The role of the hypothalamic kisspeptin/GPR54 system in the control of GnRH neurons

Summary : The hypothalamus represents less than 1 % of the total volume of the brain tissue, yet it plays a crucial role in endocrine regulations. Puberty is defined as a process leading to physical, sexual and psychosocial maturation. The hypothalamus is central to this process, via the activation of GnRH neurons. Pulsatile GnRH secretion, minimal during childhood, increases with the onset of puberty. The primary function of GnRH is to regulate the growth, development and function of testes in boys and ovaries in girls, by stimulating the pituitary gland secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Several factors contribute to the timing of puberty, including sex and ethnicity, genetics, dietary intake and energy expenditure. Kisspeptins constitute a family of small peptides arising from the proteolytic cleavage of metastin, a peptide with 54 amino acids initially purified from human placenta. These kisspeptins were the subject of much attention following their discovery because of their antimetastatic properties, but it was more recently that their determining role in the reproductive function was demonstrated. It was shown that kisspeptins are ligands of a receptor, GPR54, whose natural inactivating mutation in humans, or knockout in the mouse, lead to infertility. GnRH neurons play a pivotal role in the central regulation of fertility. Kisspeptin greatly increases GnRH release and GnRH neuron firing activity, but the neurobiological mechanisms for these actions are unknown. Gprotein-coupled receptor 54, the receptor for kisspeptin, is expressed by GnRH neurons as well as other hypothalamic neurons, suggesting that both direct and indirect effects are possible. In the first part of my thesis, we investigated a possible connection between the acceleration of sexual development induced by leptin and hypothalamic metastin neurons. However, the data generated by our preliminary experiments confirmed that the commercially available antibodies are non-specific. This finding constituted a major drawback for our studies, which relied heavily upon the neuroanatomical study of the hypothalamic metastinergic pathways to elucidate their sensitivity to exogenous leptin. Therefore, we decided to postpone any further in vivo experiment until a better antibody becomes available, and focused on in vitro studies to better understand the mechanisms of action of kisspeptins in the modulation of the activity of GnRH neurons. We used two GnRH-expressing neuronal cell lines to investigate the cellular and molecular mechanisms of action of metastin in GnRH neurons. We demonstrated that kisspeptin induces an early activation of the MAP kinase intracellular signaling pathway in both cell lines, whereas the SAP/JNK or the Akt pathways were unaffected. Moreover, we found an increase in GnRH mRNA levels after 6h of metastin stimulation. Thus, we can conclude that kisspeptin regulates GnRH neurons both at the secretion and the gene expression levels. The MAPK pathway is the major pathway activated by metastin in GnRH expressing neurons. Taken together, these data provide the first mechanism of action of kisspeptin on GnRH neurons. Résumé : L'hypothalamus est une zone située au centre du cerveau, dont il représente moins de 1 du volume total. La puberté est la période de transition entre l'enfance et l'age adulte, qui s'accompagne de transformations somatiques, psychologiques, métaboliques et hormonales conduisant à la possibilité de procréer. La fonction principale de la GnRH est la régulation de la croissance, du développement et de la fonction des testicules chez les hommes, et des ovaires chez les femmes en stimulant la sécrétion de l'hormone lutéinisante (LH) et de l'hormone folliculostimulante (FSH) par la glande hypophysaire. Plusieurs facteurs contribuent au déclanchement de la puberté, y compris le sexe et l'appartenance ethnique, la génétique, l'apport alimentaire et la dépense énergétique. Les Kisspeptines constituent une famille de peptides résultant de la dissociation proteolytique de la métastine, un peptide de 54 acides aminés initialement purifié à partir de placenta humain. Ces kisspeptines ont fait l'objet de beaucoup d'attention à la suite de leur découverte en raison de leurs propriétés anti-metastatiques, et c'est plus récemment que leur rôle déterminant dans la fonction reproductive a été démontré. Les kisspeptines sont des ligands du récepteur GPR54, dont la mutation inactivatrice chez l'homme, ou le knockout chez la souris, conduisent à l'infertilité par hypogonadisme hypogonadotrope. Les neurones à GnRH jouent un rôle central dans le règlement des fonctions reproductrices et la kisspeptine stimule l'activité des neurones à GnRH et la libération de GnRH par ces neurones. Toutefois, les mécanismes neurobiologiques de ces actions ne sont pas connus. Dans la première partie de ma thèse, nous avons étudié le lien potentiel entre l'accélération du développement sexuel induite par la leptine et les neurones hypothalamiques à metastine. Les données générées dans cette première série d'expériences ont malheureusement confirmé que les anticorps anti-metastine disponibles dans le commerce sont aspécifiques. Ceci a constitué un inconvénient majeur pour nos études, qui devaient fortement s'appuyer sur l' étude neuroanatomique des neurones hypothalamiques à metastine pour évaluer leur sensibilité à la leptine exogène. Nous avons donc décidé de focaliser nos travaux sur une étude in vitro des mécanismes d'action de la kisspeptine pour moduler l'activité des neurones à GnRH. Nous avons utilisé deux lignées de cellules neuronales exprimant la GnRH pour étudier les mécanismes d'action cellulaires et moléculaires de la metastine dans des neurones. Nous avons ainsi pu démontrer que la kisspeptine induit une activation précoce de la voie f de signalisation de la MAP kinase dans les deux lignées cellulaires, alors que nous n'avons observé aucune activation de la voie de signalisation de la P13 Kinase et de la SAP/JNK. Nous avons en outre démontré une augmentation de l'expression de la GnRH par la stimulation avec la Kisspeptine. L'ensemble de ces données contribue à élucider le mécanisme d'action avec lequel la kisspeptine agit dans les neurones à GnRH, en démontrant un effet sur l'expression génique de la GnRH. Nous pouvons également conclure que la voie de la MAPK est la voie principale activée par la metastine dans les neurones exprimant la GnRH.

