Role Of ROS In Nickel-induced Testosterone Synthesis Disturbance In Rat Leydig Cells

Objectives The aim of the present study was to investigate the level of nickel-induced reactive oxygen species?ROS?generation and testicular steroidogenic enzymes m RNA and protein expression in rat Leydig cells,and explore the role of ROS in testosterone synthesis disturbance induced by nickel sulfate?Ni SO₄?in rat Leydig cells.Methods?1?Leydig cells were prepared from the testes that were decapsulated and digested by 0.5 % collagenase ?.These cells were then purified by Percoll density gradient centrifugation at discontinuous gradient densities of Percoll?5,30,58 and 70 %?.The purity of Leydig cells was assessed by 3?-hydroxysteroid dehydrogenase?3?-HSD?staining.?2?To determine the concentration-effect of Ni SO₄ on Leydig cells,cells were seeded in 96-well flat-bottom plates?1×104 cells/well?for 0,6,12 and 24 h,contained with 0,250,500 and 1000 ?mol/L Ni SO₄ with 1 IU/ml h CG.Level of testosterone?T?in cell culture medium was detected by enzyme linked immunosorbent assay?ELISA?.RT-q PCR were performed to quantify the relative m RNA expression of testicular steroidogenic enzymes including steroidogenic acute regulatory protein?St AR?,Cytochrome P450 11A1?CYP11A1?,CYP17A1,3?-HSD and 17?-HSD.Western blot were performed to detect the relative protein expression of St AR,CYP11A1,CYP17A1,3?-HSD and 17?-HSD compared with the consistent protein expression of ?-actin.?3?To study the effects of N-acetylcysteine?NAC?and 2,2,6,6-Tetramethyl-1-piperidinyloxy?TEMPO?,Leydig cells were incubated with 5 mmol/L NAC or 1 mmol/L TEMPO respectively for 1 h before treatment of 1000 ?mol/L Ni SO₄ for 24 h.The reactive oxygen species?ROS?level was detected with DCFH-DA fluorescence staining.T level in cell culture medium was detected by ELISA.RT-q PCR were performed to quantify the relative m RNA expression of St AR,CYP11A1,CYP17A1,3?-HSD and 17?-HSD.Western blot were performed to detect the relative protein expression of these 5 mentioned testicular steroidogenic enzymes.Results?1?3?-HSD staining showed that after attachment culture for 24 h,Leydig cells purity coefficient can be observed over 95 %.?2?Compared with 0 h group,the T level was significantly increased as the treatment time?12 and 24 h?increased?P<0.05?.Compared with control group?0 ?mol/L Ni SO₄?,the T level was significantly decreased after 500 and 1000 ?mol/L Ni SO₄ treatment for 12 and 24 h?P<0.05?.Based on the above results,the exposure duration was set as 24 h.In 1000 ?mol/L Ni SO₄ group,the m RNA and protein expression of St AR,CYP11A1,CYP17A1,3?-HSD and 17?-HSD were lower than control group?P<0.05?.Based on the above results,1000 ?mol/L Ni SO₄ was chosen to be the exposure concentration for further experiments.?3?DCFH-DA fluorescence staining showed significant increase of intracellular ROS levels in Ni SO₄ group?treated with 1000 ?mol/L Ni SO₄ for 24 h?compared with control group.ROS generation was significantly alleviated by NAC and TEMPO respectively compared with Ni SO₄ group?P<0.05?.Similar trend was presented by ELISA test,T level was significantly alleviated by NAC and TEMPO respectively compared with Ni SO₄ group?P<0.05?.RT-q PCR and western blot demonstrated the consistent downregulation in the m RNA and protein expression of St AR,CYP11A1,CYP17A1,3?-HSD and 17?-HSD?P<0.05?.In addition,these abnormal changes were blocked in the presence of NAC as well as TEMPO?P<0.05?.Conclusions NiSO₄ could induce ROS generation and consequently lead to testosterone synthesis disturbance by inhibiting the expression of testicular steroidogenic enzymes in rat Leydig cells.