Molecular Mechanisms that Control Ca2+ Signaling in Human Spermatozoa

描述（由申请人提供）：细胞内Ca2+和pH值是精子与卵子受精能力的两个关键调节因子。细胞内Ca2+和pH值依次由精子离子通道控制。因此，要了解控制精子功能和男性生育能力的分子机制，我们需要对精子离子通道有更深入的了解。不幸的是，膜片钳技术应用于精子细胞的极度困难阻碍了我们对精子离子通道和控制男性生育能力的分子机制的理解。我们已经克服了这一障碍，并开发了一种将全细胞膜片钳技术应用于小鼠和人类精子的方法。令人惊讶的是，我们的膜片钳实验揭示了小鼠和人类精子中离子通道的显著差异。这些差异表明，依靠动物模型研究人类男性生育能力存在潜在的缺陷，并支持研究人类精子细胞中这些离子通道的必要性。我们的长期目标是阐明控制人类精子生育能力的离子通道信号传导机制。在这里，我们提出了三个具体的目标，以扩大我们对精子离子通道的认识。在Specific Aim 1中，我们将确定人类CatSper和Hv1通道的生理调节因子。我们假设精子活性的关键调节因子，如黄体酮、前列腺素、胆固醇和cAMP，可能通过调节CatSper或Hv1通道来调节它们对人类精子的作用。我们将使用膜片钳技术测试上述化合物对由CatSper和Hv1通道介导的电流的影响。在特异性目标2中，我们将鉴定人类精子的膜（非基因组）黄体酮受体。我们的初步结果已经确定了一组狭窄的特定蛋白质作为精子孕酮受体的候选者。我们将确定哪个候选蛋白作为黄体酮受体，并将确定其配体结合域的黄体酮。在特异性目标3中，我们将描述人类精子顶体离子通道。我们将开发一种将膜片钳技术应用于人类精子顶体的方法，然后使用这种方法来表征顶体Ca2+通道，这些通道可能从顶体释放Ca2+，并确定调节其活性的机制。从这项研究中获得的知识将有助于我们了解男性不育症的原因，并开发治疗不育症和避孕的新方法。

DESCRIPTION (provided by applicant): Intracellular Ca2+ and pH are two key regulators of the ability of sperm to fertilize an egg. Intracellular Ca2+ and pH are controlled in turn by sperm ion channels. Therefore, to understand the molecular mechanisms that control sperm function and male fertility, we need a more thorough understanding of sperm ion channels. Unfortunately, extreme difficulty in applying the patch-clamp technique to sperm cells has hampered our understanding of sperm ion channels and the molecular mechanisms controlling male fertility. We have overcome this barrier and developed a method to apply the whole-cell patch clamp technique to mouse and human spermatozoa. Surprisingly, our patch-clamp experiments revealed significant differences between ion channels in mouse and human spermatozoa. These differences indicate the potential pitfalls of relying on animal models for studying human male fertility and support the need to study these ion channels specifically in human sperm cells. Our long-term objective is to elucidate the mechanisms of ion channel-based signaling that control fertility in human spermatozoa. Here we propose three specific aims to expand our knowledge of sperm ion channels. In Specific Aim 1, we will identify the physiological regulators of human CatSper and Hv1 channels. We hypothesize that key regulators of sperm activity, such as progesterone, prostaglandins, cholesterol, and cAMP, are likely to mediate their actions on human spermatozoa by regulating CatSper or Hv1 channels. We will use the patch-clamp technique to test the effects of the above mentioned compounds on currents mediated by CatSper and Hv1 channels. In Specific Aim 2, we will identify the membrane (non- genomic) progesterone receptor of human spermatozoa. Our preliminary results have identified a narrow group of specific proteins as candidates for the sperm progesterone receptor. We will determine which of the candidate proteins serves as a progesterone receptor and will identify its ligand-binding domain for progesterone. In Specific Aim 3, we will characterize the acrosomal ion channels of human spermatozoa. We will develop a method for applying the patch-clamp technique to the acrosome of human spermatozoa and then use this method to characterize acrosomal Ca2+ channels that are likely to release Ca2+ from the acrosome and to identify the mechanisms that regulate their activity. The knowledge gained from this research will help us to understand the causes of male infertility and to develop new approaches for infertility treatment as well as contraception.