MOLECULAR REPRODUCTION AND DEVELOPMENT 29:72-76 (1991)

Hamster Oocyte Penetrability During Preovulatory Maturation PATRICIA S. CUASNICU’ AND J.M. BEDFORD’32 Departments of ‘Obstetrics and Gynecology and T e l l Biology and Anatomy, Cornell University Medical College, New York ABSTRACT In vitro fertilization techniques were used to analyze the penetrability of preovulatory hamster oocytes. The zonas of granulosa cell-free primary (GV) oocytes were penetrated in vitro in 2-3 h as readily as those of ovulated secondary oocytes (80%vs. 88%), whether inseminated separately or as mixed oocyte groups. In fact, a significantly higher (P < 0.05) mean number of perivitelline spermatozoa was present in immature (3.6)compared with secondary (1.9)oocytes,primarily reflecting a lack of the zona block to polyspermy in the immature population. By contrast, when granulosa cells remained around GV oocytes, zona penetration was low and more were penetrated, with more spermatozoa incorporated into the vitellus as a function of increasing time of oocyte recovery after hCG. We conclude, contrary to previous reports,that the zona pellucida of the hamster GV oocyte is readily penetrable by spermatozoa in vitro. However, the resumption of meiosis brings an increase in the penetrability of the granulosa cell vestment as well as the capacity for cortical granule exocytosis and the ability to decondense and transform the fertilizing sperm nucleus. The fact that the zona pellucida of the immature oocyte has proved to be penetrable in vitro and/or in vivo in all the mammals studied in this respect is discussed with particular reference to the situation in man. Key Words: Penetration, Zona pellucida, Granulosa cells INTRODUCTION Primary oocytes cannot be fertilized normally in most mammals. The potential for this usually depends on the resumption of meiosis with a breakdown of the germinal vesicle, and then on associated changes in other oocyte structures, prior to ovulation. In all species studied, the oolemma is already receptive to spermatozoa a t the germinal vesicle (GV) stage (e.g., pig, Polge and Dziuk, 1965; mouse, Iwamatsu and Chang, 1972; rat, Niwa and Chang, 1975; dog, Mahi and Yanagimachi, 1976; hamster; Usui and Yanagimachi, 1976; Moore and Bedford, 1978a,b; man, Lopata and Leung, 1988). However, the “immaturity” of the primary oocyte is certainly reflected in a n inability of the ooplasm to support sperm head decondensation (Thibault and Gerard, 1970; Iwamatsu and Chang, 1972; Niwa and Chang, 1975; Usui and Yanagimachi, 0 1991 WILEY-LISS, INC.

1976; Moore and Bedford, 1978a; Berrios and Bedford, 1979) (with the exception of canines, Mahi and Yanagimachi, 1976) and in the unresponsiveness of cortical granules to the stimulus of a fertilizing spermatozoon (Usui and Yanagimachi, 1976; Moore and Bedford, 1978a; Berrios and Bedford, 1979).The GV oocyte of the rabbit at least also characteristically fails to envelop the acrosomal region of the fusing spermatozoon with cortical folds and can not even disperse its nuclear membrane (Berrios and Bedford, 1979). In considering oocyte maturation, questions remain with regard to the functional state of the zona pellucida. The zona of the rabbit GV oocyte is as penetrable in vivo as that around ovulated oocytes (Overstreet and Bedford, 19741, and, although structural and histochemical changes occur in the zona pellucida of the maturing mouse oocyte (Kaufman et al., 1989), it can be penetrated in vitro within 1 h of insemination a t all stages of oocyte maturation (Iwamatsu and Chang, 1972). The picture is less clear in man. Primary oocytes taken from human cadaver ovaries are readily penetrated in vitro (Overstreet and Hembree, 19761,and the zonas of oocytes recovered from small follicles of unstimulated ovaries throughout the normal cycle were penetrated in vitro at all stages of meiosis (Lopata and Leung, 1988).However, one often finds that human GV and metaphase I oocytes recovered in hormone-stimulated cycles are not penetrated during clinical in vitro fertilization (IVF) procedures, and it has been proposed that a secretory contribution from the investing corona radiata (Tesarik and Kopecny, 1986) may act to “soften” and so make the human zona penetrable as the oocyte progresses beyond metaphase I (Tesarik et al., 1988). Similar inconsistencies exist for the hamster zona. The zona and vitellus of hamster oocytes with persistent GVs were penetrated over a period of 16-19 h after transfer to the oviduct of mated hamsters (Moore and Bedford, 1978a,b). However, Barros and Munoz (1974) have reported that within a more limited period the hamster GV oocyte zona is less penetrable in vitro than that of the ovulated egg. Similarly, hamster GV oocytes Received October 10, 1990; accepted January 10, 1991. Address reprint requests to P.S. Cuasnicu, Inst. de Biol. y Medicina Experimental, Vuelta de Obligado 2490, Buenos Aires 1428, Argentina.

