培养液pH值对小鼠精子体外培养过程中活力及蛋白修饰的影响
Effects of pH of Culture Medium on Motility and Protein Modifications of Mouse Sperm In Vitro

为了探究不同pH对小鼠成熟精子体外培养过程中活力参数及蛋白修饰的影响,研究采用不同pH值(6.6~7.8)培养液培养小鼠精子2 h,利用计算机辅助精子活力分析仪(CASA)、蛋白免疫印迹(WB)及免疫荧光技术,分析测定小鼠精子活力参数、蛋白修饰的变化情况,并对小鼠精子磷酸化蛋白及乙酰化蛋白进行细胞亚组分定位。结果显示,pH为7.2~7.4时精子能动性(sperm motility,MOT)、平均路径速度(path velocity,VAP)、平均直线运动速度(straight line velocity,VSL)和平均曲线运动速度(curvilinear veiocity,VCL)都有较高值,pH为6.6或7.8时精子活力受到显著抑制(P<0.05);培养液的pH为7.4处理组,小鼠精子蛋白磷酸化、乙酰化和琥珀酰化修饰均达到较高水平,论pH降低(<7.2)还是升高(>7.4)这些蛋白修饰水平都减弱,与精子活力参数变化趋势一致;免疫荧光定位结果显示,获能组磷酸化蛋白及乙酰化蛋白荧光强度明显高于未获能组,磷酸化蛋白及乙酰化蛋白遍布精子整个鞭毛区域,其中头部核区蛋白乙酰化修饰水平较高。上述研究表明pH值7.2~7.4为小鼠精子体外培养的最适pH值范围,pH值可能通过影响精子体外培养过程中的蛋白磷酸化、乙酰化及琥珀酰化作用调节精子活力及功能。本研究对探究pH值调控哺乳动物成熟精子活力的分子机理及体外培养具有重要意义。
The objective of the work was to investigate the influence of different pH values on motility and protein modifications of mouse mature sperm during culture in vitro.In this study,spermatozoa were incubated for 2 hours in Whitten’s media at different pH values(6.6-7.8).Sperm motility,protein modifications,and localization of phosphorylated as well as acetylated protein targets were analyzed using computer-assisted sperm analysis(CASA),western blot and immunofluorescence technique,respectively.The results showed that higher values of motility characteristics(MOT,VSL,VCL,VAP)were observed at pH 7.2-7.4.However,sperm motility was significantly suppressed at pH 6.6 and 7.8,with a decrease in kinetic parameters.Similarly,protein phosphorylation,acetylation and succinylation reached the highest level at pH 7.4 and decreased when sperm were incubated in media at lower(pH<7.2)or higher(pH>7.4)pHs.The fluorescence intensity of capacitation treatment group is obviously higher than that of non-capacitation control group.Phosphorylated proteins and acetylated proteins were all located in the entire flagellum area.Meanwhile,the acetylated proteins were mainly localized in the nuclear compartment of the mouse sperm.Taken together,the optimum pH for mouse sperm culture in vitro is 7.2-7.4.And pH may regulate sperm motility and functions by affecting protein phosphorylation,acetylation and succinylation modifications.The work is of great significance for revealing the molecular mechanism of pH regulation on sperm motility and for sperm incubation in vitro.