BIOCHEMISTRY OF SPERM-EGG INTERACTION AND FERTILIZATION

这项研究的目的是获得一个深细胞和 精子-卵子相互作用机制的生物化学知识 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，配子相互作用是 在成年人之外，它在几秒钟的时间内同步发生， 由于海洋无脊椎动物和 哺乳动物受精。 所获得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 该提案重点关注精子-卵子相互作用的两种现象： 海胆卵冻与精子质膜结合的机制 受体蛋白诱导精子顶体反应的机制 鲍鱼精子溶解素通过一种 非酶过程 这两种现象背后的基本机制 精卵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： 海胆精子：从卵冻中分离完整的糖蛋白， 诱导精子顶体反应。 克隆并描述这个 蛋白质，并确定它是否在高等物种中有同源物。 到 描述了这种卵冻糖蛋白的诱导性寡糖。 以确定哪些精子膜蛋白是它们的受体。 到 确定6OkD的氨基酸序列、功能和分布 海胆精子的蛋白质。 60kD和210kD精子膜蛋白 是诱导精子的卵冻糖蛋白的受体 顶体反应 6OkD蛋白与人尿调节素同源， 妊娠尿中含量高的未知功能的蛋白质。 为了克隆， 序列并确定21OkD的同源性和生理作用。 来自海胆精子膜的蛋白质。 是钙通道还是 钙通道调节剂鲍卵卵黄膜（VE）： 测定治疗前后VE糖蛋白纤维的超微结构 用粗精溶素、纯溶素、酒精或加热溶解VE。 分离单个3 - 4个VE糖蛋白并确定哪些结合 精子溶解素。 它是肽或寡糖识别结构域， 与溶素结合为什么反应物种是特定的？进行克隆和测序 与溶素结合的VE糖蛋白。 为了完全描述 显示物种特异性的VE配体结构域（寡糖或肽） 对精子溶素的亲和力。 如果是肽，则进行定点诱变 来确定关键的残留物 为了克隆、测序和表征 包含鲍鱼精子的次晶内层的蛋白质 顶体颗粒 该项目的成果对于深化 我们对动物精卵相互作用的生物化学知识 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.