In Vitro Effect Of BPA On Expression Of Occludin At Tight Junction Of Rat Testis

Objective To investigate the effects of Bisphenol A (BPA) on expression of occludin of Sertoli cell in vitro, detecting the mechanism of impairment of spermatogenesis by the BPA.Methods Primary sertoli cells were isolated and cultured from Wistar rat for4～5days, and the tight junction(TJ)-permeability barrier was established by dual-chamber culture model. With a series of concentration BPA(10-3μmol/L,10-2μmol/L,10-1μmol/L,100μmol/L,101μmol/L,102μmol/L,103μmol/L) and0.1%DMEM (without fetal bovine serum) co-incubating the Sertoli cells in vitro for24h, cell proliferative activity was assessed with CCK8assay.The median dose lethal (LD50) of BPA on Sertoli cells was150μmol/L. A series of concentration BPA(25μmol/L,50μmol/L and100μmol/L) were selected by LD50as the following experimental group and, dimethyl sulfoxide (0.1%DMSO) was used to be the control group. Cells were treated with0μmol/L (0.1%DMSO),25μmol/L,50μmol/L and100μmol/L BPA for24h, the expression of occludin was detected respectively by immunofluorescence and Western blot analysis.Results Primary Wistar rat sertoli cells were isolated and cultured successfully, TJ-permeablity barrier was established perfectly. CCK-8assay showed that, absorbance (OD value) of cells dropped significantly with the increasing drug use. Compared with the corresponding control group, Ods decreased more significantly at102μmol/L,103μmol/L BPA group (P<0.05), however,10-1μmol/L,10μmol/L,101μmol/L BPA group had no statistical significance (P<0.05). Immunofluorescent microscopy showed that, the localization of occludin was shown in each group. The intensity of occludin immunostaining was gradually decreased with the increasing doses of BPA, especially the25μmol/L and50μmol/L BPA group(P<0.05). The intensity of occludin immunostaining was dramatically decreased at25μmol/L,50μmol/L BPA group(P<0.05) and, there was no strong occludin positivity. Only a few occludin exhibited weakly positive in100μmol/L group (P<0.05). Western blotting showed that, occludin was expressed in each group, the expression of occludin decreased with the increasing BPA treatment, the expression of occludin in25μmol/L,50μmol/L,100μmol/L BPA group reduced significantly compared with that in the corresponding control group (P<0.05),50μmol/L and100μmol/L groups also had statistical significance compared with25μmol/L BPA group (P<0.05)Conclusions BPA can reduce the expression of occludin in Sertoli cells, which suggest BPA may perturb the regularly spermatogenesis though disrupting the TJ barrier of Sertoli cell.