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REGULATION OF SPERM PROACROSIN CONVERSION TO ACROSIN

精子顶体素原转化为顶体素的调节

基本信息

批准号：
3312024
负责人：
KENNETH L POLAKOSKI
金额：
$ 22.27万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
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

项目摘要

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.

这项更新提案的主要目标是充分表征顶体酶原-顶体酶系统，可以获得有关其管理的信息。两将利用生物化学和生理学方法。的生物化学表征将首先集中在确定猪顶体蛋白原的氨基酸序列，并在定义顶体酶活性位点的拓扑结构。后一项研究将通过使用亲和标记试剂来完成，确定哪些特定的氨基酸参与顶体酶的酶活性第二个目标是描述猪睾丸和附睾精子中发现的顶体酶原的前体。这样做是为了彻底了解新生的前顶体酶，使系统可以定量和调节他的转变被阐明了。这些研究将涉及前体纯化和表征以及体外猪睾丸顶体酶原mRNA的翻译。生理表征将集中在顶体蛋白原的调节上- 精子顶体酶系统。这将通过确定了特定抑制剂和顶体酶原和顶体酶的抗体对精子的能力穿透卵母细胞这些结果将通过动力学酶分析和受影响的特定形式将通过高灵敏度的明胶-SDS-PAGE酶谱仪鉴定。出于可能的临床和避孕目的，该系统将利用人类精子，然后分析它们的插入情况，没有透明带的仓鼠卵更深入的方法将侧重于充分表征的猪精子顶体蛋白原-顶体蛋白酶系统，公猪精子进行体外获能的能力，猪卵母细胞体外受精本研究将导致对分子事件的更完整的理解受精所需的，并最终可能导致可能的避孕或生育的控制手段增强的目的。此外，更深入的理解这种蛋白酶系统的调节机制会产生增加对其他可能的管制的了解蛋白水解酶系统控制着许多重要的生物流程.