Dax1 modulates ERα-dependent hypothalamic estrogen sensing in female mice.

Coupling the release of pituitary hormones to the developmental stage of the oocyte is essential for female fertility. It requires estrogen to restrain kisspeptin (KISS1)-neuron pulsatility in the arcuate hypothalamic nucleus, while also exerting a surge-like effect on KISS1-neuron activity in the AVPV hypothalamic nucleus. However, a mechanistic basis for this region-specific effect has remained elusive. Our genomic analysis in female mice demonstrate that some processes, such as restraint of KISS1-neuron activity in the arcuate nucleus, may be explained by region-specific estrogen receptor alpha (ERα) DNA binding at gene regulatory regions. Furthermore, we find that the Kiss1-locus is uniquely regulated in these hypothalamic nuclei, and that the nuclear receptor co-repressor NR0B1 (DAX1) restrains its transcription specifically in the arcuate nucleus. These studies provide mechanistic insight into how ERα may control the KISS1-neuron, and Kiss1 gene expression, to couple gonadotropin release to the developmental stage of the oocyte.

Sexually Dimorphic Neurosteroid Synthesis Regulates Neuronal Activity in the Murine Brain.

Sex steroid hormones act on hypothalamic kisspeptin neurons to regulate reproductive neural circuits in the brain. Kisspeptin neurons start to express estrogen receptors in utero, suggesting steroid hormone action on these cells early during development. Whether neurosteroids are locally produced in the embryonic brain and impinge onto kisspeptin/reproductive neural circuitry is not known. To address this question, we analyzed aromatase expression, a key enzyme in estrogen synthesis, in male and female mouse embryos. We identified an aromatase neuronal network comprising ∼6000 neurons in the hypothalamus and amygdala. By birth, this network has become sexually dimorphic in a cluster of aromatase neurons in the arcuate nucleus adjacent to kisspeptin neurons. We demonstrate that male arcuate aromatase neurons convert testosterone to estrogen to regulate kisspeptin neuron activity. We provide spatiotemporal information on aromatase neuronal network development and highlight a novel mechanism whereby aromatase neurons regulate the activity of distinct neuronal populations expressing estrogen receptors.SIGNIFICANCE STATEMENT Sex steroid hormones, such as estradiol, are important regulators of neural circuits controlling reproductive physiology in the brain. Embryonic kisspeptin neurons in the hypothalamus express steroid hormone receptors, suggesting hormone action on these cells in utero Whether neurosteroids are locally produced in the brain and impinge onto reproductive neural circuitry is insufficiently understood. To address this question, we analyzed aromatase expression, a key enzyme in estradiol synthesis, in mouse embryos and identified a network comprising ∼6000 neurons in the brain. By birth, this network has become sexually dimorphic in a cluster of aromatase neurons in the arcuate nucleus adjacent to kisspeptin neurons. We demonstrate that male aromatase neurons convert testosterone to estradiol to regulate kisspeptin neuron activity.

Optogenetic stimulation of kisspeptin neurones within the posterodorsal medial amygdala increases luteinising hormone pulse frequency in female mice.

Kisspeptin within the arcuate nucleus of the hypothalamus is a critical neuropeptide in the regulation of reproduction. Together with neurokinin B and dynorphin A, arcuate kisspeptin provides the oscillatory activity that drives the pulsatile secretion of gonadotrophin-releasing hormone (GnRH), and therefore luteinising hormone (LH) pulses, and is considered to be a central component of the GnRH pulse generator. It is well established that the amygdala also exerts an influence over gonadotrophic hormone secretion and reproductive physiology. The discovery of kisspeptin and its receptor within the posterodorsal medial amygdala (MePD) and our recent finding showing that intra-MePD administration of kisspeptin or a kisspeptin receptor antagonist results in increased LH secretion and decreased LH pulse frequency, respectively, suggests an important role for amygdala kisspeptin signalling in the regulation of the GnRH pulse generator. To further investigate the function of amygdala kisspeptin, the present study used an optogenetic approach to selectively stimulate MePD kisspeptin neurones and examine the effect on pulsatile LH secretion. MePD kisspeptin neurones in conscious Kiss1-Cre mice were virally infected to express the channelrhodopsin 2 protein and selectively stimulated by light via a chronically implanted fibre optic cannula. Continuous stimulation using 5 Hz resulted in an increased LH pulse frequency, which was not observed at the lower stimulation frequencies of 0.5 and 2 Hz. In wild-type animals, continuous stimulation at 5 Hz did not affect LH pulse frequency. These results demonstrate that selective activation of MePD Kiss1 neurones can modulate hypothalamic GnRH pulse generator frequency.

Mechanistic insights into the more potent effect of KP-54 compared to KP-10 in vivo.

