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At puberty, neurokinin B (NKB) and kisspeptin (Kiss1) may help to amplify GnRH secretion, but their precise roles remain ambiguous. We tested the hypothesis that NKB and Kiss1 are induced as a function of pubertal development, independently of the prevailing sex steroid milieu. We found that levels of Kiss1 mRNA in the arcuate nucleus (ARC) are increased prior to the age of puberty in GnRH/sex steroid-deficient hpg mice, yet levels of Kiss1 mRNA in wild-type mice remained constant, suggesting that sex steroids exert a negative feedback effect on Kiss1 expression early in development and across puberty. In contrast, levels of Tac2 mRNA, encoding NKB, and its receptor (NK3R; encoded by Tacr3) increased as a function of puberty in both wild-type and hpg mice, suggesting that during development Tac2 is less sensitive to sex steroid-dependent negative feedback than Kiss1. To compare the relative responsiveness of Tac2 and Kiss1 to the negative feedback effects of gonadal steroids, we examined the effect of estradiol (E(2)) on Tac2 and Kiss1 mRNA and found that Kiss1 gene expression was more sensitive than Tac2 to E(2)-induced inhibition at both juvenile and adult ages. This differential estrogen sensitivity was tested in vivo by the administration of E(2). Low levels of E(2) significantly suppressed Kiss1 expression in the ARC, whereas Tac2 suppression required higher E(2) levels, supporting differential sensitivity to E(2). Finally, to determine whether inhibition of NKB/NK3R signaling would block the onset of puberty, we administered an NK3R antagonist to prepubertal (before postnatal d 30) females and found no effect on markers of pubertal onset in either WT or hpg mice. These results indicate that the expression of Tac2 and Tacr3 in the ARC are markers of pubertal activation but that increased NKB/NK3R signaling alone is insufficient to trigger the onset of puberty in the mouse.

Prokineticin receptors (PROKR) are G protein-coupled receptors (GPCR) that regulate diverse biological processes, including olfactory bulb neurogenesis and GnRH neuronal migration. Mutations in PROKR2 have been described in patients with varying degrees of GnRH deficiency and are located in diverse functional domains of the receptor. Our goal was to determine whether variants in the first intracellular loop (ICL1) of PROKR2 (R80C, R85C, and R85H) identified in patients with hypogonadotropic hypogonadism interfere with receptor function and to elucidate the mechanisms of these effects. Because of structural homology among GPCR, clarification of the role of ICL1 in PROKR2 activity may contribute to a better understanding of this domain across other GPCR. The effects of the ICL1 PROKR2 mutations on activation of signal transduction pathways, ligand binding, and receptor expression were evaluated. Our results indicated that the R85C and R85H PROKR2 mutations interfere only modestly with receptor function, whereas the R80C PROKR2 mutation leads to a marked reduction in receptor activity. Cotransfection of wild-type (WT) and R80C PROKR2 showed that the R80C mutant could exert a dominant negative effect on WT PROKR2 in vitro by interfering with WT receptor expression. In summary, we have shown the importance of Arg80 in ICL1 for PROKR2 expression and demonstrate that R80C PROKR2 exerts a dominant negative effect on WT PROKR2.

Context: Mutations in TAC3 and TACR3 (encoding neurokinin B and its receptor) have been identified in Turkish patients with idiopathic hypogonadotropic hypogonadism (IHH), but broader populations have not yet been tested and genotype-phenotype correlations have not been established., Objective: A broad cohort of normosmic IHH probands was screened for mutations in TAC3/TACR3 to evaluate the prevalence of such mutations and define the genotype/phenotype relationships., Design and Setting: The study consisted of sequencing of TAC3/TACR3, in vitro functional assays, and neuroendocrine phenotyping conducted in tertiary care centers worldwide., Patients or Other Participants: 345 probands, 18 family members, and 292 controls were studied., Intervention: Reproductive phenotypes throughout reproductive life and before and after therapy were examined., Main Outcome Measure: Rare sequence variants in TAC3/TACR3 were detected., Results: In TACR3, 19 probands harbored 13 distinct coding sequence rare nucleotide variants [three nonsense mutations, six nonsynonymous, four synonymous (one predicted to affect splicing)]. In TAC3, one homozygous single base pair deletion was identified, resulting in complete loss of the neurokinin B decapeptide. Phenotypic information was available on 16 males and seven females with coding sequence variants in TACR3/TAC3. Of the 16 males, 15 had microphallus; none of the females had spontaneous thelarche. Seven of the 16 males and five of the seven females were assessed after discontinuation of therapy; six of the seven males and four of the five females demonstrated evidence for reversibility of their hypogonadotropism., Conclusions: Mutations in the neurokinin B pathway are relatively common as causes of hypogonadism. Although the neurokinin B pathway appears essential during early sexual development, its importance in sustaining the integrity of the hypothalamic-pituitary-gonadal axis appears attenuated over time.

