a sperm problem. Aside from enzymes elaborated by the
sperm, sperm motility is extremely important in achieving this end. Although
sperm may appear to swim rather aimlessly, vigorous, healthy sperm do lash
forward more or less in a straight line for some distance; ill-developed or
otherwise impaired sperm may simply swim round and round or move forward
feeblyi^n the case of flagellate sperm, the structure which makes the forward
swimming movement possible is the flagellum or tail (figs. 74, 77, 78, 79).
A two-tailed sperm or one in which the flagellate mechanism is not well developed would be at a disadvantage in this race to reach the confines of the
egg. ^rachet (’50) considers the rate of metabolism necessary to support the
acTivities of the tail or flagellum in sperm movement as directly comparable
to that of muscle.
 
An interesting peculiarity of a different type of sperm mechanism useful
iit' achieving contact with the egg’s surface is that of the so-called “rocket
sperm” of certain decapod Crustacea described by Koltzoff (fig. 75). After
attachment of the sperm to the egg by its tripod-like tips, the caudal compartment, containing a centriole and the acfosome, explodes. “Koltzoff considers
that the force of the explosion drives the sperm upon, or even into, the egg”
(Wilson, ’25, p. 299).
 
b) Role of the Acrosome in the Egg-sperm Contact. The acrosome
of the sperm (fig. 78) has long been regarded as a structure which has a
 
 
 
BEHAVIOR OF THE GAMETES
 
 
233
 
 
function in the reactions involved in fertilization. The older conception of
Waldeyer that the acrosome was a perforating device which enabled the sperm
to pass through the egg membranes and thus to enter the egg is untenable in
the light of later observation. Many years ago Bowen (’24) though admitting
a minor mechanical role for the acrosome, emphasized thati^e acrosome
essentially is a secretory product whose principal function is to initiate the
physicochemical reactions of fertilization^ It should be recalled in this connection that Hibbard (’28) and also ParatX’33, a and b) have attributed to
the acrosome of the anuran, Discoglossus, ^e ability of carrying or producing
an enzyme which enables it to reach the egg’s surface through the jelly surrounding the egg) Parat further suggested that the acrosome in this species
contains a “proteolytic enzyme” which, when introduced into the egg, results
in development.
 
The concept of a proteolytic enzyme associated with the acrosome of Discoglossus is interesting in the light of the suggestion by Leuchtenberger and
Schrader (’50) that the mucolytic enzyme, hyaluronidase, in the bull sperm
may be associated with the acrosome. Both of the above suggestions need
more work before it can be stated with certainty that the acrosome is connected with either of these enzymes in the above species. However, these suggestions do serve to emphasize the possibility that^lie acrosome may be an
enzyme-producing or enzyme-carrying device which enables the sperm to
make its way through the egg’s surroundings to the egg surface, and also, that
it may play a part in egg activation^
 
5) Summary of the Activities of the Egg and Sperm in Bringing About
the Primary or Initial Stage of the Fertilization Process, Namely, that of Egg
and Sperm Contact.
 
a) The secretion of fertilizin by the egg:  
 
(1) activates the sperm to increased motility, and
 
(2) through chemotaxis, entices the sperm to move in the direction of the
 
egg.
 
b) In moving toward the egg the lytic substances elaborated by the sperm
enable it to “plow” through the gelatinous envelopes and cellular barriers to
the surface of the egg. This movement undoubtedly is aided by movement of
the flagellum in some species, but not in all (see Discoglossus) . The acrosome
of the sperm may function at this time either as an instrument carrying lytic
substances or as one which actually manufactures these substances. The presence of large numbers of sperm near the egg may aid sperm penetration to
the egg’s surface by contributing lytic substances to the environment around
the egg which aid in the removal of membranes and other barriers surrounding
the egg.
 
c) The antifertilizin of the sperm may then unite with the fertilizin of the
 
 
 
234
 
 
FERTILIZATION
 
 
egg (probably with the agglutinin factor) ; this reaction presumably agglutinates
the sperm to the egg’s surface.
 
d) An egg-surface, liquefying factor, androgamone III, has been isolated
by Runnstrom, et al. (’44), from sea-urchin sperm (Runnstrom, ’49, p. 270).
A similar “sperm lysin” has been isolated also from mackerel testes. This work
suggests that a specific sperm lysin may be involved in the activation processes
within the egg cortex. (See theory of fertilization according to J. Loeb at end
of chapter.)
 
e) Lastly, in certain animal species, substances may be present in the
seminal fluid which induce the spawning reaction in the female, while in the
egg secretion of certain species, a factor may be present which induces spawning in the male.
 
3. Fusion of the Gametes or the Second Stage of the
Process of Fertilization
 
(Xhe actual fusion or union phase of fertilization begins once the sperm has
made contact with the egg^fig. 121 B). From this instant the rest of the fertilization story becomes essentially an egg problem. The egg up to the time
of sperm contact literally has been waiting, discharging fertilizin substances
into the surrounding medium. However, when a sperm has made successful
contact with the surface of the egg, ^e waiting period of the egg is over, its
work begins, the fusion of the two gametes ensues, and the drama of a new
life is initiated!
 
The following events of the fusion process may be listed — events which
occur quite synchronously, once the mechanisms involved in egg activation
and gametic fusion are set in motion:
 
(a) The separation of an egg membrane (fertilization membrane, vitelline
membrane, chorion, zona pellucida, etc.) from the egg’s surface and
the exudation of fluid-like substances 'from the egg’s surface.
 
(b) A fertilization cone may be elaborated in some species.
 
(c) Changes in the physicochemical activities of the egg.
 
(d) The maturation division (or divisions) is completed in most eggs.
 
(e) Profound cytoplasmic movements occur ^iC'fliany eggs which bring
about various degrees of localization of cytoplasmic substances; these
substances orient themselves into a pattern definite for the species.
In some species a cytoplasmic pattern composed of future, organforming substances is rigidly established and definitely correlated with
the first cleavage of the egg (Styela); in others it is less rigid (frog);
and in still others it appears gradually during cleavage of the egg
(teleost fishes).
 
(f) The sperm nucleus enlarges, and the middle-piece area in most animal
species develops a cleavage aster.
 
 
 
BEHAVIOR OF THE GAMETES
 
 
235
 
 
(g) The copulation movements of the egg and sperm pronuclei take place.
These movements bring about the association of the two pronuclei
near the center of the protoplasm of the egg which is actively concerned with the cleavage phenomena.
 
(h) The pronuclei may fuse to form a fusion nucleus or they may associate
less intimately. Regardless of the exact procedure of nuclear behavior,
the female and male haploid chromosome groups eventually become
associated in the first cleavage spindle to form one harmonious diploid
complex of chromosomes, composed (in most cases) of paired chromosomal mates or homologues.
 
(i) The first cleavage plane is established.
 
4. Detailed Description of the Processes Involved in
Gametic Union as Outlined Above
 
a. Separation and Importance of a Protective Egg Membrane,
Exudates, etc.
 
The term “fertilization membrane” is applied to the egg (vitelline) membrane which, in many species, becomes apparent only at the time of fertilization. In many other eggs a definite and obvious vitelline membrane is present
before the egg is fertilized and in many respects functions similarly to the
more dramatically formed fertilization membrane. Both types of membrane
fulfill definite functions during fertilization and early development. The fertilization men^brane which forms only as a distinct membrane during fertilization was observed first by Fol, in the autumn of 1876, in the starfish egg
(Fol, ’79). In the cephalochordate, Amphioxus, two definite membranes separate from the egg’s surface. One membrane forms just before the sperm enters
the egg, while the second membrane separates from the egg after the sperm
enters. Both membranes soon fuse and expand to a considerable size, leaving
a perivitelline space between them and the egg; the latter space is filled with
fluid, the perivitelline flui^(fig. 117B-F, I). In the urochordate, Styela, no
such membrane arises from the egg’s surface, but the chorion previously
formed by the follicle cells serves to fulfill the general functions of a fertilization membrane (figs. 91B,i^[J6) Jin teleost fishes, the egg emits a considerable
quantity of perivitelline fluid at the time of fertilization, effecting a slight
shrinkage in egg size with the production of a space filled with this fluid between the egg’s surface and the zona radiata (fig. 122A-C). The zona radiata
thus functions as a fertilization membrane. In the gobiid fish, Bathygobius
soporator, the chorion and/or vitelline membrane expands greatly after the
egg is discharged into sea water, and an enlarged capsule is soon formed
which assumes the size and shape of the future embryo at the time of hatching (fig. 123). (See Tavolga, ’50.) In the brook lamprey, according to
Okkelberg (’14), shrinkage of the egg at fertilization is considerable, amounting to about 14 per cent of its original volume. A slight egg shrinkage with
 
 
 
236
 
 
FERTILIZATION
 
 
 
Fig. 118. Fertilization in the guinea pig. (After Lams, Arch. Biol., Paris, 28, figures
slightly modified.) (A) Spindle of first maturation division. (B) Second maturation
division completed; head of sperm in cytoplasm beginning to swell. (C) Sperm pronucleus, with tail still attached, greatly enlarged; female pronucleus small. (D) Pronuclei
ready to fuse; chromatin material (chromosomes) evident within. (E) First cleavage
spindle. (F) First cleavage completed. Observe deutoplasmic and cytoplasmic globules
which have been exuded into the space between the blastomeres and the zona pellucida.
(G) Four-cell cleavage stage. Observe that the zona pellucida encloses the four blastomeres and the cytoplasmic globules which have been exuded. The zona functions to
keep the entire mass intact.
 
the emission of fluid is present in the amphibia and the egg thus is enabled
to revolve within a relatively thick vitelline membrane. The latter membrane
expands gradually during development, and is associated intimately with the
surrounding jelly membranes secreted by the oviduct. In the reptiles and birds,
the separation of the egg from the vitelline membrane or zona radiata and
 
 
 
BEHAVIOR OF THE GAMETES
 
 
237
 
 
the formation of the perivitelline space is less precipitous. In the egg of the
bird (e.g., pigeon or hen) (fig. 126), a vitelline space filled with fluid appears
during the latter phase of oocyte growth in the ovary which separates the
surface ooplasm of the egg from the vitelline membrane. The egg is free to
revolve in this vitelline space. In the prototherian mammals, the zona pellucida
evidently functions in a manner similar to that of the bird or reptile (figs.
46, 127) ^However, in the metatherian and eutherian mammalia, the zona
pellucida becomes separated from the ooplasm of the egg’s surface with the
subsequent development of a perivitelline space at fertilization or during early
cleavage (figs. 115, 118, 124, 125).^
 
It is to be observed, therefore, that there are two general groups of egg
or vitelline membranes in the phylum Chordata which assume an important
role at fertilization and during the earlier part of embryonic development:
 
(1 ) those membranes which become separated from the egg surface in a
somewhat dramatic manner at fertilization, and
 
(2) membranes which separate gradually during the late phases of ovarian
development and during early embryonic development.
 
In the former group are to be found the egg membranes of the eggs of
Amphioxus, teleost and many other fishes, and the amphibia; in the latter
group are the membranes of eggs of Styela, elasmobranch fishes, reptiles, birds,
and prototherian mammals. The higher mammalian eggs appear to occupy
an intermediate position. >
 
The separation of the so-called fertilization membrane has been most intensively studied in certain invertebrate forms. As a matter of interest, some
of the processes involved in membrane elevation in various invertebrate eggs
are herewith described briefly.
 
