MECHANISMS OF MAMMALIAN FERTILIZATION/POLYSPERMY BLOCK

小鼠卵子的受精引发了一种称为“卵子激活”的现象。 卵激活导致（1）皮层颗粒（CG）的刺激 胞吐作用，导致透明质酸（ZP）的修饰， 构成多精受精的嵌段，和（2）导致一种 减数分裂周期到有丝分裂周期（有丝分裂激活）。 通过类似于 在体细胞分化过程中，精子可能通过G 蛋白质介导的信号转导途径，导致产生 特定的第二信使/离子变化。 特异性第二信使[肌醇1，4，5-三磷酸（IP 3）或 蛋白激酶C（PKC）激活剂]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胞吐作用和有丝分裂激活都是由线粒体启动的。 在精子中，可以产生不同的第二信使系统， 差异调节这两种反应。 虽然G 蛋白质介导体细胞中的胞吐作用和促有丝分裂活化， 事实上，没有确凿的数据表明哺乳动物的卵子激活是 由G蛋白偶联的信号转导级联介导。 这项建议 （1）这些第二信使的作用， 调节小鼠卵子激活的这两种成分，以及（2） G蛋白在卵子激活中的作用 这些研究将使用细胞生物学 和生物化学方法，包括显微注射和体外 卵子的受精 受精后ZP的修饰，其构成了 多精受精，被认为是由皮层内容介导的 颗粒，在受精后释放。 几乎没有 然而，关于CG的内容的信息，更不用说它们的模式了 对ZP的修改。 该提案将解决 第一个描述的哺乳动物卵皮质的特征和功能 颗粒蛋白 这些研究将使用细胞生物学，生物化学， 分子方法，包括鸡蛋的代谢标记， 免疫沉淀和免疫印迹，cDNA制备和筛选 文库，以及cDNA克隆的分离和分析。 这些研究的结果将提供有关以下方面的新信息：（1） 卵激活的生化基础，（2）G蛋白在 介导精子诱导的卵激活，和（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.