FSH Is Responsible for Androgen Deprivation Therapy-Associated Atherosclerosis in Mice by Exaggerating Endothelial Inflammation and Monocyte Adhesion.

BACKGROUND: Androgen deprivation therapy (ADT) is the mainstay treatment for advanced prostate cancer. But ADTs with orchiectomy and gonadotropin-releasing hormone (GnRH) agonist are associated with increased risk of cardiovascular diseases, which appears less significant with GnRH antagonist. The difference of follicle-stimulating hormone (FSH) in ADT modalities is hypothesized to be responsible for ADT-associated cardiovascular diseases. METHODS: We administered orchiectomy, GnRH agonist, or GnRH antagonist in male ApoE-/- mice fed with Western diet and manipulated FSH levels by testosterone and FSH supplementation or FSH antibody to investigate the role of FSH elevation on atherosclerosis. By combining lipidomics, in vitro study, and intraluminal FSHR (FSH receptor) inhibition, we delineated the effects of FSH on endothelium and monocytes and the underlying mechanisms. RESULTS: Orchiectomy and GnRH agonist, but not GnRH antagonist, induced long- or short-term FSH elevation and significantly accelerated atherogenesis. In orchiectomized and testosterone-supplemented mice, FSH exposure increased atherosclerosis. In GnRH agonist-treated mice, blocking of short FSH surge by anti-FSHß antibody greatly alleviated endothelial inflammation and delayed atherogenesis. In GnRH antagonist-treated mice, FSH supplementation aggravated atherogenesis. Mechanistically, FSH, synergizing with TNF-α (tumor necrosis factor alpha), exacerbated endothelial inflammation by elevating VCAM-1 (vascular cell adhesion protein 1) expression through the cAMP/PKA (protein kinase A)/CREB (cAMP response element-binding protein)/c-Jun and PI3K (phosphatidylinositol 3 kinase)/AKT (protein kinase B)/GSK-3ß (glycogen synthase kinase 3 beta)/GATA-6 (GATA-binding protein 6) pathways. In monocytes, FSH upregulated CD29 (cluster of differentiation 29) expression via the PI3K/AKT/GSK-3ß/SP1 (specificity protein 1) pathway and promoted monocyte-endothelial adhesion both in vitro and in vivo. Importantly, FSHR knockdown by shRNA in endothelium of carotid arteries markedly reduced GnRH agonist-induced endothelial inflammation and atherosclerosis in mice. CONCLUSIONS: FSH is responsible for ADT-associated atherosclerosis by exaggerating endothelial inflammation and promoting monocyte-endothelial adhesion.

Isothiazolinone Disrupts Reproductive Endocrinology by Targeting the G-Protein-Coupled Receptor Signaling.

The unintended exposure of humans and animals to isothiazolinones has led to an increasing concern regarding their health hazards. Isothiazolinones were previously found to disrupt reproductive endocrine homeostasis. However, the long-term reproductive toxicity and underlying mechanism remain unclear. In this study, life-cycle exposure of medaka to dichlorocthylisothiazolinone (DCOIT), a representative isothiazolinone, significantly stimulated the gonadotropin releasing hormone receptor (GnRHR)-mediated synthesis of follicle stimulating hormone and luteinizing hormone in the brain. Chem-Seq and proteome analyses revealed disturbances in the G-protein-coupled receptor, MAPK, and Ca2+ signaling cascades by DCOIT. The G protein αi subunit was identified as the binding target of DCOIT. Gαi bound by DCOIT had an enhanced affinity for the mitochondrial calcium uniporter, consequently changing Ca2+ subcellular compartmentalization. Stimulation of Ca2+ release from the endoplasmic reticulum and blockage of Ca2+ uptake into the mitochondria resulted in a considerably higher cytoplasmic Ca2+ concentration, which then activated the phosphorylation of MEK and ERK to dysregulate hormone synthesis. Overall, by comprehensively integrating in vivo, ex vivo, in silico, and in vitro evidence, this study proposes a new mode of endocrine disrupting toxicity based on isothiazolinones, which is expected to aid the risk assessment of the chemical library and favor the mechanism-driven design of safer alternatives.

Evaluation of fish pituitary spheroids to study annual endocrine reproductive control.

