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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.
The effects of cadmium (Cd) on sperm motility parameters and protein tyrosine phosphorylation in mice were investigated by using in vitro culture method and CASA, WB and immunofluorescence techniques. Mouse sperm tyrosine phosphorylation protein subcellular localization.The results showed that: Cd had a significant inhibitory effect on the sperm motility of mice, and with the Cd concentration increased inhibitory effect increased when the Cd concentration reached 1.0μmol·L ~ (-1), the sperm motility (MOT) in mice was significantly lower than that in the control group. Meanwhile, Cd promoted the phosphorylation of sperm protein tyrosine in mice, especially when the concentration was ≥1.0μmol·L -1 The phosphorylation of 55 kDa protein tyrosine was significantly enhanced, and the immunofluorescence results showed that the protein was mainly located in the middle of the mouse sperm. When 30 μmol·L -1 ethylenediaminetetraacetic acid (EGTA) and 55 kDa protein did not undergo obvious tyrosine phosphorylation when 10 μmol·L -1 Cd were cultured simultaneously, but the sperm motility did not change significantly in mice, indicating that Cd could induce Tyrosinase through the induction of middle 55 kDa protein Acid phosphorylation to inhibit sperm motility, EGTA can chelate Cd ions and effectively prevent Its toxicity.Studies confirmed that Cd induced sperm-specific protein tyrosine phosphorylation increased, thereby inhibiting sperm motility.EGTA can be used for in vitro control of Cd into the cell blockers for the study of molecular mechanism of Cd reproduction toxicity provided means.