A.M.Courtot 1 and W.Lin-Tong. Materials and methods

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1 Human Reproduction vol.3 no.5 pp , 1988 Initial stages of sperm-egg interaction in a heterospecific system: fate of the post-acrosomal sheath and appearance of a particular material within the oocyte A.M.Courtot 1 and W.Lin-Tong Laboratoire d'histologie Embryologie Cytoge'n&ique, Centre Hospitalier, 78 rue du Genera] Leclerc, Kremlin-Bicgtre, France 'To whom correspondence should be addressed We describe the initial stages of the sperm-egg interaction in the human-hamster heterospecific system. The ultrastructural study reveals that fusion is initiated on both sides of the spermatozoon in the region extending from the anterior part of the post-acrosomal sheath (PAS) to the acrosome equatorial segment (AES). The splitting of the PAS and the appearance of a particular material within the oocyte subsisting until the pronuclear formation are described. The observation of the available electron micrographs suggests that this material may originate from the PAS. The role, the origin and the nature of this material are discussed. Key words: fertilization/gamete fusion/post-acrosomal sheath Introduction During fertilization, as the spermatozoon has just established contact with the egg surface, one part of the sperm membrane fuses with the oocyte membrane, while the remaining sperm components penetrate the coplasm (Szollosi and Ris, 1961; Yanagimachi and Noda, 1970a; Bedford, 1972; Soupart and Strong, 1975; Sathananthan et ai, 1986). The precise sperm head region where fusion occurs is now fairly well known (Koehler et ai, 1982; Talbot and Chacon, 1982; Moore and Bedford, 1983; Yanagimachi, 1984). By contrast, only few data are available on the fate within the oocyte of the non-nuclear material that subsists at the level of this region after the acrosome reaction. The typical structure of the acrosome equatorial segment (AES) has been identified in the ooplasm of numerous mammals within a short time of fusion (Bedford, 1972; Barros, 1977; Moore and Bedford, 1978). Concerning the postacrosomal sheath (PAS), the literature is very scant on its fate after initial contact of gamete membranes. However, the presence of this structure does not seem to be insignificant, since in infertile men a predominant anomaly of the PAS has been shown to be accompanied by a decrease in the incidence of spermatozoa attached to the oocyte membrane, as well as by a near absence of swollen sperm heads within the zona-free hamster oocyte (Courtot et ai, 1986). The purpose of the present study is to follow the fate of the sperm head region involved in the initial steps of gamete interaction, using the heterospecific system human spermatozoazona-free hamster oocytes. IRL Press Limited, Oxford, England Materials and methods The spermatozoa under study were from two ejaculates of men whose semen are routinely used in our laboratory as control samples in the hamster test. Sperm preparation After collection, semen was left for 30 min at room temperature to allow liquefaction. One millilitre of Biggers et al. (1971) medium (BWW) modified according to Courtot and Jouannet (1985) was added to 1 ml of semen in a test tube. Great care was taken to maintain an interface. The tube was placed at 37 C in a 5% CO 2 saturated atmosphere for 1 h. The upper fraction containing the motile sperm was gently removed, left at 37 C in 5 % CO2 in air for 3 h and then allowed to incubate overnight at 4 C in a closed tube. The next morning the spermatozoa were left at ambient temperature for 30 min, then at 37 C under an atmosphere of 5 % CO2 in air for 1 h before being put in contact with the oocytes. Oocyte preparation Hamster oocytes were prepared according to a procedure used routinely in this laboratory. Briefly, young female golden hamsters were induced to superovulate by intraperitoneal injection of 30 IU of pregnant mare's serum gonadotrophin (PMSG, Sigma, St Louis, MO) on day 1 and 30 IU of human chorionic gonadotrophin (HCG, Organon) on day 3. The cumulus and zona layers of the collected oocytes were removed by treatment in the BWW medium with respectively 0.1 % hyaluronidase (type I, Sigma) and 0.05% trypsin (type I, Sigma). The oocytes were washed after each treament and then put in contact with the spermatozoa. Gamete interaction Twenty oocytes were