BIOCHEMISTRY OF SPERM-EGG INTERACTION AND FERTILIZATION

精卵相互作用和受精的生物化学

• 批准号: 2635984
• 负责人: VICTOR D VACQUIER
• 金额: $ 27.17万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-06-01 至 2002-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2635984
• 关键词: Gastropoda; SDS polyacrylamide gel electrophoresis; X ray crystallography; acrosome; affinity chromatography; alternatives to animals in research; cell cell interaction; cell membrane; egg /ovum; egg proteins; exocytosis; fertilization; gel filtration chromatography; glycoproteins; high performance liquid chromatography; immunoprecipitation; membrane proteins; molecular cloning; nucleic acid sequence; oligosaccharides; polymerase chain reaction; protein structure function; sea urchins; sperm; vitellin

The objective of this research is to gain fundamental knowledge of the cell biology and biochemistry of sperm-egg interaction. Sea urchins and abalone are used because they are nonmammalian, yet exhibit striking similarities to mammalian gamete interaction. The cell biology and biochemistry of sperm-egg interaction is one of the least understood fundamental biological processes. Basic knowledge gained by this research may be used in the future development of novel methods of contraception in mammals. In sea urchins this proposal focuses on the interaction of REJ (the sperm membrane receptor for egg jelly) with ARI (the acrosome reaction inducing glycoprotein purified from egg jelly). 50% of REJ is homologous to the human polycystic kidney disease protein PKD1. Study of how REJ regulates sperm ion channels may aid in the understanding of this significant human disease. In abalone, this project focuses on the egg vitelline envelope ligand for lysin, the sperm protein which dissolves the egg envelope by a nonenzymatic mechanism. This study may aid in understanding the biochemical mechanism mammalian sperm use to penetrate the egg zona pellucida. The specific aims for sea urchin sperm are: 1. To identify sperm membrane proteins associated with the 210kDa receptor for egg jelly (REJ). 2. To clone, sequence and characterize the proteins associated with REJ. 3. To determine the functions of the protein modules of REJ. 4. To characterize the acrosome reaction inducer (ARI) from sea urchin egg jelly which binds sperm REJ. 5. To isolate and characterize the oligosaccharide of ARI which binds to REJ. 6. To use syntaxin and synaptotagmin, which we have cloned from sperm, to identify proteins associated with exocytosis of the acrosomal vesicle. The specific aims for the abalone ligand for lysin are: 7. To study the biochemistry and ultrastructure of the vitelline envelope ligand (VE-L) for lysin. To determine the affinities of homospecific and heterospecific combinations of lysins and VE-Ls. 8. To clone, sequence and characterize the cDNA for the VE-L. 9. To determine if the binding of lysin to VE-L is based on protein or carbohydrate components of the VE-L. 10. To cleave the VE-L into the smallest fragment retaining affinity for lysin. To isolate and characterize this fragment. To cocrystallize lysin complexed with this fragment for x-ray diffraction analysis. 11. To determine which residues of lysin are responsible for its species-specific dissolution of the egg vitellin envelope (VE). 12. To determine the mechanism by which lysin dissolves the VE. Is it based on the stereospecific severing of hydrogen bonds, or hydrophobic interaction?

这项研究的目的是获得基础知识 精子和卵子相互作用的细胞生物学和生物化学。 使用海胆和鲍鱼是因为它们是非哺乳动物， 却与哺乳动物的配子有着惊人的相似之处 互动 精卵细胞生物学和生物化学 相互作用是人们最不了解的基本生物学之一， 流程. 通过这项研究获得的基本知识可以用于 在未来开发新的避孕方法， 哺乳动物 在海胆中，这一建议侧重于相互作用， REJ（卵冻精膜受体）与ARI（ 顶体反应诱导糖蛋白（从卵冻中纯化）。 50%的REJ与人类多囊肾病同源 PKD 1蛋白。 REJ如何调节精子离子通道的研究可能 帮助我们了解这种重要的人类疾病。 在 鲍鱼，这个项目的重点是鸡蛋卵黄包膜 溶素的配体，溶素是溶解卵子的精子蛋白 包络 非酶机制。 这项研究可能有助于 了解哺乳动物精子的生化机制， 穿透卵透明膜。 海的具体目标 海胆精子是：1。鉴定精子膜蛋白 与210 kDa卵胶受体（REJ）相关。 2.到 克隆、测序和表征与 REJ. 3.为了确定蛋白质模块的功能， REJ. 4.为了表征顶体反应诱导剂（ARI）， 结合精子的海胆卵冻。 5.分离和 表征与REJ结合的ARI的寡糖。 6. 使用syntaxin和synaptotagmin，我们已经从 精子，以确定蛋白质相关的胞吐作用， 顶体小泡 鲍鱼配体的具体目的是 溶素为：7.研究其生化和超微结构 卵黄囊膜配体（VE-L）为溶素。 确定 同种特异性和异种特异性组合的亲和力 溶素和VE-L。 8.为了克隆、测序和表征 VE-L的cDNA。 9.为了确定溶素与 VE-L基于VE-L的蛋白质或碳水化合物成分。 10.为了将VE-L切割成最小的碎片， 对溶素的亲和力。 分离并鉴定这个碎片。 使与该片段复合的溶素共结晶， 衍射分析 11.为了确定哪些溶素残基 负责其物种特异性溶解卵 卵黄蛋白被膜（VE）。 12.为了确定 溶素溶解VE。 是基于立体定向 氢键的断裂，或者疏水作用？