In the nematode, Ascaris, the egg exudes a jelly-like substance after the
sperm has entered. This substance hardens to form a thin, tough membrane
which later thickens and expands. The egg also appears to shrink, leaving an
enlarged perivitelline space between the egg surface and the outer hardened
membrane (figs. 128, 133C-E).
 
The formation of the fertilization membrane in Echinarachnius, a genus
of sea urchins, was the subject of intensive study by Just (T9). In this species
the egg is larger than that of the sea urchin, Arbacia. According to Just’s
account, the fertilization membrane starts as a “blister” at the point of sperm
contact; from this area it spreads and rapidly becomes lifted off from the
general surface of the egg. Heilbrunn (’13) studied the fertilization membrane
of the sea urchin’s egg before fertilization and describes it as a vitelline membrane, “probably a gel or semi-gel” which is present at the surface of the
egg. It becomes visible as a distinct membrane when lifted off from the egg’s
surface after fertilization. As this elevation occurs, according to Runnstrom,
cortical granules are exuded from the surface of the egg, accompanied by a
 
 
 
 
 
 
 
 
BEHAVIOR OF THE GAMETES
 
 
239
 
 
general contraction of the egg surface. These cortical granules later become
merged with the vitelline membrane to form a relatively thick structure
(fig. 129). (See Runnstrom, ’49.) Fluid collects between the egg surface
and the fertilization membrane.
 
On the other hand, in the annelid worm, Nereis, there is a complicated
reaction at the egg’s surface at the time of fertilization (Lillie, F. R., ’12).
In this egg a definite membrane is present around the newly laid egg. When
a sperm has made an intimate contact with the egg’s surface, the cortical
layer of the egg exudes a substance which passes through the membrane to
the outside; this substance turns into jelly on coming in contact with sea water
(fig. 130B). The jelly layer carries away the excess sperm from the egg’s
surface. A striated area then appears between the vitelline membrane and
the surface of the egg. This area, shown in fig. 130B as the cortical layer,
represents the collapsed walls of small spaces of the superficial layer of the
cortex of the egg which exude their contents through the vitelline membrane
to form the surrounding jelly. The egg then forms a new ooplasmic surface
beneath the collapsed walls of the small spaces of the original cortex (fig.
13(^, ooplasmic membrane).
 
All of these changes and reactions, namely, the formation of the fertilization membrane, the exudation of cortical granules, and the emission of a fluid
or jelly together with the shrinkage of the egg result from changes which
occur in the outer layer of the egg’s protoplasm or cortex, and consequently
may be classified as cortical changesj)The activation of the egg at the time
of fertilization or during artificial stimulation thus appears to be closely integrated with cortical phenomena. It is debatable whether these changes are
the result of activation or are a part of the “cause” of activation.
 
The particular activity of egg behavior at the time of fertilization which
 
 
Fig. 119. Fertilization phenomena in the egg of Rana pipiens. (Drawings B, D-G
made from prepared slides by the courtesy of Dr. C. L. Parmenter.) (A) Semidiagrammatic representation of the egg shortly before ovulation. The germinal vesicle has
broken down, and the chromosomes in diakinesis have migrated toward the apex of the
animal pole preparatory to the first maturation spindle formation shown in (B). (B)
 
First polar spindle. Tetrad condition of chromosomes in process of separation into the
respective dyads. (C) Polar view of egg after first maturation division. Compare with
(D), which represents a section of a comparable condition. (D) Lateral view of spindle
of second maturation division. First polar body present in a slight depression at animal
pole. The egg is spawned in this condition. (E) Second polar body shown in a depression of the animal pole. Within the superficial ooplasm of the egg, the reorganized female
pronucleus is shown. (F) Meeting of the two pronuclei is shown in this section of the
egg at the bottom of the female copulation path or “egg streak,” E.S. (G) Two pronuclei
in contact (shown in F) under higher magnification. (H) Entrance and copulation
paths of sperm nucleus. (Modified from Rugh: The Frog, Philadelphia, The Blakiston
Co., 1951.) (1) Sperm-entrance path, copulation path, and meeting of pronuclei. (From
 
O. Hertwig, 1877.) (J ) First cleavage path, showing daughter nuclei. (From O. Hertwig,
1877.) (K) External, lateral view of the egg just before first cleavage. Arrows show
 
direction of pigment migration with resulting formation of gray crescent.
 
 
 
IN PER 10 OF EGGS
 
 
240
 
 
FERTILIZATION
 
 
appears to be common to the eggs of many species (sea urchin, cyclostomatous
and teleost fishes, frog, and mammal) is the contraction of the egg’s surface,
together with the exudation of various substances from the egg. (See, in this
connection, the fertilization theory of Bataillon at the end of this chapter.)
It is this behavior of the egg’s surface which makes the fertilization membranes
and other egg membranes more apparent; it represents one of the essential
and immediate activities associated with egg activation. Separation of the
various egg membranes at the time of fertilization appears to be secondary
to this primary activity.
 
Aside from the immediate functions at the time of fertilization, the activities
of the various types of vitelline membranes are concerned mainly with nutritional, environmental, and protective conditions of the early embryo. The
presence of a fluid in the perivitelline space between the membrane and the
developing egg affords a favorable environment for early developmental processes. Moreover, it permits the egg to rotate when its position is disturbed,
a proper developmental orientation being maintained. A further accommodation is evident in that it permits the developing egg to exude substances,
including yolk, into the surrounding area, which may be retained in the immediate environment of the egg and later utilized in a nutritional way. If the
surrounding vitelline membrane were not present, this material, solid or fluid,
would be dissipated. For example, in the early cleavage stage of the opossum
or guinea-pig egg, yolk material is discharged into the area surrounding the
early blastomeres (figs. 118, 125). The exuded yolk and dissolved substances
later come to lie in the cavity within the blastomeres and, thereby, may be
used for nutritional purposes. Also, in some forms, such as the opossum, the
early blastomeres utilize the zona pellucida as a framework upon which they
arrange themselves along its inner aspect during the development of the early
 
 
4  
 
 
CUMINGIA 21* WH ITA KER
 
 
CHAETOP. 21* WHITAKER
 
 
SABELLARIA 20* F.-FREMIET
 
 
NEREI S 21* WHI TAKER
 
 
ARBACIA PUNC- 21* WHITAKER^
 
PAR ACENTROTUS 2I*WARBURG“
ECHINUS 21* SHEARER'
UNFERTILIZED EGGS
 
 
^PARACENTROT US 21“RUNNSTRDM
/VSABELLARIA 20* FAURE- FREMIET
 
â–  â–  -'/.ARBACIA PUNC. 2 I* WHIT AKER
 
par AGENT ROTUS 21* WARBURG
 
", ARBACIA PUST. 20.5* WARBURG
 
B a PROTEUS 20“ EMERSON
 
/rZHIZZ'. ECHINUS MILIARIS 21* SHEARER
 
21“ WHITAKER
 
''''CUMINGIA 21* WHITAKER
\fROG skin 20“ ADOLPH
 
CHAETOPTERUS 21“ WHITAKER
 
 
F FERTILIZED EGGS
 
 
Fig. 120. Effects of fertilization on oxygen consumption in various marine eggs. (After
J. Brachet, ’50; data supplied by Whitaker.)
 
 
 
BEHAVIOR OF THE GAMETES
 
 
241
 
 
blastula. This apparent independence of the early cleavage blastomeres in the
opossum and their lack of cohesiveness is evident in other mammals, also.
The tendency of the blastomeres in mammals in general to separate from
each other emphasizes the importance of the zona as a capsule which functions to hold the blastomeres together.
 
The surrounding egg membrane, in many cases, may act osmotically to
permit a nice balance between the developing egg and the substances outside
of the membrane. For example, in birds, the egg and its contained embryo
together with its immediate environment are largely maintained as a physicochemical system due to the osmotic properties of the zona radiata or vitelline
membrane. This membrane separates the watery albumen from the nutritive
yolk material. These two substances have different osmotic conditions. Consequently, the vitelline membrane must maintain the proper conditions between these two general areas, and it performs this function in an admirable
fashion. It should be emphasized further that the vitelline membrane in the
chick’s egg is a living membrane, and consequently its osmotic properties are
different from that of a non-living membrane, such as a collodion membrane.
If the egg and albumen of the hen’s egg are separated by a thin collodion
membrane, for example, they will reach an osmotic equilibrium more rapidly
than when separated by the thin vitelline membrane. If, however, the vitelline
membrane is isolated from its normal relationships in the egg, it behaves
similarly to a collodion membrane. It is best to regard the vitelline membrane,
the yolk, and the albumen of the bird’s egg as forming an harmonious system,
in which all parts are responsible for the maintenance of the necessary conditions for development. (Consult Romanoff and Romanoff, ’49, pp. 388-391.)
 
Undoubtedly in other eggs, such as that of the frog, the delicate relationship existing between the egg, the perivitelline fluid, the vitelline membrane,
and the surrounding external medium forms a complete unit for the proper
maintenance of developmental conditions. In most eggs the vitelline or similar
membranes maintain the protective funetion until a relatively late period in
development.
 
 
b. Fertilization Cone or Attraction Cone
 
VThe fertilization cone results from specialized activity of the surface of the
egg (egg cortex) at the point of sperm contact (fig. 130)')This structure has
been described in various invertebrate eggs, such as those of th€(sea urchins,
annelid worms, mollusks, and in some of the ascidians among the protochordatay In the annelid worm. Nereis virens, as the sperm makes its way
through the egg membrane, a cone of cortical ooplasm flows out to meet the
sperm, making an intimate contact with the perforatorium (acrosome) of the
sperm (fig. 130B, C). When this contact is made, the extended cone withdraws again gradually, and appears to pull the sperm head into the egg’s
substance (fig. 130D-G).An the egg of the sea urchin, Toxopneustes varie
 
 
A.
 
 
PRIMARY —
PHASE OF
FERT ILIZATION,
IE, ACTIVATION
PROCESSES
AROUSED IN
EGG AND
SPERM WHICH
BRING ABOUT
THEIR CONTACT
 
 
B.
 
