REGULATION OF SPERM PROACROSIN CONVERSION TO ACROSIN

精子顶体素原转化为顶体素的调节

• 批准号: 3312023
• 负责人: KENNETH L POLAKOSKI
• 金额: $ 22.97万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-04-01 至 1992-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3312023
• 关键词: acrosin; acrosome; affinity chromatography; antibody; chemical structure function; enzyme mechanism; epididymis; gel filtration chromatography; genetic translation; genital secretion; hamsters; human tissue; in vitro fertilization; messenger RNA; protease inhibitor; protein sequence; sperm; swine; zymogens

The primary goal of this renewal proposal is to sufficiently characterize the proacrosin-acrosin system so that definitive information regarding its regulation can be obtained. Both biochemical and physiological approaches will be utilized. The biochemical characterization will first focus on the determination of the amino acid sequence of boar proacrosin and in defining the topography of the active site of acrosin. These latter studies will be accomplished by using affinity labeling reagents to determine which specific amino acids are involved in acrosin's enzymatic activity. The second aim will be to characterize the precursors to proacrosin found in testis and epididymal boar sperm. This will be done to obtain a thorough understanding of nascent proacrosin so that the system can be quantitated and the regulation of its conversion elucidated. These studies will involve the precursor purification and characterization as well as the in vitro translation of boar testicular proacrosin mRNA. The physiological characterization will focus on the regulation of the proacrosin- acrosin system in live sperm. This will be accomplished by determined the effectiveness that specific inhibitors and antibodies to proacrosin and acrosin have on the sperm's ability to penetrate oocytes. These results will be quantitated by a kinetic enzyme analysis and specific forms that are affected will be identified by a highly sensitive gelatin-SDS-PAGE zymograph. For possible clinical and contraceptive purposes one of the test systems will utilize human sperm and then analyze their penetration of zona-free hamster eggs. A more in depth approach will focus on the well-characterized boar sperm proacrosin-acrosin system and the boar sperm's ability to undergo in vitro capacitation and in vitro fertilization of zona intact porcine ova. This study will result in a more complete understanding of the molecular events required for fertilization and could ultimately lead to possible means of controlling it for either contraceptive or fertility enhancement purposes. In addition, a more thorough comprehension of the regulatory mechanisms of this proteinase system will produce increased understanding of the possible regulation of other proteolytic enzyme systems which control many important biological processes.

该更新提案的主要目标是充分 表征顶体素原-顶体素系统，以便确定 可以获得有关其监管的信息。 两个都 将利用生物化学和生理学方法。 这 生化表征首先侧重于测定 公猪顶体肽原的氨基酸序列及其定义 顶体蛋白活性位点的地形。 后面这些研究 将通过使用亲和标记试剂来完成 确定顶体素涉及哪些特定氨基酸 酶活性。 第二个目标是描述 在公猪的睾丸和附睾精子中发现顶体素原的前体。 这样做是为了彻底了解新生事物 顶体素原，以便可以对系统进行定量和调节 其转换过程得到阐明。 这些研究将涉及 前体纯化和表征以及体外 公猪睾丸顶体蛋白原 mRNA 的翻译。 生理学 表征将集中于顶体蛋白原的调节 活精子中的顶体素系统。 这将通过以下方式完成 确定了特定抑制剂和 顶体素原和顶体素抗体对精子的能力有影响 以穿透卵母细胞。 这些结果将通过 动力学酶分析和受影响的具体形式将 可通过高灵敏度明胶-SDS-PAGE 酶谱仪进行鉴定。 出于可能的临床和避孕目的，其中一项测试 系统将利用人类精子，然后分析其渗透力 无透明带的仓鼠蛋。 更深入的方法将重点关注 已充分表征的公猪精子顶体素原-顶体素系统和 公猪精子在体外获能的能力 完整猪卵子带的体外受精。 这项研究将 导致对分子事件有更全面的了解 受精所需，并最终可能导致 控制避孕或生育的手段 增强目的。 另外，更全面的理解 该蛋白酶系统的调节机制将产生 加深对其他可能监管的了解 蛋白水解酶系统控制许多重要的生物 流程。