Absolute Quantification of Phosphorylated ERß Amino Acids in the Hippocampus of Women and in A Rat Model of Menopause.

The rapid decline of circulating 17ß-estradiol (E2) at menopause leads to negative neurological consequences, although hormone therapy paradoxically has both harmful and positive effects depending on the age at which it is delivered. The inconsistent response to E2 suggests unappreciated regulatory mechanisms for estrogen receptors (ERs), and we predicted it could be due to age-related differences in ERß phosphorylation. We assessed ERß phosphorylation using a sensitive mass spectrometry approach that provides absolute quantification (AQUA-MS) of individually phosphorylated residues. Specifically, we quantified phosphorylated ERß in the hippocampus of women (aged 21-83 years) and in a rat model of menopause at 4 residues with conserved sequence homology between the 2 species: S105, S176, S200, and Y488. Phosphorylation at these sites, which spanned all domains of ERß, were remarkably consistent between the 2 species, showing high levels of S105 phosphorylation (80%-100%) and low levels of S200 (20%-40%). Further, S200 phosphorylation decreased with aging in humans and loss of E2 in rats. Surprisingly, Y488 phosphorylation, which has been linked to ERß ligand-independent actions, exhibited approximately 70% phosphorylation, unaltered by species, age, or E2, suggesting ERß's primary mode of action may not require E2 binding. We further show phosphorylation at 2 sites directly altered ERß DNA-binding efficiency, and thus could affect its transcription factor activity. These findings provide the first absolute quantification of ERß phosphorylation in the human and rat brain, novel insights into ERß regulation, and a critical foundation for providing more targeted therapeutic options for menopause in the future.

Epigenomic control of gonadotrophin-releasing hormone neurone development and hypogonadotrophic hypogonadism.

Mammalian reproductive success depends on gonadotrophin-releasing hormone (GnRH) neurones to stimulate gonadotrophin secretion from the anterior pituitary and activate gonadal steroidogenesis and gametogenesis. Genetic screening studies in patients diagnosed with Kallmann syndrome (KS), a congenital form of hypogonadotrophic hypogonadism (CHH), identified several causal mutations, including those in the fibroblast growth factor (FGF) system. This signalling pathway regulates neuroendocrine progenitor cell proliferation, fate specification and cell survival. Indeed, the GnRH neurone system was absent or abrogated in transgenic mice with reduced (ie, hypomorphic) Fgf8 and/or Fgf receptor (Fgfr) 1 expression, respectively. Moreover, we found that GnRH neurones were absent in the embryonic olfactory placode of Fgf8 hypomorphic mice, the putative birthplace of GnRH neurones. These observations, together with those made in human KS/CHH patients, indicate that the FGF8/FGFR1 signalling system is a requirement for the ontogenesis of the GnRH neuronal system and function. In this review, we discuss how epigenetic factors control the expression of genes such as Fgf8 that are known to be critical for GnRH neurone ontogenesis, fate specification, and the pathogenesis of KS/CHH.

Neurodevelopmental wiring deficits in the Ts65Dn mouse model of Down syndrome.

Down syndrome is the most common genetic cause of intellectual disability and occurs due to the trisomy of human chromosome 21. Adolescent and adult brains from humans with Down syndrome exhibit various neurological phenotypes including a reduction in the size of the corpus callosum, hippocampal commissure and anterior commissure. However, it is unclear when and how these interhemispheric connectivity defects arise. Using the Ts65Dn mouse model of Down syndrome, we examined interhemispheric connectivity in postnatal day 0 (P0) Ts65Dn mouse brains. We find that there is no change in the volume of the corpus callosum or anterior commissure in P0 Ts65Dn mice. However, the volume of the hippocampal commissure is significantly reduced in P0 Ts65Dn mice, and this may contribute to the impaired learning and memory phenotype of this disorder. Interhemispheric connectivity defects that arise during development may be due to disrupted axon growth. In line with this, we find that developing hippocampal neurons display reduced axon length in vitro, as compared to neurons from their euploid littermates. This study is the first to report the presence of defective interhemispheric connectivity at the time of birth in Ts65Dn mice, providing evidence that early therapeutic intervention may be an effective time window for the treatment of Down syndrome.

TET1 regulates fibroblast growth factor 8 transcription in gonadotropin releasing hormone neurons.