SECONDARY
PHASE OF —
FERTILIZATION
I.E., ACTIVATION
PROCESSES
AROUSED
IN EGG AND
SPERM WHICH
RESULT IN
FUSION OF THE
GAMETES
 
 
OVULATION AND
MIGRATION OF EGG
TO AREA WHERE
FERTILIZATION
IS TO OCCUR
 
 
SPERM DISCHARGED
FROM TESTIS AND
MIGRATE TO AREA
WHERE FERTfLIZATIO
IS TO OCCUR
 
 
/
 
 
GYNOGAMIC
SUBSTANCES
SECRETED INTO
SURROUNDING
FLUIDS FROM
 
THE EGG.
MATURATION
DIVISIONS
INITIATED IN
CERTAIN EGGS
 
 
 
 
MOTILITY
 
STIMULATED
 
 
 
 
ANDROGAMIC
 
SUBSTANCES
 
SECRETED
 
 
 
SPERM STIMULATED
TO GREATER
ACTIVITY AND
ATTRACTED TO
EGG BY GYNOGAMIC
SUBSTANCES
 
 
 
SPERM SECRETIONS
AID SPERM IN
REACHING EGG
 
 
MATURATION DIVISIONS OF EGG
COMPLETED AND SPERM DRAWN
INTO EGG; SPERM NUCLEUS ENLARGES AND ASTER FORMS IN
MIDDLE PIECE OF SPERM
FERTILIZATION MEMBRANE DEVELOPS
AND PERIVITELLINE SPACE AND
FLUID APPEARS BETWEEN EGG
AND MEMBRANE IN SOME SPECIES
(AMPHIOXUS); IN OTHER SPECIES
PERIVITELLINE SPACE AND FLUID
 
 
 
 
COMBINATION OF ACTION
OF GYNOGAMIC SUBSTANCE
AND ANDROGAMIC
SUBSTANCE POSSIBLY
BINDS SPERM TO EGG
SURFACE AND IMMOBILIZES
SPERM
 
 
GAMETES NOW READY TO
BEGIN SECOND OR FUSION
STATE OF FERTILIZATION
 
 
FORMS BETWEEN EGG AND
PREVIOUSLY FORMED MEMBRANE
(FISH, FROG, MAMMALS); EGG MAY
CONTRACT SLIGHTLY WHEN
PERIVITELLINE FLUID FORMS;
OOPLASMIC SUBSTANCES
MIGRATE TOWARD POINT WHERE
SPERM HAS ENTERED (FROG,
AMPHIOXUS, AND STYELA)
 
FUSION OF PRONUCLEI
AND ESTABLISHMENT OF
NEW DIPLOID CHROMOSOMAL
COMPLEX; CLEAVAGE
AMPHIASTER FORMS
 
 
 
OOPLASMIC MOVEMENTS
OCCUR, RESULTING IN
REORIENTATION AND
SEGREGATION OF DEFINITE
OOPLASMIC SUBSTANCES
 
 
CLEAVAGE INITIATED
 
 
Fig. 121. {See facing page for legend.)
242
 
 
 
BEHAVIOR OF THE GAMETES
 
 
243
 
 
gatus, a protoplasmic prominence appears only after a sperm begins to pass
into the egg. It persists until about the time that the pronuclei unite?( Wilson
and Mathews, 1895). (See fig. 131B-F.) A prominent fertilization cone is
found also in the starfish, Asterias forbesi (Wilson and Mathews, 1895).
In the vertebrate group, fertilization cones are not generally observed, but
the protoplasmic bridge from the egg membrane to the ooplasmic surface in
Petromyzon evidently fulfills the functions of a cone (fig. 134C).
 
(^he formation of the fertilization cone and its withdrawal again, suggests
that ooplasmic movements are concerned mainly with the sperm’s entry into
the interior of the egg. These movements appear to be aroused by some stimulus
emanating from the sperm as it contacts the egg’s surface. That is to say, although the sperm becomes immobile once it has touched the egg’s surface,
various stimuli, chemical and/or physical, issue from the sperm into the egg
substance. Here these stimuli inaugurate movements in the ooplasm which draw
the sperm into the egg. This modern view thus emphasizes motility of the
cortical area of the egg as the factor which conveys the sperm into the interior
of the egg. It suggests further that the older view of sperm entry which was
presumed to result from sperm motility alone does not agree with the actual
facts demonstrated by observation.
 
c. Some Changes in the Physiological Activities of the Egg at Fertilization
 
The separation of the egg membrane from the egg surface, the emission
of fluid substances from the egg’s surface into the perivitelline space, and
contraction of the egg’s surface have been noted above. Associated with these
immediate results of sperm contact with the egg, a pronounced movement
of cytoplasmic substances within the egg can be demonstrated in many species.
Examples of cytoplasmic movements within the ooplasm of the egg are given
below in the descriptions of the fertilization processes which occur in various
chordate species.
 
Accompanying the above-mentioned activities, pronounced changes of a
metabolic nature occur. In the egg of the frog and toad, for example, there
is little change in the oxygen consumption during fertilization, although there
 
 
Fig. 121. Two stages of fertilization in animals. (A) In the primary phase of fertilization (“external fertilization” of F. R. Lillie), the sperm is activated to greater
motility by the environmental factors encountered at the fertilization site, including the
gynogamic substances secreted by the egg. It is also drawn to the egg by a positive
chemotaxis. The lytic substances (androgamic substances) enable the stimulated sperm
to make its way more easily through the jelly membranes and ooplasmic membranes
surrounding the egg to the egg’s surface. At the egg’s surface the interaction of gynogamic
and androgamic substances brings about the agglutination of the sperm to the egg’s
surface. This initiates stage B, on the secondary phase of the fertilization process (“internal fertilization” of F. R. Lillie). (B) Secondary phase of fertilization or fusion of
the gametes. (See text for further description.) This stage begins when the sperm has
made contact with the egg and terminates when the first cleavage spindle has formed.
 
 
 
244
 
 
FERTILIZATION
 
 
 
Fig. 122. Changes during fertilization in the egg of Fundulus heteroclitus. (A) Egg
before fertilization. (B, C) Changes in the egg shortly after sperm entrance into the
egg. In (B) is shown the contraction of the egg from the vitelline membrane, the disappearance of the yolk plates, and the formation of the peri vitelline space. In (C) is shown
the migration of the peripheral cytoplasm toward the point where the sperm has entered
the egg, forming a cytoplasmic or polar cap.
 
is a pronounced drop in the respiratory quotient, presumably indicating a
change in the character of oxygen consumptioi^ (Brachet, J., ’50, p. 106).
Fertilization does not change the rate of oxygen consumption in the teleost
fish, Fundulus heteroclitus, but, in the lamprey, oxygen consumption is increased (Brachet, J., ’50, p. 108).^lso, in the egg of the sea urchin, following artificial activation or normal fertilization, there is a considerable
increase in oxygen cons umptioji3( fig. 120). In the unfertilized and fertilized
egg of the starfish (Asterias) apparently there is no change in the rate of oxygen
metabolism. (In the eggs of certain sea urchins it has been shown by Runnstrom
and co-worlOTs (Runnstrom, ’49, p. 306) that acid formation occurs following fertilization. It is of brief duration} (Constllt also Brachet, J., ’50, p. 120,
for references.) Other changes have been described, such as an increase in
viscosity of the egg (Heilbrunn, ’15), and an increase in permeability of the
egg membran#( Heilbrunn, ’15). Fertilization may produce a higher dispersity
of the egg colloidal material, at least in some specie^/ Changes of a metabolic
nature, therefore, are a part of the fertilization picture. (The reader should
consult Brachet, J., ’50, Chap. 4, for a thorough discussion of physiological
changes at fertilization.)
 
 
 
BEHAVIOR OF THE GAMETES
 
 
245
 
 
d. Completion of Maturation Divisions, Ooplasmic Movements, and
Copulatory Paths of the Male and Female Pronuclei in Eggs
of Various Chordate Species
 
A description of the maturation processes, ooplasmic movements, and the
behavior of the male and female pronuclei in the fertilization processes of
various chordate species is given below. It should be observed that all of
these events occur rather synchronously in the urochordate, Styela, and in
the egg of the frog, while in others, such as the prototherian mammal, Echidna,
they may come to pass in sequence.
 
1) Fertilization in Styela (Cynthia) partita: a) Characteristics of the
Egg Before Fertilization. The living, fully formed, primary oocyte of the
urochordate, Styela (Cynthia) partita, is about 150 /x in diameter. It possesses
at this time three areas which can be distinguished with clearness, namely, a
peripheral transparent layer which contains a sparsely distributed yellow pigment, a central mass of gray-appearing yolk, and the area of the germinal
vesicle, located near the future animal pole of the egg (fig. 132A).
 
^^he first steps leading to the maturation divisions of the chromatin material take place before sperm entrance, at the time the egg is spawned or
shortly before. At this time the wall of the germinal vesicle (i.e., the nuclear
membrane) breaks down, and the contained clear cytoplasm moves up to the
animal pole of the egg where it spreads out to form a disc. The chromosomes
then line up on the metaphase plate of the first maturation spindle; they remain thus in the metaphase of the first maturation until the sperm enters
(fig. 116A, B).
 
b) Entrance of the Sperm. The sperm enters the egg (i.e., the primary
oocyte) at the future vegetal (vegetative) pole,)either exactly at the pole or
a little to one side (fig. 116B). Sperm entrance at this pole probably is due
to a fundamental structural and physiological condition which in turn reflects
a definite polarity of the egg. (Qnly one sperm normally enters the egg, but
several sperm may penetrate through the chorion into the perivitelline space,
 
c) Cytoplasmic Segregation. A striking series of changes appear within
the cytoplasm of the egg immediately following sperm entrance. The yellowpigmented, peripheral layer of protoplasm flows toward the point of sperm
entrance (i.e., the vegetal pole) and collects into a “deep, orange-yellow
spot” which surrounds the sperm (fig. 132B, C, peripheral protoplasm). It
later spreads again and then covers most of the lower or vegetal pole of the
egg. Accompanying the flow of yellow peripheral protoplasm toward the vegetal
egg pole, most of the clear protoplasm of the germinal vesicle (i.e., the nuclear
plasm mentioned above) flows with the yellow protoplasm tovi#rd the vegetal
pole. The clear protoplasm, to some extent, tends to mingle with the yellowpigmented, peripheral protoplasm. In figure 132C, the clear protoplasm may
be observed as a clear area above the yellow-pigmented protoplasm.
 
The sperm pronucleus next moves upward away from the vegetal pole and
 
 
 
246
 
 
FERTILIZATION
 
 
toward one side of the egg to a point which marks the posterior pole of the
egg and future embryo (fig. 116M). The clear protoplasm and the yellowpigmented protoplasm move upward with the sperm (fig. 132D). The yellowpigmented protoplasm at this time forms a yellow crescent just below the
egg’s equator, and the middle point of this crescent marks the posterior end
of the future embryo (fig. 132D, E). A distinct crescent of clear protoplasm
appears just above the yellow crescent at this time (fig. 132D-F). The crescent
substance is therefore plainly differentiated at once into clear and yellow protoplasm, which remain distinct throughout the entire development (Conklin,
’05, p. 21).
 
The yolk material, which at first is centrally located in the egg, moves
toward the animal pole when the clear and yellow-pigmented protoplasms
migrate to the vegetal pole. As the yellow and clear protoplasmic crescents
are formed, the yolk material moves to occupy its ultimate position at the
vegetal pole of the egg (fig. 132D). Later when the first cleavage division
occurs, another crescentic area, the gray crescent, appears on the side of the
egg opposite the yellow crescent.
 
As a result of the segregation of ooplasmic materials, four definite areas
are localized:
 
( 1 ) a vegetal, yolk-laden area,
 
(2) a gray crescent,
 
(3) the yellow and clear protoplasmic crescents opposite the latter, and
finally
 
(4) the more or less homogeneous cytoplasm at the animal pole of the egg.
 
The movements of cytoplasmic materials in the cephalochordate, Amphioxiis, are similar to those in Styela (Conklin, ’32).
 
d) CopuLATORY Paths and Fusion of the Gametic Pronuclei. The
entrance of the sperm into the egg substance, its migratory movements in the
ooplasm, its meeting with the egg pronucleus, and final fusion or association
of the pronuclei afford an interesting problem. The factors governing the
movements of the female and male pronuclei are unknown, although the movements in many eggs are spectacular. The movements of the pronuclei in
Styela partita offer an excellent illustration of the copulatory migrations of
the pronuclei within the cytoplasm of the egg.
 
The sperm enters the egg of Styela partita, as stated previously, at the
vegetal pole near the midpolar area or a little to one side (fig. 116B). The
sperm moves inward through the yellow-pigmented protoplasm and eventually becomes surrounded with the yellow and clear protoplasms (figs. 132C;
1 1 6B-F ) . This initial pathway through the superficial protoplasm of the egg
constitutes the penetration path of the sperm (Wilhelm Roux). The sperm
head in the meantime begins to swell and becomes vesicular (figs. 116F, J;
133B-G, Ascaris). The nucleus and the middle piece of the sperm with its
 
 
 
BEHAVIOR OF THE GAMETES
 
 
247
 
 
forming aster now rotate 180 degrees, so that the aster lies anterior to the
nucleus as it migrates within the egg (fig. 116F, I, J). The sperm aster thus
precedes the pronucleus as the latter moves through the cytoplasm (fig. 1 16M) .
 
