/. Embryol. exp. Morph. Vol. 37, pp. 187-201, 1977
Printed in Great Britain
\%~]
Fertilization of immature frog eggs: cleavage and
development following subsequent activation
By RICHARD P. ELINSON1
From the Department of Zoology, University of Toronto
SUMMARY
Frog eggs are normally fertilized after reaching metaphase II. When eggs are inseminated
prior to that, several sperm enter, but entry does not activate the egg. When such inseminated,
immature eggs were maintained until they became mature and then were artificially activated,
the eggs began to cleave. The cleavage furrows were irregular and often multiple, but the eggs
developed to blastulae or partial blastulae. About 2 -> 5 % of the eggs developed to tadpoles.
Typical asters were not associated with the entering sperm; rather, asters appeared only after
activation. The sperm nucleus often formed chromosomes which were attached to small
spindles. It is clear that sperm which remain for a time in unactivated egg cytoplasm, retain
their ability to promote cleavage and development. Aster formation required not only sperm
centrioles but also activated egg cytoplasm.
Sperm which entered either near the equator or in the animal half of mature eggs usually
produced normal cleavage furrows. Sperm which entered the animal half of immature eggs
produced multiple animal half furrows when the egg was subsequently activated. In contrast,
sperm which entered near the equator of immature eggs often failed to induce furrowing on
subsequent activation or produced unusual equatorial furrows. The difference in the type of
furrow between eggs inseminated in the animal half or at the equator is interpreted as a
consequence of dissociating sperm entry from the cortical contraction which occurs on
activation.
INTRODUCTION
With mature eggs of amphibians as well as of many other animals, sperm
entry performs three functions. First, the egg is activated; secondly, the diploid
number of chromosomes is restored, and thirdly, cleavage is initiated. In contrast, when a sperm enters an immature egg, the egg is not activated and it does
not begin developing. Since there is no block to polyspermy, numerous sperm
can enter. The sperm within an immature egg undergo characteristic changes
depending on the stage of egg maturation. If the sperm enters the egg while the
egg is undergoing the first meiotic division, the sperm nucleus forms chromosomes which are attached to spindle fibers (Brachet, 1922; Bataillon, 1929;
Tchou & Chen, 1942; Iwamatsu & Chang, 1972; Das & Barker, 1976). The
formation of chromosomes by the sperm is a response to the cytoplasmic conditions existing in the egg. When nuclei from other sources are transplanted into
1
Author's address: Department of Zoology, University of Toronto, 25 Harbord Street,
Toronto, Ontario M5S1A1, Canada.
188
R. P. ELINSON
eggs undergoing the meiotic divisions, the transplanted nuclei also form chromosomes on spindles (Gurdon, 1968; Subtelny, 1968; Ziegler & Masui, 1973).
Amphibian eggs are normally fertilized after achieving and arresting at
metaphase II of meiosis. Eggs fertilized prior to metaphase II have been
examined on several occasions (Bataillon, 1929; Bataillon & Tchou, 1930;
Tchou & Chen, 1942; Tchou & Wang, 1964; Katagiri, 1974; Elinson, 1975). The
questions asked in the present work are whether an immature frog egg can
complete maturation after sperm entry and whether a sperm which enters an immature frog egg can initiate cleavage and participate in development when the
egg attains maturity. Clearly these activities are not biologically impossible
since in certain species, sperm entry occurs before the egg reaches metaphase II
(see Austin, 1965). An examination of frog gametes placed under these unusual
conditions would indicate control processes which must exist in species eggs
normally fertilized prior to metaphase II.
MATERIALS AND METHODS
Sexually mature Ranapipiens were obtained from dealers in Vermont, U.S.A.
and Quebec, Canada in the fall and stored at 4 °C until use.
Immature eggs are defined here as fully grown eggs which have been induced
hormonally to undergo maturation but which have not yet become activatable.