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.

Expanding the phenotype and genotype of female GnRH deficiency.

Context: GnRH deficiency is a rare genetic disorder of absent or partial pubertal development. The clinical and genetic characteristics of GnRH-deficient women have not been well-described. Objective: To determine the phenotypic and genotypic spectrum of a large series of GnRH-deficient women. Design, Setting, and Subjects: Retrospective study of 248 females with GnRH deficiency evaluated at an academic medical center between 1980 and 2010. Main Outcome Measures: Clinical presentation, baseline endogenous GnRH secretory activity, and DNA sequence variants in 11 genes associated with GnRH deficiency. Results: Eighty-eight percent had undergone pubarche, 51% had spontaneous thelarche, and 10% had 1-2 menses. Women with spontaneous thelarche were more likely to demonstrate normal pubarche (P = 0.04). In 27% of women, neuroendocrine studies demonstrated evidence of some endogenous GnRH secretory activity. Thirty-six percent (a large excess relative to controls) harbored a rare sequence variant in a gene associated with GnRH deficiency (87% heterozygous and 13% biallelic), with variants in FGFR1 (15%), GNRHR (6.6%), and PROKR2 (6.6%) being most prevalent. One woman had a biallelic variant in the X-linked gene, KAL1, and nine women had heterozygous variants. Conclusions: The clinical presentation of female GnRH deficiency varies from primary amenorrhea and absence of any secondary sexual characteristics to spontaneous breast development and occasional menses. In this cohort, rare sequence variants were present in all of the known genes associated with GnRH deficiency, including the novel identification of GnRH-deficient women with KAL1 variants. The pathogenic mechanism through which KAL1 variants disrupt female reproductive development requires further investigation.

Phenotypic and molecular characterization of proliferating and differentiated GnRH-expressing GnV-3 cells.

GnRH neurons provide the primary driving force upon the neuroendocrine reproductive axis. Here we used GnV-3 cells, a model of conditionally immortalized GnRH-expressing neurons, to perform an analysis of cell cycle and compare the gene expression profile of proliferating cells with differentiated cells. In the proliferation medium, 45 ± 1.5% of GnV-3 cells are in S-phase by FACS analysis. In the differentiation medium, only 9 ± 0.9% of them are in S-phase, and they acquire the characteristic bipolar shape displayed by preoptic GnRH neurons in vivo. In addition, GnV-3 cells in the differentiated state exhibit electrophysiological properties characteristic of neurons. Transcriptomic analysis identified up-regulation of 1931 genes and down-regulation of 1270 genes in cells grown in the differentiation medium compared to cells in the proliferation medium. Subsequent gene ontology study indicated that genes over-expressed in proliferating GnV-3 cells were mainly involved in cell cycle regulations, whereas genes over-expressed in differentiated cells were mainly involved in processes of differentiation, neurogenesis and neuronal morphogenesis. Taken together, these data demonstrate the occurrence of morphological and physiological changes in GnV-3 cells between the proliferating and the differentiated state. Moreover, the genes differentially regulated between these two different states are providing novel pathways potentially important for a better understanding of the physiology of mature GnRH neurons.

