Fertilization of immature frog eggs: cleavage and development following subsequent activation

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1 /. Embryol. exp. Morph. Vol. 37, pp , 1977 \%~] Printed in Great Britain Fertilization of immature frog eggs: cleavage and development following subsequent activation By RICHARD P. ELINSON 1 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.

2 188 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, 193; 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 2 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 2 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.

3 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 1 % Ringer's but they are immotile in 1 % Ringer's. Immature eggs, stored in 1 % Ringer's, retain their activatability, but they cannot be stored in 1 % Ringer's. Activated eggs can develop in 1 % Ringer's, but they do not develop in 1 % Ringer's. Accordingly, sperm in 1 % Ringer's were used to inseminate immature eggs laid dry in a dish. After a 1-min insemination the eggs were kept in 1 % Ringer's for 1-5 min. They were then transferred to 1 % Ringer's for storage until the following day when they were mature. They were electrically activated (see below) in 1 % Ringer's and transferred to 1 % Ringer's for development. Control experiments included: eggs uninseminated but stored in 1 % Ringer's and activated when mature to see if activation without insemination would lead to development; eggs inseminated and left in 1 % Ringer's to see if any were mature at the time of insemination, and eggs inseminated, stored in 1 % Ringer's, and transferred to 1 % Ringer's to see if transfer alone caused development. The eggs were shocked in a Plexiglass chamber (6 x 2 x 4 mm) with a platinum wire electrode at either end. Five pulses of 1 V lasting for 1 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 1 % Ringer's until mature, have a wrinkled surface. The 1 % 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

4 19 R. P. ELINSON Table 1. Cleavage of shocked eggs inseminated when immature v. frequency of fertilization Female Sperm concentration (x 1 5 /ml) Blastulae(%) Fertilization (%) (monospermy, %)* 81 (5) 23 (12) 74 (3) 38 (31) 98 (2) 67 (5) 93 (43) 18 (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 1 % 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 1 %. 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

5 Fertilization of immature frog eggs 191 Table 2. Re insemination of eggs inseminated when immature Column st insemination... pipiens pipiens pipiens 2nd insemination... pipiens pipiens clamitans clamitans No. Bias- No. Bias- No. Bias- No. Bias- No. Blasof tula of tula of tula of tula of tula Female eggs (%) eggs (%) eggs (%) eggs (%) eggs (%) A a* b* 5t 6 t Sum * 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 9 %. After storage in 1 % 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

6 192 R. P. ELINSON Table 3. Development of immature eggs fertilized by Rana pipiens and R. clamitans sperm R. pipiens sperm R. clamitans sperm Female No gastrulation Neurulation No gastrulation % Exogastrulation / * /o Exogastrulation V * /o Neurulation 1 1 2% Sum V / A1 /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 1 % 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 1 %, 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 1 % Ringer's for a day and then inseminated. Of 127 eggs inseminated when immature, stored, and then shocked, 3 % began neurulation and 17 % developed to tail-bud embryos (stage 17). Of 163 eggs stored and then inseminated, 67 % began neurulation and 4 % developed to tail-bud embryos. Since normal development of greater than 9 % of R. pipiens embryos can be expected, storage of the eggs has probably diminished their developmental potential. Storage, however, is not the only

7 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

8 194 R. P. ELINSON

9 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 6 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, 2 mm. Fig. 2. Two cortical spindles (S) of sperm origin. Scale line, 2 mm. Fig. 3. Metaphase I spindle (S). Scale line, -2 mm. Fig. 4. Two large areas of disrupted cytoplasm (C) caused by sperm entry. Scale line, 4 mm. Fig. 5. A spindle (S) of sperm origin found in a large area of cytoplasmic disruption. Scale line, 2mm. Fig. 6. Pigment accumulation (P) caused by a sperm in the yolk-rich cytoplasm. Scale line, 2 mm. Fig. 7. Two polar bodies {PB) forming 3 min post-activation. The adjacent section contains the bulk of one of the polar bodies Scale line, 1 mm. Fig. 8. Three asters (A) descending from the surface 3 min post activation. Scale line, 4 mm. Fig. 9. A male pronucleus (N) in an astral area and with a penetration path. Scale line, 4 mm.

