长期氯化锰腹腔注射母鼠的子代雄鼠生精细胞凋亡变化及其机制探讨

Long-term injection of manganese chloride in mother rats induces apoptosis of spermatogenic cells of offspring rats

目的探讨母代大鼠长期氯化锰腹腔注射对子代雄性大鼠睾丸组织生精细胞凋亡的影响,并探讨其机制。方法取健康雌性SD大鼠32只随机分为观察1、2、3组和对照组,各8只,分别腹腔注射2、4、8 mg/kg的MnCl_2·4H_2O和等容生理盐水,1次/d,连续注射5 d后休息2 d,共8周。各组雌鼠与正常雄鼠交配受孕后,雌鼠在妊娠期和哺乳期继续染毒,剂量不变,共染毒14周。从各组雌鼠的12周龄子代雄性大鼠中随机选取8只断头处死后取睾丸组织HE染色观察睾丸生精小管结构,TUNEL法测算生精细胞凋亡指数(AI),化学荧光法检测睾丸组织活性氧簇(ROS)水平,Western blotting法检测睾丸组织GRP78、CHOP、Caspase12和Caspase3蛋白。结果观察1、2、3组和对照组AI分别为9.05%±1.02%、15.87%±1.31%、19.59%±1.12%、5.72%±0.53%,各组AI相比,P均<0.05。观察1、2、3组和对照组雄性子鼠睾丸组织ROS水平(以荧光强度表示)分别为335.67±41.12、452.75±37.55、547.25±41.11、210.32±37.58,各组睾丸组织ROS水平相比,P均<0.05。观察1、2、3组雄性子鼠睾丸组织GRP78、CHOP、Caspase12、Caspase-3蛋白相对表达量均高于对照组(P均<0.05),观察2、3组雄性子鼠睾丸组织GRP78、CHOP、Caspase12、Caspase-3蛋白相对表达量均高于低剂量组(P均<0.05),观察3组雄性子鼠睾丸组织GRP78、CHOP、Caspase12、Caspase-3蛋白相对表达量均高于观察2组(P均<0.05)。结论长期氯化锰腹腔注射母鼠的子代雄鼠生精细胞凋亡水平较高。其机制可能是母鼠长期氯化锰染毒可以导致子代雄鼠睾丸组织ROS水平升高,激活内质网特异的CHOP/Caspase12信号通路,最终激活Caspase3。

Objective To investigate the effects of long-term administration of manganese chloride in mother rats on the apoptosis of spermatogenic cells in testes of offspring male rats. Methods Thirty-two healthy female SD rats were randomly divided into the observation groups 1,2,and 3,and the control group,with 8 rats in each group. Then 2,4,and 8 mg/kg MnCl_2·4 H_2O,and equal volume of normal saline were injected intraperitoneally for 8 weeks（ once a day,a continuous injection of 5 days,and 2 days of rest）. After mating with normal male SD rats and being identified conception,female rats were continued to be exposed to manganese during pregnancy and lactation. Eight offspring male rats of each group,12 weeks old,were randomly executed and their testes were obtained to observe the structure of seminiferous tubules by H. E staining. The apoptotic index（ AI） of spermatogenic cells was measured by TUNEL method. The levels of reactive oxygen species（ ROS） in testicles were detected by Chemiluminescence method. The expression of GRP78,CHOP,Caspase-12 and Caspase-3 proteins was detected by Western blotting in the testicles of offspring male rats. Results The AI of the observation groups 1,2,3,and control group were 9. 05% ± 1. 02%,15. 87% ± 1. 31%,19. 59% ± 1. 12%,and5. 72% ± 0. 53%,respectively; significant difference was found in AI between every two groups（ all P〈0. 05）. The ROS levels（ expressed as fluorescence intensity） of the testicular tissues in the observation groups 1,2,3,and the control group were 335. 67 ± 41. 12,452. 75 ± 37. 55,547. 25 ± 41. 11,and 210. 32 ± 37. 58,respectively; significant difference was found in ROS level between every two groups（ all P〈0. 05）. The protein expression of GRP78,CHOP,Caspase-12,and Caspase-3 in the offspring rat testicular tissues of the observation groups was higher than that of the control group（ P〈0. 05）. Compared with the observation group 1,the expression of these proteins in the observation groups 2 and 3 significantly increased（ P〈0. 05）; at the same time,the expression of these proteins in the observation group 3 was higher than that of the observation group 2（ P〈0. 05）. Conclusions After the long-term administration of manganese chloride in mother rats,the apoptosis level of spermatogenic cells increases in the offspring male rats. The underlying mechanism may be that maternal long-term Mn Cl2 exposure elevates ROS levels in testicular tissues of the offspring male rats which activates CHOP/Caspase-2,an endoplasmic reticulum specific apoptotic signaling pathway,and ultimately leads to Caspase-3 activation.