膜联蛋白5对人精子膜及DNA完整性的影响

Protective effect of Annexin 5 on human sperm membrane and DNA integrity

目的:研究膜联蛋白5(Annexin5)对人精子细胞膜及DNA完整性的影响。方法:①精子膜完整性测定:按精子浓度>20×106/ml;活率>60%选取标准收集精液标本53份,分为3组,实验组为47.5μl精液中加入2.5μl10-6mol/L的Annexin5;阴性对照组为47.5μl精液标本中加入2.5μl1mol/L的Tris-HCl(pH8.0,25℃);空白对照组为47.5μl精液标本中加入2.5μl0.01mol/L的PBS(pH7.4),作用20min后,通过精子低渗肿胀试验(HOS)检测精子膜的完整性。②DNA完整性测定:同方法①,3组实验标本作用20min后,每份标本加入2.5μl0.02mol/L的H2O2,作用60min,通过吖啶橙(AO)荧光染色检测精子核DNA的完整性。结果:An-nexin5处理20min后低渗肿胀精子百分率与空白对照组及阴性对照组比较均具有极显著差异[(66.17±12.02)%vs(58.13±13.08)%,P<0.01;(66.17±12.02)%vs(59.94±11.91)%,P<0.01];空白对照组与阴性对照组比较无显著性差异。加入H2O2后,Annexin5组的DNA碎片指数与空白对照组及阴性对照组比较均具有极显著差异[(6.39±1.07)%vs(11.16±1.16)%,P<0.01;(6.39±1.07)%vs(10.86±1.05)%,P<0.01],空白对照组与阴性对照组比较无显著性差异。结论:Annexin5蛋白能在体外提高低渗肿胀精子百分率,对精子膜的完整性具有保护作用,同时对HO作用引起的精子核DNA破坏起保护作用。

Objective：To investigate the role of Annexin 5 in protecting human sperm membrane and DNA integrity.Methods：We collected 53 semen samples based on the criteria of sperm density 20×10^6/ml and motility 60%,and divided them into an experimental group（2.5 μl 10^-6 mol/L Annexin 5 added to 47.5 μl semen）,a negative control group（2.5 μl 1 mol/L Tris-HCl [pH 8.0,25 ℃] added to 47.5 μl semen）,and a blank control group（2.5 μl 0.01 mol/L PBS [pH 7.4] added to 47.5 μl semen）.After 20 minutes of incubation,we evaluated the sperm membrane integrity using the hypoosmotic swelling test and,after another 60 minutes of treatment with H2O2 at 2.5 μl 0.02 mol/L,measured the sperm nuclear DNA integrity by acridine orange fluorescent staining.Results：After 20 minutes of treatment with Annexin 5,the experimental group showed extremely significant difference in the percentage of hypoosmotic swelling sperm（[66.17±12.02]%） from the blank control（[58.13±13.08]%,P 0.01） and the negative control group（[59.94±11.91]%,P 0.01）,but there was no significant difference between the latter two.Treatment with H2O2 remarkably increased DFI in the experimental group（6.39±1.07） as compared with the blank control（11.16±1.16） and the negative control group（10.86±1.05,P 0.01）,but no significant difference was observed between the latter two.Conclusion：Annexin 5 can increase the percentage of hypoosmotic swelling sperm in vitro and protect sperm membrane integrity,and it can also protect sperm DNA from H2O2 damage.