SPERM PENETRATION OF IMMATURE HAMSTER OOCYTES were found not t o be penetrable in vitro unless taken 4-6 h or more after an human chorionic gonadotropin (hCG)/luteinizing hormone (LH) stimulus (Mandelbaum and Plachot, 1977; Mandelbaum et al., 1977). They implied that hamster oocyte penetrability depends on an oocyte-specific factor that appears only 4-6 h after hCG and perhaps involves a changing sensitivity of the zona pellucida to acrosomal enzymes. In the face of the possibility from the existing literature that the penetrability of immature hamster oocytes differs in vitro and in vivo, we have studied the in vitro penetrability of granulosa cell-free oocytes and that of granulosa cell-invested oocytes collected at different times after hCG.

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agimachi, 1982) containing 1 mgiml PVA and 10 p,M hypotaurine were incubated in tissue culture dishes under paraffin oil. A fresh solution of epinephrine was dissolved in the medium (1.7 p,M) just prior to the addition of spermatozoa, which were then incubated in a final concentration of 1-2 106iml for up to 4 h in an atmosphere of 5% C02 in air.

In Vitro Fertilization A 300 ml aliquot of freshly prepared m-TALP was placed at the center of a plastic Petri dish, covered with mineral oil, and kept in a C02 incubator at 37“. Mature or immature eggs with or without cumulus cells were transferred into this drop and kept at 37°C for less than 20 min. Insemination was performed by adding 20 pl of the sperm suspension preincubated for 3-4 h in the capacitating medium. After 2-3 h eggs were recovered, washed in fresh medium using a smallbore pipette to remove excess spermatozoa attached to the zona surface, and mounted on wax spot slides. At this time the granulosa cells surrounding the eggs had dispersed or were finally removed by pipetting in the groups recovered 6, 9, and 15 h post-hCG. By contrast many cells remained around the eggs recovered 0 and 4 h post-hCG, and these were removed by gently shaking for 7 min in a small tube containing 1ml of 2% sodium citrate (Moore and Bedford, 1978b). The eggs were examined with a phase-contrast microscope ( ~ 4 0 0 for ) evidence of sperm penetration before and after fixation in acetic alcohol and staining in Lacmoid. Before fixation, however, a record was made of the number of supplementary spermatozoa in the perivitelline space in living oocytes. Oocytes were classified as “penetrated” when spermatozoa with clearly distinguishable free heads were found in the perivitelline space and as “fertilized” if the sperm head at least was completely within the vitellus. When the penetrability of the granulosa cell complex was evaluated, nonpenetrated oocytes with spermatozoa attached or bound to the zona pellucida were also included in the data. However, since the proportion of such oocytes was very low, the final values were not affected by inclusion of this last group.

