Gonadotropin-releasing Hormone Gene Expression Research Articles

Gonadotropin-Releasing Hormone (GnRH) and Its Agonists in Bovine Reproduction I: Structure, Biosynthesis, Physiological Effects, and Its Role in Estrous Synchronization.

GnRH is essential for the regulation of mammalian reproductive processes. It regulates the production and release of pituitary gonadotropins, thereby influencing steroidogenesis and gametogenesis. While primarily produced in the hypothalamus, GnRH is also produced in peripheral organs, such as the gonads and placenta. GnRH analogs, including agonists and antagonists, have been synthesized for the reproductive management of animals and humans. This review focuses on the functions of hypothalamic GnRH in the reproductive processes of cattle. In addition to inducing the surge release of LH, the pulsatile secretion of GnRH stimulates the pituitary gland to release FSH and LH, thereby regulating gonadal function. Various GnRH-based products have been synthesized to increase their potency and efficacy in regulating reproductive functions. This review article describes the chemical structures of GnRH and its agonists. This discussion extends to the gene expression of GnRH in the hypothalamus, highlighting its pivotal role in regulating the reproductive process. Furthermore, GnRH is involved in regulating ovarian follicular development and luteal phase support, and estrus synchronization is involved. A comprehensive understanding of the role of GnRH and its analogs in the modulation of reproductive processes is essential for optimizing animal reproduction.

The Involvement of the microRNAs miR-466c and miR-340 in the Palmitate-Mediated Dysregulation of Gonadotropin-Releasing Hormone Gene Expression.

Diets high in saturated fatty acids are associated with obesity and infertility. Palmitate, the most prevalent circulating saturated fatty acid, is sensed by hypothalamic neurons, contributing to homeostatic dysregulation. Notably, palmitate elevates the mRNA levels of gonadotropin-releasing hormone (Gnrh) mRNA and its activating transcription factor, GATA binding protein 4 (Gata4). GATA4 is essential for basal Gnrh expression by binding to its enhancer region, with Oct-1 (Oct1) and CEBP-β (Cebpb) playing regulatory roles. The pre- and post-transcriptional control of Gnrh by palmitate have not been investigated. Given the ability of palmitate to alter microRNAs (miRNAs), we hypothesized that palmitate-mediated dysregulation of Gnrh mRNA involves specific miRNAs. In the mHypoA-GnRH/GFP neurons, palmitate significantly downregulated six miRNAs (miR-125a, miR-181b, miR-340, miR-351, miR-466c and miR-503), and the repression was attenuated by co-treatment with 100 μM of oleate. Subsequent mimic transfections revealed that miR-466c significantly downregulates Gnrh, Gata4, and Chop mRNA and increases Per2, whereas miR-340 upregulates Gnrh, Gata4, Oct1, Cebpb, and Per2 mRNA. Our findings suggest that palmitate may indirectly regulate Gnrh at both the pre- and post-transcriptional levels by altering miR-466c and miR-340, which in turn regulate transcription factor expression levels. In summary, palmitate-mediated dysregulation of Gnrh and, consequently, reproductive function involves parallel transcriptional mechanisms.

Central MOTS-c infusion affects reproductive hormones in obese and non-obese rats

MOTS-c, a mitochondrial-derived peptide, acts as a systemic hormone and MOTS-c level is inversely correlated with markers of obesity. Obesity is a risk factor for male reproductive physiology and is expressed as an important cause of infertility. In this study, we aimed to determine the effects of MOTS-c, which has been proven in the hypothalamus and testicles, on the actors involved in the reproductive axis.In the study, 80 male Wistar-Albino rats were divided into two main groups, obese and non-obese (n = 40). Rats in the first main group were fed with fatty diet feed and obesity was induced. The second main group was fed with normal diet feed. Each main group was divided into 4 subgroups (Control, Sham, 10 and 100 µM MOTS-c). The lateral ventricles of the animals in the treatment groups were infused with 10 and 100 µM MOTS-c (solvent in Sham group) for 14 days. At the end of the experiment, hypothalamic Gonadotropin-Releasing Hormone (GnRH) gene expression level, serum testosterone, Luteinizing hormone (LH) and Follicle stimulating hormone (FSH) levels were determined.MOTS-c infusion caused an increase in GnRH mRNA, protein expression levels and serum testosterone, LH and FSH levels in obese and non-obese rats (p < 0.05). MOTS-c administration more significantly upregulated hormone levels in non-obese rats (p < 0.05). MOTS-c administration increases these hormones, suggesting that MOTS-c may stimulate the reproductive axis.Our results reveal that MOTS-c plays a role in the central regulation of reproduction, as well as causes increased LH, FSH and testosterone release.

