Sex steroids and leptin regulate the "first Kiss" (KiSS 1/G-protein-coupled receptor 54 system) in human gonadotropin-releasing-hormone-secreting neuroblasts.
BACKGROUND
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.


METHODS
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Morelli
Annamaria
A
Andrology Unit, Department of Clinical Physiopathology, University of Florence, Florence, Italy.
Marini
Mirca
M
Department of Anatomy, Histology and Forensic Medicine, University of Florence, Florence, Italy.
Mancina
Rosa
R
Andrology Unit, Department of Clinical Physiopathology, University of Florence, Florence, Italy.
Luconi
Michaela
M
Endocrinology Unit, Department of Clinical Physiopathology, University of Florence, Florence, Italy.
Vignozzi
Linda
L
Andrology Unit, Department of Clinical Physiopathology, University of Florence, Florence, Italy.
Fibbi
Benedetta
B
Andrology Unit, Department of Clinical Physiopathology, University of Florence, Florence, Italy.
Filippi
Sandra
S
Interdepartmental Lab of Functional and Cellular Pharmacology of Reproduction, Departments of Pharmacology and Clinical Physiopathology, University of Florence, Florence, Italy.
Pezzatini
Anna
A
Interdepartmental Lab of Functional and Cellular Pharmacology of Reproduction, Departments of Pharmacology and Clinical Physiopathology, University of Florence, Florence, Italy.
Forti
Gianni
G
Interdepartmental Lab of Functional and Cellular Pharmacology of Reproduction, Departments of Pharmacology and Clinical Physiopathology, University of Florence, Florence, Italy.
Vannelli
Gabriella B
GB
Department of Anatomy, Histology and Forensic Medicine, University of Florence, Florence, Italy.
Maggi
Mario
M
Andrology Unit, Department of Clinical Physiopathology, University of Florence, Florence, Italy;. Electronic address: m.maggi@dfc.unifi.it.
D000728:Androgens / Q000494:pharmacology; D002478:Cells, Cultured; D013196:Dihydrotestosterone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000494:pharmacology; D004967:Estrogens / Q000494:pharmacology; D015870:Gene Expression; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism; D006801:Humans; D059648:Kisspeptins; D020738:Leptin / Q000494:pharmacology*; D009297:Nasal Mucosa / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011944:Receptors, Androgen / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000187:drug effects / Q000378:metabolism*; D000074502:Receptors, Kisspeptin-1; D054411:Receptors, Leptin / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013739:Testosterone / Q000494:pharmacology; D025521:Tumor Suppressor Proteins / Q000187:drug effects / Q000378:metabolism*; D015854:Up-Regulation
D000728:Androgens; D004967:Estrogens; C106628:KISS1 protein, human; C118688:KISS1R protein, human; D059648:Kisspeptins; D020738:Leptin; D012333:RNA, Messenger; D011944:Receptors, Androgen; D043562:Receptors, G-Protein-Coupled; D000074502:Receptors, Kisspeptin-1; D054411:Receptors, Leptin; D025521:Tumor Suppressor Proteins; D013196:Dihydrotestosterone; D007987:Gonadotropin-Releasing Hormone; D013739:Testosterone; D004958:Estradiol