GnRH Cells Research Articles

NAGK Regulates the Onset of Puberty in Female Mice

This study examines the role of N-acetylglucosamine kinase (NAGK) in initiating puberty in female mice. We employed real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunofluorescence to measure NAGK expression in the hypothalamic-pituitary-ovarian axis across various developmental stages: infant, prepuberty, puberty, and adult. We further investigated the impact of Nagk gene knockdown on puberty in female mice. This included assessing the expression of puberty-related genes both in vivo and in vitro, GT1-7 cells proliferation and apoptosis, concentrations of GnRH and Kisspeptin, puberty onset timing, serum levels of progesterone (P4) and estradiol (E2), and ovarian morphology. Results revealed that Nagk mRNA is present in the hypothalamus, pituitary, and ovaries throughout different developmental stages in female mice. In the hypothalamus, Nagk mRNA levels were comparable during infant and prepuberty, lowest during puberty, and highest in adult. In the pituitary, Nagk mRNA peaked in adult, with no significant variation between infant, prepuberty, and puberty. In the ovaries, Nagk mRNA levels increased during puberty and peaked in adult. NAGK is predominantly located in the arcuate nucleus (ARC), periventricular nucleus (PeN), dorsomedial hypothalamic nucleus (DMH), paraventricular nucleus (PVN), adenohypophysis, and in the ovarian oocytes, interstitium, and granulosa cells across all developmental stages in female mice. Nagk knockdown in GT1-7 cells decreased the transcriptional level of Gnrh, Kiss1, Gpr54, Igf1 and Mapk14 mRNA and cell proliferation but increased the level of β-catenin mRNA and cell apoptosis, while reducing GnRH secretion. Following ICV injection, Nagk gene knockdown mice exhibited delayed the timing of vaginal opening (VO) and reduced hypothalamic levels of Gnrh, Kiss1, Gpr54, Igf1, Mapk14, and β-catenin mRNA. Additionally, serum concentrations of E2 in Nagk gene knockdown mice were significantly lower compared to the control group. These findings indicate that Nagk regulates the expression of Gnrh and Kiss1 mRNA in GT1-7 cells, affects hypothalamus Gnrh mRNA levels and serum E2 concentration, and that its knockdown can delay puberty onset in female mice.

Neuroendocrine effects of the duper mutation in Syrian hamsters: a role for Cryptochrome 1.

Molecular and physiological determinants of the timing of reproductive events, including the pre-ovulatory LH surge and seasonal fluctuations in fertility, are incompletely understood. We used the Cryptochrome 1-deficient duper mutant to examine the role of this core circadian clock gene in Syrian hamsters. We find that the phase of the LH surge and its stability upon shifts of the light: dark cycle are altered in duper mutants. The intensity of immunoreactive PER1 in GnRH cells of the preoptic area peaks earlier in the day in duper than wild type hamsters. We note that GnRH fibers coursing through the suprachiasmatic nucleus (SCN) contact vasopressin- and VIP-immunoreactive cells, suggesting a possible locus of circadian control of the LH surge. Unlike wild types, duper hamsters do not regress their gonads within 8weeks of constant darkness, despite evidence of melatonin secretion during the subjective night. In light of the finding that the duper allele is a stop codon in Cryptochrome 1, our results suggest important neuroendocrine functions of this core circadian clock gene.

FRI277 Temporal Coordination Of Female Reproductive Axis Activity By Vasoactive Intestinal Polypeptide (VIP) Expressing Cells In The Suprachiasmatic Nucleus

Abstract Disclosure: C. Phumsatitpong: None. S.R. Bever: None. S.L. Hu: None. M.C. Ambrose: None. L.J. Kriegsfeld: None. Circadian rhythms orchestrated by a master clock in the suprachiasmatic nucleus (SCN) are essential for ovulation in spontaneously-ovulating rodents. SCN signaling integrates with estradiol positive feedback to generate the preovulatory luteinizing hormone (LH) surge. Additionally, subordinate clocks in reproductively-relevant SCN targets temporally gate responsiveness to upstream signaling to coordinate ovulation with the active time of day. We have previously shown that the SCN communicates directly to kisspeptin (Kp) and RFamide-related peptide-3 (RFRP-3) neurons to temporally balance key stimulatory and inhibitory input to GnRH neurons required for LH surge generation. Because VIP-expressing neurons in the SCN have been implicated in the LH surge, we employed a cell-specific chemogenetic approach to determine the functional role of this cell phenotype in female reproductive axis regulation required for surge generation. We hypothesized that temporal gating by subordinate cellular clocks will prevent morning stimulation of VIP-expressing cells from increasing Kp and GnRH, and decreasing RFRP-3, cellular activity. In the afternoon, we expected inhibition of VIP-expressing cells to decrease Kp and GnRH, and increase RFRP-3, cellular activity. Specifically, female VIP-Cre mice were stereotaxically injected with either AAV-hM3Dq or hM4Di to express activating (3Dq) or inhibitory (4Di) DREADDs in VIP-expressing SCN cells. Following recovery, mice were injected clozapine n-oxide (CNO) either in the morning, prior to the LH surge (3Dq experiment), or the afternoon, around the time of the LH surge (4Di experiment). Brains were collected to examine neuronal activation in Kp, GnRH, and RFRP-3 neurons using FOS immunofluorescence as a marker. Morning stimulation of SCN VIP neurons decreased RFRP-3 neuronal activation, whereas Kp and GnRH neurons were unaffected at this time, suggesting a temporal gating mechanism at GnRH and/or Kp cells prevents premature ovulation. In contrast, afternoon inhibition of SCN VIP-expressing neurons led to an increase in neuronal activation in RFRP-3 neurons concomitant with a decrease in GnRH and Kp neurons, suggesting that VIP stimulation is required to remove RFRP-3 inhibition on the GnRH and Kp systems. We confirmed this possibility by localizing afternoon injections of CNO to RFRP-3 cells, which led to comparable decreases in GnRH and Kp cellular activity. These data indicate the importance of SCN VIP neurons in the temporally coordinated disinhibition of RFRP-3 neuronal activity required for stimulation of GnRH and Kp neurons around the time of LH surge. Supported by NIH Grant HD-050470. Presentation: Friday, June 16, 2023

