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: 3312022
• 负责人: KENNETH L POLAKOSKI
• 金额: $ 20.45万
• 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/3312022
• 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.

这项延期提案的主要目标是充分 表征顶体酶原-顶体酶系统，以便最终确定 可以获得有关其监管的信息。两者都有 将利用生物化学和生理学方法。这个 生化特征将首先集中在测定 猪前顶体酶的氨基酸序列，并在定义 顶体酶活性部位的地形图。这些后一项研究 将通过使用亲和标记试剂来实现 确定哪些特定的氨基酸与顶体酶有关 酶活性。第二个目标将是描述 在精巢和附睾猪精子中发现的顶体前体素前体。 这样做是为了彻底了解新生的 以使前顶体酶系统可以被定量和调节 阐明了其转化的原因。这些研究将涉及 前体的纯化和表征以及体外实验 猪睾丸顶体原基因的翻译。生理学 表征将集中在原顶体蛋白的调节上- 活精子中的顶体酶系统。这将通过以下方式实现 确定了特定的抑制剂和 顶体酶原和顶体酶抗体对精子的能力有影响 以穿透卵母细胞。这些结果将通过一个 受影响的动态酶分析和特定形式的Will 用高灵敏的明胶-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法鉴定。 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，然后分析他们的穿透能力 去透明带仓鼠蛋。更深入的方法将重点放在 猪精子顶体酶原-顶体酶系统和 猪精子体外获能和受精能力的研究 猪卵透明带完整的体外受精。这项研究将 导致对分子事件的更完整的理解 受精所需的，并最终可能导致 为了避孕或生育而控制它的方法 增强目的。此外，更透彻地理解 这一蛋白水解酶系统的调节机制将产生 加深对其他可能的监管的理解 控制许多重要生物的蛋白水解酶系统 流程。