The immature eggs used in the present experiments were between metaphase I
of meiosis and the acquisition of activatability. No further division of this
period was attempted. Immature eggs with jelly were obtained in two ways.
First, females were injected with pituitaries and progesterone according to
standard procedures (Di Berardino, 1967) and left at 18 °C. Eggs could be
expressed from the uterus as early as 17 h later while the eggs were not activatable until about 20 h or more. Depending on the female, several hundred immature eggs could be obtained, but frequently, few eggs were obtained prior to
activatability. In the second method, females were injected with a very small
amount of pituitary suspension (e.g. equivalent to one quarter of a male
pituitary) which is insufficient to cause ovulation. About 8 h later, the females
were given the usual pituitary and progesterone injections. Uterine eggs were
found as early as 12 h after the second injection and as immature as premetaphase I. The eggs were not activatable until 17 h or more after the second injection. This injection schedule reliably produced females with large numbers
of immature eggs. The initial small dose of pituitary apparently facilitates
ovulation without much stimulation of maturation. With either procedure,
activatability of the eggs developed by about 20 h, considerably faster than with
eggs maturing in vitro (Smith et ah 1966). Recent work has shown that ovarian
and oviducal conditions enhance the process of egg maturation (Brun, 1975;
Vitto & Wallace, 1976) and this enhancement probably accounts for the difference in timing between in vivo and in vitro acquisition of egg activatability.
Fertilization of immature frog eggs
189
Procedures for the insemination of immature eggs were designed with the
responses of the gametes to tonicity in mind. Sperm are motile and can fertilize
eggs in 10 % Ringer's but they are immotile in 100 % Ringer's. Immature eggs,
stored in 100 % Ringer's, retain their activatability, but they cannot be stored in
10 % Ringer's. Activated eggs can develop in 10 % Ringer's, but they do not
develop in 100 % Ringer's. Accordingly, sperm in 10 % Ringer's were used to
inseminate immature eggs laid dry in a dish. After a 10-min insemination the
eggs were kept in 10 % Ringer's for 10-50 min. They were then transferred to
100 % Ringer's for storage until the following day when they were mature. They
were electrically activated (see below) in 100 % Ringer's and transferred to 10 %
Ringer's for development. Control experiments included: eggs uninseminated
but stored in 100 % Ringer's and activated when mature to see if activation
without insemination would lead to development; eggs inseminated and left in
10 % Ringer's to see if any were mature at the time of insemination, and eggs
inseminated, stored in 100 % Ringer's, and transferred to 10 % Ringer's to see
if transfer alone caused development.
The eggs were shocked in a Plexiglass chamber (60 x 20 x 4 mm) with a
platinum wire electrode at either end. Five pulses of 100 V lasting for 100 msec
each were given using a Grass SD9 Stimulator (Grass Medical Instruments).
This shocking is more than sufficient to activate a mature egg, and does not
appear to interfere with normal development when applied right after sperm
entry.
Preparation of sperm, local insemination by injection, determination of
sperm entry sites and cytological procedures were the same as described previously (Elinson, 1975). True cleavage of an activated egg was judged by the
formation of a blastula or a partial blastula.
RESULTS
Cleavage following artificial activation of eggs inseminated when immature
Immature jellied frog eggs, maintained in 100 % Ringer's until mature, have
a wrinkled surface. The 100 % Ringer's is probably not the ideal medium since
uterine eggs have a smooth surface; yet, the eggs were activatable, and as discussed later could be fertilized and could develop. Upon shocking, the eggs
underwent the cortical contraction described previously (Elinson, 1975). The
contraction did not occur to the same extent and the animal-vegetal margin was
irregular compared to the contraction of mature eggs freshly squeezed from the
frog and activated. The eggs raised a fertilization membrane, rotated, and often
exhibited partial and incomplete furrowing. The furrows regressed, and the egg
cytolyzed. This is the expected response from an unfertilized, activated egg.