10 iyo R. P. ELINSON Table 4. Comparison of cleavage patterns in locally t inseminated eggs Eggs Immature Mature Immature Mature Number Cleaved Animal (%) half* Cleavage pattern (% of cleaved) Abnormal or puckersf EquatorialJ Animal half insemination Equatorial insemination * 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. 1). 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

11 Fertilization of immature frog eggs 197 Fig..1. 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, -2 mm. Fig. 11. An equatorial furrow. Scale line, -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 1 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 1-2 % 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 1 % 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.

12 198 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 1 % Ringer's probably diminished their developmental potential. Frog eggs maintained in 1 % Ringer's differ from uterine eggs in rate of protein synthesis and in morphology (Smith & Ecker, 197). 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

13 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 1 % 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, Miki- Nomura & 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.

14 2 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. REFERENCES AUSTIN, C. R. (1965). Fertilization. Englewood Cliffs, N.J.: Prentice-Hall. BATAILLON, E. (1929). Etudes cytologiques et experimentales sur les oeufs immatures de batraciens. Arch. EntwMech. Org. 117, BATAILLON, E. & TCHOU SU (193). Etudes analytiques et experimentales sur les rythmes cinetiques dan l'ceuf (Hyla arborea, Paracentrotus lividus, Bombyx mod). Archs Biol., Paris 4, BRACHET, A. (1922). Recherches sur la fecondation prematuree de l'oeuf d'oursin {Paracentrotus lividus). Archs Biol., Paris 32, BRUN, R. (1975). Oocyte maturation in vitro: contribution of the oviduct to total maturation in Xenopus laevis. Experientia 31, COGGINS, L. W. (1973). An ultrastructural and radioautographic study of early oogenesis in. the toad Xenopus laevis. J. Cell Sci. 12, DAS, N. K. & BARKER, C. (1976). Mitotic chromosome condensation in the sperm nucleus during postfertilization maturation division in Urechis egg. /. Cell Biol. 68, Di BERARDINO, M. A. (1967). Frogs. In Methods in Developmental Biology (ed. F. H. Wilt & N. K. Wessels), pp New York: Thomas Y. Crowell. ELINSON, R. P. (1971). Sperm lytic activity and its relation to fertilization in the frog Rana pipiens. J. exp. Zool. 177, ELINSON, R. P. (1973). Fertilization of frog body-cavity eggs: Rana pipiens eggs and Rana clamitans sperm. Biol. Reprod. 8, ELINSON, R. P. (1974#). A block to cross-fertilization located in the egg jelly of the frog Rana clamitans. J. Embryol. exp. Morph. 32, ELINSON, R. P. (19746). A comparative examination of amphibian sperm proteolytic activity. Biol. Reprod. 11, ELINSON, R. P. (1975). Site of sperm entry and a cortical contraction associated with egg activation in the frog Rana pipiens. Devi Biol. 47, FANKHAUSER, G. & MOORE, C. (1941). Cytological and experimental studies of polyspermy in the newt, Triturus viridescens. T. Normal fertilization /. Morphol. 68, FRANKLIN, L. E. (1965). Morphology of gamete membrane fusion and of sperm entry into oocytes of the sea urchin. /. Cell Biol. 25, FRASER, L. R. (1971). Physico-chemical properties of an agent that induces parthenogenesis in Rana pipiens eggs. /. exp. Zool. Ill, GURDON, J. B. (1968). Changes in somatic cell nuclei inserted into growing and matuiing oocytes. /. Embryol. exp. Morph. 2, HAGSTROM, B. E. & LNNING, S. (1961). Studies of the species specificity of echinoderms. Sarsia 4, HEIDEMANN, S. R. & KIRSCHNER, M. W. (1975). Aster formation in eggs of Xenopus laevis. Induction by isolated basal bodies. /. Cell Biol. 67, IWAMATSU, T. & CHANG, M. C. (1972). Sperm penetration in vitro of mouse oocytes at various times during maturation. /. Reprod. Fert. 31,

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