/. Embryol. exp. Morph. Vol. 32, 2, pp. 325-335, 1974
Printed in Great Britain
A block to cross-fertilization located in the egg
jelly of the frog Rana clamitans
By R I C H A R D P. E L I N S O N 1
From the Department of Zoology, University of Toronto
SUMMARY
The egg of Rana clamitans enrobed in its native jelly was not fertilized by sperm of R. pipiens.
However, when R. clamitans eggs were enrobed by R. pipiens jelly, they were fertilized by
R. pipiens sperm. Male pronuclei were found in the eggs, and most of the resulting embryos
were diploid. The embryos gastrulated, but most arrested at mid- to late gastrula stages.
Some begun neurulation, but none survived longer than 4 days. When R. clamitans eggs in
R. pipiens jelly were fertilized by R. clamitans sperm, the embryos developed normally
except that they failed to hatch.
In the reciprocal experiment, R. pipiens eggs were enrobed in R. clamitans jelly. The eggs
were not fertilized by R. pipiens sperm but were fertilized by R. clamitans sperm. Therefore,
the R. clamitans jelly plays a major role in preventing fertilization by sperm of other species.
The if?, clamitans jelly's block to cross-fertilization was not a block to sperm migration.
Further, the R. clamitans jelly contained factors which permitted R. pipiens sperm to fertilize
dejellied R. pipiens eggs. Dejellied eggs are usually not fertilizable in the absence of jelly
factors.
INTRODUCTION
It is clear, from the results of a variety of experimental approaches, that the
jelly which surrounds the amphibian egg plays an important role in fertilization
(see Shaver, 1966; Elinson, 1973b, for references). Species-specific differences
have been described in the morphology of the jelly coat (Salthe, 1963) as well
as in the antigens of the jelly coat (Shivers, 1965; Katagiri, 1967; Shaver, Barch
& Umpierre, 1970). The question arises as to whether the jelly coat plays any
role in the species-specificity of fertilization.
There is evidence that in many cases there is a lack of species-specificity in
amphibian fertilization, and that there is a lack of species-specific action by the
jelly coat in particular. For instance, a large number of inter-specific and intergeneric fertilizations are possible among amphibians (Montalenti, 1938; Moore,
1955). Moore (1941), in describing a series of interspecific inseminations, mentioned that there did not appear to be differences in fertilization frequencies
between homospecific and heterospecific combinations. Experiments on dejellied eggs have shown that jelly preparations from one species can support
fertilization of dejellied eggs of a second species even though, in some cases,
1
Author's address: Department of Zoology, University of Toronto, Toronto, Ontario
M5S1A1, Canada.
325
326
R. P. ELINSON
cross-fertilization does not occur between the species in question (Katagiri,
1966, 1967; Elinson, 1971a).
On the other hand, there are two reports of the jelly coat preventing fertilization by foreign sperm. Katagiri (1966) demonstrated that the failure of fertilization involving Hyla arborea japonica eggs and Rana chensinensis sperm was
du e to the failure of the Rana sperm to penetrate into the Hyla jelly coat. Experiments by Blackler & Gecking (1972) indicated that the jelly coat of Xenopus
mulleri eggs allowed few fertilizations by Xenopus laevis sperm. They hypothesized that the X. laevis sperm were unable to attach to the X. mulleri jelly coat
and hence few sperm reached the egg. In addition, Shivers (1962) and Shaver
et al. (1970) showed, using antisera against jelly, that there are species-specific
jelly antigens which play some role in fertilization.
It is likely that each cross-fertilization combination would have to be analysed
individually to see whether there is species-specificity for that combination.
The specificity may reside at the egg cell membrane, at the vitelline coat, or in
the jelly. If the specificity resides in the jelly, then a comparison of the jelly
properties in the two species would help to elucidate the mechanisms of jelly
action in fertilization.