Trial of recombinant follicle-stimulating hormone pretreatment for gnrh-induced fertility in patients with congenital hypogonadotropic hypogonadism.

CONTEXT AND OBJECTIVE: The optimal strategy for inducing fertility in men with congenital hypogonadotropic hypogonadism (CHH) is equivocal. Albeit a biologically plausible approach, pretreatment with recombinant FSH (rFSH) before GnRH/human chorionic gonadotropin administration has not been sufficiently assessed. The objective of the study was to test this method. DESIGN AND SETTING: This was a randomized, open-label treatment protocol at an academic medical center. PATIENTS AND INTERVENTIONS: GnRH-deficient men (CHH) with prepubertal testes (<4 mL), no cryptorchidism, and no prior gonadotropin therapy were randomly assigned to either 24 months of pulsatile GnRH therapy alone (inducing endogenous LH and FSH release) or 4 months of rFSH pretreatment followed by 24 months of GnRH therapy. Patients underwent serial testicular biopsies, ultrasound assessments of testicular volume, serum hormone measurements, and seminal fluid analyses. RESULTS: rFSH treatment increased inhibin B levels into the normal range (from 29 ± 9 to 107 ± 41 pg/mL, P < .05) and doubled testicular volume (from 1.1 ± 0.2 to 2.2 ± 0.3 mL, P < .005). Histological analysis showed proliferation of both Sertoli cells (SCs) and spermatogonia, a decreased SC to germ cell ratio (from 0.74 to 0.35), and SC cytoskeletal rearrangements. With pulsatile GnRH, the groups had similar hormonal responses and exhibited significant testicular growth. All men receiving rFSH pretreatment developed sperm in their ejaculate (7 of 7 vs 4 of 6 in the GnRH-only group) and showed trends toward higher maximal sperm counts. CONCLUSIONS: rFSH pretreatment followed by GnRH is successful in inducing testicular growth and fertility in men with CHH with prepubertal testes. rFSH not only appears to maximize the SC population but also induces morphologic changes, suggesting broader developmental roles.

Responsiveness to a physiological regimen of GnRH therapy and relation to genotype in women with isolated hypogonadotropic hypogonadism.

CONTEXT: Isolated hypogonadotropic hypogonadism (IHH) is caused by defective GnRH secretion or action resulting in absent or incomplete pubertal development and infertility. Most women with IHH ovulate with physiological GnRH replacement, implicating GnRH deficiency as the etiology. However, a subset does not respond normally, suggesting the presence of defects at the pituitary or ovary. OBJECTIVES: The objective of the study was to unmask pituitary or ovarian defects in IHH women using a physiological regimen of GnRH replacement, relating these responses to genes known to cause IHH. DESIGN, SETTING, AND SUBJECTS: This study is a retrospective analysis of 37 IHH women treated with iv pulsatile GnRH (75 ng/kg per bolus). MAIN OUTCOME MEASURES: Serum gonadotropin and sex steroid levels were measured, and 14 genes implicated in IHH were sequenced. RESULTS: During their first cycle of GnRH replacement, normal cycles were recreated in 60% (22 of 37) of IHH women. Thirty percent of women (12 of 37) demonstrated an attenuated gonadotropin response, indicating pituitary resistance, and 10% (3 of 37) exhibited an exaggerated FSH response, consistent with ovarian resistance. Mutations in CHD7, FGFR1, KAL1, TAC3, and TACR3 were documented in IHH women with normal cycles, whereas mutations were identified in GNRHR, PROKR2, and FGFR1 in those with pituitary resistance. Women with ovarian resistance were mutation negative. CONCLUSIONS: Although physiological replacement with GnRH recreates normal menstrual cycle dynamics in most IHH women, hypogonadotropic responses in the first week of treatment identify a subset of women with pituitary dysfunction, only some of whom have mutations in GNRHR. IHH women with hypergonadotropic responses to GnRH replacement, consistent with an additional ovarian defect, did not have mutations in genes known to cause IHH, similar to our findings in a subset of IHH men with evidence of an additional testicular defect.

