A small population of hypothalamic neurons govern fertility: the critical role of VAX1 in GnRH neuron development and fertility maintenance.

Fertility depends on the correct maturation and function of approximately 800 gonadotropin-releasing hormone (GnRH) neurons in the brain. GnRH neurons are at the apex of the hypothalamic-pituitary-gonadal axis that regulates fertility. In adulthood, GnRH neurons are scattered throughout the anterior hypothalamic area and project to the median eminence, where GnRH is released into the portal vasculature to stimulate release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary. LH and FSH then regulate gonadal steroidogenesis and gametogenesis. Absence of GnRH neurons or inappropriate GnRH release leads to infertility. Despite the critical role of GnRH neurons in fertility, we still have a limited understanding of the genes responsible for proper GnRH neuron development and function in adulthood. GnRH neurons originate in the olfactory placode then migrate into the brain. Homeodomain transcription factors expressed within GnRH neurons or along their migratory path are candidate genes for inherited infertility. Using a combined in vitro and in vivo approach, we have identified Ventral Anterior Homeobox 1 (Vax1) as a novel homeodomain transcription factor responsible for GnRH neuron maturation and fertility. GnRH neuron counts in Vax1 knock-out embryos revealed Vax1 to be required for the presence of GnRH-expressing cells at embryonic day 17.5 (E17.5), but not at E13.5. To localize the effects of Vax1 on fertility, we generated Vax1flox mice and crossed them with Gnrhcre mice to specifically delete Vax1 within GnRH neurons. GnRH staining in Vax1flox/flox:GnRHcre mice show a total absence of GnRH expression in the adult. We performed lineage tracing in Vax1flox/flox:GnRHcre:RosaLacZ mice which proved GnRH neurons to be alive, but incapable of expressing GnRH. The absence of GnRH leads to delayed puberty, hypogonadism and complete infertility in both sexes. Finally, using the immortalized model GnRH neuron cell lines, GN11 and GT1-7, we show that VAX1 is a direct regulator of Gnrh1 transcription by binding key ATTA sites within the Gnrh1 promoter. This study identifies VAX1 as a key transcription factor regulating GnRH expression and establishes VAX1 as a novel candidate gene implicated in heritable infertility

A Novel Gonadotropin-Releasing Hormone 1 (Gnrh1) Enhancer-Derived Noncoding RNA Regulates Gnrh1 Gene Expression in GnRH Neuronal Cell Models.

Gonadotropin-releasing hormone (GnRH), a neuropeptide released from a small population of neurons in the hypothalamus, is the central mediator of the hypothalamic-pituitary-gonadal axis, and is required for normal reproductive development and function. Evolutionarily conserved regulatory elements in the mouse, rat, and human Gnrh1 gene include three enhancers and the proximal promoter, which confer Gnrh1 gene expression specifically in GnRH neurons. In immortalized mouse hypothalamic GnRH (GT1-7) neurons, which show pulsatile GnRH release in culture, RNA sequencing and RT-qPCR revealed that expression of a novel long noncoding RNA at Gnrh1 enhancer 1 correlates with high levels of GnRH mRNA expression. In GT1-7 neurons, which contain a transgene carrying 3 kb of the rat Gnrh1 regulatory region, both the mouse and rat Gnrh1 enhancer-derived noncoding RNAs (GnRH-E1 RNAs) are expressed. We investigated the characteristics and function of the endogenous mouse GnRH-E1 RNA. Strand-specific RT-PCR analysis of GnRH-E1 RNA in GT1-7 cells revealed GnRH-E1 RNAs that are transcribed in the sense and antisense directions from distinct 5' start sites, are 3' polyadenylated, and are over 2 kb in length. These RNAs are localized in the nucleus and have a half-life of over 8 hours. In GT1-7 neurons, siRNA knockdown of mouse GnRH-E1 RNA resulted in a significant decrease in the expression of the Gnrh1 primary transcript and Gnrh1 mRNA. Over-expression of either the sense or antisense mouse GnRH-E1 RNA in immature, migratory GnRH (GN11) neurons, which do not express either GnRH-E1 RNA or GnRH mRNA, induced the transcriptional activity of co-transfected rat Gnrh1 gene regulatory elements, where the induction requires the presence of the rat Gnrh1 promoter. Together, these data indicate that GnRH-E1 RNA is an inducer of Gnrh1 gene expression. GnRH-E1 RNA may play an important role in the development and maturation of GnRH neurons

