Gonadotropin-releasing Hormone Gene Research Articles

Estrogen promotes gonadotropin-releasing hormone expression by regulating tachykinin 3 and prodynorphin systems in chicken

The "KNDy neurons" located in the hypothalamic arcuate nucleus (ARC) of mammals are known to co-express kisspeptin, neurokinin B (NKB), and dynorphin (DYN), and have been identified as key mediators of the feedback regulation of steroid hormones on gonadotropin-releasing hormone (GnRH). However, in birds, the genes encoding kisspeptin and its receptor GPR54 are genomic lost, leaving unclear mechanisms for feedback regulation of GnRH by steroid hormones. Here, the genes tachykinin 3 (TAC3) and prodynorphin (PDYN) encoding chicken NKB and DYN neuropeptides were successfully cloned. Temporal expression profiling indicated that TAC3, PDYN and their receptor genes (TACR3, OPRK1) were mainly expressed in the hypothalamus, with significantly higher expression at 30W than at 15W. Furthermore, overexpression or interference of TAC3 and PDYN can regulate the GnRH mRNA expression. In addition, in vivo and in vitro assays showed that estrogen (E2) could promote the mRNA expression of TAC3, PDYN, and GnRH, as well as the secretion of GnRH/LH. Mechanistically, E2 could dimerize the nuclear estrogen receptor 1 (ESR1) to regulate the expression of TAC3 and PDYN, which promoted the mRNA and protein expression of GnRH gene as well as the secretion of GnRH. In conclusion, these results revealed that E2 could regulate the GnRH expression through TAC3 and PDYN systems, providing novel insights for reproductive regulation in chickens.

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.

Central regulation of reproduction in amphibians.

This review article includes a literature review of synteny analysis of the amphibian gonadotropin-releasing hormone (GnRH) genes, the distribution of GnRH 1 and GnRH2 neurons in the central nervous system of amphibians, the function and regulation of hypophysiotropic GnRH1, and the function of GnRH1 in amphibian reproductive behaviors. It is generally accepted that GnRH is the key regulator of the hypothalamic-pituitary-gonadal axis. Three independent GnRH genes, GnRH1, GnRH2, and GnRH3, have been identified in vertebrates. Previous genome synteny analyses suggest that there are likely just two genes, gnrh1 and gnrh2, in amphibians. In three groups of amphibians: Anura, Urodela, and Gymnophiona, the distributions of GnRH1 and GnRH2 neurons in the central nervous system have also been previously reported. Moreover, these neuronal networks were determined to be structurally independent in all species examined. The somata of GnRH1 neurons are located in the terminal nerve, medial septum (MS), and preoptic area (POA), and some GnRH1 neurons in the MS and POA project into the median eminence. In contrast, the somata of GnRH2 neurons are located in the midbrain tegmentum. In amphibians, GnRH1 neurons originate from the embryonic olfactory placode, while GnRH2 neurons originate from the midbrain. The characterization and feedback regulation mechanisms of hypophysiotropic GnRH1 neurons in amphibians, the involvement of GnRH1 in amphibian reproductive behavior, and its possible mechanism of action should be elucidated in future.

Influence of phthalate and non-phthalate plasticizers on reproductive endocrine system-related gene expression profiles in Japanese medaka (Oryzias latipes)

Plasticizers containing phthalates have the potential to alter endocrine function in vertebrates. While non-phthalate plasticizers were previously considered to be environmentally friendly and safe, our research team discovered that bis-(2-ethylhexyl) adipate (DEHA) and acetyl tributyl citrate (ATBC) disrupt thyroid hormones in Japanese medaka (Oryzias latipes). We assessed reproductive- and estrogen-responsive gene expression patterns in Japanese medaka to determine whether the phthalate plasticizers bis-(2-ethylhexyl) phthalate (DEHP, positive control) and the non-phthalate plasticizers DEHA and ATBC disrupt endocrine signaling. The results showed that the levels of choriogenin H (chgH) and vitellogenin (vtg) genes increased after exposure to DEHP and ATBC, suggesting that these plasticizers may have estrogenic activity. Exposure to DEHP and DEHA resulted in the upregulation of kisspeptin (kiss), gonadotropin-releasing hormone (gnrh), and follicle-stimulating hormone beta (fshβ) genes, suggesting that these plasticizers may interfere with reproductive function. To the best of our knowledge, this is the first study to demonstrate that the non-phthalate plasticizers DEHA and ATBC can disrupt reproduction-related hormonal activity in fish.

