Gonadotropin-releasing Hormone 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.

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.

Dapagliflozin Ameliorates Ovulation Disorders via Attenuating Activated Microglia-Mediated Hypothalamic Inflammation in HFD-Fed Mice

Introduction: Sodium-glucose cotransporter 2 inhibitors (SGLT2is) have shown neuroprotective effects in obese mice. However, whether SGLT2i can ameliorate high-fat diet (HFD)-related ovulation disorders remains unknown. The aim of this research was to investigate whether dapagliflozin improves HFD-induced ovulatory dysfunction by attenuating microglia-mediated hypothalamic inflammation. Methods: C57BL/6J female mice fed HFD were treated with dapagliflozin (1 mg/kg) for 22 weeks. Plasma insulin, leptin, luteinizing hormone (LH), estradiol (E2), and IL-1β levels were also tested. Microglial morphology, cell numbers, and SGLT2 expression were evaluated using immunofluorescence. The expression of IL-1β, NLRP3, kisspeptin, gonadotropin-releasing hormone (GnRH), SGLT2, insulin, and leptin receptors in the hypothalamus was determined using immunohistochemical staining. We also examined the effects of dapagliflozin on glucose metabolism and the release of inflammatory factor in palmitic acid (PA)-treated HMC3 cells. Results: As expected, dapagliflozin improved HFD-induced metabolic disturbances, peripheral versus central insulin and leptin resistance and also restored the regular estrous cycle. Furthermore, dapagliflozin blunted microglia activation, NLRP3 inflammasome priming, hypothalamic inflammation, and increased the expression of GnRH and kisspeptin at proestrus in the hypothalamus. Additionally, dapagliflozin markedly reduced IL-6 and NO release and fat accumulation, decreased lactic acid production and glucose consumption, and inhibited mammalian target of rapamycin (mTOR) and hexokinase 2 (HK2) expression in PA-treated HMC3 cells. These effects suggest that dapagliflozin reduced the mTOR/HK2-mediated aerobic glycolysis. Conclusions: Dapagliflozin improved HFD-related ovulation disorders by regulating glucose metabolism through mTOR/HK2 signaling and attenuating microglia-mediated hypothalamic inflammation. These results validate the novel role for the neuroprotection of SGLT2i in HFD-induced obesity and ovulation disorders.

Therapeutic effects of melatonin in female mice with central precocious puberty by regulating the hypothalamic Kiss-1/Kiss1R system

In recent years, central precocious puberty (CPP) in children is becoming more common, which seriously affects their physical and psychological health and requires finding a safe and effective treatment method. The aim of this study was to investigate the therapeutic effect of melatonin on CPP. A CPP model was established by subcutaneous injection of 300 micrograms of danazol into 5-day-old female mice, followed by treatment with melatonin and leuprolide. The vaginal opening was checked daily. Mice were weighed, gonads were weighed, gonadal index was calculated, and gonadal development was observed by hematoxylin and eosin (HE) staining. Serum follicle stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2) levels were measured by ELISA. By using RT-PCR and Western blotting, the mRNA and protein expression of the hypothalamus Kiss-1, Kiss-1 receptor (Kiss1R), gonadotropin-releasing hormone (GnRH), and pituitary GnRH receptor (GnRHR) were identified. The results showed that melatonin delayed vaginal opening time and reduced body weight, gonadal weight and indices in female CPP mice. Melatonin treatment prevents uterine wall thickening and ovarian luteinization in female CPP mice. Melatonin treatment reduces serum concentrations of FSH, LH, and E2 in female CPP mice. Melatonin suppressed the expressions of Kiss-1, Kiss1R and GnRH in the hypothalamus, and the expression of GnRHR in the pituitary of the female CPP mice. Our results suggest that melatonin can inhibit the hypothalamic-pituitary-gonadal (HPG) axis by down-regulating the Kiss-1/Kiss1R system, thereby treating CPP in female mice.