Kisspeptin induces Kiss-1 and GnRH gene expression in mHypoA-55 hypothalamic cell models: Involvement of the ERK and PKA signaling pathways

mHypoA-55 cells are kisspeptin-expressing neuronal cells originating from the arcuate nucleus of the mouse hypothalamus. These cells are called KNDy neurons because they co-express kisspeptin, neurokinin B, and dynorphin A. In addition, they express gonadotropin-releasing hormone (GnRH).Here, we found that kisspeptin 10 (KP10) increased Kiss-1 (encoding kisspeptin) and GnRH gene expression in kisspeptin receptor (Kiss-1R)-overexpressing mHypoA-55 cells. KP10 greatly increased serum response element (SRE) promoter activity, which is a target of extracellular signal-regulated kinase (ERK) (20.0 ± 2.54-fold). KP10 also increased cAMP-response element (CRE) promoter activity in these cells (2.32 ± 0.36-fold). KP10-increased SRE promoter activity was significantly prevented in the presence of PD098095, a MEK kinase (MEKK) inhibitor, and KP10-induced CRE promoter activity was also inhibited by PD098059. Similarly, H89, a protein kinase A (PKA) inhibitor, significantly inhibited the KP10 induction of SRE and CRE promoters. KP10-induced Kiss-1 and GnRH gene expressions were inhibited in the presence of PD098059. Likewise, H89 significantly inhibited the KP10-induced increase in Kiss-1 and GnRH. Transfection of mHypoA-55 cells with constitutively active MEKK (pFC-MEKK) increased SRE and CRE promoter activities by 9.75 ± 1.77- and 1.36 ± 0.12-fold, respectively. Induction of constitutively active PKA (pFC-PKA) also increased SRE and CRE promoter activities by 2.41 ± 0.42- and 40.71 ± 7.77-fold, respectively. Furthermore, pFC-MEKK and -PKA transfection of mHypoA-55 cells increased both Kiss-1 and GnRH gene expression.Our current observations suggest that KP10 increases both the ERK and PKA pathways and that both pathways mutually interact in mHypoA-55 hypothalamic cells. Activation of both ERK and PKA signaling might be necessary to induce Kiss-1 and GnRH gene expressions.

Effects of benzo[a]pyrene on the reproductive axis: Impairment of kisspeptin signaling in human gonadotropin-releasing hormone primary neurons

The neuroendocrine control of reproduction is strictly coordinated at the central level by the pulsatile release of gonadotropin-releasing hormone (GnRH) by the hypothalamic GnRH neurons. Alterations of the GnRH-network, especially during development, lead to long-term reproductive and systemic consequences, also causing infertility. Recent evidence shows that benzo[a]pyrene (BaP), a diffuse pollutant that can play a role as an endocrine disruptor, affects gonadal function and gamete maturation, whereas data demonstrating its impact at hypothalamic level are very scarce. This study investigated the effects of BaP (10 μM) in a primary cell culture isolated from the human fetal hypothalamus (hfHypo) and exhibiting a clear GnRH neuron phenotype. BaP significantly decreased gene and protein expression of both GnRH and kisspeptin receptor (KISS1R), the master regulator of GnRH neuron function. Moreover, BaP exposure increased phospho-ERK1/2 signaling, a well-known mechanism associated with KISS1R activation. Interestingly, BaP altered the electrophysiological membrane properties leading to a significant depolarizing effect and it also significantly increased GnRH release, with both effects being not affected by kisspeptin addition. In conclusion, our findings demonstrate that BaP may alter GnRH neuron phenotype and function, mainly interfering with KISS1R signaling and GnRH secretion and therefore with crucial mechanisms implicated in the central neuroendocrine control of reproduction.

