温度对体外大鼠睾丸支持细胞增殖及occludin蛋白表达的影响

High temperature reduces the proliferation of and occludin expression in rat Sertoli cells in vitro

目的:通过观察不同温度对原代大鼠睾丸支持细胞增殖作用与紧密连接蛋白occludin(OCLN)表达的影响,探讨温度因素在男性生殖功能降低或男性不育中的作用机制。方法:体外分离培养雄性Wistar大鼠睾丸支持细胞(Sertoli cell,SC),油红O染色及免疫组化FasL鉴定。实验分为对照组(34℃)和实验组(35℃、36℃、37℃、38℃、39℃)。在相应的温度下培养4 d后,CCK-8法检测SC增殖作用,HE染色观察细胞形态及结构,Western印迹法和免疫荧光法检测OCLN表达水平。结果:体外培养SC的纯度为(96.20±1.95)%,CCK-8结果显示,细胞增殖活性在34～36℃时逐渐增强,36～39℃时逐渐降低,并在镜下观察发现细胞核固缩、裂解明显;免疫荧光及Western印迹显示,36℃时OCLN表达量最高;高于36℃时,OCLN表达量随着温度的增加而降低(P<0.01)。结论:温度升高(>36℃)抑制SC的增殖活性,并降低SC紧密连接蛋白的表达量。因此,高温对SC活性和紧密连接完整性的影响,可能是导致男性生育能力下降或不育的机制之一。

Objective： To explore the action mechanisms of temperature in male infertility or subfertility by observing the effects of different temperatures on the proliferation of and occludin（OCLN） expression in rat Sertoli cells in vitro.Methods： We isolated Sertoli cells from the testis of male Wistar rats,and performed oil red O staining and immunohistochemistry to identify their FasL.We cultured the Sertoli cells at 34 ℃（control group） and at 35,36,37,38 and 39 ℃（experimental groups） for 4 days.Then we measured their proliferation by CCK-8 assay,observed their morphology and structure by hematoxylin-eosin staining,and determined their OCLN expression level by Western blotting and immunofluorescence.Results： The purity of the isolated Sertoli cells was（96.20±1.95）%.CCK-8 assay indicated that the proliferation of the Sertoli cells was increased between 34 and 36 ℃,and decreased at 36-39 ℃.The pyknotic nuclei and fragmentation of the Sertoli cells were more obvious at 36℃.Western blot and immunofluorescence showed the highest level of OCLN expression at 36℃,which,however,decreased while the temperature rose above 36℃（P0.01）.Conclusion： High temperature（36℃） inhibited the proliferation of rat Sertoli cells in vitro,and decreased the expression of OCLN,which suggests that a higher temperature above 36℃ may reduce male fertility by affecting the proliferation of Sertoli cells and integrity of the tight junction among Sertoli cells or Sertoli cells and other cells.