FRI285 Evidence For ZIC1 As A Novel Gene For Isolated Hypogonadotropic Hypogonadism With Phenotypic Pleiotropic Intersection With Cerebellar Malformation

Abstract Disclosure: J. Cassin: None. D.L. Keefe: None. K.E. Sung: None. C.C. Bojo: None. G. Santiago: None. L. Plummer: None. K.B. Salnikov: None. S.B. Seminara: None. R. Balasubramania: None. P.L. Mellon: None. Isolated Hypogonadotropic Hypogonadism (IHH) is a condition marked by absent pubertal development and low sex steroids in the setting of low/normal gonadotropins. Mutations in known IHH genes only explain <50% of the genetic etiology suggesting significant missing heritability in IHH. To explore this missing IHH heritability, we examined whole exome sequencing data from approximately 1400 IHH probands for rare genetic variants in novel genes that are enriched in IHH patients. We identified four IHH probands with rare, novel heterozygous variants in ZIC1 (p.H134Rfs*21, p.Y287X, p.E299K, p.S413Y), a gene encoding C2H2-type zinc finger protein (rare variant enrichment p <0.0001 for both truncating and missense alleles). Prior literature review show that heterozygous deletions affecting the ZIC1 gene have been linked to Dandy Walker malformation [DWM], a cerebellar malformation, while gain-of-function ZIC1 single nucleotide variants have been linked to coronal craniosynostosis (premature cranial suture fusion). Notably, two IHH subjects with novel ZIC1 variants in our cohort exhibited posterior fossa abnormalities (DWM [p.H134Rfs*21] and ventriculomegaly [E299K]) but none had any cranial suture defects. Given the above genetic enrichment, phenotypic overlap between DWM and IHH, and ZIC1’s known high expression in the hypothalamus, we explored whether ZIC1 regulates the expression of hypothalamic releasing factors. We determined that ZIC1 overexpression increases GNRH1 luciferase expression in the immortalized GnRH cell line, GT1-7. We next explored whether the rare ZIC1 variants identified in the four IHH probands would affect this phenotype. We discovered that three of the four ZIC1 variants significantly reduced GNRH1-luciferase expression. Of those, one truncating mutation, p.H134Rfs*21, resulted in lower ZIC1 protein content in the cell and failure to localize to the nucleus, suggesting a hypomorphic mechanism of action. These findings implicate ZIC1 variants as putatively causal for IHH. Further studies will define the precise ZIC1 transcriptional targets that relate to ZIC1’s pleotropic effects on GnRH transcription, neuronal function, cerebellar development, and cranial suture fusion. Presentation: Friday, June 16, 2023

FRI274 Dopamine Transporter-Expressing Leptin-Sensitive Neurons In The Ventral Premammillary Nucleus Innervate Kiss1 Neurons