A role for GABA agonist in controlling the reproduction of female rats via hypothalamic ghrelin, kisspeptin, and RFRP-3 gene expression

Kisspeptin stimulates gonadotropin releasing hormone (GnRH). The GnRH neurons receive inhibitory inputs from ghrelin, RFamide related peptide-3 (RFRP-3), and gamma-aminobutyric acid (GABA) neurons. Polycystic ovary syndrome (PCOS) is associated with increased levels of GnRH/LH and kisspeptin, and decreased release of GABA, ghrelin, and RFRP-3. In the present study, the effects of GABAB receptor agonist, baclofen, were investigated on GnRH, KiSS1, RFRP-3, and ghrelin gene expression in the hypothalamus of PCOS model rats. For induction of PCOS, female Wistar rats weighing 180-200g received intra-muscular injection of estradiol valerate. Fifteen PCOS rats in three groups received intraperitoneal injections of saline, 5, or 10 mg/kg baclofen for two weeks. The hypothalamic samples were dissected. Gene expression levels of GnRH, KiSS1, RFRP-3, and ghrelin were determined by real time qPCR method. Results revealed that baclofen significantly decreased the mean relative KiSS1 gene expression compared to PCOS group. Also, the mean relative RFRP-3 gene expression significantly increased in the baclofen-receiving rats in comparison to PCOS group. Furthermore, baclofen did not change GnRH or ghrelin mRNA levels in comparison to PCOS group. According to these results it can be concluded that in PCOS condition the GABAergic signaling pathway may suppress GnRH neural activity via down or up regulation of the intra-hypothalamic neuropeptides upstream of GnRH neurons.

Mutual Interactions Between GnRH and Kisspeptin in GnRH- and Kiss-1-Expressing Immortalized Hypothalamic Cell Models.

Kisspeptin and gonadotropin-releasing hormone (GnRH) are central regulators of the hypothalamic-pituitary-gonadal axis and control female reproductive functions. Recently established mHypoA-50 and mHypoA-55 cells are immortalized hypothalamic neuronal cell models that originated from the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) regions of the mouse hypothalamus, respectively. mHypoA-50 or mHypoA-55 cells were stimulated with kisspeptin-10 (KP10) and GnRH, after which the expression of kisspeptin and GnRH was determined. Primary cultures of fetal rat brain cells were also examined. mHypoA-50 and mHypoA-55 cells expressed mRNA for Kiss-1 (which encodes kisspeptin) and GnRH as well as receptors for kisspeptin and GnRH. We found that Kiss-1 mRNA expression was significantly increased in mHypoA-50 AVPV cells by KP10 and GnRH stimulation. Kisspeptin protein expression was also increased by KP10 and GnRH stimulation in these cells. In contrast, GnRH expression was unchanged in mHypoA-50 AVPV cells by KP10 and GnRH stimulation. In mHypoA-55 ARC cells, kisspeptin expression was also significantly increased at the mRNA and protein levels by KP10 and GnRH stimulation; however, GnRH expression was also upregulated by KP10 and GnRH stimulation in these cells. KP10 and estradiol (E2) both increased Kiss-1 gene expression in mHypoA-50 AVPV cells, but combined stimulation with KP10 and E2 did not potentiate their individual effects on Kiss-1 gene expression. On the other hand, E2 did not increase Kiss-1 gene expression in mHypoA-55 ARC cells, and the KP10-induced increase of Kiss-1 gene expression was inhibited in the presence of E2 in these cells. KP10 and GnRH significantly increased c-Fos protein expression in the mHypoA-50 AVPV and mHypoA-55 ARC cell lines. In primary cultures of fetal rat neuronal cells, KP10 significantly increased Kiss-1 gene expression, whereas GnRH significantly increased GnRH gene expression. We found that kisspeptin and GnRH affected Kiss-1- and GnRH-expressing hypothalamic cells and modulated Kiss-1 and/or GnRH gene expression with a concomitant increase in c-Fos protein expression. A mutual- or self-regulatory system might be present in Kiss-1 and/or GnRH neurons in the hypothalamus.

