SPERM FUNCTION IN FERTILIZATION EVENTS

受精事件中的精子功能

• 批准号: 6182204
• 负责人: GARY E OLSON
• 金额: $ 24.19万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6182204
• 关键词: acrosome; affinity chromatography; fertilization; guinea pigs; hamsters; immunoelectron microscopy; immunofluorescence technique; immunoprecipitation; intermolecular interaction; intracellular membranes; laboratory rabbit; ligands; membrane biogenesis; membrane fusion; membrane proteins; membrane structure; molecular site; protein binding; protein isoforms; protein localization; protein structure function; sperm capacitation; syntaxin; western blottings

The sperm plasma and acrosomal membranes are partitioned into domains of distinct molecular composition which perform specific functions required for mammalian fertilization. The long range goals of this proposal are to define the molecular processes which assemble discrete membrane domains and to identify the biochemical mechanisms regulating the fusion between the periacrosomal plasma membrane and the outer acrosomal membrane during the acrosome reaction. Four aims are proposed addressing these goals. Aim one is to identify the function of the periacrosomal plasma membrane protein PM52 (sperad) in sperm capacitation and/or the acrosome reaction. Immunoprecipitation and blot overlay analyses will be performed to identify ligands for the cytoplasmic domain of PM52 and permeant peptide mimetics of the proline- rich domain of PM52 will be tested for effects on capacitation and the acrosome reaction. Aim 2 is to determine if docking interactions between the periacrosomal plasma membrane and the outer acrosomal membrane generate domain-specific protein localization. Immunoblotting and blot overlays will identify PM52 binding proteins in the outer acrosomal membrane and immunofluorescence experiments will test if disruption of PM52-outer acrosomal membrane interaction results in protein redistribution. Aim 3 is to determine if germ cell isoforms of syntaxin and synaptophysin function in the membrane fusion events of the acrosome reaction. Immunolocalization and immunoprecipitation analyses will be performed to determine if these proteins assemble into a membrane fusion complex required for acrosomal exocytosis. Aim 4 is to identify protein-protein interactions regulating the assembly of the outer acrosomal membrane-acrosomal matrix complex. Biochemical and ultrastructural experiments will be performed to identify proteins of the outer acrosomal membrane which bind to, and regulate the distribution of, acrosomal matrix assemblies within the acrosome. Completion of these studies will provide new insights into the assembly of the acrosomal segment and of its functions during mammalian fertilization and the findings have potential application to strategies for fertility regulation and/or improvement.

精浆和顶体膜被划分为不同的区域 具有不同的分子组成， 哺乳动物受精所必需的。 这一长期目标 建议是定义分子过程， 膜结构域，并确定调节的生化机制 顶体周围质膜与外膜的融合 在顶体反应期间顶体膜。 提出了四个目标 实现这些目标。 目的一是确定 精子顶体周围质膜蛋白PM 52 获能和/或顶体反应。 免疫沉淀和印迹 将进行重叠分析，以确定 PM 52的胞质结构域和脯氨酸的渗透肽模拟物， 将测试PM 52丰富结构域对获能的影响， 顶体反应 目的2是确定对接相互作用是否 顶体周质膜和顶体外膜之间 膜产生结构域特异性蛋白定位。 免疫印迹 并且印迹覆盖将鉴定PM 52结合蛋白， 顶体膜和免疫荧光实验将测试是否 PM 52-顶体外膜相互作用的破坏导致 蛋白质再分布 目的3是确定是否生殖细胞亚型的 突触融合蛋白和突触体蛋白在细胞膜融合事件中的作用 顶体反应 免疫定位和免疫沉淀分析 将进行，以确定这些蛋白质是否组装成 顶体胞吐所需的膜融合复合物。 目标4： 确定调节蛋白质组装的蛋白质-蛋白质相互作用， 顶体外膜-顶体基质复合体。 生化和 将进行超微结构实验以鉴定 顶体外膜，其结合并调节 顶体内顶体基质组装体的分布。 这些研究的完成将为大会提供新的见解 顶体节及其功能的哺乳动物 施肥和研究结果具有潜在的应用战略 用于生育调节和/或改善。