All inseminations reported between species of North American Rana lead to
fertilization, with one exception : foreign sperm are unable to fertilize the eggs of
Rana clamitans. The experiments reported here demonstrate that this failure of
cross-fertilization is due to the jelly surrounding the R. clamitans egg. The
experiments delineate a set of possible roles that the jelly could be playing in
preventing cross-fertilization.
MATERIALS AND METHODS
Sexually mature R. pipiens were obtained from J. M. Hazen and Co., Alburg,
Vermont, and the J. R. Schettle Frog Farm, Stillwater, Minnesota, during the
fall or winter. Sexually mature R. clamitans were collected near Snelgrove and
Milton, Ontario, in the late spring, and obtained from Connecticut Valley
Biological Supply Co., Southampton, Massachusetts, from Mogul-Ed, Oshkosh,
Wisconsin, and Nasco-Steinhilber, Fort Atkinson, Wisconsin, also in the late
spring. Females were induced to ovulate by injection of one to three female
pituitaries intraperitoneally, and 0-25 mg progesterone intramuscularly if
required.
Body-cavity eggs were transferred between frogs using a previously described
technique (Smith, Ecker & Subtelny, 1968). Donor eggs were stained with 0-1 %
neutral red in Ringer's solution for 1 min and washed to remove excess stain.
Following passage down the oviducts, eggs were inseminated with sperm of
R. pipiens (0-4 testes/ml of 10 % Ringer's solution) or of R. clamitans (0-2 testes/
ml of 10% Ringer's solution) following standard procedures. Donor and host
eggs were separated on the basis of the neutral red staining. In most cases,
Frog jelly and cross-fertilization
327
R. pipiens eggs were much larger than the R. clamitans eggs, and size of the egg
served as confirmation to the staining. Eggs were scored as unfertilized or
fertilized based on blastula formation. Eggs which had abortive furrows or had
puckered surfaces were not included in the data. These had been activated, but
it could not be determined whether they had been injured, or had been fertilized
but failed to develop. The experiments were carried out at room temperature
(21-23 °C).
Dejellied eggs were inseminated as described previously (Elinson, 1971 a). The
sperm concentrations used were 0-67 testes/ml for R. pipiens and 0-33 testes/ml
for JR. clamitans. Control inseminations of jellied eggs were run with each experiment to check for contamination of the sperm suspension by sperm of the other
species; no contamination was found. R. clamitans egg-water was prepared
similarly to the preparation of R. pipiens egg-water (Elinson, 1971a) except that
the former had to be separated from the eggs by filtering it through cheese
cloth, due to the nature of the R. clamitans jelly. (The outer jelly layer of the
R. clamitans jelly coat expands markedly in water filling the culture dishes, and
the eggs do not remain stuck to the dishes as do R. pipiens eggs.) Inseminated
eggs were kept overnight and scored for fertilization on the following day.
Embryos or eggs were fixed in Smith's fixative and stored in 4 % formalin.
Eggs were sectioned at 9-10 /mi, stained with Feulgen and counterstained with
light green (Moore & Ryan, 1940) for examination of the nucleus. Chromosome
preparations of late blastulae or early gastrulae were made by the method of
DiBerardino (1962).
RESULTS
Cross-insemination between R. clamitans and R. pipiens
In the course of these experiments, several thousand R. clamitans eggs in their
native jelly were inseminated by R. pipiens sperm. With one questionable
exception described later, no eggs were fertilized. This was true even when
extremely high concentrations of R.pipiens sperm were used. Sperm preparations
at concentrations of more than two orders of magnitude greater than that required to obtain 90 % fertilization of R. pipiens eggs failed to fertilize R. clamitans
eggs. The inseminated eggs did not rotate, and were not activated by R. pipiens
sperm. When eggs were sectioned 1 h post-insemination, the female nucleus was
in metaphase II, the meiotic state of an unactivated egg. Male pronuclei were not
seen, although a condensed nucleus which remained in the pigmented cortex and
formed no aster would be exceedingly difficult to detect.