Abstract Disclosure: C. Sáenz de Miera: None. L. Heikkinen: None. C. Broberger: None. C.F. Elias: None. Pubertal development is tightly regulated by energy balance, and easily disrupted in states of excess or negative energy reserves. This crosstalk between metabolism and reproduction is orchestrated by complex neural networks coordinated by circulating hormones, notably, leptin action in the hypothalamus plays a critical role. The ventral premammillary nucleus (PMv) is rich in leptin receptor (LepRb) neurons, and acts as an essential relay for leptin action on reproduction. Here, we describe a novel subpopulation of leptin sensitive neurons within the PMv that express dopamine transporter (DATPMv). We observed that in both prepubertal and adult females in diestrous, virtually all DATPMv neurons express LepRb mRNA and respond to acute leptin treatment with phosphorylation of the intracellular marker signal transducer and activator of transcription 3 (pSTAT3). Electrophyisological recordings from DAT-CRE;R26tdTomato (DAT-tdTom) mice show that DATPMv cells in adult females have a higher resting membrane potential than those in prepubertal females. DAT mRNA expression in the PMv is higher in prepubertal than in adult females. However, DAT mRNA expression was similar in adult females in diestrous, ovariectomized (OVX), and OVX treated with estradiol, indicating that the age difference observed is not due to the increase in estradiol during pubertal maturation. To trace DATPMv projections, we injected an adenoassociated virus expressing Cre-dependent channelrhodopsin (ChR2-mCherry) in the PMv of adult female DAT-Cre;Kiss1-hrGFP mice, which express GFP driven by kiss1 regulatory elements. ChR2-mCherry projections were observed in the anteroventral periventricular nucleus (AVPV) surrounding Kiss1-hrGFP neurons, a population critical for sexual maturation and positive estrogen feedback in females. No projections were observed near arcuate nucleus Kiss1-hrGFP neurons, in the medial preoptic area (MPOA) or near GnRH neurons. This contrasts with previous observations of dense projections from the overall PMv LepRb population to the MPOA, surrounding GnRH cell bodies suggesting the DATPMv subpopulation has different target areas than the non-DAT LepRb neurons. Using DAT-tdTOM females we observed that overall tdTom projections to the AVPV are denser in adult diestrous than in prepubertal females. Overall, these findings suggest that the DAT expressing LepR subpopulation in the PMv play a role in leptin regulation of sexual maturation via actions on AVPV kisspeptin neurons. Presentation: Friday, June 16, 2023

SAT591 Abnormal Levels Of Thyroid Hormones During Development Alters The Migration And Activity Of GnRH Reproductive Neurons

Abstract Disclosure: C. Quignon: None. A. Backer: None. S. Wray: None. Clinical studies have shown that thyroid hormones disorders and alteration of thyroid hormone production by environmental endocrine disruptors, have deleterious effects on reproduction. By acting in the brain on the hypothalamic component of the reproductive axis, abnormal levels of thyroid hormones can disturb sex hormones production, oestrus cycles or puberty onset, possibly leading to subfertility. Moreover, in animal models, endocrine disruptors and thyroid hormones have been shown to have long-lasting transgenerational effects with the maternal hormones influencing the development of foetal reproductive functions. Despite clear evidence of an interaction between the thyroid and reproductive systems, the effect of T3, the active form of thyroid hormones, on the GnRH neurons controlling reproduction, have been poorly studied. In this work we investigated how T3 functionally interacts with GnRH neurons and how abnormal concentration of T3 alters the migration of these neurons during development. Calcium imaging was used to study the direct effect of T3 on GnRH neuronal activity in an ex vivo nasal explants model. Acute administration of T3 (30nM) was found to stimulate the activity of GnRH cells. Dual labelling of GnRH and thyroid hormone receptors (TR) showed that the GnRH neurons express both nuclear and membrane receptors. However, the use of an antagonist that specifically blocks the TR nuclear receptors (1-850), didn’t inhibit the T3 stimulatory effect. In contrast, blockage of integrin αV/β3 membrane receptors (with cilengitide), which have been shown to be activated by T3, prevented the T3-induced increase in GnRH neuronal activity. During development GnRH neurons migrate from the nasal placodes into the brain. Acute administration of T3 for 1hr significatively decreased the migration rate of the GnRH neurons in our ex vivo model. To assess the effect of thyroid disruption during pregnancy on the development of reproductive axis in utero, pregnant females mice have been treated with methimazole to induce hypothyroidism from gestational day 6 to 13. E13 embryos have been collected from treated and control dams and the distribution of migrating GnRH neurons will be compared between both groups. Together these studies are the first to report a direct effect of thyroid hormones on GnRH neuronal activity, through the integrin αV/β3 membrane receptors and show that T3 can modulate the migration of GnRH neurons during development. These results will bring new insights on how thyroid disruption by endocrine disruptor chemicals or thyroid diseases, especially during foetal development, can lead to long term reproductive defects. Presentation: Saturday, June 17, 2023

Lesions of KNDy and Kiss1R Neurons in the Arcuate Nucleus Produce Different Effects on LH Pulse Patterns in Female Sheep.

The current model for the synchronization of GnRH neural activity driving GnRH and LH pulses proposes that a set of arcuate (ARC) neurons that contain kisspeptin, neurokinin B, and dynorphin (KNDy neurons) is the GnRH pulse generator. This study tested the functional role of ovine KNDy neurons in pulse generation and explored the roles of nearby Kiss1 receptor (Kiss1R)-containing cells using lesions produced with saporin (SAP) conjugates. Injection of NK3-SAP ablated over 90% of the KNDy cells, while Kiss-SAP (saporin conjugated to kisspeptin-54) lesioned about two-thirds of the Kiss1R population without affecting KNDy or GnRH cell number. Both lesions produced a dramatic decrease in LH pulse amplitude but had different effects on LH pulse patterns. NK3-SAP increased interpulse interval, but Kiss-SAP did not. In contrast, Kiss-SAP disrupted the regular hourly occurrence of LH pulses, but NK3-SAP did not. Because Kiss1R is not expressed in KNDy cells, HiPlex RNAScope was used to assess the colocalization of 8 neurotransmitters and 3 receptors in ARC Kiss1R-containing cells. Kiss1R cells primarily contained transcript markers for GABA (68%), glutamate (28%), ESR1 (estrogen receptor-α) mRNA, and OPRK1 (kappa opioid receptor) mRNA. These data support the conclusion that KNDy neurons are essential for GnRH pulses in ewes, whereas ARC Kiss1R cells are not but do maintain the amplitude and regularity of GnRH pulses. We thus propose that in sheep, ARC Kiss1R neurons form part of a positive feedback circuit that reinforces the activity of the KNDy neural network, with GABA or glutamate likely being involved.

