SPERM FUNCTION IN FERTILIZATION EVENTS

精子的质膜和顶体膜被分割成 具有不同分子和结构组成的结构域 在确定的受精步骤中的特定功能。长距离的 这项提议的目标是定义分子和结构 精子产生马赛克特征的机制 并确定细胞骨架组件在精子中的作用 发育和受精。有四个具体目标 这些目标。目的一是确定精子细胞细胞骨架的作用 在顶体形态发生中。速冻、深蚀刻冷冻断裂 薄层电子显微镜将被用来定义空间 顶体下f-肌动蛋白的组织和膜相互作用 仓鼠精子细胞中的网络。亲和力隔离协议将是 用于纯化精子细胞肌动蛋白结合蛋白及超微结构 将利用免疫化学来确定肌动蛋白结合的作用 顶体形态发生中的蛋白质。目标二是定义机制 它们产生了独特的分子和结构属性 顶体内膜。顶体内膜特异性单抗 抗体和超微结构免疫细胞化学将用于 确定建立特定结构域的蛋白质转运途径 蛋白质定位；特定的蛋白质-蛋白质相互作用 顶体内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 顶体外膜骨架的组装机制 并确定其在顶体功能中的作用。这种蛋白质 用于建立外层空间的贩运和分拣路径 顶体膜结构域将被确定。将确定是否 顶体膜既有固定膜的功能，又有固定膜的功能 蛋白质并将顶体基质分离成不同的 车厢。目的四是鉴定内脏的多肽 顶体膜在受精过程中起到结合小带的作用 透明质膜和卵质膜。纯化的内侧顶体 将测试膜结合透明带和卵子的能力。 并在体外阻断精子与卵子的相互作用。完成 这些具体目标将提供关于以下机制的新数据 建立顶体膜的不同结构域并提供新的 对结构域特异膜蛋白功能的洞察 精子发生和受精。

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.