With the movement of the clear and pigmented protoplasmic substances
upward toward the equator and to the point marking the future posterior
end of the embryo, the sperm pronucleus and aster move upward. This latter
movement of the sperm constitutes the copulation path, and it is formed at
a sharp angle to the penetration path (figs. 116M, 139B)^he egg chromatin
in the meantime undergoes its first and second maturation divisions (fig.
116F-L). After the second polar body has been formed, the haploid number
of chromosomes reform the egg nucleus, now called the female pronucleus
(fig. 116L, M). The latter then moves downward through the yolk along its
copulation path to meet the sperm pronucleus near the posterior pole of the
egg (figs. 116M-P; 139B). The actual meeting place in the clear cytoplasm is
about halfway between the posterior pole and the center of the egg (fig. 139B) .
 
Shortly before the pronuclei meet, the sperm aster divides, each aster
moving to opposite poles of the sperm pronucleus (fig. 116N). The two
pronuclei now meet between the amphiaster of the first cleavage (fig. 1160, P)
and thus become enclosed by the amphiaster spindle (fig. 116P). Following
this association, the entire complex migrates toward the center of the egg
together with a mass of clear cytoplasm. Some of the yellow protoplasm also
migrates slightly centerward. The latter movement of the pronuclei toward
the center of the egg is called the cleavage path. In the new position, slightly
posterior to the egg’s center, the pronuclei form an intimate association (figs.
116P, 139). The chromosomes then make their appearance, the nuclear membranes disappear, and the chromosomes line up in the metaphase plate of the
first cleavage spindle preparatory to the first cleavage (fig. 116Q). The first
cleavage plane always bisects the midplane of the future embryo and hence
bisects the yellow and clear protoplasmic crescents (figs. 116R, S; 132F, G).
 
2) Fertilization of Amphioxus. The fertilization stages of Amphioxus are
shown in figures 117A-I; 139C. The general process of fertilization in this
species appears much the same as in Styela. However, in Amphioxus the fertilization phenomena cannot be studied as readily for a pigmented material
is not formed in the peripheral cytoplasm. According to Conklin (’32), the
general movements of the cytoplasmic substances resemble those of Styela. It
is to be observed, however, that the copulation paths of the sperm and egg
pronuclei, and also the cleavage path of the two pronuclei, are different slightly
in Amphioxus from those present in Styela (fig. 139B, C).
 
3) Fertilization of the Frog’s Egg. The egg of Rana pipiens is spherical
and approximately 1.75 mm. in diameter as it lies in the uterine portion of
the oviduct just before spawning. The size, however, may vary considerably.
It has a darkly pigmented animal pole and a lightly colored vegetal pole. The
first maturation division occurs when the egg is ovulated or shortly after
 
 
 
248
 
 
FERTILIZATION
 
 
ovulation during its passage through the peritoneal cavity en route to the
oviduct (fig. 119B, C). The secondary oocyte then enters the oviduct, and
during its passage posteriad in the latter, the maturation spindle of the second
maturation division is formed (fig. 119D). The egg is in this condition when
it is spawned. Immediately upon its entrance into the water, it is fertilized by
the sperm from the amplectant male.
 
The sperm enters the egg at a point about 20 to 30 degrees down from the  
midregion of the animal pole. As it penetrates through the cortex of the egg,
a trail of dark pigment from the egg’s periphery flows in after the sperm
(fig. 119H, I). This initial entrance path of the sperm constitutes the penetration path. After making its initial entrance, the sperm begins to travel
toward its meeting place with the female pronucleus. This secondary path is
the copulation path of the sperm (fig. 1 1 91). If the sperm should continue
more or less in a straight line toward the egg pronucleus, the penetration path
and copulation path would be continuous. However, if the sperm should veer
away at an angle from the original penetration path in its journey to meet
the female pronucleus, the copulation path would be at an angle to the penetration path.
 
The second maturation division of the oocyte occurs in about 20 to 30
minutes after sperm entrance with a surrounding temperature approximating
22° C. After the female pronucleus is organized, it migrates along its copulation path toward the meeting place with the sperm pronucleus, located near
the center of the animal pole cytoplasm of the egg (fig. 1 19F, G).
 
Shortly after the sperm penetrates the egg, it revolves 180 degrees, and
the middle-piece area travels foremost. This revolving movement, whereby the
middle-piece area assumes a foremost position, is similar to that which occurs
in the protochordates. Stye la and Amphioxus. This revolving movement appears to be characteristic of all sperm after entering the egg. (See figs. 116,
117, 131.) The sperm pronucleus gradually enlarges as it continues along the
copulation path, and the first cleavage amphiaster arises in relation to the
middle-piece region.
 
Fusion of the two pronuclei occurs at about one and one-half to two hours
after fertilization at a normal room temperature of about 22° C. (fig. 119G).
At about two and three-quarter hours after fertilization the first cleavage
furrow begins (figs. 119J; 142A).
 
As stated above, the peripheral egg cytoplasm with its pigment tends to
flow into the interior of the egg, following the trail of the sperm and thus
forms a pigmented trail. The migration of the superficial cytoplasm with its
pigmented granules is general over the upper pole of the egg and its direction
of flow is toward the point of sperm penetration (see arrows, fig. 119K).
Consequently, jaX a point on the egg’s surface opposite the point of sperm
entrance, the peripheral area of the egg becomes lighter in color and assumes
 
 
 
BEHAVIOR OF THE GAMETES
 
 
249
 
 
a gray appearance. This area is crescentic in shape and is known as the gray
crescent (fig. IlOK).
 
The formation of the gray crescent occurs in the cytoplasmic area just
above the margin where the yellow-white vegetal pole material merges with
the darkly pigmented animal pole. The gray crescent is continuous with the
lighter vegetal pole material and is seen most clearly during the first cleavage
of the egg. The plane which bisects the gray crescent into two equal halves
represents the future median plane of the embryo.
 
In the frog, Rana jusca, Ancel and Vintemberger (’33) have shown that
extensive movements of egg-surface materials accompanies the formation of
the gray crescent. Sperm contact with the egg’s surface thus appears to set
in motion ooplasmic substances which fix the final symmetry of the egg and
the future embryo.
 
4) Fertilization of the Teleost Fish Egg. When the egg of the teleost fish
is spawned, the yolk lies near the center of the egg, and its yolk-free cytoplasm forms a peripheral layer. Around the egg the yolk-free cytoplasm is
somewhat more abundant in the region where the egg nuclear material is situated. This concentration of the peripheral cytoplasm at the nuclear pole is
more evident in the eggs of some species than in others. The area of nuclear
residence is situated near the micropyle in many teleost eggs, but not in all.
For example, the concentration of cytoplasm with the contained nuclear material is located in Bathygobius soporator at the opposite end to the micropyle
(Tavolga, ’50). (See fig. 123A.)
 
The sperm enters the egg through the micropyle (figs. 122, 123, 134A),
and the actual processes of fertilization are initiated when the sperm makes
contact with the peripheral ooplasm near the point where the egg nuclear
material is located. This normally occurs in about a minute or less after the
egg reaches the water. Within a few minutes the second polar body is given
off. Meanwhile, the peripheral cytoplasm flows toward the area where the
sperm has made contact, and a protoplasmic cap forms at this pole (figs.
122C; 123B-D). The remainder of the egg, with the exception of a thin
layer of surface protoplasm, contains the deutoplasmic or yolk material. The
egg is converted in this manner from a more or less centrolecithal egg into a
strongly telolecithal egg. (Compare with Styela and Amphioxus.)
 
While these events are progressing, the egg as a whole contracts slightly,
and a fluid is given off into the forming perivitelline space between the egg’s
surface and the vitelline membrane (fig. 122B, C). (However, a space between the egg membrane and the egg is evident to some extent in certain
teleost eggs before the sperm enters the egg (fig. 123A).) The egg is now
free to rotate within the perivitelline space, being cushioned and bathed by
the perivitelline fluid.
 
The expansion of the vitelline membrane of the egg in certain teleosts is
both dramatic and prophetic of the future shape of the embryo (fig. 123B-H).
 
 
 
 
 
Fig. 123. Development of the gobiid fish, Bathygobius soporator. (After Tavolga, ’50,
slightly modified.) (A) Freshly stripped egg. Adherent filaments at proximal end of
chorion; micropyle at distal end. Peripheral cytoplasm partly concentrated at the pole of
the egg containing the female nucleus. (B) Fifteen minutes after fertilization; cytoplasm concentrating at nuclear pole of the egg; chorion expanding into shape of future
embryo. (C) Twenty minutes after fertilization; second polar body given off. (D)
Twenty-five minutes after fertilization. (E) Ninety minutes after fertilization. (F)
Seventeen hours after fertilization. (G) Twenty-four hours after fertilization. (H)
Thirty hours after fertilization. (I) Thirty-six hours after fertilization. (J) Ninetysix hours after fertilization. (K) Ninety-six hours after fertilization. Hatching. (L)
Three days after hatching, temperature 27 to 29°,
 
 
250
 
 
 
BEHAVIOR OF THE GAMETES
 
 
251
 
 
In demersal eggs, that is, eggs which sink to the bottom, the protoplasmic cap
tends to assume an uppermost position. In pelagic eggs, i.e., eggs which float
in the water, the protoplasmic disc turns downward since it is the heaviest
part of the egg.
 
After the polar bodies are given off, the egg-chromatin material reforms
the female pronucleus. The latter and the sperm pronucleus migrate to a
position near the center of the protoplasmic disc. The first cleavage plane is
established within thirty minutes to an hour following sperm entrance.
 
5) Fertilization in the Egg of the Hen and the Pigeon. Fertilization in the
hen’s egg occurs without any demonstrable movement of cytoplasmic materials, as manifested in the eggs of Styela, Amphioxus, frog, and teleost fish.
The egg is strongly telolecithal, and the true protoplasm or blastodisc, which
takes part in active development, is a flattened mass about 3 mm. in width.
The germinal vesicle in the mature egg is approximately 350 /x in diameter
and about 90 /x in thickness (fig. 126A). Approximately 24 hours before
ovulation occurs, the wall of the germinal vesicle begins to break down, and
the contained nuclear sap spreads in the form of a thin sheet below the
ooplasmic membrane overlying the blastodisc (fig. 126B). (See Olsen, ’42.)
 
Changes in the chromatin material of the germinal vesicle are synchronized
with the breakdown of the membranous wall of the vesicle. The chromatin
material, extremely diffuse during the period when the yolk material was
formed and the egg as a whole was growing rapidly, contracts and assumes
the character of thickened chromosomes in the tetrad condition. The diffuse
 
 
 
Fig. 124. Fertilization stages in the rabbit egg. (A, B after Pincus, ’39.) (A) Second
 
polar body exuded; male and female pronuclei. (B) Twenty-two hours after copulation,
showing two pronuclei close together. (C) Coagulated plug in infundibular portion of
Fallopian tube, containing eggs. This plug is dissolved by sperm during fertilization
process.
 
 
 
 
Fig. 125. Fertilization in the opossum. (A after McCrady, ’38, from Duesberg; B-F
after Hartman, ’16.) (A) Conjugate sperm of opossum. (B) Ovarian egg showing
 
discus proligerus around the egg; first polar body extruded; chromosomes of egg nucleus
evident. (C) Tubal ovum. (D) Uterine ovum with pronuclei near center of the egg.
(E) First cleavage spindle of uterine egg. (F) Two-cell stage, showing zona pellucida
and exuded yolk material lying in perivitelline space.
 