Preliminary Study on Expression and Function of the Chicken W Chromosome Gene MIER3 in Embryonic Gonads.

The sex chromosomes of birds are designated Z and W. The male is homogamous (ZZ), and the female is heterogamous (ZW). The chicken W chromosome is a degenerate version of the Z chromosome and harbors only 28 protein-coding genes. We studied the expression pattern of the W chromosome gene MIER3 (showing differential expression during gonadogenesis) in chicken embryonic gonads and its potential role in gonadal development. The W copy of MIER3 (MIER3-W) shows a gonad-biased expression in chicken embryonic tissues which was different from its Z copy. The overall expression of MIER3-W and MIER3-Z mRNA and protein is correlated with the gonadal phenotype being higher in female gonads than in male gonads or female-to-male sex-reversed gonads. Chicken MIER3 protein is highly expressed in the nucleus, with relatively lower expression in the cytoplasm. Overexpression of MIER3-W in male gonad cells suggested its effect on the GnRH signaling pathway, cell proliferation, and cell apoptosis. MIER3 expression is associated with the gonadal phenotype. MIER3 may promote female gonadal development by regulating EGR1 and αGSU genes. These findings enrich our knowledge of chicken W chromosome genes and support a more systematic and in-depth understanding of gonadal development in chickens.

Autism-linked NLGN3 is a key regulator of gonadotropin-releasing hormone deficiency.

Gonadotropin-releasing hormone (GnRH) deficiency (GD) is a disorder characterized by absent or delayed puberty, with largely unknown genetic causes. The purpose of this study was to obtain and exploit gene expression profiles of GnRH neurons during development to unveil novel biological mechanisms and genetic determinants underlying GD. Here, we combined bioinformatic analyses of immortalized and primary embryonic GnRH neuron transcriptomes with exome sequencing from GD patients to identify candidate genes implicated in the pathogenesis of GD. Among differentially expressed and filtered transcripts, we found loss-of-function (LoF) variants of the autism-linked neuroligin 3 (NLGN3) gene in two unrelated patients co-presenting with GD and neurodevelopmental traits. We demonstrated that NLGN3 is upregulated in maturing GnRH neurons and that NLGN3 wild-type, but not mutant, protein promotes neuritogenesis when overexpressed in developing GnRH cells. Our data represent proof of principle that this complementary approach can identify new candidate GD genes and demonstrate that LoF NLGN3 variants can contribute to GD. This novel genotype-phenotype correlation implies common genetic mechanisms underlying neurodevelopmental disorders, such as GD and autistic spectrum disorder.

Drebrin regulates cytoskeleton dynamics in migrating neurons through interaction with CXCR4

Stromal cell-derived factor-1 (SDF-1) and chemokine receptor type 4 (CXCR4) are regulators of neuronal migration (e.g., GnRH neurons, cortical neurons, and hippocampal granule cells). However, how SDF-1/CXCR4 alters cytoskeletal components remains unclear. Developmentally regulated brain protein (drebrin) stabilizes actin polymerization, interacts with microtubule plus ends, and has been proposed to directly interact with CXCR4 in T cells. The current study examined, in mice, whether CXCR4 under SDF-1 stimulation interacts with drebrin to facilitate neuronal migration. Bioinformatic prediction of protein-protein interaction highlighted binding sites between drebrin and crystallized CXCR4. In migrating GnRH neurons, drebrin, CXCR4, and the microtubule plus-end binding protein EB1 were localized close to the cell membrane. Coimmunoprecipitation (co-IP) confirmed a direct interaction between drebrin and CXCR4 using wild-type E14.5 whole head and a GnRH cell line. Analysis of drebrin knockout (DBN1 KO) mice showed delayed migration of GnRH cells into the brain. A decrease in hippocampal granule cells was also detected, and co-IP confirmed a direct interaction between drebrin and CXCR4 in PN4 hippocampi. Migration assays on primary neurons established that inhibiting drebrin (either pharmacologically or using cells from DBN1 KO mice) prevented the effects of SDF-1 on neuronal movement. Bioinformatic prediction then identified binding sites between drebrin and the microtubule plus end protein, EB1, and super-resolution microscopy revealed decreased EB1 and drebrin coexpression after drebrin inhibition. Together, these data show a mechanism by which a chemokine, via a membrane receptor, communicates with the intracellular cytoskeleton in migrating neurons during central nervous system development.

Anti-neuroinflammatory effect of daidzein in human hypothalamic GnRH neurons in an in vitro membrane-based model.

