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Melatonin is an indole derivative that secreted rhythmically from the pineal gland, which plays essential roles in controlling reproduction and fertility in mammals.Theca cells are a type of follicular cells, and play a critical role in folliculogenesis.However, the exact effects of melatonin on mammalian ovarian theca cells are still unknown.This study was designed to investigate the effects and potential mechanisms of melatonin on porcine theca cells.In the present study, the presence and localization of melatonin receptor 2 (MT2) were identified by reverse-transcriptase PCR (RT-PCR) and immunohistochemical staining.Cell viability was assessed by MTT assay, and different melatonin concentrations were chosen to test the effects on steroid hormone production, cell proliferation, apoptosis, gene expression, and reactive oxygen species (ROS) level.Our results revealed that melatonin could inhibit the production of androstenedione and testosterone and promote apoptosis of theca cells in a dose-dependent manner, but did not affect progesterone secretion and cell proliferation.Treating the melatonin-treated cells with the general MT1 and MT2 antagonist luzindole revealed that luzindole could not counteract the melatonin-induced androgen production and theca cell apoptosis.Furthermore, melatonin was found to significantly downregulate the expression of genes encoding the steroidogenic enzymes steroidogenic acute regulatory (Star), Cyp11a1,and Hsd3β.Blocking MT2 by luzindole could neutralize the downregulated expression levels of Hsd3β but not Cyp11a1 and Star.In conclusion, exogenous melatonin was found to modulate the physiological functions of porcine theca cells by scavenging free radicals and/or binding receptor.