Scanning Electron Microscopic Observations on the Sperm Penetration through the Zona Pellucida of Mouse Oocytes Fertilized in vitro

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1 Scanning Electron Microscopic Observations on the Sperm Penetration through the Zona Pellucida of Mouse Oocytes Fertilized in vitro Masatsugu MOTOMURA and Yutaka TOYODA School of Veterinary Medicine and Animal sciences, Kitasato University, Towada-shi 034 (Received February 4, 1980) Abstract Superovulated mouse oocytes were fertilized in vitro with pre-incubated cauda epididymal spermatozoa, and the process of sperm penetration through the zona pellucida was studied by scanning electron microscopy. By 5min after insemination, the spermatozoa had passed through the cumulus oophorus, reached the surface of the zona pellucida and had begun to penetrate the zona pellucida. Spermatozoa in the act of penetrating the zona pellucida were frequently found in the oocytes fixed 11min after insemination. The sperm penetration through the zona pellucida was completed within 20min in most oocytes leaving 'sperm penetration hole' on the zona pellucida. The sperm penetration holes were not completely repaired by 4hr after insemination. Jpn. J. Zootech. Sci., 51 (8): , 1980 Early stages of mammalian fertilization have been studied extensively by light and transmission electron microscopy in hamster1-5), guinea pig6), mouse7, 8), rate9), rabbit10-14), pig15) and human16). So far, however, detailed morphological and chronological descriptions have not been made concerning the process of sperm penetration through the zona pellucida, although scanning electron microscopic observations on the process of sperm incorporation into ooplasm have recently been made in hamster 17, 18), rat19-21) and mouse22). The purpose of the present study is to observe the morphological features of sperm penetration through the zona pellucida of mouse oocytes fertilized in vitro by means of scanning electron microscopy. Materials and Methods The procedure for in vitro fertilization used throughout this study was essentially the same as described by TOYODA et al.23). The medium used was a modified Krebs- Ringer bicarbonate solution containing 5.55mM glucose, 1.0mM Na-pyruvate, 4mg/ Jpn. J. Zootech. Sci., 51 (8):

2 MOTOMURA and TOYODA Fig. 1. Classified figures of the process of sperm penetration through the zona pellucida. Table 1. Sperm penetration through the zona pellucida of mouse oocytes in vitro. * Fig. 1. Freshly ovulated oocytes were obtained from the adult female mice of the same strain (weighing 27-38g) which had been induced to superovulate by i. p. injections of 5 i.u. PMSG followed 48hr later with 5 i. u. hcg and killed 16-17hr after injection of hcg. The oocytes in cumulus were released from excised oviducts into fertilization medium between 9 and 19min after dilution of spermatozoa, and then incubated for For scanning electron microscopy, the oocytes were picked up from the fertilization medium and immediately transferred into a medium containing 0.6% glutaraldehyde in order to stop the fertilization process. The cumulus cells surrounding the oocytes were removed by the addition of 0.1% hyaluronidase in medium and the oocytes were then transferred into 2.5% glutaraldehyde in 0.1M phosphate buffer 596

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4 MOTOMURA and TOYODA After drying, the paper bags were opened very carefully and looked for the ova under a dissecting microscope. The ova on the filter papers were transferred onto doublestick cellophane tape on specimen mounts, sputter-coated with platinum, and were examined with Hitachi S-450 scanning electron microscope operated at 15 kv. 598

