SPERM FUNCTION IN FERTILIZATION EVENTS

受精事件中的精子功能

• 批准号: 2392362
• 负责人: GARY E OLSON
• 金额: $ 20.28万
• 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 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2392362
• 关键词: SDS polyacrylamide gel electrophoresis; acrosome; actin binding protein; affinity chromatography; cell adhesion; cytoskeletal proteins; cytoskeleton; egg /ovum; electron microscopy; fertilization; freeze etching; guinea pigs; hamsters; immunocytochemistry; intermolecular interaction; intracellular membranes; laboratory mouse; laboratory rabbit; membrane biogenesis; membrane proteins; membrane structure; microfilaments; monoclonal antibody; myosins; protein purification; protein structure function; protein transport; spermatogenesis; western blottings

The spermatozoan plasma and acrosomal membranes are partitioned into domains of distinct molecular and structural composition which perform specific functions at defined steps of fertilization. The long range goals of this proposal are to define the molecular and structural mechanisms which generate the mosaic character of the spermatozoan membranes and to identify the role of cytoskeletal assemblies in sperm development and fertilization. Four specific aims are directed towards these goals. Aim one is to define the role of the spermatid cytoskeleton in acrosome morphogenesis. Quick-freeze, deep-etch freeze-fracture and thin section electron microscopy will be employed to define the spatial organization and membrane interactions of the subacrosomal f-actin network in hamster spermatids. Affinity-isolation protocols will be utilized to purify spermatid actin-binding proteins and ultrastructural immunochemistry will be utilized to define the role of actin-binding proteins in acrosome morphogenesis. Aim two is to define the mechanisms which generate the unique molecular and structural properties of the inner acrosomal membrane. Inner acrosomal membrane-specific monoclonal antibodies and ultrastructural immunocytochemistry will be employed to identify protein trafficking pathways which establish domain-specific protein localizations; specific protein-protein interactions between the inner acrosomal membrane and perinuclear cytoskeleton which function in membrane domain formation will be identified. Aim three is to identify the mechanism of assembly of the membrane skeleton of the outer acrosomal membrane and to define its roles in acrosome function. The protein trafficking and sorting pathways utilized to establish the outer acrosomal membrane domain will be identified. It will be determined if the acrosomal lamina functions both to immobilize integral membrane proteins and to segregate the acrosomal matrix into distinct compartments. Aim four is to identify polypeptides of the inner acrosomal membrane which function during fertilization to bind the zona pellucida and the egg plasma membrane. Purified inner acrosomal membranes will be tested for their ability to bind the zona and the egg plasma membrane and to block sperm-egg interactions in vitro. Completion of these specific aims will provide new data on the mechanisms which establish the different domains of the acrosomal membrane and provide new insights into the function of domain-specific membrane proteins during spermiogenesis and fertilization.

精子血浆和粘膜膜被划分为 执行的不同分子和结构组成的域 在定义的受精步骤下的特定功能。 远距离 该建议的目标是定义分子和结构 产生精子的镶嵌特征的机制 膜并确定细胞骨架组件在精子中的作用 发育和施肥。 四个具体目标是针对的 这些目标。 目的是定义精子细胞骨架的作用 在高级体形态发生中。 快速冻结，深蚀刻冻结裂缝和 将采用薄部分电子显微镜来定义空间 乳粒F-肌动蛋白的组织和膜相互作用 仓鼠精子中的网络。 亲和力 - 隔离协议将是 用于纯化精子肌动蛋白结合蛋白和超微结构 免疫化学将用于定义肌动蛋白结合的作用 蛋白质中的蛋白质形态发生。 目标两个是定义机制 它产生了独特的分子和结构特性 内部杂色膜。 内部粘膜膜特异性单克隆 将采用抗体和超微结构免疫细胞化学化学 确定蛋白质运输途径，以建立特定域特异性 蛋白质局部；特定的蛋白质 - 蛋白质相互作用 内部粘膜膜和核周细胞骨架，在 将确定膜结构域的形成。 目标三是确定 外顶杂色体膜骨架的组装机制 膜并定义其在Acrosos功能中的作用。 蛋白质 贩运和分类途径用于建立外部 将识别Arosomal膜结构域。 将确定是否 AROSOMAL LAMINA功能既可以固定整体膜 蛋白质并将谷子基质分离为不同 车厢。 目标四是识别内部的多肽 在受精过程中起作用以结合Zona的粘膜膜 Pellucida和卵质膜。 纯化的内cro体 将测试膜的结合能力和卵子 质膜并在体外阻断精子 - 蛋的相互作用。 完成 这些特定目的将提供有关机制的新数据 建立杂色膜的不同领域并提供新的 洞悉域特异性膜蛋白的功能 精子发生和受精。