The Roles Of Pten/Akt And Mitochondrial Signaling Pathway In Rat Testicular Apoptosis Induced By BPA

Part I Study of the effects of BPA on Sertoli cells and mechanisms involved in apoptosis induced by BPAObjective:Bisphenol-A (BPA) is one of endocrine-disrupting chemicals and exists extensively around our environment. It could affect the male reproduction but the mechanisms of reproductive toxicity were limited. The aim of this study was to explore the effects of BPA on Sertoli cells and mechanisms involved in apoptosis induced by BPA.Methods:Sertoli cells were treated with0,30,50,70μM BPA for24h.Then MTT assay was applied to measure the viability of Sertoli cells; the level of ROS was measured by ELISA reader; the levels of MDA and the activity of SOD were detected following with the protocol of assay kit; RT-PCR and Western blotting were used to detect Pten, Pdkl, Akt cytochrome c、Bax and procaspase-8mRNA levels and protein abundances of Pten, phospho-Akt, Bax, cytochrome c, bcl-2and the apoptosis-related protein levels of procaspase-9and cleaved caspase-3; The apoptosis of Sertoli cells was determined by flow cytometric analysis; the apoptotic morphological change of cells was observed by AO/EB and Hoechst33258staining.Results:The viability of Sertoli cells was reduced when exposed to BPA (p<0.05).; The activity of SOD was significantly decreased while the level of MDA was remarkably increased (p<0.05); Compared with the control, BPA exposure induced significant increasing of ROS production (p<0.05), which could be neutralized by preincubation with NAC. The changes of mRNA expression were also observed, the mRNA expression levels of Pten, Pdkl, Akt, cytochrome c,Bax and procaspase8were increased (P<0.05). But the antioxidant NAC treatment could reverse the mRNA expression of cytochrome c Bax and procaspase-8.Compared with the control,the observed results showed the levels of Pten, Bax and cytochrome c were remarkably elevated in cytosol however the abundance of phospho-Akt and Bcl-2protein was reduced (P<0.05); The level of apoptosis-related protein cleaved caspase-3was remarkable increased while procaspase-9was significantly deceased (p<0.05). Moreover, the levels of Bax and cytochrome c could also be ameliorated by antioxidant NAC, but NAC treatment made little change about Bcl-2protein expression.The apoptosis was examined by flow cytometric analysis, Hoechst and AO/EB staining. BPA exposure could cause apoptotic cell death and the Hoechst and AO/EB staining indicated the BPA exposure resulted in the morphological change of Sertoli cell including nuclear condensation and fragmentation, which also could be inhibited by preincubation of NAC.Conclusion:BPA exposure could decrease the viability of Sertoli cells, induce cellular ROS accumulation and apoptosis of Sertoli cells; The Pten/Akt signaling pathway might play a role in this apoptoic procession; BPA induced apoptosis of Sertoli cells through ROS mediated mitochondrial signaling pathway.PartⅡ Study of the mechanisms involved in BPA-induced testicular apoptosis in prepubertal ratsObjective:Based on the results of cultured Sertoli cells, this study is to investigate the mechanisms involved in BPA-induced testicular apoptosis in prepubertal rats.Methods:20-day-old prepubertal male rats were given different doses of BPA by intraperitoneal injection and the rats exposure experiment as following:solvent control group,2mg/kg b.wt,10mg/kg b.wt and50mg/kg b.wt respectively.The injection was taken every other day for10days. All animals were sacrificed after the BPA injection was finished. In the present study, Western blotting were used to detect protein abundances of Pten, phospho-Akt, cytochrome c, Bax and the apoptosis-related protein levels of procaspase-3and cleaved caspase-3; The method of TUNEL was used to investigate the effect of BPA on testicular apoptosis; the HE staining was used to observe the histological changes in seminiferous tubules.Results:The observed results showed BPA exposure resulted in the significant increase of Pten, Bax, cytochrome c and FasL protein levels (P<0.05), while the level of procaspase-3significantly reduced (P<0.05). And a significant elevation of cleaved caspase-3was also observed. But phospho-Akt protein was no significantly higher than that of the control group (P>0.05).The results of TUNEL showed the increasing apoptosis of germ cells among the seminiferous tubules (P<0.05).HE staining indicated the inflammatory infiltration of testis tissues and the counts and layers of spermatogenic cells were obviously decreased.Conclusion:BPA could induce apoptosis of testicular germ cells through mitochondrial signaling pathway, the upregulated expression of Pten was also involved in testicular apoptosis exposed to BPA, but the mechanism should be further investigated, the Fas/FasL signaling pathway might be also involved in this apoptotic procession.