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Analysis of molecules for fertilization on human spermatozoa and oocytes

人类精子和卵母细胞受精分子的分析

基本信息

批准号：
09671679
负责人：
OHASHI Kazutomo
金额：
$ 1.86万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671679/
关键词：
human spermatozoa human oocyte acrosome reaction fertilization CD46

项目摘要

CD46 (membrane cofactor protein) acts to down-regulate complement cascade activity by binding complement components C3b and also acts as the cell surface receptor for measles virus.The extra-cytoplasmic components of the molecule are composed of four short consensus repeat (SCR) regions.SCR-1 is related to the down-regulation of complement activity and SCR-2 is a main domain of the measles virus fusion receptor.In our study, we revealed a novel SCR-3 related function in fertilization.We performed the hamster oocyte penetration test using the addition of antibodies against SCR-1, 2, and 3 of CD46 molecule.The anti-SCR-3 monoclonal antibodies reduced the penetration of human spermatozoa to the hamster oocytes in a dose-dependent manner, but did not inhibit the sperm binding to the oocytes.We concluded that CD46, especially SCR-3, was a fusion receptor of human spermatozoa to the oocyte and a marker of the fertilization capacity of human spermatozoa.We then developed a new method to assess the fertilization capacity of human spermatozoa by a two-color staining using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and anti-CD46 monoclonal antibody.The two-color staining revealed four staining patterns of acrosomal region.We defined PSA-positive/CD46 negative spermatozoa as acrosome-intact, PSA-negative/CD46 positive spermatozoa as acrosome-reacted, PSA-negative/CD46 negative spermatozoa as acrosome-damaged.The percentage of acrosome-reacted spermatozoa increased more rapidly than those of acrosome-damaged spermatozoa with the incubation time.The percentage of acrosome-reacted spermatozoa at the 4-hour incubation was related to fertilization in the clinical human IVF.

CD46膜辅因子蛋白（membrane cofactor protein）通过与补体C3 b结合下调补体级联反应活性，同时也是麻疹病毒的细胞表面受体。该分子的胞质外组分由四个短的一致重复序列（SCR）区域组成。SCR-1与补体活性的下调有关，SCR-2是麻疹病毒融合受体的一个主要结构域。在我们的研究中，我们发现了一种新的与SCR-3相关的受精功能。我们使用添加抗CD 46分子的SCR-1、2和3的抗体进行仓鼠卵母细胞穿透试验。抗SCR-3单克隆抗体以剂量依赖的方式减少人精子对仓鼠卵母细胞的穿透，但不抑制精子与卵母细胞的结合。我们得出结论，CD 46，尤其是SCR-3，在此基础上，我们建立了一种用荧光素异硫氰酸酯标记的豌豆凝集素（Pisumsativumagglutinin）双色染色法检测人精子受精能力的新方法用荧光素标记前列腺特异性抗原（FITC-PSA）和抗CD 46单克隆抗体，对顶体区进行双色染色，发现顶体区有4种染色模式。顶体反应精子百分率随孵育时间的延长而增加，顶体反应精子百分率随孵育时间的延长而增加，顶体反应精子百分率与临床IVF受精率相关。