/. Embryol. exp. Morph. Vol. 32, 2, pp. 325-335, 1974 325 Printed in Great Britain

A block to cross-fertilization located in the jelly of the frog Rana clamitans

By RICHARD P. ELINSON1 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 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 inter- generic fertilizations are possible among amphibians (Montalenti, 1938; Moore, 1955). Moore (1941), in describing a series of interspecific inseminations, men­ tioned that there did not appear to be differences in fertilization frequencies between homospecific and heterospecific combinations. Experiments on de­ jellied 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. 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 fertiliza­ tion by foreign sperm. Katagiri (1966) demonstrated that the failure of fertiliza­ tion 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. Experi- ments by Blackler & Gecking (1972) indicated that the jelly coat of Xenopus mulleri eggs allowed few fertilizations by Xenopus laevis sperm. They hypothe­ sized 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 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 , 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 experi­ ment 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 re­ quired 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) Host's eggs (R. pipiens)

, " \ i •> No. of No. % No. of No. % Sperm eggs fertilized fertilized eggs fertilized fertilized R. pipiens 3050 517 17 5258 4823 92 R. clamitans 1367 1090 80 2374 2285 96 observations were made. First, the eggs were inseminated with high concentra­ tions 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 inject­ ing 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 ovi­ ducts 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 egg- jelly combination ...

No. of No. % No. of No. % Sperm eggs fertilized fertilized eggs 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

No. of No. % Sperm* Medium eggs fertilized fertilized R. pipiens 10% Ringer's solution 403 25 6-2 R. pipiens egg-water 329 189 57 R. pipiens 10% Ringer's solution 359 22 61 R. clamitans egg-water 397 82 21 R. clamitans 10% Ringer's solution 264 12 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 con­ tamination 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 com­ bination, 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 nor­ mally until hatching. R. clamitans eggs fertilized by R. pipiens sperm developed through the blastula stage and began gastrulating. They formed large 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 unincor­ porated 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 fertiliza­ tion 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 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 un­ expected 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 ovi- ducal 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 fertiliza­ tion 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. ELINSON I would like to thank Dr Yoshio Masui for his comments, Richard Hall for technical assist­ ance, 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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