To Study The Role Of Fas-mediated Signaling Pathway In ZEA Induced Apoptosis Of Sertoli Cells

ZEA is an estrogen-like toxin produced by the sickle fungus,which is widely found in cereal grains around the world.Studies have shown that ZEA structure is similar to 17[3-estradiol5 which can antagonize the binding and regulation of estrogen and receptors,affect the secretion of gonadotropin-releasing hormone(GnRH),and lead to a decline in the reproductive capacity of animals and humans.In recent years,the phenomenon that ZEA can cause reproductive dysfunction in male animals has attracted attention,but the mechanism is unknown.In order to explore the mechanism of male reproductive toxicity in ZEA,this study used the isolated primary Sertoli cells of rat as a material for the induction of Fas-mediated apoptosis of testicular Sertoli cells in vitro by ZEA.1.Effect of ZEA induced Apoptosis via Fas/FasL pathway in Sertoli CellsTo investigate the effect of ZEA on Fas/FasL apoptosis signaling pathway in Sertoli cells,the primary Sertoli cells of rat were used as the experiment material,exposed to different concentration of ZEA(0,5,10,20 μmol/L)for 24 h.AnnexinV-FITC/PI method was used to detect the apoptosis rate;Western blot and qRT-PCR were used to detect the expression and transcription of Fas/FasL related protein in the death receptor pathway.The cells were treated with 20 μmol/L ZEA,and the apoptosis proteins Fas,FasL,FADD,cleaved-caspase-8 were detected by Western blot at different exposure times(0,4,8,16 h).The results of the flow cytometry showed that compared with the control group,the apoptotic rate of each group was significantly increased(P<0.05 or,P<0.01).The qRT-PCR results showed that the level of gene transcription of Fas and FasL was increased significantly with 10,20 μmol/L of ZEA(P<0.05 or P<0.01),FADD and caspase-8 were increased significantly with 20 μmol/L of ZEA(P<0.01).Compared with the control group,western-blot analysis showed that the expression of Fas,FasL,FADD,cleaved-caspase8 were increased of 5.moI/L ZEA(P<0.05 or P<0.01).With the increase of ZEA exposure time,the expression of Fas,FasL,FADD,cleaved-caspase8 were significantly or significantly higher than the control group(P<0.05 or P<0.01).All these results clarified that ZEA would induce the apoptosis of Sertoli cell via the Fas/FasL pathway.2.The role of Fas-mediated signaling pathway in ZEA induced mitochondrial apoptosis in Sertoli cellsTo investigate the role of Fas-mediated signaling pathway in ZEA induced mitochondrial apoptosis in Sertoli cells,the primary Sertoli cells were exposed to different concentration of ZEA(0,5,10,20 μmol/L)or 20 μmol/L and 10 μmol/L Z-IETD(caspase-8 inhibitor)for 24 h.The apoptosis genes Bax and Bcl-2 were detected by qRT-PCR.The expression of Bax,Bcl-2 and distribution of cytochrome C,activation of BID,caspase-9 and caspase-3 were detected by Western blot.AnnexinV-FITC/PI method was used to detect the apoptosis rate.The JC-1 probe was loaded,then the change of mitochondrial membrane potential(△ψm)was detected by immunofluorescence and flow cytometry.The results showed that the treatment of ZEA would significantly promote the transcriptional level of Bax gene(P<0.05),as well as significantly suppress the transcriptional level of Bcl-2 gene(P<0.05).The results of Western blot showed that,compared with the control group,the expression of Bax ascended,the expression of Bcl-2 descended,the ratio of Bax/Bcl-2 increased highly significant(P<0.01).The activation of BID was increased significantly with the ZEA groups(P<0.05 or P<0.01).The release of cytochrome C from mitochondrial to cytoplasm,the activation of caspase-9 and caspase-3 were increased significantly with the ZEA groups(P<0.05 or P<0.01).Compared with the control group,the Z-IETD-FMK group had no obvious significance in cell-related indicators(P>O.05).Compared with 20 μmol/L ZEA,the apoptotic rate had a significantly decreased in the 20 μmol/L ZEA and 10μmol/L Z-IETD-FMK exposure group;The △ψm had a significantly increased in the 20 μmol/L ZEA and 10 μmol/L Z-IETD-FMK exposure group.The release of cytochrome C and the activation of BID,caspase-9,caspase-3 in the Sertoli cells treated with 10 μmol/L Z-IETD-FMK and 20 μmol/L ZEA were significantly decreased(P<0.01).All these results clarified that Fas-mediated signaling pathway in ZEA induced mitochondrial apoptosis on Sertoli cells3.Regulatory relationship between Fas/FasL and JNK signaling pathway during testicular Sertoli cell apoptosis induced by ZEATo investigate the regulatory relationship between Fas/FasL pathways and JNK pathways in apoptosis of Sertoli cells induced by ZEA,the primary Sertoli cells exposed to different concentration of ZEA(0,5,10,20 μmol/L)or 20 μmol/L ZEA and 2.5 μg/μL Anti-FasL,10μmol/L Z-IETD,or 1 μg/μL DAXX siRNA for 24 h.AnnexinV-FITC/PI was used to detect the apoptosis rate.Western blot was used to detect FADD,Cleaved-caspase-8,Cleaved-caspase-9,DAXX,ASK-1,JNK3,and p-JNK.The results showed that compared with the control group,there were no significant difference in the anti-FasL group(P>0.05).Compared with the 20 μmol/L ZEA group,the anti-FasL and ZEA group could significantly inhibit the apoptosis rate in the Sertoli cells.(P<0.01);The expression of FADD,Cleeaved capase-8,Cleaved caspase-9,Cleaved caspase-3 was deceased significantly(P<0.05 or P<0.01).Compared with the control group,the expression of DAXX,ASK-1 and JNK3/p-JNK were increased significantly(P<0.05 or P<0.01).Compared with the control group,there were no significant difference in the cell-associated indexes between the caspase-8 inhibitors Z-IETD-FMK group and the DAXX siRNA group(P>0.05).Compared with the 20 μmol/L ZEA group,the expression of DAXX,ASK-1 and JNK3/p-JNK was significantly decreased when the Sertoli cell were exposed to the Z-IETD-FMK and ZEA group(P<0.05 or P<0.01).The expression of cleaved caspase-8 and cleaved caspase-3 were significantly decreased when the Sertoli cell was exposed to the DAXX siRNA and ZEA group(P<0.05 or P<0.01).The above results indicate that,the apoptosis on Sertoli cells induced by ZEA could be Fas/FasL pathways and JNK pathways could regulate each other.It is concluded that the male reproductive toxicity of ZEA may affect the development of spermatozoa with the apoptosis of Sertoli cells.ZEA could induce the apoptosis of rat Sertoli cells by Fas-mediated signaling pathway.Fas-mediated apoptosis signaling pathway could directly affect the development and maturation of sperm cells in Sertoli cells and spermatogenic cells.