GnRH Neuronal Migration Research Articles

RNF216 Regulates the Migration of Immortalized GnRH Neurons by Suppressing Beclin1-Mediated Autophagy.

RNF216, encoding an E3 ubiquitin ligase, has been identified as a causative gene for Gordon Holmes syndrome, characterized by ataxia, dementia, and hypogonadotropic hypogonadism. However, it is still elusive how deficiency in RNF216 leads to hypogonadotropic hypogonadism. In this study, by using GN11 immature GnRH neuronal cell line, we demonstrated an important role of RNF216 in the GnRH neuron migration. RNA interference of RNF216 inhibited GN11 cell migration, but had no effect on the proliferation of GN11 cells or GnRH expression. Knockdown of RNF216 increased the protein levels of its targets, Arc and Beclin1. RNAi of Beclin1, but not Arc, normalized the suppressive effect caused by RNF216 knockdown. As Beclin1 plays a critical role in the autophagy regulation, we further demonstrated that RNAi of RNF216 led to increase in autophagy, and autophagy inhibitor CQ and 3-MA rescued the GN11 cell migration deficit caused by RNF216 knockdown. We further demonstrated that pharmacological increase autophagy by rapamycin could suppress the GN11 cell migration. We thus have identified that RNF216 regulates the migration of GnRH neuron by suppressing Beclin1 mediated autophagy, and indicated a potential contribution of autophagy to the hypogonadotropic hypogonadism.

Prevalence and associated phenotypes of PLXNA1 variants in normosmic and anosmic idiopathic hypogonadotropic hypogonadism.

Idiopathic hypogonadotropic hypogonadism (IHH) can be divided into two major forms, normosmic IHH and Kallmann syndrome (KS). Genetic mutations are responsible for the majority of IHH. PLXNA1 has recently been implicated in the GnRH neuron migration and the etiology of KS. We aimed to investigate the prevalence and associated phenotypes of PLXNA1 variants in a large cohort of IHH patients. We screened the whole exome data of 215 IHH patients in a single center for causative PLXNA1 variants. Our studies showed eight novel (p.Arg836His, p.Lys1451Arg, p.Val287Met, p.Val536Ile, p.Ser1850Arg, p.Ile1701Val, p.Arg319Trp, and p.Pro485Leu) and two previously described (p.Arg528Trp and p.Gly720Glu) heterozygous PLXNA1 variants in nine affected individuals from seven unrelated families. Only three of nine patients were anosmic (KS) while the remaining patients showed normal olfactory function (nIHH). Seven of nine patients (77.7%) harbored additional one or two variants in other nIHH/KS-associated genes, including PROKR2, IGSF10, HS6ST1, SEMA3E, CCDC141, FGFR1, NRP1, POLR3A, and SRA1. Our findings indicate that PLXNA1 variants cause not only anosmic but also normosmic IHH with a relatively high prevalence (3.9%). Heterozygous missense PLXNA1 variants appear to be involved together with other IHH gene variants in bringing about the IHH disease phenotype.

ERRATUM FOR "Serotonin Receptor 1A (HTR1A), a Novel Regulator of GnRH Neuronal Migration in Chick Embryo".

ERRATUM FOR "Serotonin Receptor 1A (HTR1A), a Novel Regulator of GnRH Neuronal Migration in Chick Embryo".

Etiology of Central (Hypogonadotropic) Female Hypogonadism: Genetic, Organic and Functional Causes

Central (hypogonadotropic) hypogonadism (CH) can be mentioned among the causes of menstrual and fertility problems in women. This clinical syndrome is characterized by decreased sex steroid secretion due to the impairment of basal and/or impulse secretion of gonadotropins. It can be congenital or acquired, organic or functional (i.e. without visible anatomic damages of the hypothalamo-hypophyseal area), isolated or combined with other pituitary hormone deficits (hypopituitarism). All causes of central female hypogonadism can be conditionally divided into some subgroups: genetic factors (lead to congenital CH), organic factors (lead to acquired CH), and idiopathic (lead functional CH). Congenital CH can be caused by genes that control ontogenesis and migration of GnRH neurons, regulate secretion of GnRH and provide its’ biological effects on gonadotropins. The potential organic causes of CH include pituitary tumors and some other types of tumors with similar localization, consequences of surgical or radiation intervention in connection with these tumors, brain injury as well as autoimmune, infiltrative and other rare diseases. Classical risk factors for functional CH include stress, excessive physical exercises and critically low body mass, however, variety of hormonal changes (including metabolic hormones) was observed and genetic predisposition was also shown for these patients. It is very important to understand complex pathogenetic factors that will help to improve diagnosis of this rare disease.

