MKRN3 inhibits puberty onset via interaction with IGF2BP1 and regulation of hypothalamic plasticity.

Makorin ring finger protein 3 (MKRN3) was identified as an inhibitor of puberty initiation with the report of loss-of-function mutations in association with central precocious puberty. Consistent with this inhibitory role, a prepubertal decrease in Mkrn3 expression was observed in the mouse hypothalamus. Here, we investigated the mechanisms of action of MKRN3 in the central regulation of puberty onset. We showed that MKRN3 deletion in hypothalamic neurons derived from human induced pluripotent stem cells was associated with significant changes in expression of genes controlling hypothalamic development and plasticity. Mkrn3 deletion in a mouse model led to early puberty onset in female mice. We found that Mkrn3 deletion increased the number of dendritic spines in the arcuate nucleus but did not alter the morphology of GnRH neurons during postnatal development. In addition, we identified neurokinin B (NKB) as an Mkrn3 target. Using proteomics, we identified insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) as another target of MKRN3. Interactome analysis revealed that IGF2BP1 interacted with MKRN3, along with several members of the polyadenylate-binding protein family. Our data show that one of the mechanisms by which MKRN3 inhibits pubertal initiation is through regulation of prepubertal hypothalamic development and plasticity, as well as through effects on NKB and IGF2BP1.

The Congenital and Acquired Mechanisms Implicated in the Etiology of Central Precocious Puberty.

The etiology of central precocious puberty (CPP) is multiple and heterogeneous, including congenital and acquired causes that can be associated with structural or functional brain alterations. All causes of CPP culminate in the premature pulsatile secretion of hypothalamic GnRH and, consequently, in the premature reactivation of hypothalamic-pituitary-gonadal axis. The activation of excitatory factors or suppression of inhibitory factors during childhood represent the 2 major mechanisms of CPP, revealing a delicate balance of these opposing neuronal pathways. Hypothalamic hamartoma (HH) is the most well-known congenital cause of CPP with central nervous system abnormalities. Several mechanisms by which hamartoma causes CPP have been proposed, including an anatomical connection to the anterior hypothalamus, autonomous neuroendocrine activity in GnRH neurons, trophic factors secreted by HH, and mechanical pressure applied to the hypothalamus. The importance of genetic and/or epigenetic factors in the underlying mechanisms of CPP has grown significantly in the last decade, as demonstrated by the evidence of genetic abnormalities in hypothalamic structural lesions (eg, hamartomas, gliomas), syndromic disorders associated with CPP (Temple, Prader-Willi, Silver-Russell, and Rett syndromes), and isolated CPP from monogenic defects (MKRN3 and DLK1 loss-of-function mutations). Genetic and epigenetic discoveries involving the etiology of CPP have had influence on the diagnosis and familial counseling providing bases for potential prevention of premature sexual development and new treatment targets in the future. Global preventive actions inducing healthy lifestyle habits and less exposure to endocrine-disrupting chemicals during the lifespan are desirable because they are potentially associated with CPP.

Hypothalamic Overexpression of Makorin Ring Finger Protein 3 Results in Delayed Puberty in Female Mice.

Makorin ring finger protein 3 (MKRN3) is an important neuroendocrine player in the control of pubertal timing and upstream inhibitor of gonadotropin-releasing hormone secretion. In mice, expression of Mkrn3 in the hypothalamic arcuate and anteroventral periventricular nucleus is high early in life and declines before the onset of puberty. Therefore, we aimed to explore if the persistence of hypothalamic Mkrn3 expression peripubertally would result in delayed puberty. Female mice that received neonatal bilateral intracerebroventricular injections of a recombinant adeno-associated virus expressing Mkrn3 had delayed vaginal opening and first estrus compared with animals injected with control virus. Subsequent estrous cycles and fertility were normal. Interestingly, male mice treated similarly did not exhibit delayed puberty onset. Kiss1, Tac2, and Pdyn mRNA levels were increased in the mediobasal hypothalamus in females at postnatal day 28, whereas kisspeptin and neurokinin B protein levels in the arcuate nucleus were decreased, following Mkrn3 overexpression, compared to controls. Cumulatively, these data suggest that Mkrn3 may directly or indirectly target neuropeptides of Kiss1 neurons to degradation pathways. This mouse model suggests that MKRN3 may be a potential contributor to delayed onset of puberty, in addition to its well-established roles in central precocious puberty and the timing of menarche.

