母鼠长期低剂量锰染毒对子代大鼠睾丸氧化应激状态及生精细胞凋亡的影响

Effect of maternal chronic exposure of low-dose manganese on spermatogenic cells of offspring rats

目的探讨氯化锰长期低剂量染毒对子代大鼠睾丸中氧化应激状态及生精细胞凋亡的影响。方法健康雌性SD大鼠32只随机分为对照组,低、中、高剂量组,每组8只。锰染毒组分别腹腔注射2、4和8 mg/kg MnCl_2·4H_2O,对照组腹腔注射等容生理盐水,1次/d,5 d/周,共8周。与正常雄性SD大鼠交配受孕后,雌鼠在妊娠期和哺乳期继续染毒。每组随机取8只12周龄子代雄性大鼠断头处死后取材,HE染色观察睾丸生精小管结构;q RT-PCR、免疫组织化学、Western blot分别检测叉头蛋白转录因子O 3a(Fox O3a)、与Bcl-2相互作用的细胞死亡调节子(Bim)、半胱天冬酶9(Caspase-9)m RNA和蛋白的表达;TUNEL技术检测生精细胞凋亡。结果 (1)HE染色可见各锰染毒组睾丸生精小管呈现不同程度的形态学改变。随着锰染毒剂量的增加,生精细胞层数逐渐减少,各级生精细胞排列紊乱、数量减少或缺如,管腔内成熟的精子数目减少;(2)中、高剂量组精子密度、精子活动率降低,而精子畸形率上升(P<0.05);(3)各锰染毒组大鼠睾丸组织内超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化酶(GSH-Px)活性随锰染毒剂量的增加而降低(P<0.05);而睾丸组织内丙二醛(MDA)含量和活性氧(ROS)水平随锰染毒剂量的增加逐渐升高(P<0.05);(4)在m RNA和蛋白水平,Fox O3a的表达随锰染毒剂量的增加逐渐上升(P<0.05);低剂量组Bim m RNA表达量未见变化,但在中、高剂量组则降低,而细胞死亡调节因子表达量则随锰染毒剂量的增加逐渐增加(P<0.05);(5)Caspase-9 m RNA和蛋白表达量在各锰染毒组均升高,且随锰染毒剂量的增加逐渐增加(P<0.05);(6)各锰染毒组大鼠均可见生精细胞凋亡,其凋亡指数高于对照组,且随锰染毒剂量的增加而上升(P<0.05)。结论长期低剂量锰染毒可抑制子代大鼠睾丸内抗氧化酶活性或(和)增加MDA含量,提高睾丸组织ROS水平,促进Fox O3a和Bim表达,启动Caspase-9信号通路,最终导致子代大鼠生精细胞的凋亡。

Objective To investigate the effect of chronic exposure of low-dose manganese chloride on spermatogenic cells of offspring rats.Methods Totally 32 healthy female SD rats were randomly divided into control group,low dose group,middle dose group and high dose group(n=8).Rats in manganese exposure group received different doses of manganese chloride(2,4,and 8 mg/kg in low dose group,middle dose group and high dose group,respectively)once a day,5 days a week in a total of 8 weeks.Rats in control group were treated with saline for 8 weeks.After mating with normal male SD rats,female rats were continuously exposed to manganese during pregnancy and lactation as mentioned above.Testes of 12 week old offspring were harvested.HE staining was performed to observe the structure of seminiferous tubules.The expression levels of FoxO3a,Bim,Caspase-9 mRNA,and protein in testis were identified by real-time PCR,immunohistochemistry,and Western blotting.Apoptosis rate of spermatogenic cells was detected by TUNEL.Results Morphological changes of seminiferous tubules in manganese exposure groups were observed including decrease of spermatogenic cell,disorder of spermatogenic cell structure and reduction of mature sperms.Abnormality was more severe along with increase of manganese exposure.Density and activity of sperms decreased while deformity ratio increased significantly in middle and high dose groups when compared with control group(P<0.05).Activity of SOD,CAT and GSH-Px in the testis of rats exposed to manganese decreased dramatically while levels of MDA and ROS increased in dose dependent manner when compared with control group(P<0.05).The expression levels of FoxO3a,Bim and Caspase-9 increased in dose dependent manner when compared with control group(P<0.05).Apoptosis of spermatogenic cells in manganese exposed groups were significantly enhanced in dose dependent manner compared with control group(P<0.01).Conclusion Chronic maternal exposure of manganese leads to apoptosis of spermatogenic cells in offspring rats through activating oxidation mediated Caspase-9 signaling pathway.