Fibroblast growth factor 8 (FGF8) is a potent morphogen that regulates the ontogenesis of gonadotropin-releasing hormone (GnRH) neurons, which control the hypothalamus-pituitary-gonadal (HPG) axis, and therefore reproductive success. Indeed, FGF8 and FGFR1 deficiency severely compromises vertebrate reproduction in mice and humans and is associated with Kallmann Syndrome (KS), a congenital disease characterized by hypogonadotropic hypogonadism associated with anosmia. Our laboratory demonstrated that FGF8 signaling through FGFR1, both of which are KS-related genes, is necessary for proper GnRH neuron development in mice and humans. Here, we investigated the possibility that non-genetic factors, such as the epigenome, may contribute to KS onset. For this purpose, we developed an embryonic explant model, utilizing the mouse olfactory placode (OP), the birthplace of GnRH neurons. We show that TET1, which converts 5-methylcytosine residues (5mC) to 5-hydroxymethylated cytosines (5hmC), controls transcription of Fgf8 during GnRH neuron ontogenesis. Through MeDIP and ChIP RT-qPCR we found that TET1 bound to specific CpG islands on the Fgf8 promoter. We found that the temporal expression of Fgf8 correlates with not only TET1 binding, but also with 5hmC enrichment. siRNA knockdown of Tet1 reduced Fgf8 and Fgfr1 mRNA expression. During this time period, Fgf8 also switched histone status, most likely via recruitment of EZH2, a major component of the polycomb repressor complex-2 (PRC2) at E13.5. Together, these studies underscore the significance of epigenetics and chromatin modifications to temporally regulated genes involved in KS.

The Regulation and Function of Fibroblast Growth Factor 8 and Its Function during Gonadotropin-Releasing Hormone Neuron Development.

Over the last few years, numerous studies solidified the hypothesis that fibroblast growth factor (FGF) signaling regulates neuroendocrine progenitor cell proliferation, fate specification, and cell survival and, therefore, is critical for the regulation and maintenance of homeostasis of the body. One important example that underscores the involvement of FGF signaling during neuroendocrine cell development is gonadotropin-releasing hormone (GnRH) neuron ontogenesis. Indeed, transgenic mice with reduced olfactory placode (OP) Fgf8 expression do not have GnRH neurons. This observation indicates the requirement of FGF8 signaling for the emergence of the GnRH neuronal system in the embryonic OP, the putative birth place of GnRH neurons. Mammalian reproductive success depends on the presence of GnRH neurons to stimulate gonadotropin secretion from the anterior pituitary, which activates gonadal steroidogenesis and gametogenesis. Together, these observations are critical for understanding the function of GnRH neurons and their control of the hypothalamus-pituitary-gonadal (HPG) axis to maintain fertility. Taken together, these studies illustrate that GnRH neuron emergence and hence HPG function is vulnerable to genomic and molecular signals that abnormally modify Fgf8 expression in the developing mouse OP. In this short review, we focus on research that is aimed at unraveling how androgen, all-trans retinoic acid, and how epigenetic factors modify control mouse OP Fgf8 transcription in the context of GnRH neuronal development and mammalian reproductive success.

Perinatal midline astrocyte development is impaired in fibroblast growth factor 8 hypomorphic mice.

Our previous studies showed that Fgf8 mutations can cause Kallmann syndrome (KS), a form of congenital hypogonadotropic hypogonadism, in which patients do not undergo puberty and are infertile. Interestingly, some KS patients also have agenesis of the corpus callosum (ACC) suggesting that KS pathology is not limited to reproductive function. Here, we asked whether FGF8 dysfunction is the underlying cause of ACC in some KS patients. Indeed, early studies in transgenic mice with Fgf8 mutations reported the presence of failed or incomplete corpus callosum formation. Additional studies in transgenic mice showed that FGF8 function most likely prevents the prenatal elimination of glial fibrillary acidic protein (GFAP)-immunoreactive (IR) glial cells in the indusium griseum (IG) and midline zipper (MZ), two anterior-dorsal midline regions required for corpus callosum formation (i.e., between embryonic days (E) 15.5-18.5). Here, we tested the hypothesis that FGF8 function is critical for the survival of the GFAP-IR midline glial cells. First, we measured the incidence of apoptosis in the anterior-dorsal midline region in Fgf8 hypomorphic mice during embryonic corpus callosum formation. Second, we quantified the GFAP expression in the anterior-dorsal midbrain region during pre- and postnatal development, in order to study: 1) how Fgf8 hypomorphy disrupts prenatal GFAP-IR midline glial cell development, and 2) whether Fgf8 hypomorphy continues to disrupt postnatal GFAP-IR midline glial cell development. Our results indicate that perinatal FGF8 signaling is important for the timing of the onset of anterior-dorsal Gfap expression in midline glial cells suggesting that FGF8 function regulates midline GFAP-IR glial cell development, which when disrupted by Fgf8 deficiency prevents the formation of the corpus callosum. These studies provide an experimentally-based mechanistic explanation as to why corpus callosum formation may fail in KS patients with deficits in FGF signaling.

