Gonadotropin-releasing Hormone Expression Research Articles

Gonadotropin-Releasing Hormone Stimulates Biliary Proliferation by Paracrine/Autocrine Mechanisms

During cholestatic liver disease, there is dysregulation in the balance between biliary growth and loss in bile duct-ligated (BDL) rats modulated by neuroendocrine peptides via autocrine/paracrine pathways. Gonadotropin-releasing hormone (GnRH) is a trophic peptide hormone that modulates reproductive function and proliferation in many cell types. We evaluated the autocrine role of GnRH in the regulation of cholangiocyte proliferation. The expression of GnRH receptors was assessed in a normal mouse cholangiocyte cell line (NMC), sham, and BDL rats. The effect of GnRH administration was evaluated in normal rats and in NMC. GnRH-induced biliary proliferation was evaluated by changes in intrahepatic bile duct mass and the expression of proliferation and function markers. The expression and secretion of GnRH in NMC and isolated cholangiocytes was assessed. GnRH receptor subtypes GnRHR1 and GnRHR2 were expressed in cholangiocytes. Treatment with GnRH increased intrahepatic bile duct mass as well as proliferation and function markers in cholangiocytes. Transient knockdown and pharmacologic inhibition of GnRHR1 in NMC decreased proliferation. BDL cholangiocytes had increased expression of GnRH compared with normal rats, accompanied by increased GnRH secretion. In vivo and in vitro knockdown of GnRH decreased intrahepatic bile duct mass/cholangiocyte proliferation and fibrosis. GnRH secreted by cholangiocytes promotes biliary proliferation via an autocrine pathway. Disruption of GnRH/GnRHR signaling may be important for the management of cholestatic liver diseases.

Gonadotropin-releasing hormone induces biliary proliferation by both paracrine and autocrine mechanisms

During cholestatic liver disease, there is dysregulation in the balance between biliary growth and loss. In bile ductligated (BDL) rats, this balance is modulated by neuroendocrine peptides via autocrine/paracrine pathways (1). Gonadotropinreleasing hormone (GnRH) is a trophic peptide hormone that modulates reproduc- tive function and proliferation in many cell types (2). We evaluated the autocrine role of GnRH in the regulation of cholangiocyte growth. The expression of GnRH receptors was evaluated in a normal mouse cholangiocyte cell line (NMC), sham and BDL rats. The effect of GnRH administration was evaluated in normal rats and in NMC. GnRH-induced biliary proliferation was evaluated by changes in intrahepatic bile duct mass (IBDM) by CK19 specific staining, the expression of proliferation and function markers. The expression and secretion of GnRH in NMC and isolated cholangiocytes was assessed. GnRH receptor subtypes GnRHR1 and GnRHR2 were expressed in biliary epithelium. Treatment with GnRH increased IBDM as well as proliferation and function markers in cholangiocytes. Transient knockdown and pharmacologic inhibition of GnRHR1 in NMC decreased proliferation. BDL cholangiocytes had increased expression of GnRH compared with normal rats, accompanied by increased GnRH secretion. In vivo and in vitro knockdown of GnRH decreased intrahepatic bile duct mass/cholangiocyte proliferation and fibrosis. GnRH secreted by cholangiocytes promotes biliary proliferation via an autocrine pathway. Disruption of GnRH/ GnRHR signaling may be important for the management of cholestatic liver diseases.

Tumor necrosis factor αlpha impairs kisspeptin signaling in human fetal hypothalamic neurons