When immature eggs were inseminated, they showed no activation responses
and appeared as if they were unfertilized. Electrical shocking of these inseminated immature eggs did not activate them. When eggs inseminated when
13
EMB
37
190
R. P. ELINSON
Table 1. Cleavage of shocked eggs inseminated when immature v.
frequency of fertilization
Female
1
2
3
4
Sperm concentration
(x 105/ml)
Blastulae(%)
3-2
0-2
5-4
0-2
5-5
0-2
240
0-4
33
0
41
14
69
38
36
12
Fertilization (%)
(monospermy, %)*
81
23
74
38
98
67
93
18
(50)
(12)
(30)
(31)
(20)
(50)
(43)
(12)
* The percentage of feitilization is the percentage of the total number of eggs which had
at least one sperm entry site. The percentage of monospermy is the percentage of the total
number of eggs which had only one sperm entry site. The percentage of polyspermy would
be the percentage of monospermy subtracted from the percentage of fertilization.
immature were allowed to mature and were then transferred to 10 % Ringer's,
few if any eggs began to cleave. The few cleaved eggs probably resulted from
activation by cortical injury when the eggs were transferred. The eggs were quite
flaccid due to the digestion of the vitelline coat by the sperm, and the cortex was
easily injured.
When eggs inseminated when immature were allowed to mature and were
then shocked, many of them began to cleave (Table 1). The cleavage furrows
were usually irregular and often multiple; however, the furrows were true
furrows since the eggs developed into blastulae or partial blastulae. The time
between insemination and shocking was usually about a day. It is clear, therefore, that a sperm retains its ability to induce cleavage after residing in
unactivated egg cytoplasm for a day.
The frequency of shocked inseminated immature eggs which began cleavage
leading to blastula or partial blastula formation was less than 100 %. In order
to compare the frequency of blastula formation with the frequency of fertilization, some inseminated eggs were tested for later cleavage ability while others
were fixed, bleached, and scored for sperm entry sites. When immature eggs
were fixed 1 h after insemination and bleached, small dots or smudges of
accumulated pigment were seen on the surface, corresponding to sperm entry
sites. The dots differed from the streaks seen on fertilized mature eggs (Elinson,
1975). This difference was due to the fact that the cortex of the immature egg
does not contract or shift in response to sperm entry unlike the cortex of the
mature egg. Since the immature egg was not activated, there was no obvious
block to polyspermy, and bleached immature eggs had multiple sperm entry
sites. The percentage of fertilization is compared to the percentage of blastula
Fertilization of immature frog eggs
191
Table 2. Re insemination of eggs inseminated when immature
Column
1st insemination
2nd insemination
Female
1
2
3
A a*
b*
...
...
1
pipiens
—
2
—
pipiens
No. Bias- No.
of
tula
of
eggs (%) eggs
3
pipiens
pipiens
Bias- No.
tula
of
(%) eggs
4
—
clamitans
Bias- No.
tula
of
(%) eggs
5
pipiens
clamitans
Bias- No. Blastula
of
tula
(%) eggs
(%)
38
30
28
21
23
24
8
0
0
5
17
8
75
43
51
50
50
40
20
44
82
48
48
45
83
36
34
43
41
32
8
0
9
28
10
0
70
36
42
44
44
20
47
31
95
98
98
100
52
31
30
41
38
20
6
0
13
80
47
40
t
28
11
34
21
21
33
47
81
17
100
Sum
192
6-8
293
43
270
11
259
71
229
36
5t
6
* In this trial, different R. pipiens sperm suspensions were used for the first insemination in
Aa and 46, but the same sperm suspensions were used for the reinsemination.
t The same R. clamitans sperm suspension was used for the reinsemination of eggs from
females 5 and 6.
formation in Table 1. Only about half of the eggs into which sperm entered
formed blastulae, and some blastulae must have developed from polyspermic
eggs. The latter is clear in those cases where the percentage of blastula formation
is greater than the percentage of monospermy.