Kisspeptins regulate the mammalian reproductive axis by stimulating release of gonadotrophin releasing hormone (GnRH). Different length kisspeptins (KP) are found of 54, 14, 13 or 10 amino-acids which share a common C-terminal 10-amino acid sequence. KP-54 and KP-10 have been widely used to stimulate the reproductive axis but data suggest that KP-54 and KP-10 are not equally effective at eliciting reproductive hormone secretion after peripheral delivery. To confirm this, we analysed the effect of systemic administration of KP-54 or KP-10 on luteinizing hormone (LH) secretion into the bloodstream of male mice. Plasma LH measurements 10 min or 2 hours after kisspeptin injection showed that KP-54 can sustain LH release far longer than KP-10, suggesting a differential mode of action of the two peptides. To investigate the mechanism for this, we evaluated the pharmacokinetics of the two peptides in vivo and their potential to cross the blood brain barrier (BBB). We found that KP-54 has a half-life of ~32 min in the bloodstream, while KP-10 has a half-life of ~4 min. To compensate for this difference in half-life, we repeated injections of KP-10 every 10 min over 1 hr but failed to reproduce the sustained rise in LH observed after a single KP-54 injection, suggesting that the failure of KP-10 to sustain LH release may not just be related to peptide clearance. We tested the ability of peripherally administered KP-54 and KP-10 to activate c-FOS in GnRH neurons behind the blood brain barrier (BBB) and found that only KP-54 could do this. These data are consistent with KP-54 being able to cross the BBB and suggest that KP10 may be less able to do so.

Kisspeptin-GPR54 signaling in mouse NO-synthesizing neurons participates in the hypothalamic control of ovulation.

Reproduction is controlled in the brain by a neural network that drives the secretion of gonadotropin-releasing hormone (GnRH). Various permissive homeostatic signals must be integrated to achieve ovulation in mammals. However, the neural events controlling the timely activation of GnRH neurons are not completely understood. Here we show that kisspeptin, a potent activator of GnRH neuronal activity, directly communicates with neurons that synthesize the gaseous transmitter nitric oxide (NO) in the preoptic region to coordinate the progression of the ovarian cycle. Using a transgenic Gpr54-null IRES-LacZ knock-in mouse model, we demonstrate that neurons containing neuronal NO synthase (nNOS), which are morphologically associated with kisspeptin fibers, express the kisspeptin receptor GPR54 in the preoptic region, but not in the tuberal region of the hypothalamus. The activation of kisspeptin signaling in preoptic neurons promotes the activation of nNOS through its phosphorylation on serine 1412 via the AKT pathway and mimics the positive feedback effects of estrogens. Finally, we show that while NO release restrains the reproductive axis at stages of the ovarian cycle during which estrogens exert their inhibitory feedback, it is required for the kisspeptin-dependent preovulatory activation of GnRH neurons. Thus, interactions between kisspeptin and nNOS neurons may play a central role in regulating the hypothalamic-pituitary-gonadal axis in vivo.

Kisspeptin neurons co-express met-enkephalin and galanin in the rostral periventricular region of the female mouse hypothalamus.

It is now well established that the kisspeptin neurons of the hypothalamus play a key role in regulating the activity of gonadotropin-releasing hormone (GnRH) neurons. The population of kisspeptin neurons residing in the rostral periventricular region of the third ventricle (RP3V), encompassing the anteroventral periventricular (AVPV) and periventricular preoptic nuclei (PVpo), are implicated in the generation of the preovulatory GnRH surge mechanism and puberty onset in female rodents. The present study examined whether these kisspeptin neurons may express other neuropeptides in the adult female mouse. Initially, the distribution of galanin, neurotensin, met-enkephalin (mENK), and cholecystokinin (CCK)-immunoreactive cells was determined within the RP3V of colchicine-treated mice. Subsequent experiments, using a new kisspeptin-10 antibody raised in sheep, examined the relationship of these neuropeptides to kisspeptin neurons. No evidence was found for expression of neurotensin or CCK by RP3V kisspeptin neurons, but subpopulations of kisspeptin neurons were observed to express galanin and mENK. Dual-labeled RP3V kisspeptin/galanin cells represented 7% of all kisspeptin and 21% of all galanin neurons whereas dual-labeled kisspeptin/mENK cells represented 28-38% of kisspeptin neurons and 58-68% of the mENK population, depending on location within the AVPV or PVpo. Kisspeptin neurons in the arcuate nucleus were also found to express galanin but not mENK. These observations indicate that, like the kisspeptin population of the arcuate nucleus, kisspeptin neurons in the RP3V also co-express a range of neuropeptides. This pattern of co-expression should greatly increase the dynamic range with which kisspeptin neurons can modulate the activity of their afferent neurons.

The testosterone-dependent and independent transcriptional networks in the hypothalamus of Gpr54 and Kiss1 knockout male mice are not fully equivalent.