Connecting nutritional deprivation and pubertal inhibition via GRK2-mediated repression of kisspeptin actions in GnRH neurons.

BACKGROUND: Perturbations in the timing of puberty, with potential adverse consequences in later health, are increasingly common. The underlying neurohormonal mechanisms are unfolded, but nutritional alterations are key contributors. Efforts to unveil the basis of normal puberty and its metabolic control have focused on mechanisms controlling expression of Kiss1, the gene encoding the puberty-activating neuropeptide, kisspeptin. However, other regulatory phenomena remain ill-defined. Here, we address the putative role of the G protein-coupled-receptor kinase-2, GRK2, in GnRH neurons, as modulator of pubertal timing via repression of the actions of kisspeptin, in normal maturation and conditions of nutritional deficiency. METHODS: Hypothalamic RNA and protein expression analyses were conducted in maturing female rats. Pharmacological studies involved central administration of GRK2 inhibitor, ßARK1-I, and assessment of gonadotropin responses to kisspeptin or phenotypic and hormonal markers of puberty, under normal nutrition or early subnutrition in female rats. In addition, a mouse line with selective ablation of GRK2 in GnRH neurons, aka G-GRKO, was generated, in which hormonal responses to kisspeptin and puberty onset were monitored, in normal conditions and after nutritional deprivation. RESULTS: Hypothalamic GRK2 expression increased along postnatal maturation in female rats, especially in the preoptic area, where most GnRH neurons reside, but decreased during the juvenile-to-pubertal transition. Blockade of GRK2 activity enhanced Ca+2 responses to kisspeptin in vitro, while central inhibition of GRK2 in vivo augmented gonadotropin responses to kisspeptin and advanced puberty onset. Postnatal undernutrition increased hypothalamic GRK2 expression and delayed puberty onset, the latter being partially reversed by central GRK2 inhibition. Conditional ablation of GRK2 in GnRH neurons enhanced gonadotropin responses to kisspeptin, accelerated puberty onset, and increased LH pulse frequency, while partially prevented the negative impact of subnutrition on pubertal timing and LH pulsatility in mice. CONCLUSIONS: Our data disclose a novel pathway whereby GRK2 negatively regulates kisspeptin actions in GnRH neurons, as major regulatory mechanism for tuning pubertal timing in nutritionally-compromised conditions.

Puberty, A Sensitive Window of Hypothalamic Development and Plasticity.

Puberty is a developmental period characterized by a broad range of physiologic changes necessary for the acquisition of adult sexual and reproductive maturity. These changes mirror complex modifications within the central nervous system, including within the hypothalamus. These modifications result in the maturation of a fully active hypothalamic-pituitary-gonadal (HPG) axis, the neuroendocrine cascade ensuring gonadal activation, sex steroid secretion, and gametogenesis. A complex and finely regulated neural network overseeing the HPG axis, particularly the pubertal reactivation of gonadotropin-releasing hormone (GnRH) secretion, has been progressively unveiled in the last 3 decades. This network includes kisspeptin, neurokinin B, GABAergic, and glutamatergic neurons as well as glial cells. In addition to substantial modifications in the expression of key targets, several changes in neuronal morphology, neural connections, and synapse organization occur to establish mature and coordinated neurohormonal secretion, leading to puberty initiation. The aim of this review is to outline the current knowledge of the major changes that neurons secreting GnRH and their neuronal and glial partners undergo before and after puberty. Emerging mediators upstream of GnRH, uncovered in recent years, are also addressed herein. In addition, the effects of sex steroids, particularly estradiol, on changes in hypothalamic neurodevelopment and plasticity are discussed.

Hypothalamic miR-30 regulates puberty onset via repression of the puberty-suppressing factor, Mkrn3.

