镉对小鼠精子蛋白酪氨酸磷酸化修饰的影响及EGTA的保护作用  

Effects of cadmium on protein tyrosine phosphorylation of mouse spermatozoa and the protective role of EGTA

作　　者：王立蕊 杨强震 李玉华[1] 李思思[1] 张宇坤[1] 付杰丽 甄林青 李新红[1] 

机构地区：[1]上海交通大学农业与生物学院上海市兽医生物技术重点实验室,上海200240

出　　处：《应用生态学报》2016年第12期4045-4051,共7页Chinese Journal of Applied Ecology

基　　金：上海市科技兴农攻关项目(2014-2-5);上海交通大学国家级大学生科创项目(12166)资助~~

摘　　要：采用体外培养的方法,利用精子活力分析软件(CASA)、蛋白免疫印迹(WB)及免疫荧光技术,探讨镉(Cd)对小鼠精子活力参数、蛋白酪氨酸磷酸化修饰的影响,并对小鼠精子酪氨酸磷酸化蛋白进行细胞亚组分定位.结果表明:Cd对小鼠精子活力具有明显抑制作用,且随着Cd浓度的增加抑制作用增强,当Cd浓度达到1.0μmol·L^(-1)时,小鼠精子活力(MOT)显著低于对照组;同时,Cd促进小鼠精子蛋白酪氨酸磷酸化,当浓度≥1.0μmol·L^(-1)时,尤其分子量约为55 kDa的蛋白酪氨酸磷酸化程度显著增强,且免疫荧光结果显示主要集中在小鼠精子中段;当用30μmol·L^(-1)乙二醇二乙醚二胺四乙酸(EGTA)和10μmol·L^(-1)Cd同时培养时,55 kDa蛋白并未发生明显的酪氨酸磷酸化修饰,而且小鼠精子活力变化不显著.表明Cd可能是通过诱导中段55 kDa蛋白发生酪氨酸磷酸化修饰从而抑制精子活力,EGTA能螯合Cd离子并有效防止其毒性作用.研究证实,Cd诱导精子特异性蛋白酪氨酸磷酸化增强,进而抑制精子活力.EGTA可以用于体外控制Cd进入细胞的阻断剂,为Cd繁殖毒性分子机制的研究提供了研究手段.In this study, we explored the effects of cadmium （Cd） on mouse sperm motility parame-ters, protein tyrosine phosphorylation and the location of tyrosinephosphorylated targets using computerassisted sperm analysis （CASA）, western blot （WB） and immunofluorescence technique coupled to sperm in vitro culture method, respectively. The results showed sperm motility was inhibi-ted by Cd in a dosedependent manner and when Cd increased to 1.0 μmol·L^-1, sperm motility was inhibited significantly （P〈0.05）. Simultaneously, protein tyrosine phosphorylation was enhanced by Cd and in particular, the tyrosine phosphorylation of ～55 kDa proteins was greatly promoted when Cd concentrations were greater or equal to 1.0 μmol·L^-1 （P〈0.05）. Importantly, these tyrosinephosphorylated proteins were mainly localized in the middle piece of mouse sperm. However, when sperm was incubated with 30 μmol·L^-1 ethylene glycol tetraacetic acid （EGTA） and 10 μmol·L^-1 Cd concurrently, both the tyrosine phosphorylation of ～55 kDa proteins and sperm motility were not changed obviously （P〉0.05）. These results suggested that Cd may inhibit sperm motility by inducing the tyrosine phosphorylation of ～55 kDa proteins in the middle piece and EGTA could chelate Cd ions to relieve its toxicity. This study demonstrated that Cd induced the tyrosine phosphorylation of a specific subset of proteins and thus decreased sperm motility. Interestingly, EGTA acted as an inhibitor to block Cd from entering the sperm, which provided a novel research method for revealing the molecular mechanisms of reproductive toxicity caused by Cd.

关 键 词：重金属 信号通路 繁殖毒性 阻断剂 

分 类 号：S859.8[农业科学—临床兽医学]