SPERM FUNCTION IN FERTILIZATION EVENTS

这项建议的长期目标是确定结构性的 和特定膜结构域的生化性质以及 哺乳动物精子膜相关集合体的研究 这些膜结构域和膜相关组件如何发挥作用 在受精过程中。在本提案中，将注意 重点关注参与顶体反应的膜，在 为了确定有助于膜融合过程的机制， 和顶体后段的质膜上，以便 描述它在配子融合中的作用。 本提案的具体目标是：1)描述 大分子复合体(模糊涂层)，仅定位于 顶体外膜的管腔面；2)确定如何 毛皮复合体与外膜整体蛋白结合 顶体细胞膜；3)确定绒毛-被毛复合体在 顶体反应的膜囊化过程；4)利用 抗模糊涂层复合体的抗体用于鉴定顶体反应 精子；5)确定毛茸茸组件在精子过程中是否被修改 附睾成熟；6)鉴定相关酶活性 膜结构域参与顶体反应；7)至 描述顶体后鞘的特征；8)识别细胞内 哺乳动物精子中细胞骨架蛋白的分布。氮气 将使用空化和选择性提取程序来破坏 精子和纯化的亚细胞部分，包括特定的 膜结构域或膜相关组件将通过 各种离心法。结合了超微结构， 生化和免疫学技术，这些技术以前是 用于鉴定膜相关蛋白的功能性质 其他单元格类型中的组件，将用于实现这些特定的 目标。这些具体目标的解决将有助于我们的 对膜生理学的理解，对我们对重大事件的理解 在受精过程中对男性生殖生物学的影响。

The long-range objectives of this proposal are to identify the structural and biochemical properties of specific membrane domains and membrane-associated assemblies of mammalian spermatozoa and to determine how these membrane domains and membrane-associated assemblies function during the fertilization process. In the present proposal attention will be focused upon the membranes participating in the acrosome reaction, in order to identify mechanisms contributing to the membrane fusion process, and upon the plasma membrane of the postacrosomal segment, in order to characterize its role in gamete fusion. The specific objectives of this proposal are 1) to characterize the macromolecular complex (fuzzy-coat) which is exclusively localized on the luminal face of the outer acrosomal membrane; 2) to determine how the fuzzy-coat complex associates with integral membrane proteins of the outer acrosomal membrane; 3) to define the role of the fuzzy-coat complex in the membrane vesiculation process of the acrosome reaction; 4) to utilize antibodies against the fuzzy-coat complex to identify acrosome reacting sperm; 5) to determine if the fuzzy-coat assembly is modified during sperm maturation in the epididymis; 6) to identify enzyme activities associated with membrane domains participating in the acrosome reaction; 7) to characterize the postacrosomal sheath; and 8) to identify the intracellular distribution of cytoskeletal proteins in mammalian spermatozoa. Nitrogen cavitation and selective extraction procedures will be employed to disrupt spermatozoa and purified subcellular fractions consisting of specific membrane domains or membrane-associated assemblies will be isolated by a variety of centrifugation protocols. A combination of ultrastructural, biochemical and immunological techniques, which have previously been utilized to identify the functional properties of membrane-associated assemblies in other cell types, will be employed to pursue these specific aims. A resolution of these specific aims will contribute to our understanding of membrane physiology, to our understanding of major events in the fertilization process and to male reproductive biology.