FOLLICLE CELLS
 
 
VITELLINE 1 IP. B
 
 
 
Fig. 126. Maturation and fertilization in the hen’s egg. (Drawings from photomicrographs by Olsen, ’42.) (A) Cross section of germinal vesicle of almost mature egg,
 
showing the general position and condition of the intact germinal vesicle. (B) Egg just
prior to ovulation. Germinal vesicle spreading laterally as a thin layer below the ooplasmic
membrane. (C) Chromatin material near center of disintegrating germinal vesicle
(G.V.) of an egg estimated to be one hour prior to ovulation. (D) First polar body
(1 P.B.) of recently ovulated egg. (E) Cross section of blastodisc of recently ovulated
egg showing male pronucleus (^), female pronucleus (9), and second polar body
(2 P.B.).
 
 
252
 
 
BEHAVIOR OF THE GAMETES
 
 
253
 
 
diplotene state thus passes into the diakinesis stage (figs. 126C; 135A, show
the breakdown of the nuclear wall and appearance of chromosomes in the
pigeon).
 
The first maturation division occurs and the first polar body is extruded
shortly before ovulation (fig. 126D). The second maturation spindle is then
formed. In this state the egg is ovulated. From four to six sperm penetrate
into the egg shortly after it enters the infundibulum of the oviduct. The latter
events are consummated within fifteen minutes after ovulation. The second
maturation division then occurs, followed by the discharge of the second polocyte, which becomes manifest about the time of, or shortly before, the fusion
of a single male pronucleus with the female pronucleus (fig. 126E). Thus,
although polyspermy is the rule, only one sperm pronucleus takes part in the
syngamic process.
 
After the two pronuclei become closely associated, the chromosomes become evident, the nuclear membranes disintegrate, and the first cleavage spindle
is formed in about five and one-quarter hours after the sperm enters the egg
(Olsen, ’42).
 
In the egg of the pigeon, according to Harper (’04), the germinal vesicle
breaks down, and the first polar spindle forms in the egg just before ovulation
(fig. 135A, B). Fertilization then occurs just as the egg (in reality the primary
oocyte) enters the oviduct. Normally from 15 to 20 sperm enter the blastodisc
of the pigeon’s egg. However, only one sperm pronucleus associates with the
female pronucleus. Consequently, unlike the condition in the hen’s egg, both
maturation divisions occur and the first and second polar bodies are given
off after sperm entrance (fig. 135C, D). Following the maturation divisions,
the two pronuclei proceed to associate (fig. 135E, F). The first cleavage
nucleus is shown in fig. 135G with two accessory sperm nuclei shown to the
extreme left of the figure.
 
6) Fertilization in the Rabbit. In the rabbit, ovulation occurs around 10
to 1 1 hours after copulation. It takes about four hours for the sperm to travel
to the upper parts of the Fallopian tube. (See Chap. 4.) The sperm thus lie
waiting for about six to seven hours before the eggs are ovulated. When the
eggs are discharged from the ovary, each egg is surrounded by its cumulus
cells. The latter form the corona radiata, surrounding the zona pellucida
(fig. 124A). As the eggs are discharged from their follicles, an albuminous
substance from the follicles forms a clot, and several eggs are included within
this clot (fig. 124C). A sperm, therefore, must make its way through the substance of the clot, as well as between the cells of the corona radiata, and then
through the zona pellucida to reach the egg. This feat is accomplished partly
by its own swimming efforts and partly also by means of an enzyme (or
enzymes) which dissolves a pathway for the sperm. (See hyaluronidase, etc.,
mentioned on pp. 229.) The ferment hyaluronidase, associated with the sperm,
frees the eggs from the albuminous clot and aids in the dissolution of the
 
 
 
 
 
 
 
 
Fig. 127. Maturation and fertilization in Echidna. (Courtesy, Flynn and Hill, ’39.)
(A) Oocyte, diameter 3.9 by 3.6 mm. Section of upper pole of egg showing saucer-shaped
germinal vesicle lying in the germinal disc. (B) First polar spindle of egg just previous
to ovulation. (C) First polar body and chromatin of female nucleus just previous to
formation of second polar spindle shown in (D). (D) Second polar spindle of newly
 
ovulated egg. Sperm presumably enters germinal disc at this time but possibly may wait
until condition shown in (E) in some instances. (E) Second polar body and female
pronucleus. (F) Male and female pronuclei. (G, H) Fusion stages of pronuclei.
 
 
 
BEHAVIOR OF THE GAMETES
 
 
255
 
 
corona radiata cells, so that each egg lies free in the Fallopian tube, surrounded by the zona pellucida. It may be that some other lytic substance associated with the sperm also is active in aiding the sperm to reach the egg’s
surface.
 
The first maturation division of the egg occurs as the egg is being ovulated.
The egg remains in this condition until the sperm enters, which normally
occurs within two hours after ovulation. Thus, sperm entrance into the rabbit’s
egg presumably is much slower than in the case of the hen’s egg, possibly due
to the albuminous and cellular barriers mentioned above. Several sperm may
penetrate through the zona pellucida into the perivitelline space, but only one
succeeds in becoming attached to the egg’s surface (Pincus, ’39). The sperm
tail is left behind in the perivitelline fluid, and the sperm head and middle
piece “appear to be drawn into the egg cytoplasm rather rapidly” (Pincus
and Enzmann, ’32). The second polar body is then extruded, a process which
ordinarily is completed about the thirteenth hour following copulation (fig.
124A). About three or four hours later (that is, about 17 hours after copulation) the two pronuclei are formed and begin to approach one another,
and at 20 to 23 hours after copulation the pronuclei have expanded to full
size and come to lie side by side (fig. 124B). The migration of the pronuclei
to the center of the egg thus consumes about four to six hours. The spindle
for the first cleavage division generally is found from 21 to 24 hours after
copulation (Pincus, ’39). (Consult also Gregory, ’30; Lewis and Gregory, ’29.)
 
7) Fertilization in the Echidna^ a Prototherian Mammal. The egg of the
Tasmanian anteater, Echidna, when it reaches the pouch is about 15 by 13
mm. in diameter. This measurement, of course, is only approximate, and it
includes the egg proper plus its external envelopes of albumen and the leathery
shell. (The egg of Ornithorhynchus is slightly larger, approximating 17 by 14
mm.) At the time of fertilization in the upper portion of the Fallopian tube,
the fresh ovum of Echidna without its external envelopes measures about four
to 4.5 mm. in diameter.
 
The fully developed eggs of the monotreme (prototherian) mammals are
strongly telolecithal, with a small disc of true protoplasm situated at one pole
as in the bird or reptile egg. In Echidna aculeata this disc measures about
0.7 mm. in diameter during the maturation stages. Just before the onset of
the maturation divisions of the nucleus, the germinal vesicle is saucer-shaped
and lies in the midportion of the upper part of the disc (fig. 127A). The first
maturation division (fig. 127B, C) occurs before ovulation, while the second
maturation division (fig. 127D, E) occurs after ovulation. There is some evidence that the second maturation division, in some cases, may precede the
actual entrance of the sperm into the germinal disc (Flynn and Hill, ’39). In
figure 127F-H, the stages in the association of the male and female pronuclei
are shown.
 
As fertilization is accomplished, a rearrangement and movement of the
 
 
 
256
 
 
FERTILIZATION
 
 
ooplasmic substances of the germinal disc occur. As a result, the blastodisc,
circular during the maturation period, becomes transformed into an ovalshaped affair with the polar bodies situated on one end (fig. 136). The first
plane of cleavage is indicated by numerals I-I, and the second plane of cleavage
by numerals II-II. A distinct bilateral symmetry thus is established by the
rearrangement of ooplasmic materials during the fertilization process. (Compare with Styela, Amphioxus, and frog.)
 
£. Significance of the Maturation Divisions of the Oocyte in Relation
to Sperm Entrance and Egg Activation
 
As indicated in the foregoing pages, the maturation divisions of the oocyte
vary greatly in different animal species. Figure 137 shows that the time of
sperm entrance in the majority of eggs occurs either before or during the
maturation divisions, that is, when the female gamete is in the primary or
secondary oocyte condition. In some animals, however, the sperm enters
normally after the two maturation divisions are completed.
 
The correlation between the maturation period of the egg and sperm entrance
indicates that the breakdown of the germinal vesicle and the accompanying
maturation divisions has a profound effect upon the egg. This conclusion is
substantiated by experimental data. For example, A. Brachet (’22) and
Runnstrbm and Monne (’45, a and b), working on the sea-urchin egg, found
 
 
 
Fig. 128. Formation of the vitelline membrane in the egg of A scans after fertilization.
(After Collier, ’36.) (A) Heavy cell wall (vitelline membrane) is beginning to thicken.
 
(B) Cell wall is reaching condition of maximum thickness. (C) Egg contracts away
from vitelline membrane, leaving perivitelline space filled with fluid-like substance, forming the typical fertilized egg of Ascaris as ordinarily observed.
 
 
 
MICROPYLES
 
 
257
 
 
that several sperm enter the egg in the sea urchin if insemination is permitted
before the maturation divisions occur. The immature egg, therefore, lacks the
mechanism for the control of sperm entrance. Moreover, A. Brachet (’22)
and Bataillon (’29) demonstrated that the sperm nuclei and asters behave
abnormally under these conditions, and normal development is impossible.
Runnstrom and Monne have further shown for the sea-urchin egg that the
normal fertilization process, permitting the entrance of but one sperm, requires
a mechanism which is built up gradually by degrees during the time when
the maturation divisions of the egg occur, even extending to a necessary short
period after the divisions are completed. Not only is the mechanism which
permits but one sperm to enter the egg established at this time in the seaurchin egg, but Runnstrom and Monne further conclude, p. 25, “that the
cytoplasmic maturation” which occurs at the period of the maturation divisions, “involves the accumulation at the egg surface of substances which
participate in the activating reactions.” It appears, therefore, that the breakdown of the germinal vesicle together with the phenomena associated with the
maturation divisions is an all-important period of oocyte development, controlling sperm entrance on the one hand and, on the other, presumably being
concerned with formation of substances which permit egg activation.
 
F. Micropyles and Other Physiologically Determined Areas for
Sperm Entrance
 
A micropyle is a specialized structural opening in the membrane or membranes surrounding many eggs which permits the sperm to enter the egg.
For example, in the eggs of teleostean fishes or in the eggs of cyclostomatous
fishes, a small opening through the vitelline membrane (or chorion) at one
pole of the egg permits the sperm to enter (figs. 93 A; 134A-F). On the
other hand, many chordate eggs do not possess a specialized micropyle through
the egg membrane. The latter condition is found in the protochordates, Styela
and Arnphioxus, and in vertebrates in general other than the fish group. In
Styela and Arnphioxus the sperm enters the vegetal pole of the egg, i.e., the
pole opposite the animal or nuclear pole. In most of the vertebrate species
the sperm enters the animal or nuclear pole of the egg usually to one side of
the area where the maturation divisions occur (figs. 115, 118, 1191). In urodele
amphibians, the passage of several sperm into the egg at the time of fertilization complicates the picture. However, the sperm which finally conjugates
with the egg pronucleus is the one nearest the area where the egg pronucleus
is located. The several sperm entering other parts of the egg ultimately degenerate (fig. 138). Presumably this condition is present in reptiles and birds
where many sperm normally enter the egg at fertilization.
 