The failure of JR. pipiens sperm to fertilize R. clamitans eggs was not due to a
failure of sperm migration through the jelly to the eggs. It is often difficult to find
amphibian sperm near the egg surface using sperm concentrations which are
sufficient to ensure fertilization. This is due in part to the large size of the egg.
In order to see if R. pipiens sperm migrated through R. clamitans jelly, two
328
R. P. ELINSON
Table 1. Fertilization of R. clamitans eggs transferred to R. pipiens body cavities
The results are the sum of a number of experiments in which 11 donors, 16 hosts, MR. pipiens
sperm suspensions, and 8 R. clamitans sperm suspensions were used in total.
Donor's eggs (R. clamitans)
,
Sperm
R. pipiens
R. clamitans
"
\
Host's eggs (R. pipiens)
i
•>
No. of
eggs
No.
fertilized
%
fertilized
No. of
eggs
No.
fertilized
%
fertilized
3050
1367
517
1090
17
80
5258
2374
4823
2285
92
96
observations were made. First, the eggs were inseminated with high concentrations of sperm. In these inseminations, a very large number of sperm were seen
near the egg surface. Secondly, the application of sperm was localized by injecting sperm into an area of the outer jelly layer. Sperm were observed to move
from the injection point through the jelly and to reach the egg surface. Despite
the presence of far larger numbers of sperm near the egg surface than seen
normally, no fertilizations occurred in these two experiments.
It did not appear that the R. pipiens sperm penetrated the egg's vitelline coat.
Previous experiments have shown that when sperm digest through the vitelline
coat but fail to activate eggs, a bleb forms on the egg surface (Elinson, 1971 b).
Blebs were never seen on R. clamitans eggs inseminated by R. pipiens sperm.
In the reciprocal cross, R. pipiens eggs were fertilized by R. clamitans sperm,
and the resulting embryos invariably arrested at the end of the blastula stage.
A slight invagination indicating the start of gastrulation was occasionally
noticed, but the embryos developed no further. These results confirm Moore's
(1941, 1949) results.
Reciprocal egg transfers between R. clamitans and R. pipiens
In order to examine the role of the jelly in the inability of R. pipiens sperm
to fertilize R. clamitans eggs, R. clamitans body cavity eggs were transferred to
the body cavities of R. pipiens females. The eggs travelled down the host's oviducts and acquired a jelly coat characteristic of R. pipiens. Upon insemination
with R. pipiens sperm, the R. clamitans eggs coated with foreign jelly began
development (Table 1).
The blastulae which formed as a result of this insemination could have
Frog jelly and
cross-fertilization
329
Table 2. Fertilization ofR. pipiens eggs transferred to R. clamitans body cavities
The results are the sum of a number of experiments in which 6 donors, 8 hosts, 4 R. pipiens
sperm suspensions, and 4 R. clamitans sperm suspensions were used in total.
Donor's eggs (/?. pipiens)
Host's eggs (R. clamitans)
Diagram of eggjelly combination . . .
Sperm
No. of
eggs
No.
fertilized
%
fertilized
No. of
eggs
No.
fertilized
%
fertilized
R. pipiens*
380
3
0-8
1328
1
008
R. clamitans
280
215
77
1632
1605
98
* To control for sperm quality, normally jellied R. pipiens eggs were inseminated with the
R. pipiens sperm used in these experiments. Of 491 eggs, 468 (= 95%) were fertilized.
developed from eggs into which spenrfpenetrated, or from eggs which in some
way were stimulated parthenogenetically without sperm entry. To rule out this
latter possibility, eggs were fixed 1 h after insemination and sectioned for nuclear
examination. Of 103 eggs sectioned, 82 had a metaphase II spindle and no
evidence of sperm entry; 11 had a male pronucleus and the metaphase II block
was broken, and 10 had gross disruptions of pigment and cytoplasm. Of 138 eggs
from the same groups which were allowed to develop, 110 remained unactivated,
15 developed to the blastula stage, and 13 cytolysed. These results indicate
that the R. pipiens sperm did enter the JR. clamitans eggs. The eggs with gross
pigment disruptions were probably injured prior to insemination, and clearly
would have cytolysed if left unfixed. The results strongly suggest that the eggs
which began development were the eggs into which the sperm entered. Control
experiments indicated that the fertilizations were not a result of the transfer
procedure or of sperm contamination.
