A novel mechanism for the melatonin inhibition of testosterone secretion by rat Leydig cells: reduction of GnRH-induced increase in cytosolic Ca2+

The site of inhibition, by melatonin, of GnRH-dependent testosterone secret ion was investigated in adult rat Leydig cells cultured in vitro. The vario us effects downstream of the binding of GnRH to its own receptor were isola ted and mimicked by specific drugs. Testosterone secretion was then evaluat ed after 3h stimulation with GnRH, thapsigargin phorbol-12-myristate-13-ace tate (100nM), arachidonic acid (20 mu M), and ionomycin (1 mu M) in the pre sence or absence of melatonin (215nM). The effect of melatonin on the GnRH- induced changes in cytoplasmic calcium concentration ([Ca2+](i)) was also s tudied, using Fura-2 as fluorescent Ca2+ indicator. Melatonin attenuated th e increase in [Ca2+](i) and inhibited the testosterone secretion induced by GnRH, but not that induced by ionomycin. Both ionomycin and thapsigargin p otentiated GnRH-induced testosterone secretion; however, ionomycin, but not thapsigargin, partially prevented the inhibitory effect of melatonin on ce lls stimulated with GnRH. The effect of melatonin was probably dependent on the binding of melatonin to its Gi-protein-coupled receptor, as the inhibi tory effect on GnRH-induced secretion was supressed in cells pretreated wit h pertussis toxin in a concentration of 180 ng/ml for 20h. Assay of 17-hydr oxy-progesterone showed that, irrespective of the treatment, cells cultured with melatonin secreted greater amounts than controls. We conclude that me latonin reduces GnRH-induced testosterone secretion by 1) decreasing [Ca2+] (i), through impairment of the GnRH-dependent release of Ca2+ from intracel lular stores and 2) blocking 17-20 desmolase enzymatic activity, an effect that occurs irrespective of changes in [Ca2+](i).