Effects Of Selenium On The In Vitro Proliferation And Testosterone Production Of Leydig Cells In Sheep

Selenium is an essential trace element, which plays an important role in cell proliferation, apoptosis and cell function. Recently, researches have suggested that selenium can mediate the male fertility and the proliferation of germ cells. However, few studies have reported the effect of selenium on leydig cell. Therefore, the present study was conducted to determine the influence of selenium on in vitro proliferation, apoptosis and testosterone production of leydig cells in sheep. In order to provide some experimental basis for clarifying the regulation role of selenium on spermatogenesis of male animals, the molecular mechanism of selenium mediate testosterone biosynthesis has also been determined.The leydig cells were separated and divided into four experimental groups which treated with different concentration of selenium (0,2,4 or 8?mol/L). After cultured 48 h, leydig cells of each group were collected to detect the proliferation and apoptosis using MTT and Flow cytometric assay. The SOD and GSH-Px activities and ROS content were tested by colorimetry. Testosterone production of leydig cells were measured by ELISA. The mRNA expression level of apoptosis, cell cycle and testosterone production related genes were also detected by Real-time PCR. The protein expression level of cell cycle related genes, testosterone production related genes and ERK1/2 were measured by western blot.The results showed that the proliferation of leydig cells and the expression of CDK1 was decreased (P<0.05) as the concentration of selenium rise. The percentage of apoptotic leydig cells and the mRNA expression of CASPASE 3, CASPASE 8, P21, P53 and BCL-2 and the protein expression of P21 and P53 were significantly (P<0.05) increased with the increase of selenium level. The proliferation of leydig cells in the control was significantly decreased (P<0.05) compared with the 2 ?mol/L and 4?mol/L groups. No significant difference of the percentage of apoptotic leydig cells was found between control and 2?mol/L groups. The highest (P<0.05) GSH-Px activities and the lowest (P<0.05) ROS content were observed in the 2 ?mol/L group. The 2 ?mol/L selenium treatment significantly increased (P<0.05) the testosterone production of sheep Leydig cells and the expression of testosterone biosynthesis related genes (StAR, 3?-HSD). The 2 ?mol/L selenium treatment could significantly increase (P<0.05) the level of p-ERK1/2 protein. After adding the inhibitor PD0325901 of p-ERK1/2, the content of testosterone and the protein expression of StAR,3?-HSD with inhibitor treatment group was significantly reduced (P<0.05) than uninhibited treatment group. There was no significantly different between the content of testosterone in 2 ?mol/L group and the control group after both were treated with the inhibitor.These results demonstrated that selenium can affect proliferation and apoptosis of leydig cells by regulating the antioxidant enzyme activities and the expressions of cell cycle and apoptosis related genes. Selenium treatment could regulate the expression of StAR and 3?-HSD by activing the ERK signaling pathway which resulting in the testosterone biosynthesis was enhance in sheep leydig cells.