Iida H, Yoshinaga Y, Tanaka S, Toshimori K, Mori T
Faculty of Agriculture, Kyushu University, Higashi-ku, Fukuoka, 812-8581, Japan.
Dev Biol. 1999 Jul 1;211(1):144-55. doi: 10.1006/dbio.1999.9302.
The acrosome reaction is a membrane fusion event that is prerequisite for sperm penetration through the zona pellucida. To elucidate the molecular mechanisms involved in membrane fusion, the expression and localization of Rab proteins, a subfamily of small GTPases that have been shown to play key roles in regulation of intracellular membrane traffic and exocytosis, were examined in rat testis and sperm. Reverse transcription polymerase chain reaction, immunoblot analysis, and immunofluorescence microscopy revealed that Rab3A protein, which is thought to be involved in regulation of exocytosis in neurons and endocrine cells, is associated with the sperm acrosome. The protein was undetectable in acrosome-free heads prepared by sucrose density gradient centrifugation. Immunogold electron microscopy performed on ultrathin cryosections provided further evidence that Rab3A protein is associated with the acrosomal membrane. Acrosome reaction assays revealed that synthetic peptide of the Rab3 effector domain inhibited acrosomal exocytosis triggered by calcium ionophore A23187 in a concentration-dependent fashion, suggesting that Rab3A acts as an inhibitory regulator in the acrosome reaction. In view of the putative role of Rab3A protein in membrane fusion systems, these results suggest that Rab3A could be involved in regulating the mammalian acrosome reaction by controlling the membrane fusion system in sperm.
顶体反应是一种膜融合事件，是精子穿透透明带的前提条件。为了阐明参与膜融合的分子机制，我们检测了Rab蛋白（已证明在调节细胞内膜运输和胞吐作用中起关键作用的小GTP酶亚家族）在大鼠睾丸和精子中的表达及定位。逆转录聚合酶链反应、免疫印迹分析和免疫荧光显微镜检查显示，Rab3A蛋白（被认为参与神经元和内分泌细胞的胞吐作用调节）与精子顶体相关。在通过蔗糖密度梯度离心制备的无顶体头部中未检测到该蛋白。对超薄冰冻切片进行的免疫金电子显微镜检查提供了进一步证据，表明Rab3A蛋白与顶体膜相关。顶体反应试验表明，Rab3效应结构域的合成肽以浓度依赖性方式抑制钙离子载体A23187触发的顶体胞吐作用，这表明Rab3A在顶体反应中起抑制调节作用。鉴于Rab3A蛋白在膜融合系统中的假定作用，这些结果表明Rab3A可能通过控制精子中的膜融合系统参与调节哺乳动物的顶体反应。
