| Spermatozoa are further matured after transit through the caput and corpus epididymis and then stored at the cauda epididymis.During storage in the lumen of the cauda epididymis,imbalance of the free radical production and the antioxidant system can lead to oxidative stress,which can affect sperm structure,survival and fertilization rates.In this study,179 rams of Hu sheep were raised to 6 months old in the same environment and divided into low fertility group（L）and high fertility group（H）according to epididymal weight,sperm density,sperm acrosome integrity and histomorphology.Gas chromatography,q RT-PCR,western blot and immunohistochemistry were used to analyse the fatty acid composition and antioxidant activity of cauda epididymis of Hu sheep with different fertility.Then,the key genes regulating cauda epididymis lipid metabolism and antioxidation were screened by RNA-Seq technology Results are as follows:（1）Epididymis weight of 179 six-month-old Hu sheep was significantly and positively correlated with testicular weight,body weight and sperm density.According to the epididymis weight,sperm density,acrosome integrity and cauda epididymis histomorphology,nine rams each in group H and group L were selected for analysis.The diameter of epididymal lumen,and the sperm concentration in group H was significantly higher than those in group L（P < 0.05）.（2）Fatty acid research found that the content of crude fat,total fatty acid,total n-3 PUFA and DHA of cauda epididymis in group H was significantly higher than those in group L（P < 0.05）.The ratio of n-6/n-3 PUFA was significantly lower than that in group L（P < 0.05）.Immunohistochemical results showed that SREBP1 and ELOVL2 were expressed in pseudostratified columnar ciliated epithelium and smooth muscle cells.The m RNA expression of SREBP1 in group H were up-regulated（P = 0.09）.The m RNA expression of ELOVL2 in group H was significantly increased（P < 0.05）,protein expression was also up-regulated.（3）Compared with group L,group H had higher antioxidant activity with GSHPx and T-AOC increased significantly（P < 0.05）,while MDA content,the average optical density of ROS in epididymal epithelial cells and sperm,and mitochondrial copy number were significantly lower（P < 0.05）.Immunohistochemical results showed that GPX5,GPX3,GPX4 and SOD were widely distributed in cauda epididymides,and the expression intensity in group H was significantly higher than those in group L（P <0.05）.The gene expression of GPX5 and GPX3 was significantly up-regulated in group H（P < 0.05）.（4）RNA sequencing showed that there were 1404 DEGs.Compared with group L,735 genes were up-regulated and 669 genes were down-regulated in group H.Genes involved in lipid metabolism and antioxidation were screened by GO enrichment analysis.In KEGG results,pathways related to lipid metabolism and antioxidant activity were enriched.Key genes related to lipid metabolism and antioxidation including CYCS,COX6A1,ERO1 LB,SMOX,GPX3,GPX5,GPX7,CYP4V2,ALDH3A2 were selected.In conclusion,compared with low fertility group,the cauda epididymis of high fertility group had higher n-3 PUFA content,and lower ROS level and stronger antioxidant activity to protect sperm from ROS.Key genes,such as CYCS,COX6A1,ERO1 LB,SMOX,GPX3,GPX5,GPX7,CYP4V2 and ALDH3A2,may involve in the regulation of lipid metabolism and antioxidant capacity of Hu sheep epididymis. |
