Effects Of P, P'-DDE, β-BHC And Their Combination On The Expression Of Vimentin, N-cadherin And FSHR In Rat Sertoli Cells

p, p'-DDE and ?-BHC were the major metabolites of organochlorine pesticide DDT and hexachlorocyclohexane (BHC), because of the characteristics that they are difficult to degradate and have a long residual period, there are still a large number of residues in the environment. Studies have shown that p, p'-DDE possesses anti-androgen effect and can bind to androgen receptor competitively,β-BHC can imitate the effect of estrogen, both of which are environmental endocrine disruptors and can interfere with normal body internal secretion, particularly the impact on the reproductive system have been obtained more and more people's attention.Sertoli cells are the only somatic cells in spermatogenic epithelium, they can provide nutrition and support for spermatogenesis, secrete necessary bioactive factors for germ cells, participate in the formation of blood-testis barrier, help the translocation of germ cells, expel the sperm and so on. The functional proteins of Sertoli cells play a very important role in the process of spermatogenesis. It is reported that the Sertoli cells'cytoskeletal protein Vimentin plays a critical role in maintaining the structural integrity of seminiferous epithelium, it is important for the proliferation of germ cell, also related with cell signaling and apoptosis. N-cadherin is a calcium-dependent adhesion molecule, plays an important role in the interaction between Sertoli cells and germ cells, differentiation of tissue and formation during embryonic development, and it can inhibit apoptosis of cells. Changes of the expression of N-cadherin can disturb signal transduction and affect the spermatogenesis. Spermatogenesis is regulated by FSH and testosterone, FSH plays it's role by binding to the FSHR of Sertoli cells, so the expression of FSHR is important to research the function of Sertoli cells. In this research, Sertoli cells were administrated by p, p'-DDE,β-BHC separately and their combination, the aim is to investigate the effects of p, p'-DDE andβ-BHC on the expression of Vimentin, N-cadherin and FSHR of rat testicle Sertoli cells, and reveal the possible molecular mechanism of organochlorine pesticides on male reproductive toxicity.PartⅠThe toxic action of p, p'-DDE,β-BHC and their combination on rat Sertoli cells in vitroObjective: To explore the effect of p, p'-DDE andβ-BHC on viability rat Sertoli cells, determine the dose of administration on cultured sertoli cells.Method: Sertoli cells separated from testicular of SD rats of 18~20 days age were cultured in DMEM media with 10% fetal bovine serum at 35℃in a humidified incubator of 5%CO2. After cultured for 24h, the cells were treated with 10,30,50,70μmol/Lp, p'-DDE,β-BHC and their combination for 24h, then we used MTT method to determine their absorbencies under 490nm wave-length.Results: With the increasing of the concentration of p, p'-DDE,β-BHC and their combination, the absorbance decreased gradually. Compared with DMSO group, the the rate of cell survivor decreased significantly when the concentration of p, p'-DDE,β-BHC and their combination reached to 50μmol/L p, p'-DDE, and there were significant differences (P<0.05). The results also shown that between 50μmol/L p, p'-DDE orβ-BHC separately and 50μmol/L their combination groups, and there were significant differences.Conclusion: p, p'-DDE,β-BHC and their combination could do harm to Sertoli cells when the concentration of p, p'-DDE,β-BHC and their combination were above 50μmol/L, and the combination had greater impact. PartⅡEffects of p, p'-DDE,β-BHC and their combination on the expression of Vimentin, N-cadherin and FSHR in rat Sertoli cells in vitroObjective: To investigate the effect of p, p'-DDE,β-BHC and their combination on Vimentin, N-cadherin and FSHR mRNA and protein expression in Sertoli cells.Method: The real-time fluorescence quantitative PCR and Western blottng methods were used to test the Vimentin, N-cadherin and FSHR mRNA and protein expression in Sertoli cells in vitro.Results: Compared with the control, the levels of Vimentin mRNA in Sertoli cells increased significantly in the 10?mol/Lβ-BHC group. With the increasing doses of each poison, Vimentin