Reinsemination of eggs inseminated when immature
Since eggs inseminated when immature retain their unfertilized appearance,
the question arises as to whether they can be refertilized on achieving maturity.
Can sperm rather than an electric shock be used to activate the eggs once
mature? Immature eggs were inseminated with R. pipiens sperm at a concentration sufficient to expect a frequency of fertilization of more than 90 %. After
storage in 100 % Ringer's for a day, the eggs were reinseminated with very high
concentrations of R. pipiens sperm or of R. clamitans sperm. The latter was used
since due to its high acrosomal protease activity, R. clamitans sperm are capable
of fertilizing eggs under conditions where R. pipiens sperm cannot (Elinson,
1973; 1974a, b). As seen in Table 2, eggs reinseminated with R. pipiens sperm
(column 3) formed blastulae at a frequency marginally higher than found
for non-reinseminated control eggs (column 1). Eggs reinseminated with
R. clamitans sperm (column 5), however, formed blastulae at a frequency higher
than control eggs (column 1). This experiment demonstrates that eggs which
were inseminated (and which were probably entered by sperm) when immature
can be stimulated by reinsemination to cleave when mature.
The frequency of blastula formation following reinsemination by either
13-2
192
R. P. ELINSON
Table 3. Development of immature eggs fertilized by Rana pipiens
and R. clamitans sperm
R. clamitans sperm
R. pipiens sperm
Female
1
2
3
Sum
No
gastrulation
9
6
25
40
52 V
No
Exogastrulation
Neurulation gastrulation
3
13
5
21
27 °/
12
3
1
16
21 °/
*••
A1
/o
/o
18
15
23
56
97%
Exogastrulation
Neurulation
0
1
0
1
2V
1
0
0
1
2%
*• /o
R. pipiens or R. clamitans sperm is low. One possible reason for this is that jellied
eggs hydrated for periods of time lose fertilizability (Elinson, 1971). To control
for this decline, some immature eggs from each trial were stored in 100 %
Ringer's and inseminated only at the time that the experimental eggs were
reinseminated (Table 2, columns 2 and 4). Although the frequency of blastula
formation was considerably less than 100 %, it was higher than that of reinseminated eggs (compare column 2 with column 3, column 4 with column 5).
Therefore, the low level of activation achieved on reinsemination cannot be
attributed solely to hydration changes of the jellied egg. Rather, insemination
when the eggs were immature made fertilization more difficult when the eggs
were mature.
Development following artificial activation of eggs inseminated when immature
The inseminated eggs which began cleaving due to the electric shock could
develop into normal-looking blastulae, although generally more than half
developed as partial blastulae. The latter had uncleaved areas in the animal half
or much of the vegetal half uncleaved. Most of the embryos were arrested as a
blastula or an exogastrula (Table 3). A variable but low frequency began
neurulation (see Table 3) and fewer than 5 % of the eggs which cleaved initially
developed to swimming tadpoles. The frequency of development was not
obviously increased by using more dilute sperm suspensions.
The poor development was due in part to the condition of the eggs at the time
of activation. The development of eggs inseminated when immature and
shocked 1 day later was compared to that of eggs stored in 100 % Ringer's for a
day and then inseminated. Of 127 eggs inseminated when immature, stored, and
then shocked, 30 % began neurulation and 17 % developed to tail-bud embryos
(stage 17). Of 163 eggs stored and then inseminated, 67 % began neurulation and
40 % developed to tail-bud embryos. Since normal development of greater than
90 % of R. pipiens embryos can be expected, storage of the eggs has probably
diminished their developmental potential. Storage, however, is not the only
Fertilization of immature frog eggs
193
source of decreased developmental ability in the eggs which were inseminated
when immature and then shocked.
