Calcium signaling nanodomains in sperm motility and fertility

项目摘要 自2001年发现以来，鞭毛Ca ~（2+）通道CatSper一直是唯一一个遗传上依赖于Ca ~（2+）的通道。 突变导致男性不育。目前对功能的理解存在重大的知识差距， 调节这一至关重要的通道及其信号通路，以触发超活化，一种不对称的 雄性生殖力所需的鞭毛运动。长期目标是加深对精子生理学的理解 并发现可以用来评估和控制精子活力和生育力的新途径。 CatSper通道在四个线性“赛车条纹”纳米结构域中形成独特的多蛋白质Ca 2+信号传导复合物 沿着精子的尾巴。这种空间有序的纳米结构域的存在可以作为成功精子的标志 过度活跃本申请的总体目标是阐明CatSper通道复合物是如何在细胞内形成的。 分子定义和结构组织整合信号转导途径，导致机制， 过度激活所需的生理转变中心假设是CatSper内的Ca 2+信号状态 纳米结构域决定精子的活力和生育能力。确定其分子机制的基本原理 CatSper渠道监管是，这方面的知识可能会提供一个强大的科学框架， 可以开发出改变精子运动性从而改变雄性生育力的药理学策略。本申请 提出表征新型CatSper相互作用分子，其与Ca 2+结合和膜运输有关， 在显微镜下观察准备受精的精子中的Ca 2+信号状态。核心假设将 通过追求三个具体目标进行测试：1）定义新的CatSper组件在调节 2）确定新分子在纳米结构域形成中的作用;以及 3)阐明精子中实现受精的结构域整合信号通路。电生理 将采用记录和鞭毛波形分析来评估功能丧失表型对 通道活性和精子活力。对于第二个目标，从敲除的不同阶段的生精细胞- OUT模型将用于确定它们在通道复合物组装和纳米畴形成中的作用。为 第三个目标，将使用运动相关超分辨率和原位分子成像方法来研究 具有已证实的运动性和生育力的单个精子细胞的信号状态和结构域组织。 申请人认为，该研究提案具有创新性，因为它侧重于新的CatSper组件 使用新的动物模型来测试将信号域组织与通道活动耦合的原始概念 在调节Ca 2+信号方面的作用，并将新的方法引入精子生物学领域。拟议研究 是重要的，因为它预计将提供新的机械见解CatSper通道活动和精子 准备受精的精子的运动调节和分子特性。最终，这些知识 为辅助生殖、男性不育症诊断、 以及制定避孕药具的新目标。

PROJECT SUMMARY Since discovery in 2001, flagellar Ca2+ channel CatSper has remained the only Ca2+ channel in which genetic mutations cause male infertility. There are critical knowledge gaps in the current understanding of the functional regulation of this critically important channel and its signaling pathways to trigger hyperactivation, an asymmetric flagellar motion required for male fertility. The long-term goal is to deepen the understanding of sperm physiology and to uncover new pathways that could be exploited to assess and control sperm motility and fertility. CatSper channels form unique multi-protein Ca2+ signaling complexes in four linear “racing stripe” nanodomains along the sperm tail. The presence of this spatially ordered nanodomains serves as a marker of successful sperm hyperactivation. The overall objective of this application is to elucidate how the CatSper channel complex is molecularly defined and structurally organized to integrate signal transduction pathways leading to the mechan- ical transitions required for hyperactivation. The central hypothesis is that the Ca2+ signaling state within CatSper nanodomains determines sperm motility and fertility. The rationale for determining the molecular mechanisms of CatSper channel regulation is that this knowledge will likely offer a strong scientific framework whereby new pharmacological strategies to alter sperm motility, and thus male fertility, can be developed. This application proposes to characterize novel CatSper-interacting molecules implicated in Ca2+ binding and membrane traffick- ing and microscopically visualize the Ca2+ signaling state in sperm ready to fertilize. The central hypothesis will be tested by pursuing three specific aims: 1) Define the function of the novel CatSper components in regulating channel activity and sperm motility; 2) Determine the role of the novel molecules in nanodomain formation; and 3) Elucidate the domain-integrated signaling pathways in sperm that achieve fertilization. Electrophysiological recording and flagellar waveform analysis will be employed to evaluate loss-of-function phenotype effects on channel activity and sperm motility. For the second aim, a different stage of spermatogenic cells from the knock- out models will be used to determine their role in channel complex assembly and nanodomain formation. For the third aim, motility-correlation super-resolution and in situ molecular imaging methods will be used to investigate the signaling state and domain organization of individual sperm cells with proven motility and fertility. The research proposal is innovative, in the applicant’s opinion, because it focuses on new CatSper components using new animal models to test an original concept of coupling signaling domain organization to channel activity in regulating Ca2+ signaling, and incorporates new methods into the sperm biology field. The proposed research is significant because it is expected to provide new mechanistic insights into CatSper channel activity and sperm motility regulation and molecular properties of sperm that are ready to fertilize. Ultimately, such knowledge has the potential to offer new opportunities for improvements in assisted reproduction, diagnosis of male infertility, and development of new targets for contraceptives.