Postnatal feeding with high-fat combined with high-glucose diet induces precocious puberty in Sprague‒Dawley rat pups

With economic development and overnutrition, including high-fat diets (HFD) and high-glucose diets (HGD), the incidence of obesity in children is increasing, and thus, the incidence of precocious puberty is increasing. Therefore, it is of great importance to construct a suitable animal model of overnutrition-induced precocious puberty for further in-depth study. Here, we fed a HFD, HGD, or HFD combined with a HGD to pups after P-21 weaning, while weaned pups fed a normal diet served as the control group. The results showed that HFD combined with a HGD increased the body weight (BW) of weaned rat pups. In addition, a HFD, HGD, and HFD combined with a HGD lowered the age at which vaginal opening occurred and accelerated the vaginal cell cycle. Furthermore, a HFD combined with a HGD increased the weight of the uterus and ovaries of weaned rat pups. Additionally, a HFD combined with a HGD promoted the development of reproductive organs in weaned female rat pups. Ultimately, a HFD combined with a HGD was found to elevate the serum levels of gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), follicle stimulating hormone (FSH), leptin, adiponectin, and oestradiol (E2) and increase hypothalamic GnRH, Kiss-1, and GPR54 expression levels in weaned female rat pups. The current study found that overnutrition, such as that through a HFD combined with HGD, could induce precocious puberty in weaned female rat pups. In addition, a rat model of overnutrition-induced precocious puberty was established.

Physiological role of dietary energy in the sexual maturity: clues of body size, gonad development, and serum biochemical parameters of Chinese indigenous chicken

Sexual maturity is a crucial factor in the formation and development of poultry reproductive capacity. The nutritional status has been confirmed to play an important role in the regulation of sexual maturity. To investigate the effect of dietary energy levels on sexual maturity in chicken, diets with 3 energy levels (group L: 2,573 kcal/kg, group C: 2,836 kcal/kg, group H: 3,122 kcal/kg) were implemented to feed Guangyuan Gray chickens. During this trial, body weight, body size, organ development, sexual maturity, reproductive performance and blood biochemical parameters were monitored. The earlier sexual maturity was observed in group H, as well as a heavier first egg weight, larger interpubic distance and higher total cholesterol (T-CHO) content at sexual maturity. The dietary energy levels had no significant effect on body weight at first egg and egg production at 300 d of age. Although dietary energy levels had a significant effect on body weight, comb length, tibia length and girth, abdominal fat weight, oviduct weight and length, T-CHO, triglyceride (TG) content and estradiol (E2) level during the rearing period. No significant difference of gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH) level among 3 groups was observed during the trial. The dietary energy levels had effects on mRNA expression of GnRH, estrogen receptor 1 (ESR1), estrogen receptor 2 (ESR2) in hypothalamus, gonadotropin inhibitory hormone receptor (GnIHR) in pituitary and luteinizing hormone receptor (LHR), ESR2 in ovary. The GnIHR/GnRHR ratio in pituitary was higher before sexual maturity and decreased at sexual maturity. The results of correlations analysis found that all the body size, carcass traits, serum biochemical parameters negatively correlated with age at first egg except for interpubic distance and serum blood glucose content. Collectively, dietary energy levels had effects on sexual maturity of chicken, which may be achieved by affecting body weight, gonad development, endocrine and the mRNA expression of genes related to hypothalamus-pituitary-gonad axis. These results further set our understanding of how dietary energy regulates sexual maturity.

Long-COVID cognitive impairments and reproductive hormone deficits in men may stem from GnRH neuronal death

