Effects Of P, P'-dde And β-bhc In The Expression Ofcalcium,erk And Bcl-2 In Rat Sertoli Cells

the scholars believed that environmental estrogen and other EDCs caused thereproductive abnormalities. DDT (2,2-bis(4-chlorophenyl)-1,1,1-trichloroethane) andBHC (benzene-hexachloride) were persistent organic pollutants(POPs), theseorganochlorine pesticides were left over in the adipose tissue and blood lipids throughbiomagnifications of the food chain for decades. p,p'-DDE andβ-BHC are the majormetabolites of DDT and BHC respectively, they have a high level in human and otheranimals. The researches indicate that these organochlorine pesticides and theirmetabolites have the interference of endocrine, especially to male reproductivefunction.Recently, studies on the effects of organochlorine pesticides on signaltransduction pathway became hotspot. Some research reported that Mitogen-activatedprotein kinase (MAPK ) play a major role in the maintenance and control ofspermatogenesis, which can regulate cell proliferation, differentiation and apoptosis intesticle tissues of mammal animals. The previous studies of our laboratory havedemonstrated that p,p'-DDE could result in the phosphorylation of ERK and inducethe activation of the downstream pathway in order to regulate rat Sertoli cells. But thecombined effect of p,p'-DDE,β-BHC on ERK MAPK signal transduction pathway,Bcl-2 and Ca²⁺ of rat Sertoli cells remains unclear. The aim of this research is toinvestigate the possible mechanism of combined effects of p,p'-DDE andβ-BHC onrat testicle Sertoli cells. PartⅠThe cell cultured in vitro and the toxicity of Combinedeffectof p,p'-DDE andβ-BHC on rat Sertoli cellsSertoli cells were separated from testicular of SD rats of 18-20 days age, theywere cultured in DMEM media with 5% fetal bovine serum at 35℃in a humidifiedincubator of 5% CO2.After for 24h ,the cells were treated with 10μmol/L,30μmol/L,50μmol/L,70μmol/Lp,p'-DDE ,β-BHC and their combination for 24h, then we usedMTT method to determine their absorbencies under the 490nm wave-length. Theresults show that with increasing of the concentration of p'-DDE ,β-BHC and theircombination, the absorbency all decreased. The effects of p,p'-DDE ,β-BHC and theircombination at the concentration of 50μmol/L all have a significant differencescompared with DMSO group (P<0.05).The results also show that among p,p'-DDE ,β-BHC and their combination group,there are a significant difference at the concentration of 50μmol/L.In a word, theresults indicated that p,p'-DDE ,β-BHC and their combination could do harm toSertoli cells when the concentration of p,p'-DDE ,β-BHC and their combination wereabove 50μmol/L.PartⅡStudy on the effect of Ca²⁺ of rat Sertoli cells treated withp,p'-DDE,β-BHC and their combinationSertoli cells were cultured 2 days after separating from testicular tissue of rats,and then were divided into four groups respectively incubated with DMSO (used ascontrol group), p,p'-DDE,β-BHC and their combination under the concentration of10, 30, 50μmol/1 for 24h, After inspecting the Fluorescence intensities of Ca²⁺ in thedifferent concentration groups, they increased along with the increasing concentrationof p,p'-DDE,β-BHC and their combination. The results show that they are harmful to the Sertoli cells of p,p'-DDE,β-BHC and their mixture.PartⅢStudy on the expression of erk, bcl-2 mRNA of rat Sertolicells treated with p,p'-DDE,β-BHC and their combined effectsSertoli cells were cultured 2 days after separating from testicular tissue of rats,and then were divided into four groups respectively incubated with DMSO (used ascontrol group), p,p'-DDE,β-BHC and their combination under the concentration of10, 30, 50μmol/1 for 24h, After that we used Real Time PCR method to study thecombined effect of p,p'-DDE,β-BHC on the express of erk,bcl-2 mRNA of rat Sertolicells. The results show that p,p'-DDE,β-BHC and their combination can all descendthe expression of erk,bcl-2 mRNA .PartⅣStudy on the Combined effect of p,p'-DDE,β-BHC on theexpress of p-Erk, Erk of rat Sertoli cellsSertoli cells were cultured 2 days after separating from testicular tissue of rats,and then they were divided into four groups respectively incubated with DMSO (usedas control group), p,p'-DDE,β-BHC and their combination under the concentration of10, 30, 50μmol/L for 24h. Firstly by using western blotting method,we study thecombined effects of p,p'-DDE,β-BHC on expression of p-Erk, Erk proteins indifferent concentrations . Secondly, Sertoli cells were incubated at the highconcentration of 50μmol/1, we used western blotting method to study the combinedeffects of p,p'-DDE,β-BHC on expression of p-Erk, Erk proteins in differenttime.The results show that there was a persistent increasing, then descended,after thatincreased again of p-Erk protein expression in different concentrations of p,p'-DDE.At the same time the expressions of p-Erk protein in different concentrations ofβ-BHC and the combination of p,p'-DDE andβ-BHC were descending along with the concentrations increased . However the expressions of Erk proteins had no differencesin the four groups.Sertoli cells were cultured in different time with the same high concentration(50μmol/L) , the expressions of p-Erk protein in the groups of p,p'-DDE ,β-BHC andtheir combination were firstly increasing , had its maximum at the 6h point , and thenthe expressions of p-Erk protein were descending .The results indicated that p,p'-DDE,β-BHC and their combination can result in the phosphorylation of Erk protein andinduce the activation of ERK/MAPK pathway .The activation of ERK/MAPKpathway could be a kind of favorable effect on male reproductive function.