Sperm Motility Modulations Crucial to Fertilization

描述(申请人提供)：为了到达卵母细胞并使其受精，精子不仅必须具有强大的运动性，还必须适当地调节鞭毛跳动模式。它们必须过度激活，这涉及切换到高幅度、不对称的跳动，CatSper缺失突变小鼠精子无法受精就证明了这一点。此外，有证据表明，精子会对趋化因子做出反应，以引导它们向卵母细胞移动。虽然钙信号参与了这两个过程，但过度激活与趋化的关系仍是一个谜。在排卵之前，精子在输卵管下部的精子库中变得过度活跃，因此很可能是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，过度激活被调节为将精子引向卵母细胞。这个R03试点项目的目标是为过度激活的调制获得确凿的证据。目的1：确定精子在输卵管内的鞭毛运动方式。通过对交配小鼠输卵管的透照来分析精子从输卵管下部的储存库逃逸并向卵母细胞移动时的运动，将首次在体内研究过度激活与趋化性的关系。将评估不对称折弯的对称性和方向。目的：区分钙离子通过CatSper通道的内流和冗余核膜(RNE)储存区的钙释放。CatSper通道位于鞭毛主段的质膜上，RNE位于鞭毛的底部。CatSper通道的激活会产生深的正钩弯曲(与小鼠精子头的钩的方向相同)，而刺激RNE释放钙则会产生深的反钩弯曲。从深前钩弯曲到深反钩弯曲可以帮助调整精子朝向卵母细胞的路线。钙离子成像和蛋白质组学方法将用于区分精子的反应。目的3：检测趋化信号对过度激活精子行为的影响。如果有证据支持这一假说，那么我们将寻求确定精子运动调节剂的来源，并进一步阐明调节精子运动的细胞信号通路。这一发现将为治疗不孕不育和开发避孕方法提供参考。 与公共卫生相关：大约7%的美国夫妇报告说，他们在没有使用避孕药的一年后没有怀孕。大约一半的不育病例是由于男性因素，特别是精子数量和/或活动率低。为了受精，精子必须能够高度激活，并对趋化因子做出反应，以引导它们向卵子移动。目前尚不清楚过度激活与趋化作用之间的关系。目的是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，从而使它们能够到达卵子，从而阐明这种关系。

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.