MATERIALS AND METHODS Collection of Eggs Oviductal oocytes were obtained from 4-5-week-old females injected with 30 IU pregnant mare’s serum gonadotropin (PMSG) (s.c) followed by 40 IU hCG (i.p.1 48-60 h later. About 15-20 ova in cumulus oophorus were released from the ampulla of each oviduct 15-17 h after hCG injection. These were then placed for 1-2 min in Tyrode’s medium containing bovine serum albumin (BSA) (3 mgiml) and 0.1% hyaluronidase (Sigma type IV) to remove cumulus cells. Primary oocytes with a germinal vesicle (GV oocytes) were recovered by pricking antral follicles of the PMSG-treated females at 0 h post-hCG. In some cases, the adhering granulosa cells were removed by shaking the oocytes for 1min in a small tube containing 1ml of 2% sodium citrate. The naked GV oocytes were then set aside in fresh Tyrode’s solution. When there was a need to distinguish mature oocytes from GV oocytes, one or the other population was first labeled by incubation for 1 min in Tyrode’s solution containing 0.1% fluorescein isothiocyanate (FITC), then washed three times in Tyrode’s medium. In studies of the penetrability of the granulosa layer as a function of the stage of oocyte maturation, immature cumulus-oocyte complexes were recovered from follicles at 0, 4, 6, and 9 h after the hCG injection. After washing in fresh medium, these were exposed directly Statistical Analysis to capacitated spermatozoa as described below. As controls, mature oocytes recovered in cumulus from the Data were analyzed for statistical significance by the oviduct 15 h after hCG injection were inseminated in x2 test for the percentage of sperm penetration in vitro at the same time. mature and immature oocytes and by the Wilcoxon signed rank-test for the number of penetrated spermaIn Vitro Capacitation tozoa per oocyte or egg. Spermatozoa released from the cauda epididymidis of adult golden hamsters (3-6 months) were allowed to RESULTS disperse in SWM (PBS:sucrose 0.3 M 1:l viv; 1 mgiml Penetrability of Granulosa Cell-Free GV Oocytes polyvinyl alcohol, 10 pm hypotaurine) at 37”C,and the The results of in vitro insemination of immature suspension was centrifuged at 200g for 5-7 min. The pellet was then resuspended in 1ml of SWM and highly cumulus-free GV oocytes with capacitated spermatozoa motile cells were separated from immotile or dead are presented in Table 1. At the time of final examispermatozoa, by passage through a column of 250-300 nation, the immature oocytes had not resumed meiosis, pm glass beads (Sigma) (Lui et al., 1979). Aliquots of as judged by the persistent presence of a germinal 0.5 ml of modified Tyrode’s medium (m-TALP) (Yan- vesicle and the absence of polar bodies. Nonetheless,

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P.S. CUASNICU AND J.M. BEDFORD TABLE 1. In Vitro Penetrability of Granulosa Cell-Free Hamster GV Oocytes and Secondary Oocytes No. of No. of Percent penetration experiments oocytes through ZP Secondary oocytesa Primary (GV) oocytesa Secondary oocytesb Primary (GV) oocytesb

88 80" 77 70"

157 146 76 77

6 3

aOocytes inseminated as separate populations. bOocytes inseminated as a mixed population in a dish, one being labeled randomly with FITC. CSpermatozoawere judged to be incorporated into the vitellus in 250% of GV oocytes, but invariably the sperm nucleus failed to decondense.

TABLE 2. Penetration In Vitro of Granulosa Cell-Invested Oocytes Recovered at Various Intervals After hCG Hours post-hCG 0 4 6 9 15

No. of oocytes

Percent penetrationa (range)

Percent with sperm in the vitellus

Mean no. of perivitelline sperm/oocyte

Mean no. of vitelline sperm/oocyte

213 272 120 118 224

33 (0-59) 33 (0-83) 43 (19-70) 69 (30-97) 84 (60-100)

7 12 27 58 82

1.30 0.98 0.38 0.25 0.08

0.36 0.38 0.77 1.07 1.40

aIncludes oocytes with sperm in perivitelline space or vitellus.

there was no statistical difference in the zona penetration (80% vs. 88%)of immature and mature eggs. As another approach to detection of any possible difference in the penetrability of the zonas of secondary and GV oocytes, these were mixed, one population was labeled with FITC, and were inseminated together in one dish with capacitated spermatozoa. As is shown in Table 1, there was no statistical difference in the proportion of mature (77%) and GV oocytes (70%) penetrated in the same culture dish. In both experiments, however, the mean number of spermatozoa penetrating the zona pellucida was always significantly higher (P< 0.05) for immature (3.6) than for mature (1.9) eggs. Sperm penetration into the vitellus occurred in more than 50% of the penetrated GV oocytes. However, in no case did the sperm nucleus swell and decondense in the ooplasm of a GV oocyte.

remained the same in oocytes recovered 4 h post-hCG (33%),but thereafter it increased steadily as a function of time in oocytes recovered 6-15 h after hCG injection (4344%).The percentage with spermatozoa incorporated by the vitellus increased in parallel also as a function of the stage of their preovulatory maturation.