Sexually dimorphic distribution of Prokr2 neurons revealed by the Prokr2-Cre mouse model.

Prokineticin receptor 2 (PROKR2) is predominantly expressed in the mammalian central nervous system. Loss-of-function mutations of PROKR2 in humans are associated with Kallmann syndrome due to the disruption of gonadotropin releasing hormone neuronal migration and deficient olfactory bulb morphogenesis. PROKR2 has been also implicated in the neuroendocrine control of GnRH neurons post-migration and other physiological systems. However, the brain circuitry and mechanisms associated with these actions have been difficult to investigate mainly due to the widespread distribution of Prokr2-expressing cells, and the lack of animal models and molecular tools. Here, we describe the generation, validation and characterization of a new mouse model that expresses Cre recombinase driven by the Prokr2 promoter, using CRISPR-Cas9 technology. Cre expression was visualized using reporter genes, tdTomato and GFP, in males and females. Expression of Cre-induced reporter genes was found in brain sites previously described to express Prokr2, e.g., the paraventricular and the suprachiasmatic nuclei, and the area postrema. The Prokr2-Cre mouse model was further validated by colocalization of Cre-induced GFP and Prokr2 mRNA. No disruption of Prokr2 expression, GnRH neuronal migration or fertility was observed. Comparative analysis of Prokr2-Cre expression in male and female brains revealed a sexually dimorphic distribution confirmed by in situ hybridization. In females, higher Cre activity was found in the medial preoptic area, ventromedial nucleus of the hypothalamus, arcuate nucleus, medial amygdala and lateral parabrachial nucleus. In males, Cre was higher in the amygdalo-hippocampal area. The sexually dimorphic pattern of Prokr2 expression indicates differential roles in reproductive function and, potentially, in other physiological systems.

The mystery of puberty initiation: genetics and epigenetics of idiopathic central precocious puberty (ICPP).

Puberty is a major developmental stage. Damaging mutations, considered as "mistakes of nature", have contributed to the unraveling of the networks implicated in the normal initiation of puberty. Genes involved in the abnormal hypothalamic-pituitary-gonadal (HPG) axis development, in the normosmic idiopathic hypogonadotropic hypogonadism (nIHH), in the X-linked or autosomal forms of Kallmann syndrome and in precocious puberty have been identified (GNRH1, GNRHR, KISS1, GPR54, FGFR1, FGF8, PROK2, PROKR2, TAC3, TACR3, KAL1, PROK2, PROKR2, CHD7, LEP, LEPR, PC1, DAX1, SF-1, HESX-1, LHX3, PROP-1). Most of them were found to play critical roles in HPG axis development and regulation, the embryonic GnRH neuronal migration and secretion, the regulation and action of the hypothalamic GnRH. However, the specific neural and molecular mechanisms triggering GnRH secretion remain one of the scientific enigmas. Although GnRH neurons are probably capable of autonomously generating oscillations, many gonadal steroid-dependent and -independent mechanisms have also been proposed. It is now well proven that the secretion of GnRH is regulated by kisspeptin as well as by permissive or opposing signals mediated by neurokinin B and dynorphin. These three supra-GnRH regulators compose the kisspeptin-neurokinin B-dynorphin neuronal (KNDy) system, a key player in pubertal onset and progression. Moreover, an ongoing increasing number of inhibitory, stimulatory and permissive networks acting upstream on GnRH neurons, such as GABA, NPY, LIN28B, MKRN3 and others integrate diverse hormonal and peripheral signals and have been proposed as the "gate-keepers" of puberty, while epigenetic modifications play also an important role in puberty initiation.