The approximate chromosome numbers of a few embryos derived from eggs
inseminated when immature were determined. Of nine blastulae, five were close to
diploid, three were close to triploid, and one had spreads ranging from 22 to 65
chromosomes.Of four tail-bud embryos,one had more than the diploid number and
the remainder had between the triploid and pentaploid number of chromosomes.
Although the exact number of chromosomes was not determined, it is clear that
the embryos have at least the diploid number. This strongly suggests that the
sperm contributed chromosomes to the embryo.
To support this conclusion, advantage was taken of the fact that when R.
pipiens eggs are fertilized by R. clamitans sperm, the embryos arrest as blastulae.
Accordingly, some immature eggs were inseminated with R. pipiens sperm and
some with R. clamitans sperm. After subsequent maturation and activation by
shocking, about half of the eggs inseminated with R. pipiens sperm failed to
gastrulate while the rest exogastrulated or neurulated (Table 3). In contrast,
97 % of the eggs inseminated with R. clamitans sperm failed to gastrulate. These
results combined with the ploidy levels indicate that sperm chromosomes can
survive in the egg cytoplasm and participate in the development of the embryo.
Appearance of sperm nuclei in egg cytoplasm
One hour after insemination of immature eggs, condensed sperm nuclei could
be found well sunk into the cytoplasm. They were associated with a small
granule-free area of cytoplasm but a typical aster had not formed. On the
following day, sperm nuclei were found in four different configurations. Condensed nuclei were sometimes seen embedded in the cortical pigment. They were
found only because associated with them was a small area of cytoplasm which
was free of any pigment granules or other inclusions and which was stained with
light green. This cytoplasmic area was generally on the outer surface of the
pigmented cortex (Fig. 1). Since these areas were usually contained in one 9 /im
section, the nucleus was often obscured by pigment and its structure was not
determined.
In addition to these nuclei, some sperm nuclei condensed into chromosomelike forms and were arranged on small cortical spindles (Fig. 2). The spindles
were similar in size and form to the female meiotic metaphase spindles (Fig. 3),
but were often less well formed. In some cases, the area of cytoplasm surrounding the spindle was disrupted with patches of cytoplasm interrupted by
unstained areas.
Besides the sperm nuclei in the cortical region, two forms of nuclei were found
in the cytoplasm. The form depended on whether the nucleus was in the cytoplasm containing small or large yolk platelets. Among the small yolk platelets
(yolk-free area), large areas of disrupted cytoplasm were found (Fig. 4), similar
to those associated with cortical spindles described above. The disrupted areas
194
R. P. ELINSON
Fertilization of immature frog eggs
195
often contained one or more patches of light green stained, granule-free cytoplasm. A sperm spindle could be found within the disrupted area. The spindle
was usually small and irregular, and the sperm chromatin had condensed to form
chromosomes. In a few instances, a regularly shaped spindle was found (Fig. 5).
Among the medium or large yolk platelets (yolk-rich area), the nuclei were
obscured by small dense, pigment granule accumulations which occupied only
one or two sections (Fig. 6). No extensive area of cytoplasmic disruption was
found with the pigment accumulation.
The factors involved in determining whether a sperm nucleus was in the
cortex or in the cytoplasm have not been ascertained. Whether one position or
the other predominated, varied between experiments. Some eggs had sperm
nuclei in both positions.
Upon shocking of eggs inseminated when immature, the eggs were activated
and multiple polar bodies formed (Fig. 7). The extra polar bodies were assumed
to be from spindles derived from sperm, since normally only one polar body
forms on activating a frog egg. In addition, asters formed and began migrating
into the cytoplasm from the site of polar body formation (Fig. 8). The female
pronucleus never has an aster associated with it. By 60 min after activation,
several large astral areas with nuclei were present in the eggs. The nuclei (Fig. 9)
looked the same as nuclei found in inseminated mature eggs. In the yolk-rich
cytoplasm, the nuclei were surrounded by, at most, a radial cytoplasmic
organization suggestive of an aster. However, a typical astral area did not form.