Fibroblast Growth Factor 8 Expression in GT1-7 GnRH-Secreting Neurons Is Androgen-Independent, but Can Be Upregulated by the Inhibition of DNA Methyltransferases.

Fibroblast growth factor 8 (FGF8) is a potent morphogen that regulates the embryonic development of hypothalamic neuroendocrine cells. Indeed, using Fgf8 hypomorphic mice, we showed that reduced Fgf8 mRNA expression completely eliminated the presence of gonadotropin-releasing hormone (GnRH) neurons. These findings suggest that FGF8 signaling is required during the embryonic development of mouse GnRH neurons. Additionally, in situ hybridization studies showed that the embryonic primordial birth place of GnRH neurons, the olfactory placode, is highly enriched for Fgf8 mRNA expression. Taken together these data underscore the importance of FGF8 signaling for GnRH emergence. However, an important question remains unanswered: How is Fgf8 gene expression regulated in the developing embryonic mouse brain? One major candidate is the androgen receptor (AR), which has been shown to upregulate Fgf8 mRNA in 60-70% of newly diagnosed prostate cancers. Therefore, we hypothesized that ARs may be involved in the regulation of Fgf8 transcription in the developing mouse brain. To test this hypothesis, we used chromatin-immunoprecipitation (ChIP) assays to elucidate whether ARs interact with the 5'UTR region upstream of the translational start site of the Fgf8 gene in immortalized mouse GnRH neurons (GT1-7) and nasal explants. Our data showed that while AR interacts with the Fgf8 promoter region, this interaction was androgen-independent, and that androgen treatment did not affect Fgf8 mRNA levels, indicating that androgen signaling does not induce Fgf8 transcription. In contrast, inhibition of DNA methyltransferases (DNMT) significantly upregulated Fgf8 mRNA levels indicating that Fgf8 transcriptional activity may be dependent on DNA methylation status.

Fibroblast growth factor 8 regulates postnatal development of paraventricular nucleus neuroendocrine cells.

BACKGROUND: Fibroblast growth factors (FGFs) are crucial signaling molecules that direct the development of the vertebrate brain. FGF8 gene signaling in particular, may be important for the development of the hypothalamus-pituitary-adrenal (HPA)-axis. Indeed, newborn Fgf8 hypomorphic mice harbor a major reduction in the number of vasopressin (VP) neurons in the paraventricular nucleus (PVN), the central output component of the HPA-axis. Additionally, recent studies indicated that adult heterozygous ((+/neo)) Fgf8 hypomorphic mice exhibit more anxiety-like behaviors than wildtype (WT) mice. These studies led us to investigate whether Fgf8 hypomorphy abrogated VP and/or corticotropin-releasing hormone (CRH) neuronal development in the postnatal day (PN) 21 and adult mouse PVN. Furthermore, we studied whether Fgf8 hypomorphy disrupted HPA responsiveness in these mice. METHODS: Using immunohistochemistry, we examined the development of VP and CRH neurons located in the PVN of PN 21 and adult Fgf8 (+/neo) mice. Moreover, we used a restraint stress (RS) paradigm and measured corticosterone levels with enzyme immunoassays in order to assess HPA axis activation. RESULTS: The number of VP neurons in the PVN did not differ between WT and Fgf8 (+/neo) mice on PN 21 and in adulthood. In contrast, CRH immunoreactivity was much higher in Fgf8 (+/neo) mice than in WT mice on PN 21, this difference was no longer shown in adult mice. RS caused a higher increase in corticosterone levels in adult Fgf8 (+/neo) mice than in WT mice after 15 min, but no difference was seen after 45 min. CONCLUSIONS: First, Fgf8 hypomorphy did not eliminate VP and CRH neurons in the mouse PVN, but rather disrupted the postnatal timing of neuropeptide expression onset in PVN neurons. Second, Fgf8 hypomorphy may, in part, be an explanation for affective disorders involving hyperactivity of the HPA axis, such as anxiety.

