卵細胞膜と精子の相互作用 : ヒト卵細胞膜多精子受精防御機構の解析の視点から(1 受精機構とその異常)

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An effective block to multiple sperm penetration in mammalian oocytes is critical for normal fertilization and subsequent embryonic development. It has been speculated that the human oocyte relies primarily on the zona reaction to prevent polyspermy. However, the role and the mechanisms concerning the oocyte plasma membrane block in human oocyte are poorly understood. We firstly investigate a main mechanism of polyspermy block in human oocytes. Secondary, the existence, time course and mechanisms of the human plasma membrane block to polyspermy were investigated by an in vitro fertilization assay using zona pellucida-free unfertilized oocytes. The total incidence of polypronuclear fertilization in our IVF program was 6.2%. A significantly higher polyspermic fertilization was observed after SUZI(34.5%), indicating that zona reaction mainly involved in polyspermy block in human oocytes. Sperm penetration assay using the zona-free human oocytes demonstrated that number of penetrating spermatozoa reached a maximum by 2 h after insemination. A reinsemination experiment also revealed that the number of penetrating and binding spermatozoa were not significantly defferent between control and reinsemination oocytes, suggesting that the plasma membrane block plays an important role in the prevention of polyspermy in the human oocyte. It has not been determined if there is a requirement for sperm-oocyte membrane fusion for the establishment of a block to sperm penetration at the oocyte plasma membrane level in the human. Therefore, we investigated whether the incorporation of the sperm plasma membrane into the oolemma contributes to the plasma membrane block to polyspermy, using zona-free oocytes fertilized by intracytoplasmic sperm injection(ICSI)or activated by parthenogenetic activation. Only two of the 35 pronuclear oocytes fertilized spermatozoa(control)demonstrated one single penetrating spermatozoa. In contrast, ICSI or parthenogenetically activated oocytes at pronuclear atage could still be penetrated by and fuse with excess spermatozoa, and this did not result in a block response. These findings suggest that the establishment of the plasma membrane block to soerm penetration in the human oocyte may require a fusion process between sperm and oocyte plasma membrane. The plasma membrane block to polyspermy does not involve cortical granule exocytosis, because of CG contents was observed in both control and non-sperm-fertilized oocytes. To clarify the role of CD9 or integrins concerning the polyspermy block at oolemma, we investigated expression of CD9 and integrins on plasma membrane in unfertilized oocytes, sperm-fertilized and ICSI fertilized pronuclear oocytes. CD9 protein was expressed on plasma membrane in unfertilized oocytes and ICSI fertilized pronuclear oocytes, however, was not observed in sperm-fertilized pronuclear oocytes. Similarly, 4 of 7 α-subunits and 3 of 6β-subunits examined were detected in unfertilized and ICSI fertilized oocytes but not sperm-fertilized oocytes. Treatment of oocytes with anti-CD9 antibody inhibited fusion and penetration processes of spermatozoa. Thus, changes of expression of CD9 or integrins may contribute to oocyte plasma membrane block to sperm fusion and penetration. Heterologous fertilization was performed to assess the chromosomal normality of mouse-human hybrid embryo at the first cleavage metaphase after ICSI. ICSI procedure do not cause an increase in the frequency of aneuploidy and structural aberrations in both paternal and maternal chromosomes. In conclusion, we have clarified the existence and mechanism of human oosyte plasma membrane block to sperm penetration. These results would facilitate understanding of the mechanism of human sperm-oosyte interaction.
2001-09-01

卵細胞膜と精子の相互作用 : ヒト卵細胞膜多精子受精防御機構の解析の視点から(1 受精機構とその異常)

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