Phytoestrogens can control high-fat diet-induced hypothalamic inflammation that is associated with severe consequences, including obesity, type 2 diabetes, cardiovascular and neurodegenerative diseases. However, the phytoestrogen anti-neuroinflammatory action is poorly understood. In this study, we explored the neuroprotection mediated by daidzein in hypothalamic neurons by using a membrane-based model of obesity-related neuroinflammation. To test the daidzein therapeutic potential a biohybrid membrane system, consisting of hfHypo GnRH-neurons in culture on PLGA membranes, was set up. It served as reliable in vitro tool capable to recapitulate the in vivo structure and function of GnRH hypothalamic tissue. Our findings highlighted the neuroprotective role of daidzein, being able to counteract the palmitate induced neuroinflammation. Daidzein protected hfHypo GnRH cells by downregulating cell death, proinflammatory processes, oxidative stress, and apoptosis. It also restored the proper cell morphology and functionality through a mechanism which probably involves the activation of ERβ and GPR30 receptors along with the expression of GnRH peptide and KISS1R.

Expression of genes for Kisspeptin (KISS1), Neurokinin B (TAC3), Prodynorphin (PDYN), and gonadotropin inhibitory hormone (RFRP) across natural puberty in ewes.

Expression of particular genes in hypothami of ewes was measured across the natural pubertal transition by in situ hybridization. The ewes were allocated to three groups (n = 4); prepubertal, postpubertal and postpubertally gonadectomized (GDX). Prepubertal sheep were euthanized at 20 weeks of age and postpubertal animals at 32 weeks. GDX sheep were also euthanized at 32 weeks, 1 week after surgery. Expression of KISS1, TAC3, PDYN in the arcuate nucleus (ARC), RFRP in the dorsomedial hypothalamus and GNRH1 in the preoptic area was quantified on a cellular basis. KISS1R expression by GNRH1 cells was quantified by double‐label in situ hybridization. Across puberty, detectable KISS1 cell number increased in the caudal ARC and whilst PDYN cell numbers were low, numbers increased in the rostral ARC. TAC3 expression did not change but RFRP expression/cell was reduced across puberty. There was no change across puberty in the number of GNRH1 cells that expressed the kisspeptin receptor (KISS1R). GDX shortly after puberty did not increase expression of any of the genes of interest. We conclude that KISS1 expression in the ARC increases during puberty in ewes and this may be a causative factor in the pubertal activation of the reproductive axis. A reduction in expression of RFRP may be a factor in the onset of puberty, removing negative tone on GNRH1 cells. The lack of changes in expression of genes following GDX suggest that the effects of gonadal hormones may differ in young and mature animals.

Understanding the trade-off between the environment and fertility in cows and ewes.

The environment contributes to production diseases that in turn badly affect cow performance, fertility and culling. Oestrus intensity is lower in lame cows, and in all cows 26% potential oestrus events are not expressed (to avoid getting pregnant). To understand these trade-offs, we need to know how animals react to their environment and how the environment influences hypothalamus-pituitary-adrenal axis (HPA) interactions with the hypothalamus-pituitary-ovarian axis (HPO). Neurotransmitters control secretion of GnRH into hypophyseal portal blood. GnRH/LH pulse amplitude and frequency drive oestradiol production, culminating in oestrus behaviour and a precisely-timed GnRH/LH surge, all of which are disrupted by poor environments. Responses to peripheral neuronal agents give clues about mechanisms, but do these drugs alter perception of stimuli, or suppress consequent responses? In vitro studies confirm some neuronal interactions between the HPA and HPO; and immuno-histochemistry clarifies the location and sequence of inter-neurone activity within the brain. In both species, exogenous corticoids, ACTH and/or CRH act at the pituitary (reduce LH release by GnRH), and hypothalamus (lower GnRH pulse frequency and delay surge release). This requires inter-neurones as GnRH cells do not have receptors for HPA compounds. There are two (simultaneous, therefore fail-safe?) pathways for CRH suppression of GnRH release via CRH-Receptors: one being the regulation of kisspeptin/dynorphin and other cell types in the hypothalamus, and the other being the direct contact between CRH and GnRH cell terminals in the median eminence. When we domesticate animals, we must provide the best possible environment otherwise animals trade-off with lower production, less intense oestrus behaviour, and impaired fertility. Avoiding life-time peri-parturient problems by managing persistent lactations in cows may be a worthy trade-off on both welfare and economic terms – better than the camouflage use of drugs/hormones/feed additives/intricate technologies? In the long term, getting animals and environment in a more harmonious balance is the ultimate strategy.

