Mechanism Of Nickel-induced Mitochondrial Damage In Mouse Primary Leydig Cells

Objectives:To establish primary cells culture system of Leydig cells (LCs) from mice testis and explore the mechanism of mitochondrial damage induced by nickel sulfate (NiSO4) in vitro.Methods:(1) LCs were isolated by using collagenase of type four, and purified by the means of the density gradient centrifugation with Percoll separating medium and adherent culture in mice. The purity of LCs was identified by3beta-hydroxysteroid dehydrogenase (3β-HSD) staining assay.(2) The LCs were treated by NiSO4at doses of0,250,500and l000μmol/L for24hours when LCs reached80%～85%confluence, respectively. The level of reactive oxygen species (ROS) was measured by using DCFH-DA fluorescent marking assay and the content of adenosine-5’-triphosphate (ATP) was detected by the phosphomolybdic acid assay.(3) The Real-time Quantitative PCR (FQ-PCR) and Western blot techniques were used to measure the levels of mRNA and protein expressions in NADH dehydrogenase subunit1(ND1), Cytochrome b(Cyt-b) and Cytochrome oxidase II (COX II), which is the key genes of Electron Respiratory Chain in the mitochondrial of LCs.Results:(1) The purity of cultured LCs was above90%by using3β-HSD staining assay.(2) Compared with the control group after treatment with NiSO4at different doses for24hours, the level of ROS in LCs increased (P<0.05) and the content of ATP decreased (P<0.05) in500and1000μmol/L NiSO4groups.(3) The results of FQ-PCR and Western blot showed that the expression of ND1, Cyt-b and COX II genes at mRNA and protein level decreased significantly in500and1000μmol/L NiSO4groups compared with the control group (P<0.05).Conclusions:The method of separation, purification and culture of mouse LCs set up successfully in vitro. Summary, the present study suggests that the NiSO4-induced decrease of ATP level is related to the increase of ROS levels and down regulation of the mRNA and protein expressions of ND1, Cyt-b and COX II in mouse primary LCs.