酪氨酸磷酸化蛋白在体外获能豚鼠精子上的分布与表达  

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

作　　者：孔丽娟[1] 李忠浩[1] 黄静燕[1] 代婷婷[1] 王根林[1] 

机构地区：[1]南京农业大学动物科技学院,南京210095

出　　处：《中国生物工程杂志》2007年第4期89-93,共5页China Biotechnology

基　　金：国家自然科学基金资助项目(30471243)

摘　　要：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 aim of this study was to detect the localization and level of tyrosine phosphorylated proteins during in vitro capacitation of guinea pig sperm. Sperm from mature guinea pigs were incubated in modified TALP under 5% CO2 in air at 37 ℃. The capacitation effect was assessed by chlortetracycline （CTC） staining. Western blotting and indirect immunofluorescence were used to analyze the level and localization of tyrosine phosphorylation. The results showed that guinea pig sperm underwent a time-dependent increase in protein tyrosine phosphorylation during the in vitro capacitation and the percentage of protein tyrosine phosphorylated sperm increased from 36% to 92% from the beginning of incubation to 7h incubation. Also, there was a shift in the site of phosphotyrosine-specific fluorescence from the head of sperm to both the head and the flagellum of sperm. Moreover, there were three proteins phosphorylated in this experiment. After 0 to 0.5h incubation, the protein of 40kDa was detected by anti-phosphotyrosine monoclonal antibody, and the intensity of this protein increased in the following incubation. Then, after lh incubation, another protein of 80kDa was found and the level of this protein reached the highest point at 3h. Also, in 3h incubation, a protein of 45kDa was detected and the intensity of this protein increased in the following incubation.

关 键 词：精子获能 酪氨酸磷酸化 豚鼠 

分 类 号：Q25[生物学—细胞生物学]