FIGURES
1-9
Except for Fig. 3, these sections are from eggs inseminated when immature and
fixed one day later. The eggs in Figs. 1-6 were not activated while the eggs in
Figs. 7-9 were activated.
Fig. 1. Two cortical disruptions (D) caused by sperm entry. Scale line, 002 mm.
Fig. 2. Two cortical spindles (S) of sperm origin. Scale line, 002 mm.
Fig. 3. Metaphase I spindle (S). Scale line, 0-02 mm.
Fig. 4. Two large areas of disrupted cytoplasm (C) caused by sperm entry. Scale
line, 004 mm.
Fig. 5. A spindle (S) of sperm origin found in a large area of cytoplasmic disruption.
Scale line, 002mm.
Fig. 6. Pigment accumulation (P) caused by a sperm in the yolk-rich cytoplasm. Scale
line, 002 mm.
Fig. 7. Two polar bodies {PB) forming 30 min post-activation. The adjacent section
contains the bulk of one of the polar bodies Scale line, 001 mm.
Fig. 8. Three asters (A) descending from the surface 30 min post activation. Scale
line, 004 mm.
Fig. 9. A male pronucleus (N) in an astral area and with a penetration path. Scale
line, 004 mm.
R. P. ELINSON
iyo
inseminated eggs
Table 4. Comparison of cleavage patterns in locally
t
Cleavage pattern
(% of cleaved)
Eggs
Immature
Mature
Immature
Mature
Number
Cleaved
(%)
Animal
half*
Abnormal
or
puckersf
EquatorialJ
270
114
Animal half insemination
90
85
9-5
94
94
2-8
4-5
0-9
294
184
Equatorial insemination
59
36
13
92
88
12
51
0
* An animal half furrow in an inseminated mature egg is a normal furrow dividing the
egg in half. An animal half furrow in an inseminated immature egg is one or more furrows
cutting through the animal half.
f Puckers refer to small surface contractions. Abnormal furrows refer to furrows only in
inseminated mature eggs which fail to divide the egg in half.
t An equatorial furrow is seen in Fig. 11.
Local insemination of immature eggs
Previous experiments had indicated that the cortical contraction associated
with activation played a role in moving the entering sperm nearer to the female
pronucleus (Elinson, 1975). Since the sperm entering an immature egg does not
induce the cortical contraction, it was of interest to see whether sperm entering
near the equator led to different consequences than sperm entering near the
animal pole.
When mature eggs were locally inseminated, the frequency of fertilization and
the time of first cleavage were the same regardless of whether the insemination
site was over the equator or well within the animal half (Elinson, 1975; present
results). With inseminations in the animal half, the first cleavage was normal in
almost all eggs (Table 4). With inseminations at the equator, about 12 % of the
cleavages were abnormal. The abnormal eggs often had two furrows not at
right angles. In addition, another 12 % of the eggs delayed completing the
cleavage on the side of the egg opposite to the site of sperm entry, but the furrow
was normal once completed. On eggs inseminated at the equator, part of the
paternal streak representing the sperm entry site (Elinson, 1975), could be seen
on living eggs (Fig. 10). When the eggs cleaved, the furrow was usually close to
the streak.
In contrast to mature eggs, when immature eggs were locally inseminated and
subsequently electrically activated, fewer eggs inseminated at the equator began
cleaving within 3 h (Table 4). (The 3-h criterion was used since the first cleavage
of normally inseminated eggs occurs at 2-5 h and since abortive, incomplete
Fertilization of immature frog eggs
197
Fig. .10. Part of a paternal streak (S) representing the sperm entry site on a living
egg. This mature egg was inseminated at the equator. The streak is close to the
cleavage furrow (F). Scale line, 0-2 mm.
