The Study On Regulation Mechanism Of Androgen On Antioxidation Of Glutathione Peroxidase 5 In Epididymis Of Sheep

The microenvironment of epididymal lumen formed by epididymal epithelium cells(EECs)plays a key role in maintaining the normal maturation of spermatozoa.Reactive oxygen species are the byproducts of redox metabolism of cells.An appropriate amount of ROS in epididymis plays an important role in the physiological process of sperm maturation and capacitation through regulating c AMP level.However,excessive ROS can damage spermatozoa by oxidative stress.Therefore,maintaining the redox balance of epididymal microenvironment plays a major role in sperm maturation.Glutathione Peroxidase 5(GPX5)is highly expressed in epididymis.At present,there is no report on the function and regulation of GPX5 in epididymal epithelial cells of sheep.Therefore,in this study,on the basis of optimizing the culture conditions of sheep EECs in vitro,and the regulation mechanism of GPX5 antioxidant function in EECs of sheep was studied.The results are as follows:1.The effect of EGF on the proliferation of sheep EECs in vitro.The cell growth was determined by CCK-8 the expression of GPX5 and AR genes in EECs was detected by RT-PCR,the cell cycle and apoptosis of cells were detected by flow cytometry,and the phosphorylation levels of EGFR,Akt and FOXO1 were detected by Western blot.The results showed that 50 ng/m L EGF can significantly promote EECs proliferation,and the GPX5 and AR proteins could be normally expressed in different passages of EECs.EGF extremely significantly increased the proportion of cells at the S stage(P<0.01)and significantly decreased the apoptosis index(P<0.05).Compared with the control group,the addition of Gefitinib,an EGFR inhibitor,extremely significantly reduced the proportion of cells in S phase(P<0.01),and extremely significantly increased the apoptosis index(P<0.01);the addition of LY294002,a PI3K/Akt inhibitor,significantly reduced the proportion of cells in S phase(P<0.05),but the apoptosis index did not significantly change(P>0.05).Compared with the control group,EGF extremely significantly increased the EGFR,AKT and FOXO1 phosphorylation levels of EECs(P<0.01).Compared with EGF group,EGFR,Akt and FOXO1 phosphorylation level in EGF+gefitinib group were extremely significantly decreased(P<0.01),while Akt and FOXO1 phosphorylation level in EGF+LY294002 group was extremely significantly decreased(P<0.01).2.The effect of trehalose on the proliferation of sheep EECs in vitro.The proliferation of EECs was detected by microplate reader.RT-PCR was used to detect EECs functional markers from different passages.Cell cycle and apoptosis were detected by flow cytometry.ROS level was detected by DCFH-DA probe,and the main antioxidant levels were detected by enzyme chemical method.The expression of GPX5 in cells were detected by q RT-PCR and ELISA.The results showed that 100 mmol/L trehalose effectively improved the growth ability of EECs,extremely significantly increased the proportion of cells in S phase(P<0.01),and extremely significantly reduced the apoptosis rate(P<0.01);100 mmol/L trehalose cultured EECs could be continuously passaged for 14times in vitro,with normal GPX5 and AR genes profiles.Compared with the control group,the ROS of cells treated with 100 mmol/L trehalose decreased extremely significantly(P<0.01),the CAT and GPXs activities showed extremely significant(P<0.01)and significant(P<0.05)increases,and the m RNA and protein expression of GPX5 showed extremely significant(P<0.01)and significant(P<0.05)up regulation,respectively.3.Antioxidant effect of GPX5 in sheep EECs.si RNA was used to interfere GPX5gene expression in sheep EECs and treatment divided into si RNA-GPX5,si RNA-NC transfected control group and CK control group.The proliferation of cells treated with different concentrations of H₂O₂was detected by CCK-8,the intracellular ROS level was detected by DCFH-DA,the intracellular MDA content was detected by TBA method,and the intracellular 8-OHd G level was detected by immunofluorescence,and q RT-PCR was used to detect