DISCUSSION

Several studies noted in the Introduction have demonstrated that the zona pellucida of the primary oocyte is receptive t o spermatozoa in vivo and in vitro in a variety of mammals. The present experiments indicate that the hamster is no exception. The results of insemination of mixed oocyte populations particularly show the hamster zona to be equally penetrable in the GV and the ovulated oocyte, at least in the IVF system used here. The greater mean number of spermatozoa in the perivitelline space of the naked GV oocytes (3.6vs 1.9) Penetrability of Follicular Cell-Invested Oocytes mirrors results obtained after transfer of diplotene at Various Stages of Preovulatory Maturation oocytes in vivo (Moore and Bedford, 1978b) and in the Since the results in Table 1 made it clear that the main probably reflects their lack of a zona block to zona is readily penetrable even in the GV oocyte, polyspermy. granulosa cell-invested eggs recovered from ovarian Subtle qualitative changes have been reported to follicles at different times after hCG injection were occur in the ability of the zona to induce an acrosome inseminated similarly to assess the influence on zona reaction as the hamster oocyte matures (Uto et al., penetration of the presence of the granulosa cell layer 19881,and certain visible characteristics of the hamster at various stages of oocyte maturation. If recovered at zona pellucida change on entering the oviduct (Yang the time of hCG injection, 33% of the granulosa cell- and Yanigamachi, 1989). Although we have observed covered primary oocytes were penetrated by capaci- no difference either in the in vitro penetrability of the tated spermatozoa on examination 2-3 h after in vitro zona in granulosa cell-free mature ovarian and ovuinsemination (Table 2). That penetration percentage lated hamster oocytes (data not shown), the observa-

SPERM PENETRATION OF IMMATURE HAMSTER OOCYTES tions of (Uto et al., 1988) and Yang and Yanigamachi (1989) suggest that the zonas of mature oocytes may induce the acrosome reaction faster than those of immature oocytes. It might be argued that the greater sperm numbers used in the IVF system could tend to obscure a subtle in vivo difference between immature and mature oocytes in the penetrability of the zona. However, in a previous experiment, a high proportion (95%)of diplotene hamster oocytes was penetrated even when exposed to the moderate sperm numbers that populate the tubal ampulla after natural mating (Moore and Bedford, 1978b). In comparing the results in Tables 1and 2, it becomes clear that the granulosa cell layer around the GV oocyte represents a significant impediment. The fact that the penetrability of this layer increases as a function of time after hCG may well depend on a coincident cumulus expansion and mucification (Dekel et al., 1978,1979; Eppig, 1982) and on a disappearance of gap junctions within the cumulus (Dekel et al., 1981; Larsen et al., 1981; Eppig, 1982). It seems less likely that it also reflects a parallel increase in hyaluronidase sensitivity of the cumulus (Barros and Munoz, 1973; Dekel et al., 19781, since capacitated hamster spermatozoa do not need to undergo an acrosome reaction in order to pass through mature cumulus to the zona surface (Corselli and Talbot, 1987). Despite earlier negative findings (Barros and Munoz, 19731, the hamster vitellus seems to be generally receptive even before GV breakdown (Usui and Yanagimachi, 1976; Moore and Bedford, 1978a,b). Here, spermatozoa were judged to have fused with more than 50% of the penetrated cell-free GV oocytes, but with only about 20% of those invested with granulosa cells 2-3 h after insemination. However, the increase here in granulosa-invested oocytes paralleled with some lag time the developing penetrability of the oocyte vestment (Table 2), and together the results indicate that the hamster oolemma is receptive in principle at all maturation stages. To conclude, in the hamster and other mammals whose oocytes are ovulated at metaphase 11, preovulatory maturation emerges as a process involving change within the ooplasm and cortex (reflected especially in a failure of sperm nucleus decondensation and also the block to polyspermy), together with improvement in penetrability of the cumulus oophorus as meiosis progresses. On the other hand, as confirmed here for the hamster, there appears to be no significant change in penetrability of the zona pellucida of the primary oocyte with resumption of meiosis or following ovulation. Interestingly, this penetrability is maintained for a long time. For example, the zonas of hamster oocytes aged in the oviduct for 54 h after ovulation were still readily penetrated when transferred into a periovulatory tubal ampulla that contained capacitated spermatozoa (P. Viriyapanich and J.M. Bedford, unpublished observations). The fact that the zona has proved to be readily penetrable at the primary oocyte stage in all other

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mammals studied focuses attention on man as a possible exception. Immature human oocytes aspirated from stimulated ovaries in the hours before ovulation sometimes become more fertilizable after culture in vitro, probably as a result of further cumulus mucification and expansion of the intercellular matrix involving secreted proteoglycans (Tesarik and Kopecny, 1986). It has been suggested that human oocyte penetrability also may require functional maturation changes in the zona, brought about by such secretions in the preovulatory period (Tesarik et al., 1988). However, this idea conflicts with the observation that spermatozoa can consistently penetrate the zona around immature human oocytes obtained from nonstimulated ovaries (Overstreet and Hembree, 1976; Nishimoto et al., 1982; Lopata and Leung, 1988). In considering this dilemma, it can be noted that there is a subpopulation of oocytes representing about 1 5 2 0 % of those aspirated for IVF, whose zonas appear to be inherently impenetrable, even though they have spermatozoa deep within the substance of the zona and often have reached metaphase I1 (J.M.B., personal observations). It is likely that many of the GV or metaphase I human oocytes not penetrated during IVF also belong to this group. In human oocyte populations recovered for IVF, the zonas of many remaining at the GV or metaphase I stage may be resistant to penetration because they are inherently abnormal, not because they are immature.

