Gonadotropin-releasing Hormone Receptor Expression Research Articles

GnRH receptor mediates lipid storage in female adipocytes via AMPK pathway.

Objective: Due to high levels of serum gonadotropin-releasing hormone (GnRH), perimenopausal or menopausal women, girls with central precocious puberty, women of polycystic ovary syndrome, and females receiving long-term GnRH agonist (GnRHa) treatment are at substantially higher risk of developing obesity. However, it remains poorly understood how GnRH affects body weight. Here, we explored whether the gonadotropin-releasing hormone receptor (GnRHR) was expressed in adipocytes and how GnRHR mediated lipid accumulation and the development of obesity.Methods: The samples were from 18 patients with benign tumors operated between 01/2018 and 06/2018 at the Women's Hospital School of Medicine Zhejiang University. Immunofluorescence, Western Blotting, and RT-PCR were used to detect whether the GnRH receptor was expressed in the specimens and human preadipocytes-subcutaneous (HPA-s). The GnRH receptor agonist diphereline with different concentrations was used to stimulate the HPA-s cells for 24, 48, and CCK-8 was used to detect cell proliferation. Oil red-O staining was used to detect lipid droplets in mature adipocytes. The phosphorylation of AMPK-Ser485/Thr172 was detected by Western Blotting.Results: GnRH receptor was expressed in all 18 human subcutaneous adipose tissue specimens. Cultured HPA-s expressed the GnRH receptor, and the expression increased during the process of cell maturation. The GnRH receptor agonist diphereline can stimulate the proliferation of HPA-s cells, which can advance the earliest occurrence of lipid droplets in HPA-s cells and the occurrence of lipid droplets in 50% cells by 1-2 days. Diphereline can stimulate the increase in the number of lipid droplets in mature adipocytes. The phosphorylation level of AMPK-Ser485/Thr172 in mature adipocytes was decreased by diphereline.Conclusion: The GnRH receptor was expressed in adipocytes. As adipocytes mature, GnRH receptor expression gradually increased. GnRHa stimulates the proliferation of HPA-s, promotes adipocyte maturation, increases the formation of lipid droplets in mature adipocytes, and inhibits the activation of the AMPK pathway in adipocytes. Our findings may elucidate the mechanism of obesity in these female populations and provide some evidence on how GnRH contributes to obesity. Additionally, these results provide theoretical support for further research on the mechanisms of obesity, thus enhancing our understanding of the functional diversity of GnRH and establishing a new theoretical basis for the impact of GnRH on metabolism.

The Effects of Zearalenone on the Localization and Expression of Reproductive Hormones in the Ovaries of Weaned Gilts.

This study aims to investigate the effects of zearalenone (ZEA) on the localizations and expressions of follicle stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), gonadotropin releasing hormone (GnRH) and gonadotropin releasing hormone receptor (GnRHR) in the ovaries of weaned gilts. Twenty 42-day-old weaned gilts were randomly allocated into two groups, and treated with a control diet and a ZEA-contaminated diet (ZEA 1.04 mg/kg), respectively. After 7-day adjustment, gilts were fed individually for 35 days and euthanized for blood and ovarian samples collection before morning feeding on the 36th day. Serum hormones of E2, PRG, FSH, LH and GnRH were determined using radioimmunoassay kits. The ovaries were collected for relative mRNA and protein expression, and immunohistochemical analysis of FSHR, LHR, GnRH and GnRHR. The results revealed that ZEA exposure significantly increased the final vulva area (p < 0.05), significantly elevated the serum concentrations of estradiol, follicle stimulating hormone and GnRH (p < 0.05), and markedly up-regulated the mRNA and protein expressions of FSHR, LHR, GnRH and GnRHR (p < 0.05). Besides, the results of immunohistochemistry showed that the immunoreactive substances of ovarian FSHR, LHR, GnRH and GnRHR in the gilts fed the ZEA-contaminated diet were stronger than the gilts fed the control diet. Our findings indicated that dietary ZEA (1.04 mg/kg) could cause follicular proliferation by interfering with the localization and expression of FSHR, LHR, GnRH and GnRHR, and then affect the follicular development of weaned gilts.

Effective accumulative temperature affects gonadal maturation by controlling expression of GnRH, GnRH receptor, serotonin receptor and APGWamide gene in Pacific abalone, Haliotis discus hannai during broodstock conditioning in hatcheries.