Reciprocal transfers of body cavity eggs between the two species produced
R. pipiens eggs coated with R. clamitans jelly. Although these eggs were fertilized
by R. clamitans sperm, R. pipiens sperm did not fertilize them (Table 2). This
result, coupled with the result of the other transfer series, demonstrates that the
R. clamitans jelly plays a major role in preventing cross-fertilization.
As can be seen in Table 2, three R. pipiens eggs with R. clamitans jelly and one
host R. clamitans egg were apparently fertilized by R. pipiens sperm. The three
fertilized R. pipiens eggs were raised to the tadpole stage. All hatched, developed
330
R. P. ELINSON
Table 3. Fertilization of dejellied R. pipiens eggs with
components from R. pipiens and R. clamitans
Sperm*
Medium
No. of
eggs
No.
fertilized
%
fertilized
R. pipiens
10% Ringer's solution
403
25
6-2
329
189
R. pipiens egg-water
57
R. pipiens
10% Ringer's solution
359
22
61
397
82
R. clamitans egg-water
21
264
12
R. clamitans
10% Ringer's solution
4-5
R. pipiens egg-water
239
201
84
R. clamitans
10% Ringer's solution
295
11
3-7
R. clamitans egg-water
337
305
91
* To control for sperm quality, normally jellied R. pipiens eggs were inseminated with the
sperm used in these experiments. R. pipiens sperm fertilized 95 % of 745 eggs and R. clamitans
sperm fertilized 97% of 725 eggs.
normally, and appeared to be diploid. They could not have resulted from contamination of the R. pipiens sperm suspension with R. clamitans sperm since
R. pipiens eggs fertilized by R. clamitans sperm invariably arrest at gastrula. It
is not certain whether the R. clamitans egg which began development was
fertilized, or whether it developed parthenogenetically. Cleavage in the vegetal
region was irregular, and the embryo failed to gastrulate. It was clearly a R.
clamitans egg on the basis of size and lack of staining. If the embryo resulted
from fertilization, it represents a unique case of a R. clamitans egg in its native
jelly being fertilized by a foreign sperm.
Fertilization of dejellied R. pipiens eggs with sperm and egg-water
from R. pipiens and R. clamitans
Eggs without jelly are generally not fertilized when placed with sperm.
However, the presence of materials leached from fully jellied eggs (egg-water)
ensures the fertilization of eggs which have had their jelly removed (Elinson,
1971 a). It is possible that the factors in egg-water are species-specific, and that
the inability of foreign sperm to utilize R. clamitans factors is the cause of their
failure to fertilize R. clamitans eggs. To test this, fertilization of dejellied R.
pipiens eggs in the presence of R. clamitans egg-water was attempted.
As seen in Table 3, dejellied R. pipiens eggs were fertilized by R. pipiens sperm
in the presence of egg-water from either R. pipiens or R. clamitans at frequencies
which were considerably higher than the frequencies in 10% Ringer's solution.
Four different R. pipiens and six different R. clamitans egg-water preparations
all had some activity. Although the R. pipiens sperm-i?. clamitans egg-water
combination gave the lowest frequency of fertilization of an experimental combination, it is clear that preparations can be made from R. clamitans jelly which
support fertilization by R. pipiens sperm.
