Direct action of melatonin in human granulosa-luteal cells

The direct involvement of melatonin in modulation of ovarian steroidogenesi s, the high levels of melatonin found in human follicular fluid, and the pr esence of melatonin binding sites in the ovary led us to hypothesize that m elatonin acts as a modulator of ovarian function. In contrast to the hypoth alamus and pituitary, the mechanism of melatonin action at the level of the ovary is still poorly understood. In the present study, we investigated th e gene expression of the two different forms of melatonin receptors in huma n granulosa-luteal cells, using RT-PCR. PCR products corresponding to the e xpected sizes of the melatonin receptor subtypes, mt(1)-R and MT2-R, were o btained from granulosa-luteal cells, and the authenticity of the PCR produc ts was confirmed by Southern blot hybridization with cDNA probes. Subsequen t cloning and sequence analysis revealed that the ovarian mt(1)-R and MT2-R cDNAs are identical to their brain counterparts. Because gonadotropins and GnRH acting through specific receptors in the human ovary regulate cellula r functions, we investigated the role of melatonin in the regulation of FSH receptor, LH receptor, GnRH, and GnRH receptor levels. Treatment with mela tonin (10 pM-100 nm) significantly increased LH receptor mRNA levels withou t altering the expression of the FSH receptor gene. Both GnRH and GnRH rece ptor mRNA levels were significantly decreased, to 61% and 45% of control le vels, respectively, after melatonin treatment. Melatonin treatment alone ha d no effect on basal progesterone production but enhanced the effects of hu man CG-stimulated progesterone production. Because MAPKs are activated in r esponse to a diverse array of extracellular stimuli leading to the regulati on of cell growth, division, and differentiation, and because melatonin has been shown to modulate cellular proliferation and differentiation, in this study, we demonstrated that melatonin activated MAPK in a dose- and time-d ependent manner. In summary, our studies demonstrate, for the first time, t hat melatonin can regulate progesterone production, LH receptor, GnRH, and GnRH receptor gene expression through melatonin receptors in human granulos a-luteal cells, which may be mediated via the MAPK pathway and activation o f Elk-1. Our results support the notion that melatonin plays a direct role in regulating ovarian function.