Water temperature is a crucial environmental factor that influences reproductive function of abalone. Broodstock conditioning exposed to effective accumulative temperature (EAT) is a common practice in abalone hatcheries. To understand the molecular mechanism underlying the regulation of gonadal maturation and reproduction of Haliotis discus hannai exposed to EAT and induced spawning period, changes in expression of neuroendocrine genes encoding two gonadotropin releasing hormone (Hdh-GnRH, GnRH-like peptide), GnRH receptor (HdhGnRH-R), serotonin receptor (5-HTHdh) and Hdh-APGWamide in neural ganglia and gonadal tissues were examined. Gonadosomatic index (GSI) was significantly increased with increasing EAT °C-days. Expression levels of Hdh-GnRH, GnRH-like peptide, HdhGnRH-R, 5-HTHdh and Hdh-APGWamide mRNA were significantly increased with increasing EAT °C-days in ganglion (where the gene synthesized) and gonadal tissues. The significant increase in mRNA expression of each examined gene started from EAT 500 to 750°C-days, reached an initial peak at 1000°C-days, suggesting gonadal maturation started from the onset of EAT and slowly continued until 750°C-days, then at 1000°C-days reached to initial peak developmental period. The maturation reached to spawning state at 1000°C-days and peaked at 1500°C-days. Hdh-GnRH showed significantly higher mRNA expression in pleuropedal ganglion and branchial ganglion, whereas GnRH like peptide showed higher expression in cerebral ganglion, and HdhGnRH-R, 5-HTHdh and Hdh-APGWamide showed higher expression in pleuropedal ganglion. All genes were expressed higher at higher EAT °C-days. During induced spawning period, higher mRNA expression of examined genes was observed at the time of spawning; however, a sharp decrease occurred after spawning, suggesting that these genes are involved in spawning activities. Taken together, these results indicate that an increase of EAT °C-days can increase expression of neuroendocrine genes and enhance gonadal maturation. Besides all these genes are involved in the process of spawning induction, and increase of GSI has a positive correlation with the increase of gene expression.

Thermal Stress Affects Gonadal Maturation by Regulating GnRH, GnRH Receptor, APGWamide, and Serotonin Receptor Gene Expression in Male Pacific Abalone, Haliotis discus hannai During Breeding Season

Temperature has crucial effects on gonadal development and reproduction of abalone. To understand the impact of thermal stress on molecular and physiological processes triggering the regulation of reproduction, changes in the mRNA expression of neuroendocrine genes encoding two abalone gonadotropin-releasing hormone (Hdh-GnRH, Hdh-GnRH-like peptide), GnRH receptor (Hdh-GnRH-R), Hdh-APGWamide, serotonin receptor (5-HThdh), and a heat shock protein HSP70 were examined in ganglia and testis of male Pacific abalone (Haliotis discus hannai). Abalone were exposed to low water temperature (LWT) and high water temperature (HWT) in early and peak breeding seasons for 7 days. Then, gonadosomatic index (GSI) was calculated, relative gene expression was measured by qRT-PCR, and levels of testosterone in hemolymph were also measured by ELISA during the peak breeding season. GSI did not show any significant changes during the early breeding season. However, it was significantly decreased in LWT- or HWT-exposed abalone compared to the normal water temperature (NWT) group during the peak breeding season. In the early breeding season, changes of mRNA expression of all five genes were significant between LWT and HWT groups on day-7. In the peak breeding season, compared to the NWT group, the mRNA expressions of different genes were significantly decreased in different tissues both in LWT and HWT groups of abalone, such as Hdh-GnRH-like peptide in the cerebral ganglion (CG) and testis; Hdh-GnRH in the pleuropedal ganglion (PPG) and branchial ganglion (BG); Hdh-GnRH-R in the CG, PPG, and testis; and Hdh-APGWamide in the PPG and testis. Interestingly, the expression of 5-HThdh was significantly increased in the HWT group but decreased in the LWT group. Expression of HSP70 was significantly increased in the testis after exposure to HWT. Hemolymph levels of testosterone were significantly decreased in the HWT group compared to those in the NWT group. Altogether, these results denote that thermal stress has a repressive effect on gonadal maturation and reproduction by regulating the expression of Hdh-GnRH-like peptide, Hdh-GnRH, Hdh-GnRH-R, Hdh-APGWamide, 5-HThdh, and HSP70 genes and levels of hemolymph testosterone.

