Sperm Motility Modulations Crucial to Fertilization

描述（由申请人提供）：为了到达卵母细胞并使其受精，精子不仅必须是活跃的，而且还必须适当地调节鞭毛跳动模式。它们必须过度激活，这涉及切换到高振幅，不对称的跳动，正如CatSper无效突变小鼠精子不能过度激活受精所证明的那样。此外，有证据表明，精子响应趋化因子，以指导其向卵母细胞的运动。虽然Ca 2+信号参与了这两个过程，超活化趋化性的关系是一个谜。在排卵前，精子在输卵管下部的精子库中变得过度活化，因此很可能在暴露于来自卵块的趋化信号之前。我们的总体工作假设是，超激活被调节以引导精子朝向卵母细胞。R 03试验项目的目的是获得调节过度激活的确凿证据。目的1：研究精子鞭毛在输卵管内的运动规律。超活化与趋化性的关系将首次在体内研究，使用交配小鼠输卵管的透照来分析精子从输卵管下部的储库中逃逸并向卵母细胞移动时的运动。将评价不对称弯曲的对称性和方向。目的2：区分Ca 2+通过CatSper通道内流的作用和多余核膜（RNE）储存的Ca 2+释放的作用。CatSper通道被限制在鞭毛的主要部分的质膜上，RNE位于鞭毛的基部。CatSper通道的激活产生深的亲钩弯曲（与小鼠精子头部的钩相同的方向），而刺激RNE的Ca 2+释放诱导深的反钩弯曲。从深亲钩到深反钩弯曲的转换可以调整精子朝向卵母细胞的过程。Ca 2+成像和蛋白质组学方法将用于区分精子的反应。目的3：探讨趋化信号对超活化精子行为的影响。如果获得支持该假设的证据，那么我们将寻求确定精子活力调节剂的来源，并进一步阐明调节精子活力的细胞信号通路。研究结果应有助于治疗不孕症和开发避孕方法。 公共卫生相关性：大约7%的美国夫妇报告说，他们在不使用避孕药一年后没有怀孕。大约一半的不孕症病例归因于男性因素，特别是精子数量和/或活力低。为了受精，精子必须能够过度激活，并对趋化因子做出反应，以引导它们向卵子运动。关于超活化与趋化性的关系还不清楚。目的是阐明这种关系，通过分析精子在输卵管中的行为，并使用细胞生物学和蛋白质组学方法来确定精子运动是如何调节的，使它们能够到达卵子。

DESCRIPTION (provided by applicant): In order to reach the oocyte and fertilize it, sperm must not only be vigorously motile, but must also modulate flagellar beating patterns appropriately. They must hyperactivate, which involves switching to high-amplitude, asymmetrical beating, as evidenced by the failure to fertilize of CatSper null mutant mouse sperm that cannot hyperactivate. Furthermore, there is evidence that sperm respond to chemotactic factors in order to direct their movement toward the oocyte. Although Ca2+ signaling is involved in both processes, the relationship of hyperactivation to chemotaxis is a mystery. Sperm become hyperactivated in the sperm reservoir in the lower oviduct before the time of ovulation and thus very likely before exposure to chemotactic signals from the egg mass. Our overall working hypothesis is that hyperactivation is modulated to direct sperm toward the oocyte. The objective of this R03 pilot project is to gain solid evidence for modulation of hyperactivation. Aim 1: To determine the flagellar movement patterns of sperm in the oviduct. The relationship of hyperactivation to chemotaxis will be studied for the first time in vivo using transillumination of oviducts of mated mice to analyze the movements of sperm as they escape from the reservoir in the lower oviduct and move toward oocytes. The symmetry and direction of asymmetrical bends will be evaluated. Aim 2: To distinguish the effects of Ca2+ influx through CatSper channels from those of Ca2+ release from redundant nuclear envelope (RNE) stores. CatSper channels are confined to the plasma membrane of the principal piece of the flagellum and the RNE is located at the base of the flagellum. Activation of CatSper channels produces deep pro-hook bends (in the same direction as the hook of the mouse sperm head), whereas stimulation of Ca2+ release from the RNE induces deep anti-hook bends. Switching from deep pro-hook to deep anti-hook bends could serve to adjust the course of sperm toward the oocyte. Ca2+ imaging and proteomic approaches will be used to distinguish the responses of sperm. Aim 3: To test the effects of putative chemotactic signals on behavior of hyperactivated sperm. If evidence is obtained in support of the hypothesis, then we will seek to identify the sources of modulators of sperm motility and further elucidate the cell signaling pathways that modulate sperm motility. Findings should inform efforts to treat infertility and to develop contraceptive methods. PUBLIC HEALTH RELEVANCE: Approximately 7% of US couples reported that they did not get pregnant after a year without contraceptive use. Roughly half of the cases of infertility are attributed to a male factor, particularly low sperm quantity and/or motility. To fertilize, sperm must be able to hyperactivate and also to respond to chemotactic factors to direct their movement toward the egg. Nothing is known about the relationship of hyperactivation to chemotaxis. The objective is to elucidate this relationship, by analyzing behavior of sperm in the oviduct and using cell biological and proteomic approaches to determine how sperm movement is modulated to enable them to reach the egg.