Effect of anti-Müllerian hormone in hypothalamic Kiss-1- and GnRH-producing cell models

Purpose: Anti-Müllerian hormone (AMH) is one of the local factors involved in follicle development. In addition, AMH and its receptor are broadly expressed throughout the body. In this study, we examined how AMH modifies gene expression of Kiss-1 and GnRH. Materials and methods: mHypoA-50 and mHypoA-55 cells were originated from the hypothalamic anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC), respectively, and these cells are known as Kiss-1 (which encodes kisspeptin) expressing cell models. These cells also express gonadotropin-releasing hormone (GnRH) genes. Our experiments were performed useing these cell models. Results: Both mHypoA-50 and mHypoA-55 hypothalamic cells expressed AMH and AMH receptor type 2 (AMHR2). Exogenous AMH failed to alter the expression levels of the Kiss-1 gene in both cell models but significantly increased GnRH gene expression by 1.73 ± 0.2-fold at 100 pM in mHypoA-50 AVPV cells and by 1.74 ± 0.17-fold at 1 nM in mHypoA-55 ARC cells. AMH also augmented GnRH protein expression in both cell models. Similar to the phenomenon observed in the hypothalamic cell lines, 100 pM AMH significantly increased GnRH, but not Kiss-1, mRNA expression in primary cultures of fetal rat brain cells. Kisspeptin-10 (KP10) increased Kiss-1 gene expression in mHypoA-55 ARC cells but this was blocked by AMH. AMH did not alter the expression of the kisspeptin receptor (Kiss1R) or that of neurokinin B or dynorphin A in mHypoA-55 ARC cells. Conclusions: It was demonstrated that AMH participates in hypothalamic-pituitary-gonadal axis control by stimulating GnRH expression. In addition, AMH might be a potent repressor of Kiss-1 gene expression induced by KP10.

The epigenetic role of HTR1A antagonist in facilitating GnRH expression for pubertal initiation control

Serotonin (5-hydroxytryptamine [5-HT]), a metabolite of tryptophan, acts on the components of the hypothalamus-hypophysis-gonad axis and induces puberty delay in mammals via 5-HT receptor 1A (HTR1A). However, the roles of HTR1A in the hypothalamus in pubertal regulation of gene expression are not fully understood. In the current study, the upregulated gonadotropin-releasing hormone (GnRH) expression in GT1-7 GnRH neuronal cells induced by the HTR1A antagonist WAY-100635 maleate was observed in vitro. Furthermore, RNA sequencing (RNA-seq) showed decreased expression of chromobox 4 (CBX4), a member of the polycomb-repressive complex 1 (PRC1), and the loss of RING2 and YY1 interaction with CBX4, suggesting the degradation of the PRC1 in GT1-7 cells treated with maleate. Chromatin immunoprecipitation sequencing (ChIP-seq) showed that the genome-wide occupancy of CBX4 and histone H2A lysine-119 ubiquitination (H2AK119ub) was compromised, especially on the promoter of GnRH. Finally, we determined that inactivation of phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) contributed to CBX4 downregulation. Taken together, we concluded that HTR1A antagonists could enhance GnRH transcription via PRC1 degradation and H2AK119ub loss driven by reduced CBX4 expression through PI3K/Akt and MAPK/ERK pathway suppression in GT1-7 cells and provided a potential epigenetic mechanism of action of HTR1A on GnRH gene expression for mammalian puberty onset.

