THE EPIDIDYMAL INITIATION OF SPERM MOTILITY

本提案旨在全面介绍 哺乳动物精子在附睾中运动能力的发育 过境。 它是基于现有的知识的作用， 钙离子、环磷酸腺苷和pH在调节成熟大鼠运动中的作用 和未成熟的精子，以及我们证明腺苷可能 在刺激成熟和成熟的运动中发挥关键作用， 未成熟精子由于其降低细胞质水平的能力 钙离子。 我们在提案中提供了证据， 牛的一系列生化事件的描述 尾精子从腺苷导致的环 AMP水平和运动的开始。 我们将检验这个假设 腺苷刺激成熟牛精子的运动， 降低细胞质钙离子水平， 刺激腺苷酸环化酶，抑制环AMP 磷酸二酯酶，提高环AMP水平，增加蛋白质 磷酸化，并改变钙调蛋白依赖的磷酸化 蛋白激酶底物。 我们还将检验以下假设： 牛头精子是不动的，因为这一系列的事件 不可能发生。 这一观点与我们获得的数据一致 这表明牛的头部精子中 钙离子水平（0.8- 0.9 μ M），低水平的腺苷和 环磷酸腺苷和pHi约0.4 pH单位低于能动尾 精子 我们将通过一次操作一个 细胞内腺苷、钙、环磷酸腺苷和pHi水平 同时测量所有精子的水平 其他因素和监测精子活力。 在这个和所有 在提案中的研究，我们将监测精子头部和 鞭毛运动参数的全计算机化方法 在上一个资助期内在我们实验室开发的。 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 运动性是逐渐改变，通过相互作用的精子和 附睾主动脉雄激素依赖性分泌蛋白 细胞，上述因素的水平对水平发现， 成熟精子 要做到这一点，我们将首先确定所有的水平， 附睾连续段的因素及其相关性 这些水平与完整的运动概况。 后续我们 将共同培养附睾主细胞，先前培养在 附睾区域雄激素的存在和缺乏 其中钙，腺苷， 环磷酸腺苷，pHi或运动模式的发生，并决定 是否在上皮细胞的影响下分离的精子 分泌蛋白在培养物中经历相同的变化， 在完整的附睾中发现 如果情况属实，我们会 分离、纯化和表征这些蛋白质， 已知的、预定的功能的优点。 成功 执行本建议中所述的目标将导致 对成熟精子运动机制的新描述， 精子如何在附睾中活动的新观点。

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.