THE EPIDIDYMAL INITIATION OF SPERM MOTILITY

本提案的目的是用化学术语界定以下方面的基础： 当精子穿过牛时， 附睾 该提案起源于我们国家的发展， 体外模型系统的实验室，其中已经可以 启动牛头部不动精子的前向运动 附睾的cAMP水平的同时升高与高 浓度的磷酸二酯酶抑制剂和添加特定的 附睾蛋白，我们称之为前向运动蛋白（FMP）。 最近，我们提供了第一个证据， 调节哺乳动物精子中的cAMP水平和运动性（最有可能是通过 Ra质膜受体），并且响应于 腺苷在牛精子附睾成熟过程中显著增加。 我们还提供了第一个证据，表明在一个发展的变化， 鞭毛胞质溶胶的pH值是在成熟增加之前所必需的， cAMP表现为运动性，并且这种pH变化显著增加 头部精子对腺苷的敏感性， 启动运动所需的PDE抑制剂水平。 我们还 最近描述的，通过电影摄影，运动特性， 头部精子被不纯的FMP制剂改变，并已证实， 这些制剂改变了头部精子的许多运动参数， 包括渐进性，以接近成熟的尾 精子 在本提案中，我们计划详细记录腺苷在以下方面的作用： 附睾发育的动力学，提供化学证据， 膜上存在腺苷受体，表明腺苷在管腔内 事实上，液体确实可以调节精子的活力，测量精子的pH值变化， 在开发过程中和用碱体外活化后， pH值变化使头部精子呈“尾状”的机制， 开发一种计算机辅助测定方法， 在存在和不存在FMP的情况下发育中精子的运动模式， 用此方法纯化FMP，建立FMP的RIA，用RIA研究 蛋白质的附睾结合位点和细胞结合位点，以及， 最终确定是否涉及蛋白质羧甲基化反应 在运动起始中，由于它们影响cAMP代谢的能力。

The purpose of this proposal is to define, in chemical terms, the basis for development of the capacity for motility as sperm traverse the bovine epididymis. The proposal has its origin in the development in our laboratory of an in vitro model system in which it has been possible to initiate progressive motility in immotile sperm from the bovine caput epididymis by simultaneous elevation of cAMP levels with high concentrations of phosphodiesterase inhibitors and addition of a specific epididymal protein, which we have called Forward Motility Protein (FMP). Recently, we have provided the first evidence that extracellular adenosine regulates cAMP levels and motility in mammalian sperm (most likely through an Ra plasma membrane receptor) and that the capacity to respond to adenosine increases markedly in bovine sperm during epididymal maturation. We have also provided the first evidence that a developmental change in the pH of the flagellar cytosol is required before maturational increases in cAMP are manifested as motility and that this pH change markedly increases the sensitivity of caput sperm to adenosine and dramatically lowers the levels of PDE inhibitors required to initiate motility. We have also recently described, by cinematography, the motility characteristics of caput sperm altered by impure preparations of FMP and have confirmed that these preparations change a number of motility parameters of caput sperm, including progressivity, to values approximating those of mature caudal sperm. In this proposal, we plan to document in detail the role of adenosine in the epididymal development of motility, provide chemical evidence for the existence of a membrane adenosine receptor, show that adenosine in luminal fluids does, in fact, regulate motility, measure the pH changes in sperm during development and after in vitro activation with bases, determine the mechanism(s) by which the pH change renders caput sperm "caudal-like", develop a computer-assisted assay with which to quantify the complex motility patterns of developing sperm in the presence and absence of FMP, use this assay to purify FMP, develop an RIA for FMP, use the RIA to study the epididymal binding site, and cellular binding site of the protein, and, finally determine whether protein carboxymethylation reactions are involved in motility initiation by virtue of their ability to affect cAMP metabolism.