incubated in 200 /xl of BWW medium containing spermatozoa previously diluted to a final concentration of 2.5 x 10 6 sperm/ml of medium. Incubation was for 3 h at 37 C under a saturated atmosphere of 5% CO 2 in air. Finally, the zona-free oocytes were washed three times in the culture medium. Fixation and embedding in araldite were according to Bisson and David's technique (1975), except for fixation in glutaraldehyde which lasted for 2 3 h compared with 1 h in die standard procedure. Processing for the electron microscopy study Ultrathin sections were cut with a Reichert Om U 3 ultramicrotome, put onto single-slot, formvar-coated grids, men stained with 651

2 A.M.Courtot and W.Iin-Tong Figs 1 5. Fig. 1. Light microscope observation of spermatozoa attached to the oocyte membrane and swollen sperm heads within the ooplasm. Fig. 2. Electron micrograph of a human sperm attached to the hamster oocyte surface: the spermatozoon has already undergone the acrosome reaction; only the equatorial segment of the acrosome (AES) is still visible; the post-acrosomal sheath (PAS) can be clearly seen on one side of the spermatozoon; the spermatozoon appears to be closely associated with some oocyte microvilli. Fig. 3. Electron micrograph illustrating the earliest detectable stage of human sperm hamster oocyte interaction: a fusion-involved region including the anterior part of the PAS and the AES forms a belt around the sperm head on both sides of the spermatozoon; the PAS splitting can be clearly observed at this stage. Fig. 4. Electron micrograph of a spermatozoon in the same stage of fusion: an aggregate of particles (EFM) appears in the vicinity of the PAS. Fig. 5. Electron micrograph of a spermatozoon in the initial stage of nuclear decondensation: chromatin decondensation is initiated at the level of the fusion-involved region. An aggregate of particles (EFM) can be seen. uranyl acetate and lead citrate for examination under a Siemens Elmiskop CT 150 transmission electron microscope. 652 For this study, only the micrographs were retained which show- ed spermatozoa with well-constituted heads and whose incidence of cutting allowed examination of the anterior part of the acrosome, the AES and the PAS.

3 Gamete fusion in the hamster test 0M (uh v>4» / & * <i 7 S efm Figs 6-8. Fig. 6. Electron micrograph showing a swollen sperm head within the ooplasm: an aggregate of particles (EFM) is visible in the vicinity of the decondensing sperm head. Fig. 7. Electron micrograph of a spermatozoon in a further stage of chromatin decondensation. The presence of EFM can be observed. Fig. 8. Electron micrograph of a spermatozoon in a pronuclear stage. An aggregate of particles (EFM) can be seen outside the pronuclear membrane. Results Figure 1 shows spermatozoa attached to the oocyte membrane and swollen sperm heads within the ooplasm as observed under the light microscope. Transmission electron microscope examination of the spermatozoa revealed that all attached spermatozoa had undergone the acrosome reaction, leaving the AES intact (Figures 2-5 ). As shown on Figure 2, the plasma membrane overlying the AES and the PAS is not always visible. The electron micrographs illustrating the earliest detectable stages of sperm-egg interaction reveal that fusion is initiated in the sperm region including the AES and the anterior part of the PAS. This results from the examination of the same section that shows both aspects of the spermatozoon are involved in 653

4 A.M.Courtot and W.Iin-Tong fusion. Some sections exhibit the presence of cortical granules in the ooplasm on both sides of the sperm. This observation suggests the existence of a protrusion of the ooplasm rather than that of mere oocyte microvilli that would surround the spermatozoon (Figures 3 and 5). At this early stage of fusion, the anterior part of the PAS has lost its classical aspect and is split into particles visible on both aspects of the spermatozoon (Figures 3 and 4). Particles of the same size as those from the split PAS can be seen in the vicinity within both the ooplasm and the protrusion of oocyte cytoplasm that surrounds the spermatozoon (Figure 4). Those particles form round or ovoid aggregates whose mean diameter is 0.45 /tin. Several aggregates may be observed around one spermatozoon. For the purpose of the study, this material, which has not yet been described was called 