THE EPIDIDYMAL INITIATION OF SPERM MOTILITY

本提案的目的是用化学术语定义 精子在牛体内运动能力的发展 附睾症。这项建议源于我们国家的发展 一种体外模型系统的实验室，在该系统中可以 牛头肌静止精子的渐进性运动 同时升高cAMP和高cAMP引起附睾症 磷酸二酯酶抑制剂的浓度和添加特定的 附睾蛋白，我们称之为前向运动蛋白(FMP)。 最近，我们首次提供了细胞外腺苷的证据 调节哺乳动物精子中的cAMP水平和运动能力(很可能是通过 Ra质膜受体)和对 牛精子中的腺苷在附睾成熟过程中显著增加。 我们还提供了第一个证据，表明人类的发育变化 鞭毛胞浆的pH在成熟的增加之前是必需的 CAMP表现为运动性，这种pH变化明显增加 头部精子对腺苷的敏感度显著降低 启动运动所需的PDE抑制剂水平。我们还有 最近，通过电影摄影技术，描述了 被不纯的FMP制剂改变的头部精子并已证实 这些制剂改变了头部精子的一些运动参数， 包括进行性，接近于成熟尾部的数值 精子。 在这项提案中，我们计划详细记录腺苷在 附睾发育的运动性，为生理学研究提供化学依据 膜上腺苷受体的存在，表明腺苷在腔内 事实上，液体确实调节精子的活力，测量精子的酸碱度变化 在发育过程中和用碱基体外激活后，确定 (S)pH变化使头部精子“尾状”的机理(S)， 开发一种计算机辅助分析方法来定量该复合体 FMP存在和不存在时精子发育的运动模式， 用此方法纯化FMP，建立FMP的RIA，并用RIA进行研究 附睾结合部位和蛋白质的细胞结合部位，以及， 最后确定是否涉及蛋白质的羧甲基化反应 通过它们影响cAMP代谢的能力而启动运动。

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.