Insulin and NPY pathways and the control of GnRH function and puberty onset.

Energy balance exerts a critical influence on reproductive function. Leptin and insulin are among the metabolic factors signaling the nutritional status of an individual to the hypothalamus, and their role in the overall modulation of the activity of GnRH neurons is increasingly recognized. As such, they participate to a more generalized phenomenon: the signaling of peripheral metabolic changes to the central nervous system. The physiological importance that the interactions occurring between peripheral metabolic factors and the central nervous system bear for the control of food intake is increasingly recognized. The central mechanisms implicated are the focus of attention of very many research groups worldwide. We review here the experimental data that suggest that similar mechanisms are at play for the metabolic control of the neuroendocrine reproductive function. It is appearing that metabolic signals are integrated at the levels of first-order neurons equipped with the proper receptors, ant that these neurons send their signals towards hypothalamic GnRH neurons which constitute the integrative element of this network.

When genetic load does not correlate with phenotypic spectrum: lessons from the GnRH receptor (GNRHR).

CONTEXT: A broad spectrum of GnRH-deficient phenotypes has been identified in individuals with both mono- and biallelic GNRHR mutations. OBJECTIVE: The objective of the study was to determine the correlation between the severity of the reproductive phenotype(s) and the number and functional severity of rare sequence variants in GNRHR. SUBJECTS: Eight hundred sixty-three probands with different forms of GnRH deficiency, 46 family members and 422 controls were screened for GNRHR mutations. The 70 subjects (32 patients and 38 family members) harboring mutations were divided into four groups (G1-G4) based on the functional severity of the mutations (complete or partial loss of function) and the number of affected alleles (monoallelic or biallelic) with mutations, and these classes were mapped on their clinical phenotypes. RESULTS: The prevalence of heterozygous rare sequence variants in GNRHR was significantly higher in probands vs. controls (P < 0.01). Among the G1-G3 groups (homozygous subjects with successively decreasing severity and number of mutations), the hypogonadotropic phenotype related to their genetic load. In contrast, subjects in G4, with only monoallelic mutations, demonstrated a greater diversity of clinical phenotypes. CONCLUSIONS: In patients with GnRH deficiency and biallelic mutations in GNRHR, genetic burden defined by severity and dose is associated with clinical phenotype. In contrast, for patients with monoallelic GNRHR mutations this correlation does not hold. Taken together, these data indicate that as-yet-unidentified genetic and/or environmental factors may combine with singly mutated GNRHR alleles to produce reproductive phenotypes.

Expression of the GABAA receptor associated protein Gec1 is circadian and dependent upon the cellular clock machinery in GnRH secreting GnV-3 cells.

The timely regulation of gonadotropin-releasing hormone (GnRH) secretion requires a GABAergic signal. We hypothesized that GEC1, a protein promoting the transport of GABA(A) receptors, could represent a circadian effector in GnRH neurons. First, we demonstrated that gec1 is co-expressed with the GABA(A) receptor in hypothalamic rat GnRH neurons. We also confirmed that the clock genes per1, cry1 and bmal1 are expressed and oscillate in GnRH secreting GnV-3 cells. Then we could show that gec1 is expressed in GnV-3 cells, and oscillates in a manner temporally related to the oscillations of the clock transcription factors. Furthermore, we could demonstrate that these oscillations depend upon Per1 expression. Finally, we observed that GABA(A) receptor levels at the GnV-3 cell membrane are timely modulated following serum shock. Together, these data demonstrate that gec1 expression is dependent upon the circadian clock machinery in GnRH-expressing neurons, and suggest for the first time that the level of GABA(A) receptor at the cell membrane may be under timely regulation. Overall, they provide a potential mechanism for the circadian regulation of GnRH secretion by GABA, and may also be relevant to the general understanding of circadian rhythms.

Deciphering genetic disease in the genomic era: the model of GnRH deficiency.