CCDC141 Mutations in Idiopathic Hypogonadotropic Hypogonadism.

Gonadotropin-releasing hormone neurons originate outside the central nervous system in the olfactory placode and migrate into the central nervous system, becoming integral components of the hypothalamic-pituitary-gonadal axis. Failure of this migration can lead to idiopathic hypogonadotropic hypogonadism (IHH)/Kallmann syndrome (KS). We have previously shown that CCDC141 knockdown leads to impaired migration of GnRH neurons but not of olfactory receptor neurons. The aim of this study was to further describe the phenotype and prevalence of CCDC141 mutations in IHH/KS. Using autozygosity mapping, candidate gene screening, whole-exome sequencing, and Sanger sequencing, those individuals carrying deleterious CDCD141 variants and their phenotypes were determined in a cohort of 120 IHH/KS families. No interventions were made. Our studies revealed nine affected individuals from four independent families in which IHH/KS is associated with inactivating CCDC141 variants, revealing a prevalence of 3.3%. Affected individuals (with the exception of those from family 1 who concomitantly have FEZF1 mutations) have normal olfactory function and anatomically normal olfactory bulbs. Four affected individuals show evidence of clinical reversibility. In three of the families, there was at least one more potentially deleterious variant in other known puberty genes with evidence of allelic heterogeneity within respective pedigrees. These studies confirm that inactivating CCDC141 variants cause normosmic IHH but not KS. This is consistent with our previous in vitro experiments showing exclusively impaired embryonic migration of GnRH neurons upon CCDC141 knockdown. These studies expand the clinical and genetic spectrum of IHH and also attest to the complexity of phenotype and genotype in IHH.

Serotonin Receptor 1A (HTR1A), a Novel Regulator of GnRH Neuronal Migration in Chick Embryo.

The hypothalamic GnRH neurons are a small group of cells that regulate the reproductive axis. These neurons are specified within the olfactory placode, delaminate from this structure, and then migrate to enter the forebrain before populating the hypothalamus. We have used microarray technology to analyze the transcriptome of the olfactory placode at a number of key time points for GnRH ontogeny using the chick embryo. This resulted in the identification of a large number of genes whose expression levels change significantly over this period. This repertoire includes those genes that are known to be important for GnRH neuronal development as well as many novel genes, such as the serotonin receptor 1A, HTR1A. We find that HTR1A is expressed in the region of the olfactory placode that generates GnRH neurons. We further show that when this receptor is inactivated using a selective HTR1A antagonist as well as a gene knockdown approach using RNAi, this resulted in delayed migration causing the GnRH neurons to stall just outside the forebrain. These findings implicate HTR1A as being important for GnRH neuronal migration from the olfactory placode to the forebrain. Our study thus extends the repertoire of genes involved in GnRH neuron biology and thus identifies new candidate genes that can be screened for in patients who do not show mutations in any of the previously identified hypogonadotrophic hypogonadism/Kallmann syndrome genes.

MRNA analysis of genes responsible for idiopathic hypogonadotrophic hypogonadism

Background. Hypogonadotropic hypogonadism (HH) is a disorder characterized by delayed or absent pubertal development due to pathology of the hypothalamic-pituitary-gonadal axis. HH may be both congenital (Kallmann’s syndrome) and sporadic. Congenital or isolated HH is divided into with anosmia/hyposmia (KS) and with normal olfaction (nIHH). Nowadays several tens of genes involved in the functioning of the reproductive axis are known. However DNA lesions can be found just in 5-15% of such cases of HH.Aim. So we decided to measure mRNA expression of several genes which can be found in leukocytes of peripheral blood - namely GNRHR and GNRH1 (are necessary for adequate biological effect of GnRH); PROK2 and CHD7 (are responsible for the migration of GnRH neurons), WDR11 and DUSP6 (are involved in normal sexual development).Methods. A quantitative determination of mRNA expression of these genes were comlpeted in the fresh peripheral blood sample by PCR in real time.Results. Examined patients: 9 women with hypogonadotropic hypogonadism (age from 18 to 28 y.o.); duration of the disease from 2 to 15 years; 3 of them – amenorrhea I and 6 – amenorrhea II. Reasons of amenorrhea II were: stress, excessive exercises, rapid body weight loss, past use of oral contraceptives. The control group: 19 healthy women; age from 19 to 37 y.o.; with regular ovalutory menstrual cycle, some of them have children. mRNA expression of examined genes differed from normal patterns in each case of hypogonadotropic hypogonadism. Changes in GNRHR, GNRH1 and DUSP6 mRNA expression were found in most of cases. However variations of mRNA expression were multidirectional in each case and there was no similarity among expression profiles of patients according to amenorrhea type or anamnestic factors.Conclusions. According to our preliminary results, in women with hypogonadotropic hypogonadism the functional activity damage of “reproductive-responsible” genes could be found in each case. Probably mRNA expression measuring could be a perspective method for proving hypothalamo-pituitary level of reproductive disorders and may help to determine which genes should be tested for DNA impairment.