Ontogenesis of gonadotropin-releasing hormone neurons: a model for hypothalamic neuroendocrine cell development.

The vertebrate hypothalamo-pituitary-gonadal axis is the anatomical framework responsible for reproductive competence and species propagation. Essential to the coordinated actions of this three-tiered biological system is the fact that the regulatory inputs ultimately converge on the gonadotropin-releasing hormone (GnRH) neuronal system, which in rodents primarily resides in the preoptic/hypothalamic region. In this short review we will focus on: (1) the general embryonic temporal and spatial development of the rodent GnRH neuronal system, (2) the origin(s) of GnRH neurons, and (3) which transcription - and growth factors have been found to be critical for GnRH neuronal ontogenesis and cellular fate-specification. Moreover, we ask the question whether the molecular and cellular mechanisms involved in GnRH neuronal development may also play a role in the development of other hypophyseal secreting neuroendocrine cells in the hypothalamus.

MicroRNAs in the aging female brain: a putative mechanism for age-specific estrogen effects.

Menopause is characterized by the rapid age-related decline of circulating 17ß-estradiol (E(2)) levels in women, which can sometimes result in cognitive disorders such as impaired memory and increased anxiety. Hormone therapy (HT) is a widely used treatment for the adverse effects associated with menopause; however, evidence suggests that HT administered to postmenopausal women age 65 years and over can lead to increased risks for cognitive disorders. We hypothesized that these age-related changes in E(2) action are due to posttranscriptional gene regulation by microRNAs (miRNAs). miRNAs are a class of small noncoding RNAs that regulate gene expression by binding to the 3'-untranslated region of target mRNAs and subsequently target these transcripts for degradation. In the present study, 3- and 18-month-old female rats were oophorectomized (OVX) and treated 1 week after surgery with 2.5 µg E(2) once per day for 3 days. Total RNA was isolated from the ventral and dorsal hippocampus, central amygdala, and paraventricular nucleus. Our results showed that E(2) differentially altered miRNA levels in an age- and brain region-dependent manner. Multiple miRNA target prediction algorithms revealed putative target genes that are important for memory and stress regulation, such as BDNF, glucocorticoid receptor, and SIRT-1. Indeed, quantitative RT-PCR analyses of some of the predicted targets, such as SIRT1, showed that the mRNA expression levels were the inverse of the targeting miRNA, thereby confirming the prediction algorithms. Taken together, these data show that E(2) regulates miRNA expression in an age- and E(2)-dependent manner, which we hypothesize results in differential gene expression and consequently altered neuronal function.

Gender differences in neurodevelopment and epigenetics.

The concept that the brain differs in make-up between males and females is not new. For example, it is well established that anatomists in the nineteenth century found sex differences in human brain weight. The importance of sex differences in the organization of the brain cannot be overstated as they may directly affect cognitive functions, such as verbal skills and visuospatial tasks in a sex-dependent fashion. Moreover, the incidence of neurological and psychiatric diseases is also highly dependent on sex. These clinical observations reiterate the importance that gender must be taken into account as a relevant possible contributing factor in order to understand the pathogenesis of neurological and psychiatric disorders. Gender-dependent differentiation of the brain has been detected at every level of organization--morphological, neurochemical, and functional--and has been shown to be primarily controlled by sex differences in gonadal steroid hormone levels during perinatal development. In this review, we discuss howthe gonadal steroid hormone testosterone and its metabolites affect downstream signaling cascades, including gonadal steroid receptor activation, and epigenetic events in order to differentiate the brain in a gender-dependent fashion.

Opposite-sex housing reactivates the declining GnRH system in aged transgenic male mice with FGF signaling deficiency.