The pituitary gland is a small endocrine gland located below the hypothalamus. This gland releases several important hormones and controls the function of many other endocrine system glands to release hormones. Fish pituitary hormonal cells are controlled by neuroendocrine and sex steroid feedback. To study the complex pituitary function in vivo, we established an in vitro pituitary spheroid assay and evaluated its suitability for monitoring the annual reproductive physiological conditions in Takifugu rubripes, also known as torafugu, is one of the most economically important species distributed in the northwestern part of the Pacific Ocean, in the western part of the East China Sea, and in more northern areas near Hokkaido, Japan. Fish pituitary spheroids can be easily constructed in liquid or solid plates. The culture medium (L-15) made the aggregation faster than MEM (Hank's). A Rho-kinase inhibitor (Y-27632, 10 µM) and/or fish serum (2.5 %) also promoted spheroid formation. Laser confocal microscopy analysis of spheroids cultured with annual serum of both sexes revealed that luteinizing hormone (LH) synthesis has the highest peak in the final maturation stage (3 years old, May) in accordance with the highest serum sex steroid levels; in contrast, follicle stimulating hormone (FSH) synthesis has no correlation with the dose of serum or nutrients. Similarly, 3D cell propagation assays using female serum showed that total pituitary cells displayed the highest proliferation at puberty onset (2 years old, October) before half a year of the spawning season. These results indicate that pituitary spheroids are useful in vitro models for monitoring the reproductive physiological status of fish in vivo and may be applicable to the in vitro screening of environmental chemicals and bioactive compounds affecting reproductive efficiency in aquaculture.

PLXNA1 and PLXNA3 cooperate to pattern the nasal axons that guide gonadotropin-releasing hormone neurons.

Gonadotropin-releasing hormone (GnRH) neurons regulate puberty onset and sexual reproduction by secreting GnRH to activate and maintain the hypothalamic-pituitary-gonadal axis. During embryonic development, GnRH neurons migrate along olfactory and vomeronasal axons through the nose into the brain, where they project to the median eminence to release GnRH. The secreted glycoprotein SEMA3A binds its receptors neuropilin (NRP) 1 or NRP2 to position these axons for correct GnRH neuron migration, with an additional role for the NRP co-receptor PLXNA1. Accordingly, mutations in SEMA3A, NRP1, NRP2 and PLXNA1 have been linked to defective GnRH neuron development in mice and inherited GnRH deficiency in humans. Here, we show that only the combined loss of PLXNA1 and PLXNA3 phenocopied the full spectrum of nasal axon and GnRH neuron defects of SEMA3A knockout mice. Together with Plxna1, the human orthologue of Plxna3 should therefore be investigated as a candidate gene for inherited GnRH deficiency.

EAP1 regulation of GnRH promoter activity is important for human pubertal timing.

The initiation of puberty is orchestrated by an augmentation of gonadotropin-releasing hormone (GnRH) secretion from a few thousand hypothalamic neurons. Recent findings have indicated that the neuroendocrine control of puberty may be regulated by a hierarchically organized network of transcriptional factors acting upstream of GnRH. These include enhanced at puberty 1 (EAP1), which contributes to the initiation of female puberty through transactivation of the GnRH promoter. However, no EAP1 mutations have been found in humans with disorders of pubertal timing. We performed whole-exome sequencing in 67 probands and 93 relatives from a large cohort of familial self-limited delayed puberty (DP). Variants were analyzed for rare, potentially pathogenic variants enriched in case versus controls and relevant to the biological control of puberty. We identified one in-frame deletion (Ala221del) and one rare missense variant (Asn770His) in EAP1 in two unrelated families; these variants were highly conserved and potentially pathogenic. Expression studies revealed Eap1 mRNA abundance in peri-pubertal mouse hypothalamus. EAP1 binding to the GnRH1 promoter increased in monkey hypothalamus at the onset of puberty as determined by chromatin immunoprecipitation. Using a luciferase reporter assay, EAP1 mutants showed a reduced ability to trans-activate the GnRH promoter compared to wild-type EAP1, due to reduced protein levels caused by the Ala221del mutation and subcellular mislocation caused by the Asn770His mutation, as revealed by western blot and immunofluorescence, respectively. In conclusion, we have identified the first EAP1 mutations leading to reduced GnRH transcriptional activity resulting in a phenotype of self-limited DP.