Mammary gland-specific regulation of GNRH and GNRH-receptor gene expression is likely part of a local autoregulatory system in female vizcachas (Rodentia: Chinchillidae)

In addition to key mammotrophic hormones such as the pituitary prolactin (PRL) and the ovarian steroids progesterone and estradiol, there are local factors that modulate the tissue dynamics of the mammary glands during pregnancy and lactation. By immunohistochemistry and RT-PCR, we found local transcription and translation of gonadotropin-releasing hormone (GNRH), GNRH receptor (GNRHR), PRL and PRL receptor (PRLR) in mammary glands of adult vizcachas during pregnancy and lactation. Both GNRH and GNRHR showed a lag between protein expression and gene transcription throughout the gestational period: while the highest transcription levels of these genes were recorded at early-pregnancy, the epithelial immunoexpressions of both showed their maximum during lactation. RIA results corroborated the presence of GNRH in mammary glands at all the analyzed stages and confirmed the maximum amount of this peptide in the lactating group. Significant amounts of GNRH were detected in milk samples as well. Conversely, PRL and PRLR shared similar protein and gene expression profiles, all exhibiting maximum values during lactation. GNRH peptide content in mammary glands of females with sulpiride-induced hyperprolactinemia (HP) was significantly lower than that of control females (CT). Although PRL mRNA levels remained unchanged, there was a marked increase in theα-lactalbumin (LALBA) transcription in mammary glands of HP- vs CT-females. These results suggest that after targeting mammary glands, PRL stimulates the expression of milk protein genes, but also, tempers the local expression of GNRH. Mammary gland-explantssupplemented with a GNRH analogue (GN-explants) had no differences in terms of PRLR orLALBA transcription levels compared to CT-explants, so the mammary PRLR signaling would not appear to be modulated by GNRH. Yet, mRNA expression levels of both GNRH and the GNRHR-downstream factor, EGR1, were significantly higher in GN-explants compared to that of CT which would point to a GNRH-positive feedback mechanism. In summary, the local coupled expression of GNRH, GNRHR and EGR1 in the mammary gland throughout pregnancy of vizcachas, the PRL-dependent mammary GNRH secretion as well as the GNRH positive feedback on its own transcription suggest an autocrine-paracrine regulatory mechanism and propose an active role for GNRH in mammary gland tissue remodeling.

SUN-035 A Role for GNRH-II in the Control of Puberty?

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons represent the primary neuroendocrine link between the brain and the reproductive system. Although they play a key role in stimulating the release of FSH and LH from the anterior pituitary gland, the underlying mechanism by which they trigger the onset of puberty is unclear. To address this issue, RT-PCR, in situ hybridization histochemistry, and Affymetrix gene arrays were used to profile hypothalamic GnRH gene expression in prepubertal and adult rhesus macaques (Macaca mulatta). Like humans, these nonhuman primates express two molecular forms of GnRH (GnRH-I and GnRH-II), both of which are highly effective at stimulating gonadotropin release via the same GnRHR1 receptor. However, only GnRH-II shows increased hypothalamic expression in the presence of elevated estrogen concentrations (i.e., positive feedback), whereas GnRH-I expression either remains the same or decreases (i.e., negative feedback). In the present study, the hypothalamic expression levels of GnRH-I and GnRHR1 were found to be no different between prepubertal and adult animals, despite marked differences in circulating sex-steroid hormone levels, whereas the hypothalamic expression level of GnRH-II was significantly higher in the adults than in the juveniles. Therefore, although the traditional GnRH-I neurons are likely to play a fundamental role in initiating FSH and LH release during the early stages of pubertal development, GnRH-II neurons may play an important role in maintaining elevated gonadotropin release during the final stages (i.e., at a time when the GnRH-I neurons are subjected to increasing negative sex-steroid feedback from the maturing gonads). Taken together, the data suggest that sexual maturation in primates is likely to be orchestrated by the concerted action of two distinct GnRH neuronal subtypes that respond differentially to sex-steroid feedback.

Role of RFRP-3 in the Regulation of Kiss-1 Gene Expression in the AVPV Hypothalamic Cell Model mHypoA-50.