We have recently demonstrated a causal link between loss of gonadotropin-releasing hormone (GnRH), the master molecule regulating reproduction, and cognitive deficits during pathological aging, including Down syndrome and Alzheimer's disease. Olfactory and cognitive alterations, which persist in some COVID-19 patients, and long-term hypotestosteronaemia in SARS-CoV-2-infected men are also reminiscent of the consequences of deficient GnRH, suggesting that GnRH system neuroinvasion could underlie certain post-COVID symptoms and thus lead to accelerated or exacerbated cognitive decline. We explored the hormonal profile of COVID-19 patients and targets of SARS-CoV-2 infection in post-mortem patient brains and human fetal tissue. We found that persistent hypotestosteronaemia in some men could indeed be of hypothalamic origin, favouring post-COVID cognitive or neurological symptoms, and that changes in testosterone levels and body weight over time were inversely correlated. Infection of olfactory sensory neurons and multifunctional hypothalamic glia called tanycytes highlighted at least two viable neuroinvasion routes. Furthermore, GnRH neurons themselves were dying in all patient brains studied, dramatically reducing GnRH expression. Human fetal olfactory and vomeronasal epithelia, from which GnRH neurons arise, and fetal GnRH neurons also appeared susceptible to infection. Putative GnRH neuron and tanycyte dysfunction following SARS-CoV-2 neuroinvasion could be responsible for serious reproductive, metabolic, and mental health consequences in long-COVID and lead to an increased risk of neurodevelopmental and neurodegenerative pathologies over time in all age groups. European Research Council (ERC) grant agreements No 810331, No 725149, No 804236, the European Union Horizon 2020 research and innovation program No 847941, the Fondation pour la Recherche Médicale (FRM) and the Agence Nationale de la Recherche en Santé (ANRS) No ECTZ200878 Long Covid 2021 ANRS0167 SIGNAL, Agence Nationale de la recherche (ANR) grant agreements No ANR-19-CE16-0021-02, No ANR-11-LABEX-0009, No. ANR-10-LABEX-0046, No. ANR-16-IDEX-0004, Inserm Cross-Cutting Scientific Program HuDeCA, the CHU Lille Bonus H, the UK Medical Research Council (MRC) and National Institute of Health and care Research (NIHR).

Consumption of the nonnutritive sweetener acesulfame potassium increases central precocious puberty risk

The prevalence of precocious puberty and the consumption of nonnutritive sweeteners (NNS) is rapidly growing worldwide. However, the effects of NNSs on precocious puberty remain unclear. We examined the impact of acesulfame potassium (AceK), one of the most widely used NNS, on central precocious puberty (CPP) development using ex vivo and in vitro studies. 884 girls aged 6–12 were enrolled with complete AceK consumption data and CPP outcome assessment in the Taiwan Pubertal Longitudinal Study from 2018 to 2022. After adjustment for confounders, compared with no AceK consumption, AceK consumption at more than the median dose was associated with higher CPP risk in girls (odds ratio = 1.88, 95% confidence interval = 1.16–3.06; p for trend = 0.003). In rats, AceK consumption from in-utero to post-weaning stages accelerated puberty onset, accompanied by increased brain gonadotropin-releasing hormone (GnRH) expression. Intracerebroventricular AceK injection also induced early puberty onset in rats. In N44 hypothalamic neuron cells, AceK treatment increased reactive oxygen species production, which led to protein kinase A (PKA) activation and increased GnRH expression. These findings suggest that prepubertal girls should consume soft drinks or food products containing AceK more cautiously.

Identification and characterization of gonadotropin-releasing hormone (GnRH) in Zhikong scallop Chlamys farreri during gonadal development.

Gonadotropin-releasing hormone (GnRH) controls synthesis of sex steroid hormones through hypothalamic-pituitary-gonadal (HPG) axis in vertebrates. But in mollusks, research on neuroendocrine control of gonadal function, such as the function of GnRH during gonadal development is limited. In this study, we investigated the morphology and structure of the nerve ganglia of Zhikong scallop Chlamys farreri by physiological and histological observations. We also cloned the ORF and studied the expression patterns of GnRH in the scallop. Tissue expression analysis showed that GnRH was highly expressed in parietovisceral ganglion (PVG). The in situ hybridization result further confirmed that GnRH mRNA only distributed in some good-sized neurons in the posterior lobe (PL) and some pint-sized neurons in the lateral lobe (LL). In addition, by examining the expression of GnRH during gonadal development in ganglia, we found GnRH displayed higher expression in the female scallops, and showed significant high expression at the growing stage of female scallops in PVG. This study would contribute to gaining insight into the mechanism underlying reproduction regulation by GnRH in the scallop and help to provide a better understanding of reproductive neuroendocrine in mollusks.