Comparison of antimüllerian hormone levels and antral follicle count as predictor of ovarian response to controlled ovarian stimulation in good-prognosis patients at individual fertility clinics in two multicenter trials

Objective To compare antimüllerian hormone (AMH) and antral follicle count (AFC) as predictors of ovarian response to controlled ovarian stimulation at individual fertility clinics. Design Retrospective analysis of individual study center data in two multicenter trials. Centers that provided >10 patients were included in the analysis. Setting A total of 19 (n = 519 patients) and 18 study centers (n = 686 patients) participating in a long GnRH agonist trial (MERIT) and a GnRH antagonist trial (MEGASET), respectively. Patient(s) Infertile women of good prognosis. Intervention(s) Long GnRH agonist or GnRH antagonist cycles. Main Outcome Measure(s) Correlation between AMH and AFC, and oocyte yield by each study center for each trial. Results(s) Antimüllerian hormone was more strongly correlated with oocyte yield than AFC: r = 0.56 vs. r = 0.28 in the GnRH agonist cohort, and r = 0.55 vs. r = 0.33 in the GnRH antagonist cohort. The correlation was numerically higher for AMH than for AFC at a significantly higher proportion of study centers: 17 (89%) and 15 (83%) centers in the long GnRH agonist and GnRH antagonist trial, respectively. Assessment of the relative capacity of AMH and AFC for predicting oocyte yield demonstrated that AMH dominated the model: AMH, R2 = 0.29 and 0.23; AFC: R2 = 0.07 and 0.07; AMH + AFC: R2 = 0.30 and 0.23 for long GnRH agonist and GnRH antagonist trials, respectively. Conclusions(s) Antimüllerian hormone was a stronger predictor of ovarian response to gonadotropin therapy than AFC at the study center level in both randomized trials utilizing GnRH agonist and GnRH antagonist protocols. Antral follicle count provided no added predictive value beyond AMH.</p

Leukemia Inhibitory Factor Represses GnRH Gene Expression via cFOS during Inflammation in Male Mice.

BackgroundThe mechanisms whereby neuroinflammation negatively affects neuronal function in the hypothalamus are not clear. Our previous study determined that obesity-mediated chronic inflammation elicits sex-specific impairment in reproductive function via reduction in spine density in gonadotropin-releasing hormone (GnRH) neurons. Neuroinflammation and subsequent decrease in GnRH neuron spine density was specific for male mice, while protection in females was independent of ovarian estrogens.MethodsTo examine if neuroinflammation-induced cytokines can directly regulate GnRH gene expression, herein we examined signaling pathways and mechanisms in males in vivo and in GnRH-expressing cell line, GT1-7.ResultsGnRH neurons express cytokine receptors, and chronic or acute neuroinflammation represses GnRH gene expression in vivo. Leukemia inhibitory factor (LIF) in particular represses GnRH expression in GT1-7 cells, while other cytokines do not. STAT3 and MAPK pathways are activated following LIF treatment, but only MAPK pathway, specifically p38α, is sufficient to repress the GnRH gene. LIF induces cFOS that represses the GnRH gene via the -1,793 site in the enhancer region. In vivo, following high-fat diet, cFOS is induced in GnRH neurons and neurons juxtaposed to the leaky blood brain barrier of the organum vasculosum of the lamina terminalis, but not in the neurons further away.ConclusionOur results indicate that the increase in LIF due to neuroinflammation induces cFOS and represses the GnRH gene. Therefore, in addition to synaptic changes in GnRH neurons, neuroinflammatory cytokines directly regulate gene expression and reproductive function, and the specificity for neuronal targets may stem from the proximity to the fenestrated capillaries

Ghrelin Decreases Firing Activity of Gonadotropin-Releasing Hormone (GnRH) Neurons in an Estrous Cycle and Endocannabinoid Signaling Dependent Manner.