the m RNA expression of the main antioxidant gene in EECs.The results showed that with the increase of H₂O₂concentration,the proliferation of si RNA-GPX5group decreased compared with CK control group.Compared with CK control group,ROS content and MDA production in si RNA-GPX5 cells showed extremely significant(P<0.01)and significant(P<0.05)increases,respectively,while 8-OHd G level in si RNA-GPX5 cells increased significantly.The m RNA expression of GPX1 and GPX3 in the si RNA-GPX5+T group was significantly higher than that in the si RNA-GPX5treatment group(P<0.05),and the amount of SOD and GPX4 m RNA expression was extremely significant higher than that of the si RNA-GPX5 treatment group(P<0.01).4.Regulation of testosterone,AR and coregulators on GPX5 in sheep EECs.CCK-8was used to detect the proliferation of EECs treated with different concentrations of testosterone.q RT-PCR was used to detect the m RNA expression of GPX5,AR,SRC-1,CBP,P300 and NCOR2.The expression of GPX5,AR,SRC-1,CBP,p300 and NCOR2were detected by immunofluorescence and Western blot.Dual luciferase reporter gene was used to detect the binding of AR to GPX5,the binding site and AR transcription activity.The results showed that exogenous testosterone showed concentration dependence on the proliferation of EECs,in which the cell growth of 100 nmol/L testosterone group was better.The m RNA and protein expression of GPX5 in 100 nmol/L testosterone group was extremely significantly increased(P<0.01),while the m RNA and protein expression of GPX5 in 1000 nmol/L testosterone group was not significantly different from that in the control group(P>0.05),but extremely significantly(P<0.01)and significantly(P<0.05)lower than that in 100 nmol/L testosterone group,respectively.Compared with the control group,the expression of AR in the cell nucleus was significantly increased in the 100nmol/L testosterone group(P<0.05),The m RNA and protein expression levels of AR were extremely significantly increased(P<0.01)and significantly increased(P<0.05),respectively,but in the 1000 nmol/L testosterone group,the m RNA and protein expression of AR was decreased significantly(P<0.05).In the 1000 nmol/L testosterone group,the expression of CBP,P300 and SRC-1 was extremely significantly increased(P<0.01),especially in the nucleus.There was no significant difference in GPX5 m RNA and protein expression between si RNA-AR group and control group(P>0.05).The expression of GPX5 m RNA and protein in pc DNA3.1-AR group was significantly higher than that in control group(P<0.01).The luciferase activity of p GL3-GPX5-pro-wt+pc DNA3.1-AR group was significantly higher than that of p GL3-GPX5-pro-wt+pc DNA3.1 group(P<0.05).Compared with p GL3-GPX5-pro-wt,the luciferase activity of p GL3-GPX5-pro-mut1 group was significantly lower(P<0.05).The expression of SRC-1and P300 protein in si RNA-AR group was extremely significantly higher than that in control group(P<0.01).The expression of GPX5 m RNA and protein in si RNA-SRC-1group and that in si RNA-p300 group was significantly lower than that in control group(P<0.05).There was no significant difference in AR protein expression between si RNA-SRC-1 and si RNA-p300 groups and control group(P>0.05),but the transcription activity of AR in si RNA-SRC-1 and si RNA-p300 groups was significantly decreased(P<0.05).In conclusion,EGF up-regulated the phosphorylation of FOXO1 through EGFR/PI3K/Akt pathway and enhanced the growth ability of sheep EECs in vitro.Trehalose could promote the proliferation of EECs in vitro by increasing the activities of CAT and GPXs and the expression of GPX5.GPX5 can effectively protect sheep EECs from lipid peroxidation and DNA oxidative damage.Testosterone could regulate the expression of GPX5,AR and its coregulators in sheep EECs in a concentration dependent manner.Testosterone regulated GPX5 expression through AR and its coregulators SRC-1 and p300/CBP.AR promoted GPX5 transcription in a targeted way.SRC-1 and p300/CBP could increase the transcription activity of AR and maintain the expression of GPX5 when AR expression was significantly decreased.