Sex Steroids and Leptin Regulate the “First Kiss” (KiSS 1/G-Protein-Coupled Receptor 54 System) in Human Gonadotropin-Releasing-Hormone-Secreting Neuroblasts

Abstract

The G-protein-coupled receptor 54 (GPR54) and its ligand kisspeptin, encoded by the KiSS-1 gene, have been involved in the molecular mechanisms underlying the reawakening of gonadotropin-releasing hormone (GnRH) neurons at puberty. GPR54 mutations cause hypogonadotropic hypogonadism in human and mice. Aim. Our aim was to study regulation of the KiSS-1/GPR54 system using a previously characterized primary culture of human fetal GnRH-secreting neuroblasts, FNC-B4. KiSS-1/GPR54 gene and protein expressions in FNC-B4 were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), immunocytochemistry, and Western blot. Expression of kisspeptin and GPR54 in fetal olfactory mucosa (OM), from which FNC-B4 cells were derived, was analyzed with confocal microscopy. Regulation of KiSS-1/GPR54 expression in FNC-B4 was evaluated in response to sexual steroids and leptin. Effect of kisspeptin on GnRH secretion and migration in FNC-B4 was also investigated. Kisspeptin and GPR54 were immunolocalized and co-expressed with GnRH in OM and FNC-B4 cells. Kisspeptin (1 microM, 24 hours) induced GnRH secretion, but not gene expression, and inhibited migration (IC(50) = 6.28 +/- 3.71 nM) in FNC-B4. The 24-hour exposure to increasing concentrations of 17-beta-estradiol (0.01-1 nM) significantly and dose-dependently decreased, whereas androgens (dihydrotestosterone [DHT], 0.01-1 nM) significantly stimulated KiSS-1/GPR54 mRNA. Testosterone (1 nM) showed a stimulatory effect only after blocking its aromatization with letrozole. In addition, leptin (1 nM, 24 hours), an adipocyte-derived hormone acting on the reproductive axis, significantly increased KiSS-1/GPR54 expression in FNC-B4. Immunocytochemistry and Western blot analysis confirmed the regulatory effects found with qRT-PCR. Interestingly, leptin (1 nM, 24 hours) also significantly increased both leptin receptor (LEPR) and androgen receptor (AR) mRNA. DHT (0.01-1 nM) also up-regulated LEPR and AR genes, suggesting a synergistic action between leptin and androgens aimed to up-regulate the KiSS-1/GPR54 system, which, in contrast, was inhibited by estrogens. Our results indicate that an interplay between metabolic and sexual hormones may trigger the KiSS-1/GPR54 signaling to GnRH neurons suggesting new mechanisms which regulate puberty onset.