'early fusion material' (EFM), as it appears in the earliest detectable stage of gamete interaction. The EFM can be observed as soon as sperm nucleus decondensation is initiated at the level of this fusioninvolved sperm region (Figure 5). It remains in the vicinity of the spermatozoon when this has penetrated the ooplasm further (Figure 6) and the chromatin completely achieves decondensation (Figure 7). The EFM is still visible when the pronucleus is formed, a process corresponding to the last observable stage of gamete interaction in the hamster test; it is then located outside the pronuclear membrane in the vicinity of the nucleus (Figure 8). Discussion The results from this study show that gamete interaction is initiated in the sperm region including the AES and the PAS, a finding that is similar to the observations made by Talbot and Chacon (1982) and Koehler et al. (1982). Since images of fusion could be observed on both aspects of the same spermatozoon, it is assumed that the sperm region implicated in the initial stages of sperm egg interaction forms a belt around the sperm head. All around this belt, the PAS material disintegrates, and soon one or several aggregates of particles (EFM) appear within the ooplasm in the immediate proximity of the sperm. Those aggregates of EFM are visible until formation of the male pronucleus. In a heterospecific system it is not possible to follow their evolution after the pronucleus stage. One might think that the events described above occur only in the human-hamster heterospecific system. However, similar observations have been made in the literature in the homospecific system. In a study on fertilization in the hamster based on the use of zona-free oocytes, Yanagimachi and Noda (1970b) described the sudden appearance of several aggregates of small particles, simlar to the EFM, within and around the decondensing sperm nucleus. These authors then raised the question as to whether those particles would be dispersed by the sperm itself, or whether their presence would result from the interaction of a substance released by the sperm with the ooplasm. Da-Yvan and Longo (1983) have reported the presence of similar particles characteristically associated with the male pronucleus within mono- and dispermic hamster oocytes. Such observations do not seem to be restricted to the hamster oocyte, since similar structures have been described in the mouse during fertilization (D.Szollosi, personal communication). Although Sathananthan et al. (1986) do not mention the existence of such a material in man, it is interesting to note the presence of similar structures in the vicinity of a swollen sperm head on one of the electron micrographs illustrating their paper (Figure 5, p. 323). The origin and the nature of the EFM remain to be elucidated. No similar material has ever been observed in non-fertilized oocytes or sperm. According to these experiments, the EFM appears in the early stages of gamete interaction, as the AES and the anterior part of the PAS have just established contact with the oocyte surface. Moreover, the splitting of the PAS seems to be related to the presence of the EFM, as suggested by these micrographs. It has been reported by D.Szollosi (personal communication) that under certain physiological conditions a similar material may be observed within the oocyte of the mouse after sperm penetraton and in the absence of nuclear decondensation. On the basis of these observations, it would appear that the EFM is totally or partially of non-nuclear sperm origin. In that respect, the presence of sperm cytoplasmic components subsisting within the oocyte after fertilization has already been reported in invertebrates and in the mouse (Shapiro, 1984). A comprehensive study on labelled proteins carried out in the sea urchin could demonstrate the existence of a category of 'embryo forms of sperm polypeptides' induced by proteolysis in one cell stage and persisting until gastrulation (Gundersen and Shapiro, 1984). Would the EFM correspond to a sperm material originating from the PAS? Has this material already undergone oocyte alterations? Which are its relationships with the oocyte cytoskeleton, and how can those induce its concentration and its preservation in the vicinity of the pronucleus? Would the EFM play a part in the early stage of development? It has recently been demonstrated