| In the process of sperm transport in the epididymis,the epididymal epithelium and sperm metabolism will produce a series of reactive oxygen species(ROS)in the epididymal lumen microenvironment.An appropriate amount of ROS can be involved in regulating the normal physiological function of sperm,while excessive ROS will lead to oxidative stress in sperm,so it is very important to maintain a normal physiological level of ROS in epididymis.There are different forms of antioxidant enzymes in the epididymis,but how these enzymes work has not been clarified.Therefore,this experiment systematically analyzed the expression,activity and distribution of antioxidant enzymes in different parts of sheep epididymis,so as to provide a theoretical basis for further elucidating the regulation mechanism of antioxidant function of epididymis.1.Expression of antioxidant enzymes in different parts of epididymis.Q-PCR and Western Blot were used to detect the expression of Cu,Zn-SOD,CAT,GPX5 and PRDX6 in the caput,corpus and cauda of epididymis.The results showed that the mRNA and protein levels of Cu,Zn-SOD increased from the caput to the cauda epididymis,and the level of cauda epididymis was extremely significantly higher than these of caput and corpus epididymis(P<0.01).The expression of CAT mRNA in the caput epididymis was extremely significantly higher than these in the corpus and cauda epididymis(P<0.01),and the protein level of CAT in corpus epididymis was the highest(P<0.01).The mRNA and protein of GPX5 and PRDX6 decreased from caput to cauda epididymis,and the expression of mRNA and protein in caput epididymis was the highest,which was extremely significantly higher than that in corpus and cauda epididymis(P<0.01).This shows that the demand for various antioxidant enzymes is different in different parts of epididymal tissue,so it presents region special expression.2.Expression of antioxidant enzymes in epididymal fluid in different parts.The activities of total antioxidant capacity(TAC),superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GPXs)and peroxidase(PRDXs)in different parts of epididymal fluid were detected by enzyme activity detection kit,and the expressions of Cu,Zn-SOD,CAT,GPX5 and PRDX6 in caput,corpus and cauda epididymal fluid were detected by Western Blot test.The results showed that the activities of TAC,GPXs and PRDXs in cauda epididymal fluid were extremely significantly higher than those of caput and corpus epididymal fluid(P<0.01).The activity of SOD in the cauda epididymal fluid was extremely significantly higher than that of the caput epididymal fluid(P<0.01),and was significantly higher than that of corpus epididymal fluid(P<0.05).There was no significant difference in CAT activity in different parts of epididymal fluid.The expression of Cu,Zn-SOD protein in epididymal fluid increased from caput to cauda epididymis,and the expression of cauda epididymal fluid was extremely significantly higher than these in caput and corpus epididymal fluid(P<0.01).The protein level of CAT in epididymal fluid from the caput to cauda epididymis increased at first and then decreased.The protein level of CAT in corpus epididymal fluid was the highest,and it was extremely significantly higher than that cauda epididymal fluid(P<0.01),but The protein level of CAT in cauda epididymal fluid was significantly higher than that in caput epididymal fluid(P<0.05).The expression of GPX5 and PRDX6 protein in epididymal fluid decreased from caput to cauda epididymis,and the expression in caput epididymal fluid was the highest,which was significantly higher than that in corpus and cauda epididymal fluid(P<0.01).This indicates that the cauda epididymis has a higher TAC in the lumen microenvironment provided by the epididymal tissue for sperm.The expression trend of antioxidant enzymes in epididymal fluid was consistent with that in epididymal tissue.3.Expression of antioxidant enzymes in epididymal sperm in different parts.The activities of TAC,SOD,CAT,GPXs and PRDXs in different parts of epididymal sperm were detected by enzyme activity detection kit,then the protein expressions of Cu,Zn-SOD,CAT,GPX5 and PRDX6 in caput,corpus and cauda epididymal sperm were detected by Western Blot,and the distribution of antioxidant proteins were detected by immunofluorescence.The results showed that the activities of TAC,GPXs and PRDXs in sperm from caput to cauda epididymis increased,and the activity of sperm in cauda epididymis was significantly higher than these in caput and corpus epididymis(P<0.01).howerver,the SOD activity in sperm from caput to cauda epididymis decreased,and the SOD activity in caput epididymal sperm was significantly higher than that in the corpus epididymal sperm(P<0.05).CAT activity was not detected in epididymal sperm.The expression of Cu,Zn-SOD protein in sperm from caput to cauda epididymis increased,and the expression of Cu,Zn-SOD protein in e cauda pididymal sperm was extremely significantly higher than that in caput epididymal sperm and corpus epididymal sperm(P<0.01).In addition,for different parts of epididymal sperm,Cu,Zn-SOD positive signals were distributed on the whole sperm.The expression of GPX5 protein decreased caput to cauda epididymal sperm,and the expression in caput epididymal sperm was the highest,which was extremely significantly higher than that in corpus and cauda epididymal sperm(P<0.01).The results of immunofluorescence showed that GPX5 was strongly expressed in the postnuclear cap,neck,the middle and principal piece of the sperm,and slightly expressed in the end piece of sperm;the expression of PRDX6 protein decreased from the caput to the cauda epididymal sperm,and the expression of PRDX6 protein in caput epididymal sperm was extremely significantly higher than these in corpus and cauda epididymal sperm(P<0.01),while the expression of PRDX6 protein in corpus epididymal sperm was extremely significantly higher than that in cauda epididymal sperm(P<0.01).The immunofluorescence results of PRDX6 showed that the PRDX6 in the caput epididymal sperm was strongly expressed only in the head of the sperm,but not in the tail of the sperm.As the sperm enters the corpus and cauda epididymis,PRDX6 was expressed in the sperm haed,middle and principal piece of the sperm,but not in the end piece of the sperm.In addition,the CAT protein was not detected in epididymal sperm in different parts of epididymis by Western blot or immunofluorescence detection.In conclusion,these results suggest that Cu,Zn-SOD,GPX5 and PRDX6 have a similar change trend in epididymal tissue,epididymal fluid and epididymal sperm.but the antioxidant function of CAT is not necessary.The cauda epididymis has higher antioxidant enzyme activity,which requires a more complex antioxidant system to protect sperm in cauda epididymis from oxidative damage. |
