MAMMALIAN SPERM SURFACE MANNOSIDASE

该提案侧重于鉴定、纯化和化学 存在于相对物上的互补分子的表征 启动哺乳动物精卵识别和结合的配子 物种 这是我们关注的焦点，因为我们描述了一种新的α-D- 甘露糖苷酶对几个物种的精子。酶是一种内在的 质膜（PM）组件及其催化域是定向 朝向完整精子的外表面。 几份报告， 包括我们最近在小鼠中的研究，表明这种酶具有 在识别和结合高甘露糖/杂合物中的受体样作用 型寡糖（OS）链，我们已经证明了对小鼠骨骼肌 件. 针对精子亚型的多克隆抗体 表面甘露糖苷酶与存在于 洗涤剂溶解精子PM。 这种抗体将用于检查 精子表面甘露糖苷酶的化学性质，并评估其作用 精子与卵子结合和受精。 这项建议旨在 确定精子表面相互作用的分子机制 甘露糖苷酶与大鼠睾丸上存在的互补分子 透明质酸（ZP）糖缀合物。提案的具体目标 l）动力学性质、化学性质的检查，以及 α-D-甘露糖苷酶的定位。 2)检查其膜 拓扑学和精子发生过程中的阶段特异性合成 成熟 3)精子表面甘露糖苷酶在精子发育中的作用 精卵结合和受精。 4)识别和 编码精子PM α-D-甘露糖苷酶的cDNA的表征。 第五章） 来自人肝癌细胞的配体样分子的鉴定和表征 大鼠ZP。 6)化学性质、数量和结构的测定 配体分子上的OS链。 老鼠将是主要的物种 在这项研究中使用。 然而，如果α-D-甘露糖苷酶的抗体显示 与其他物种精子酶的交叉反应，我们将研究 精子甘露糖苷酶在其他物种中的作用。 拟议的研究将提供关于下列物质的化学性质的资料： 精子表面甘露糖苷酶及其互补分子 透明的 结合起来，这些研究将进一步加深我们对 精子-卵子相互作用和受精的分子机制 老鼠，并将提供深入了解的原因，高度的 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.