Changes in Both Neuron Intrinsic Properties and Neurotransmission Are Needed to Drive the Increase in GnRH Neuron Firing Rate during Estradiol-Positive Feedback.

Central output of gonadotropin-releasing hormone (GnRH) neurons controls fertility and is sculpted by sex-steroid feedback. A switch of estradiol action from negative to positive feedback initiates a surge of GnRH release, culminating in ovulation. In ovariectomized mice bearing constant-release estradiol implants (OVX+E), GnRH neuron firing is suppressed in the morning (AM) by negative feedback and activated in the afternoon (PM) by positive feedback; no time-of-day-dependent changes occur in OVX mice. In this daily surge model, GnRH neuron intrinsic properties are shifted to favor increased firing during positive feedback. It is unclear whether this shift and the observed concomitant increase in GABAergic transmission, which typically excites GnRH neurons, are independently sufficient for increasing GnRH neuron firing rate during positive feedback or whether both are needed. To test this, we used dynamic clamp to inject selected previously recorded trains of GABAergic postsynaptic conductances (PSgs) collected during the different feedback states of the daily surge model into GnRH neurons from OVX, OVX+E AM, and OVX+E PM mice. PSg trains mimicking positive feedback initiated more action potentials in cells from OVX+E PM mice than negative feedback or OVX (open feedback loop) trains in all three animal models, but the positive-feedback train was most effective when applied to cells during positive feedback. In silico studies of model GnRH neurons in which >1000 PSg trains were tested exhibited the same results. These observations support the hypothesis that GnRH neurons integrate fast-synaptic and intrinsic changes to increase firing rates during positive feedback.SIGNIFICANCE STATEMENT Infertility affects 15%-20% of couples; failure to ovulate is a common cause. Understanding how the brain controls ovulation is critical for new developments in both infertility treatment and contraception. Ovarian estradiol alters both the intrinsic properties of gonadotropin-releasing hormone (GnRH) neurons and synaptic inputs to these cells coincident with production of sustained GnRH release that ultimately triggers ovulation. We demonstrate here using dynamic clamp and mathematical modeling that estradiol-induced shifts in synaptic transmission alone can increase firing output, but that the intrinsic properties of GnRH neurons during positive feedback further poise these cells for increased response to higher frequency synaptic transmission. These data suggest that GnRH neurons integrate fast-synaptic and intrinsic changes to increase firing rates during the preovulatory GnRH surge.

The Origin of GnRH Pulse Generation: An Integrative Mathematical-Experimental Approach.

Fertility critically depends on the gonadotropin-releasing hormone (GnRH) pulse generator, a neural construct comprised of hypothalamic neurons coexpressing kisspeptin, neurokoinin-B and dynorphin. Here, using mathematical modeling and in vivo optogenetics we reveal for the first time how this neural construct initiates and sustains the appropriate ultradian frequency essential for reproduction. Prompted by mathematical modeling, we show experimentally using female estrous mice that robust pulsatile release of luteinizing hormone, a proxy for GnRH, emerges abruptly as we increase the basal activity of the neuronal network using continuous low-frequency optogenetic stimulation. Further increase in basal activity markedly increases pulse frequency and eventually leads to pulse termination. Additional model predictions that pulsatile dynamics emerge from nonlinear positive and negative feedback interactions mediated through neurokinin-B and dynorphin signaling respectively are confirmed neuropharmacologically. Our results shed light on the long-elusive GnRH pulse generator offering new horizons for reproductive health and wellbeing.SIGNIFICANCE STATEMENT The gonadotropin-releasing hormone (GnRH) pulse generator controls the pulsatile secretion of the gonadotropic hormones LH and FSH and is critical for fertility. The hypothalamic arcuate kisspeptin neurons are thought to represent the GnRH pulse generator, since their oscillatory activity is coincident with LH pulses in the blood; a proxy for GnRH pulses. However, the mechanisms underlying GnRH pulse generation remain elusive. We developed a mathematical model of the kisspeptin neuronal network and confirmed its predictions experimentally, showing how LH secretion is frequency-modulated as we increase the basal activity of the arcuate kisspeptin neurons in vivo using continuous optogenetic stimulation. Our model provides a quantitative framework for understanding the reproductive neuroendocrine system and opens new horizons for fertility regulation.