CCDC141 Mutation Identified in Anosmic Hypogonadotropic Hypogonadism (Kallmann Syndrome) Alters GnRH Neuronal Migration.

The first mutation in a gene associated with a neuronal migration disorder was identified in patients with Kallmann Syndrome, characterized by hypogonadotropic hypogonadism and anosmia. This pathophysiological association results from a defect in the development of the GnRH and the olfactory system. A recent genetic screening of Kallmann Syndrome patients revealed a novel mutation in CCDC141. Little is known about CCDC141, which encodes a coiled-coil domain containing protein. Here, we show that Ccdc141 is expressed in GnRH neurons and olfactory fibers and that knockdown of Ccdc141 reduces GnRH neuronal migration. Our findings in human patients and mouse models predict that CCDC141 takes part in embryonic migration of GnRH neurons enabling them to form a hypothalamic neuronal network to initiate pulsatile GnRH secretion and reproductive function.

Spectrum of phenotype and genotype of congenital isolated hypogonadotropic hypogonadism in Asian Indians.

Congenital isolated hypogonadotropic hypogonadism (IHH) is caused due to defect in GnRH neuronal development, migration and action. Although genetic aetiology of IHH is increasingly being studied, Asian Indian data on phenotypic spectrum and genetic basis are scarce. To investigate the phenotypic and genotypic spectrum of IHH in Asian Indian subjects. A cohort of 135 IHH probands were characterized phenotypically for reproductive and nonreproductive features and screened for rare sequence variations (RSVs) in five genes KAL1, FGFR1, FGF8, GNRHR and KISS1R. Of 135 probands [56 normosmic IHH (nIHH) and 79 Kallmann syndrome (KS)], 20 were familial cases. KS group had more male dominance (M:F ratio of 8:1) as compared to nIHH group (M:F ratio of 1·5:1). Complete absence of puberty was more prevalent in KS probands (81% in KS vs 46% in nIHH). The prevalence of MRI abnormalities was more in anosmic group (92·8%) as compared to hyposmic (37·5%) and normosmic groups (15·4%). No particular nonreproductive phenotypic predominance was seen in any group. Genotyping revealed rare sequence variation (RSV) detection rate of 15·5% in five genes studied: (KAL1 - 4·4%, FGFR1 - 4·4%, GNRHR - 6·7%, oligogenicity - 1·5%). Prevalence of RSV was more common in familial cases (35%) as compared to sporadic (12·2%). GNRHR RSV p.C279Y (not reported in patients of ethnicities other than south Asians) was recurring in four unrelated patients. In our cohort, 60% were KS with majority of males and a severe reproductive phenotype as against nIHH. Contribution of the genetic burden for the five genes studied was 15·5%. RSV p.C279Y in GNRHR may have a founder effect originating from south Asia. This study provides a model for molecular and phenotypic representation of Asian Indian subjects with IHH.