The continued presence of gonadotropin-releasing hormone (GnRH) neurons is required for a healthy reproductive lifespan, but factors that maintain postnatal GnRH neurons have not been identified. To begin to understand these factors, we investigated whether 1) fibroblast growth factor (FGF) signaling and 2) interactions with the opposite sex are involved in the maintenance of the postnatal GnRH system. A transgenic mouse model (dnFGFR mouse) with the targeted expression of a dominant-negative FGF receptor (dnFGFR) in GnRH neurons was used to examine the consequence of FGF signaling deficiency on postnatal GnRH neurons. Male dnFGFR mice suffered a significant loss of postnatal GnRH neurons within the first 100 days of life. Interestingly, this loss was reversed after cohabitation with female, but not male, mice for 300-550 days. Along with a rescue in GnRH neuron numbers, opposite-sex housing in dnFGFR males also increased hypothalamic GnRH peptide levels, promoted a more mature GnRH neuronal morphology, facilitated litter production, and enhanced testicular morphology. Last, mice hypomorphic for FGFR3 exhibited a similar pattern of postnatal GnRH neuronal loss as dnFGFR males, suggesting FGF signaling acts, in part, through FGFR3 to enhance the maintenance of the postnatal GnRH system. In summary, we have shown that FGF signaling is required for the continued presence of postnatal GnRH neurons. However, this requirement is not absolute, since sexual interactions can compensate for defects in FGFR signaling, thereby rescuing the declining GnRH system. This suggests the postnatal GnRH system is highly plastic and capable of responding to environmental stimuli throughout adult life.

Fibroblast growth factor signaling deficiencies impact female reproduction and kisspeptin neurons in mice.

Fibroblast growth factor (FGF) signaling is essential for the development of the gonadotropin-releasing hormone (GnRH) system. Mice harboring deficiencies in Fgf8 or Fgf receptor 1 (Fgfr1) suffer a significant loss of GnRH neurons, but their reproductive phenotypes have not been examined. This study examined if female mice hypomorphic for Fgf8, Fgfr1, or both (compound hypomorphs) exhibited altered parameters of pubertal onset, estrous cyclicity, and fertility. Further, we examined the number of kisspeptin (KP)-immunoreactive (ir) neurons in the anteroventral periventricular/periventricular nuclei (AVPV/PeV) of these mice to assess if changes in the KP system, which stimulates the GnRH system, could contribute to the reproductive phenotypes. Single hypomorphs (Fgfr1(+/-) or Fgf8(+/-)) had normal timing for vaginal opening (VO) but delayed first estrus. However, after achieving the first estrus, they underwent normal expression of estrous cycles. In contrast, the compound hypomorphs underwent early VO and normal first estrus, but had disorganized estrous cycles that subsequently reduced their fertility. KP immunohistochemistry on Postnatal Day 15, 30, and 60 transgenic female mice revealed that female compound hypomorphs had significantly more KP-ir neurons in the AVPV/PeV compared to their wild-type littermates, suggesting increased KP-ir neurons may drive early VO but could not maintain the cyclic changes in GnRH neuronal activity required for female fertility. Overall, these data suggest that Fgf signaling deficiencies differentially alter the parameters of female pubertal onset and cyclicity. Further, these deficiencies led to changes in the AVPV/PeV KP-ir neurons that may have contributed to the accelerated VO in the compound hypomorphs.

Fibroblast growth factor signaling in the developing neuroendocrine hypothalamus.

Fibroblast growth factor (FGF) signaling is pivotal to the formation of numerous central regions. Increasing evidence suggests FGF signaling also directs the development of the neuroendocrine hypothalamus, a collection of neuroendocrine neurons originating primarily within the nose and the ventricular zone of the diencephalon. This review outlines evidence for a role of FGF signaling in the prenatal and postnatal development of several hypothalamic neuroendocrine systems. The emphasis is placed on the nasally derived gonadotropin-releasing hormone neurons, which depend on neurotrophic cues from FGF signaling throughout the neurons' lifetime. Although less is known about neuroendocrine neurons derived from the diencephalon, recent studies suggest they also exhibit variable levels of dependence on FGF signaling. Overall, FGF signaling provides a broad spectrum of cues that ranges from genesis, cell survival/death, migration, morphological changes, to hormone synthesis in the neuroendocrine hypothalamus. Abnormal FGF signaling will deleteriously impact multiple hypothalamic neuroendocrine systems, resulting in the disruption of diverse physiological functions.

Abnormal hypothalamic oxytocin system in fibroblast growth factor 8-deficient mice.