Kisspeptin, encoded by the Kiss-1 gene, plays a crucial role in reproductive function by governing the hypothalamic-pituitary-gonadal axis. The recently established Kiss-1-expressing cell model mHypoA-50 displays characteristics of neuronal cells of the anteroventral periventricular (AVPV) region of the mouse hypothalamus. Because Kiss-1 gene expression in these cells is upregulated by estradiol (E2), mHypoA-50 cells are regarded as a valuable model for the study of Kiss-1-expressing neurons in the AVPV region. These cells also express RFamide-related peptide-3 (RFRP-3), a mammalian homolog of gonadotropin inhibitory hormone. The RFRP-3 expression in mHypoA-50 cells was increased by melatonin stimulation. In addition, E2 stimulation increased RFRP-3 expression in these cells. Treatment of the mHypoA-50 cells with exogenous RFRP-3 resulted in the increase of Kiss-1 messenger RNA expression within the cells; however, RFRP-3 did not modify gonadotropin-releasing hormone or kisspeptin-induced Kiss-1 gene expression in these cells. In addition, we found that RFRP-3 stimulation increased the expression of corticotropin-releasing hormone, which may be involved in E2-induced positive feedback in mHypoA-50 cells. Our observations suggest that RFRP-3 might be involved in positive feedback regulation by directly or indirectly increasing Kiss-1 gene expression.

SUN-475 Analysis of Palmitate- and Bisphenol A-Mediated Changes in MicroRNAs Targeting the Novel Reproductive Peptide Phoenixin and Its Receptor Gpr173 in Hypothalamic Cell Lines

Phoenixin (PNX) is a recently identified peptide that forms part of the hypothalamic-pituitary-gonadal axis. It is a highly conserved, 14 or 20 amino acid peptide expressed at high levels in the hypothalamus. Within hypothalamic neurons, PNX increases gene expression of gonadotropin-releasing hormone (GnRH) and kisspeptin, two critical reproductive peptides. In relation to reproductive function, knockdown of either PNX or its receptor, GPR173, extends the estrous cycle. As PNX and GPR173 are relatively understudied, delineating how this peptide and its putative receptor are regulated at the gene level is essential for further definition of functional significance. We have previously reported that palmitate, a saturated fatty acid, increases Pnx and reduces Gpr173 mRNA levels over 24 hours in immortalized hypothalamic clonal cell lines. We have also found that bisphenol A (BPA), an endocrine disrupting chemical widely thought to inhibit reproductive function, reduces Pnx and Gpr173 gene expression. The mechanism by which these compounds alter gene expression is the subject of current investigation. One layer of gene regulation is microRNAs (miRNAs), short segments of RNA that decrease mRNA expression levels. Palmitate and BPA are known to alter microRNA (miRNA) levels in tissues outside of the hypothalamus; therefore, we hypothesized that levels of miRNAs that target Pnx and Gpr173 in the hypothalamus may be contributing to the observed palmitate- and BPA-mediated changes in mRNA levels. Using the predictive online resources, miRwalk 3.0, TargetScan and miRanda, we have identified 112 miRNAs with putative binding sites in the Pnx 3’UTR, as well as 300 miRNAs that can bind the Gpr173 3’UTR. To identify miRNAs affected by palmitate or BPA exposure, the mHypoE-46 and the mHypoA-59 cell lines were treated with 50 μM palmitate or 100 μM BPA, respectively, for 4 and 24 hours. The miRNA profiles after these treatments are being assessed on the Affymetrix GeneChip miRNA 4.0 array, which includes over 30,000 probe sets for mature miRNAs. Whether the miRNAs mediate the alterations in Pnx and Gpr173 gene expression will be assessed through the use of antagomirs and mimics. Knowledge of miRNAs controlling Pnx and Gpr173 expression contributes to our understanding of how nutrients and environmental chemicals affect the hypothalamus, potentially representing new targets for therapeutics.