Mkrn3, the maternally imprinted gene encoding the makorin RING-finger protein-3, has recently emerged as putative pubertal repressor, as evidenced by central precocity caused by MKRN3 mutations in humans; yet, the molecular underpinnings of this key regulatory action remain largely unexplored. We report herein that the microRNA, miR-30, with three binding sites in a highly conserved region of its 3' UTR, operates as repressor of Mkrn3 to control pubertal onset. Hypothalamic miR-30b expression increased, while Mkrn3 mRNA and protein content decreased, during rat postnatal maturation. Neonatal estrogen exposure, causing pubertal alterations, enhanced hypothalamic Mkrn3 and suppressed miR-30b expression in female rats. Functional in vitro analyses demonstrated a strong repressive action of miR-30b on Mkrn3 3' UTR. Moreover, central infusion during the juvenile period of target site blockers, tailored to prevent miR-30 binding to Mkrn3 3' UTR, reversed the prepubertal down-regulation of hypothalamic Mkrn3 protein and delayed female puberty. Collectively, our data unveil a novel hypothalamic miRNA pathway, involving miR-30, with a prominent role in the control of puberty via Mkrn3 repression. These findings expand our current understanding of the molecular basis of puberty and its disease states.

Pulsatile GnRH Therapy May Restore Hypothalamus-Pituitary-Testis Axis Function in Patients With Congenital Combined Pituitary Hormone Deficiency: A Prospective, Self-Controlled Trial.

Context: The effectiveness of pulsatile gonadotropin-releasing hormone (GnRH) therapy in patients with congenital combined pituitary hormone deficiency (CCPHD) has not been investigated because of the limited number of patients, as well as these patients' presumed pituitary hypoplasia, poor gonadotrophic cell reserve, and impaired gonadotrophic response to GnRH. Objective: To assess the pituitary response to pulsatile GnRH therapy in men with CCPHD. Design: Prospective, self-controlled, 3-month clinical trial. Settings: University endocrine clinic. Patients: Men with hypogonadotropic hypogonadism caused by CCPHD. Intervention: Pulsatile GnRH was administered subcutaneously for 3 months. Main outcome measures: Primary endpoints were total serum testosterone, testicular volume, and luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels. Secondary endpoints included occurrence of spermatogenesis. Results: A total of 40 men with CCPHD completed the study. Of these, 60% (24 of 40) showed a good response to pulsatile GnRH treatment (response group). At 3 months, their LH and FSH levels increased to within the normal range and their testosterone levels increased to 8.67 ± 4.83 nmol/L. Of the patients in the response group, 33.3% (8 of 24) of them achieved spermatogenesis. The remaining 40% (16 of 40) of patients had a poor response to pulsatile GnRH treatment. Magnetic resonance imaging (MRI) did not reveal any correlation between pituitary response and pituitary height and/or integrity of the pituitary stalk. Conclusions: This study suggests that gonadotrophs in patients with CCPHD can exist and be functional-even with MRI evidence of pituitary hypoplasia or dysplasia. Pulsatile GnRH therapy restored pituitary-testis axis function in 60% of patients with CCPHD. These results may directly guide the clinical therapeutic choice.

GnRH Neuron-Specific Ablation of Gαq/11 Results in Only Partial Inactivation of the Neuroendocrine-Reproductive Axis in Both Male and Female Mice: In Vivo Evidence for Kiss1r-Coupled Gαq/11-Independent GnRH Secretion.