Clock control of mammalian reproductive cycles: Looking beyond the pre-ovulatory surge of gonadotropins.

Several aspects of the physiology and behavior of organisms are expressed rhythmically with a 24-h periodicity and hence called circadian rhythms. Such rhythms are thought to be an adaptive response that allows to anticipate cyclic events in the environment. In mammals, the circadian system is a hierarchically organized net of endogenous oscillators driven by the hypothalamic suprachiasmatic nucleus (SCN). This system is synchronized by the environment throughout afferent pathways and in turn it organizes the activity of tissues by means of humoral secretions and neuronal projections. It has been shown that reproductive cycles are regulated by the circadian system. In rodents, the lesion of the SCN results on alterations of the estrous cycle, sexual behavior, tonic and phasic secretion of gonadotropin releasing hormone (GnRH)/gonadotropins and in the failure of ovulation. Most of the studies regarding the circadian control of reproduction, in particular of ovulation, have only focused on the participation of the SCN in the triggering of the proestrus surge of gonadotropins. Here we review aspects of the evolution and organization of the circadian system with particular focus on its relationship with the reproductive cycle of laboratory rodents. Experimental evidence of circadian control of neuroendocrine events indispensable for ovulation that occur prior to proestrus are discussed. In order to offer a working model of the circadian regulation of reproduction, its participation on aspects ranging from gamete production, neuroendocrine regulation, sexual behavior, mating coordination, pregnancy and deliver of the product should be assessed experimentally.

Roles of copper in neurokinin B and gonadotropin-releasing hormone structure and function and the endocrinology of reproduction.

Copper is a metal ion present in all organisms, where it has well-known roles in association with proteins and enzymes essential for cellular processes. In the early decades of the twentieth century copper was shown to influence mammalian reproductive biology, and it was subsequently shown to exert effects primarily at the level of the pituitary gland and/or hypothalamic regions of the brain. Furthermore, it has been reported that copper can interact with key neuropeptides in the hypothalamic-pituitary-gonadal axis, notably gonadotropin-releasing hormone (GnRH) and neurokinin B. Interestingly, recent phylogenetic analysis of the sequences of GnRH-related peptides indicates that copper binding is an evolutionarily ancient property of this neuropeptide family, which has been variously retained, modified or lost in the different taxa. In this mini-review the metal-binding properties of neuropeptides in the vertebrate reproductive pathway are reviewed and the evolutionary and functional significance of copper binding by GnRH-related neuropeptides in vertebrates and invertebrates are discussed.

The role of FSH and LH in prostate cancer and cardiometabolic comorbidities.

INTRODUCTION: In this article we advance a potential explanation for the incidence of cardiovascular (CV) and cardiometabolic risk in patients undergoing androgen deprivation therapy (ADT) for prostate cancer. Our conceptual model involves the differential impact of gonadotropin-releasing hormone (GnRH) agonists and antagonists on the follicle-stimulating hormone (FSH) system. MATERIALS AND METHODS: Authors searched online repositories and meeting abstract databases for relevant materials. RESULTS: Mounting evidence links FSH with development and progression of prostate cancer. What is also becoming clear is that the differential effects of GnRH agonists and antagonists on FSH may at least partially explain the differing effects these agents have on CV risk during ADT. While GnRH antagonists immediately suppress FSH, GnRH agonists provoke a transient surge in FSH that may contribute to the higher CV risk observed with these agents. Additionally, recent studies suggest that GnRH antagonists may significantly reduce CV risk compared to GnRH agonists, particularly in men with pre-existing CV disease. CONCLUSIONS: Patients with cardiovascular risk factors who require ADT may benefit from the better control of FSH provided by GnRH antagonists. ADT itself appears to heighten CV risk, and data suggest that FSH may at least partly drive this risk by promoting inflammation, atherosclerosis, insulin resistance, adipocyte rearrangement and plaque instability.

RFamide-related peptide-3 (RFRP-3) suppresses sexual maturation in a eusocial mammal.