Musashi as a Regulator of the Follicle-Stimulating Hormone in the Gonadotropes

The cyclic expression of gonadotropin releasing-hormone receptors (GnRHR), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) by pituitary gonadotropes is critical in the female reproductive process. We have shown that the translational regulator Musashi (MSI) binds to Gnrhr mRNA and inhibits its translation, and the gonadotrope-specific deletion of Msi1 and Msi2 (Gon-Msi-null) leads to increased pituitary GnRHR protein levels. An in silico analysis of gonadotropin mRNAs revealed 5 different MSI binding elements in the 3’UTR of Fshb mRNA. We hypothesize that, in addition to Gnrhr, MSI may also bind and repress Fshb mRNA translation in the gonadotropes. To test if MSI does target the Fshb transcript in the pituitary, we performed RNA immunoprecipitation (IP) on pooled control female mouse pituitaries using a MSI1 antibody and measured Fshb mRNA by qRT-PCR. To study the in vivo effects of MSI on Fshb, we harvested the pituitaries of the Gon-Msi-null (MUT) female mice and their littermate controls (CTL) during the estrous cycle. We collected serum and protein for EIAs to measure the levels of FSH and LH, and RNA for Fshb qRT-PCR. We harvested proestrous ovaries and fixed them for embedding, sectioning, and H&E staining. Our RNA IP experiments show a 7-fold enrichment for Fshb with the MSI1 antibody. The Gon-Msi-null females have significantly higher pituitary FSH protein content than controls on estrous morning (MUT: 4.8±1.3 vs. CTL: 1.8±2.6 ng/ml/μg protein, p<0.0001, n=9-10/group). These mice also have increased serum FSH levels (MUT: 56.9±6.4 vs. CTL: 44.5±9.6 ng/ml, p=0.0147, n=9-10/group). No changes at the Fshb mRNA level were detected. Analysis of Gon-Msi-null ovaries revealed a 50% decrease in the number of follicles, with significant decreases in the average numbers of maturing follicles (p<0.0175) and corpora lutea (p<0.0215). Interestingly, the LH levels in these mice were also altered. The Gon-Msi-null females show a decrease in the pituitary LH protein content in the evening of proestrus (MUT: 11.8±1.4 vs. CTL: 15.1±2.0 ng/ml/μg protein, p=0.0333, n=7/group), in addition to a delayed and blunted LH surge (MUT: 2.6±1.9 vs. CTL: 7.3±3.5 ng/ml, p=0.0089, n=7-11/group). Taken together, our data indicate that Fshb is a Musashi target in the gonadotropes. By deleting MSI from the pituitary gonadotropes, we observe an increase in FSH protein content and serum levels. These Gon-Msi-null female mice have significantly fewer maturing follicles and corpora lutea, which might suggest lower levels of estrogens and progesterone. This, together with the increased GnRHR pituitary protein content, affects LH secretion, leading to a blunted LH surge in the Gon-Msi-null females. Our studies thus reveal a novel translational regulatory mechanism to govern levels of critical reproductive hormones in the pituitary.

The Gut Microbiome's Influence on Hormones and Fertility in Zebrafish

One in eight couples of reproductive age will experience difficulties conceiving and having children. A woman's fertility is largely contributed to by estrogen production. Recent research indicates that the composition of the gut microbiome may contribute to estrogen levels and thus play a role in reproductive health and fertility. However, currently there is little to no work connecting the microbiome to the hormonal imbalances associated with infertility. This study investigates the effects of gut microbiome modulation on hormones in the hypothalamic-pituitary-gonadal (HPG) axis and fertility. Total bacterial DNA in the gut, expression of GnRH receptor, and expression of estrogen receptor were examined via qRT-PCR in three groups of fish: untreated, antibiotic-treated with zero-day reacclimation period, and antibiotic-treated with 14-day reacclimation period. There was over a 50% reduction in bacterial abundance in antibiotic treated fish (n=4, p< 0.05). In females, this corresponded to a 17-fold increase of GnRH receptor expression in antibiotic treated fish with a 14-day reacclimation (n= 6, p< 0.05) while estrogen receptor expression showed no significant differences in any groups. Collectively, these data indicate the gut microbiota may play a role in modulation of reproductive hormones, thus influencing fertility in zebrafish.