The gonadotropin-releasing hormone (GnRH) is the master regulator of fertility and kisspeptin (KP) is a potent trigger of GnRH secretion from GnRH neurons. KP signals via KISS1R, a Gαq/11-coupled receptor, and mice bearing a global deletion of Kiss1r (Kiss1r(-/-)) or a GnRH neuron-specific deletion of Kiss1r (Kiss1r(d/d)) display hypogonadotropic hypogonadism and infertility. KISS1R also signals via ß-arrestin, and in mice lacking ß-arrestin-1 or -2, KP-triggered GnRH secretion is significantly diminished. Based on these findings, we hypothesized that ablation of Gαq/11 in GnRH neurons would diminish but not completely block KP-triggered GnRH secretion and that Gαq/11-independent GnRH secretion would be sufficient to maintain fertility. To test this, Gnaq (encodes Gαq) was selectively inactivated in the GnRH neurons of global Gna11 (encodes Gα11)-null mice by crossing Gnrh-Cre and Gnaq(fl/fl);Gna11(-/-) mice. Experimental Gnaq(fl/fl);Gna11(-/-);Gnrh-Cre (Gnaq(d/d)) and control Gnaq(fl/fl);Gna11(-/-) (Gnaq(fl/fl)) littermate mice were generated and subjected to reproductive profiling. This process revealed that testicular development and spermatogenesis, preputial separation, and anogenital distance in males and day of vaginal opening and of first estrus in females were significantly less affected in Gnaq(d/d) mice than in previously characterized Kiss1r(-/-) or Kiss1r(d/d) mice. Additionally, Gnaq(d/d) males were subfertile, and although Gnaq(d/d) females did not ovulate spontaneously, they responded efficiently to a single dose of gonadotropins. Finally, KP stimulation triggered a significant increase in gonadotropins and testosterone levels in Gnaq(d/d) mice. We therefore conclude that the milder reproductive phenotypes and maintained responsiveness to KP and gonadotropins reflect Gαq/11-independent GnRH secretion and activation of the neuroendocrine-reproductive axis in Gnaq(d/d) mice. SIGNIFICANCE STATEMENT: The gonadotropin-releasing hormone (GnRH) is the master regulator of fertility. Over the last decade, several studies have established that the KISS1 receptor, KISS1R, is a potent trigger of GnRH secretion and inactivation of KISS1R on the GnRH neuron results in infertility. While KISS1R is best understood as a Gαq/11-coupled receptor, we previously demonstrated that it could couple to and signal via non-Gαq/11-coupled pathways. The present study confirms these findings and, more importantly, while it establishes Gαq/11-coupled signaling as a major conduit of GnRH secretion, it also uncovers a significant role for non-Gαq/11-coupled signaling in potentiating reproductive development and function. This study further suggests that by augmenting signaling via these pathways, GnRH secretion can be enhanced to treat some forms of infertility.

A new pathway in the control of the initiation of puberty: the MKRN3 gene.

Pubertal timing is influenced by complex interactions among genetic, nutritional, environmental, and socioeconomic factors. The role of MKRN3, an imprinted gene located in the Prader-Willi syndrome critical region (chromosome 15q11-13), in pubertal initiation was first described in 2013 after the identification of deleterious MKRN3 mutations in five families with central precocious puberty (CPP) using whole-exome sequencing analysis. Since then, additional loss-of-function mutations of MKRN3 have been associated with the inherited premature sexual development phenotype in girls and boys from different ethnic groups. In all of these families, segregation analysis clearly demonstrated autosomal dominant inheritance with complete penetrance, but with exclusive paternal transmission, consistent with the monoallelic expression of MKRN3 (a maternally imprinted gene). Interestingly, the hypothalamic Mkrn3 mRNA expression pattern in mice correlated with a putative inhibitory input on puberty initiation. Indeed, the initiation of puberty depends on a decrease in factors that inhibit the release of GnRH combined with an increase in stimulatory factors. These recent human and animal findings suggest that MKRN3 plays an inhibitory role in the reproductive axis to represent a new pathway in pubertal regulation.

The integrated hypothalamic tachykinin-kisspeptin system as a central coordinator for reproduction.

Tachykinins are comprised of the family of related peptides, substance P (SP), neurokinin A (NKA), and neurokinin B (NKB). NKB has emerged as regulator of kisspeptin release in the arcuate nucleus (ARC), whereas the roles of SP and NKA in reproduction remain unknown. This work explores the roles of SP and NKA in the central regulation of GnRH release. First, central infusion of specific agonists for the receptors of SP (neurokinin receptor 1, NK1R), NKA (NK2R) and NKB (NK3R) each induced gonadotropin release in adult male and ovariectomized, estradiol-replaced female mice, which was absent in Kiss1r(-/-) mice, indicating a kisspeptin-dependent action. The NK2R agonist, however, decreased LH release in ovariectomized-sham replaced females, as documented for NK3R agonists but in contrast to the NK1R agonist, which further increased LH release. Second, Tac1 (encoding SP and NKA) expression in the ARC and ventromedial nucleus was inhibited by circulating estradiol but did not colocalize with Kiss1 mRNA. Third, about half of isolated ARC Kiss1 neurons expressed Tacr1 (NK1R) and 100% Tacr3 (NK3R); for anteroventral-periventricular Kiss1 neurons and GnRH neurons, approximately one-fourth expressed Tacr1 and one-tenth Tacr3; Tacr2 (NK2R) expression was absent in all cases. Overall, these results identify a potent regulation of gonadotropin release by the SP/NK1R and NKA/NK2R systems in the presence of kisspeptin-Kiss1r signaling, indicating that they may, along with NKB/NK3R, control GnRH release, at least in part through actions on Kiss1 neurons.

