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ENDOCRINE AND REPRODUCTIVE

Functional Melatonin Receptors in Rat Ovaries at Various Stages of the Estrous Cycle

Jose M. Soares, Jr.¹, Monica I. Masana, Çaatay Erahin², and Margarita L. Dubocovich

Departments of Molecular Pharmacology and Biological Chemistry (J.M.S., M.I.M., C.E., M.L.D.) and Psychiatry and Behavioral Science (M.L.D.), Northwestern Drug Discovery Program (C.E., M.L.D.), Northwestern University Feinberg School of Medicine, Chicago, Illinois

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]GTPS) 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]GTPS 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 17-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 17-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.

Address correspondence to: Dr. Margarita L. Dubocovich, Department of Molecular Pharmacology and Biological Chemistry (S215), Northwestern University Feinberg School of Medicine, 303 East Chicago Ave., Chicago, IL 60611-3008. E-mail: mdubo{at}northwestern.edu