Prokineticins in central and peripheral control of human reproduction

Prokineticin 1 (PROK1) and (PROK2), are two closely related proteins that were identified as the mammalian homologs of their two amphibian homologs, mamba intestinal toxin (MIT-1) and Bv8. PROKs activate two G-protein linked receptors (prokineticin receptor 1 and 2, PROKR1 and PROKR2). Both PROK1 and PROK2 have been found to regulate a stunning array of biological functions. In particular, PROKs stimulate gastrointestinal motility, thus accounting for their family name "prokineticins". PROK1 acts as a potent angiogenic mitogen, thus earning its other name, endocrine gland-derived vascular endothelial factor. In contrast, PROK2 signaling pathway has been shown to be a critical regulator of olfactory bulb morphogenesis and sexual maturation. During the last decade, strong evidences established the key roles of prokineticins in the control of human central and peripheral reproductive processes. PROKs act as main regulators of the physiological functions of the ovary, uterus, placenta, and testis, with marked dysfunctions in various pathological conditions such as recurrent pregnancy loss, and preeclampsia. PROKs have also been associated to the tumor development of some of these organs. In the central system, prokineticins control the migration of GnRH neurons, a key process that controls reproductive functions. Importantly, mutations in PROK2 and PROKR2 are associated to the development of Kallmann syndrome, with direct consequences on the reproductive system. This review describes the finely tuned actions of prokineticins in the control of the central and peripheral reproductive processes. Also, it discusses future research directions for the use of these cytokines as diagnostic markers for several reproductive diseases.

Mutations in IGSF10 cause self-limited delayed puberty, via disturbance of GnRH neuronal migration

Mutations in IGSF10 cause self-limited delayed puberty, via disturbance of GnRH neuronal migration

Point mutations in KAL1 and the mitochondrial gene MT-tRNA(cys) synergize to produce Kallmann syndrome phenotype.

Kallmann syndrome (KS) is an inherited developmental disorder defined as the association of hypogonadotropic hypogonadism and anosmia or hyposmia. KS has been shown to be a genetically heterogeneous disease with different modes of inheritance. However, variants in any of the causative genes identified so far are only found in approximately one third of KS patients, thus indicating that other genes or pathways remain to be discovered. Here, we report a large Han Chinese family with inherited KS which harbors two novel variants, KAL1 c.146G>T (p.Cys49Phe) and mitochondrial tRNAcys (m.5800A>G). Although two variants can’t exert obvious effects on the migration of GnRH neurons, they show the synergistic effect, which can account for the occurrence of the disorder in this family. Furthermore, the disturbance of the mitochondrial cysteinyl-tRNA pathway can significantly affect the migration of GnRH cells in vitro and in vivo by influencing the chemomigration function of anosmin-1. Our work highlights a new mode of inheritance underlay the genetic etiology of KS and provide valuable clues to understand the disease development.

The molecular control of GnRH neuron development.

Fertility critically depends by a small number of hypothalamic neurons secreting the neurohormone GnRH. During development GnRH neurons migrate from the nasal placode to the hypothalamus by following the migratory path formed by the vomeronasal axons. Developmental defects of this process can cause congenital GnRH deficiency (GD), characterized by absent/delayed puberty and consequent infertility. The underlying mutated loci are for the majority of GD cases unknown, partially because of a poor understanding of the molecular mechanisms that control the development of these crucial neuroendocrine cells. Here we provide evidence of the importance of class 3 semaphorins and their receptors in this process. Specifically, I will explain how two different semaphorins play distinct roles during the migration of GnRH neurons. Thus, semaphorin3A affects the migration of GnRH neurons in the nasal compartment, via the co-receptors Neuropilin-1 and 2, whereas semaphorin3E via its receptor plexind1 controls the survival of GnRH neurons once positioned in the hypothalamus. Accordingly, disrupted semaphorin signalling may be involved in the aethiopathogenesis of genetic diseases characterized by GnRH deficiency.

Gonadotropin and kisspeptin gene expression, but not GnRH, are impaired in cFOS deficient mice

cFOS is a pleiotropic transcription factor, which binds to the AP1 site in the promoter of target genes. In the pituitary gonadotropes, cFOS mediates induction of FSHβ and GnRH receptor genes. Herein, we analyzed reproductive function in the cFOS-deficient mice to determine its role in vivo. In the pituitary cFOS is necessary for gonadotropin subunit expression, while TSHβ is unaffected. Additionally, cFOS null animals have the same sex-steroid levels, although gametogenesis is impeded. In the brain, cFOS is not necessary for GnRH neuronal migration, axon targeting, cell number, or mRNA levels. Conversely, cFOS nulls, particularly females, have decreased Kiss1 neuron numbers and lower Kiss1 mRNA levels. Collectively, our novel findings suggest that cFOS plays a cell-specific role at multiple levels of the hypothalamic–pituitary–gonadal axis, affecting gonadotropes but not thyrotropes in the pituitary, and kisspeptin neurons but not GnRH neurons in the hypothalamus, thereby contributing to the overall control of reproduction.

