MAMMALIAN SPERM SURFACE MANNOSIDASE

该提案侧重于鉴定、纯化和化学 对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 种。 自从我们表征了一种新型 alpha-D- 以来，这一直是我们的重点 几种物种精子中的甘露糖苷酶。酶是一种内在的 质膜（PM）成分及其催化域是定向的 朝向完整精子的外表面。 多份报告， 包括我们最近对小鼠的研究表明，该酶具有 在高甘露糖/杂种的识别和结合中具有类似受体的作用 我们已经在小鼠透明带上证明了类型寡糖（OS）链 组件。 针对精子亚型产生的多克隆抗体 表面甘露糖苷酶与存在于中的甘露糖苷酶活性发生交叉反应 洗涤剂溶解了精子PM。 该抗体将用于检查 精子表面甘露糖苷酶的化学性质并评估其作用 精子与卵子的结合和受精。 该提案旨在 确定精子表面相互作用的分子机制 甘露糖苷酶与大鼠透明带上存在的互补分子 透明 (ZP) 糖复合物。提案的具体目标 是： 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) 膜的检查 精子发生和精子过程中的拓扑结构和特定阶段的合成 成熟。 3) 精子表面甘露糖苷酶的作用评价 精子与卵子的结合和受精。 4) 身份识别和 编码精子 PM α-D-甘露糖苷酶的 cDNA 的表征。 5） 类配体分子的鉴定和表征 大鼠 ZP。 6) 化学性质、数量和结构的测定 配体分子上的 OS 链。 老鼠将是主要物种 本研究中使用。 然而，如果 α-D-甘露糖苷酶抗体显示 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 透明带。 结合起来，这些研究将进一步加深我们对 精卵相互作用和受精的分子机制 老鼠，并将深入了解高程度的原因 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.