酪氨酸磷酸化蛋白在体外获能豚鼠精子上的分布与表达

为研究豚鼠精子获能过程中蛋白酪氨酸磷酸化的变化规律，将豚鼠精子悬浮于改良的TALP获能培养基中，在5% CO2 孵箱37 ℃培养，以精子与金霉素(CTC) 荧光结合类型为指标评价精子获能状态，用免疫荧光技术和Western blot方法检测酪氨酸磷酸化的蛋白在精子上的分布以及酪氨酸磷酸化水平的变化。结果显示，随着获能的进行，发生蛋白酪氨酸磷酸化的精子占总精子的百分比增加，由未获能前的36％增至获能7h时的92％。酪氨酸磷酸化的蛋白分布变广，由精子头部扩增至精子头部、鞭毛主段和中段。另外，有80，45，40kDa的三种蛋白发生酪氨酸磷酸化，其中40kDa的蛋白酪氨酸磷酸化水平自精子体外培养后呈递增趋势，45kDa的蛋白酪氨酸磷酸化自培养3h后发现并呈递增趋势，而80kDa的蛋白酪氨酸磷酸化水平在精子培养3h时最高，后呈递减趋势。

The Localization and Expression of Tyrosine Phosphorylated Proteins During In Vitro Capacitation of Guinea Pig Spermatozoa

The spermatozoa from mature guinea pigs were incubated in modified TALP under 5% CO2 in air at 37 ℃. The capacitation condition was assessed by chlortetracycline (CTC) staining, the localization and level of tyrosine phosphoryalted proteins during the capacitation were detected by indirect immunofluorescence or Western blot. The results showed that the number of sperm tyrosine protein phosphorylated increased from 31% to 92% during capacitation period. The site of phosphorylated tyrosine-specific fluorescence was expanded from the head of spermatozoa to the whole head and principal and mid-piece areas of the flagellum. 40kDa protein in sperm extracts taken at 0 to 0.5h after incubation was detected by anti-phosphotyrosine monoclonal antibody. 1h after incubation, another protein of 80kDa was found and the level of this protein reached the highest point at 3h. 3h after incubation, three proteins with relative molecular weight of 40, 45, 80kDa were detected.