BIOCHEMISTRY OF SPERM-EGG INTERACTION AND FERTILIZATION

精卵相互作用和受精的生物化学

• 批准号: 2673444
• 负责人: VICTOR D VACQUIER
• 金额: $ 27.61万
• 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/2673444
• 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; molecular cloning; nucleic acid sequence; oligosaccharides; polymerase chain reaction; protein structure function; sea urchins; sperm; syntaxin; 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 精卵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， 但与哺乳动物配子表现出惊人的相似之处 相互作用。 精卵的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。 通过本研究获得的基本知识可用于 未来新型避孕方法的发展 哺乳动物。 在海胆中，该提案侧重于相互作用 REJ（卵果冻的精子膜受体）与 ARI（ 顶体反应诱导从蛋冻中纯化的糖蛋白）。 REJ 50%与人类多囊肾同源 蛋白 PKD1。 REJ 如何调节精子离子通道的研究可能 帮助了解这种重要的人类疾病。 在 鲍鱼，该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 通过非酶机制形成包膜。 这项研究可能有助于 了解哺乳动物精子的生化机制 穿透卵子透明带。 海上的具体目标 海胆精子有： 1. 识别精子膜蛋白 与蛋冻 (REJ) 的 210kDa 受体相关。 2. 到 克隆、测序并表征与相关的蛋白质 拒绝。 3. 确定蛋白质模块的功能 拒绝。 4. 表征顶体反应诱导物 (ARI) 结合精子的海胆卵果冻 REJ。 5. 隔离和 表征与 REJ 结合的 ARI 寡糖。 6. 使用我们克隆的syntaxin和synaptotagmin 精子，识别与胞吐作用相关的蛋白质 顶体小泡。 鲍鱼配体的具体目标 溶素有： 7. 研究溶素的生物化学和超微结构 溶素的卵黄膜配体（VE-L）。 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. 克隆、测序和表征 VE-L 的 cDNA。 9. 确定溶素是否与 VE-L是基于VE-L的蛋白质或碳水化合物成分。 10. 将VE-L切割成保留的最小片段 对溶素的亲和力。 分离并表征该片段。 共结晶与该片段复合的溶素以用于 X 射线 衍射分析。 11. 确定哪些溶素残基 负责其物种特异性的卵溶解 卵黄蛋白包膜（VE）。 12. 确定机制 溶素溶解 VE。 是否基于立体定向 氢键断裂，还是疏水相互作用？