Abstract

This study investigated the receptor mechanism(s) by which the hormone melatonin directly affects ovarian function. Expression of MT₁ and MT₂ melatonin receptor mRNA was detected in the rat ovaries both by reverse transcriptase-polymerase chain reaction and in situ hybridization with digoxigenin-labeled oligoprobes. Specific 2-[¹²⁵I]iodomelatonin binding was significantly higher in ovarian tissue from animals sacrificed during proestrus than in metestrus, suggesting regulation of melatonin receptors by estrogens. Additionally, basal and melatonin-mediated stimulation of guanosine 5′-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) binding to ovarian sections was higher in proestrus compared with metestrus. During proestrus, both luzindole (0.1 μM) and 4-phenyl-2-propionamidotetraline (4P-PDOT) (0.1 μM), acting as inverse agonists, inhibited basal [³⁵S]GTPγS binding to ovarian sections, suggesting the presence of MT₁ constitutively active melatonin receptors. In primary cultures of ovarian granulosa cells, melatonin inhibited forskolin-stimulated cAMP accumulation through activation of G_i-coupled melatonin receptors. This inhibition was blocked by both, luzindole, and 4P-PDOT, acting as competitive receptor antagonists. Exposure of granulosa cells in culture to 17β-estradiol seems to alter the state of melatonin receptor coupling. Indeed, the efficacy of 4P-PDOT on forskolin-stimulated cAMP formation was reversed from an MT₂ partial agonist in vehicle-treated cells to that of an MT₁ inverse agonist in 17β-estradiol (0.1 μM)-treated granulosa cells. We conclude that MT₁ and MT₂ melatonin receptors expressed in antral follicles and corpus luteum may affect steroidogenesis through cAMP-mediated signaling. These results underscore the implications of the levels of ovarian estrogen when melatonin receptor ligands are used as therapeutic agents.