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MOLECULAR ANALYSIS OF ACROSOME REACTION OF MAMMALIAN SPERMATOZOA

哺乳动物精子顶体反应的分子分析

基本信息

批准号：
12640665
负责人：
IIDA Hiroshi
金额：
$ 2.3万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12640665/
关键词：
Syntaxin acrosome reaction spermatozoa small GTPase fertilization 先体形成受精

项目摘要

The acrosome reaction includes a membrane fusion event that is pre-requisite for sperm penetration through the zona pellucida and subsequent fertilization. Since SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins have been shown to be key players in membrane fusion during regulated exocytosis in nerve terminals and secretory cells, and since the acrosome reaction has some features in common with regulated exocytosis, we hypothesized that SNARE proteins might also regulate acrosomal exocytosis. RT-PCR analysis demonstrated the expression of SNARE proteins, three isoforms of syntaxin 2 (2A, 2B and 2C) and syntaxin 4A, in rat testes. Immunoblot analysis with anti-syntaxin 2 antibody showed that the protein was expressed in rodent spermatozoa, and that it was associated with membrane components of spermatozoa prepared by sucrose density gradient centrifugation. Confocal laser scanning microscopy with double immunolabeling revealed that syntaxin 2 was co-localized with acrin 1, a 90 kDa acrosomal protein, over the acrosomal region of spermatozoa but was not associated with the posterior half of head or tail. Localization of syntaxin 2 over the acrosomal region was supported by the finding that it was shed from sperm heads during an acrosome reaction induced by calcium ionophore A23187 in vitro. In view of putative role of syntaxin proteins in other membrane fusion systems, these data suggest that syntaxin 2 may be involved in regulating the acrosomal reaction in rodent spermatozoa. We also found that small GTPase Rab6 was assosiciated with a developing acrosome. The GTPase was, however, disappeared in a complete acrosome of spermatozoa, suggesting that it might be not involved in the acrosome reaction.

顶体反应包括膜融合事件，这是精子穿透透明膜和随后受精的先决条件。由于SNARE（可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体）蛋白已被证明是在调节胞吐过程中的神经末梢和分泌细胞的膜融合的关键球员，因为顶体反应有一些共同的功能与调节胞吐，我们假设，SNARE蛋白也可能调节顶体胞吐。RT-PCR分析证实了SNARE蛋白，syntaxin 2的三种亚型（2A，2B和2C）和syntaxin 4A，在大鼠睾丸中的表达。抗syntaxin 2抗体的免疫印迹分析表明，该蛋白在啮齿动物精子中表达，并且它与蔗糖密度梯度离心制备的精子的膜成分相关。共聚焦激光扫描显微镜双免疫标记显示，syntaxin 2共定位与acrin 1，一个90 kDa的顶体蛋白，在精子的顶体区域，但不与后半部分的头部或尾部。syntaxin 2在顶体区域的定位得到以下发现的支持：在体外由钙离子载体A23187诱导的顶体反应期间，syntaxin 2从精子头部脱落。鉴于syntaxin蛋白在其他膜融合系统中的假定作用，这些数据表明syntaxin 2可能参与调节啮齿动物精子的顶体反应。我们还发现小GTTRab 6与发育中的顶体有关。而在完整的精子顶体中，GT3蛋白则消失，提示其可能不参与顶体反应。