Olfactory Agenesis in Kallmann Syndrome (KS).

Kallmann syndrome (KS) is a heterogeneous genetic disorder characterised by isolated gonadotrophic deficiency and anosmia. Defective migration of GnRH neurons along with olfactory neurons results in hypoplasia or agenesis of olfactory bulb and or sulci and gonadotrophin deficiency. We hereby report a young female who presented with features of hypogonadism and anosmia. A 17-year-old female presented to our endocrine clinic with primary amenorrhea. She was born of non-consanguineous marriage and her birth history was unremarkable and developmental mile stones were normal. On questioning, she admitted to the fact that she was not very appreciative of smell from the childhood when compared to other family members. Her stature was at par with her peers and there was neither a history of any chronic illness nor any medication. She had one male elder sibling whose growth and pubertal development were normal according to age. On examination, her height was 162 cm with a mid-parental height of 159 cm. There was no pallor or oedema or thyromegaly. There were no dysmorphic features. Her Tanner staging for breasts was prepubertal (Tanner 1) and pubic hair was also Tanner stage 1. Cardiovascular, respiratory and neurological examinations were within normal limits. Her blood investigations were as follows: TSH – 2 uIU/ml (Normal: 0.8-4), FT4-1.1 ng/dl (Normal: 0.8-1.8), FSH-0.2mIU/ml, LH-0.2mIU/ml, Prolactin-12 ng/ml (Normal<20), morning cortisol -16 μg/dl (Normal: 12-15), Hemoglobin- 13.2 gm/dl, Creatinine- 1.1 mg/dl (Normal: 0.8-1.3). She underwent MRI scanning of the brain [Table/Fig-1] which revealed the absence of olfactory bulb and olfactory sulci [Table/Fig-1]. The MRI brain of a normal subject is shown in [Table/Fig-2] for comparison. Her low gonadotropin levels in the absence of normal pubertal development and imaging features were suggestive of KS, a form of hypogonadotropic hypogonadism. There were no skeletal or renal abnormalities. She was initiated on cyclical oestrogen – progesterone pills with which she developed secondary sexual characters along with menstrual bleeding at one year follow up. [Table/Fig-1]: MRI brain of the patient showing absence of olfactory sulci and bulb [Table/Fig-2]: MRI brain of a normal subject displaying olfactory sulci and bulb KS is a heterogeneous genetic disorder which include hypogonadism and anosmia due to hypoplasia or agenesis of olfactory bulb and sulci along with GnRH deficiency [1]. It is a genetically heterogenous condition, with X-linked, autosomal dominant and recessive modes of inheritance due to mutataions in KAL 1, FGF 8/ FGFR1 and PROK2/PROK2R respectively [2]. The X linked forms may be associated with other phenotypic features. The autosomal forms of KS (A-KS) exhibit no consistent phenotype apart from gonadotropin deficiency and anosmia, although a heterogeneous collection of defects are seen in a minority of cases. The associated defects include cleft palate or other craniofacial and dental defects, unilateral renal agenesis or aplasia; cryptorchidism, micropenis, neural hearing defects and synkinesis or mirror movements of hand [3]. Since GnrH pulsation studies may not be possible at all centres, MRI with 1 mm cut is an important diagnostic tool for diagnosis of KS. Management of these subjects include screening for associated anomalies and monitoring for progressive worsening secondary to organ defects. The goal of the treatment is two folds. First, hormone replacement therapy is needed to induce puberty and to maintain gonadal hormones at near normal physiological levels till fifth decade of life. Secondly, fertility treatment involves administering gonadotrophins to induce ovulation.