Oxytocin (OT) is a nonapeptide essential for maternal care. The development of the OT neuroendocrine system is a multi-step process dependent on the action of many transcription factors, but upstream signaling molecules regulating this process are still poorly understood. In this study, we examined if fibroblast growth factor 8 (FGF8), a signaling molecule critical for forebrain development, is essential for the proper formation of the OT system. Using immunohistochemistry, we showed a significant reduction in the number of neurons immunoreactive for the mature OT peptide in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) in the hypothalamus of homozygous (HOMO) FGF8 hypomorphic mice compared to wild-type mice. The number of neurons positive for oxyphysin prohormone in the SON but not the PVN was also significantly reduced in FGF8 HOMO hypomorphs. However, steady-state mRNA levels of the oxyphysin prohormone were not significantly different between FGF8 hypomorphs and WT mice. These data suggest that a global reduction in FGF8 signaling leads to an overall reduction of mature OT and oxyphysin prohormone levels that may have resulted from defects in multiple stages of the hormone-synthesis pathway. Since proper hormone synthesis is a hallmark of mature OT neurons, this study suggests that FGF8 signaling may contribute to the phenotypic maturation of a neuroendocrine system that originates within the diencephalon.

Role of fibroblast growth factor signaling in gonadotropin-releasing hormone neuronal system development.

There is growing evidence demonstrating that fibroblast growth factor (FGF) signaling is important for the development of the gonadotropin-releasing hormone (GnRH) neuronal system. In humans, loss-of-function mutations in FGF receptor 1 (Fgfr1) and Fgf8 lead to hypogonadotropic hypogonadism (HH) with or without anosmia. Insights into how FGF signaling deficiency disrupts the GnRH system in humans are beginning to emerge from studies using transgenic mouse models. In this review, we summarize GnRH system defects in several lines of FGF signaling-deficient mice. We showed that FGF signaling is critically required for olfactory placode induction, differentiation, and GnRH neuronal fate specification and postnatal maintenance. Extrapolating from these transgenic mouse data, we suggest that idiopathic HH in patients harboring loss-of-function Fgfr1 and/or Fgf8 mutations is not merely a result of defective GnRH neuronal migration, but also insults accumulated in the GnRH system during fate specification and postnatal development.

Differential fibroblast growth factor 8 (FGF8)-mediated autoregulation of its cognate receptors, Fgfr1 and Fgfr3, in neuronal cell lines.

Fibroblast growth factors (FGFs) mediate a vast range of CNS developmental processes including neural induction, proliferation, migration, and cell survival. Despite the critical role of FGF signaling for normal CNS development, few reports describe the mechanisms that regulate FGF receptor gene expression in the brain. We tested whether FGF8 could autoregulate two of its cognate receptors, Fgfr1 and Fgfr3, in three murine cell lines with different lineages: fibroblast-derived cells (3T3 cells), neuronal cells derived from hippocampus (HT-22 cells), and neuroendocrine cells derived from hypothalamic gonadotropin-releasing hormone (GnRH) neurons (GT1-7 cells). GnRH is produced by neurons in the hypothalamus and is absolutely required for reproductive competence in vertebrate animals. Several lines of evidence strongly suggest that Fgf8 is critical for normal development of the GnRH system, therefore, the GT1-7 cells provided us with an additional endpoint, Gnrh gene expression and promoter activity, to assess potential downstream consequences of FGF8-induced modulation of FGF receptor levels. Results from this study suggest that the autoregulation of its cognate receptor represents a common downstream effect of FGF8. Further, we show that Fgfr1 and Fgfr3 are differentially regulated within the same cell type, implicating these two receptors in different biological roles. Moreover, Fgfr1 and Fgfr3 are differentially regulated among different cell types, suggesting such autoregulation occurs in a cell type-specific fashion. Lastly, we demonstrate that FGF8b decreases Gnrh promoter activity and gene expression, possibly reflecting a downstream consequence of altered FGF receptor populations. Together, our data bring forth the possibility that, in addition to the FGF synexpression group, autoregulation of FGFR expression by FGF8 represents a mechanism by which FGF8 could fine-tune its regulatory actions.

Arginine vasopressin regulation in pre- and postpubertal male rats by the androgen metabolite 3beta-diol.