Neuroendocrine mechanisms underlying social inhibition of puberty are not well understood. Here, we use a model exhibiting the most profound case of pubertal suppression among mammals to explore a role for RFamide-related peptide-3 [RFRP-3; mammalian ortholog to gonadotropin-inhibitory hormone (GnIH)] in neuroendocrine control of reproductive development. Naked mole rats (NMRs) live in sizable colonies where breeding is monopolized by two to four dominant animals, and no other members exhibit signs of puberty throughout their lives unless they are removed from the colony. Because of its inhibitory action on the reproductive axis in other vertebrates, we investigated the role of RFRP-3 in social reproductive suppression in NMRs. We report that RFRP-3 immunofluorescence expression patterns and RFRP-3/GnRH cross-talk are largely conserved in the NMR brain, with the exception of the unique presence of RFRP-3 cell bodies in the arcuate nucleus (Arc). Immunofluorescence comparisons revealed that central expression of RFRP-3 is altered by reproductive status, with RFRP-3 immunoreactivity enhanced in the paraventricular nucleus, dorsomedial nucleus, and Arc of reproductively quiescent NMRs. We further observed that exogenous RFRP-3 suppresses gonadal steroidogenesis and mating behavior in NMRs given the opportunity to undergo puberty. Together, our findings establish a role for RFRP-3 in preserving reproductive immaturity, and challenge the view that stimulatory peptides are the ultimate gatekeepers of puberty.

Gonadotropin-releasing hormone-independent effects of recombinant vertebrate ancient long opsin in the goldfish Carassius auratus reveal alternative reproduction pathways.

Vertebrate ancient long (VAL)-opsin is a green-sensitive photoreceptor that shows high sequence similarity to vertebrate ancient opsin, which is considered to play a role in sexual maturation via gonadotropin-releasing hormone (GnRH); however, the role of VAL-opsin in vertebrate sexual maturity remains unclear. Therefore, we investigated the possible role of VAL-opsin in reproduction in the goldfish Carassius auratus under a state of GnRH inhibition. Goldfish were injected with recombinant VAL-opsin protein (0.5 µg/g body mass) and/or the GnRH antagonist cetrorelix (0.5 µg/fish), and changes in the mRNA expression levels of genes associated with goldfish reproduction were measured by quantitative polymerase chain reaction, including those involved in the hypothalamus-pituitary-gonad (HPG) axis, VAL-opsin, GnRH, the gonadotropins (GTHs) luteinizing hormone and follicle-stimulating hormone, and estrogen receptor (ER). Moreover, the fish were irradiated with a green light-emitting diode (520 nm) to observe the synergistic effect on the HPG axis with VAL-opsin. Green LED exposure significantly and slightly increased the VAL-opsin and GnRH levels, respectively; however, these effects were blocked in groups injected with cetrorelix at all time points. Cetrorelix significantly decreased the mRNA levels of GTHs and ER, whereas these hormones recovered by co-treatment with VAL-opsin. These results indicate that green LED is an effective light source to promote the expression of sex hormones in fish. Moreover, VAL-opsin not only affects activity of the HPG axis but also appears to act on the pituitary gland directly to stimulate a new sexual maturation pathway that promotes the secretion of GTHs independent of GnRH.

Gonadotropin-Releasing Hormone (GnRH) Neuron Excitability Is Regulated by Estradiol Feedback and Kisspeptin.

