苯甲酸雌二醇暴露对小鼠睾丸组织的氧化损伤

Estradiol benzoate-induced oxidative damage in testicular tissue of mice

目的探讨外源性雌激素苯甲酸雌二醇(estradiol benzoate,EB)产生氧化应激诱导雄性小鼠的生精功能障碍。方法将39只清洁级4周龄雄性昆明小鼠随机分为3组,分别为对照组(玉米油)和5、10 mg/kg EB染毒组,每组13只。采用肌肉注射方式进行染毒,染毒容量为5μl/g,隔天1次,持续28 d。染毒结束后,每组随机选3只雄性小鼠按1∶2比例与雌性小鼠合笼,记录配对雌性小鼠受孕数和产仔数。采用流式细胞术检测睾丸细胞周期各时相细胞所占比例。睾丸组织匀浆,采用化学比色法检测睾丸组织内超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)、一氧化氮合酶(NOS)活力和丙二醛(MDA)、一氧化氮(NO)含量。采用实时荧光定量PCR(q RT-PCR)技术检测睾丸组织SOD、CAT、谷胱甘肽过氧化物酶4(GPx4)及caspese-3、Bax m RNA表达水平,采用Western blot技术检测睾丸组织Caspese-3、Bax蛋白表达水平。结果与对照组相比,各剂量EB染毒组小鼠睾丸组织G0/G1期和S期细胞所占比例均下降,而G2/M期细胞所占比例均升高,差异有统计学意义(P<0.05);且随着EB染毒剂量的升高,小鼠睾丸G0/G1期和S期细胞所占比例均呈下降趋势,而G2/M期细胞所占比例呈上升趋势。与对照组相比,各剂量EB染毒组小鼠睾丸组织SOD、CAT、GSH-Px活力均降低,而NOS活力及MDA、NO含量均升高,差异有统计学意义(P<0.05);且随着EB染毒剂量的升高,小鼠睾丸组织SOD、CAT、GSH-Px活力均呈下降趋势,而NOS活力及MDA、NO含量均呈上升趋势。与对照组相比,各剂量EB染毒组小鼠睾丸组织SOD、CAT、GPx4 m RNA的表达水平均降低,而Caspase-3蛋白和m RNA的表达水平均升高,差异有统计学意义(P<0.05);且随着EB染毒剂量的升高,小鼠睾丸组织SOD、CAT、GPx4 m RNA的表达水平均呈下降趋势,而caspase-3、Bax蛋白和m RNA的表达水平均呈上升趋势。结论 EB可阻滞小鼠生精细胞周期,产生氧化损伤并上调caspase-3和Bax表达,这可能是EB引起雄性小鼠生精功能障碍的重要途径。

Objective To explore the spermatogenesis dysfunction induced by exogenous estradiol benzoate （EB） through oxidative stress in male mice. Methods A total of 39 clean grade male Kunming mice aged 28 days were randomly divided into three groups,thirteen in each, the mice of the control group were injected with corn oil only by an every other day intramuscular injection for 28 days; Meantime, the mice of the treatment groups were injected with EB at the doses of 5 or 10 mg/kg, respectively. After the experiment, three mice of each group were kept for fertility detection respectively, and two female were randomly set for cohabiting with each male mouse. In the end,the litter size of each female mouse was counted by using the fertility statistics. The proportion of cells in each phase of testicular cells was measured. The activity of superoxide dismutase （SOD）, catalase （CAT）, glutathione peroxidase （GSH-Px）, nitric oxide synthase （NOS） and the contents of malondialdehyde （MDA）, nitric oxide （NO） in testicular tissue were detected by chemistry colorimetry. The mRNA expression of SOD, CA T, glutathione peroxidase 4 （GPx4） and easpase-3, Box in the testes was detected by qRT-PCR, the protein expression of caspase-3 and Bax were detected by Western blot. Results Compared with the control group,the proportions of Go/G1 and S phase cells reduced significantly, the cells of G2/M phase increased significantly （P〈0.05） with a does-dependent relationship in EB exposure groups. The activities of SOD, CAT, GSH-Px were significantly decreased with a dose-dependent relationship, the activities of NOS and the contents of MDA and NO of each EB exposure group were significantly higher than the control group （P〈0.05） with a dose-dependent relationship. The mRNA expression levels of SOD, CA T, and GPx4 were significantly downregulated in testes,while easpase-3 and Box were significantly upregulated （P〈0.05） in EB exposure groups. With the increase of EB exposure doses, SOD, CA T, and GPx4 expression had a declining trend,but caspase-3 and Bax had a rising trend. Conclusion Estradiol benzoate can block the spermatogenic cell cycle, induce oxidative damage as well as up-regulate the caspase-3 and Bax expression, which was the important cause of EB-induced spermatogenesis dysfunction in male mice.