Frog jelly and
cross-fertilization
331
Development ofK. clamitans eggs fertilized by R. pipiens sperm
The development of embryos from some of the donor R. clamitans females
was followed. (All R. clamitans eggs to be discussed in this section had R. pipiens
jelly coats.) R. clamitans eggs fertilized by R. clamitans sperm developed normally until hatching. R. clamitans eggs fertilized by R. pipiens sperm developed
through the blastula stage and began gastrulating. They formed large yolk plugs,
but appeared to be lagging behind the control embryos. On the second day
most of the R. clamitans $ x R. pipiens $ embryos had large amounts of unincorporated yolk and were dead or dying. Some of them showed various degrees of
external neural differentiation. This ranged from a short neural plate to more or
less normal neural folds. All of the embryos died by the third day except those
from one donor. These remaining embryos were shaped like stunted tail-bud
embryos, had little external morphology, and were dead by the fourth day.
Approximate counts of chromosome numbers were done on some R. clamitans
$ x R. pipiens <$ embryos to determine their ploidy. Of 40 late blastulae or
gastrulae scored, 31 appeared to be diploid, 5 appeared to be haploid, and 3
appeared to be triploid. One embryo appeared to be a mosaic. Of 10 figures
examined from this embryo, 6 had the haploid number of chromosomes, and
4 had a very large number of chromosomes (about the pentaploid level).
Attempts to demonstrate the presence or absence of R. pipiens genetic material
in these embryos through a preliminary karyotype analysis and through isozyme
analysis for lactic dehydrogenase and 6-phosphogluconate dehydrogenase have
not been successful. The developmental arrest is probably due to R. pipiens
genetic material, but the presence of R. pipiens genetic material in the eggs after
1 h post-insemination has not been demonstrated.
Failure ofR. clamitans embryos to hatch from R. pipiens jelly
The development of the R. clamitans $ x R. clamitans S embryos in R. pipiens
jelly was unusual in that, although the embryos appeared normal, they failed to
hatch. Under the conditions used, normal R. pipiens embryos in R. pipiens jelly
and normal R. clamitans embryos in R. clamitans jelly hatch on the third day
following insemination. Prior to hatching, the perivitelline chamber surrounding
these normal embryos increases in volume, thus providing more room for the
embryo. In contrast, the R. clamitans embryos in the R. pipiens jelly were still
tightly coiled in the perivitelline chamber on the third day. The R. pipiens jelly
surrounding the JR. clamitans embryos remained intact until the fifth day when
most of the jelly appeared to have dissolved. Although many of the unhatched
embryos died by about the fifth day, several were raised for 10 days, at which time
the normal R. clamitans embryo was a swimming tadpole (Fig. 1). R. clamitans
embryos in the R. pipiens jelly failed to hatch even when the outer two visible
jelly layers, V 2 and V 3, were removed, leaving the embryo surrounded by the
fertilization membrane and some of the inner visible jelly layer, V 1. A similar
332
R. P. ELINSON
Fig. 1. R. clamitans ?x ƒ?. clamitans $ embryo in R. pipiens jelly, 10 days after
insemination. A R. clamitans tadpole of the same age is present for comparison
with the unhatched experimental embryo. Scale-line = 1 mm.
hatching failure has been observed when embryos of the toad Bufo americanus
developed within the jelly of R. pipiens.
DISCUSSION
The experiments reported here demonstrate that the jelly plays a critical role
in preventing fertilization of the R. clamitans egg by a foreign sperm. To my
knowledge, this is the first case reported in which foreign sperm can migrate
through the jelly, and yet the jelly prevents cross-fertilization. (It is likely that
the cell membrane or the vitelline coat of the R. clamitans egg also contributes
to the failure of cross-fertilization; even with a R. pipiens jelly coat, the R.
clamitans'eggs were fertilized at a low frequency by R. pipiens sperm.) We can
consider two explanations for the failure of R. clamitans jelly to support fertilization by foreign sperm.