BACKGROUND: Humans and mice with loss of function mutations in GPR54 (KISS1R) or kisspeptin do not progress through puberty, caused by a failure to release GnRH. The transcriptional networks regulated by these proteins in the hypothalamus have yet to be explored by genome-wide methods. RESULTS: We show here, using 1 million exon mouse arrays (Exon 1.0 Affymetrix) and quantitative polymerase chain reaction (QPCR) validation to analyse microdissected hypothalamic tissue from Gpr54 and Kiss1 knockout mice, the extent of transcriptional regulation in the hypothalamus. The sensitivity to detect important transcript differences in microdissected RNA was confirmed by the observation of counter-regulation of Kiss1 expression in Gpr54 knockouts and confirmed by immunohistochemistry (IHC). Since Gpr54 and Kiss1 knockout animals are effectively pre-pubertal with low testosterone (T) levels, we also determined which of the validated transcripts were T-responsive and which varied according to genotype alone. We observed four types of transcriptional regulation (i) genotype only dependent regulation, (ii) T only dependent regulation, (iii) genotype and T-dependent regulation with interaction between these variables, (iv) genotype and T-dependent regulation with no interaction between these variables. The results implicate for the first time several transcription factors (e.g. Npas4, Esr2), proteases (Klk1b22), and the orphan 10-transmembrane transporter TMEM144 in the biology of GPR54/kisspeptin function in the hypothalamus. We show for the neuronal activity regulated transcription factor NPAS4, that distinct protein over-expression is seen in the hypothalamus and hippocampus in Gpr54 knockout mice. This links for the first time the hypothalamic-gonadal axis with this important regulator of inhibitory synapse formation. Similarly we confirm TMEM144 up-regulation in the hypothalamus by RNA in situ hybridization and western blot. CONCLUSIONS: Taken together, global transcriptional profiling shows that loss of GPR54 and kisspeptin are not fully equivalent in the mouse hypothalamus.

Gpr54-/- mice show more pronounced defects in spermatogenesis than Kiss1-/- mice and improved spermatogenesis with age when exposed to dietary phytoestrogens.

Mice with mutations in the kisspeptin signaling pathway (Kiss1(-/-) or Gpr54(-/-)) have low gonadotrophic hormone levels, small testes, and impaired spermatogenesis. Between 2 and 7 months of age, however, the testes of the mutant mice increase in weight and in Gpr54(-/-) mice, the number of seminiferous tubules containing spermatids/spermatozoa increases from 17 to 78%. In contrast, the Kiss1(-/-) mice have a less severe defect in spermatogenesis and larger testes than Gpr54(-/-) mice at both 2 and 7 months of age. The reason for the improved spermatogenesis was investigated. Plasma testosterone and FSH levels did not increase with age in the mutant mice and remained much lower than in wild-type (WT) mice. In contrast, intratesticular testosterone levels were similar between mutant and WT mice. These data indicate that age-related spermatogenesis can be completed under conditions of low plasma testosterone and FSH and that intratesticular testosterone may contribute to this process. In addition, however, when the Gpr54(-/-) mice were fed a phytoestrogen-free diet, they showed no age-related increase in testes weight or improved spermatogenesis. Thus, both genetic and environmental factors are involved in the improved spermatogenesis in the mutant mice as they age although the mice still remain infertile. These data show that the possible impact of dietary phytoestrogens should be taken into account when studying the phenotype of mutant mice with defects in the reproductive axis.

Kisspeptin can stimulate gonadotropin-releasing hormone (GnRH) release by a direct action at GnRH nerve terminals.

The G protein-coupled receptor GPR54, and its peptide ligand kisspeptin (Kp), are crucial for the induction and maintenance of mammalian reproductive function. GPR54 is expressed by GnRH neurons and is directly activated by Kp to stimulate GnRH release. We hypothesized that Kp may be able to act at the GnRH nerve terminals located in the mediobasal hypothalamus (MBH) region. To test this hypothesis, we used organotypic culture of MBH explants challenged with Kp, followed by RIA to detect GnRH released into the cultured medium. Kp stimulation for 1 h induced GnRH release from wild-type male MBH in a dose-dependent manner, whereas this did not occur in MBH explants isolated from Gpr54 null mice. Continuous Kp stimulation caused a sustained GnRH release for 4 h, followed by a decrease of GnRH release, suggesting a desensitization of GPR54 activity. Tetrodotoxin did not alter the Kp-induced GnRH release, indicating that Kp can act directly at the GnRH nerve terminals. To localize Gpr54 expression within the MBH, we used transgenic mice, in which Gpr54 expression is tagged with an IRES-LacZ reporter gene and can be visualized by beta-galactosidase staining. Gpr54 expression was detected outside of the median eminence, in the pars tuberalis. In conclusion, our results provide evidence for a potent stimulating effect of Kp at GnRH nerve terminals in the MBH of the mouse. This study suggests a new point at which Kp can act on GnRH neurons.

The GPR54 gene as a regulator of puberty.

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