Post-Transcriptional Regulation of Gnrhr: A Checkpoint for Metabolic Control of Female Reproduction.

The proper expression of gonadotropin-releasing hormone receptors (GnRHRs) by pituitary gonadotropes is critical for maintaining maximum reproductive capacity. GnRH receptor expression must be tightly regulated in order to maintain the normal pattern of expression through the estrous cycle in rodents, which is believed to be important for interpreting the finely tuned pulses of GnRH from the hypothalamus. Much work has shown that Gnrhr expression is heavily regulated at the level of transcription. However, researchers have also discovered that Gnrhr is regulated post-transcriptionally. This review will discuss how RNA-binding proteins and microRNAs may play critical roles in the regulation of GnRHR expression. We will also discuss how these post-transcriptional regulators may themselves be affected by metabolic cues, specifically with regards to the adipokine leptin. All together, we present evidence that Gnrhr is regulated post-transcriptionally, and that this concept must be further explored in order to fully understand the complex nature of this receptor.

Changes in mRNA and protein levels of gonadotropin releasing hormone and receptor in ovine thymus, lymph node, spleen, and liver during early pregnancy

There exists maternal immunological modification in maternal immune organs during early pregnancy in mammals. Gonadotropin releasing hormone (GnRH) is widely distributed in vertebrate tissues, including immune organs. However, it is unclear that early pregnancy induces expression of GnRH and GnRH receptor (GnRHR) in ovine immune organs. The objective of this study was to explore the expression of GnRH and GnRHR in main immune organs (thymus, lymph node, spleen, and liver) during early pregnancy in sheep. Ovine thymus, lymph node, spleen and liver were sampled at day 16 of estrous cycle, and days 13, 16, and 25 of pregnancy. The expression of GnRH and GnRHR was detected through real-time quantitative PCR, Western blot and immunohistochemistry analysis. The results indicated that early pregnancy induced upregulation of mRNA and protein levels of GnRH and GnRHR in the maternal lymph node, spleen and liver, and mRNA and protein of GnRH in the maternal thymus, but mRNA and protein of GnRHR decreased in the maternal thymus during early pregnancy. In summary, the mRNA and protein levels of GnRH and GnRHR were changed in maternal thymus, lymph node, spleen and liver in a tissue specific manner during early pregnancy in sheep.

Evaluation of the Hypothalamic Kisspeptin System throughout the Estrous Cycle in Gilts

Kisspeptin has been demonstrated to affect reproductive cyclicity and the attainment of puberty in multiple species, presumably through its actions on gonadotropin releasing hormone and luteinizing hormone. Kisspeptin administration causes increased plasma concentrations of LH in pigs, sheep, and rats. The objective of this experiment was to evaluate changes in the hypothalamic kisspeptin system throughout the estrous cycle in gilts. Estrus was synchronized in forty crossbred gilts (191 d, 121 kg) and estrus detection was performed by exposing gilts to a mature boar. The first day gilts stood immobile was denoted d 1 of the estrous cycle. Blood samples were collected via jugular venipuncture on d 1, 4, 7, 9, 14, 16, and 19 of the estrous cycle. Ten animals were slaughtered on d 1, 9, 14, and 21 of the estrous cycle when medial basal hypothalami, anterior pituitary glands, and blood were collected. Relative expression of hypothalamic kisspeptin (KISS1), kisspeptin receptor (KISS1R), estrogen receptors-a, anterior pituitary gland GnRH receptor, β-actin, and GAPDH was determined using real-time reverse transcriptase PCR. Fold changes in relative expression were determined using the Relative Expression Software Tool. Relative expression of KISS1 was increased (P = 0.006) 3.2 fold on d 1 versus d 21 and 2.3 fold (P = 0.003) on d 9 versus d 21 of the estrous cycle, but was not different (P > 0.05) among the remaining days of the estrous cycle. Relative expression of estrogen receptor-b was decreased (P = 0.05) 0.8 fold on d 9 versus d 21 and (P = 0.005) 0.7 fold on d 14 versus d 21, but was not different (P > 0.05) among the remaining days. Relative expression of anterior pituitary gland GnRH receptor was increased (P

Gonadotropin-Releasing Hormone Receptor Expression in Human Spinal Cord.

