MINORITY PREDOCTORAL FELLOWSHIP PROGRAM

描述(由申请人提供)：我们的目标是阐明与精子过度激活有关的信号通路。受精时，在输卵管中发现一种活跃的精子运动类型--过度激活。由于精子过度激活的能力通常是精子生育能力的指标，确定过度激活运动的调节机制可能会导致开发提高精子生育能力的治疗方法，或者导致针对特定精子运动调节器的避孕药物的开发。我们的具体目的是利用普鲁卡因诱导的公牛精子过度激活来确定：1)蛋白质磷酸化在过度激活精子中的作用，应用蛋白质印迹分析和间接免疫荧光。2)应用钙离子荧光指示剂和单细胞成像技术研究细胞内钙离子在高激活运动中的作用。3)用pH敏感的荧光探针和单细胞图像分析技术测量细胞内的pH，发现胞质碱化在调节细胞的过度激活运动中起作用。我们认为，过度激活是通过鞭毛蛋白磷酸化的信号通路介导的，这种信号通路依赖于胞浆中钙离子的内流和碱化。

DESCRIPTION (provided by applicant): Our goal is to elucidate the signaling pathway involved in sperm hyperactivation. Hyperactivation, a vigorous type of sperm motility, is found in the oviduct at time of fertilization. Because the ability of sperm to hyperactivate is often an indictor of sperm fertility, determining the mechanism by which hyperactivated motility is regulated can lead to development of treatments to promote sperm fertility or alternatively, result in development of contraceptives targeting specific modulators of sperm motility. Our specific aims are to use procaine-induced hyperactivated bull sperm to determine the role of: 1) Protein phosphorylation in hyperactivated sperm using western blot analysis and indirect immunofluorescence. 2) Intracellular Ca 2+ in hyperactivated motility using fluorescent Ca 2+ indictors and single cell imaging. 3) Cytoplasmic alkalization in modulating hyperactivated motility by measuring intracellular pH using pH sensitive fluorescent probes and single cell image analysis. We propose that hyperactivation is mediated by a signaling pathway involving phosphorylation of flagellar proteins, which is dependent on Ca 2+ influx and alkalization of the cytosol.