Context: Kisspeptin, encoded by the KISS1 gene, is a key stimulatory factor of GnRH secretion and puberty onset. Inactivating mutations of its receptor (KISS1R) cause isolated hypogonadotropic hypogonadism (IHH). A unique KISS1R-activating mutation was described in central precocious puberty (CPP)., Objective: Our objective was to investigate KISS1 mutations in patients with idiopathic CPP and normosmic IHH., Patients: Eighty-three children with CPP (77 girls) and 61 patients with IHH (40 men) were studied. The control group consisted of 200 individuals with normal pubertal development., Methods: The promoter region and the three exons of KISS1 were amplified and sequenced. Cells expressing KISS1R were stimulated with synthetic human wild-type or mutant kisspeptin-54 (kp54), and inositol phosphate accumulation was measured. In a second set of experiments, kp54 was preincubated in human serum before stimulation of the cells., Results: Two novel KISS1 missense mutations, p.P74S and p.H90D, were identified in three unrelated children with idiopathic CPP. Both mutations were absent in 400 control alleles. The p.P74S mutation was identified in the heterozygous state in a boy who developed CPP at 1 yr of age. The p.H90D mutation was identified in the homozygous state in two unrelated girls with CPP. In vitro studies revealed that the capacity of the P74S and H90D mutants to stimulate IP production was similar to the wild type. After preincubation of wild-type and mutant kp54 in human serum, the capacity to stimulate signal transduction was significantly greater for P74S compared with the wild type, suggesting that the p.P74S variant is more stable. Only polymorphisms were found in the IHH group., Conclusion: Two KISS1 mutations were identified in unrelated patients with idiopathic CPP. The p.P74S variant was associated with higher kisspeptin resistance to degradation in comparison with the wild type, suggesting a role for this mutation in the precocious puberty phenotype.

Previously, we have reported that 17beta-estradiol (E(2)) induces an increase in firing activity of primate LH-releasing hormone (LHRH) neurons. The present study investigates whether E(2) alters LHRH release as well as the pattern of intracellular calcium ([Ca(2+)](i)) oscillations and whether G protein-coupled receptor 30 (GPR30) plays a role in mediating the rapid E(2) action in primate LHRH neurons. Results are summarized: 1) E(2), the nuclear membrane-impermeable estrogen, estrogen-dendrimer conjugate, and the plasma membrane-impermeable estrogen, E(2)-BSA conjugate, all stimulated LHRH release within 10 min of exposure; 2) whereas the estrogen receptor antagonist, ICI 182,780, did not block the E(2)-induced LHRH release, E(2) application to cells treated with pertussis toxin failed to induce LHRH release; 3) GPR30 mRNA was expressed in olfactory placode cultures, and GPR30 protein was expressed in a subset of LHRH neurons; 4) pertussis toxin treatment blocked the E(2)-induced increase in [Ca(2+)](i) oscillations; 5) knockdown of GPR30 in primate LHRH neurons by transfection with small interfering RNA (siRNA) for GPR30 completely abrogated the E(2)-induced changes in [Ca(2+)](i) oscillations, whereas transfection with control siRNA did not; 6) the estrogen-dendrimer conjugate-induced increase in [Ca(2+)](i) oscillations also did not occur in LHRH neurons transfected with GPR30 siRNA; and 7) G1, a GPR30 agonist, resulted in changes in [Ca(2+)](i) oscillations, similar to those observed with E(2). Collectively, E(2) induces a rapid excitatory effect on primate LHRH neurons, and this rapid action of E(2) appears to be mediated, in part, through GPR30.