Molecular regulation and genetic basis of gonadotropin-releasing hormone genes: A review

This review systematically introduces basic information on the hypothalamic pituitary-gonad axis and provides knowledge on the regulation, location, function, reproduction, gene mutations, disorders, sexual behavior, life cycle, and effect of environmental factors on the gonadotropin-releasing hormone gene. On the other hand, this review focused on the GnRH gene, regulations, receptor structures, and their signaling pathways, in addition to its related genes and its effect on crucial hormones such as follicle-stimulating hormone, luteinizing hormone, testosterone, estradiol, and progesterone. Additionally, gonadotropin-inhibiting hormone and its related peptides, such as R-Famide peptides, were found to decrease hormone secretion by working on the hypothalamic pituitary gonads axis to inhibit the biosynthesis process of gonadotropin alpha and beta subunits. Additionally, the roles of crucial hormones in reproduction and fertility, as well as the disruption, resulted from mutations. Special characteristics of several hormones and pulsatile secretion of gonadotropin-releasing hormone were also summarized.

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.

Common and female-specific roles of protein tyrosine phosphatase receptors N and N2 in mice reproduction

Simultaneous knockout of the neuroendocrine marker genes Ptprn and Ptprn2, which encode the protein tyrosine phosphatase receptors N and N2, causes infertility in female mice while males are fertile. To elucidate the mechanism of the sex-specific roles of Ptprn and Ptprn2 in mouse reproduction, we analyzed the effects of their double knockout (DKO) on the hypothalamic-pituitary–gonadal axis. In DKO females, delayed puberty and lack of ovulation were observed, complemented by changes in ovarian gene expression and steroidogenesis. In contrast, testicular gene expression, steroidogenesis, and reproductive organs development were not significantly affected in DKO males. However, in both sexes, pituitary luteinizing hormone (LH) beta gene expression and LH levels were reduced, as well as follicle-stimulating hormone beta gene and gonadotropin-releasing hormone (GnRH) gene, while the calcium-mobilizing and LH secretory actions of GnRH were preserved. Hypothalamic Gnrh1 and Kiss1 gene expression was also reduced in DKO females and males. In parallel, a significant decrease in the density of immunoreactive GnRH and kisspeptin fibers was detected in the hypothalamic arcuate nucleus of DKO females and males. The female-specific kisspeptin immunoreactivity in the rostral periventricular region of the third ventricle was also reduced in DKO females, but not in DKO males. These data indicate a critical role of Ptprn and Ptprn2 in kisspeptin-GnRH neuronal function and sexual dimorphism in the threshold levels of GnRH required to preserve reproductive functions.

Research Note: Polymorphisms of gonadotrophin-releasing hormone gene and their association with growth traits in quail (Coturnix Coturnix)

This study aimed to identify polymorphisms of gonadotropin-releasing hormone (GnRH) gene and their association with growth traits in quail by PCR and direct sequencing. Genomic DNA was extracted from quail blood samples of 36 from Savimalt (SV) and 49 from French Giant (FG). Growth traits were measured and used for candidate gene analysis, as body weight (BW), shank length (SL), chest width (CW), chest depth (CD), breastbone length (BBL), body length (BL), and shank circumference (SC). The results showed that a total of 20 SNPs were detected in GnRH gene, whereas 8 SNPs were significantly associated with growth traits (P < 0.05). The T215C, G279A, C458T, A520G, and C547G were significantly associated with SL at 3 wk of age in the FG strain, whereas A583T was significantly related to BBL and BL, and C591T was significantly related to SL, BBL, and BL, whereas A592G was significantly correlated with SL, CW, CD, BBL, and BL (P < 0.05). The 8 SNPs were significantly related to CW, CD, and BBL at 3 wk of age in the SV strain, whereas A583T, C591T, and A592G were significantly associated with BW (P < 0.05). The G279A showed significant correlations with SL at 5 wk of age in FG, whereas A583T showed significant associations with SC in FG, and C591T was significantly associated with BW and SC in FG, whereas A592T was significantly related to BW, SL, and CD in FG (P < 0.05). The T215C, G279A, C458T, A520G, and C547G were significantly correlated with BW, CW, BBL, and BL at 5 wk of age in SV, whereas A583T, C591T, and A592G were significantly related to BW, SL, CW, BBL, and BL (P < 0.05). Haplotypes based on 8 SNPs showed significant correlation with BW, SL, CW, CD, BBL, BL, and SC in FG (P < 0.05). In conclusion, the GnRH gene could be used as a molecular genetic marker to provide theoretical foundation to improve growth traits in quail.