GnRH pulse frequency-dependent differential regulation of LH and FSH gene expression.

The pituitary gonadotropin hormones, FSH and LH, are essential for fertility. Containing an identical α-subunit (CGA), they are comprised of unique ß-subunits, FSHß and LHß, respectively. These two hormones are regulated by the hypothalamic decapeptide, GnRH, which is released in a pulsatile manner from GnRH neurons located in the hypothalamus. Varying frequencies of pulsatile GnRH stimulate distinct signaling pathways and transcriptional machinery after binding to the receptor, GnRHR, on the cell surface of anterior pituitary gonadotropes. This ligand-receptor binding and activation orchestrates the synthesis and release of FSH and LH, in synergy with other effectors of gonadotropin production, such as activin, inhibin and steroids. Current research efforts aim to discover the mechanisms responsible for the decoding of the GnRH pulse signal by the gonadotrope. Modulating the response to GnRH has the potential to lead to new therapies for patients with altered gonadotropin secretion, such as those with hypothalamic amenorrhea or polycystic ovarian syndrome.

Central precocious puberty caused by mutations in the imprinted gene MKRN3.

BACKGROUND: The onset of puberty is first detected as an increase in pulsatile secretion of gonadotropin-releasing hormone (GnRH). Early activation of the hypothalamic-pituitary-gonadal axis results in central precocious puberty. The timing of pubertal development is driven in part by genetic factors, but only a few, rare molecular defects associated with central precocious puberty have been identified. METHODS: We performed whole-exome sequencing in 40 members of 15 families with central precocious puberty. Candidate variants were confirmed with Sanger sequencing. We also performed quantitative real-time polymerase-chain-reaction assays to determine levels of messenger RNA (mRNA) in the hypothalami of mice at different ages. RESULTS: We identified four novel heterozygous mutations in MKRN3, the gene encoding makorin RING-finger protein 3, in 5 of the 15 families; both sexes were affected. The mutations included three frameshift mutations, predicted to encode truncated proteins, and one missense mutation, predicted to disrupt protein function. MKRN3 is a paternally expressed, imprinted gene located in the Prader-Willi syndrome critical region (chromosome 15q11-q13). All affected persons inherited the mutations from their fathers, a finding that indicates perfect segregation with the mode of inheritance expected for an imprinted gene. Levels of Mkrn3 mRNA were high in the arcuate nucleus of prepubertal mice, decreased immediately before puberty, and remained low after puberty. CONCLUSIONS: Deficiency of MKRN3 causes central precocious puberty in humans. (Funded by the National Institutes of Health and others.).

Metabolic influences on neuroendocrine regulation of reproduction.

PURPOSE OF REVIEW: Reproduction is a tightly regulated function in which many mechanisms contribute to ensure the survival of the species. Among those, due to the elevated energy requirements of reproduction, metabolic factors exert a pivotal role in the control of hypothalamic-pituitary-gonadal axis. Although this control may occur at multiple levels of the axis, the majority of interactions between metabolic and reproductive systems take place in the hypothalamus. In this article, we present an overview of the state-of-the-art knowledge regarding the metabolic regulation of reproduction at the central level. We aim to identify the neuroanatomical location where both functions interconnect by discussing the likelihood of each component of the neuronal hierarchical network controlling gonadotropin-releasing hormone (GnRH) release to be first-order responders to metabolic cues, especially the peripheral metabolic signals leptin, insulin, and ghrelin. RECENT FINDINGS: Latest evidence suggests that the primary action of leptin, insulin, and ghrelin to regulate reproduction is located upstream of the main central elicitors of gonadotropin release, Kiss1 and GnRH neurons, and neuroanatomically separated from their metabolic action. SUMMARY: The study of the neuronal interactions between the mechanisms governing metabolism and reproduction offers the platform to overcome or treat a number of prevailing metabolic and/or reproductive conditions.

Human GnRH deficiency: a unique disease model to unravel the ontogeny of GnRH neurons.

