Molecular Mechanisms that Control Ca2+ Signaling in Human Spermatozoa

描述（由申请人提供）：细胞内Ca 2+和pH是精子与卵子受精能力的两个关键调节因子。细胞内的Ca ~（2+）和pH值依次由精子离子通道控制。因此，为了了解控制精子功能和男性生育力的分子机制，我们需要更深入地了解精子离子通道。不幸的是，将膜片钳技术应用于精子细胞的极端困难阻碍了我们对精子离子通道和控制男性生育力的分子机制的理解。我们已经克服了这一障碍，并开发了一种方法，应用全细胞膜片钳技术的小鼠和人类精子。令人惊讶的是，我们的膜片钳实验揭示了小鼠和人类精子离子通道之间的显着差异。这些差异表明，依赖动物模型研究人类男性生育能力的潜在陷阱，并支持需要研究这些离子通道，特别是在人类精子细胞。我们的长期目标是阐明控制人类精子生育力的基于离子通道的信号转导机制。在这里，我们提出了三个具体的目标，以扩大我们的精子离子通道的知识。在具体目标1中，我们将确定人类CatSper和Hv 1通道的生理调节剂。我们推测，精子活动的关键调节因子，如孕酮，肾上腺素，胆固醇和cAMP，可能通过调节CatSper或Hv 1通道介导其对人类精子的作用。我们将采用膜片钳技术检测上述化合物对CatSper和Hv 1通道介导的电流的影响。在第二章中，我们将鉴定人精子膜上的（非基因组）孕酮受体.我们的初步结果已经确定了一个狭窄的特定蛋白质组作为候选人的精子孕酮受体。我们将确定哪些候选蛋白质作为孕酮受体，并将确定其配体结合域孕酮。在特定目标3中，我们将描述人类精子顶体离子通道的特征。我们将开发一种方法应用膜片钳技术的顶体的人精子，然后使用这种方法来表征顶体的Ca 2+通道，可能会释放Ca 2+从顶体和确定的机制，调节其活动。从这项研究中获得的知识将有助于我们了解男性不育的原因，并开发不育治疗和避孕的新方法。

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.