SPERM FUNCTION IN FERTILIZATION EVENTS

受精事件中的精子功能

• 批准号: 2198014
• 负责人: GARY E OLSON
• 金额: $ 18.03万
• 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/2198014
• 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.

精子的质膜和顶体膜被分为 不同的分子和结构组成的域， 在特定的施肥步骤中发挥特定的作用。 远程 该提案的目标是定义分子和结构 产生精子镶嵌特征的机制 膜，并确定细胞骨架组装在精子中的作用 发育和受精。 四个具体目标是针对 这些目标。 目的一是明确精子细胞骨架的作用 顶体形态发生 快速冷冻，深蚀刻冷冻断裂和 薄切片电子显微镜将被用来定义空间 顶体下肌动蛋白的组织和膜相互作用 仓鼠精子细胞的网络。 亲和隔离协议将被 用于纯化精细胞肌动蛋白结合蛋白和超微结构 免疫化学将被用来确定肌动蛋白结合的作用， 顶体形态发生中的蛋白质。 目标二是明确机制 其产生所述聚合物的独特的分子和结构特性， 顶体内膜 顶体内膜特异性单克隆抗体 抗体和超微结构免疫细胞化学将被用来 鉴定建立结构域特异性 蛋白质定位;蛋白质之间的特异性蛋白质-蛋白质相互作用 顶体内膜和核周细胞骨架， 将鉴定膜结构域形成。 目标三是识别 顶体外膜骨架的组装机制 膜，并确定其在顶体功能中的作用。 蛋白质 利用贩运和分类途径建立外层空间 将鉴定顶体膜结构域。 将确定是否 顶体板层既起保护完整膜作用， 蛋白质和分离顶体基质成不同的 隔间 目的四是鉴定内分泌多肽 在受精过程中起作用的顶体膜， 透明体和卵质膜。 纯化内顶体 膜将被测试其结合昆虫和卵的能力 质膜和阻断精卵相互作用。 完成 这些具体目标将提供有关机制的新数据， 建立顶体膜的不同区域，并提供新的 深入了解结构域特异性膜蛋白在 精子发生和受精。