Alterations of estradiol-induced histone H3 acetylation in the preoptic area and anteroventral periventricular nucleus of middle-aged female rats

In this study we investigated the characteristics of histone H3 acetylation in the anterior hypothalamus under E2 positive feedback to gain a better understanding of the mechanism underlying reduced GnRH neuron activation and altered gene expression in female reproductive aging. Young and middle-aged female rats were ovariectomized (OVX) and treated with estradiol (E2) or oil. C-Fos expression, the number of GnRH neurons co-localized with c-Fos in the preoptic area (POA), and the number of acetylated histone H3 cells in the POA and anteroventral periventricular nucleus (AVPV) were quantified at the time of the expected GnRH neuron activation. We used real-time PCR to evaluate the expression of Esr1 target genes including Kiss1 and VGluT2 and genes known as Esr1 coregulators in the anterior hypothalamus. Our results show that in the young females, E2 markedly increased histone H3 acetylation in the POA and AVPV, coincident with increased c-Fos and GnRH neuron activation in the POA. In middle-aged females, E2-induced histone H3 acetylation was reduced in the POA but was not significantly altered in the AVPV. This occurred in association with a reduction of c-Fos expression and the number of GnRH cells expressing c-Fos in the POA as well as a down-regulation of Kiss1 and VGluT2 mRNA expression in the anterior hypothalamus of the animals. E2 caused significant decreases in Ncoa2 and Crebbp mRNA expression in the anterior hypothalamus of young, but not middle-aged females. Taken together, these data suggest that alterations of histone H3 acetylation in the POA and AVPV and the inability of Ncoa2 and Crebbp to respond to E2 in the middle-aged anterior hypothalamus partially contribute to the decline of GnRH neuron activation and E2 target gene expression changes that occur in female along with reproductive aging.

Gonadal hormone-dependent vs. -independent effects of kisspeptin signaling in the control of body weight and metabolic homeostasis

BackgroundKisspeptins, encoded by Kiss1, have emerged as essential regulators of puberty and reproduction by primarily acting on GnRH neurons, via their canonical receptor, Gpr54. Mounting, as yet fragmentary, evidence strongly suggests that kisspeptin signaling may also participate in the control of key aspects of body energy and metabolic homeostasis. However, characterization of such metabolic dimension of kisspeptins remains uncomplete, without an unambiguous discrimination between the primary metabolic actions of kisspeptins vs. those derived from their ability to stimulate the secretion of gonadal hormones, which have distinct metabolic actions on their own. In this work, we aimed to tease apart primary vs. secondary effects of kisspeptins in the control of key aspects of metabolic homeostasis using genetic models of impaired kisspeptin signaling and/or gonadal hormone status. MethodsBody weight (BW) gain and composition, food intake and key metabolic parameters, including glucose tolerance, were comparatively analyzed, in lean and obesogenic conditions, in mice lacking kisspeptin signaling due to global inactivation of Gpr54 (displaying profound hypogonadism; Gpr54−/−) vs. Gpr54 null mice with selective re-introduction of Gpr54 expression only in GnRH cells (Gpr54−/−Tg), where kisspeptin signaling elsewhere than in GnRH neurons is ablated but gonadal function is preserved. ResultsIn male mice, global elimination of kisspeptin signaling resulted in decreased BW, feeding suppression and increased adiposity, without overt changes in glucose tolerance, whereas Gpr54−/− female mice displayed enhanced BW gain at adulthood, increased adiposity and perturbed glucose tolerance, despite reduced food intake. Gpr54−/−Tg rescued mice showed altered postnatal BW gain in males and mildly perturbed glucose tolerance in females, with intermediate phenotypes between control and global KO animals. Yet, body composition and leptin levels were similar to controls in gonadal-rescued mice. Exposure to obesogenic insults, such as high fat diet (HFD), resulted in exaggerated BW gain and adiposity in global Gpr54−/− mice of both sexes, and worsening of glucose tolerance, especially in females. Yet, while rescued Gpr54−/−Tg males displayed intermediate BW gain and feeding profiles and impaired glucose tolerance, rescued Gpr54−/−Tg females behaved as controls, except for a modest deterioration of glucose tolerance after ovariectomy. ConclusionOur data support a global role of kisspeptin signaling in the control of body weight and metabolic homeostasis, with a dominant contribution of gonadal hormone-dependent actions. However, our results document also discernible primary effects of kisspeptin signaling in the regulation of body weight gain, feeding and responses to obesogenic insults, which occur in a sexually-dimorphic manner. Summary of translational relevanceKisspeptins, master regulators of reproduction, may also participate in the control of key aspects of body energy and metabolic homeostasis; yet, the nature of such metabolic actions remains debatable, due in part to the fact that kisspeptins modulate gonadal hormones, which have metabolic actions on their own. By comparing the metabolic profiles of two mouse models with genetic inactivation of kisspeptin signaling but different gonadal status (hypogonadal vs. preserved gonadal function), we provide herein a systematic dissection of gonadal-dependent vs. -independent metabolic actions of kisspeptins. Our data support a global role of kisspeptin signaling in the control of body weight and metabolic homeostasis, with a dominant contribution of gonadal hormone-dependent actions. However, our results document also discernible primary effects of kisspeptin signaling in the regulation of body weight gain, feeding and responses to obesogenic insults, which occur in a sexually-dimorphic manner. These data pave the way for future analyses addressing the eventual contribution of altered kisspeptin signaling in the development of metabolic alterations, especially in conditions linked to reproductive dysfunction.