Identification of a promoter element mediating kisspeptin-induced increases in GnRH gene expression in sheep

Gonadotropin-releasing hormone (GnRH) plays an important role in regulating the activities of other components downstream of the hypothalamic-pituitary-gonadal (HPG) axis and maintaining the normal reproductive cycle of animals. However, the molecular mechanisms by which GnRH synthesis and secretion are regulated in sheep remains unclear. In this study, a series of eight recombinant vectors with deletion fragments were constructed and cotransfected with pGL3-Basic and pRL-SV40 into sheep hypothalamic neuronal cells. After treatment with 1 nM kisspeptin, the core promoter of the sheep GnRH gene was identified to be in the region of −1912 bp to −1461 bp by dual-luciferase reporter assay. Bioinformatics analysis showed that there was a binding site for the transcription factor Otx-2 in the core promoter region (−1786 to −1770 bp) that was highly conserved among different species. The expression patterns of Kiss-1, Otx-2 and GnRH in the sheep hypothalamus were the same, and the expression of Kiss-1, Otx-2 and GnRH was significantly higher in the breeding season than in nonbreeding season (P < 0.01). In addition, when hypothalamic neurons were cultured in vitro with kisspeptin, kisspeptin induced the expression of GnRH and Otx-2. In conclusion, these results provide evidence that the core promoter region (−1786 to −1770 bp) of the GnRH gene is involved in the regulation of hypothalamic activity by kisspeptin and that binding of the transcription factor Otx-2 mediates this activation.

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

Background: The 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. Methods: To 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. Results: GnRH 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. Conclusion: Our 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.

Various responses of male pituitary–gonadal axis to different intensities of long-term exercise: Role of expression of KNDY-related genes

The essential role of regular physical activity has been emphasized for maintaining a healthy life. However, unfortunately, during the last few decades, the lifestyle of people has led to a decrease in physical activity. Research studies have shown that exercise of different intensities is applied on reproductive performance indices, luteinizing hormone (LH) and testosterone (T), with different effects. Nevertheless, the molecular and cellular mechanisms underlying its function are not completely understood. Therefore, this study aimed to evaluate the role of kisspeptin, neurokinin-B and pro-dynorphin (KNDY) gene-expression changes located in the upstream of GnRH neurons in transferring the effects of different long-term exercise intensities on male reproductive axis. Twenty-one adult Wistar rats were randomly divided into control, 6-month regular moderate exercise (RME-6) and 6-month regular intensive exercise (RIE-6). In moderate and intensive exercise groups, rats were treated 5 days a week for 60 min, at 22 and 35 m/min, respectively. Finally, the hypothalamic arcuate nucleus was isolated and the relative gene expression of kisspeptin (Kiss1), neurokinin-B (Nkb), pro-dynorphin (Pdyn) and gonadotropin-releasing hormone (Gnrh) genes were measured by realtime polymerase chain reaction method. The results showed that RIE-6 treatment decreased Gnrh and increased Pdyn mRNA levels in the arcuate nucleus. Furthermore, although RME-6 treatment decreased Nkb and increased Pdyn mRNA levels, the Gnrh mRNAwas not affected. Regarding the Gnrh mRNA levels and serum concentrations of reproductive indices (LH and T), moderate exercise did not impose harmful effects on the hypothalamic-pituitary-gonadal axis than intensive exercise. The different impacts of diverse long-term exercise intensities on the male pituitary-gonadal axis maybe relay by the various changes in hypothalamic Nkb and Pdyn gene expressions.

Obesity-related cellular stressors regulate gonadotropin releasing hormone gene expression via c-Fos/AP-1.

Obesity is a risk factor for infertility, but mechanisms underlying this risk are unclear. Fertility is regulated by hypothalamic gonadotropin-releasing hormone, encoded by the Gnrh1 gene. Because obesity promotes endoplasmic reticulum (ER) stress, we sought to determine how tunicamycin-induced ER stress affected Gnrh1 gene expression in the mouse hypothalamic cell line GT1-7. Tunicamycin repressed expression of Gnrh1 in a PKC- and JNK-dependent manner, while upregulating expression of a known Gnrh1 repressor, Fos. Obesity is associated with increased circulating free fatty acids, and exposure to palmitate promoted ER stress and inflammation. Fos expression increased with palmitate dose, but Gnrh1 expression was upregulated with low-dose palmitate and repressed with high-dose palmitate. Using a small molecule inhibitor, we determined that AP-1 was required for Gnrh1 repression by high-dose palmitate or tunicamycin-induced ER stress. These findings suggest that hypogonadism driven by decreased hypothalamic GnRH may be a component of obesity-related infertility.