The expression of the gonadotrophin-releasing hormone receptor expression on pituitary gonadotrophs in humans is well characterized. In nervous system they have also been found in hippocampi and cerebral cortex. However, gonadotrophin-releasing hormone receptor expression in human spinal cord has not been reported. This study was to analyze the gonadotrophin-releasing hormone receptor expression in human spinal cord by immunohistochemistry, mRNAs by reverse transcriptase polymerase chain reaction, cDNA cloning and Western blot. The results show immunoreactive material to gonadotrophin-releasing hormone receptor in motoneurons of the spinal cord. Further, the study revealed that spinal cord expressed the gonadotrophin-releasing hormone receptor mRNA. The amplicon sequence corresponds to 100% of identity to GenBank. In Western blot, a band of 37kDa were found in extracts of spinal cord and placenta as a control. In conclusion, human spinal cord expresses gonadotrophin-releasing hormone receptor analyzed through immunohistochemistry, the expression of its mRNA, cloning its cDNA and Western blot analysis. The presence of gonadotrophin-releasing hormone receptor in the spinal cord suggests the possibility of an extrapituitary functional role independent of reproductive system.

Molecular Basis for the Evolved Instability of a Human G-Protein Coupled Receptor

Membrane proteins are prone to misfolding and degradation. This is particularly true for mammalian forms of the gonadotropin-releasing hormone receptor (GnRHR). Though they function at the plasma membrane, mammalian GnRHRs tend to accumulate within the secretory pathway. Their apparent instability is believed to have evolved in response to selection for attenuated GnRHR activity. Nevertheless, the structural basis of this adaptation remains unclear. We find that this adaptation coincides with a C-terminal truncation and an increase in the polarity of its transmembrane (TM) domains. This enhanced polarity compromises the translocon-mediated cotranslational folding of two TM domains. Moreover, replacing a conserved polar residue in TM6 with an ancestral hydrophobic residue partially restores GnRHR expression with minimal impact on function. An evolutionary analysis suggests variations in the polarity of this residue are associated with reproductive differences. Our findings suggest the marginal energetics of cotranslational folding can be exploited to tune membrane protein fitness.

Effect of Acute and Prolonged Inflammation on the Gene Expression of Proinflammatory Cytokines and Their Receptors in the Anterior Pituitary Gland of Ewes.

An acute and prolonged inflammation inhibits the reproduction process by the disruption of the neurohormonal activity of the hypothalamic-pituitary-gonadal axis. It is thought that these changes may be caused by proinflammatory cytokines, i.e., interleukin (IL) -1β, IL-6 and tumor necrosis factor (TNF) α. The aim of this study was to determine the effect of an acute and prolonged inflammation on the expression of genes encoding cytokine and their receptors, gonadotropin releasing hormone receptor (GnRHR), beta subunits of luteinizing hormone (LHβ) and follicle-stimulating (FSHβ) in the anterior pituitary (AP). Moreover, the circulating concentration of LH and FSH was also assayed. Two experiments were carried out on adult ewes which were divided into two control groups and treated with lipopolysaccharide (LPS; 400 ng / kg). Acute inflammation was caused by a single injection of LPS into the external jugular vein, while the chronic inflammation was induced by seven times LPS injection (one a day). In both experiments, animals were euthanized 3h after the last LPS / NaCl injection and the blood samples collected 15 min before euthanasia. An acute inflammation stimulates the expression of the IL-1β, IL-6 and TNFα genes and their receptors in the AP of sheep. Prolonged inflammation increased TNFα gene expression and both types of TNFα and IL-6 receptors. Both an acute and prolonged inflammation inhibited LHβ gene expression in the AP and reduced LH level in blood. A sevenfold LPS injection raises FSH concentration. The gene expression of GnRHR was reduced in the ovine AP only after a single injection of endotoxin. Our results suggest that there are important differences in the way how an acute and prolonged inflammation influence proinflammatory cytokines and their receptors gene expression in the AP of anestrous ewes, which could be reflected by differences in the AP secretory activity during these states.

