MOLECULAR ANALYSIS OF ACROSOME REACTION OF MAMMALIAN SPERMATOZOA

顶体反应包括膜融合事件，这是精子穿透透明带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)蛋白在神经末梢和分泌细胞的调节胞吐过程中是膜融合的关键角色，而且顶体反应与调节胞吐有一些共同的特征，我们推测SNARE蛋白也可能调节顶体的胞吐。RT-PCR分析表明，SNARE蛋白在大鼠睾丸中有表达，它们分别是Synaxin 2的三种亚型(2A、2B和2C)和Synaxin 4A。免疫印迹分析表明，该蛋白在啮齿动物精子中有表达，并与蔗糖密度梯度离心法制备的精子膜组分有关。激光共聚焦扫描显微镜双重免疫标记显示，Synaxin 2与90 kDa顶体蛋白acrin 1共定位于精子顶体区域，但与头部或尾部的后半部分无关。在体外，在钙离子载体A23187诱导的顶体反应中，Synaxin 2从精子头部脱落的发现支持了Synaxin 2在顶体区域的定位。鉴于Synaxin蛋白在其他膜融合系统中的可能作用，这些数据表明Synaxin 2可能参与调节啮齿动物精子的顶体反应。我们还发现，小的GTP酶Rab6与一个发育中的顶体相结合。然而，GTP酶在精子的一个完整顶体中消失，这表明它可能不参与顶体反应。

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.