Season-dependent effect of water temperatures on circadian expression of kisspeptin and gonadotropin-releasing hormone genes in Clarias magur

Season-dependent effect of water temperatures on circadian expression of kisspeptin and gonadotropin-releasing hormone genes in Clarias magur

Perinatal exposure to low doses of cypermethrin induce the puberty-related hormones and decrease the time to puberty in the female offspring.

Pyrethroid insecticides are ubiquitously detected in environmental media, food, and urine samples. Our previous epidemiological study reported a correlation between increased pyrethroid exposure and delayed pubertal development in Chinese girls. In this study, we further investigated the effects of perinatal exposure to low doses of cypermethrin (CP) on pubertal onset and hypothalamic-pituitary-ovarian axis in the female mice offspring. The treatment of CP with 60μg/kg/day from gestation day 6 (GD6) to postnatal day 21 (PND21) significantly decreased the time to puberty in the female offspring. Exposure of CP increased the serum levels of gonadotropin-releasing hormone (GnRH) and the expression of GnRH genes in a dose-dependent manner in the female offspring. CP also induced the serum levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), as well as the expression of gonadotropin subunit genes [LHβ, FSHβ, and chorionic gonadotropin α (Cgα)]. Furthermore, CP induced serum estradiol (E2) levels and the expression of steroidogenesis-related genes [steroidogenic acute regulatory (StAR) and Cytochrome p 450, family 11, subfamily A, polypeptide 1 (CYP11A1)] in the ovary. In accordance with the in vivo tests, administration of CP (6.7, 20, and 60μg/L) stimulated a dose-dependent increase in the synthesis and secretion of the puberty-related hormones in the explants of hypothalamus, pituitary, and ovary. The interference with calcium channels in the ovary may be responsible for CP-induced pubertal onset. Our study provided evidence that perinatal exposure to low doses of CP induced puberty-related hormones and decreased the time to puberty in the female offspring.

Editorial introductions

Editorial introductions

Expression and functional analysis of GnRH at the onset of puberty in sheep

Gonadotropin-releasing hormone (GnRH) is a key factor at the onset of puberty. This decapeptide has been found in mammalian ovaries, but its regulatory mechanism in the ovary of sheep at the onset of puberty is not clear. This study investigated the coding sequence (CDS) of the GnRH gene in the ovary of Duolang sheep and the expression of GnRH mRNA in different tissues at the onset of puberty, and analyzed the effect of GnRH on ovarian granulosa cells (GCs) of Duolang sheep. The results showed that the GnRH CDS of sheep was cloned, the full length of the GnRH CDS in sheep ovary was 279 bp, and the nucleotide sequence was completely homologous to that in the hypothalamus. The expression of GnRH mRNA was highest in the hypothalamus and ovary. The expression of related hormones and receptors in GCs of Duolang sheep treated with different concentrations of GnRH for 24 h was affected. GnRH significantly inhibited LH synthesis and LHR expression in GCs. Low concentration (100 ng mL) had the most obvious therapeutic effect on follicle-stimulating hormone (FSH) and FSHR. Higher concentration (250 ng mL) significantly promoted estradiol and ER mRNA. These findings provide strong evidence that ovarian GnRH is an important regulatory factor at the onset of puberty in sheep.

Gene Editing of the Catfish Gonadotropin-Releasing Hormone Gene and Hormone Therapy to Control the Reproduction in Channel Catfish, Ictalurus punctatus.