In conclusion, therefore, it may be stated that the point of sperm entrance
in chordate eggs in general appears to be definitely related to one area of
the egg, either by the presence of a morphologically developed micropyle or
 
 
 
 
(TELLINE
MEMBRANE
CORTICAL
GRANULES
"^P L A S M A
2 SURFACE
 
 
 
FERTIL IZATION
 
membrane
 
 
/PL A SMA
SURFACE
 
3
 
 
Fig. 129. Formation of the fertilization membrane in the mature egg of the sea urchin.
(A) Surface of the egg and surrounding jelly coat before fertilization. (After Runnstrom,
’49, p. 245.) (Bl, 2, 3, 4, 5) Point x marks the point of sperm contact. The fertilization
membrane separates from the egg at the point of sperm contact and spreads rapidly
around the egg from this point. (After Just, ’39, p. 106.) (C) Membrane formation in
 
greater detail. (After Runnstrom, ’49, p. 276.) (I) As the vitelline membrane is lifted
 
off from the plasma surface of the egg, cortical granular material is exuded from the
egg cortex and passes out across the perivitelline space toward the fertilization membrane. (2) Cortical granules begin to consolidate with the vitelline membrane. (3)
Fully developed fertilization membrane is formed by a union of the vitelline membrane
with the cortical granules derived from the egg cortex.
 
 
258
 
 
 
IMPORTANCE OF THE SPERM ASTER
 
 
259
 
 
by some physiological condition inherent in the organization of the egg. In
the majority of chordate eggs, the place of sperm penetration is at that pole
of the egg which contains the egg nuclear material, although in some, such
as in the gobiid fish (fig. 123), the micropyle, permitting the sperm to get
through the egg membrane, may be situated at a point opposite the nuclear
pole of the egg.
 
 
G. Monospermic and Polyspermic Eggs
 
In the eggs of most animal species only one sperm normally enters the egg.
Such eggs are known as monospermic eggs. Among the chordates, the eggs
of Styela, Amphioxus, frog, toad, and mammals are monospermic. Abnormal
cleavage and early death of the embryo is the general result of dispermy and
polyspermy in frogs (Brachet, A., ’12; Herlant, ’ll). In those chordates
whose eggs possess much yolk, the eggs are normally polyspermic, and several
sperm enter the egg at fertilization, although only one male pronucleus enters
into syngamic relationship with the egg pronucleus; the other sperm soon degenerate and die in most cases (fig. 138). (See Fankhauser, ’48.) In some
urodele amphibia, it appears that syngamic conjugation of more than one
sperm pronucleus with the egg pronucleus may occur in certain instances and
may give origin to heteroploidy, and development may be quite normal
(Fankhauser, ’45). Examples of normal polyspermic eggs are: birds, reptiles,
tailed amphibia, elasmobranch fishes, and possibly some teleost fishes. Among
the invertebrates, polyspermy is found in some insects and in the Bryozoa.
In the sea urchin, polyspermy may occur, but abnormal embryos are the rule
in such cases as indicated above. Similar conditions are found in certain other
invertebrates (Morgan, ’27, pp. 84-86).
 
Two explanations of normal polyspermy are suggested:
 
( 1 ) The presence of a superabundance of yolk hinders the operation of
the mechanism whereby the egg inhibits the entrance of extra sperm;
the egg, therefore, falls back upon a second line of defense within its
own substance which excludes the sperm from taking part in or hindering the normal functioning of the syngamic nucleus in its relation
to development; and
 
(2) a large amount of yolk makes it advantageous to the egg for extra
sperm to enter, as they may contribute enzymes or other substances
which enable the egg better to carry on the metabolism necessary in
utilizing yolk material.
 
H. Importance of the Sperm Aster and the Origin of the First
Cleavage Amphiaster
 
One of the older views of fertilization maintained that the egg possessed
the cytoplasm but lacked a potent centrosome or “cell center” capable of
 
 
 
 
Fig, 130. Fertilization in Nereis. (After F. R. Lillie, ’12.) (A) Sperm of Nereis,
 
entire. (B) Egg of Nereis, 15 minutes after insemination. The fertilization cone is evident below point of sperm contact. Observe that the intact germinal vesicle is present
in the center of the egg. It will break down as the sperm enters the egg (G). The cortical
substance from the empty cortical compartments in the cortical layer shown around the
periphery of the egg has passed out through the vitelline membrane to form the jelly
layer around the egg. (C-G) Entrance of the sperm head into the ooplasm of the egg
as the fertilization cone substance is withdrawn inward from the vitelline membrane.
(C) Fifteen minutes after insemination. (D) Thirty-seven minutes after insemination.
(E) Forty-eight and one-half minutes after insemination. (F) Fifty-four minutes after
insemination. (G) Sperm head has completed penetration. Observe that the middle
piece of the sperm remains outside, attached to the vitelline membrane. Anaphase of
first maturation division.
 
 
260
 
 
 
IMPORTANCE OF THE SPERM ASTER
 
 
261
 
 
giving origin to the first cleavage amphiaster, whereas the sperm possessed a
dynamic centrosome with its included centriole but lacked sufficient cytoplasm
for division or cleavage. Consequently, fertilization brought together a relationship necessary for cleavage and development. This idea was first set forth
by Boveri (see fertilization theories at the end of this chapter).
 
In the majority of animals, the central body (i.e., the centrosome) with
its surrounding aster, which ultimately divides and gives origin to the first
cleavage amphiaster, does not arise until after the sperm has entered the egg.
In these cases the aster complex arises in the middle piece of the sperm in
close proximity to the nucleus. These facts are well illustrated in figures 116,
117, and 131. Many studies of the fertilization process and early cleavage
bolster this general conclusion. There are some exceptions, however, to this
rule. For example, Wheeler ( 1 895) in his studies of fertilization in Myzostoma
glabrum demonstrated that the centrioles of the egg near the germinal vesicle
give origin to the amphiaster concerned with polar body formation. Following
the maturation divisions, the female pronucleus with its centrioles and forming
amphiaster, migrates along the copulation path to meet the sperm pronucleus.
The amphiaster and centrioles are closely adherent to the egg pronucleus
during the migration of the latter. In the honeybee, Nachtsheim (M3) found
a similar situation, while in the mollusk, Crepidula plana, Conklin (’04) found
evidence which suggests that one aster of the cleavage amphiaster arises from
the egg, whereas the other aster arises from the sperm, “although there is
not positive evidence that they are directly derived from egg and sperm
centrosomes.”
 
Where the egg develops as a result of artificial stimulation the first cleavage
spindle arises without the aid of the sperm middle piece. In these instances
the amphiaster probably is derived from the central body of the last maturation division, or, it may be, from certain asters or cytasters artificially induced
in the egg cytoplasm by the activation process. The production of numerous
asters in the cytoplasm of the egg by artificial stimulation has long been
known (Mead, 1897; Morgan, 1899, ’00).
 
The general conclusion to be extracted from the evidence at hand, therefore, suggests that the central body from the last maturation spindle or other
artificially induced asters in the egg cytoplasm may form the amphiaster of
the first cleavage spindle in the case of an emergency. Such an emergency
arises in normal parthenogenesis or in cases of artificial activation (artificial
parthenogenesis) of the egg. However, under the conditions of normal fertilization the sperm aster fulfills the role of developing the first cleavage
amphiaster.
 
Regardless of the fact that the first cleavage amphiaster appears to be derived from the middle piece of the sperm, the influence of the egg protoplasm
is undoubtedly an important factor in its formation. In the normal polyspermy
of the newt, Triton (fig. 138; Fankhauser, ’48), the sperm aster nearest the
 
 
 
262
 
 
FERTILIZATION
 
 
egg pronucleus enlarges and develops the amphiaster, whereas the more distantly located sperm asters fade and disintegrate. This fact suggests that some
influence from the egg pronucleus stimulates the further development of the
amphiaster in the sperm nearest the egg pronucleus. In experiments on insemination of egg fragments in the urodele, Triton, Fankhauser (’34) found
that the sperm aster in that fragment which did not contain the egg nucleus
failed to reach the size of the aster in the fragment containing the egg nucleus.
He concludes, p. 204, “The interactions between the sperm complex and the
cytoplasm of the egg seem, therefore, to be stimulated in the presence of the
egg nucleus.”
 
On the other hand, the experiments on androgenesis by Whiting (’49) in
Habrobracon, and the insemination of the “red halves” of the sea-urchin egg
by Harvey (’40) demonstrate that the sperm aster can, without the egg
pronucleus, produce the first cleavage amphiaster. However, the presence of
a nucleoplasmic substance in both of these cases cannot be ruled out. For
example, A. Brachet (’22) and Bataillon (’29), the former working on the
sea-urchin egg and the latter on the eggs of two amphibian species, demonstrated that large, normal sperm asters and large vesicular sperm nuclei do
not form until after the germinal vesicle breaks down and the egg becomes
mature. Premature fertilization results in polyspermy, small sperm nuclei, and
small sperm asters. In normal fertilization, therefore, it is very probable that
the development of the sperm aster into a normal cleavage amphiaster is
dependent:
 
( 1 ) upon the egg cytoplasm, and
 
(2) upon some factor contributed to the egg cytoplasm by the nuclear sap
or from the chromosomes of the female nucleus at the time of the breakdown of the germinal vesicle or during the maturation divisions.
 
I. Some Related Conditions of Development Associated with the
Fertilization Process
 
1. Gynogenesis
 
The word gynogenesis means “female genesis.” Therefore, gynogenesis is
the development of the egg governed by the female pronucleus alone. The
male gamete may enter the egg but plays no further role (Sharp, ’34, p. 406;
Wilson, ’25, p. 460). In the nematode, Rhabdites aberrans, the egg produces
but one polar body, and diploidy is retained. The egg is penetrated by the
sperm which takes no part in later development, as it degenerates upon entering
the egg.
 
In the above instance, it is doubtful whether or not sperm is necessary to
activate the egg. However, in the nematode, Rhabdites pellio, the egg is penetrated by the sperm which plays no further role in development. Nevertheless,
in the latter instance, sperm entrance appears to be necessary for egg acti
 
 
CONDITIONS ASSOCIATED WITH FERTILIZATION PROCESS 263
 
 
 
Fig. 131. Fertilization in the sea urchin, Toxopneustes variegatus. (After Wilson and
Mathews, 1895.) (A) Sperm head and middle piece. (B) Fertilization cone (attrac
tion cone; “cone of exudation” of Fol). The fertilization cone forms after the sperm
head and middle piece have entered the egg, and persists through (F) when the pronuclei
begin to come together. (C-J) Different stages in the fusion of the pronuclei. Observe
that the sperm rotates at about 180® and that the sperm aster appears near base of nucleus
(D, E). The aster grows rapidly (F, G) as the sperm pronucleus advances toward the
female pronucleus, and appears between the two pronuclei in (G). In (H) the aster has
divided, and the daughter asters are found at either end of the two fusing pronuclei.
In (I, J) the two asters are at either end of the fusion nucleus. (J) Fusion nucleus
between the amphiaster of the first cleavage.
 
vation. A somewhat similar phenomenon may also occur in other animal
species taking part in hybrid crosses, where some or all of the paternal chromosomes may be eliminated; activation normally occurs in these instances, and
development results. Gynogenesis is experimentally produced in amphibia by
radiating the sperm before fertilization. Development is carried on by the
female pronucleus in the latter instance, although it may produce larvae which
ultimately die. Parthenogenesis, natural and artificial, in all its essential features in a sense may be regarded as gynogenesis.
 