Evolutionary survival of a species is largely a function of its reproductive fitness. In mammals, a sparsely populated and widely dispersed network of hypothalamic neurons, the gonadotropin-releasing hormone (GnRH) neurons, serve as the pilot light of reproduction via coordinated secretion of GnRH. Since it first description, human GnRH deficiency has been recognized both clinically and genetically as a heterogeneous disease. A spectrum of different reproductive phenotypes comprised of congenital GnRH deficiency with anosmia (Kallmann syndrome), congenital GnRH deficiency with normal olfaction (normosmic idiopathic hypogonadotropic hypogonadism), and adult-onset hypogonadotropic hypogonadism has been described. In the last two decades, several genes and pathways which govern GnRH ontogeny have been discovered by studying humans with GnRH deficiency. More importantly, detailed study of these patients has highlighted the emerging theme of oligogenicity and genotypic synergism, and also expanded the phenotypic diversity with the documentation of reversal of GnRH deficiency later in adulthood in some patients. The underlying genetic defect has also helped understand the associated nonreproductive phenotypes seen in some of these patients. These insights now provide practicing clinicians with targeted genetic diagnostic strategies and also impact on clinical management.

Pubertal impairment in Nhlh2 null mice is associated with hypothalamic and pituitary deficiencies.

Pubertal development is impaired in mice lacking the basic helix-loop-helix transcription factor Nhlh2. The mechanisms underlying changes in reproduction in Nhlh2-deficient mice (Nhlh2(-/-)) are unclear. Here we show that hypothalamic GnRH-1 content is reduced in adult Nhlh2(-/-) mice as is the number of GnRH-1 neurons localized to mid- and caudal hypothalamic regions. This reduction was detected postnatally after normal migration of GnRH-1 neurons within nasal regions had occurred. Phenotype rescue experiments showed that female Nhlh2(-/-) mice were responsive to estrogen treatment. In contrast, puberty could not be primed in female Nhlh2(-/-) mice with a GnRH-1 regimen. The adenohypophysis of Nhlh2(-/-) mice was hypoplastic although it contained a full complement of the five anterior pituitary cell types. GnRH-1 receptors (GnRHRs) were reduced in Nhlh2(-/-) pituitary gonadotropes as compared with wild type. In vitro assays indicated that Nhlh2 expression is regulated in parallel with GnRHR expression. However, direct transcriptional activity of Nhlh2 on the GnRHR promoter was not found. These results indicate that Nhlh2 plays a role in the development and functional maintenance of the hypothalamic-pituitary-gonadal axis at least at two levels: 1) in the hypothalamus by regulating the number and distribution of GnRH-1 neurons and, 2) in the developing and mature adenohypophysis.

GPR54 and KiSS-1: role in the regulation of puberty and reproduction.

The finding of inactivating mutations in GPR54 in IHH patients and the lack of reproductive maturation of the GPR54 null mouse have uncovered a previously unrecognized role for GPR54 and KiSS-1 in the physiologic regulation of puberty and reproduction. This newly identified function for GPR54 and its cognate ligand, kisspeptin, has led to additional studies that have localized GPR54 and KiSS-1 mRNA in the hypothalamus, colocalized GPR54 in GnRH neurons, demonstrated GnRH-dependent activation of LH and FSH release by kisspeptin, and shown increased hypothalamic KiSS-1 and GPR54 mRNA levels at the time of puberty. Taken together, these findings establish the role of the kisspeptin-GPR54 system in the stimulation of GnRH neurons during puberty. The mechanisms by which kisspeptin activates GnRH release, as well as the trigger for this pathway at the onset of puberty, are yet to be elucidated. In the future, modulators of GPR54 activity, including kisspeptin, may prove valuable in clinical applications in the fields of both cancer therapy and reproductive medicine.

KiSS-1 and GPR54 as new players in gonadotropin regulation and puberty.