GnRH protective effects against amyloid β-induced cognitive decline: A potential role of the 17β-estradiol

IntroductionThe 17β-estradiol (E2) enhances hippocampal dendritic spine synapses, facilitates learning processes, and exerts neuroprotection. Brain estrogen decline has been reported in Alzheimer's disease. The role of GnRH in modulating steroid biosynthesis convinced us to examine whether hippocampal GnRH administration could enhance the local E2 levels and overcome the development of cognition decline in amyloid β (Aβ) neurotoxicity. To explore if GnRH acts through regulating E2 synthesis, letrozole, an aromatase inhibitor, has been applied in combination with GnRH. MethodsFemale rats received an intracerebroventricular injection of Aβ. The GnRH and, or letrozole were injected into the CA1 for 14 consecutive days. Working memory, novel object recognition memory, and anxiety-like behavior were evaluated. Serum and hippocampal E2 levels were measured. Hippocampal mRNA expression of GnRH (GnRH-R) and E2 (ERα and ERβ) receptors was assessed. GnRH effect on the excitability of pyramidal cells was studied by in vivo single-unit recording. ResultsGnRH increased hippocampal E2 levels, evoked an increase in the spontaneous firing of pyramidal neurons, and caused mRNA overexpression of hippocampal GnRH receptors. GnRH prevented the adverse effects of Aβ on working memory, NOR index, and anxiogenic behavior. Letrozole did not reverse GnRH modulatory effects on hippocampal E2 levels and neuroprotection. ConclusionGnRH prevented the Aβ-induced memory deficit, which may be mediated through hippocampal E2 levels enhancement. The electrophysiological analysis revealed the enhanced neuronal excitability in the CA1 region. All these data suggest that GnRH might be a promising candidate that reduces anxiety and improves memory indices in the context of Aβ neurotoxicity.

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-238 Estrogen Modulates Expression Levels of Gonadotropin-Releasing Hormone Receptor (GNRHR) in Immortalized Kisspeptin Neurons in Vitro

In female animals, ovarian estradiol (E2) can act as both a negative feedback inhibitor of GnRH secretion, as well as a positive feedback stimulator at the time of ovulation. Both of these E2-regulated mechanisms work via stimulation or repression of two distinct neuronal populations of Kisspeptin (KP)-synthesizing neurons. While it is clear that AVPV KP neurons increase kiss1 expression during the preovulatory surge on proestrus, subsequent secretory mechanisms required for potentiation of GnRH surge release remain unclear. Two KP-secreting cell lines, KTaV-3, which demonstrate increased kiss1 expression under high E2 exposure, and KTaR-1, which exhibit kiss1 suppression under low E2 exposure, were used to probe the presence of GnRH receptor (GnRHR) expression under different E2 exposure conditions.KTaV-3 and KtaR-1 cells were treated with a range of doses of E2 (5-100pM) and/or progesterone (20nM) for varying durations (4-96h), exposed to steroid hormones either constitutively or via modulating levels over time, approximating concentration changes found during the murine estrous cycle. Following RNA isolation, cDNAs were probed with primers for gnrhr. Preliminary results in KTaV-3 cells reveal the expression of gnrhr is induced only following elevated (50-100pM) E2 treatment for 18-24h. These same E2 exposure conditions were also found to increase expression of the homeobox protein dlx3, a transcription factor required for GnRHR expression in pituitary gonadotropes. In Arc-derived KTaR-1 cells, gnrhr expression was observed only following decreases in E2 concentration, while dlx3 remained constitutively elevated in this cell line. While reciprocal GnRH-Kisspeptin connections have not yet been observed in vivo, these observations suggest the potential for Kisspeptin neurons to respond to GnRH secretory changes under particular E2 exposure conditions, by modulating receptivity to GnRH at the level of the AVPV and/or Arcuate nuclei. We are continuing to explore the temporal parameters of this induction of GnRHR in KP cells, and if exposure of immortalized KP neurons to GnRH in vitro elicits expression and signaling changes in a time- and E2-dependent manner. Results will provide a more complete understanding of positive and negative feedback mechanisms required for normal neuroendocrine regulation of reproduction.