Female-specific pituitary gonadotrope dysregulation in mice with chronic focal epilepsy

Gonadotropin hormone release from the anterior pituitary is critical to regulating reproductive endocrine function. Clinical evidence has documented that people with epilepsy display altered levels of gonadotropin hormones, both acutely following seizures and chronically. Despite this relationship, pituitary function remains a largely understudied avenue in preclinical epilepsy research. Recently, we showed that females in the intrahippocampal kainic acid (IHKA) mouse model of temporal lobe epilepsy displayed changes in pituitary expression of gonadotropin hormone and gonadotropin-releasing hormone (GnRH) receptor genes. Circulating gonadotropin hormone levels, however, have yet to be measured in an animal model of epilepsy. Here, we evaluated the circulating levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), GnRH receptor (Gnrhr) gene expression, and sensitivity to exogenous GnRH in IHKA males and females. Although no changes in overall dynamics of pulsatile patterns of LH release were found in IHKA mice of either sex, estrus vs. diestrus changes in basal and mean LH levels were larger in IHKA females with prolonged, disrupted estrous cycles. In addition, IHKA females displayed increased pituitary sensitivity to GnRH and higher Gnrhr expression. The hypersensitivity to GnRH was observed on diestrus, but not estrus. Chronic seizure severity was not found to be correlated with LH parameters, and FSH levels were unchanged in IHKA mice. These results indicate that although there are changes in pituitary gene expression and sensitivity to GnRH in IHKA females, there may also be compensatory mechanisms that aid in maintaining gonadotropin release in the state of chronic epilepsy in this model.

Hypothalamic GnRH expression and pulsatility depends on a balance of prolactin receptors in the plains vizcacha, Lagostomus maximus.

In mammals, gestation is considered a physiological hyperprolactinemia status. Prolactin (PRL) is one of the modulators of gonadotropin-releasing hormone (GnRH) neurons function. The South American plains vizcacha (Lagostomus maximus) is a unique model to study the regulation of hypothalamic GnRH neurons by direct and indirect steroid-dependent pathways. The aim was to characterize the hypothalamic expression of endocrine markers in vizcacha during gestation as well as their response to experimental induced hyperprolactinemia. The possible involvement of PRL regulatory pathways on GnRH in the context of hypothalamic and pituitary reactivation in mid-gestating vizcachas was discussed. Using two in vivo approaches, we determined changes in the hypothalamic expression and distribution of prolactin receptor (PRLR), tyrosine hydroxylase (TH), and dopamine type 2 receptor. A significant increment in the number of tuberoinfundibular dopaminergic (TIDA) neurons was determined in the arcuate nucleus from early to term pregnancy. On the other hand, at preoptic area, the number of both TH+PRLR+ and GnRH+PRLR+ double-labeled neurons significantly decreased at mid-pregnancy probably allowing the recovery of GnRH expression indicating that both types of neurons may represent the key points of PRL indirect and direct pathways modulating GnRH. Moreover, in a model of induced hyperprolactinemic vizcachas, the inhibitory effect of PRL on GnRH at both expression and delivery levels were confirmed. These results suggest the concomitant participation of both PRL regulatory pathways on GnRH modulation and pinpoint the key role of PRL on GnRH expression enabling the recovery of the hypothalamic activity during the gestation in this species.

Synthetic microfiber exposure negatively affects reproductive parameters in male medaka (Oryzias latipes)

Microplastics not only accumulate in the bodies of fishes and cause damage to the organs, but also cause many other problems, such as reduced reproductive capacity, by acting directly or indirectly on the hypothalamus-pituitary-gonad axis (HPG axis). In this study, we investigated the changes in HPG axis-related genes in male medaka (Oryzias latipes) exposed to fiber-type microplastics. We confirmed the progression of vitellogenesis, a sign of endocrine disruption, in male fish. In the microfiber-exposed group, microfiber accumulation was confirmed in the gills and intestines. One week after exposure to two different concentrations of microfibers (500 and 1,000 fibers/L), the fish showed increased expression of gonadotropin-releasing hormone (GnRH) and luteinizing hormone receptor (LH-R) mRNA. From day 10 of exposure to the microfibers, there was an increase in the expression of the gonadotropin-inhibitory hormone (GnIH) mRNA and a decrease in the expression of GnRH and LH-R mRNA. There was an increase in the cytochrome P450 aromatase (CYP19a) mRNA expression and plasma estradiol (E2) concentration in the 1,000 fibers/L exposure group. High vitellogenin (VTG) mRNA expression was confirmed seven days after exposure in the 1,000 fibers/L group, which was consistent with the VTG mRNA expression signals detected in the liver using in situ hybridization. These results suggest that microfiber ingestion may cause short-term endocrinal disruption of the HPG axis in male medaka, which in turn may interfere with their normal maturation process.

