Effect of melatonin on steroidogenesis-related enzymes expression and testosterone synthesis following CoCl(2)-induced hypoxia in TM3 Leydig cells

OBJECTIVE(S): This study examined the effects of melatonin treatment on steroidogenesis dysfunction and testosterone impairment, following CoCl(2)-induced hypoxia in TM3 Leydig cells. MATERIALS AND METHODS: The TM3 cells were divided into four groups. The first group received no treatment. The MLT group was treated with a concentration of 1 mM melatonin. In the CoCl(2) group, 0.2 mM CoCl(2) was added to the medium to induce Hif1α overexpression. The MLT+CoCl(2) group received 0.2 mM CoCl(2) and 1 mM melatonin. After 24 hr treatment, the cells and supernatants were collected and used for further determination. The MTT assay was performed to estimate the decrease in cell viability throughout the CoCl(2) and melatonin treatment. The mRNA and the protein levels were evaluated using Real-time PCR and Western blot analysis. The ELISA assay kit was used to detect the testosterone content. RESULTS: CoCl(2) treatment caused Hif1α overexpression in TM3 Leydig cells. Moreover, CoCl(2) treatment of these cells led to considerable downregulation of Star, Hsd3b1, and Gata4 well as Mtnr1a and Mtnr1b mRNA/protein expression coupled with testosterone content repression in the cell culture medium. Melatonin administration in cells treated with CoCl(2), decreased Hif1α mRNA/protein expression, but had no significant effect on Star, Hsd3b1, Gata4, Mtnr1a mRNA/protein expression, and the testosterone level in the cell culture medium. Melatonin caused recovery of decrease in the Mtnr1b gene and protein expression. CONCLUSION: There was no significant effect on steroidogenesis-related genes, proteins, and testosterone synthesis in the absence of gonadotropin treatment plus melatonin following CoCl(2)-induced hypoxia in TM3 Leydig cells.