5 Sperm Penetration through Zona Pellucida Results and Discussions Uninseminated oocytes The zona pellucida of the uninserninated oocytes as viewed by scanning electron microscopy showed an intricated network of interconnecting fibers and a porous sponge-like structure. The zona pellucida had no structure equivalent to a sperm penetration hole described below. Inseminated oocytes The process of sperm penetration through the zona pellucida was classified into 6 stages as follows (see Fig. 1). Stage 0: No spermatozoon attaches to the surface of zona pellucida. Stage 1: One or more spermatozoa attach to the zona pellucida but sperm penetration of the zona pellucida is not observed. Stage 2: One or more spermatozoa have begun to penetrate through the zona pellucida and the anterior half of the sperm head is embedded in the zona pellucida. Stage 3: One or more spermatozoa are in the act of penetrating through the zona pellucida and the posterior half of the sperm head is embedded in the zona pellucida. Stage 4: The whole sperm head is embedded in the zona pellucida. Stage 5: The sperm penetration through the zona pellucida is completed and one or more holes made by fertilizing spermatozoa (referred to as 'sperm penetration hole') are observed in the zona pellucida. The incidence of these stages at each time of fixation is presented in Table 1. All oocytes incubated for 5min with spermatozoa had the spermatozoa attached to the zona pellucida as shown in Fig. 2. These adhering spermatozoa did not lie flat upon the outer zonal surface, but attached at an angle to the surface with their anterior tip of the dorsal curvature of the head. The spermatozoa had begun to penetrate through the zona pellucida in a half of the oocytes (6/12) at 5min after insemination and 85% of the oocytes (17/20) had spermatozoa penetrating through the zona pellucida at 11min after insemination (Figs. 3A, 3B and 3C). These microscopic photographs (Figs. 2, 3B and 3C) suggest that sticky zonal material disolved by the penetrating spermatozoa is holding the sperm head to the zona pellucida at the earliest and following stages of sperm penetration. The spermatozoa which were in the act of penetrating through the zona pellucida seem to have lost their acrosomes (Figs. 3B and 3C). The sperm penetration holes in the zona pellucida were observed in a small proportion of the oocytes (3/20) examined 11min after insemination, and more than half of the oocytes (54%; 7/13) had the sperm penetration hole (s) at 15min after insemination. The percentages of the oocytes having the sperm penetration hole (s) were 80 (8/10), 79 (15/19), 100 (21/21) and 91% (10/11) at 20, 26, 28 and 35min after insemination, respectively, (Figs. 4A and 4B). The sperm penetration holes were also observed in all of 19 oocytes which were examined after incubation for 4hr with spermatozoa (Figs. 5A and 5B). From these findings, it is suggested that a very short period, probably less than 599

6 MOTOMURA and TOYODA 5min is required for spermatozoa to pass through the cumulus oophorus and to begin to penetrate the zona pellucida, and less than about 20min is required to complete penetration through the zona pellucida. The sperm penetration holes are not very different from one another, having elongated, rounded form with one sharp edge. The holes become smaller in the deeper portion of the zona pellucida, being narrowest at the bottom (inner surface of the zona oellucida). Long and short diameter of the holes found in the oocytes fixed 26min the outer surface of the zona pellucida. It is inferred from these observations that zona penetration by a spermatozoon is effected by a sperm-borne zona lysin and by a mechanical shearing by sharp edge of the sperm head (Figs. 3, 4 and 5), but the motility pattern of penetrating spermatozoa is not yet fully understood. Since the sperm penetration holes were observed in the zona pellucida of the oocytes fixed at 4hr after insemination and were not different in size (long and short at 26min, the holes are not completely repaired by this time. References 1) YANAGIMACHI, R. and Y. D. NODA, Am. J. Anat., 128: ) YANAGIMACHI, R. and Y. D. NODA, J. Ultrast. Res., 31: ) FRANKLIN, L. E., C. BARROS and E. N. FUSSELL, Biol. Reprod., 3: ) OURA, C. and F. YASUZUMI, Gunma Symp. Endocrinol., 13: ) OURA, C., Metabolism and Disease, 16: ) NODA, Y. D. and R. YANAGIMACHI, Develop. Grow. Differen., 18: ) STEFANINI, M., C. OURA and L. ZAMBONI, J. Submicr. Cytol., 1: ) THOMPSON, R. S., D. M. SMITH and L. ZAMBONI, Fert. Steril., 25: ) SZOLLOSI, D. G. and H. RIS, J. Biophys. Biochem. Cytol., 10: ) HADEK, R., J. Ultrast. Res., 8: ) BEDFORD, J. M., J. Reprod. Fert., Suppl., 2: ) BEDFORD, J. M., Am. J. Anat., 123: ) BEDFORD, J. M., Biol. Reprod., Suppl. 2: ) BEDFORD, J. M., Am. J. Anat., 133: ) SZOLLOSI, D. G. and R. H. F. HUNTER, J. Anat., 116: ) SOUPART, P. and P. A. STRONG, Fert. Steril., 25: ) YANAGIMACHI, R. and Y. D. NODA, Experientia, 28: ) NODA, Y. D. and Y. HIDAKA, HITACHI Sci. Inst. News, 20: ) SUGAWARA, S. and S. TAKEUCHI, Japan. J. Anim. Reprod., 19: ) SHALGI, R., D. M. PHILLIPS and P. F. KRAICER, Gamete Res., 1: ) HORIUCHI, T., J. TAKAHASHI, S. SUGAWARA and J. MASAKI, Japan. J. Anim. Reprod., 25: ) NICOSIA, S. V., D. P. WOLF and L. MASTROIANNI, Jr., Gamete Res., 1: ) TOYODA, Y., M. YOKOYAMA and T. Host, Japan. J. Anim. Reprod., 16:

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