The orexigenic peptide, ghrelin is known to influence function of GnRH neurons, however, the direct effects of the hormone upon these neurons have not been explored, yet. The present study was undertaken to reveal expression of growth hormone secretagogue receptor (GHS-R) in GnRH neurons and elucidate the mechanisms of ghrelin actions upon them. Ca(2+)-imaging revealed a ghrelin-triggered increase of the Ca(2+)-content in GT1-7 neurons kept in a steroid-free medium, which was abolished by GHS-R-antagonist JMV2959 (10µM) suggesting direct action of ghrelin. Estradiol (1nM) eliminated the ghrelin-evoked rise of Ca(2+)-content, indicating the estradiol dependency of the process. Expression of GHS-R mRNA was then confirmed in GnRH-GFP neurons of transgenic mice by single cell RT-PCR. Firing rate and burst frequency of GnRH-GFP neurons were lower in metestrous than proestrous mice. Ghrelin (40nM-4μM) administration resulted in a decreased firing rate and burst frequency of GnRH neurons in metestrous, but not in proestrous mice. Ghrelin also decreased the firing rate of GnRH neurons in males. The ghrelin-evoked alterations of the firing parameters were prevented by JMV2959, supporting the receptor-specific actions of ghrelin on GnRH neurons. In metestrous mice, ghrelin decreased the frequency of GABAergic mPSCs in GnRH neurons. Effects of ghrelin were abolished by the cannabinoid receptor type-1 (CB1) antagonist AM251 (1µM) and the intracellularly applied DAG-lipase inhibitor THL (10µM), indicating the involvement of retrograde endocannabinoid signaling. These findings demonstrate that ghrelin exerts direct regulatory effects on GnRH neurons via GHS-R, and modulates the firing of GnRH neurons in an ovarian-cycle and endocannabinoid dependent manner

GnRH Receptor Expression and Reproductive Function Depend on JUN in GnRH Receptor‒Expressing Cells.

Gonadotropin-releasing hormone (GnRH) from the hypothalamus regulates synthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary gonadotropes. LH and FSH are heterodimers composed of a common α-subunit and unique β-subunits, which provide biological specificity and are limiting components of mature hormone synthesis. Gonadotrope cells respond to GnRH via specific expression of the GnRH receptor (Gnrhr). GnRH induces the expression of gonadotropin genes and of the Gnrhr by activation of specific transcription factors. The JUN (c-Jun) transcription factor binds to AP-1 sites in the promoters of target genes and mediates induction of the FSHβ gene and of the Gnrhr in gonadotrope-derived cell lines. To analyze the role of JUN in reproductive function in vivo, we generated a mouse model that lacks JUN specifically in GnRH receptor‒expressing cells (conditional JUN knockout; JUN-cKO). JUN-cKO mice displayed profound reproductive anomalies such as reduced LH levels resulting in lower gonadal steroid levels, longer estrous cycles in females, and diminished sperm numbers in males. Unexpectedly, FSH levels were unchanged in these animals, whereas Gnrhr expression in the pituitary was reduced. Steroidogenic enzyme expression was reduced in the gonads of JUN-cKO mice, likely as a consequence of reduced LH levels. GnRH receptor‒driven Cre activity was detected in the hypothalamus but not in the GnRH neuron. Female, but not male, JUN-cKO mice exhibited reduced GnRH expression. Taken together, our results demonstrate that GnRH receptor‒expression levels depend on JUN and are critical for reproductive function

Down Regulation with Luteal GnRH Agonist Therapy in Euploid Embryo Transfers Does Not Impact Pregnancy Rates