Gonadotropin-releasing hormone (GnRH) neurons produce the central output controlling fertility and are regulated by steroid feedback. A switch from estradiol negative to positive feedback initiates the GnRH surge, ultimately triggering ovulation. This occurs on a daily basis in ovariectomized, estradiol-treated (OVX+E) mice; GnRH neurons are suppressed in the morning and activated in the afternoon. To test the hypotheses that estradiol and time of day signals alter GnRH neuron responsiveness to stimuli, GFP-identified GnRH neurons in brain slices from OVX+E or OVX female mice were recorded during the morning or afternoon. No differences were observed in baseline membrane potential. Current-clamp revealed GnRH neurons fired more action potentials in response to current injection during positive feedback relative to all other groups, which were not different from each other despite reports of differing ionic conductances. Kisspeptin increased GnRH neuron response in cells from OVX and OVX+E mice in the morning but not afternoon. Paradoxically, excitability in kisspeptin knock-out mice was similar to the maximum observed in control mice but was unchanged by time of day or estradiol. A mathematical model applying a Markov Chain Monte Carlo method to estimate probability distributions for estradiol- and time of day-dependent parameters was used to predict intrinsic properties underlying excitability changes. A single identifiable distribution of solutions accounted for similar GnRH neuron excitability in all groups other than positive feedback despite different underlying conductance properties; this was attributable to interdependence of voltage-gated potassium channel properties. In contrast, redundant solutions may explain positive feedback, perhaps indicative of the importance of this state for species survival.SIGNIFICANCE STATEMENT Infertility affects 15%-20% of couples; failure to ovulate is a common cause. Understanding how the brain controls ovulation is critical for new developments in both infertility treatment and contraception. Gonadotropin-releasing hormone (GnRH) neurons are the final common pathway for central neural control of ovulation. We studied how estradiol feedback regulates GnRH excitability, a key determinant of neural firing rate using laboratory and computational approaches. GnRH excitability is upregulated during positive feedback, perhaps driving increased neural firing rate at this time. Kisspeptin increased GnRH excitability and was essential for estradiol regulation of excitability. Modeling predicts that multiple combinations of changes to GnRH intrinsic conductances can produce the firing response in positive feedback, suggesting the brain has many ways to induce ovulation.

Has GnRH a direct role in AMH regulation?

OBJECTIVE: Anti-Mullerian hormone (AMH) together with luteinizing hormone (LH) and follicle-stimulating hormone (FSH) plays crucial roles in gonadal functions. However, the possible effects of GnRH on AMH via the hypothalamic-pituitary-gonadal (HPG) axis remain unexplored. We aimed to explore the changes in AMH levels after bolus GnRH stimulation and understand the relationship of AMH with FSH and LH in healthy subjects. METHODS: Thirty-one prepubertal children (15 males/16 females) and 78 adults (36 males/42 females) were included. We collected basal (0 minute) samples for determining levels of hormones. After GnRH treatment at a dose of 2.5 µg/kg body weight (maximum of 100 µg/kg body weight) intravenously, blood was collected at 30 minutes intervals for 120 minutes. Serum LH, FSH and AMH were measured by electrochemiluminometric assays. RESULTS: After injection of GnRH, AMH levels were significantly decreased in 30 minutes (P < 0.001) in all groups with parallel increase of FSH and LH. In the second 30 minutes, all hormones levels reversed. There was also a moderate correlation between AMH and FSH (r = -0.430, P < 0.001). CONCLUSIONS: GnRH lowers serum AMH levels, which have a negative correlation with the increase in gonadotrophins. These data pinpoint GnRH as an important factor of the AMH regulation, leading new opportunities for the understanding of AMH role in reproductive function and dysfunction.

Chronic administration of gonadotropin releasing-hormone improves learning in old gonadectomized rats.

The aim of this study was to determine whether chronic administration of GnRH improves performance of learning tasks and expression of spinophilin in the hippocampus of gonadectomized old rats. Eighteen-month-old male Wistar rats were used and divided into three groups: control (intact rats); gonadectomized; and gonadectomized + GnRH. The latter group was injected intramuscularly with GnRH (100 nM) twice a day for five weeks. The learning tasks we used were the novel object recognition task (NOR), elevated T-maze (ETM) and active avoidance test (AAT). Results showed that in NOR and ETM learning tasks, gonadectomized rats treated with GnRH had a significantly better performance than gonadectomized rats without treatment. GnRH-treated gonadectomized rats displayed performance comparable to that of intact rats. Furthermore, the expression of spinophilin in the hippocampus of gonadectomized rats treated with GnRH increased with respect to untreated gonadectomized rats. In conclusion, the chronic administration of GnRH improves learning in old gonadectomized rats. It is possible that the mechanism could involve a greater number of dendritic contacts associated with a higher expression of spinophilin.

c-JUN Dimerization Protein 2 (JDP2) Is a Transcriptional Repressor of Follicle-stimulating Hormone ß (FSHß) and Is Required for Preventing Premature Reproductive Senescence in Female Mice.