MON-009 GLUT1-Mediated Glycolysis Facilitates GnRH-Induced Secretion of Luteinizing Hormone

Reproduction requires intensive energy expenditure, and energy availability impacts the function of the reproductive endocrine HPG-axis. Accordingly, the reproductive axis is suppressed during hypoglycemia. Circulating blood glucose can directly interact with gonadotropes within the highly vascular pituitary. Therefore, it is possible that gonadotropes may sense energy availability via the presence of glucose in the circulation and integrate this status with input from GnRH neurons to regulate hormone production. Gonadotropes dominantly express glucose transporter 1 (GLUT1) and increase glucose uptake in response to GnRH. Thus, we hypothesized that gonadotropes engage glycolysis in response to GnRH stimulation due to the high energy demand of protein synthesis required for LH production. We developed an approach to sort and successfully culture primary gonadotropes from wild type mice. Using this approach, we performed extracellular flux analysis and found that gonadotropes respond to GnRH by inducing GLUT1-mediated glycolysis that is independent of mitochondrial respiration. Knock-down of GLUT1 expression in the LβT2 gonadotrope cell line, glucose restriction, or treatment with the competitive inhibitor of glycolysis, 2-DG, diminished GnRH-induced LH secretion, indicating GLUT1 expression is necessary for maximal GnRH-induced LH secretion. We confirmed this observation in primary female mouse gonadotropes by limiting glucose availability which resulted in diminished basal LH and FSH secretion. Lastly, GLUT1 expression in the pituitary correlates with GnRH receptor expression and is increased during the LH surge in a mouse model. These results implicate glucose uptake through GLUT1 as permissive for gonadotrope secretion of LH and therefore reproductive function, especially the LH surge. We conclude that GLUT1-mediated glucose uptake is an important rate-limiting step in gonadotropin synthesis and operation of the HPG-axis.

Gonadotropin-Releasing Hormone Receptor-Targeted Near-Infrared Fluorescence Probe for Specific Recognition and Localization of Peritoneal Metastases of Ovarian Cancer.

Background: Peritoneal dissemination is common in advanced ovarian cancer. The completeness of cytoreduction is an independent prognostic factor. The intraoperative fluorescence imaging via tumor-specific near-infrared fluorophore might improve staging and surgical completeness. A promising target for ovarian cancer is the gonadotropin-releasing hormone receptor (GnRHR). This study aimed to develop a GnRHR-targeted near-infrared imaging probe for the detection of peritoneal metastases of ovarian cancer.Methods: Indocyanine green (ICG) was conjugated with GnRH antagonist peptide to develop an ovarian cancer-selective fluorescence probe GnRHa-ICG. GnRHR expression was detected in ovarian cancer tissues. The binding capacity of GnRHa-ICG and ICG was detected in both cancer cell lines and mouse models of peritoneal metastatic ovarian cancer using fluorescence microscopy, flow cytometry, and near-infrared fluorescence imaging.Results: Tissue microarray analysis revealed the overexpression of GnRHR in ovarian cancer. GnRH-ICG exhibited the binding capacity in a panel of cancer cell lines with different expression levels of GnRHR. In ovarian cancer mouse models, GnRHa-ICG signals were detected in peritoneal tumor lesions rather than normal peritoneal and intestines tissues. ICG showed intensive fluorescence signals in intestines. The tumor-to-muscle ratio and tumor-to-intestine ratio of GnRHa-ICG was 7.41 ± 2.82 and 4.37 ± 1.66, higher than that of ICG (4.60 ± 0.50 and 0.57 ± 0.06) at 2 h post administration. The fluorescence signal of peritoneal metastases peaked in intensity at 2 h and maintained for up to 48 h. ICG also showed a weak signal in the tumor lesions due to the enhanced permeability and retention effect, but the intensity decreased quickly within 48 h.Conclusions: The developed GnRHR-targeted imaging agent GnRHa-ICG could specifically detected peritoneal tumor lesions from normal peritoneal and intestines tissues because of the modification of GnRHa to ICG. The plateau period of GnRHa-ICG accumulation may be feasible for clinical applications in fluorescence-guided surgery. Our GnRHR imaging concept may be effective in other hormone-related tumors with upregulated GnRHR expression.

The Therapeutic Effect of Shugan Xiehuo Formula in the Female Rat Model with Central Precocious Puberty.