2. Androgenesis
 
This form of development is experimentally produced by removal of egg
pronucieus with a small pipette before nuclear syngamy occurs (Porter, ’39)
or by treating the egg with x-rays before fertilization (Whiting, ’49). The
male pronucleus seems incapable of bringing about normal and full development in amphibia, but in wasps, where the egg pronucleus has been destroyed
by radiation, it has been successful (Whiting, ’49).
 
 
 
 
 
Fig. 132. Movement of ooplasmic substances in the egg of Styela (Cynthia) partita
at the time of fertilization. (All figures after Conklin, ’05.) (A) Unfertilized egg after
 
the disappearance of the nuclear membrane of the germinal vesicle. The gray yolk is
shown in the center of the egg, surrounded by the yellow-pigmented cytoplasm. The test
cells and chorion surround the egg. (B) Egg five minutes after fertilization, showing
the streaming of the peripheral protoplasm indicated by arrows toward the vegetal pole,
where the sperm has entered. The gray yolk is shown in the upper part of the egg below
the nuclear material. The clear protoplasm, derived from the nuclear sap of the germinal
vesicle, also flows down with the peripheral protoplasm. (C) Side view of an egg after
peripheral protoplasm has migrated to the vegetal pole of the egg. The clear protoplasm
is shown at the upper edge of the yellow cap. The polar bodies are forming in the
midpolar area (MP.) at the animal pole. (D) Side view of the egg showing the yellow
crescent and the area of clear protoplasm above the yellow crescent. The yolk material
is shown at the vegetal pole below and to one side of the yellow crescent. (E) Yellow
crescent and clear protoplasm viewed from the posterior pole of the egg. Animal pole
is above the crescent; yolk material is below. (F) Same view as (E) a little later,
showing the external beginnings of the first cleavage. The polar bodies are shown above
the crescent material. (G) View similar to that of (E, F) a little later. The first cleavage
is complete. Observe that the clear protoplasm and the yellow crescent have been bisected
equally. The cleavage plane corresponds to the median axis of the embryo.
 
 
264
 
 
 
CONDITIONS ASSOCIATED WITH FERTILIZATION PROCESS
 
 
265
 
 
3. Merogony
 
Merogony is the development of part of the egg, that is, an egg fragment.
Egg fragments are obtained by shaking the egg to pieces, by cutting with a
sharp instrument, or by the use of centrifugal force. Andromerogony is the
development of a non-nucleate egg fragment after it has been fertilized by
 
 
 
Fig. 133. Stages in the fertilization of Ascaris. (After Boveri, 1887.) (A) Ameboid
 
sperm on the periphery of the egg; germinal vesicle in center of primary oocyte. (B)
Sperm entered egg substance; germinal vesicle broken down and tetrads becoming evident.
 
(C) Sperm in center of the egg; first maturation division, showing tetrads on spindle.
 
(D) Second maturation division; first polar body at egg’s surface. (E) First and second
polar bodies shown; sperm aster forming in relation to sperm. (F) Second polar body;
male and female pronuclei. (G) Male and female haploid chromosomes evident;
amphiaster forming. (H) Chromosomes distinct, showing haploid condition. Observe
amphiaster. (I) Amphiaster complete; metaphase of first cleavage.
 
 
 
266
 
 
FERTILIZATION
 
 
GERMINAL VESICLE
 
\ MICROPYLAR CANAL
 
\ I VILLOUS LAYER
 
 
 
Fig. 134. Micropyle and egg membranes of certain fishes. (A) Micropyle, egg membranes, and germinal vesicle in Lepidosteus. (Modified from three figures drawn by
E. L. Mark, Bull. Mus. Comp. Zool. at Harvard College, 19: No. 1.) (B-F) Micropyle
 
and egg membranes of the cyclostome, Petromyzon planeri. (Slightly modified from
Calberla, Zeit. Wiss. Zool., 30.) (B) Mature, unfertilized egg. (C) Sperm passes
 
through the micropyle and enters the protoplasmic strand, P.S. (D) Higher power view
of sperm in protoplasmic strand; also observe that the egg is shrinking away from the
egg membrane, forming the peri vitelline space. (E, F) Egg contracts away from the
egg membrane, leaving the egg free to revolve within the membrane.
 
sperm. Development is not normal and does not go beyond the larval condition. Parthenogenetic merogony is the development of non-nucleate parts
of the egg which have been artificially activated. Artificial activation of nonnucleate parts of the egg of the sea urchin, Arbacia, is possible by immersion
of these parts of the egg for 10 to 20 minutes in sea water, concentrated to
about one half of the original volume, or by the addition of sodium chloride
to sea water to bring it to a similar hypertonicity (Harvey, ’36, ’38, ’51).
 
 
 
THEORIES OF FERTILIZATION
 
 
â– 267
 
 
These parthenogenetic merogons develop to the blastula stage only. Gynomerogony is the parthenogenetic development of an egg fragment containing
the egg pronucleus.
 
Theories of Fertilization and Egg Activation
 
Boveri, T., 1887, 1895. In somatic mitoses, the division center or centrosome is handed down from cell to cell. In the development of the female
gamete, the division center degenerates or becomes physiologically incapable
of continuing the division of the egg either before or after the maturation
divisions. The mature egg thus contains all the essentials for development
other than a potent division center. The sperm, on the other hand, lacks the
 
 
 
Fig. 135. Fertilization phenomena in the pigeon. (After Harper, ’04.) (A) Germinal
 
vesicle of late ovarian egg. The chromatin material is shown in the center of the vesicle;
the nuclear wall is beginning to break down. (B) Spindle of first maturation division.
Egg just ovulated and entering the oviduct. Sperm enters the egg at this time. (C)
Second polar spindle and first polar body. (D) First and second polar bodies; egg
pronucleus reorganizing. (E) Two pronuclei approaching, preparatory to fusion. Sperm
nucleus to the left. (F) Two pronuclei fusing. (G) Accessory sperm nuclei to the
left of this figure; fusion nucleus to the right.
 
 
268
 
 
FERTILIZATION
 
 
H CLEAVAGE
 
 
 
Fig. 136. Organization of germinal disc of the Echidna egg following fertilization.
 
(After Flynn and Hill, ’39.)
 
cytoplasmic conditions necessary for development, but possesses an active
division center which it introduces into the egg at fertilization. Fertilization,
therefore, restores the diploid number of chromosomes to the egg and introduces an active division center.
 
Loeb, J., ’13. Loeb believed that two factors were involved in egg activation: (a) Superficial cytolysis of the egg cortex which leads to a sudden increase in the oxidation processes of the egg, and (b) a factor which corrects
cytolysis and excess oxidation, thus restoring the egg to normal chemical
conditions. He placed great emphasis on superficial cytolysis of the cortex
with the resultant elevation of the fertilization membrane.
 
Loeb suggested that in normal fertilization the sperm brings in a lytic principle which brings about cortical cytolysis, and a second substance which
regulates oxidation.
 
For discussion of this theory, consult J. Brachet, ’50, p. 138.
 
Bataillon, E., ’10, ’ll, ’13, ’16. Like Loeb, Bataillon emphasized two
steps in the activation process of the egg: (a) First treatment, whether it is
the puncture of the frog’s egg by a fine needle or the butyric acid treatment
of the egg of the sea urchin, according to the method of Loeb, causes; ( 1 ) elevation of fertilization membrane and the excretion of toxic substances from
the egg, and (2) the formation of a monaster, (b) Second treatment, whether
 
 
 
THEORIES OF FERTILIZATION
 
 
269
 
 
it is blood, in the case of the frog, or hypertonic sea water, as used by Loeb
in the sea-urchin egg, introduces a catalyzer which converts the monaster
into an amphiaster, and in this way renders the egg capable of cleavage.
 
Bataillon placed great emphasis upon the exudation (excretion) of substances into the perivitelline space and the elevation of the fertilization membrane. He believed that the unfertilized egg was inhibited because of an
accumulation of metabolic products and that activation or fertilization led
to a release of these substances to the egg’s exterior.
 
For discussion, consult Wilson, ’25, p. 484; J. Brachet, ’50, p. 144.
 
Lillie, F. R., ’14, ’19. This author postulated that a substance, fertilizin,
carried in the cortex of the egg, exerts two kinds of actions in the activation
process: (1) An activating, attracting, and agglutinating action on the sperm,
and (2) an aetivating effect on the egg itself. In essence, the egg is selffertilizing, for the fertilizing substance is present in the egg. The procedure
is somewhat as follows: At the period optimum for fertilization, inactive
fertilizin (i.e., inactive from the viewpoint of possessing the ability to activate
the egg) is produced by the egg. Released into the surrounding water, it
activates, attracts, and agglutinates the sperm at the egg’s surface. As the
sperm touches the egg, it unites with a part of the fertilizin molecule. The
 
 
 
FIRST
 
MATURATION
 
DIVISION
 
SECOND
 
MATURATION
 
DIVISION—
 
 
Fig. 137. Maturation divisions of the oocyte relative to time of sperm entrance. (A)
Sperm enters the primary oocyte before maturation divisions. In some, e.g.. Nereis,
Thalassema, Ascaris, Fluty nereis, Myzostonui, etc., the sperm enters before the germinal
vesicle breaks down; in Styela, Chaetopterus, pigeon, etc., the first maturation spindle is
formed or forming; in the dog, the condition is somewhat similar to Nereis, Ascaris, etc.
(B) Sperm enters the egg after first maturation division, i.e., in secondary oocyte stage
(Asterias (starfish), Amphioxus, hen, rabbit, man, frog, salamander, newt, most vertebrates). (C) Sperm enters the egg after maturation divisions are completed, i.e., in
the mature egg {Arhacia and other sea urchins; possibly in monotreme. Echidna, on
occasion).
 
 
 
270
 
 
FERTILIZATION
 
 
 
Fig. 138. Polyspermy in the European newt, Triton. (After Fankhauser, ’48.) (A)
 
Ten minutes after insemination at 23® C. Metaphase of second maturation division; four
sperm have entered the egg, one of which is at the vegetal pole of the egg, and another
between the two poles of the egg. (B) One hour and 30 minutes; second polar body
given off; small egg pronucleus moves toward nearest sperm nucleus. The latter will
become the principal sperm nucleus. Observe that accessory sperm nuclei are enlarging
and a sperm aster is developed relative to each. (C) Two hours and 30 minutes. Egg
and principal sperm pronuclei in contact; maximum development of sperm asters. (D)
Three hours. Fusion of egg pronucleus and principal sperm pronucleus. Accessory sperm
nucleus nearest to fusion nucleus shows signs of degeneration. Accessory sperm asters
remain undivided, while principal sperm aster has formed an amphiaster. (E) Three
hours and 30 minutes. Metaphase of first cleavage; all accessory sperm nuclei degenerating. (F) Four hours. Early telophase of first cleavage; remnant of accessory nuclei
being pushed out of animal pole region by amphiaster and spindle of first cleavage division.
 
fertilizin molecule plus the sperm then have the ability to unite with an egg
receptor, and the union of the fertilizin-sperm complex with the egg receptor,
releases the activating principle within the egg, which spreads “with extreme
rapidity” around the egg cortex. The activating principle activates the egg as
a whole, setting it in motion toward development. It is thought to work especially upon the cortex of the egg, producing cortical changes, including the
formation of a fertilization membrane. Further, it agglutinates or immobilizes
all other sperm around the egg. Consequently, polyspermy may be hindered
by this agglutination effect and by the fertilization membrane. In regard to
polyspermy, Lillie also postulated another substance, antifertilizin, within the
egg which unites with the remaining fertilizin molecules in the egg the instant
that one sperm has made successful union with a molecule of fertilizin, thus
preventing other sperm from entering the egg.
 