Follicle-stimulating hormone (FSH) regulates follicular growth and stimulates estrogen synthesis in the ovaries. FSH is a heterodimer consisting of an α subunit, also present in luteinizing hormone, and a unique ß subunit, which is transcriptionally regulated by gonadotropin-releasing hormone 1 (GNRH). Because most FSH is constitutively secreted, tight transcriptional regulation is critical for maintaining FSH levels within a narrow physiological range. Previously, we reported that GNRH induces FSHß (Fshb) transcription via induction of the AP-1 transcription factor, a heterodimer of c-FOS and c-JUN. Herein, we identify c-JUN-dimerization protein 2 (JDP2) as a novel repressor of GNRH-mediated Fshb induction. JDP2 exhibited high basal expression and bound the Fshb promoter at an AP-1-binding site in a complex with c-JUN. GNRH treatment induced c-FOS to replace JDP2 as a c-JUN binding partner, forming transcriptionally active AP-1. Subsequently, rapid c-FOS degradation enabled reformation of the JDP2 complex. In vivo studies revealed that JDP2 null male mice have normal reproductive function, as expected from a negative regulator of the FSH hormone. Female JDP2 null mice, however, exhibited early puberty, observed as early vaginal opening, larger litters, and early reproductive senescence. JDP2 null females had increased levels of circulating FSH and higher expression of the Fshb subunit in the pituitary, resulting in elevated serum estrogen and higher numbers of large ovarian follicles. Disruption of JDP2 function therefore appears to cause early cessation of reproductive function, a condition that has been associated with elevated FSH in women.

Knockout of the Gnrh genes in zebrafish: effects on reproduction and potential compensation by reproductive and feeding-related neuropeptides.

Gonadotropin-releasing hormone (GNRH) is known as a pivotal upstream regulator of reproduction in vertebrates. However, reproduction is not compromised in the hypophysiotropic Gnrh3 knockout line in zebrafish (gnrh3-/-). In order to determine if Gnrh2, the only other Gnrh isoform in zebrafish brains, is compensating for the loss of Gnrh3, we generated a double Gnrh knockout zebrafish line. Surprisingly, the loss of both Gnrh isoforms resulted in no major impact on reproduction, indicating that a compensatory response, outside of the Gnrh system, was evoked. A plethora of factors acting along the reproductive hypothalamus-pituitary axis were evaluated as possible compensators based on neuroanatomical and differential gene expression studies. In addition, we also examined the involvement of feeding factors in the brain as potential compensators for Gnrh2, which has known anorexigenic effects. We found that the double knockout fish exhibited upregulation of several genes in the brain, specifically gonadotropin-inhibitory hormone (gnih), secretogranin 2 (scg2), tachykinin 3a (tac3a), and pituitary adenylate cyclase-activating peptide 1 (pacap1), and downregulation of agouti-related peptide 1 (agrp1), indicating the compensation occurs outside of Gnrh cells and therefore is a noncell autonomous response to the loss of Gnrh. While the differential expression of gnih and agrp1 in the double knockout line was confined to the periventricular nucleus and hypothalamus, respectively, the upregulation of scg2 corresponded with a broader neuronal redistribution in the lateral hypothalamus and hindbrain. In conclusion, our results demonstrate the existence of a redundant reproductive regulatory system that comes into play when Gnrh2 and Gnrh3 are lost.

Enhancement of a robust arcuate GABAergic input to gonadotropin-releasing hormone neurons in a model of polycystic ovarian syndrome.