Arginine vasopressin (AVP) is a nonapeptide expressed in several brain regions. In addition to its well-characterized role in osmoregulation, AVP regulates paternal behavior, aggression,circadian rhythms, and the stress response. In the bed nucleus of the stria terminalis (BST), AVP gene expression is tightly regulated by gonadal steroid hormones. However, the degree by which AVP is regulated by gonadal steroid hormones in the suprachiasmatic nucleus (SCN) and medial amygdala (MeA) is unclear. Previous studies have shown that AVP expression in the brain of gonadectomized rats is restored with testosterone, 17beta-estradiol, and 5alpha-dihydrotestosterone(DHT) replacement. In addition, we have demonstrated that 3beta-diol, a metabolite of DHT,increased AVP promoter activity in a neuronal cell line and that the effects of 3beta-diol on AVP promoter activity were mediated by estrogen receptor-beta. To test whether 3beta-diol has a physiological role in the regulation of central AVP expression in vivo, we gonadectomized pre- and postpubertal male rats and followed with once daily injections of estradiol benzoate (EB),DHT-propionate, 3beta-diol-dipropionate, or vehicle. The SCN, BST, and MeA were analyzed for AVP mRNA expression using in situ hybridization. In the BST, intact juveniles had significantly fewer AVP-expressing cells than adults. GDX abolished all AVP mRNA expression in the BST in both age groups, whereas treatment with EB restored >80% and DHTP <10% of the AVP expression. Interestingly, 3beta-diol-proprionate was more effective at inducing AVP expression in juveniles than in adults, suggesting that the regulation of AVP by 3beta-diol might be age dependent [corrected].

Decreased FGF8 signaling causes deficiency of gonadotropin-releasing hormone in humans and mice.

Idiopathic hypogonadotropic hypogonadism (IHH) with anosmia (Kallmann syndrome; KS) or with a normal sense of smell (normosmic IHH; nIHH) are heterogeneous genetic disorders associated with deficiency of gonadotropin-releasing hormone (GnRH). While loss-of-function mutations in FGF receptor 1 (FGFR1) cause human GnRH deficiency, to date no specific ligand for FGFR1 has been identified in GnRH neuron ontogeny. Using a candidate gene approach, we identified 6 missense mutations in FGF8 in IHH probands with variable olfactory phenotypes. These patients exhibited varied degrees of GnRH deficiency, including the rare adult-onset form of hypogonadotropic hypogonadism. Four mutations affected all 4 FGF8 splice isoforms (FGF8a, FGF8b, FGF8e, and FGF8f), while 2 mutations affected FGF8e and FGF8f isoforms only. The mutant FGF8b and FGF8f ligands exhibited decreased biological activity in vitro. Furthermore, mice homozygous for a hypomorphic Fgf8 allele lacked GnRH neurons in the hypothalamus, while heterozygous mice showed substantial decreases in the number of GnRH neurons and hypothalamic GnRH peptide concentration. In conclusion, we identified FGF8 as a gene implicated in GnRH deficiency in both humans and mice and demonstrated an exquisite sensitivity of GnRH neuron development to reductions in FGF8 signaling.

Fibroblast growth factor 8 signaling through fibroblast growth factor receptor 1 is required for the emergence of gonadotropin-releasing hormone neurons.

GnRH neurons are essential for the onset and maintenance of reproduction. Mutations in both fibroblast growth factor receptor (Fgfr1) and Fgf8 have been shown to cause Kallmann syndrome, a disease characterized by hypogonadotropic hypogonadism and anosmia, indicating that FGF signaling is indispensable for the formation of a functional GnRH system. Presently it is unclear which stage of GnRH neuronal development is most impacted by FGF signaling deficiency. GnRH neurons express both FGFR1 and -3; thus, it is also unclear whether FGFR1 or FGFR3 contributes directly to GnRH system development. In this study, we examined the developing GnRH system in mice deficient in FGF8, FGFR1, or FGFR3 to elucidate the individual contribution of these FGF signaling components. Our results show that the early emergence of GnRH neurons from the embryonic olfactory placode requires FGF8 signaling, which is mediated through FGFR1, not FGFR3. These data provide compelling evidence that the developing GnRH system is exquisitely sensitive to reduced levels of FGF signaling. Furthermore, Kallmann syndrome stemming from FGF signaling deficiency may be due primarily to defects in early GnRH neuronal development prior to their migration into the forebrain.