EPIDIDYMAL INITIATION OF SPERM MOTILITY

细胞内钙离子([Ca~(2+)]i)、pH([pH]_i)和cAMP是关键的调节因子 精子活力。我们已经记录了精子cAMP水平的增加， [CVa2+]i在附睾期急剧下降，[pH]i升高 成熟。精子[pH]i升高，同时[Ca~(2+)=]i降低，也是一种 允许将cAMP级别的提升转换为 在体外转化为附睾头精子的运动能力。我们建议 附睾转运过程中[钙离子]i的临界变化，进而增加 CAMP水平通过钙调素介导的机制，并此外放大 CAMP在蛋白磷酸化水平的作用。我们的结果 明确提示精子内[Ca~(2+)]_i、[pH]_i和cAMP相互作用促进 附睾发育对精子活力的影响。我们将确定 的机械基础和功能后果 小鼠精子成熟过程中这些关键调控因子之间的相互关系 牛的附睾体。 在第一个目标中，我们将量化对精确变化的动力反应。 在精子内水平上，无论是单独的还是选定的组合， CAMP、Ca~(2+)和pH。需要闪光光解设备才能发挥作用 这些监管机构的级别变动已经完成， 这项新技术在精子中的可行性现在已经被证明。一个 全自动电脑化系统，也是在这个实验室开发的， 将用于分析精子头部和鞭毛的运动。目的 这一目的是为了确定满足以下条件的充要条件 不活动的附睾头精子的运动诱导。 在第二个目标中，我们将确定[pH]i和[Ca~(2+)]i是如何在两者中被调节的 成熟和未成熟的精子，在附睾精子过程中发生变化 成熟。我们对这些调解人的水平是如何的了解 在哺乳动物精子中的调节程度令人惊讶地差。我们将决定如何 [pH]i诱导线粒体钙循环和Na+/Ca2+的变化 反转运机制在精子成熟过程中降低[Ca~(2+)]i。最后，我们 将记录这些离子是如何参与调节种子内的 营地的级别。 在最终目标中，我们将扩展我们对 冈田酸敏感蛋白磷酸酶对成熟精子的调节作用 运动和附睾运动发育。蛋白磷酸酶是 细胞内秒之间串扰中的重要介体 体细胞中的信使。我们将使用生化和生物化学的组合 鉴定对精子重要的磷酸酶的分子方法 发展。 这些目标的成功执行将提供第一个描述 小鼠精子活力发育的生化基础 任何物种的附属物。

Intracellular calcium ([Ca2+]i), ph ([pH]i), and cAMP are key regulators of sperm motility. We have documented that sperm cAMP levels increase, [CVa2+]i dramatically declines, and [pH]i increases during epididymal maturation. An increase in sperm [pH]i, while lowering [Ca2=]i, is also a permissive event that enables the elevation of cAMP levels to be translated into motility in caput epididymal sperm in vitro. We suggest that the critical changes in [Ca2+]i during epididymal transit, in turn, increase cAMP levels by calmodulin-mediated mechanisms and in addition amplify the effect of cAMP at the level of protein phosphorylation. Our results clearly indicate the [Ca2+]i, [pH]i, and cAMP interact in sperm to promote epididymal development of sperm motility. We will determine the mechanistic basis for and the functional consequences of the interrelationships among these key regulators during sperm maturation in the bovine epididymis. In aim one, we will quantify the motility response to precise alterations in the intra-sperm levels, either individually or in selected combination, of cAMP, Ca2+, and pH. The flash-photolysis equipment needed to effect alterations in the levels of these regulators has been assembled and the feasibility of this novel technique in sperm has now been documented. A fully automated computerized system, also developed in this laboratory, will be used for analysis of sperm head and flagellar motion. The purpose of this aim is to identify the necessary and sufficient conditions for motility induciton in immotile caput apididymal sperm. In aim two, we will determine how [pH]i and [Ca2+]i are regulated in both mature and immature sperm and are altered during epididymal sperm maturation. Our knowledge of how the levels of these mediators are regulated in mammalian sperm is surprisingly poor. We will determine how [pH]i induced changes in mitochondrial calcium cycling and Na+/Ca2+ antiport mechanisms lower [Ca2+]i during sperm maturation. Finally, we will document how these ions are involved in regulation of intrasperm levels of cAMP. In the final aim, we will extend our novel observations ont he role of okadaic acid-sensitive protein phosphatases in regulating mature sperm motility and epididymal motility development. Protein phosphatases are important mediators in the cross-talk between intracellular second messengers in somatic cells. We will use a combination of biochemical and molecular approaches to identify the phosphatases important to sperm development. Successful execution of these aims will provide the first description of the biochemical basis for the development of sperm motility in the epididymis of any species.