Isolated gonadotropin-releasing hormone (GnRH) deficiency is a treatable albeit rare form of reproductive failure that has revealed physiological mechanisms controlling human reproduction, but despite substantial progress in discovering pathogenic single-gene defects, most of the genetic basis of GnRH deficiency remains uncharted. Although unbiased genetic investigations of affected families have identified mutations in previously unsuspected genes as causes of this disease in some cases, their application has been severely limited because of the negative effect of GnRH deficiency on fertility; moreover, relatively few of the many candidate genes nominated because of biological plausibility from in vitro or animal model experiments were subsequently validated in patients. With the advent of exciting technological platforms for sequencing, homozygosity mapping, and detection of structural variation at the whole-genome level, human investigations are again assuming the leading role for gene discovery. Using human GnRH deficiency as a paradigm and presenting original data from the screening of numerous candidate genes, we discuss the emerging model of patient-focused clinical genetic research and its complementarities with basic approaches in the near future.

The fetal hypothalamus has the potential to generate cells with a gonadotropin releasing hormone (GnRH) phenotype.

BACKGROUND: Neurospheres (NS) are colonies of neural stem and precursor cells capable of differentiating into the central nervous system (CNS) cell lineages upon appropriate culture conditions: neurons, and glial cells. NS were originally derived from the embryonic and adult mouse striatum subventricular zone. More recently, experimental evidence substantiated the isolation of NS from almost any region of the CNS, including the hypothalamus. METHODOLOGY/FINDINGS: Here we report a protocol that enables to generate large quantities of NS from both fetal and adult rat hypothalami. We found that either FGF-2 or EGF were capable of inducing NS formation from fetal hypothalamic cultures, but that only FGF-2 is effective in the adult cultures. The hypothalamic-derived NS are capable of differentiating into neurons and glial cells and most notably, as demonstrated by immunocytochemical detection with a specific anti-GnRH antibody, the fetal cultures contain cells that exhibit a GnRH phenotype upon differentiation. CONCLUSIONS/SIGNIFICANCE: This in vitro model should be useful to study the molecular mechanisms involved in GnRH neuronal differentiation.

Complete hypogonadotropic hypogonadism associated with a novel inactivating mutation of the gonadotropin-releasing hormone receptor.

In this study, we describe a patient with a phenotype of complete hypogonadotropic hypogonadism who presented primary failure of pulsatile GnRH therapy, but responded to exogenous gonadotropin administration. This patient bore a novel point mutation (T for A) at codon 168 of the gene encoding the GnRH receptor (GnRH-R), resulting in a serine to arginine change in the fourth transmembrane domain of the receptor. This novel mutation was present in the homozygous state in the patient, whereas it was in the heterozygous state in both phenotypically normal parents. When introduced into the complementary DNA coding for the GnRH-R, this mutation resulted in the complete loss of the receptor-mediated signaling response to GnRH. In conclusion, we report the first mutation of the GnRH-R gene that can induce a total loss of function of this receptor and is associated with a phenotype of complete hypogonadotropic hypogonadism.

In vitro neuroendocrine effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the AhR-expressing hypothalamic rat GnV-3 cell line.

The aryl hydrocarbon receptor (AhR) is involved in a wide variety of biological and toxicological responses, including neuroendocrine signaling. Due to the complexity of neuroendocrine pathways in e.g. the hypothalamus and pituitary, there are limited in vitro models available despite the strong demand for such systems to study and predict neuroendocrine effects of chemicals. In this study, the applicability of the AhR-expressing rat hypothalamic GnV-3 cell line was investigated as a novel model to screen for neuroendocrine effects of AhR ligands using 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as reference compound. The qRT-PCR analyses demonstrated the presence of several sets of neurotransmitter receptors in the GnV-3 cells. TCDD (10nM) altered neurotransmitter signaling by up-regulation of glutamate (Grik2), gamma-amino butyric acid (Gabra2) and serotonin (Ht2C) receptor mRNA levels. However, no significant changes in basal and serotonin-evoked intracellular Ca(2+) concentration ([Ca(2+)]i) or serotonin release were observed. On the other hand, TCDD de-regulated period circadian protein homolog 1 (Per1) and gonadotropin releasing hormone (Gnrh) mRNA levels within a 24-h time period. Both Per1 and Gnrh genes displayed a similar mRNA expression pattern in GnV-3 cells. Moreover, the involvement of AhR in TCDD-induced alteration of Neuropeptide Y (Npy) gene expression was found and confirmed by using siRNA targeted against Ahr in GnV-3 cells. Overall, the combined results demonstrate that GnV-3 cells may be a suitable model to predict some mechanisms of action and effects of AhR ligands in the hypothalamus.