THE EPIDIDYMAL INITIATION OF SPERM MOTILITY

本提案旨在提供对 哺乳动物精子在附睾期的运动发育 中转。它基于对以下角色的现有知识 钙离子、环磷酸腺苷和pH对成虫运动的调节作用 和未成熟的精子，根据我们的证明，腺苷可能 在激发成熟期和成熟期的运动性方面发挥关键作用 未成熟精子具有降低胞浆水平的能力 钙离子。我们在提案中提供了证据，允许 牛的一系列生化事件的描述 尾部精子由腺苷引导至环状突起 AMP水平与运动开始时间的关系。我们将检验这一假设 腺苷通过以下途径刺激成熟牛精子的活力 降低细胞内钙离子水平，进而降低细胞内钙离子水平 刺激腺苷环化酶，抑制环磷酸腺苷 磷酸二酯酶，提高环磷酸腺苷水平，增加蛋白质 磷酸化，并改变钙调素依赖的磷酸化 蛋白激酶底物。我们还将检验这一假设 牛头精子是静止的，因为这一系列事件 不可能发生。这一观点与我们获得的数据是一致的 这表明牛头精液中含有异常高的 钙离子水平(0.8-0.9um)，低水平的腺苷和 环状AMP和a phi比活动尾部低约0.4个pH单位 精子。我们将通过一次一个地操作 细胞内腺苷、钙、环磷酸腺苷和PHI的水平 同时测量所有精子的水平 其他因素和监测精子活动率。在这一切中 在提案中的研究中，我们将监测精子头和 鞭毛运动参数的全计算机化方法 这是我们实验室在上一次资助期间开发的。在 在项目的最后阶段，我们将测试假设 牛附睾在精子发育中的作用 运动性是指通过精子和精子的相互作用逐渐改变 附睾主分泌蛋白的雄激素依赖性 细胞，上述因子的水平与在 成熟的精子。要做到这一点，我们将首先确定所有 附睾连续节段的影响因素及其相互关系 这些级别具有完整的运动特征。随后，我们 将与之前培养的附睾主细胞共培养 附睾区雄激素的存在和缺失 其中钙、腺苷水平的显著变化 循环AMP、Phi或运动模式的发生和决定 分离的精子是否受上皮细胞的影响 分泌蛋白在培养过程中会发生相同的变化 记录在完整的附睾体中。如果发现是这样，我们将 通过提取、分离、纯化和鉴定这些蛋白质 利用已知的、预定的功能。成功 本建议书中描述的目标的执行将从 成熟精子运动机制的新描述 关于精子如何在附睾处活动的新观点。

This proposal is designed to provide a comprehensive view of the development of motility by mammalian sperm during epididymal transit. It is based upon existing knowledge of the roles of calcium ion, cyclic AMP, and pH in regulating motility in mature and immature sperm and on our demonstration that adenosine may play a key role in stimulating motility in both mature and immature sperm by virtue of its ability to lower cytosolic levels of calcium ion. We provide evidence in the proposal which allows the description of a sequence of biochemical events in bovine caudal sperm leading from adenosine to the elevation of cyclic AMP levels and the onset of motility. We will test the hypothesis that adenosine stimulates motility in mature bovine sperm by lowering the level of cytoplasmic calcium ion and that this in turn stimulates adenylate cyclase, inhibits cyclic AMP phosphodiesterase, elevates cyclic AMP levels, increases protein phosphorylation, and alters the phosphorylation of CaM-dependent protein kinase substrates. We will also test the hypothesis that bovine caput sperm are immotile because this sequence of events cannot occur. This view is consistent with data we have obtained which shows that bovine caput sperm contain exceptionally high levels of calcium ion (0.8-0.9muM), low levels of adenosine and cyclic AMP and a pHi some 0.4 pH units lower than motile caudal sperm. We will test this view by manipulating, one at a time, the intracellular levels of adenosine, calcium, cyclic AMP, and pHi in caput sperm while simultaneously measuring the levels of all other factors and monitoring sperm motility. In this and all studies in the proposal we will monitor both sperm head and flagellar movement parameters by a fully computerized method developed in our laboratory during the last funding period. In the final stages of the project we will test the hypothesis that the function of the bovine epididymis in the development of sperm motility is to progressively alter, by interaction of sperm and androgen-dependent secretory proteins of epididymal principal cells, the levels of the above factors toward the levels found in mature sperm. To do this we will first determine the levels of all factors in successive segments of the epididymis and correlate these levels with complete motility profiles. Subsequently, we will coculture epididymal principal cells, previously cultured in the presence and absence of androgens, from epididymal regions where significant changes in the levels of calcium, adenosine, cyclic AMP, pHi or the pattern of motility occur and determine whether isolated sperm under the influence of epithelial cell secretory proteins undergo the same changes in culture as were noted in the intact epididymis. If this is found to be so, we will isolate, purify, and characterize these proteins by taking advantage of known, predetermined functions. Successful execution of the aims described in this proposal will lead from a new description of the mechanisms of motility in mature sperm to a new view of how sperm become motile in the epididymis.