EPIDIDYMAL INITIATION OF SPERM MOTILITY

细胞内钙（[Ca 2 +] i）、pH（[pH] i）和cAMP是细胞内钙离子浓度的关键调节因子。 精子活力 我们已经证明精子cAMP水平增加， [CVa2+] i急剧下降，[pH] i增加， 成熟 精子[pH] i的增加，同时[Ca 2 =] i的降低，也是 允许事件，使cAMP水平的升高被翻译 附睾头精子活力。 我们建议 附睾转运过程中[Ca2 +] i的临界变化反过来增加 cAMP水平的钙调素介导的机制，并此外放大 cAMP在蛋白磷酸化水平的作用。 我们的结果 清楚地表明[Ca 2 +] i，[pH] i和cAMP在精子中相互作用，以促进 附睾发育对精子活力的影响。 康贝特人将以 的机制基础和功能后果， 精子成熟过程中这些关键调节因子之间的相互关系 牛的附睾 在目标一，我们将量化运动反应，以精确的改变 在精子内水平，单独或以选定的组合， cAMP、Ca~（2+）和pH值的影响。 这些调节剂水平的改变已经组装， 这种新技术在精子中的可行性现已被证明。 一 全自动计算机化系统，也是在这个实验室开发的， 将用于分析精子头部和鞭毛运动。 目的 这一目标的目的是确定必要和充分条件， 不动精子顶精子运动诱导 在目标二中，我们将确定[pH] i和[Ca 2 +] i在两种细胞中是如何调节的， 成熟和未成熟的精子，并在附睾精子 成熟 我们对这些介质水平的了解 在哺乳动物精子中的调节是令人惊讶的差。 我们将决定如何 [pH]i引起线粒体钙循环和Na +/Ca2+的变化 反向转运机制降低精子成熟过程中的[Ca2 +] i。 最后我们 将记录这些离子是如何参与调节精子内分泌的。 cAMP水平。 在最后的目标，我们将扩大我们的新的意见对他的作用， 冈田酸敏感蛋白磷酸酶在调节成熟精子中的作用 运动和附睾运动发育。 蛋白磷酸酶是 在细胞内第二个细胞之间的串扰中的重要介质 体细胞中的信使 我们将使用生物化学和 鉴定精子重要磷酸酶的分子方法 发展 成功地实现这些目标将提供第一个描述， 精子活力的发展的生化基础 任何物种的附睾。

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.