ACKNOWLEDGMENTS P.S.C. was supported by a postdoctoral fellowship from the Fogarty International Center, N.I.H. This study was supported by NIH grant AG02947. REFERENCES Barros C, Munoz G (1973): Sperm-egg interaction in immature hamster oocytes. J Exp Zoo1 186:73-78. Barros C, Munoz G (1974): Sperm penetration through the zona pellucida of immature hamster oocytes. Acta Physiol Latinoam 24:612-615. Berrios M, Bedford JM (1979): Oocyte maturation: Aberrant postfusion responses of the rabbit primary oocyte to penetrating spermatozoa. J Cell Sci 39:l-12. Corselli J, Talbot P (1987): In vitro penetration of hamster oocytecumulus complexes using physiological numbers of sperm. Dev Biol 1221227-242. Dekel N, Hillensjo T, Kraicer P F (1979): Maturational effects of gonadotropins on the cumulus-oocyte complex of the rat. Biol Reprod 20:191-197. Dekel N, Kraicer PF, Phillips DM, Sanchez RS, Segal SJ (1978): Cellular associations in the rat oocyte-cumulus cell complex: Morphology and ovulatory changes. Gamete Res 1:47-57. Dekel N, Lawrence TS, Gillula NB, Beers WH (1981): Modulation of cell to cell communication in the cumulus-oocyte complex and the regulation of oocyte maturation by LH. Dev Biol 86:356-362. Eppig JJ (1982): The relationship between cumulus cell-oocyte coupling, oocyte maturation and cumulus expansion. Dev Biol89:268272. Iwamatsu T, Chang MC (1972): Sperm penetration in vitro of mouse oocytes at various times during maturation. J Reprod Fertil31:237248. Kaufman MH, Fowler RE, Barratt E, McDougall RD (1989): Ultrastructural and histochemical changes in the murine zona pellucida during the final stages of oocyte maturation prior to ovulation. Gamete Res 24:35-48.

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the egg vestments in follicular oocytes, unfertilized and fertilized ova of the rabbit. Dev Biol 41:185-192. Overstreet JW, Hembree WC (1976):Penetration of the zona pellucida of non-living human oocytes by human spermatozoa in vitro. Fertil Steril 27:815-831. Polge E, Dziuk P (1965): Recovery of immature eggs penetrated by spermatozoa following induced ovulation in pig. J Reprod Fertil 9:357358. Tesarik J , Kopecny V (1986): Late preovulatory synthesis of proteoglycans by the human oocyte and cumulus cells and their secretion into the oocyte-cumulus-complex extracellular matrices. Histochemistry 85523-528. Tesarik J, Pilka L, Travnik P (1988): Zona pellucida resistance to sperm penetration before the completion of human oocyte maturation. J Reprod Fertil 83:487-495. Thibault C, Gerard M (1970): Facteur cytoplasmique necessaire a la formation du pronucleus male dans l‘oocyte de lapine. CR Hebd Seanc Acad Sci Paris 270:2025-2026. Usui N, Yanagimachi R (1976) Behaviour of hamster sperm nuclei incorporated into eggs at various stages of maturation, fertilization and early development. J Ultrastruct Res 57:276-288. Uto N, Yoshimatsu N, Lopata A, Yanagimachi R (1988):Zona induced acrosome reaction of hamster spermatozoa. J Exp Zool 248:113-120. Yanagimachi R (1982):In vitro sperm capacitation and fertilization of golden hamster eggs in a chemically defined medium: In ESE Hafez, K Semm (eds): “In vitro fertilization and Embryo Transfer.” Lancaster: MTP Press, pp 65-76. Yang CH, Yanagimachi R (1989): Differences between mature ovarian and oviductal oocytes: A study using the golden hamster. Hum Reprod 4:63-7 1.

Hamster oocyte penetrability during preovulatory maturation.

In vitro fertilization techniques were used to analyze the penetrability of preovulatory hamster oocytes. The zonas of granulosa cell-free primary (GV...
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