长期低剂量锰染毒对子代大鼠生精细胞细胞周期阻滞和凋亡的影响

Effects of chronic low-dose manganese exposure on cell cycle arrest and apoptosis in spermatogenic cells of rat offspring

目的探讨氯化锰长期低剂量染毒对子代大鼠生精细胞细胞周期及细胞凋亡的影响。方法健康雌性SD大鼠32只随机分为对照组和锰染毒组,每组8只。锰染毒组腹腔注射2、4和8 mg/kgMnCl_2·4H_2O,对照组腹腔注射等容生理盐水,1次/d,5次/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;Western Blot检测FOXO3A、P16、P21、CDK2、CDK4、CDK6、BIM和Caspase-9的表达;TU-NEL法检测生精细胞凋亡。结果随锰染毒剂量的增加,生精小管呈现生精细胞层数减少、生精细胞排列紊乱、数量减少等变化;随锰染毒剂量的增加,睾丸组织FOXO3A的表达逐渐上升;P16、P21表达量逐渐增加;CDK2、CDK4则逐渐降低,CDK6的表达未见明显改变;BIM、Caspase-9表达及生精细胞凋亡指数均随锰染毒剂量的增加逐渐增加。结论长期低剂量锰染毒可能通过诱导FOXO3A表达,引起子代大鼠生精细胞的细胞周期阻滞和细胞凋亡。

Objective To investigate the effect of chronic low-dose manganese chloride exposure on cellcycle and apoptosis of spermatogenic cells in offspring rat. Methods 32 healthy female SD rats were randomlydivided into control and manganese exposure groups(n = 8). The manganese exposure group respectively received2,4 and 8 mg/Kg MnCl_2·4 H_2O,while the control group was treated with equal volume saline for 8 weeks(1 time/d,5 d/w). After mating with normal male SD rats and determining conception,female rats were continued to be ex-posed to manganese during pregnancy and lactation. There were. 8 offspring male rats in each group. In the 12 th week,the offspring rates were randomly executed and their testes were used to observe the structure of seminifer-ous tubules by HE staining. The expression of FOXO3 A,cell cycle associated proteins P16,P21,CDK2,CDK4,CDK6 and apoptosis related proteins BIM,Caspase-9 in the testis of offspring male rats were detected bywestern blotting. In addition,the apoptosis of spermatogenic cells was detected by TUNEL. Results(1)Withthe increase of administered-Mn Cl2 dosage,the seminiferous tubule showed various changes,including layers ofspermatogenic cell decreased,spermatogenic cells arranged in disorder,the number of mature sperm in thelumen of seminiferous tubule significantly reduced.(2) The expression of FOXO3 A,P16 and P21 increasedwhile CDK2 and CDK4 decreased gradually with the increase manganese exposure dose. CDK6 expression wasnot significantly changed in all groups.(3)The expression of BIM,Caspase-9 and apoptotic index of spermatogen-ic cells increased gradually with the increase of manganese dose. Conclusion Chronic low-dose of manga-nese exposure could induce expression of FOXO3 A,further cause cell cycle arrest and apoptosis of spermatogen-ic cells in offspring rat.