Variation in Hypothalamic GnIH Expression and Its Association with GnRH and Kiss1 during Pubertal Progression in Male Rhesus Monkeys (Macaca mulatta).

Modulation of pulsatile gonadotropin-releasing hormone (GnRH) secretion across postnatal development in higher primates is not fully understood. While gonadotropin-inhibitory hormone (GnIH) is reported to suppress reproductive axis activity in birds and rodents, little is known about the developmental trajectory of GnIH expression in rhesus monkeys throughout the pubertal transition. This study was aimed at examining the variation in GnIH immunoreactivity (-ir) and associated changes among GnIH, GnRH, and Kiss1 mRNA expression in the hypothalamus of infant, juvenile, prepubertal, and adult male rhesus monkeys. The brains from rhesus macaques were collected from infancy until adulthood and were examined using immunofluorescence and RT-qPCR. The mean GnIH-ir was found to be significantly higher in prepubertal animals (p < 0.01) compared to infants, and significantly reduced in adults (p < 0.001). Significantly higher (p < 0.001) GnRH and Kiss1 mRNA expression was noted in adults while GnIH mRNA expression was the highest at the prepubertal stage (p < 0.001). Significant negative correlations were seen between GnIH-GnRH (p < 0.01) and GnIH-Kiss1 (p < 0.001) expression. Our findings suggest a role for GnIH in the prepubertal suppression of the reproductive axis, with disinhibition of the adult reproductive axis occurring through decreases in GnIH. This pattern of expression suggests that GnIH may be a viable target for the development of novel therapeutics and contraceptives for humans.

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.

Exogenous Melatonin Regulates Puberty and the Hypothalamic GnRH-GnIH System in Female Mice.

In recent years, the age of children entering puberty is getting lower and the incidence of central precocious puberty is increasing. It is known that melatonin plays an increasingly important role in regulating animal reproduction, but the specific role and mechanism of melatonin in regulating the initiation of puberty remain unclear. The purpose of the current study was to investigate the effect of subcutaneous melatonin injection on pubertal development in female mice and its mechanism of action. Female mice that were 22 days old received 1 mg/kg doses of melatonin subcutaneously every day for 10, 15 and 20 days. The vaginal opening was checked daily. Hematoxylin and eosin (HE) stain was used to determine the growth of the uterus and ovaries. Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of follicle-stimulating hormone (FSH), gonadotropin-inhibiting hormone (GnIH), and gonadotropin-releasing hormone (GnRH) in serum. By using RT-PCR and Western blotting, the mRNA and protein expression of the hypothalamus GnRH, GnIH, Kisspeptin (Kp), Proopiomelanocortin (POMC), Neuropeptide Y (NPY), as well as G protein-coupled receptor 147 (GPR147) were identified. The findings demonstrated that melatonin could suppress ovarian follicle and uterine wall growth as well as delay vaginal opening, decrease serum levels of GnRH and FSH and increase levels of GnIH. Melatonin increased GnIH and GPR147 expression in the hypothalamus in comparison to the saline group, while decreasing the expression of GnRH, Kisspeptin, POMC, and NPY. In conclusion, exogenous melatonin can inhibit the onset of puberty in female mice by modulating the expression of hypothalamic GnRH, GnIH, Kisspeptin, POMC and NPY neurons and suppressing the hypothalamic-pituitary-gonadal axis.

Effects of Fuyou Formula on GnRH Secretion and Related Gene Expression in Treating Precocious Puberty.

The Fuyou (Fy) formula is an in-hospital preparation consisting of traditional Chinese medicine (TCM) that has been used for treating precocious puberty (PP) for more than 20 years. In this study, we aimed to clarify the effect of the Fy formula and its major components on PP. To confirm the effect of the Fy formula on the release of hypothalamic gonadotropin-releasing hormone (GnRH), GT1-7 cells were treated with estrogen to build the model group and subsequently treated with the Fy formula and its major components to explore their effects on the secretion of GnRH. The level of GnRH in GT1-7 cells was determined using enzyme-linked immunosorbent assay. The results illustrated that, compared to the model group, the Fy formula inhibited the release of GnRH. In addition, the expression levels of proteins related to GnRH secretion, including GnRH, gonadotropin-releasing hormone receptor (GnRHR), Kiss-1 metastasis-suppressor (Kiss1), G-protein coupled receptor 54 (GPR54), estrogen receptor α (ERα), insulin-like growth factor-1 (IGF-1), and insulin-like growth factor-1 receptor (IGF-1R), were detected by real-time polymerase chain reaction (RT-qPCR). The results demonstrated that the Fy formula significantly reduced the level of GnRH secretion in the GT1-7 cell lines compared with the model group. Moreover, it significantly downregulated the expression of GnRH, GnRHR, Kiss1, GPR54, ERα, IGF-1, and IGF-1R. In summary, our results indicate that the Fy formula and its major components may inhibit the effects of estrogen, which alleviates PP through transcriptional regulation of target genes.

