The effects of melatonin and gonadal steroids on gonadotropin-releasing hormone (GnRH) regulation in hypothalamic GT1-7 neuronal cells

Gonadotropin-releasing hormone (GnRH) is a central factor of the hypothalamic-pituitary-gonadal (HPG) axis. Using the GT1-7 cell line, an 'in vitro' model of GnRH-secreting neurons of the hypothalamus, the direct regulation of GnRH by the hormones melatonin, estrogen and androgen was investigated. The pineal hormone melatonin is involved in the regulation of circadian rhythms and reproduction. GT1-7 cells were found to express membrane-bound mt1 and MT2 melatonin receptors, as well as putative nuclear melatonin receptors RORÃ and RZRß. Interestingly, melatonin downregulates GnRH gene expression in a 24 h cyclical pattern. This effect appears to occur at the transcriptional level and can be mapped to five specific regions within the GnRH neuron-specific enhancer including -1827/-1819, -1780/-1772, -1746/-1738, -1736/-1728 and -1697/-1689. The region located at -1736/-1728 appears to include consensus binding sites for ROR/RZR orphan nuclear receptors. However, this region does not appear to bind the RORÃ receptor. The signal transduction mechanisms involved in melatonin-mediated regulation of GnRH were found to include the PKA, PKC and ERK 1/2 MAPK pathways. Melatonin inhibited forskolin-stimulated cAMP accumulation in GT1-7 cells through a PTX-sensitive mechanism, induced an increase in PKC activity to approximately 1.65-fold over basal levels, increased the phosphorylation of ERK 1 and ERK 2 proteins, and increased both 'c-fos' and 'junB' mRNA expression. Using the PKA inhibitor H-89, PKC inhibitor bisindolylmaleimide, and ERK 1/2 MAPK inhibitor PD98059, melatonin-mediated downregulation of GnRH mRNA expression appears to involve the PKC and MAPK pathways, but not the PKA pathway. Melatonin suppressed GnRH secretion by 45% through signal transduction mechanisms involving the PKA, PKC and MAPK pathways. The gonadal steroids, estrogen and androgen, are known to provide important hormonal signals and sustain the intricate balance within the feedback loop of the HPG axis. The GT1-7 cells were found to express the androgen receptor and both estrogen receptor (ER)-Ã and ERß mRNAs and proteins. 5Ã-dihydrotestosterone (DHT) and 17ß-estradiol were both found to downregulate GnRH mRNA levels to approximately 55% of basal levels. Regulation by estradiol requires ERÃ as the methoxychlor metabolite HPTE, which is an ERÃ-specific agonist/ERß-specific antagonist also downregulated GnRH gene expression. Repression of GnRH mRNA levels by estradiol appears to occur at the transcriptional level. Saturation binding analysis confirmed the presence of a high affinity low capacity androgen binding entity (Kd = 0.06 nm, Bmax = 12.4 fmol/mg protein) in the GT1-7 cells. Repression of GnRH mRNA by DHT is due to the action of the classic AR as the effect is blocked by hydroxyflutamide, a specific AR antagonist. Furthermore, the ligand-activated AR expressed in the GT1-7 cells is capable of regulating reporter gene activity. These studies provide the first evidence that melatonin, estrogen, and androgen may mediate their control of reproductive physiology, in part, though direct regulation of the GnRH gene in this important population of neurons within the hypothalamus.