Watanabe Toshihiko, Kubo Hideo, Takeshima Shinya, Nakagawa Mami, Ohta Manami, Kamimura Saori, Takayama-Watanabe Eriko, Watanabe Akihiko, Onitake Kazuo
Department of Biology, Faculty of Science, Yamagata University, Yamagata, Japan.
Int J Dev Biol. 2010;54(4):591-7. doi: 10.1387/ijdb.092894tw.
Motility initiation is a key event during internal fertilization of female-stored sperm, although the underlying mechanisms remain unclear. In internally fertilizing urodeles, quiescent sperm initiate motility on the surface of the egg-jelly, a thick extracellular matrix that accumulates around the egg in oviduct. By immunizing mice with egg-jelly extracts, we successfully generated an alpha34 monoclonal antibody (mAb) which neutralized sperm motility-initiating activity in the egg-jelly of the newt, Cynops pyrrhogaster, in a dose-dependent manner. The alpha34 mAb recognized an unglycosylated 34 kDa protein in the outermost of the six layers that comprise egg-jelly. Under nonreducing conditions, immunoblotting with alpha34 mAb produced many bands in addition to the 34 kDa protein, suggesting that the 34 kDa protein associates not only with the jelly matrix itself, but also with additional substances present in the matrix. Our current results are compatible with the supposed features of sperm motility-initiating substance (SMIS), indicating that the 34 kDa protein itself, or a complex consisting of the 34 kDa protein and some other molecules, is the SMIS in C. pyrrhogaster. Immunofluorescence staining further indicated that SMIS was distributed in a dot-like pattern in the outermost jelly layer and was fully covered with acrosome reaction-inducing substance (ARIS). Immunocytochemical and scanning electron microscopic examinations of the outermost jelly layer strongly suggests that the 34 kDa protein localized in granules (2 microm) and that ARIS was distributed covering the granules and in the sheet-like structure above the granules. These data suggest that the initiation of sperm motility is mediated by the acrosome reaction.
在雌性储存精子的体内受精过程中，精子活力的启动是一个关键事件，但其潜在机制仍不清楚。在进行体内受精的有尾两栖动物中，静止的精子在卵胶膜表面开始运动，卵胶膜是一种厚厚的细胞外基质，在输卵管中围绕卵子积累。通过用卵胶膜提取物免疫小鼠，我们成功制备了一种α34单克隆抗体（mAb），该抗体以剂量依赖的方式中和了蝾螈（Cynops pyrrhogaster）卵胶膜中的精子活力启动活性。α34 mAb识别构成卵胶膜的六层最外层中的一种未糖基化的34 kDa蛋白质。在非还原条件下，用α34 mAb进行免疫印迹除了产生34 kDa蛋白质条带外，还产生了许多条带，这表明34 kDa蛋白质不仅与胶状基质本身相关，还与基质中存在的其他物质相关。我们目前的结果与精子活力启动物质（SMIS）的推测特征相符，表明34 kDa蛋白质本身，或由34 kDa蛋白质和其他一些分子组成的复合物，是蝾螈中的SMIS。免疫荧光染色进一步表明，SMIS以点状模式分布在最外层胶状层中，并完全被顶体反应诱导物质（ARIS）覆盖。对最外层胶状层的免疫细胞化学和扫描电子显微镜检查强烈表明，34 kDa蛋白质定位于颗粒（2微米）中，并且ARIS分布在颗粒上并覆盖颗粒以及颗粒上方的片状结构。这些数据表明精子活力的启动是由顶体反应介导的。