protein expression decreased gradually. Compared with the control, the descent of allβ-BHC groups and 50μmol/L combined group have statistically significant difference (P<0.05).With the increasing doses of each poison, N-cadherin mRNA expression was firstly increased and then decreased, witch 30?mol/L p, p'-DDE, 30?mol/Lβ-BHC and 10?mol/L combination groups increased significantly compared with the control(P<0.05). The protein levels of N-cadherin in 30,50μmol/L p, p'-DDE, 50μmol/Lβ-BHC and all combined groups were significantly lower than the control (P <0.05).FSHR mRNA expression was firstly increased and then decreased, compared with the control, the increase of 10μmol/L p, p'-DDE and 10μmol/Lβ-BHC groups had a statistically significant difference (P<0.05). Compared with the control, the mRNA levels in 50μmol/Lβ-BHC and 50μmol/L combined group decreased significantly (P<0.05). Compared with the control, the FSHR protein expression in 10, 50μmol/L p, p'-DDE, allβ-BHC and 50μmol/L combination groups decreased significantly(P<0.05). There were statistically significant difference between 10μmol/L combined group and the same concentration of the p, p'-DDE orβ-BHC separately. 30μmol/L combined group decreased significantly compared with the same concentration ofβ-BHC (P<0.05).Conclusion: Lower doses ofβ-BHC can stimulate Vimentin mRNA expression and high doses of p, p'-DDE,β-BHC and their combination can inhibit Vimentin protein expression.Lower doses of p, p'-DDE,β-BHC and their combination can increase the expression of N-cadherin mRNA, but p, p'-DDE,β-BHC and their combination can decrease the protein expression of N-cadherin. Low doses p, p'-DDE,β-BHC can stimulate the FSHR mRNA expression and high doses can inhibit its mRNA and protein expressions.PartⅢEffects of p, p'-DDE,β-BHC and their combination on Vimentin, N-cadherin and FSHR in adolescent SD rat testisObjective: To investigate the effects of p, p'-DDE,β-BHC and their combination on Vimentin, N-cadherin and FSHR mRNA and protein expressions in adolescent SD rat testis.Method: The real-time fluorescence quantitative PCR and Western blotting methods were used to test the Vimentin, N-cadherin and FSHR mRNA and protein expressions in adolescent SD rat testis.Results: In p, p'-DDE exposure groups, Vimentin mRNA expression was firstly increased and then decreased, compared with the control, 60mg/kg group increased significantly (P<0.05), Vimentin protein expression gradually decreased with the increasing of the concentration, 60, 100mg/kg group decreased significantly.Compared with the control, both of Vimentin mRNA and protein expressions decreased significantly inβ-BHC and the combination (P<0.05).Compared with the control, N-cadherin mRNA expression level was no significant difference, but the protein levels of N-cadherin decreased gradually with the increasing p, p'-DDE dose, the 60, 100 mg/kg groups were significantly decreased. The protein level inβ-BHC and the combination were significantly reduced, compared with the same concentration of separate group, the combined group had no significant difference.FSHR mRNA expression level of testicular tissue in p, p'-DDE exposure groups was firstly increased and then decreased, the group of 60mg/kg increased significantly, FSHR mRNA expression level decreased significantly inβ-BHC group. Compared with the control, FSHR protein expression in the 100mg/kg p, p'-DDE,β-BHC and the combination groups were significantly reduced.Conclusion: Lower doses of p, p'-DDE can stimulate Vimentin mRNA expression.β-BHC can reduce Vimentin mRNA expression. Both p, p'-DDE andβ-BHC could reduced the Vimentin protein expression in adolescent rat testis. p, p'-DDE,β-BHC had no apparent effect on N-cadherin mRNA expression in pubertal rat testis, but could decrease its protein expression.Lower doses of p, p'-DDE can stimulate the mRNA expression of FSHR,β-BHC can reduce the mRNA expression of FSHR. Both of p, p'-DDE andβ-BHC can reduce the protein expression of FSHR in pubertal rat testis.The study showed that the results in vitro were consistent with that in vivo. we get the following conclusion: p, p'-DDE andβ-BHC can interfere with the expression of Vimentin, N-cadherin and FSHR in Sertoli cells, and affect the function of male reproductive system.