ヒト精子CD46分子を介する精子・卵細胞融合機構の解明とその異常の検出法に関する研究(1 受精機構とその異常)

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Membrane cofactor protein(MCP or CD46)is expressed on the inner acrosomal membrane of acrosomereacted human sperm. CD46 is a widely distributed C3b/C4b-binding cell surface glycoprotein that serves as an inhibitor of complement activation on host cell. CD46 also acts as a cellular fusion receptor for measles virus. However, the function of CD46 molecules on acrosome-reacted human sperm remains to be unknown. We performed the hamster egg penetration assay using four anti-CD46 antibodies(anti-SCR1, 2 and 3). Two anti-SCR3 antibodies reduced fertilization and sperm penetration but no reduction in sperm binding was observed in the anti-CD46 antibody treated sperm. These results indicated that SCR3 of CD46 on human acrosome-reacted sperm contributed to the sperm-egg fusion. To distiguish between the functional arosome reaction and acrosomal loss, acrosomal status was assessed with two-color fluorescence staining using fluorescein isothiocyanate-conjugated Pisium sativum agglutinin(FITC-PSA)and MH61(anti-CD46 monoclonal antibody)with Texas-red conjugated antimouse immunoglobulin G antiserum. Two-color fluorescence staining revealed four staining patterns of the acrosomal region : PSA-positive/CD46-nagative(acrosomal intact), PSA-negative/CD46-positive(functional acrosome reaction), PSA-negative/CD46-negative(acrosomal loss), and PSA-positive/CD46-positive(antifact). The percentage of functional acrosome reaction increased to a greater extent than that of acrosomal loss with the duration of in vitro incubation of human sperm. Two-color fluorescence staining of sperm obtained from 34 patients with normozoospermia who were participating in IVF program was performed. The percentage of functional acrosome reaction in fertilization group was significantly higher than that in the no-fertilization group(5.8%±2.4% versus 3.4%±1.6%), indicating that the percentage of functional acrosome-reacted sperm can predict fertilization in human IVF. Two-color fluorescence staining with FITC-PSA and the anti-CD46 monoclonal antibody may be useful for distinguishing between the functional acrosome reaction and acrosomal loss. Acrosomal status was evaluated using immunobeads coated with MH61 monoclonal antibody(Acrobeads). When viable sperm was co-incubated with Acrobeads for an appropriate lenght of time, the number of sperm binding to Acrobeads increased with the progression of the acrosome reaction. The formation of a sperm-bead complex was observed with a phase-contrast microscope, and we developed the Acrobeads test to assess sperm function using this sperm-bead agglutination. We compared with the results in the Acrobeads test with those in the sperm penetration assay(SPA)using zona-free hamster oocytes. The sperm whose Acrobeads score 0 at both 6 and 24 hours of incubation showed significantly poor potential to penetrate the zona-free hamster oocytes in comparison with the other group. Consequently, we determined an abnormal score in the Acrobeads test as 0 at 24 hours of incubation. To determined the effectiveness of the Acrobeads test for predicting the outcome of IVF, we carried out the Acrobeads test and SPA within 3 months before IVF and we analyzed the results in relation to IVF outcome. The negative predictive value of the Acrobeads test was 100%, whereas that of the SPA was 73%, indicating the Acrobeads test may be used to evaluate the fertilizing capacity of human sperm. To further examination, we evaluated the usefulness of Acrobeads test as a test kit for diagnosing the fertilizing capacity of human sperm using 210 semen samples from male partners of infertile couples in IVF program at 20 university hospital. We defined the criteria for assessment of fertilizing capacity in the Acrobeads test as follows : 0 was poor, 1 to 3 were good, and 4 was excellent. We also used the Acrobeads to separate the acrosome-reacted sperm and applied this technique to analyze the mechanism of acrosome reaction. The Acrobeads-binding method could become a
社団法人日本産科婦人科学会の論文
2001-09-01

ヒト精子CD46分子を介する精子・卵細胞融合機構の解明とその異常の検出法に関する研究(1 受精機構とその異常)

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