First, the R. clamitans jelly may not contain certain factors which are normally
used by Rana sperm in fertilization. Sperm of many different species undergo an
acrosome reaction in response to the egg investments. Since R. pipiens and
R. clamitans sperm have acrosomes (Poirier & Spink, 1971), it is likely that they
undergo an acrosome reaction. However, this reaction has never been described
for amphibian sperm. It is known from several lines of experimentation that the
jelly does affect the sperm in a functional sense (Shivers & James, 1970, 1971;
Elinson, 1971 a, b; Wolf & Hedrick, 1971) and this functional change could
include the acrosome reaction. One test available for examining the activity of
jelly factors in this functional change is to see whether or not they can support
fertilization of dejellied eggs. This test indicated that R. pipiens sperm can indeed
utilize R. clamitans jelly factors.
The question is then raised as to why R. pipiens sperm cannot utilize intact
R. clamitans jelly to fertilize eggs. One explanation is that the concentration or
Frog jelly and cross-fertilization
333
distribution of jelly factors in the intact jelly is different from that in egg-water.
Shaver et al. (1970) demonstrated a difference in the distribution of certain jelly
components between JR. pipiens and R. clamitans. The inactivation of these
components with antibodies depressed the frequency of fertilization, but no
difference which could account for the failure of foreign sperm to fertilize R.
clamitans eggs was noted.
A second possibility for the jelly's failure to support foreign sperm fertilization
is that the jelly may affect fertilization in ways other than those demonstrated by
the dejellied egg test. For instance, the jelly or other oviduct secretions may act
on the vitelline coat of the egg, making the coat more penetrable by sperm.
There is evidence that some substances from the oviduct become localized on the
egg surface or on the vitelline coat (Nace, Suyama & Smith, 1960; Humphries,
1970), and these substances could be important in fertilization. We could
postulate that R. clamitans jelly does not alter the vitelline coat in ways that
other Rana jelly does. As a result, foreign sperm cannot fertilize R. clamitans
eggs since they cannot penetrate the vitelline coat. This hypothesis requires that
the R. clamitans sperm have a greater capability for penetrating the vitelline
coat.
Recent experiments have demonstrated that the R. clamitans sperm does
contain more proteolytic activity directed against the vitelline coat than do
sperm of other amphibians (Elinson, 1973 c). In addition, it has been shown that
R. pipiens body cavity eggs required an experimental alteration of their vitelline
coat before they could be fertilized by R. pipiens sperm (Elinson, 1973a) but
required no alteration before R. clamitans sperm could fertilize them (Elinson,
1973 b). These observations support the hypothesis that the jelly affects the
vitelline coat with respect to fertilization.
The failure of R. clamitans embryos in R. pipiens jelly to hatch was an unexpected side-result of these experiments. The failure was not due to the bulk
of the jelly but rather to the jelly near the fertilization membrane or to the
fertilization membrane itself. Prior to actual hatching, there is an increase in
the volume of the perivitelline chamber (Cambar & Willaume, 1954; Kobayashi,
1954; Salthe, 1965). The mechanism of this increase is not known, but in the
hatching failure reported here, the perivitelline chamber did not show its usual
increase, indicating an early failure in the hatching process. If, in fact, an oviducal secretion affects the vitelline coat, the type of jelly applied to the egg could
influence hatching. However, it is premature to suggest a basis for the hatching
failure.
In summary, the R. clamitans jelly forms a species-specific block to fertilization which is not a block to foreign sperm migration. Despite this block, the
jelly contains factors which can support fertilization by foreign sperm. The
possibility is raised that the jelly can have an effect on the egg or its vitelline coat
and that this effect is important in fertilization.
334
R. P . E L I N S O N
I would like to thank Dr Yoshio Masui for his comments, Richard Hall for technical assistance, Donata Zulys for preliminary work on karyotypes and isozymes, and the Jay family for
their help in collecting frogs. This work was supported by grant No. A 6356 from the National
Reseach Council of Canada.
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{Received 10 December 1973; revised 28 January 1974)
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