BIOCHEMISTRY OF SPERM-EGG INTERACTION AND FERTILIZATION

这项研究的目标是获得深层次的细胞和 精子-卵子相互作用机制的生化知识 受精。海洋无脊椎动物、海胆和 鲍鱼，在这项研究中使用，因为它们是非哺乳动物， 便宜，它们产生大量的配子，配子相互作用 在成虫之外，它在几秒钟的时间跨度内同步发生， 由于海洋无脊椎动物和 哺乳动物受精。所学到的基本知识可应用于 非激素避孕新方法的发展前景 哺乳动物。这项建议关注精子-卵子相互作用的两个现象： 海胆卵冻与精子质膜结合的机制 诱导精子顶体反应的受体蛋白及其机制 鲍鱼精子裂解素通过一种 非酶过程。这两种现象背后的基本机制 精子-卵子相互作用是非激素的潜在靶点 避孕。该项目的具体目标如下：SEA 海胆精子：从蛋冻中分离完整的糖蛋白 引发精子顶体反应。克隆并描述这一点 蛋白质，并确定它在高等物种中是否有同源物。至 鉴定这种蛋冻糖蛋白的诱导性低聚糖。 以确定哪些精子膜蛋白是它们的受体。至 测定6okD的氨基酸序列、功能和分布 海胆精子的蛋白质。6okD和210okD精子膜蛋白 是诱导精子的卵冻糖蛋白的受体 顶体反应。6okD蛋白与人尿调蛋白同源，是一种 孕妇尿液中含量较高的未知功能蛋白质。为了克隆， 210KD的序列测定及其同源性和生理功能的测定 来自海胆精子膜的蛋白质。是钙离子通道还是 钙通道调节剂？鲍鱼卵黄信封(VES)：至 VE糖蛋白纤维前后超微结构的测定 VE与粗制精子赖氨酸、纯赖氨酸、酒精或热能一起溶解。 分离单独的3-4 VE糖蛋白并确定与哪一种结合 精子裂解素。它是一个多肽还是寡糖识别结构域 与赖氨酸结合？为什么反应物种是特定的？克隆并测序 与赖氨酸结合的VE糖蛋白。要完整地描述 VE配基结构域(寡糖或肽)，显示物种特异性 对精子裂解素的亲和力。IF多肽，进行定点突变 以确定关键的残留物。克隆、测序和鉴定 构成鲍鱼精子晶状衬里的蛋白质 顶体颗粒。这一项目的成果对深化 我们对动物精卵相互作用生物化学的认识 受精。

The objective of this research is to acquire a deep cellular and biochemical knowledge of the mechanisms of sperm-egg interaction during fertilization. Marine invertebrate animals, the sea urchin and the abalone, are used in this research because they are nonmammalian, inexpensive, they yield vast quantities of gametes, gamete interaction is external to the adults, it occurs synchronously in a time span of seconds, and because of the striking similarities between marine invertebrate and mammalian fertilization. The basic knowledge gained may be applied in the future to the development of novel methods of nonhormonal contraception in mammals. This proposal focuses on two phenomena of sperm-egg interaction: The mechanism by which sea urchin egg jelly binds to sperm plasma membrane receptor proteins to induce the sperm acrosome reaction; and the mechanism by which abalone sperm lysin dissolves the egg vitelline envelope by a nonenzymatic process. The basic mechanisms underlying these two phenomena of sperm-egg interaction are potential targets for nonhormonal contraception. The specific aims of this project are the following: Sea urchin sperm: To isolate the intact glycoprotein from egg jelly that induces the sperm acrosome reaction. To clone and characterize this protein and to determine if it has homologs in higher species. To characterize the inductive oligosaccharides of this egg jelly glycoprotein. To determine which sperm membrane proteins are receptors for them. To determine the amino acid sequence, function and distribution of the 6OkD protein of sea urchin sperm. The 6OkD and 2lOkD sperm membrane proteins are the receptors for the egg jelly glycoprotein which induces the sperm acrosome reaction. The 6OkD protein is homologous to human uromodulin, a protein of unknown function that is high in pregnancy urine. To clone, sequence and determine the homology and physiological role of the 2lOkD protein from sea urchin sperm membranes. Is it a calcium channel or a regulator of calcium channels? Abalone egg vitelline envelopes (VEs): To determine the ultrastructure of the VE glycoprotein fibers before and after dissolution of VE with crude sperm lysins, pure lysins, alcohol or heat. To isolate the individual 3-4 VE glycoproteins and determine which bind to sperm lysin. Is it a peptide or an oligosaccharide recognition domain that binds to lysin? Why is the reaction species specific? To clone and sequence the VE glycoproteins which bind to lysin. To completely characterize the VE ligand domains (oligosaccharide or peptide) which show species specific affinity for sperm lysin. If peptide, to perform site directed mutagenesis to determine the crucial residues. To clone, sequence and characterize the protein comprising the paracrystalline lining of the abalone sperm acrosomal granule. The results of this project are central to deepening our knowledge of the biochemistry of sperm-egg interaction in animal fertilization.