Simple SummaryGonadotropin-releasing hormone (GnRH) plays a pivotal role in fish reproduction. In the present study, transcription activator-like effector nuclease (TALEN) plasmids targeting the catfish gonadotropin-releasing hormone (cfGnRH) gene were delivered into fertilized eggs with double electroporation to sterilize channel catfish (Ictalurus punctatus). After low fertility was observed, application of luteinizing hormone-releasing hormone analog (LHRHa) hormone therapy resulted in good spawning and hatch rates for mutants (individuals with human-induced sequence changes at the cfGnRH locus). Gene editing of channel catfish for the reproductive confinement of gene-engineered, domestic, and invasive fish to prevent gene flow into the natural environment appears promising.Transcription activator-like effector nuclease (TALEN) plasmids targeting the channel catfish gonadotropin-releasing hormone (cfGnRH) gene were delivered into fertilized eggs with double electroporation to sterilize channel catfish (Ictalurus punctatus). Targeted cfGnRH fish were sequenced and base deletion, substitution, and insertion were detected. The gene mutagenesis was achieved in 52.9% of P1 fish. P1 mutants (individuals with human-induced sequence changes at the cfGnRH locus) had lower spawning rates (20.0–50.0%) when there was no hormone therapy compared to the control pairs (66.7%) as well as having lower average egg hatch rates (2.0% versus 32.3–74.3%) except for one cfGnRH mutated female that had a 66.0% hatch rate. After low fertility was observed in 2016, application of luteinizing hormone-releasing hormone analog (LHRHa) hormone therapy resulted in good spawning and hatch rates for mutants in 2017, which were not significantly different from the controls (p > 0.05). No exogenous DNA fragments were detected in the genome of mutant P1 fish, indicating no integration of the plasmids. No obvious effects on other economically important traits were observed after the knockout of the reproductive gene in the P1 fish. Growth rates, survival, and appearance between mutant and control individuals were not different. While complete knock-out of reproductive output was not achieved, as these were mosaic P1 brood stock, gene editing of channel catfish for the reproductive confinement of gene-engineered, domestic, and invasive fish to prevent gene flow into the natural environment appears promising.

Genetic polymorphisms of reproductive hormones and their receptors in assisted reproduction technology for patients with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is one of the most common endocrinopathies in women of childbearing, which is defined by the accumulation of multiple, small fluid-filled ovarian cysts without the selection of a single dominant follicle. Most PCOS phenotypes are characterized by the absence of spontaneous ovulation, resistance toward ovulation inductors, the production of a large immature oocytes number, and the high prevalence of ovarian hyperstimulation syndrome, resulting in reduced assisted reproductive technologies (ART) programs effectiveness. The review analyses current data about the relationship between polymorphism genotypes of KISS genes, follicle stimulating hormone (FSH), luteinizing hormone (LH), anti-Müllerian hormone (AMH) and their receptors genes, gonadotropin-releasing hormone (GnRH), estrogen, and progesterone receptors genes, the PCOS risk and the features of ovarian response to stimulation during ART cycles. The use of single nucleotide polymorphisms (SNPs) as prognostic markers of ART programs outcomes would provide a personalized approach to the drugs and doses choice for ovarian stimulation and significantly increase the chance of pregnancy.

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Editorial introductions

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.

Central µ-Opioid Receptor Antagonism Blocks Glucoprivic LH Pulse Suppression and Gluconeogenesis/Feeding in Female Rats.

Energetic status often affects reproductive function, glucose homeostasis, and feeding in mammals. Malnutrition suppresses pulsatile release of the gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) and increases gluconeogenesis and feeding. The present study aims to examine whether β-endorphin-μ-opioid receptor (MOR) signaling mediates the suppression of pulsatile GnRH/LH release and an increase in gluconeogenesis/feeding induced by malnutrition. Ovariectomized female rats treated with a negative feedback level of estradiol-17β (OVX + low E2) receiving 2-deoxy-D-glucose (2DG), an inhibitor of glucose utilization, intravenously (iv) were used as a malnutrition model. An administration of D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), a selective MOR antagonist, into the third ventricle blocked the suppression of the LH pulse and increase in gluconeogenesis/feeding induced by iv 2DG administration. Histological analysis revealed that arcuate Kiss1 (kisspeptin gene)-expressing cells and preoptic Gnrh1 (GnRH gene)-expressing cells co-expressed little Oprm1 (MOR gene), while around 10% of arcuate Slc17a6 (glutamatergic marker gene)-expressing cells co-expressed Oprm1. Further, the CTOP treatment decreased the number of fos-positive cells in the paraventricular nucleus (PVN) in OVX + low E2 rats treated with iv 2DG but failed to affect the number of arcuate fos-expressing Slc17a6-positive cells. Taken together, these results suggest that the central β-endorphin-MOR signaling mediates the suppression of pulsatile LH release and that the β-endorphin may indirectly suppress the arcuate kisspeptin neurons, a master regulator for GnRH/LH pulses during malnutrition. Furthermore, the current study suggests that central β-endorphin-MOR signaling is also involved in gluconeogenesis and an increase in food intake by directly or indirectly acting on the PVN neurons during malnutrition in female rats.

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.