Previous studies in both human and animal models have linked overnutrition-related metabolic dysfunctions to hypothalamic inflammation (1, 2). Moreover, metabolic disorders are often associated with male hypogonadotropic hypogonadism, suggesting that inflammatory pathways may impair central regulatory networks involving gonadotropin releasing hormone (GnRH) neuron activity. However, studies in humans are strongly hampered by the anatomical distribution of these neurons, scattered within the preoptic area of the hypothalamus. This study was aimed at investigating the effects of inflammatory stimuli in human fetal hypothalamic (hfHypo) primary cell cultures. These cells have shown evident GnRH neuron features, as demonstrated by quantitative gene expression profile, immunohistochemistry, flow cytometry and ELISA detection of GnRH peptide in the culture medium. Moreover, hfHypo cells express KISS1R and accordingly respond to kisspeptin, the main central regulator of GnRH neuron function. Exposing hfHypo cells to TNFα (10ng/ml, 5h) determined nuclear translocation of NFkB, as well as a significant increase of COX2 mRNA expression, thus demonstrating the induction of inflammatory signaling. Prolonged exposure (24 h) to TNFα strongly down-regulated KISS1R mRNA without changing GnRH expression. Moreover, we know that kisspeptin is able to depolarize GnRH neurons through the activation of a canonical transient receptor potential (TRPC)-like cationic channel. Electrophisiological studies demonstrated that kisspeptin (1 μM) induced a clear TRPC-mediated depolarizing response in hfHypo cells. Pretreating cells with TNFα (10 ng/ml, 24h) significantly inhibited kisspeptin-sensitive TRPC currents. Our results indicate that inflammatory pathways may impair GnRH neuron function by interfering with the ability of these neurons in responding to kisspeptin.

Involvement of prolactin in the meloxicam-dependent inflammatory response of the gonadotropic axis to prolonged lipopolysaccharide treatment in anoestrous ewes.

An immune challenge can affect the reproductive process in females. Peripheral administration of bacterial endotoxin (lipopolysaccharide; LPS) decreases LH secretion and disrupts ovarian cyclicity. The aim of the present study was to determine the effects of a cyclo-oxygenase (COX)-2 inhibitor (meloxicam) on gonadotropin-releasing hormone (GnRH) and LH secretion in anoestrous ewes during systemic inflammation induced by LPS. LPS (400ngkg-1 per day) suppressed LH release. In three individuals, meloxicam (500μgkg-1, i.v.) abolished LPS-induced LH suppression. In another three ewes LH was ineffective. Similar changes were observed in hypothalamic GnRH expression. The effect of meloxicam depended on the circulating level of prolactin: meloxicam abolished inflammatory-dependent suppression of GnRH and LH secretion when plasma prolactin levels were similar to those in untreated animals, but was ineffective in those with elevated levels of prolactin. We conclude that COX-2 inhibitors minimise the negative effect of inflammation on the reproductive system but that this effect may be antagonised by prolactin.

Second generation anti-epileptic drugs adversely affect reproductive functions in young non-epileptic female rats

Reproductive endocrine disturbances are a major health concern in women with epilepsy due to their long term use of antiepileptic drugs (AEDs). Second generation AEDs such as topiramate (TPM) and gabapentin are frequently used for the treatment of epilepsy as well as migraine, bipolar disorder etc. Despite the widespread clinical complications, however the definitive mechanism(s) mediating the side effects of TPM and gabapentin remain obscure. The present study was aimed to evaluate the long term effects of TPM and gabapentin on reproductive functions in young female Wistar rats. Estrous cyclicity, ovarian histology as well as estradiol, LH, leptin and insulin hormones level were studied to elucidate the long-term effect of these AEDs monotherapy on reproductive functions in non-epileptic animals. Further to explore the effects on gonadotropin releasing hormone (GnRH) neuroendocrine plasticity, the expression of GnRH, gamma-amino butyric acid (GABA), glutamic acid decarboxylase (GAD), glial fibrilliary acidic protein (GFAP) and polysialylated form of neural cell adhesion molecule (PSA-NCAM) was studied in median eminence (ME) region of these animals by immunohistochemistry, Western blot hybridization and RT-PCR. Our results demonstrate that TPM and gabapentin treatment for 8 weeks cause reproductive dysfunction as ascertained by disturbed hormonal levels and estrous cyclicity as well as alterations in GABAergic system and GnRH neuronal–glial plasticity. Our findings suggest that treatment with TPM and gabapentin disrupts the complete hypothalamo-hypophyseal-gonadal axis (HPG) through GnRH pulse generator in hypothalamus.

