SPERM FUNCTION IN FERTILIZATION EVENTS

精子的质膜和顶体膜是分开的 不同分子和结构组成的区域， 在规定的施肥步骤中显示特定的功能。 的 该提案的长期目标是定义分子和 产生马赛克特征的结构机制 精子膜和确定细胞骨架的作用 组装在精子发育和受精中。 首先我们将 确定细胞骨架组件是否在分离中起作用 膜蛋白的选择域通过识别血浆 与细胞骨架相关的膜组分和制备 针对它们的抗体免疫定位它们的表面 分布。 膜下组装， 以及它们的细胞骨架联系在 精子发育和受精事件将由 使用电子显微镜和免疫定位技术。 其次，我们将定义的分子和结构特性的 从经处理的质膜获得的精子膜骨架 与非离子洗涤剂;我们将采用电子显微镜， 确定其超微结构和电泳技术，以确定 其多肽组成和特异性蛋白质-蛋白质 与整合膜蛋白的相互作用。 第三，我们将 利用细胞分级分离顶体细胞骨架 程序，并确定其多肽组成和相互作用 与质膜的特定成分结合。 第四，我们 表征洗涤剂稳定的气溶胶基质组分， 关于它们的多肽组成，并定义它们在 分离顶体内的顶体水解酶。 最后我们 将定义细胞骨架组装的蛋白质组成 仅限于精子中段，并确定它是否在 引导线粒体形成中段， 精子发生或维持线粒体分布， 成熟精子。 这些具体目标的实现将为 新的数据机制的基础马赛克建设 精子质膜，并提供所需的分子数据， 特定施肥事件的基础。

The spermatozoan plasma and acrosomal membranes are partitioned into domains of distinct molecular and structural composition which display specific functions at defined fertilization steps. 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. First we will determine if cytoskeletal assemblies function in the segregation of membrane proteins to selected domains by identifying plasma membrane components associated with the cytoskeleton and preparing antibodies against them to immunolocalize their surface distributions. The submembranous assembly with which they associate and changes in their cytoskeletal associations during sperm development and fertilization events will be determined the using electron microscopy and immunolocalization techniques. Second we will define the molecular and structural properties of the sperm membrane skeleton obtained from plasma membranes treated with nonionic detergents; we will employ electron microscopy to define its ultrastructure and electrophoretic techniques to define its polypeptide composition and specific protein-protein interactions with integral membrane proteins. Third we will isolate the acrosomal cytoskeleton utilizing cell fractionation procedures and define its polypeptide composition and interaction with specific components of the plasma membrane. Fourth we will characterize the detergent-stable aerosomal matrix components with respect to their polypeptide composition and define their role in segregating acrosomal hydrolases within the acrosome. Finally we will define the protein composition of a cytoskeletal assembly restricted to the sperm midpiece and determine if it functions in directing the mitochondria to the forming midpiece during spermiogenesis or in maintaining mitochondrial distribution in mature spermatozoa. Completion of these specific aims will provide new data on the mechanisms underlying the mosaic construction of the sperm plasma membrane and provide needed data on the molecular basis of specific fertilization events.