The recent identification of loss-of-function mutations in the gene encoding GPR54, the receptor for the KiSS-1-derived peptides, kisspeptins, has highlighted a previously unrecognized pathway in the physiologic regulation of puberty and reproduction. Patients with loss-of-function mutations in GPR54 have idiopathic hypogonadotropic hypogonadism, and mice lacking GPR54 similarly fail to undergo puberty and have immature reproductive organs and low levels of sex steroids and gonadotropins. These observations have led to the hypothesis that kisspeptins activate hypothalamic GnRH release, thereby serving as a pivotal factor in the pubertal activation of the reproductive cascade. This hypothesis is supported by subsequent studies in rodent and primate models that have demonstrated localization of KiSS-1 mRNA in the hypothalamus, colocalization of GPR54 in GnRH neurons, GnRH-dependent activation of LH and FSH release by intracerebroventricular or peripheral administration of kisspeptin, and increased hypothalamic KiSS-1 and GPR54 mRNA levels at the onset of puberty. Taken together, these findings weave a compelling case for a role of the kisspeptin-GPR54 system in the activation of GnRH neurons at the time of pubertal awakening of the reproductive axis.

Regulation of gonadotropins by inhibin and activin.

Inhibin, activin, and follistatin were first identified as gonadal hormones that could exert selective effects on follicle-stimulating hormone (FSH) secretion without affecting luteinizing hormone (LH). Although the primary source of inhibin remains the gonad, both activin and follistatin are produced in extragonadal tissues and can exert effects on FSH through an autocrine-paracrine mechanism. These proteins can effect the regulation of the gonadotropins at many levels. First, activin can directly stimulate FSH biosynthesis and release from the gonadotrope cells of the pituitary gland. Second, activin up-regulates gonadotropin-releasing hormone receptor (GnRHR) gene expression, leading to alterations in the synthesis and release of both gonadotropins in response to GnRH. Third, activin can stimulate GnRH release from GnRH neurons in the hypothalamus and thereby affect FSH and LH secretion. Both inhibin and follistatin can negatively regulate these effects by preventing activin binding to the activin receptor at the cell membrane and blocking activation of downstream signal transduction pathways. This review concentrates on the mechanisms through which inhibin, activin, and follistatin regulate the gonadotropins. We discuss the expression of inhibin/activin subunits and receptors throughout the hypothalamus and pituitary and their role in the regulation of FSH and LH. The mechanisms of inhibin and activin signaling are also reported, with particular attention to developments in our understanding of inhibin receptor action and activin-induced transcriptional regulation of the FSHbeta gene promoter. Finally, we present recent findings that other members of the transforming growth factor beta superfamily may also play an important role in transcriptional regulation of the pituitary gonadotropins.

The GPR54 gene as a regulator of puberty.

BACKGROUND: Puberty, a complex biologic process involving sexual development, accelerated linear growth, and adrenal maturation, is initiated when gonadotropin-releasing hormone begins to be secreted by the hypothalamus. We conducted studies in humans and mice to identify the genetic factors that determine the onset of puberty. METHODS: We used complementary genetic approaches in humans and in mice. A consanguineous family with members who lacked pubertal development (idiopathic hypogonadotropic hypogonadism) was examined for mutations in a candidate gene, GPR54, which encodes a G protein-coupled receptor. Functional differences between wild-type and mutant GPR54 were examined in vitro. In parallel, a Gpr54-deficient mouse model was created and phenotyped. Responsiveness to exogenous gonadotropin-releasing hormone was assessed in both the humans and the mice. RESULTS: Affected patients in the index pedigree were homozygous for an L148S mutation in GPR54, and an unrelated proband with idiopathic hypogonadotropic hypogonadism was determined to have two separate mutations, R331X and X399R. The in vitro transfection of COS-7 cells with mutant constructs demonstrated a significantly decreased accumulation of inositol phosphate. The patient carrying the compound heterozygous mutations (R331X and X399R) had attenuated secretion of endogenous gonadotropin-releasing hormone and a left-shifted dose-response curve for gonadotropin-releasing hormone as compared with six patients who had idiopathic hypogonadotropic hypogonadism without GPR54 mutations. The Gpr54-deficient mice had isolated hypogonadotropic hypogonadism (small testes in male mice and a delay in vaginal opening and an absence of follicular maturation in female mice), but they showed responsiveness to both exogenous gonadotropins and gonadotropin-releasing hormone and had normal levels of gonadotropin-releasing hormone in the hypothalamus. CONCLUSIONS: Mutations in GPR54, a G protein-coupled receptor gene, cause autosomal recessive idiopathic hypogonadotropic hypogonadism in humans and mice, suggesting that this receptor is essential for normal gonadotropin-releasing hormone physiology and for puberty.