Katafuchi K, Mori T, Toshimori K, Iida H
Department of Zoology, Faculty of Agriculture, Kyushu University, Higashi-ku, Fukuoka 812-8581, Japan.
Mol Reprod Dev. 2000 Dec;57(4):375-83. doi: 10.1002/1098-2795(200012)57:4<375::AID-MRD10>3.0.CO;2-Z.
The acrosome reaction includes a membrane fusion event that is a prerequisite 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 colocalized 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 the 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.
顶体反应包括膜融合事件，这是精子穿透透明带并随后受精的先决条件。由于可溶性N - 乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）蛋白已被证明是神经末梢和分泌细胞中调节性胞吐作用期间膜融合的关键参与者，并且由于顶体反应与调节性胞吐作用有一些共同特征，我们推测SNARE蛋白可能也调节顶体胞吐作用。逆转录 - 聚合酶链反应（RT - PCR）分析表明，SNARE蛋白、 syntaxin 2的三种同工型（2A、2B和2C）以及syntaxin 4A在大鼠睾丸中表达。用抗syntaxin 2抗体进行的免疫印迹分析表明，该蛋白在啮齿动物精子中表达，并且与通过蔗糖密度梯度离心制备的精子膜成分相关。双重免疫标记的共聚焦激光扫描显微镜显示，syntaxin 2与顶体蛋白1（一种90 kDa的顶体蛋白）在精子顶体区域共定位，但与头部后半部或尾部无关。体外钙离子载体A23187诱导的顶体反应过程中，syntaxin 2从精子头部脱落，这一发现支持了syntaxin 2在顶体区域的定位。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。
