The Effect And Mechanism Of Maternal Chronic Low Dose Manganese Chloride Exposure On Apoptosis Of Spermatogenic Cells In Offspring Rats

Objective: To observe the effects of long-term chronic manganese(Mn) on the spermatogenic cells of male offspring through poisoning on parent female SD rats. Methods: 32 female SD rats(weighting 90-120 g) were randomly divided into 4 groups: control group, 2 mg/kg Mn Cl2 group, 4 mg/kg Mn Cl2 group and 8 mg/kg Mn Cl2 group. 8 rats/group. Mn exposure groups and control group were given normovolemic Mn Cl2·4H2O and Saline(NS) respectively by intraperitoneal injection(5 d/w, 1 time/d) for eight weeks. The female rats were mated with normal male SD rats. Pregnancy were determined by vaginal suppository, and then followed by continuing Mn poisoning in pregnancy(3 w) and lactation(3 w) period. Serum and testicular of the male offspring at sexual maturity phase(8 w) were detected by the following tests: 1. The testicular structural changes were observed by Hematoxylin-eosin(HE) staining; 2. Real-time PCR technology was used to detect m RNA expression of apoptosis-related genes OPA1, DRP1, Caspase 9, Bim, FOXO3 A in male offspring testis; 3. Immunohistochemistry and Western blot techniques were used to detect localization and quantitative expression of the above apoptosis-related protein; 4. The total serum testosterone levels in offspring rats was examined by chemiluminescence method; 5. The serum SOD activity in offspring rats was detected by WST-1; 6. Visible method was used to detect serum CAT vitality of offspring rats; 7. Serum GSH-Px vigor of offspring rats was detected by enzymatic reaction. Results: 1. The testicular seminiferous tube of male offspring of control group are basically the same size, and spermatogenic cells were arranged orderly. Interstitials were normal, no congestion, hemorrhage and inflammatory cell infiltration. On the contrary, the testis structure shows different degrees of morphological changes in Mn exposure groups, and the change degrees increased in a Mn-dose dependent manner.2. The effect of Mn exposure on expression DRP1, Caspase 9, Bim and FOXO3 A of spermatogenic cells in male offspring: compared with the control group, the expression levels of DRP1, Caspase 9, Bim and FOXO3 A m RNA, the positive-cell-rate expression and protein levels were significantly higher(P<0.05) in a Mn exposure dose-dependent manner(P<0.05).3. The effect of Mn exposure on OPA1 in spermatogenic cells of male offspring: compared with the control group, the expression level of OPA1 m RNA, OPA1 positive-cell-rate and protein levels of the manganese exposure groups were lower(P<0.05). Among Mn exposure groups, OPA1 expression levels reduced in a Mn dose-dependent manner(P<0.05).4. The effect of Mn on total serum testosterone of male offspring: compared with the control group, the total serum testosterone level were higher in Mn exposure groups(P<0.05). And the total serum testosterone is increased in a Mn dose-dependent manner(P<0.05).5. The impact of Mn exposure on serum antioxidant enzyme system of male offspring: compared with the control group, serum SOD, CAT and GSH-Px activity of Mn exposure groups were significantly lower(P<0.05). And the serum antioxidant enzymes are increased in a Mn dose-dependent manner(P<0.05). Conclusion: Maternal exposed Mn could infiltrate into testis of offspring through the placental barrier and the blood-testis barrier, which induces spermatogenic cell apoptosis and spermatogenesis dysfunction by following mechanisms:1.Decrease the serum testosterone level of male offspring rats, and arrest sperm development of the cell cycle by acting on the hypothalamus-pituitary-gonad axis.2. Induce apoptosis by reducing the activity of serum antioxidant enzymes of offspring rats, increasing the oxidative stress, and inducing membrane lipid peroxidation of spermatogenic cells.3. The germ cell apoptosis may be achieved by up-regulating expression of FOXO3 A, Bim, and/or up-regulating expression of DRP1 while down-regulating expression of OPA1,breaking the mitochondrial fusion/fission balance, starting the mitochondrial apoptosis signaling pathway, and finally inducing apoptosis through up-regulating the expression of the downstream effecter Caspase 9.