LKB1 alleviates high glucose‑ and high fat‑induced inflammation and the expression of GnRH and sexual precocity‑related genes, in mouse hypothalamic cells by activating the AMPK/FOXO1 signaling pathway.

Precocious puberty (PP) is a developmental disorder. Hypothalamic cells can produce gonadotropin-releasing hormone (GnRH), the final output of neuroendocrine regulation that occurs during puberty. The aim of the present study was to investigate the role of live kinase B1 (LKB1), also known as serine/threonine kinase, in the progression of PP and identify the underlying mechanisms. First, the levels of LKB1 in peripheral blood and peripheral blood mononuclear cells of children with PP were detected by reverse transcription-quantitative (RT-q) PCR or western blotting. After the GT1-7 mouse hypothalamus cell line was treated with high glucose (HG) and high fat (HF), the expression of LKB1 and GnRH was tested. LKB1 was overexpressed by transfection with a pcDNA3.1 plasmid and the levels of inflammatory factors, GnRH, PP-related factors and proteins in the AMP-activated protein kinase (AMPK)/forkhead box protein O1 (FOXO1) pathway were determined using RT-qPCR or western blot analysis. Subsequently, Compound C, an inhibitor of AMPK/FOXO1 signaling, was used to clarify whether the effects of LKB1 on PP were mediated by the regulation of this pathway. Results indicated that children with PP exhibited a lower LKB1 expression. In addition, HG and HF culture resulted in an enhanced GnRH expression and a reduced LKB1 expression in GT1-7 cells. LKB1 overexpression inhibited the contents of TNF-α, IL-6 and GnRH in in GT1-7 cells exposed to HG and HF and reduced the expression of PP-related proteins, including estrogen receptor-β, cluster of differentiation 36 and G-protein-coupled receptor. In addition, the expression of phosphorylated (p)-AMPK and p-FOXO1 was markedly downregulated following LKBI overexpression. Furthermore, compound C intervention partially diminished the inhibitory effects of LKB1-mediated upregulation on the levels of inflammation and PP-related factors. In conclusion, these results demonstrated that LKB1 alleviated HG- and HF-induced inflammation, as well as the expression of GnRH and sexual precocity-related genes, in GT1-7 cells by activating the AMPK/FOXO1 signaling pathway.

Effects of melatonin on the production of GnRH and LH in luteal cells of pregnant sows.

Effects of melatonin on the release and synthesis of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) at the hypothalamus and pituitary levels have been explored in some species, but a similar study in the corpora lutea (CL) has not yet been conducted. In this study, the immunostaining for GnRH and LH was observed in luteal cells of porcine CL during pregnancy, and a significant effect of pregnant stage on the level of GnRH and LH was found; higher values for GnRH and LH immunostaining and mRNA were detected in the early and mid-stages CL than in the later-stage CL (P < 0.01). Furthermore, the patterns of melatonin membrane receptors (MT1 and MT2) expression were consistent with those of GnRH and LH expression in the CL of pregnant sows; the relative levels of MT1 and MT2 in the early and mid-stages were significantly higher than those in the later-stage (P < 0.01). In luteal cells, melatonin dose-dependently increased in GnRH and LH secretion and mRNA expression. Melatonin also increased the GnRH-induced accumulation of LH and the LH-induced secretion of P4 in luteal cells. Additionally, the effects of melatonin on luteal GnRH and LH production were blocked by luzindole, a non-selective MT1 and MT2 receptor antagonist. Our results demonstrate the stimulatory effects of melatonin on GnRH and LH production in luteal cells of pregnant sows, suggesting a potential role for melatonin in luteal function through regulating the release and synthesis of GnRH and LH in luteal cells.