Effect of CD14/TLR4 antagonist on GnRH/LH secretion in ewe during central inflammation induced by intracerebroventricular administration of LPS

BackgroundImmune stress induced by lipopolysaccharide (LPS) influences the gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) secretion. Presence of LPS interacting Toll-like receptor (TLR) 4 in the hypothalamus may enable the direct action of LPS on the GnRH/LH secretion. So, the aim of the study was to investigate the influence of intracerebroventricular (icv) injection of TLR4 antagonist on GnRH/LH secretion in anestrous ewes during LPS-induced central inflammation. Animals were divided into three groups icv-treated with: Ringer-Locke solution, LPS and TLR4 antagonist followed by LPS.ResultsIt was demonstrated that TLR4 antagonist reduced LPS-dependent suppression of GnRH gene expression in the preoptic area and in the medial basal hypothalamus, and suppression of receptor for GnRH gene expression in the anterior pituitary gland. It was also shown that TLR4 antagonist reduced suppression of LH release caused by icv injection of LPS. Central administration of LPS stimulated TLR4 gene expression in the medial basal hypothalamus.ConclusionsIt was indicated that blockade of TLR4 prevents the inhibitory effect of centrally acting LPS on the GnRH/LH secretion. This suggests that some negative effects of bacterial infection on the hypothalamic-pituitary-gonadal axis activity at the hypothalamic level may be caused by central action of LPS acting through TLR4.

Phoenixin: uncovering its receptor, signaling and functions.

Phoenixin (PNX) is a newly discovered peptide that has been linked to reproductive function, both in the hypothalamus and pituitary. This review will focus on the most recent discoveries related to this novel neuropeptide. Initially, it was found that PNX increased gonadotropin releasing hormone (GnRH)-stimulated luteinizing hormone (LH) release from pituitary cells. Importantly, knockdown of PNX in female rats extended the estrous cycle by 2.3 days. Using novel hypothalamic cell lines, we found that PNX has a stimulatory role on kisspeptin (Kiss) and GnRH gene expression and secretion. The PNX receptor was uncovered using siRNA knockdown of GPR173, an orphan receptor postulated to bind PNX. We have found that the PNX-R signaling through protein kinase A (PKA) in hypothalamic neurons. Althuogh a number of studies demonstrate that PNX plays an important role in reproductive function, there is also evidence that it may have other functions, regulating the heart, feeding, memory, and anxiety, both in the brain and the periphery.

Transcriptional interaction between cFOS and the homeodomain-binding transcription factor VAX1 on the GnRH promoter controls Gnrh1 expression levels in a GnRH neuron maturation specific manner

Gonadotropin-releasing hormone (GnRH) is required for pubertal onset and reproduction, thus the control of GnRH transcription is tightly regulated during development and adulthood. GnRH neuron development depends on transcription factors of the homeodomain family. For example, Ventral anterior homeobox 1 (Vax1) is necessary to maintain GnRH expression after embryonic day 13 in the mouse. To further our understanding of the mechanisms by which VAX1 regulates GnRH gene expression, we asked whether VAX1 interacts with other transcription factors to modify GnRH expression levels. Using the GnRH cell lines, GN11 and GT1-7, we found that activation of PKC enhances expression of the immediate early gene cFos in both GN11, and GT1-7, and represses expression of Vax1 in GT1-7. Further, VAX1 interacts with cFOS while bound to the GnRH promoter. In immature GN11 cells, VAX1 and cFOS enhance GnRH expression, whereas VAX1 and cFOS have a repressive role in the mature GT1-7 cells.