Effects of selenium on the proliferation, apoptosis and testosterone production of sheep Leydig cells in vitro

Theriogenology. 2017 Apr 15:93:24-32. doi: 10.1016/j.theriogenology.2017.01.022. Epub 2017 Jan 16.

Abstract

The objective of this study was to investigate the effects of selenium (Se) on in vitro proliferation, apoptosis and testosterone production of sheep Leydig cells and its underlying mechanism. Leydig cells were collected from 8-month-old sheep and divided into four treatment groups (0, 2.0, 4.0 and 8.0 μmol/L Se). After treatment with Se for 48 h, the MTT and flow cytometric assay were used to detect cell proliferation and apoptosis. Testosterone level in the culture medium was determined by ELISA. The mRNA expression and protein abundance of cell cycle, apoptosis and testosterone synthesis-related genes were detected using real-time PCR and western blot analysis. The results showed that the highest percentage of live and apoptotic cells was obtained in the 2.0 and 8.0 μmol/L group, respectively. In the Se treatment groups, the proliferation rate of Leydig cells and the expression of cell cycle-related genes were decreased with the increasing Se supplementation in the culture medium. The percentage of apoptotic cells was increased with the increasing Se level, which was consistent with the expression of pro-apoptosis genes. The highest GSH-Px activity and lowest ROS content were also observed in the 2.0 μmol/L group. Appropriate Se level (2.0 μmol/L) can significantly increase the expression of p-ERK1/2, StAR and 3β-HSD, and improve the testosterone synthesis. Compared with the control group, PD0325901 could significantly inhibit the production of testosterone and the protein abundance of p-ERK1/2, StAR and 3β-HSD. Se treatment can mitigate the inhibition effect of PD0325901 and the testosterone secretion between the 2.0 μmol/L and control group was not significantly different. These results demonstrate that Se can affect the proliferation and apoptosis of Leydig cells by regulating cellular oxidative stress and the expressions of cell cycle and apoptosis-related genes. Se can also enhance the testosterone production of Leydig cells by activating the ERK signaling pathway and the expression of its downstream genes (StAR and 3β-HSD), which could be closely related to the regulating roles of Se in male fertility and spermatogenesis.

Keywords: Apoptosis; Leydig cells; Proliferation; Selenium; Testosterone.

MeSH terms

  • 3-Hydroxysteroid Dehydrogenases / analysis
  • 3-Hydroxysteroid Dehydrogenases / genetics
  • Animals
  • Apoptosis / drug effects*
  • Apoptosis / genetics
  • CDC2 Protein Kinase / analysis
  • CDC2 Protein Kinase / genetics
  • Caspases / analysis
  • Caspases / genetics
  • Cell Cycle
  • Cell Proliferation / drug effects*
  • Cells, Cultured
  • Culture Media, Conditioned / chemistry
  • Cyclin-Dependent Kinase Inhibitor Proteins / analysis
  • Cyclin-Dependent Kinase Inhibitor Proteins / genetics
  • Dose-Response Relationship, Drug
  • Leydig Cells / drug effects*
  • Leydig Cells / physiology*
  • MAP Kinase Signaling System / drug effects
  • Male
  • Phosphoproteins / analysis
  • Phosphoproteins / genetics
  • RNA, Messenger / analysis
  • Selenium / pharmacology*
  • Sheep
  • Testosterone / biosynthesis*
  • Testosterone / genetics

Substances

  • Culture Media, Conditioned
  • Cyclin-Dependent Kinase Inhibitor Proteins
  • Phosphoproteins
  • RNA, Messenger
  • steroidogenic acute regulatory protein
  • Testosterone
  • 3-Hydroxysteroid Dehydrogenases
  • CDC2 Protein Kinase
  • Caspases
  • Selenium