Selenium triggers AMPK-mTOR pathway to modulate autophagy related to oxidative stress of sheep Leydig cells

Abstract

The objective of this study was to investigate the effect of oxidative stress induced by excessive Se on autophagy of sheep Leydig cells and its underlying mechanism. Leydig cells isolated from the testis of 8-month-old sheep were purified using a discontinuous Percoll density gradient. Cells were divided into four treatment groups (0, 2.0, 4.0 and 8.0 μmol/L of Se). After treatment with Se for 48 h, cell proliferation was detected by CCK-8 assay kit. The biochemical methods were used to evaluate the antioxidant status of Leydig cells. The mRNA transcript and protein abundance related to the AMPK-mTOR pathway and autophagy were detected by real-time PCR and western blot analysis.

The results showed that the Leydig cells treated with 8.0 μmol/L Se have the lowest cell viability. The greater ROS content and lower GSH-Px activity were also observed in the Se_8.0 group. The inclusion of 2.0 μmol/L Se in the medium did not affect the autophagy of Leydig cells. However, the relative abundance of ATG5 protein and LC3II/I ratio were elevated in the Se_8.0 group. Oxidative stress induced by excessive Se (8.0 μmol/L) dramatically improved the abundance of key proteins related to AMPK-mTOR pathway and led to an increase of phosphorylated AMPK, mTOR and ULK1. Compared with the Se_8.0 group, compound C could significantly inhibit the key molecules of AMPK-mTOR signaling pathway and mitigate the autophagy of Leydig cells induced by excessive Se. These results indicate that appropriate Se (2.0 μmol/L) can enhance the viability of sheep Leydig cells. Oxidative stress caused by Se excess can induce cell autophagy via activating AMPK-mTOR signaling pathway. The existed crosstalk between autophagy and apoptosis could decide the fate of Leydig cells. This process could play a decisive role in the maintenance of normal male fertility and spermatogenesis by affecting the number of Leydig cells in testis.