SAT-407 Evidence for Impaired Kisspeptin Signaling During Metabolic Stress in Female Mice

Stress impairs reproduction, at least in part, by suppressing gonadotropin secretion. One common metabolic stressor is hypoglycemia, which occurs in a variety of pathologies and in healthy people, particularly athletes. Insulin-induced hypoglycemia (IIH) is a repeatable, reliable, and quantifiable acute model of metabolic stress that inhibits pulsatile luteinizing hormone (LH) secretion, though little is known about the underlying mechanism. We have observed that IIH robustly suppresses LH pulses in female mice, which could be mediated by either a pituitary or hypothalamic effect. In the present study, we performed a kisspeptin challenge to determine whether downstream cells involved in LH pulsatility are impaired during IIH. Adult mice (C57/BL6, n = 8/group) were ovariectomized and acclimated to handling for tail-tip blood collection and i.p. injection. On the day of each challenge, mice were fasted for 5 hr and then received an i.p. injection of either saline or insulin (0.75mU/kg). All animals then received an injection of kisspeptin (2μg/g), 45 min later. This time point represents a consistent period of LH pulse suppression following IIH. Frequent blood samples were collected for 30 min before and after kisspeptin injection. Two-weeks later, the experiment was repeated with each mouse receiving the alternate treatment (i.e. insulin or saline) in a cross-over design. Mean LH concentrations were significantly increased after kisspeptin injection in animals treated with saline (saline, pre: 3.8 ± 0.6 vs. post: 7.8 ± 0.5 ng/ml). Interestingly, the LH response to kisspeptin in animals treated with insulin was also markedly increased (insulin, pre: 3.2 ± 0.6 vs. post: 6.4 ± 0.3 ng/ml), but not significantly different from animals treated with saline. We conclude from this study that impaired GnRH or gonadotrope cell function is not the primary cause of reduced LH secretion in this model of metabolic stress. Since kisspeptin neurons in the arcuate nucleus likely form the GnRH/LH pulse generator, we next determined whether arcuate kisspeptin cells were inhibited during IIH. For this purpose, we used a mouse model expressing GFP in kisspeptin cells (Kiss1-Cre-GFP). Mice were fasted and injected with insulin or saline, as above, and fixed neural tissue was collected for assessment of c-Fos immunoreactivity in arcuate Kiss1 cells. Two hours following saline injection ~20% of Kiss1 neurons contained c-Fos. In contrast, only 6.6 ± 0.5% of Kiss1 neurons contained c-Fos in animals treated with insulin. Thus, reduced gonadotropin secretion in IIH may be mediated by an inhibition of the ARC kisspeptin cells. Together these data support the hypothesis that inhibition of LH pulses by metabolic stress is mediated by neural factors including arcuate kisspeptin cells. Future work will investigate kisspeptin cell afferents that convey the inhibitory signal initiated by metabolic stressors.

SAT-425 NKB Signaling in the Posterodorsal Medial Amygdala Stimulates Gonadotropin Release in a Kisspeptin-Independent Manner in Female Mice

Female reproduction is regulated by a complex signaling network, the function of which is dependent on the level of circulating estrogens. Neurokinin B (NKB) colocalizes with kisspeptin in Kiss1 neurons of the arcuate nucleus (ARC). The autosynaptic action of NKB via the neurokinin 3 receptor (NK3R) in these neurons induces kisspeptin release in the presence of sex steroids. This has led us to believe that NKB stimulates LH secretion in a kisspeptin-dependent manner despite the latter mechanism having been investigated in the persistent hypogonadal state [e.g. Kiss1r knock out (KO) mice and agonadal monkeys]. Here, we aimed to assess whether in the presence of estradiol, senktide, a NK3R specific agonist, can stimulate LH release in a kisspeptin-independent manner. Adult WT female mice were ovariectomized (OVX) or OVX and estradiol-treated (OVX+E2) and studied in parallel to Kiss1KO or Kiss1KO+E2 littermates (n=10/group). Intracerebroventricular (icv) injections of senktide (600pmol) were performed and blood samples were collected before and 25 min after icv injection. Interestingly, the presence of E2 in Kiss1KO females resulted in LH increase like that observed in WT animals. Subsequently, we aimed to locate the brain area which senktide may be acting to stimulate LH release in Kiss1KO+E2 mice. We mapped the distribution of NK3R in the mouse hypothalamus and moreover investigated the anatomical relationship of NK3R and GnRH expression with dual-label immunohistochemistry as a likely pathway of the kisspeptin-independent stimulation of LH release exerted by NKB. We observed no instances of co-expression between NK3R and GnRH cell bodies (>100 cells analyzed from a total of 16 mice) and only minimal close appositions between the two proteins in the ARC and medial septum. Thus, the kisspeptin-independent action of NKB cannot be attributed to direct stimulation of NK3R located on GnRH neurons. Interestingly, we observed that NK3R immunopositive cells in the posterodorsal medial amygdala (MePD), unlike the ARC, were upregulated in the presence of E2. Next, WT OVX+E2 and Kiss1KO+E2 females were injected with senktide (600pmol; n=5/group) in the ARC, the AVPV/PeN or the MePD; areas found to contain NK3R-expressing neurons, and blood samples were collected before and then every 15 min for 45 min. Senktide significantly stimulated LH secretion in all three areas in WT OVX+E2, but only in the MePD of Kiss1KO+E2 mice. Conversely, in the absence of E2, Kiss1KO females injected with senktide in the MePD did not show any alteration in LH, mimicking the results we obtained after icv administration. These data demonstrate that activation of NK3R in the amygdala stimulates LH release in a kisspeptin independent manner in the presence of estrogen. This novel pathway for LH release, that bypasses the kisspeptin neuron, may play an important role in linking amygdala regulated social cues with gonadotropin release.