The Effect of Photoperiodic Conditions on GnRH/LH Secretion in Ewes.

Simple SummaryDuring the course of evolution, animals have evolved biological rhythms that are associated with changes in the lighting and temperature of their environment. Females in most breeds of sheep are seasonal breeders, with ovulatory cycles occurring in the autumn and winter and anovulation in the spring and summer. Secretion of gonadotropin releasing hormone and luteinizing hormone, the main hormones regulating reproduction in females, displays a circadian pattern; however, data concerning the day/night differences in their secretion in ewes are incomplete. The aim of the undertaken study was to determine the day/night differences in the secretion of gonadotropin releasing hormone and luteinizing hormone in follicular phase and anestrous ewes. It was demonstrated that secretion of investigated hormones is subject to diurnal and seasonal changes. The observed reduction in luteinizing hormone release, a few hours after the sunset, seems to be universal for both the anestrus and follicular phase. It could be concluded that the nocturnal suppression of luteinizing hormone secretion in follicular phase ewes may be a mechanism moving this hormone surge to the early morning.Secretion of gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH) displays a circadian pattern. Data concerning differences in daily GnRH/LH secretion during different seasons in sheep are fragmentary. The aim of the study was to determine day/night differences in GnRH/LH secretion in the follicular phase and in the anestrous ewes. The studies were performed on Blackhead ewes (n = 24). Ewes from each season were divided into two groups of six animals (day and night group). The animals were euthanized 5 h after sunset or 5 h after sunrise and blood was taken to determine LH and melatonin concentrations. In the hypothalamus, the expression of GnRH and gonadotropin releasing hormone receptor (GnRHR) was determined. In the anterior pituitary, the expression of mRNA encoding subunit β of LH (LHβ) and GnRHR was assayed. Our study showed that GnRH/LH secretion is subject to diurnal and seasonal changes. The observed reduction in LH release, a few hours after the sunset, seems to be universal for both the anestrus and follicular phase, when the processes occurring at the hypothalamus are more equivocal. It could be concluded that the nocturnal suppression of LH secretion in follicular phase ewes may be a mechanism moving the LH surge to the early morning.

Targeting GNAQ in hypothalamic nerve cells to regulate seasonal estrus in sheep

Kazakh sheep are typical seasonal estrus animals. Their reproductive system regulation mainly involves the complex regulation of the hypothalamic–pituitary–gonadal axis (HPGA), which is also closely related to reproductive hormone secretion. Gonadotropin-releasing hormone (GnRH), synthesized and secreted by the hypothalamus, is the key to controlling sheep reproductive activity. We studied how GNAQ (G protein subunit alpha q) regulates estrus in sheep by controlling GnRH expression and secretion. We used hypothalamic nerve cells as the research model. GNAQ overexpression and RNA interference vectors were constructed and transfected into the hypothalamic nerve cells of fetal Kazakh sheep. qPCR, western blotting, and enzyme-linked immunosorbent assay were used to detect GNAQ gene expression in Kazakh ewe tissues and analyze its regulatory effect on GnRH expression in the hypothalamic nerve cells. The fetal sheep hypothalamic nerve cells were successfully isolated and cultured. qPCR and cell immunofluorescence showed that the purity of positive cells was >95%. The tissue expression profile showed that there were different degrees of GNAQ gene expression in the Kazakh ewe tissue. Expression levels were relatively higher in the hypothalamus, pituitary, brain, and uterine tissues. When GNAQ expression was downregulated in the hypothalamic nerve cells, the upstream genes KISS1 (kisspeptin), GPR54 (KISS1 receptor), and ER (estrogen receptor) were all upregulated, as were the downstream genes PLCB1 (phospholipase C beta 1), PRKCB (protein kinase C beta), and GNRH. At the same time, GnRH secretion levels were also upregulated. GNAQ regulated its downstream gene PLCB1 in the hypothalamic nerve cells, and directly regulated GnRH expression and secretion through the calcium and PRKC signaling pathways. GNAQ also regulated kisspeptin expression, subsequently regulating GnRH expression and secretion indirectly through the kisspeptin–GPR54 signaling pathway. Our results are of great importance for improving the reproductive performance of seasonal-estrus sheep.