Expression And Regulation Of Glutathione Peroxidase 5 In The Epididymis Of Inner Mongolia Cashmere Goats

Epididymis is the main place for sperm maturation.and the detailed study on mechanism of regulation has important theoretical guiding significance in improving male reproductive capacity.Glutathione peroxidase 5(GPx5),as a epididymis specific protein,plays an important role in the protection of sperm against oxidative stress and maintenance of epididymal microenvironment.However,there was no detailed study of GPx5 in goat epididymis at present.We detected the expression of GPx5 in testis and different parts of epididymis of inner mongolia cashmere goats in three reproductive physiology stages:puberty,mature stage and aged stage.In this study,culture system of cashmere goat of epididymal epithelial cell with high purity and good stability was established for the first time,the influence and possible mechanism of steroid hormones on proliferation of epididymal epithelial cells were investigated,and the regulation of GPx5 expression in epididymal epithelial cells was studied in vitro.The results were as follows.1.The expression of GPx5 mRNA and protein in testis and epididymis of cashmere goats in three reproductive physiology stages(puberty,mature stage and aged stage)were detected by fluorescence quantitative PCR,western blotting and immunohistochemistry.The results showed that GPx5 was expressed in the epididymis of cashmere goats in all reproductive physiology stages,but not in testis.However,there were differences in the expression region and quantity among the three stages.At the puberty stage,GPx5 mRNA and protein were mainly expressed in epididymal caput.At the body mature stage,the expression of GPx5 mRNA and protein in each epididymal region was significantly higher than these in other stages.Initial segment and epididymal caput were the main expression regions.At the aged stage,GPx5 mRNA and protein were mainly expressed in epididymal caput and a significant reduction was detected compared to that in body mature stage.Immunohistochemistry results showed that GPx5 protein mainly existed in epididymal epithelial cell and obvious GPx5 positive signals in epididymal sperm of cashmere goats at different ages.These results indicated that the expression region and quantity of GPx5 in epididymis were directly related to the reproductive physiology stage of cashmere goats.2.The primary cells were obtained from epididymis caput of 10-12 months cashmere goat using enzymatic dispersion and improved tissue-piece digestion method.The cells were identified by immunocytochemistry and immunofluorescence methods.Cell purity and proliferation were also investigated respectively by flow cytometry and Cell Counting Kit(CCK)method.Ultrastructures of epididymial epithelial cells were observed under transmission electron microscope.The results showed that the cells obtained by both methods were in island-like clonal growth and good proliferation ability,and possessed active secretory and metabolic function.The expression of epithelial special cytokeratin 18(CK18)and epididymial special GPx5 and the result of flow cytometry showed that both methods can obtain high purity epididymal epithelial cells.The primary cells obtained by improved tissue-piece digestion method took a long time culture,compared to that the primary cells obtained by enzyme digestion method took a short time culture with more cells.These results provided a good foundation for further study on the function of epididymis of cashmere goat.3.The effects of testosterone and hydrocortisone on the cell proliferation were detected by CCK method.The androgen receptor(AR)was detected by immunocytochemistry in epididymal epithelial cells cultured in vitro,and the expression level of AR mRNA and protein was detected by fluorescence quantitative PCR and ELISA.The results showed that,compared with the control group,low concentration testosterone(100 nmol · L-1)could significantly promote the proliferation of epididymal epithelial cells,high concentrations of testosterone(1000 nmol.L-1)had no effect on cell proliferation,which indicated that testosterone could promote the proliferation of epididymal epithelial cells in a dose-dependent manner.200 nmol.L-1 hydrocortisone also could promote the proliferation of epididymal epithelial cells.The synergistic effect of hydrocortisone and testosterone was observed on the proliferation of cells.100 nmol.L-1 testosterone and 200 nmol.L-1 hydrocortisone could both up-regulated the mRNA and protein expression of AR in epididymal epithelial cells,but the mRNA and protein expression of AR were significantly increased when they co-existed.In addition,the immunocytochemistry result showed that AR protein was detected in epididymal epithelial cells,and the expression pattern was similar to that in tissues.4.The effects of testosterone on reactive oxygen species(ROS)in epididymal epithelial cells were detected by flow cytometry,and the effects of testosterone on the expression of GPx5 and AR mRNA and protein in epididymal epithelial cells were detected by fluorescence quantitative PCR and ELISA respectively.The results showed that low concentrations testosterone could decrease the level of intracellular ROS and high concentrations(1000 nmol L-1)had no effect.100 nmol L-1 significantly increased the expression of GPx5,but this effect could be blocked by AR receptor blockers.High concentration of testosterone had no significant difference in the expression of GPx5 compared with the control group.100 nmol L-1 testosterone significantly increased the expression of AR,and a decreased expression of AR in high concentration of testosterone compared with the control group.The results showed that low concentration of testosterone could increase the expression of AR and GPx5 in epididymal epithelial cells,which could reduce the level of ROS and protect the epididymal epithelial cells.