Sirt1-Deficient Mice Have Hypogonadotropic Hypogonadism due to Defective GnRH Neuronal Migration

Hypogonadatropic hypogonadism (HH) can be acquired through energy restriction or may be inherited as congenital hypogonadotropic hypogonadism and its anosmia-associated form, Kallmann's syndrome. Congenital hypogonadotropic hypogonadism is associated with mutations in a group of genes that impact fibroblast growth factor 8 (FGF8) function. The Sirt1 gene encodes a nicotinamide adenine dinucleotide-dependent histone deacetylase that links intracellular metabolic stress to gene expression. Herein Sirt1(-/-) mice are shown to have HH due to failed GnRH neuronal migration. Sirtuin-1 (Sirt1) catalytic function induces GnRH neuronal migration via binding and deacetylating cortactin. Sirt1 colocalized with cortactin in GnRH neurons in vitro. Sirt1 colocalization with cortactin was regulated in an FGF8/fibroblast growth factor receptor-1 dependent manner. The profound effect of Sirt1 on the hormonal status of Sirt1(-/-) mice, mediated via defective GnRH neuronal migration, links energy metabolism directly to the hypogonadal state. Sirt1-cortactin may serve as the distal transducer of neuronal migration mediated by the FGF8 synexpression group of genes that govern HH.

Loss of Growth arrest specific gene 6 (Gas6) results in altered GnRH neuron migration, delayed vaginal opening and sexual maturation in mice

Prior work has shown the importance of TAM (Tyro3, Axl, Mer) receptor tyrosine kinases in GnRH neuronal development and reproductive function. It is unclear if TAM receptor actions are dependent on ligand activation for their functional effects; thus, we characterized reproductive phenotype of ligand Growth arrest specific gene (Gas6) null mice. Gas6 null mice showed delayed vaginal opening and delayed first estrus. Animals eventually attained normal estrous cycles as adults. The GnRH neuronal population was significantly decreased in Gas6 null adults and embryos, but the final positioning of cell bodies in the hypothalamus was normal. Vaginal tissue showed up-regulation of TAM receptor mRNAs in the absence of the ligand. These data confirm that Gas6 plays a role in early GnRH neuronal development and during vaginal opening. The phenotype of Gas6 KO mice suggests that TAMs function in a ligand-dependent and independent manner to control GnRH neuron development to modulate normal reproductive function.

Functional Consequences ofAXLSequence Variants in Hypogonadotropic Hypogonadism

Prior studies showed that Axl /Tyro3 null mice have delayed first estrus and abnormal cyclicity due to developmental defects in GnRH neuron migration and survival. The objective of the study was to test whether the absence of Axl would alter reproductive function in mice and that mutations in AXL are present in patients with Kallmann syndrome (KS) or normosmic idiopathic hypogonadotropic hypogonadism (nIHH). The sexual maturation of Axl null mice was examined. The coding region of AXL was sequenced in 104 unrelated, carefully phenotyped KS or nIHH subjects. Frequency of mutations was compared with other causes of GnRH deficiency. Functional assays were performed on the detected mutations. Axl null mice demonstrated delay in first estrus and the interval between vaginal opening and first estrus. Three missense AXL mutations (p.L50F, p.S202C, and p.Q361P) and one intronic variant 6 bp upstream from the start of exon 5 (c.586-6 C>T) were identified in two KS and 2 two nIHH subjects. Comparison of the frequencies of AXL mutations with other putative causes of idiopathic hypogonadotropic hypogonadism confirmed they are rare variants. Testing of the c.586-6 C>T mutation revealed no abnormal splicing. Surface plasmon resonance analysis of the p.L50F, p.S202C, and p.Q361P mutations showed no altered Gas6 ligand binding. In contrast, GT1-7 GnRH neuronal cells expressing p.S202C or p.Q361P demonstrated defective ligand dependent receptor processing and importantly aberrant neuronal migration. In addition, the p.Q361P showed defective ligand independent chemotaxis. Functional consequences of AXL sequence variants in patients with idiopathic hypogonadotropic hypogonadism support the importance of AXL and the Tyro3, Axl, Mer (TAM) family in reproductive development.