Fig. 11. An equatorial furrow. Scale line, 0-2 mm.
furrows form in uninseminated eggs at later times. The abortive furrows could
be easily confused with irregular sperm induced furrows). The lower frequency
of cleavage was not necessarily due to a lower frequency of sperm entry. Fourteen
eggs inseminated at the equator which failed to cleave within 3 h after activation
were fixed, sectioned and examined for male pronuclei; 12 had pronuclei. The
pronuclei were dividing in the large yolk area, but they did not have an extensive
astral system.
The immature eggs inseminated at the equator which did cleave generally
began cleavage at least 10 min after eggs inseminated in the animal half. This
delay, however, is not responsible for the lower frequency of cleavage scored at
3 h for eggs inseminated at the equator. When eggs were scored for blastula
formation, the frequencies were 10-20 % lower than when scored for cleavage
at 3 h with eggs inseminated either at the equator or in the animal half.
The cleavage furrow pattern was different on immature eggs inseminated in
the animal half or at the equator. Most of the eggs inseminated in the animal
half had one or more furrows in the animal half (Table 4). About 10 % of the
cleaving eggs had animal half puckers rather than furrows. These may not have
been caused by sperm since similar puckers appear on activated unfertilized
eggs. Only 5 % of the cleaved eggs had a furrow which could be called an
equatorial furrow (Fig. 11). In contrast, about half of the cleaving eggs which
were inseminated at the equator had an equatorial furrow on subsequent
activation (Table 4).
In summary, immature eggs inseminated at the equator differed from immature eggs inseminated in the animal half and from locally inseminated
mature eggs. They showed delays in first cleavage and had a lower frequency of
cleavage. They also frequently have an equatorial type of furrow.
198
R. P. ELINSON
DISCUSSION
The results demonstrate that sperm which have resided in unactivated egg
cytoplasm for a day retain their ability to promote cleavage and to provide
chromosomes. There are a number of reasons why the eggs, which were fertilized
when immature, later showed low frequencies of cleavage and development.
First, the storage of eggs for a day in 100 % Ringer's probably diminished their
developmental potential. Frog eggs maintained in 100 % Ringer's differ from
uterine eggs in rate of protein synthesis and in morphology (Smith & Ecker,
1970). A second explanation is that since the eggs were polyspermic, multiple
cleavage centers or centrioles were present in the egg cytoplasm. Multiple
centers would lead to multipolar divisions, abnormal cleavage patterns, and
unequal distribution of chromosomes among the cells. Development of polyspermic frog eggs is generally poor (see Morgan, 1927). However, even at low
sperm concentrations where the frequency of monospermy should be higher,
development was poor. An additional explanation is that the sperm's chromatin
is induced to form chromosomes without undergoing DNA synthesis. Sperm
nuclei placed in eggs prior to metaphase II do not synthesize DNA (Skoblina,
1974), but they do condense. Upon activation, the sperm spindles described in
the present report would segregate unreplicated chromosomes, some of which
would be thrown out into polar bodies or otherwise lost. The utilization of the
remaining chromosomes to form the zygote nucleus would produce aneuploid
nuclei with resultant abnormalities.
Besides the above explanations, the sperm nuclei were found in abnormal
locations within the immature eggs. In particular, nuclei were found in the yolkrich cytoplasm and these nuclei failed to alter the surrounding cytoplasm as did
nuclei in yolk-poor cytoplasm. Similar observations have been made on nuclei
in polyspermic urodele eggs (Fankhauser & Moore, 1941). Nuclei in yolk-rich
cytoplasm were capable of dividing, but they did not move to the normal area of
pronuclear association. The nuclei and their associated centrioles frequently
failed to induce cleavage or induced an abnormal furrow. Since a furrow is a
result of an astral-cortical interaction (Kubota, 1966; Rappaport, 1971), the
small or absent asters associated with nuclei in yolk-rich cytoplasm probably
cannot reach the cortex to induce it to furrow.