Gamma amino-butyric acid and the control of GnRH secretion in sheep

The release of GnRH from nerve terminals in the median eminence into the portal vessels is influenced by factors in the internal and external environment of the animal. In the former category are the gonadal steroid hormones oestrogen and progesterone which alter the characteristics of GnRH secretion during the oestrous and seasonal cycles. These cannot exert their actions directly on the GnRH neurones as they do not possess hormone receptors. Therefore, some other steroid-sensitive neuronal system must relay this information to the GnRH neurones. Gamma amino-butyric acid (GABA) neurones are good candidates for this role as they contain steroid hormone receptors and synapse on GnRH neurones. Recent studies in ewes have sought to identify a role for GABA in mediating the actions of both oestrogen and progesterone on GnRH release. The technique of microdialysis was used to monitor GABA concentrations in areas containing GnRH cell bodies during the oestrogen-induced surge of GnRH and during progesterone negative feedback. Concentrations of this inhibitory neurotransmitter have been shown to fall in the former situation where GnRH release is being stimulated, but to be increased when progesterone is depressing GnRH release. GABA may also be important in mediating the seasonal switch in the negative feedback actions of oestradiol. During the anoestrous season, when oestradiol is a potent inhibitor of GnRH secretion, specific GABA receptor antagonists can stimulate neurohormone release, an action that is not observed in the breeding season when oestrogen is much less potent.

Leptin actions on the reproductive neuroendocrine axis in sheep

There is a growing literature on the role of leptin in appetite and neuroendocrine regulation in domestic ruminants. Circulating leptin concentration is higher in fat than in thin sheep, is reduced by chronic underfeeding and is higher in sheep subjected to long-day rather than short-day photoperiods. Leptin is reduced acutely by fasting and increases after meals so that there are long- and short-term components to the systemic leptin signal. Nutritional stimulation of reproductive neuroendocrine output is associated with increased circulating concentrations of leptin; peripheral leptin administration restores LH secretion in fasted sheep, and leptin is permissive (although not a trigger) for puberty. Intracerebroventricular (i.c.v.) pharmacological leptin infusion stimulates LH in underfed but not in well-fed sheep, and reduces food intake in well-fed sheep. A single i.c.v. pharmacological injection or physiological infusion of leptin stimulates LH in well-fed sheep, with or without a concomitant decrease in appetite. Furthermore, these appetite and LH responses are differentially affected by photoperiod, indicating that different neuronal pathways may mediate the two responses. Hypothalamic leptin receptors co-localize with orexigenic and anorexigenic neurones, some of which contact GnRH cells, but the confluence of leptin signalling with photoperiod (melatonin) signalling remains unresolved. Photoperiod-entrained sheep provide potential models of altered central leptin sensitivity, in which downstream mechanisms regulating appetite and GnRH may be dissociated.

GnRH expression and cell proliferation are associated with seasonal breeding and food hoarding in Mongolian gerbils (Meriones unguiculatus)

Seasonal brain plasticity contributes to a variety of physiological and behavioral processes. We hypothesized that variations in GnRH expression and cell proliferation facilitated seasonal breeding and food hoarding. Here, we reported seasonal changes in sexual and social behavior, GnRH expression and brain cell proliferation, and the role of photoperiod in inducing seasonal breeding and brain plasticity in Mongolian gerbils (Meriones unguiculatus). The gerbils captured in April and July had more mature sexual development, higher exploratory behavior, and preferred novelty much more than those captured in September. Male gerbils captured in April and July had consistently higher GnRH expression than those captured in September. GnRH expression was also found to be suppressed by food-induced hoarding behavior in the breeding season. Both subadult and adult gerbils from April and July had higher cell proliferation in SVZ, hypothalamus and amygdala compared to those in September. However, adult gerbils captured in September preferred familiar objects, and no seasonal differences were found in cell proliferation in hippocampal dentate gyrus among the three seasons. The laboratory study showed that photoperiod alone did not alter reproductive traits, behavior, cell proliferation or cell survival in the detected brain regions. These findings suggest that the structural variations in GnRH expression in hypothalamus and cell proliferation in hypothalamus, amygdala and hippocampus are associated with seasonal breeding and food hoarding in gerbils. It gives a new insight into the proximate physiological and neural basis for these seasonal life-history traits of breeding and food hoarding in small mammals.