Effects Of P, P'-dde, β-bhc And Their Combination On Jnk Mapk Signal Transduction Pathway In Rat Testicular Tissues In Vitro

Studies have showed that, discharging of a large quantity of endocrine disturbance chemicals (EDCs) and their persistent existence have caused serious reproductive harm to both animals and human. Organochloride pesticides are a typical kind of EDCs. 2,2-bis(4-chlorophenyl)-1,1,1-trichloroethane (DDT) and benzene- hexachloride (BHC) in organochloride pesticides family were produced and used widely last century. Although production and usage had been banned after the discovery of their adverse effect in environment and living beings, the residue level is still high, because of the characters of slow degradation, easy to be accumulation and biomagnification through food chain. p,p'-DDE andβ-BHC are the main metabolic products of DDT and BHC respectively, which accumulate in adipose tissues and hardly discharge. Considering the prolonged effect in endocrine disturbance, p,p'-DDE andβ-BHC become hot spots in research.Recent years, accompanied with the progress of cellular signal transduction pathway studies, especially the discovery of mitogen-activated protain kinase (MAPK) signal transduction pathway affecting cellular proliferation, differentiation and apoptosis, researchers pay close attention to the progress that organochloride pesticides activate MAPK pathway so as to disturbance spermatogenesis. Our laboratory has proved that, p,p'-DDE,β-BHC as well as their combination can induce apoptosis of Sertoli cells by activating JNK and p38 pathway in MAPK. Based on our previous studies, this research aims at to explore the mechanism of p,p'-DDE andβ-BHC affecting JNK pathway, taking testicular tissues as object. The coculture system of Sertoli cells and spermatogenic cells in tissue level is the innovative point of this experiment. PartⅠRat Testicular Tissue Culture in vitro, Morphological Identification and Apoptosis AnalysisObjective: To establish a testicular tissue culture method in vitro (Sertoli cells and spermatogenic cells coculture system in tissue level). Explore the effect p,p'-DDE,β-BHC and their combination on this system morphologically.Method: Rat testicular tissue were cut into approximately 2mm3 fragments and cultured in DMEM culture medium, divided tissues into 5 groups and cultured them for 1,2,3,4,5 days respectively. They were fixed and embedded, and then underwent HE staining and TUNEL staining. Treated tissues with 10μmol/L, 30μmol/L, 50μmol/L p,p'-DDE,β-BHC or their 1:1 combination respectively for 24h, detect apoptosis condition by TUNEL staining.Result: Normal tissues survived well in this culture model, with few cell apoptosis. Tissues treated with organochloride pesticides showed changes in morphology, deep in yellow staining, decrease in cell count compared with control group.Conclusion: Coculture model of Sertoli cells and spermatogenic cells can be established in tissue culture. p,p'-DDE,β-BHC and the combination are harm to testicular cells in the model.Part II Effects of p,p'-DDE andβ-BHC on jnk mRNA Expression in Rat Testicular TissueObjective: To explore the effect p,p'-DDE andβ-BHC on total jnk mRNA expression in testicular tissues.Method: Detect the expression of total jnk mRNA under different concentration of p,p'-DDE,β-BHC and their combination by fluorescent quantitative real time PCR (FQ-PCR).Result: All treated group expressed high mRNA ratio of jnk/β-actin compared with control group, p<0.05. In p,p'-DDE orβ-BHC treated groups, along with the increased concentration, mRNA ratio decreased first and then increased, however, when treated with both p,p'-DDE andβ-BHC, it showed an opposite tendency. The effect ofβ-BHC was less severe compared with the others.Conclusion: There were different reaction mechanisms between treated Sertoli cells and spermatogenic cells. 30μmol/L p,p'-DDE orβ-BHC can induce inhibition of mRNA transcription in spermatogenic cells, however, this concentration of their mixture would induce mRNA expression significantly.PartⅢEffects of p,p'-DDE andβ-BHC on Jnk Expression in Rat Testicular TissueObjective: To explore the effect p,p'-DDE andβ-BHC on Jnk protein expression in testicular tissues.Method: Detect the expression of total Jnk1/2 and p-Jnk1/2 under different concentration of p,p'-DDE,β-BHC and their combination by western blotting.Result: All treated group showed high p-Jnk1/2 expression compared with control group, p<0.05, whereas there were no changes in the expression of Jnk1/2. In p,p'-DDE orβ-BHC treated groups, along with the increased concentration, p-Jnk1/2 expression decreased first and then increased, and 30μmol/L p,p'-DDE orβ-BHC treated groups showed significant differences compared with their 10μmol/L and 50μmol/L groups, (p<0.01), however, when treated with both p,p'-DDE andβ-BHC, it showed an opposite tendency, also with a significant difference in 30μmol/L treated group. p-Jnk2 were highly expressed compared with p-Jnk1. In lower concentration groups, the mixture group showed the weakest effect; in other concentration groups,β-BHC was the least severe one.Conclusion: This result confirms the conclusion showed in last part, and when treated with 50μmol/L mixture, large amounts of spermatogenic cells were already apoptotic early than Sertoli cells.The results above indicate that, Organochloride pesticides p,p'-DDE andβ-BHC can induce apoptosis of spermatogenic epithelial cells in rat testicular tissues by activating JNK MAPK cellular signal transduction pathway, which consequently cause a adverse effect of reproductive system, however, there are some differences between various kinds of cells when induced by this pathway.