I had previously suggested that the cortical contraction associated with frog
egg activation serves to bring the sperm nucleus towards the egg nucleus, thus
helping the nuclei to reach each other (Elinson, 1975). The present experiments
support this hypothesis. Sperm entrance into immature eggs occurs without the
cortical contraction. More than half of the immature eggs inseminated at the
equator which cleaved on shocking had their initial cleavage furrow near
the equator rather than near the animal pole. This unusual furrowing would be
expected if the sperm nucleus were not shifted towards the animal pole. In eggs
where the sperm responsible for cleavage was sunk deep into the cytoplasm, the
Fertilization of immature frog eggs
199
cortical contraction which occurred upon electrical shocking would not be
expected to move it. Its equatorial position would lead to an equatorial cleavage.
In eggs where the sperm remained in the cortex, the later shock should have
moved it and the resulting cleavage should have been less equatorial. However,
even when the sperm remained in the cortex, it may have led to an equatorial
cleavage since the cortical contraction of the shocked eggs stored in 100 %
Ringer's did not appear to be as extensive as that of freshly squeezed eggs. In
either case, there is a clear defect of the positioning of sperm nuclei which induces
furrowing in equatorially inseminated immature eggs and the results are consistent with the idea that the defect is the separation in time of the cortical
contraction from sperm entry.
In amphibian fertilization, the sperm must bring in the cleavage centers
(which are probably centrioles), as demonstrated by the following evidence. If
an unfertilized egg is activated artificially, it is only capable of abnormal furrows
which regress. If an egg is fertilized and the female nucleus removed, cleavage
proceeds normally and the embryo develops as an androgenetic haploid. When
an egg is activated and supplied with microtubular elements in frog (Fraser,
1971), the eggs will cleave parthenogenetically. The microtubular elements
provided by the experimenter substitute for the cleavage centers supplied by the
sperm. Similar results have been obtained with fish eggs (Iwamatsu, MikiNomura & Ohta, 1976). Recent experiments indicate that the cleavage-initiating
substances may in fact be centrioles (Heidemann & Kirschner, 1975; Mailer
et al. 1976).
It is not known if the frog egg possesses centrioles associated with the meiotic
spindle. Xenopus oocytes at the pachytene stage have centrioles (Coggins, 1973).
Although mammalian eggs at pachytene also have centrioles, the meiotic
spindles lack them (Szollosi, Calarco & Donahue, 1972). The experiments here
demonstrate that the sperm's centrioles can survive in the unactivated egg
cytoplasm for a day, so that residence in the egg cytoplasm for this period of
time is insufficient to inactivate the centrioles. The fate of the egg's centrioles
remains unknown.
The action of the centriole in organizing an aster or a spindle is dependent
upon the cytoplasmic conditions. When oocytes prior to germinal vesicle breakdown are provided with centrioles by fertilization or by injection, asters do not
form (Hagstrom & Lonning, 1961; Franklin, 1965; Heidemann & Kirschner,
1975). Variable results are obtained when eggs undergoing meiotic divisions are
provided with centrioles. In sea urchin (Hagstrom & Lonning, 1961) and frog
(Bataillon, 1929), sometimes asters form and sometimes anastral spindles
develop. In the present experiments, the sperm induced the formation of small
spindles and caused disruptions of the cytoplasm in immature eggs but typical asters did not form. The disrupted cytoplasmic areas may be regions of
irregularly assembled microtubular elements. Upon activation after the eggs
achieved maturity, typical asters developed. This result indicates two things.
200
R. P. ELINSON
First, polymerization of microtubules to form the small meiotic spindles can
occur under conditions where polymerization to form typical asters does not
occur. Secondly, the failure of asters to form in eggs undergoing the meiotic
divisions prior to activation is not due to the lack of centrioles. When provided
with centrioles, typical asters do not form. Rather the cytoplasmic conditions
of these eggs do not permit aster formation. These conditions are altered on
activation of the egg.
I would like to thank James Norton for his suggestions and observations, and Malka
Goldenberg for her techrical assistance. The work was supported by the National Research
Council of Canada.
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