Conclusion
There was no significant effect on steroidogenesis-related genes, proteins, and testosterone synthesis in the absence of gonadotropin treatment plus melatonin following CoCl2-induced hypoxia in TM3 Leydig cells.
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 CoCl2 group, 0.2 mM CoCl2 was added to the medium to induce Hif1α overexpression. The MLT+CoCl2 group received 0.2 mM CoCl2 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 CoCl2 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
CoCl2 treatment caused Hif1α overexpression in TM3 Leydig cells. Moreover, CoCl2 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 CoCl2, 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.