MAMMALIAN SPERM SURFACE MANNOSIDASE

该提案的重点是鉴定、提纯和化学物质 反面存在的互补分子的表征 哺乳动物中启动精卵识别和结合的配子 物种。自从我们描述了一种新的α-D- 几种植物精子上的甘露糖苷酶。这种酶是一种内在的 质膜成分及其催化域的定位 朝向完整精子的外表面。几份报告， 包括我们最近在小鼠身上的研究表明，这种酶具有 受体在识别和结合高甘露糖/杂交种中的作用 我们在小鼠卵泡带上展示的类型寡糖链(OS) 组件。一种针对精子异构体的多克隆抗体 表面甘露糖苷酶与存在的甘露糖苷酶活性发生交叉反应 洗涤剂增溶精子PM这种抗体将被用来检测 精子表面甘露糖苷酶的化学性质及其作用评价 在精子-卵子结合和受精方面。这项提议旨在 确定精子表面相互作用的分子机制 甘露糖苷酶与大鼠卵泡带上的互补分子 透明质酸(ZP)糖结合物(S)。该提案的具体目标 是：L)动力学性质、化学性质和 α-D-甘露糖苷酶的定位。2)膜的检查 精子发生和精子发生过程中的拓扑结构和阶段特异性合成 成熟。3)评价精子表面甘露糖苷酶在 精卵结合和受精。4)识别和 精子质膜α-D-甘露糖苷酶编码基因的特性5) 人血清白蛋白中配体样分子(S)的鉴定与鉴定 大鼠ZP。6)确定化合物的化学性质、数量和结构 配基分子上的OS链(S)。老鼠将成为主要物种 在这项研究中使用。然而，如果对α-D-甘露糖苷酶的抗体显示 与其他物种的精子酶的交叉反应，我们将检查 精子甘露糖苷酶在其他物种中的作用。 拟议的研究将提供有关化学性质的信息。 精子表面甘露糖苷酶及其互补分子S 透明带。总而言之，这些研究将加深我们对 人精子与卵子相互作用和受精的分子机制 老鼠，并将提供洞察原因的高度 在哺乳动物物种中观察到的物种特异性。

The proposal focuses on identification, purification, and chemical characterization of complementary molecules present on the opposite gametes which initiate sperm-egg recognition and binding in mammalian species. This has been our focus since we characterized a novel alpha-D- mannosidase on spermatozoa of several species. The enzyme is an intrinsic plasma membrane (PM) component and its catalytic domain is oriented towards the outer surface of the intact spermatozoa. Several reports, including our recent study in the mouse, suggest that the enzyme has a receptor-like role in the recognition and binding to high mannose/hybrid type oligosaccharide (OS) chains which we have demonstrated on mouse zona components. A polyclonal antibody raised against an isoform of sperm surface mannosidase cross-reacts with the mannosidase activity present in the detergent solubilized sperm PM. This antibody will be used to examine the chemical nature of the sperm surface mannosidase and evaluate its role in sperm-egg binding and fertilization. This proposal is designed to determine the molecular mechanisms of the interaction of sperm surface mannosidase with the complementary molecules present on the rat zona pellucida (ZP) glycoconjugate(s). The specific objectives of the proposal are: l) Examination of kinetic properties, chemical nature, and localization of the alpha-D-mannosidase. 2) Examination of its membrane topology and stage-specific synthesis during spermatogenesis and sperm maturation. 3) Evaluation of the role of the sperm surface mannosidase in sperm-egg binding and fertilization. 4) Identification and characterization of cDNA encoding sperm PM alpha-D-mannosidase. 5) Identification and characterization of ligand-like molecule(s) from the rat ZP. 6) Determination of the chemical nature, number, and structure of the OS chains on ligand molecule(s). Rats will be the primary species used in this study. However, if the antibody to alpha-D-mannosidase shows cross-reactivity with the sperm enzyme from other species, we will examine the role of the sperm mannosidase in other species. The proposed studies will provide information on the chemical nature of the sperm surface mannosidase and its complementary molecule(s) on the zona pellucida. Combined, the studies will further our understanding of the molecular mechanisms of sperm-egg interactions and fertilization in the rat, and will provide insight into the reasons for the high degree of species-specificity observed in mammalian species.