Wilson C. J. Chung

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.

Sex differences in the hypothalamus in the different stages of human life

Quite a number of structural and functional sex differences have been reported in the human hypothalamus and adjacent structures that may be related to not only reproduction, sexual orientation and gender identity, but also to the often pronounced sex differences in prevalence of psychiatric and neurological diseases. One of the recent focuses of interest in this respect is the possible beneficial effect of sex hormones on cognition in Alzheimer patients. The immunocytochemical localization of estrogen receptors (ER) alpha, beta and androgen receptors has shown that there are indeed numerous targets for sex hormones in the adult human brain. Observations in the infundibular nucleus have, however, indicated that in this brain area the hyperactivity resulting from a lack of estrogens in the menopause seems to protect females against Alzheimer changes, in contrast to males. It is thus quite possible that estrogen replacement therapy may, in these brain areas, lead to inhibition of neuronal metabolism and thus to the same proportion of Alzheimer changes as are observed in men. Knowledge about the functional sex differences in the brain and the effect of sex hormones on neuronal metabolism may thus provide clues not only for the possible beneficial effects of these hormones (e.g., on cognition or hypertension), but also on possible central side effects of estrogen replacement therapy.

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 thepathogenesis 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.

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.

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

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.

Compound Deficiencies in Multiple Fibroblast Growth Factor Signalling Components Differentially Impact the Murine Gonadotrophin‐Releasing Hormone System

Gonadotrophin‐releasing hormone (GnRH) neurones control the onset and maintenance of fertility. Aberrant development of the GnRH system underlies infertility in Kallmann syndrome [KS; idiopathic hypogonadotropic hypogonadism (IHH) and anosmia]. Some KS patients harbour mutations in the fibroblast growth factor receptor 1 (Fgfr1) and Fgf8 genes. The biological significance of these two genes in GnRH neuronal development was corroborated by the observation that GnRH neurones were severely reduced in newborn transgenic mice deficient in either gene. In the present study, we hypothesised that the compound deficiency of Fgf8 and its cognate receptors, Fgfr1 and Fgfr3, may lead to more deleterious effects on the GnRH system, thereby resulting in a more severe reproductive phenotype in patients harbouring these mutations. This hypothesis was tested by counting the number of GnRH neurones in adult transgenic mice with digenic heterozygous mutations in Fgfr1/Fgf8, Fgfr3/Fgf8 or Fgfr1/Fgfr3. Monogenic heterozygous mutations in Fgfr1, Fgf8 or Fgfr3 caused a 30–50% decrease in the total number of GnRH neurones. Interestingly, mice with digenic mutations in Fgfr1/Fgf8 showed a greater decrease in GnRH neurones compared to mice with a heterozygous defect in the Fgfr1 or Fgf8 alone. This compounding effect was not detected in mice with digenic heterozygous mutations in Fgfr3/Fgf8 or Fgfr1/Fgfr3. These results support the hypothesis that IHH/KS patients with digenic mutations in Fgfr1/Fgf8 may have a further reduction in the GnRH neuronal population compared to patients harbouring monogenic haploid mutations in Fgfr1 or Fgf8. Because only Fgfr1/Fgf8 compound deficiency leads to greater GnRH system defect, this also suggests that these fibroblast growth factor signalling components interact in a highly specific fashion to support GnRH neuronal development.

Fibroblast Growth Factor Signaling Deficiencies Impact Female Reproduction and Kisspeptin Neurons in Mice

ABSTRACT 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.