MECHANISMS OF MAMMALIAN FERTILIZATION/POLYSPERMY BLOCK

小鼠卵子受精会引发一种称为“卵子激活”的现象。 EGG的激活导致(1)皮质颗粒(CG)的刺激 胞吐作用导致透明带(ZP)的修饰 构成块到多精受精，以及(2)导致一个 减数分裂周期转变为有丝分裂周期(有丝分裂激活)。以…为类比 分化的体细胞，精子可能通过G 蛋白介导的信号转导途径导致了 特定的第二信使/离子变化。 特定的第二信使[肌醇1，4，5-三磷酸(IP3)或 蛋白激酶C(PKC)激活剂]可引起ZP修饰，但 不会导致有丝分裂激活。这些观察表明，尽管 CG胞吐和有丝分裂激活都是通过受精启动的 精子，不同的第二信使系统可能会产生 对这两种反应进行差异化调控。此外，虽然G 蛋白质介导体细胞的胞吐和有丝分裂激活， 事实上，没有确凿的数据表明哺乳动物卵子的激活 由G蛋白偶联的信号转导通路介导。这项建议 我将解决(1)这些第二信使在不同方面的作用 调节小鼠卵子激活的这两个成分，以及(2)作用 G蛋白在卵子激活中的作用。这些研究将使用细胞生物学 和生化方法，包括微量注射和体外 卵子受精。 受精后ZP的修饰，构成块体 对于多精受精，被认为是由皮层的内容物介导的 颗粒，在受精后释放。几乎没有什么 然而，关于CG的内容的信息，更不用说他们的模式了 对ZP修改的行动。这项提案将解决 首次描述的哺乳动物卵皮质区的特性和功能 颗粒蛋白。这些研究将使用细胞生物学、生化和 分子方法，包括鸡蛋的新陈代谢标记， 免疫沉淀和免疫印迹、cDNA的制备和筛选 文库的构建，以及克隆的分离和分析。 这些研究的结果将提供关于以下方面的新信息： 卵子激活的生化基础，(2)G蛋白在卵子激活中的作用 介导精子诱导的卵子激活，以及(3)表征和 第一个哺乳动物皮质颗粒抗原的克隆。

Fertilization of mouse eggs initiates a phenomenon termed "egg activation". Egg activation results in (1) the stimulation of cortical granule (CG) exocytosis that results in modifications of the zona pellucida (ZP) that constitute the block to polyspermy, and (2) results in the conversion of a meiotic cycle to a mitotic one (mitotic activation). By analogy to differentiated somatic cells, the sperm may initiate these events by a G protein-mediated signal transduction pathway that results in the generation of specific second messengers/ionic changes. Specific second messengers[inositol 1, 4, 5-triphosphate (IP3) or activators of protein kinase C (PKC)] elicit the ZP modifications but do not result in mitotic activation. These observations suggest that although both CG exocytosis and mitotic activation are initiated by fertilizing sperm, different second messenger systems may be generated that differentially regulate these two responses. In addition, although G proteins mediate exocytosis and mitogenic activation in somatic cells, there are in fact no conclusive data that mammalian egg activation is mediated by a G-protein-coupled signal transduction cascade. This proposal will address (1) the role of these second messengers in differentially regulating these two components of mouse egg activation, and (2) the role of G proteins in egg activation. These studies will use cell biological and biochemical approaches, including microinjection and in vitro fertilization of eggs. The modification of the ZP after fertilization, which constitutes the block to polyspermy, is thought to be mediated by the contents of the cortical granules, which are released following fertilization. There is little information, however, regarding the contents of CGs, let alone their mode of action on the ZP modification. This proposal will address the characterization and function of the first described mammalian egg cortical granule protein. These studies will use cell biological, biochemical, and molecular approaches, including metabolic labeling of eggs, immunoprecipitation and immunoblotting, preparation and screening of cDNA libraries, and isolation and analysis of cDNA clones. Results of these studies will provide new information regarding (1) the biochemical basis of egg activation, (2) the role of G proteins in mediating sperm-induced egg activation, and (3) the characterization and cloning of the first mammalian cortical granule antigen.