through nuclear transfer experiments on a colony of DDK mice that the male pronucleus interacts with the ooplasm in the one-cell stage (Renard and Babinet, 1986). It would be interesting to know whether the EFM remains associated with the pronucleus during transfer experiments. So far, no biochemical study has been carried out to determine the nature of this material with the exception of Da-Yvan and Longo's investigations (1983), which showed that cytoplasm granular aggregations were not associated with reaction products when the ammoniacal silver reaction was used. Further investigations were therefore needed to determine the nature and the real origin of the EFM. If this material originated from the PAS, as is assumed in this study, this structure should play an important role in fertilization. This would also explain why PAS anomalies considerably alter the fertilizing ability of spermatozoa. Acknowledgements The authors thank Professor G.David for his careful supervision of the manuscript and his invaluable advice, Dr D.Szollosi for his helpful discussions, Professor P.Jouannet, Dr J.P.Renard and Dr J.Barra for their constructive criticism. Special thanks are due to Mr P.Caut for his technical assistance with the electron microscope. Mrs F.Siryani for the translation, Mrs V.Cepisul and Mrs E.Reimann for typing the manuscript. 654

5 Gamete fusion in the hamster test References Barros.C. (1977) The fertile mammalian spermatozoon. Rev. Microsc. Elect., 4, 107. Bedford.J.M. (1972) An electron microscopic study of sperm penetration into the rabbit egg after natural mating. Am. J. Anat., 133, Biggers,J.D., Whitten.W.K. and Whittingham.D.P. (1971) The culture of mouse embryos in vitro. In Daniel,J.C. (ed.), Methods in Mammalian Embryology. W.H.Freeman, San Francisco, pp BissonJ.P. and David.G. (1975) Anomalies morphologiques du spermatozoide humain. 2. Etude ultrastructurale. J. Gynecol. Obstet. Biol. Reprod., 1, Courtot.A.M. and Jouannet,P. (1985) Zona-free hamster ovum penetration by human spermatozoa: influence of various sperm factors. Reprod. Nutr. Dev., 25, Courtot.A.M., Escalier,D., Jouannet,P. and David,G. (1986) Impaired ability of human spermatozoa to penetrate zona-free hamster oocytes: Is a post-acrosomal sheath anomaly involved? Gamete Res., 17, Da-Yvan,C. and Longo,F.J. (1983) A cytochemical study of nuclear changes in fertilized hamster egg. Anat. Rec, 207, Gundersen.G.G. and Shapiro,B.M. (1984) Sperm surface proteins persist after fertilization. J. Cell Biol., 99, KoehlerJ.K., De CurtisJ., Stenchever.M.A. and Smith.D. (1982) Interaction of human sperm zona-free hamster eggs. Gamete Res., 6, Moore.H.D.M. and Bedford.J.M. (1978) Ultrastructure of equatorial segment of hamster spermatozoa during penetration of oocytes. J. Ultrastruct. Res., 62, Moore,H.D.M. and Bedford.J.M. (1983) The interaction of mammalian gametes in the female. In Hartmann,J.F. (ed.), Mechanism and Control of Animal Fertilization. Academic Press, New York, pp RenardJ.P. and Babinet,Ch. (1986) Identification of a paternal developmental effect on the cytoplasm of one-cell-stage mouse embryos. Proc. Natl. Acad. Sci. USA, 83, Sathananthan.A.H., Edirisinghe.R., Ratman,S.S. and Wong,P.C. (1986) Human sperm egg interaction in vitro. Gamete Res., 15, Shapiro.B.M. (1984) Molecular aspects of sperm-egg fusion. In Cell Fusion, Ciba Foundation Symposium, Vol. 103, pp Soupart,P. and Strong,P.A. (1975) Ultrastructural observations on polyspermic penetration of zona pellucida-free human oocytes inseminated in vitro. Fertil. Sterii, 26, Szollosi.D. and Ris,H. (1961) Observations on sperm penetration in the rat. J. Biophys. Biochem. Cytol., 10, Talbot.P. and Chacon,R.S. (1982) Ultrastructural observations on binding and membrane fusion between human sperm and zona pellucidafree hamster oocytes. Fertil. Sterii., 37, Yanagimachi.R. (1984) Zona-free hamster eggs: their use in assessing fertilizing capacity and examining chromosomes of human spermatozoa. Gamete Res., 10, Yanagimachi.R. and Noda,Y.D. (1970a) Ultrastructural changes in the hamster sperm head during fertilization. J. Ultrastruct. Res., 31, Yanagimachi.R. and Noda.Y.D. (1970b) Electron microscope studies of sperm incorporation into the golden hamster egg. Am. J. Anat., 128, Received on December 4, 1987; accepted on February 17,

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