Central precocious puberty (CPP) severely affects children's physical and mental health and needs to be treated promptly and effectively. This article aimed to research the therapeutic effect of Shugan Xiehuo Formula (SXF) on CPP. A female CPP rat model was established and then treated with leuprolide and different doses of SXF. Sex organ volume and index were measured. Ovaries and uteri were visualized by hematoxylin-eosin staining. The concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), and estradiol (E2) in peripheral blood were determined. The expression levels of gonadotropin-releasing hormone (GnRH), gonadotropin-releasing hormone receptor (GnRHR), estrogen receptor alpha (ERα), and G protein-coupled receptor 30 (GPR30) in the hypophysis were investigated by Real-Time Quantitative Reverse Transcription PCR and western blot. GnRH expression in the hypothalamus and GnRHR expression in the ovary were detected by immunohistochemistry. SXF reduced the volume of the bilateral ovaries, as well as the volumes of the uterus, hypothalamus, and hypophysis in the female CPP rats and diminished the index of the ovary, uterus, hypothalamus, and hypophysis in the female CPP rats (P < 0.05 or P < 0.01). SXF treatment inhibited follicle maturation and uterine wall thickening in the female CPP rats. SXF decreased the concentrations of FSH, LH, PRL, and E2 in the peripheral blood in the female CPP rats (P < 0.01 or P < 0.001). SXF suppressed the expressions of GnRH, GnRHR, ERα, and GPR30 in the hypophysis (P < 0.05), the expression of GnRH in the hypothalamus (P < 0.01), and the expression of GnRHR in the ovaries (P < 0.001) of the female CPP rats. Overall, our study revealed that SXF had therapeutic effects on CPP in female rats. This is worthy of promoting clinically.

Gpr120 mRNA expression in gonadotropes in the mouse pituitary gland is regulated by free fatty acids.

GPR120 is a long-chain fatty acid (LCFA) receptor that is specifically expressed in gonadotropes in the anterior pituitary gland in mice. The aim of this study was to investigate whether GPR120 is activated by free fatty acids in the pituitary of mice and mouse immortalized gonadotrope LβT2 cells. First, the effects of palmitate on GPR120, gonadotropic hormone b-subunits, and GnRH-receptor expression in gonadotropes were investigated in vitro. We observed palmitate-induced an increase in Gpr120 mRNA expression and a decrease in follicle-stimulating hormone b-subunit (Fshb) expression in LβT2 cells. Furthermore, palmitate exposure caused the phosphorylation of ERK1/2 in LβT2 cells, but no significant changes were observed in the expression levels of luteinizing hormone b-subunit (Lhb) and gonadotropin releasing hormone-receptor (Gnrh-r) mRNA and number of GPR120 immunoreactive cells. Next, diurnal variation in Gpr120 mRNA expression in the male mouse pituitary gland was investigated using ad libitum and night-time restricted feeding (active phase from 1900 to 0700 h) treatments. In ad libitum feeding group mice, Gpr120 mRNA expression at 1700 h was transiently higher than that measured at other times, and the peak blood non-esterified fatty acid (NEFA) levels were observed from 1300 to 1500 h. These results were not observed in night-time-restricted feeding group mice. These results suggest that GPR120 is activated by LCFAs to regulate follicle stimulating hormone (FSH) synthesis in the mouse gonadotropes.

Study on the Mechanism of Sarsasapogenin in Treating Precocious Puberty by Regulating the HPG Axis.

The present study aims to investigate the effects and mechanisms of sarsasapogenin resistance to precocious puberty. Female Sprague Dawley rats were divided into a normal (N) group, model (M) group, leuprolide (L) group, and sarsasapogenin (Sar) group. Rats at 5 days of age were given a single subcutaneous injection of 300 micrograms of danazol to establish the precocious puberty model. After 10 days of modeling, drug intervention was started. The development of the uterus and ovary was observed by hematoxylin and eosin (HE) staining. The levels of the serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estradiol (E2) were determined by radioimmunoassay. Also, the expressions of the hypothalamic gonadotropin releasing hormone (GnRH), Kiss-1, G protein-coupled receptor 54 (GPR54), and pituitary gonadotropin releasing hormone receptor (GnRH-R) were detected by RT-PCR. The results showed that compared with the model group, sarsasapogenin could significantly delay the opening time of vaginal, decreased uterine and ovarian coefficients, and reduced uterine wall thickness. Moreover, it can significantly downregulate the levels of serum hormones and reduce the expression of GnRH, GnRH-R, and kiss-1. In summary, our results indicate that sarsasapogenin can regulate the HPG axis through the kiss-1/GPR54 system for therapeutic precocious puberty.