Introduction : Gonadotropin-releasing hormone (GnRH) agonists have been used during assisted reproductive technology (ART) treatment for pituitary suppression and stimulation. Currently, clinical opinion is divided about whether GnRH agonist therapy improves pregnancy rates when used for luteal down-regulation in a frozen euploid embryo transfer (FET). This study evaluated the clinical utility of GnRH agonist down-regulation in single, euploid FET cycles. Methods : A retrospective analysis was performed, using data from patients who underwent a single, euploid FET cycle from 2012 to 2019. Patients were segregated into two cohorts: Group A: single, euploid FET with down-regulation using GnRH agonist; Group B: single, euploid FET without down-regulation using GnRH agonist. Primary outcomes include pregnancy rates among cohorts. Results : Group A demonstrated a pregnancy rate of 72.92% in 96 cycles. Group B demonstrated a pregnancy rate of 73.27% in 5,668 cycles. There was no difference in pregnancy rates between groups, X2(2, N = 5764) = .0061, p = .94. A subgroup of patients (n=5) with endometriosis in Group A achieved an 80% (4/5) pregnancy rate. Discussion : Single, euploid FET cycle pregnancy rates were not affected by the use of down-regulation with a GnRH agonist. Increased pregnancy rates found with prolonged GnRH agonist use in other studies weren’t seen with short term use for FET cycles. Future research should focus on molecular markers and gene transcription signatures to attempt to define whether there is an ideal population of patients who would benefit from GnRH agonist down-regulation prior to FET

Afferent Neuronal Control of Type-I Gonadotropin Releasing Hormone Neurons in the Human.

Understanding the regulation of the human menstrual cycle represents an important ultimate challenge of reproductive neuroendocrine research. However, direct translation of information from laboratory animal experiments to the human is often complicated by strikingly different and unique reproductive strategies and central regulatory mechanisms that can be present in even closely related animal species. In all mammals studied so far, type-I gonadotropin releasing hormone (GnRH) synthesizing neurons form the final common output way from the hypothalamus in the neuroendocrine control of the adenohypophysis. Under various physiological and pathological conditions, hormonal and metabolic signals either regulate GnRH neurons directly or act on upstream neuronal circuitries to influence the pattern of pulsatile GnRH secretion into the hypophysial portal circulation. Neuronal afferents to GnRH cells convey important metabolic-, stress-, sex steroid-, lactational-, and circadian signals to the reproductive axis, among other effects. This article gives an overview of the available neuroanatomical literature that described the afferent regulation of human GnRH neurons by peptidergic, monoaminergic, and amino acidergic neuronal systems. Recent studies of human genetics provided evidence that central peptidergic signaling by kisspeptins and neurokinin B (NKB) play particularly important roles in puberty onset and later, in the sex steroid-dependent feedback regulation of GnRH neurons. This review article places special emphasis on the topographic distribution, sexual dimorphism, aging-dependent neuroanatomical changes, and plastic connectivity to GnRH neurons of the critically important human hypothalamic kisspeptin and NKB systems

Ovarian Dynamic in Ongole Grade Cattle After GnRH Injection in Ovsynch Protocol Based on Progesterone Device

PO cattle have weaknesses to show clear estrus signs which cause difficulty in artificial insemination implementation. The present study was designed to obtain ovarian dynamic as effect of GnRH injection in ovsynch protocol based on progesterone intravaginal device. Heifers (18) and cows (n= 12) were allocated to one of three groups. Cuemate-PGF2α (CP) group inserted with a Cuemate on day 0-7 and injected with prostaglandin on day 7. Cuemate-PGF2α-GnRH (CPG) group was treated as CP group with the addition of GnRH injection on day 9. GnRH-Cuemate-PGF2α-GnRH (GCPG) group was treated as CPG group with addition of GnRH injection on day 0. Ultrasonography was performed on days 0-3, day 7 until ovulation and 7 days after ovulation. Percentage of ovulation synchronization increased significantly (P&lt;0.01) between CP, CPG, and GCPG, respectively, both in heifers (16%, 50%, and 85%, respectively) and cows (0%, 60%, and 100%, respectively), on day 11. Preovulatory follicle diameters between CP, CPG, and GCPG treatments were not different significantly both in heifers (11.9±0.5, 11.9±0.5, and 12.1±0.6 mm, respectively) and cows (11.7±0.4, 11.8±0.7, and 11.1±0.6 mm, respectively). This study concluded that GCPG protocol increased the synchrony of ovulation rate both in cows and heifers, without affecting the follicle preovulatory and CL diameters