For discussion, see J. Brachet, ’50, p. 143; Dalcq, ’28.
 
 
 
THEORIES OF FERTILIZATION
 
 
271
 
 
Lillie, R. S., ’41. Like Loeb, R. S. Lillie conceived of cortical changes as
being the main aspect of activation, particularly changes such as a decrease
in viscosity which permits interaction of various substances which normally
are kept separated in the unactivated egg. Lillie’s hypothesis may be stated
as follows: An activating substance, comparable to a growth hormone or
auxin, is formed in the egg. This substance may be called (A). The formation of (A) results from the interaction of two substances, (S) and (B),
present in low concentrations in the egg. One of these substances, (S), is
synthesized in the egg by treating the egg in various ways, such as immersion
in sea water in the presence of oxygen. The other substance, (B), is freed
from pre-existing combination by a simple splitting (hydrolytic) process initiated or catalyzed by acid. This reaction is independent of oxygen. The
union of the two substances, (S) and (B), forms the activating substance,
(A). Lillie thus believes in a single factor as the initiator of development.
Complete activation of the egg results when (A) is produced in adequate
concentration; partial activation occurs when it is present in quantity below
the optimum concentration.
 
, For discussion, see Brachet, J., ’50, p. 141.
 
Hcilbrunn, L. V., ’15, ’28, ’43. This author believes that an increase in
viscosity with resultant coagulation or gelation of egg cytoplasm is involved
directly with the initiation of development. Heilbrunn regards this gelation
process to be similar to the clotting of blood. He also regards calcium as
the main agent in bringing about this effect, and therefore believes calcium
to be concerned directly with egg activation. According to this view, calcium
is bound to the proteins localized in the egg cortex. At the time of activation,
artificially or by sperm contact, part of this calcium is liberated which in turn
produces a coagulation of the cytoplasm, initiating development. Dalcq and
his associates also have emphasized the importance of calcium in the activation process.
 
For discussion, see Brachet, J., ’50, p. 146; Dalcq, ’28; Runnstrom, ’49.
 
Runnstrom, J., ’49. Runnstrom more recently has contended that an inhibitor of proteolytic enzymes may be present in the vitelline membrane and
cortex of the egg. He assumes that the inhibitor, possibly fertilizin, may be
identical with a heparin-like substance. He further assumes that the inhibitor
is bound to a kinase and is released when protein substances associated with
the sperm unite with the inhibitor. “A kinase acting on a proenzyme may then
be released”; the latter, i.e., the kinase, acts upon the proenzyme in the cortex
of the egg, giving origin to an enzyme or enzymes which initiate development.
Runnstrdm’s position in essence is a modern statement of the inhibition theory
of F. R. Lillie (see J. Morphol., vol. 22).
 
 
 
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Galtsoff, P. S. 1938. Physiology of reproduction of Ostrea virgitiica. il. Stimulation of spawning in the female oyster.
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Gregory, P. W. 1930. The early embryology of the rabbit. Contrib. Embryol. 21:
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Guyer, M. F. 1907. The development of
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Hammond, J. 1934. The fertilisation of
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Harding, D. 1951. Initiation of cell division in the Arbacia egg by injury substances. Physiol. Zool. 24:54.
 
Harper, E. H. 1904. Fertilization and early
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Hartman, C. G. 1916. Studies in the development of the opossum Didelphys
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, Kuhn, R., Schartau, O., and Wal
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274
 
 
FERTILIZATION
 
 
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Hibbard, H. 1928. Contribution a I’etude
# The transportation of the gametes to the site normal for the species where environmental conditions are suitable for gametic union (Chap. 4), and
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# Fertilization or the union of the gametes (Chap. 5).
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Just, E. E. 1919. The fertilization reaction
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Lewis, W. H. and Gregory, P. W. 1929.
==Transportation of the Gametes==
Cinematographs of living developing
[[Book - Comparative Embryology of the Vertebrates 2-4|'''4. Transportation of the Gametes (Sperm and Egg) from the Germ Glands to the Site where Fertilization Normally Occurs''']]
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Columbia University Press, New York.  
1. Definitions


2. Transportation of the egg in those forms where fertilization occurs in the anterior portion of the oviduct


a. Birds


BIBLIOGRAPHY
b. Mammals


3. Transportation of the egg in those species where fertilization is effected in the


275
caudal portion of the oviduct or in the external medium


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==Fertilization==
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2. Artificial activation of the egg
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1938. A propOs du determinisme
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de la symetrie bilaterale chez les amphibiens anoures. Conditions qui provoquent I’apparition du croissant gris.
b. Some of the procedures used in artificial activation of the egg
Compt. rend. Soc. de biol. 129:59.  


Pinciis, G. 1939. The comparative behavior of mammalian eggs in vivo and in
c. Results obtained by the work on artificial parthenogenesis
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on the activation of rabbit eggs. Proc.
Am. Philos. Soc. 83:631.


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Runnstrom, J. 1949. The mechanism of  
b. Characteristics of the male gamete
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— , , and Tiselius, A. 1944.
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b) Spawning-inducing substances
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36A: No. 18, 1.


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276
5) Summary of the activities of the egg and sperm in bringing about the primary or initial stage of the fertilization process, namely, that of egg and sperm contact




FERTILIZATION
3. Fusion of the gametes or the second stage of the process of fertilization


4. Detailed description of the processes involved in gametic union as outlined above


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Compt. rend. Acad. d. Sc. 188:97.

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Nelsen OE. Comparative embryology of the vertebrates (1953) Mcgraw-Hill Book Company, New York.

   Comparative Vertebrate Embryology 1953: 1. The Period of Preparation | 2. The Period of Fertilization | 3. The Development of Primitive Embryonic Form | 4. Histogenesis and Morphogenesis of the Organ Systems | 5. The Care of the Developing Embryo | Figures
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Part II - The Period of Fertilization

Part II - The Period of Fertilization: 4. Transportation of the Gametes (Sperm and Egg) from the Germ Glands to the Site where Fertilization Normally Occurs | 5. Fertilization


The period of fertilization involves:

  1. The transportation of the gametes to the site normal for the species where environmental conditions are suitable for gametic union (Chap. 4), and
  2. Fertilization or the union of the gametes (Chap. 5).


The union of the gametes may be divided into two phases, viz.:

  1. The primary phase which is terminated when the sperm has made intimate contact with the egg's surface, and
  2. The secondary phase or the fusion of the two gametes resulting in the initiation of development.


Transportation of the Gametes

4. Transportation of the Gametes (Sperm and Egg) from the Germ Glands to the Site where Fertilization Normally Occurs


A. Introduction

1. Activities of the male and female gametes in their migration to the site of fertilization

B. Transportation of the sperm within the male accessory reproductive structures

1. Transportation of sperm from the testis to the external orifice of the genital duct in the mammal

a. Possible factors involved in the passage of the seminal fluid from the testis to the main reproductive duct

1) Accumulated pressure within the seminiferous tubules

2) Activities within the efferent ductules of the testis

b. Movement of the semen along the epididymal duct

1) Probable immotility of the sperm

2) Importance of muscle contraction, particularly in the vas deferens

3) Summary of factors which propel the seminal fluid from the testis to the external orifice of the reproductive duct in the mammal

2. Transportation of sperm in other vertebrates with a convoluted reproductive duct

3. Transportation of sperm from the testis in vertebrates possessing a relatively simple reproductive duct

C. Transportation of sperm outside of the genital tract of the male

1. Transportation of sperm in the external watery medium

2. Transportation of sperm in forms where fertilization of the egg is internal

a. General features relative to internal fertilization

1) Comparative numbers of vertebrates practicing internal fertilization

2) Sites or areas where fertilization is effected

3) Means of sperm transfer from the male genital tract to that of the female

b. Methods of sperm transport within the female reproductive tract

1) When fertilization is in the lower or posterior portion of the genital tract

2) When fertilization occurs in the upper extremity of the oviduct

3) When fertilization occurs in the ovary

D. Sperm survival in the female genital tract

E. Sperm survival outside the male and female tracts

1, In watery solutions under spawning conditions

2. Sperm survival under various artificial conditions; practical application in animal breeding

F. Transportation of the egg from the ovary to the site of fertilization

1. Definitions

2. Transportation of the egg in those forms where fertilization occurs in the anterior portion of the oviduct

a. Birds

b. Mammals

3. Transportation of the egg in those species where fertilization is effected in the

caudal portion of the oviduct or in the external medium

a. Frog

b. Other amphibia

c. Fishes

G. Summary of the characteristics of various mature chordate eggs together with the site of fertilization and place of sperm entrance into the egg

Fertilization

5. Fertilization

A. Definition of fertilization

B. Historical considerations concerning gametic fusion and its significance.

C. Types of egg activation

1. Natural activation of the egg

2. Artificial activation of the egg

a. Object of studies in artificial parthenogenesis

b. Some of the procedures used in artificial activation of the egg

c. Results obtained by the work on artificial parthenogenesis

D. Behavior of the gametes during the fertilization process

1. General condition of the gametes when deposited within the area where fertilization is to occur

a. Characteristics of the female gamete

1) Oocyte stage of development

2) Inhibited or blocked condition

3) Low level of respiration

4) Loss of permeability

b. Characteristics of the male gamete

2. Specific activities of the gametes in effecting physical contact of the egg with the sperm

a. Activities of the female gamete in aiding sperm and egg contact

1) Formation of egg secretions which influence the sperm

a) Fertilizin complex

b) Spawning-inducing substances

b. Activities of the male gamete in aiding the actual contact of the two gametes

1) Sperm secretions

a) Secretions producing lysis

b) Secretions related specifically to the fertilization reactions

c) Secretions which induce the spawning reaction in the female

2) Relation and function of sperm number in effecting the contact of the sperm with the egg

3) Influences of the seminal plasma in effecting sperm contact with the egg

4) Roles played by specific structural parts of the sperm in effecting contact with the egg

a) Role of the flagellum

b) Role of the acrosome in the egg-sperm contact

5) Summary of the activities of the egg and sperm in bringing about the primary or initial stage of the fertilization process, namely, that of egg and sperm contact


3. Fusion of the gametes or the second stage of the process of fertilization

4. Detailed description of the processes involved in gametic union as outlined above

a. Separation and importance of a protective egg membrane, exudates, etc.

b. Fertilization cone or attraction cone

c. Some changes in the physiological activities of the egg at fertilization

d. Completion of maturation divisions, ooplasmic movements, and copulatory paths of the male and female pronuclei in eggs of various chordate species

1) Fertilization in Styela (Cynthia) partita

a) Characteristics of the egg before fertilization

b) Entrance of the sperm

c) Cytoplasmic segregation

d) Copulatory paths and fusion of the gametic pronuclei

2) Fertilization of Amphioxus

3) Fertilization of the frog’s egg

4) Fertilization of the teleost fish egg

5) Fertilization in the egg of the hen and the pigeon

6) Fertilization in the rabbit

7) Fertilization in the Echidna, a prototherian mammal

E. Significance of the maturation divisions of the oocyte in relation to sperm entrance and egg activation

F. Micropyles and other physiologically determined areas for sperm entrance

G. Monospermic and polyspermic eggs

H. Importance of the sperm aster and the origin of the first cleavage amphiaster

I. Some related conditions of development associated with the fertilization process

1. Gynogenesis

2. Androgenesis

3. Merogony

J. Theories of fertilization and egg activation

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