Polycystic ovarian syndrome (PCOS), the leading cause of female infertility, is associated with an increase in luteinizing hormone (LH) pulse frequency, implicating abnormal steroid hormone feedback to gonadotropin-releasing hormone (GnRH) neurons. This study investigated whether modifications in the synaptically connected neuronal network of GnRH neurons could account for this pathology. The PCOS phenotype was induced in mice following prenatal androgen (PNA) exposure. Serial blood sampling confirmed that PNA elicits increased LH pulse frequency and impaired progesterone negative feedback in adult females, mimicking the neuroendocrine abnormalities of the clinical syndrome. Imaging of GnRH neurons revealed greater dendritic spine density that correlated with increased putative GABAergic but not glutamatergic inputs in PNA mice. Mapping of steroid hormone receptor expression revealed that PNA mice had 59% fewer progesterone receptor-expressing cells in the arcuate nucleus of the hypothalamus (ARN). To address whether increased GABA innervation to GnRH neurons originates in the ARN, a viral-mediated Cre-lox approach was taken to trace the projections of ARN GABA neurons in vivo. Remarkably, projections from ARN GABAergic neurons heavily contacted and even bundled with GnRH neuron dendrites, and the density of fibers apposing GnRH neurons was even greater in PNA mice (56%). Additionally, this ARN GABA population showed significantly less colocalization with progesterone receptor in PNA animals compared with controls. Together, these data describe a robust GABAergic circuit originating in the ARN that is enhanced in a model of PCOS and may underpin the neuroendocrine pathophysiology of the syndrome.

The gonadotropin-inhibitory hormone (Lpxrfa) system's regulation of reproduction in the brain-pituitary axis of the zebrafish (Danio rerio).

Gonadotropin-inhibitory hormone (GNIH) was discovered in quail with the ability to reduce gonadotropin expression/secretion in the pituitary. There have been few studies on GNIH orthologs in teleosts (LPXRFamide (Lpxrfa) peptides), which have provided inconsistent results. Therefore, the goal of this study was to determine the roles and modes of action by which Lpxrfa exerts its functions in the brain-pituitary axis of zebrafish (Danio rerio). We localized Lpxrfa soma to the ventral hypothalamus, with fibers extending throughout the brain and to the pituitary. In the preoptic area, Lpxrfa fibers interact with gonadotropin-releasing hormone 3 (Gnrh3) soma. In pituitary explants, zebrafish peptide Lpxrfa-3 downregulated luteinizing hormone beta subunit and common alpha subunit expression. In addition, Lpxrfa-3 reduced gnrh3 expression in brain slices, offering another pathway for Lpxrfa to exert its effects on reproduction. Receptor activation studies, in a heterologous cell-based system, revealed that all three zebrafish Lpxrfa peptides activate Lpxrf-R2 and Lpxrf-R3 via the PKA/cAMP pathway. Receptor activation studies demonstrated that, in addition to activating Lpxrf receptors, zebrafish Lpxrfa-2 and Lpxrfa-3 antagonize Kisspeptin-2 (Kiss2) activation of Kisspeptin receptor-1a (Kiss1ra). The fact that kiss1ra-expressing neurons in the preoptic area are innervated by Lpxrfa-ir fibers suggests an additional pathway for Lpxrfa action. Therefore, our results suggest that Lpxrfa may act as a reproductive inhibitory neuropeptide in the zebrafish that interacts with Gnrh3 neurons in the brain and with gonadotropes in the pituitary, while also potentially utilizing the Kiss2/Kiss1ra pathway.

Brain endothelial cells control fertility through ovarian-steroid-dependent release of semaphorin 3A.

Neuropilin-1 (Nrp1) guides the development of the nervous and vascular systems, but its role in the mature brain remains to be explored. Here we report that the expression of the 65 kDa isoform of Sema3A, the ligand of Nrp1, by adult vascular endothelial cells, is regulated during the ovarian cycle and promotes axonal sprouting in hypothalamic neurons secreting gonadotropin-releasing hormone (GnRH), the neuropeptide controlling reproduction. Both the inhibition of Sema3A/Nrp1 signaling and the conditional deletion of Nrp1 in GnRH neurons counteract Sema3A-induced axonal sprouting. Furthermore, the localized intracerebral infusion of Nrp1- or Sema3A-neutralizing antibodies in vivo disrupts the ovarian cycle. Finally, the selective neutralization of endothelial-cell Sema3A signaling in adult Sema3aloxP/loxP mice by the intravenous injection of the recombinant TAT-Cre protein alters the amplitude of the preovulatory luteinizing hormone surge, likely by perturbing GnRH release into the hypothalamo-hypophyseal portal system. Our results identify a previously unknown function for 65 kDa Sema3A-Nrp1 signaling in the induction of axonal growth, and raise the possibility that endothelial cells actively participate in synaptic plasticity in specific functional domains of the adult central nervous system, thus controlling key physiological functions such as reproduction.