冷冻保存对人精子膜蛋白PH-20和透明质酸酶活性的影响  

Effect of cryopreservation on the expression of sperm surface protein PH-20 and the activity of hyaluronidase in human sperm

作　　者：马晓萍[1,2] 高晓勤[2] 丁贤胜 周桦[4] Ma Xiaoping Gao Xiaoqin Ding Xiansheng Zhou Hua(Department of Histology and Embryology, Guizhou Medical University, Cruiyang 550004 Department of Histology and Embryology, Zunyi Medical and Pharmaceutical College, Zunyi 563006 Department of Pathology, First People＇s Hospital of Liupanshui , Liupanshui 553000 Reproductive Medicine Center, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, China)

机构地区：[1]贵州医科大学组织学与胚胎学教研室,贵阳550004 [2]遵义医药高等专科学校组织学与胚胎学教研室,遵义563006 [3]六盘水市人民医院病理科,六盘水553000 [4]贵州医科大学附属医院生殖医学中心,贵阳550004

出　　处：《解剖学杂志》2016年第5期561-564,615,共5页Chinese Journal of Anatomy

基　　金：贵州省科技创新人才团队项目(黔科合人才团队[2014]4005号);贵州省科技计划项目(黔科合LH字[2015]7567号)

摘　　要：目的：探讨冷冻保存对人精子膜蛋白PH-20和顶体内透明质酸酶活性的影响。方法：24例正常生育力精液标本行冷冻保存。免疫印迹检测PH-20蛋白在人精子中的表达，免疫荧光观察PH-20蛋白在人精子上的定位，采用改良透明质酸钠明胶底物膜法检测精子顶体内透明质酸酶（HYD）活性（HYD阳性反应率、HYD活性强度）。结果：免疫印迹显示正常标本冷冻前、后精子均有PH-20蛋白的表达，其PH-20/βactin平均光密度在正常组冷冻前为0.41±0.15；正常组解冻后为024±0.11，两者的平均光密度差异有统计学意义；间接免疫荧光染色后在荧光显微镜下观察冷冻前PH-20阳性率达72.88%±8.04%；解冻后PH-20阳性率降至46.97%±8.38%，差异有统计学意义；解冻后HYD活性与冷冻前比较均有显著性下降。结论：冷冻解冻过程可引起精子PH-20蛋白表达减少和精子PH-20阳性率降低，以及受精过程中的关键酶HYD活性减弱。Objective： To investigate the effect of cryopreservation on the expression and location of sperm surface protein PH- 20 and the activity of hyaluronidase in human sperm. Methods： Semen samples were obtained from fertile men. Western blotting was used to detect the PH-20 protein expression in human spermatozoa. Its location on human spermatozoa was determined by indirect immunofluorescent staining using PH-20 antibody. The positive rate and activity intensity of hyaluronidase（HYD） were detected by modified sodium hyaluronate-gelatin membrane. Results： Western blotting showed that average optic density of PH-20/β-actin was 0.41 ±0.15 before cryopreservation and 0. 24±0. 11 after being thawed. The percentage of PH-20 positive rate was 72.88%± 8.04% in frozen group and that was decreased to 46.97% ± 8.38% after being thawed detected by irelirect immunofluorescence staining. The positive reaction rate and activity of HYD were significantly lower in the thawed group than that in fresh sperm. Conclusion： Cryopreservation causes significant reduction of PH-20 protein expression, the percentage of PH-20 positive rate and the HYD activity（positive reaction rate of HYD, activity intensity of HYD） in human sperm.

关 键 词：精子 冷冻保存 PH-20 透明质酸酶 

分 类 号：R321[医药卫生—人体解剖和组织胚胎学]