Objective: To investigate the role of pH2AX in the reversibility of mouse testicular reproductive function impaired by single heat stress.
Methods: Twenty-four C57 male mice were randomly divided into heat stress and control groups and immersed in water at 43℃ and 25℃, respectively, for 15 minutes. At 1, 7, and 14 days of heat exposure, all the mice were sacrificed and their testis tissues collected for determining the apoptosis of the germ cells by TUNEL and measuring the expression level of the pH2AX protein by immunohistochemistry and Western blot.
Results: The highest percentage of apoptotic cells were found in the seminiferous tubules of the mice in the heat stress group on the 1st day of the exposure and almost no apoptosis was observed at 7 and 14 days. The pH2AX protein was expressed in the nuclei of the basement membrane of adjacent seminiferous tubules. Compared with the control group, the expression of pH2AX was significantly increased on the 1st day of exposure (0.47 ± 0.02 vs 1.61 ± 0.04, P <0.01), then decreased at 7 days (0.85 ± 0.03) in comparison with that on the 1st day (P <0.01), and again elevated at 14 days (1.72 ± 0.02) as compared with either those at 1 and 7 days (P <0.01) or that of the control (P <0.01).
Conclusions: Heat stress causes dynamic changes of the pH2AX expression in the testis of the mouse, which are associated with heat stress-induced proliferation and division of the testicular spermatogenic cells.
目的： 探讨pH2AX在单次热应激致小鼠睾丸生殖功能可逆性中的表达变化。方法： C57雄性小鼠24只，随机按1、7、14 d分为对照组(n = 6)和热应激组(n = 6)。热应激组和对照组小鼠下腹部分别置于43 ℃和25 ℃水浴中干预15 min，于处理后1、7、14 d 取睾丸组织。TUNEL检测睾丸细胞凋亡情况，免疫组化染色检测pH2AX蛋白定位及表达，Western 印迹检测pH2AX蛋白表达水平。结果： TUNEL结果显示，在热应激后的第1天生精小管中的凋亡细胞数量最多，第7、14天细胞凋亡基本消失。免疫组化显示，pH2AX蛋白在睾丸生精小管基底膜处细胞核表达。Western印迹检测显示，与对照组相比，热应激后的第1天，pH2AX蛋白表达明显增加（0.47±0.02 vs 1.61±0.04，P<0.01）；第7天（0.85±0.03）与热应激处理第1天相比pH2AX蛋白表达显著减少（P<0.01）；第14天（1.72±0.02）与热应激处理第1、7天及对照组相比pH2AX蛋白表达显著增加（P均<0.01）。结论： 小鼠睾丸热应激处理后pH2AX蛋白表达量在睾丸中出现动态变化，这种变化与热应激后睾丸生精细胞的增殖分裂相关。.
Keywords: mouse; pH2AX; reproductive function; reversibility; testis; single heat stress.