A Critical Point of Male Gonad Development: Neuroendocrine Correlates of Accelerated Testicular Growth in Rats during Early Life

Testis growth during early life is important for future male fertility and shows acceleration during the first months of life in humans. This acceleration coincides with the peak in gonadotropic hormones in the blood, while the role of hypothalamic factors remains vague. Using neonatal rats to assess this issue, we found that day 9 of life is likely critical for testis development in rats. Before this day, testicular growth was proportional to body weight gain, but after that the testes showed accelerated growth. Hypothalamic kisspeptin and its receptor mRNA levels begin to elevate 2 days later, at day 11. A significant increase in the mRNA levels for gonadotropin-releasing hormone (GnRH) receptors in the hypothalamus between days 5 and 7 was followed by a 3-fold decrease in GnRH mRNA levels in this brain region during the next 2 days. Starting from day 9, hypothalamic GnRH mRNA levels increased significantly and positively correlated with accelerated testicular growth. Triptorelin, an agonist of GnRH, at a dose that had no effect on testicular growth during “proportional” period, increased testis weights during the period of accelerated growth. The insensitivity of testicular growth to GnRH during “proportional” period was supported by inability of a 2.5-fold siRNA knockdown of GnRH expression in the hypothalamus of the 7-day-old animals to produce any effect on their testis weights. GnRH receptor blockade with cetrorelix was also without effect on testis weights during “proportional” period but the same doses of this GnRH antagonist significantly inhibited “accelerated” testicular growth. GnRH receptor mRNA levels in the pituitary as well as plasma LH concentrations were higher during “accelerated” period of testicular growth than during “proportional” period. In general, our data defined two distinct periods in rat testicular development that are primarily characterized by different responses to GnRH signaling.

Effects of NELL2 on the regulation of GnRH expression and puberty in female rats.

Neural tissue-specific epidermal growth factor-like repeat domain-containing protein (NELL2) was previously found to play an important role in nerve growth, neural differentiation, neural elasticity, synaptic transport, and vesicle release. In this study, we examined the effect of NELL2 on gonadotropin-releasing hormone (GnRH) neurons and the initiation of puberty in female rats. We studied changes in NELL2 mRNA and protein expression at different stages of sexual development (postnatal days 30, 35, and 45) in female rats to determine the impact of NELL2 on GnRH mRNA expression. We also investigated the influence on the vulva-opening age by inhibiting NELL2 mRNA expression through lentiviral vector-mediated RNA interference. The intraventricular administration of an NELL2-interfering virus reduced NELL2 and GnRH expression at multiple stages of sexual development and delayed the age of vulva-opening in female rats. These results demonstrate that lentiviral-mediated RNA interference technology can be used for targeted regulation of sexual development in vivo. In addition, we found that NELL2 regulated the initiation of puberty in female rats.

Variation in Progesterone Receptors and GnRH Expression in the Hypothalamus of the Pregnant South American Plains Vizcacha, Lagostomus maximus (Mammalia, Rodentia)1

In mammals, elevated levels of progesterone (P4) throughout gestation maintain a negative feedback over the hypothalamic-hypophyseal-gonadal (H-H-G) axis, avoiding preovulatory follicular growth and preventing ovulation. Recent studies showed that in the South American plains vizcacha (Lagostomus maximus) folliculogenesis progresses to preovulatory stages during gestation, and an ovulatory process seems to occur at midgestation. The aim of this work was to analyze hypothalamic gonadotropin-releasing hormone (GnRH) and P4 receptors (PR) expression and luteinizing hormone (LH) secretion and correlate these with the functional state of the ovary in nonovulating and ovulating females and gestating females with special emphasis in the supposedly ovulating females at midgestation. We investigated P4 and LH serum levels as well as the distribution, localization, and expression of PR and GnRH in the hypothalamus of L. maximus at different time points during gestation and in nongestating, ovulating and nonovulating, females. A significant increment in GnRH, P4, and LH was detected in midpregnant vizcachas with respect to early-pregnant and to ovulating females. PR was also significantly increased in midpregnant animals. PR was detected in neurons of the preoptic and hypothalamic areas. Coexistence of both PR and GnRH in neurons of medial preoptic area and supraoptic nucleus was detected. Midpregnant animals showed increased number of PR immunoreactive cells at median eminence, localized adjacently to GnRH immunoreactive fibers. High expression of hypothalamic GnRH and PR, despite an increased level of P4, was correlated with the presence of antral, preovulatory follicles, and luteinized unruptured follicles at midgestation that suggest a possible role of the H-H-G axis in the modulation of ovulation during gestation in L. maximus.

Maternal Exposure to Dioxin Imprints Sexual Immaturity of the Pups through Fixing the Status of the Reduced Expression of Hypothalamic Gonadotropin-Releasing Hormone

Our previous studies have shown that treatment of pregnant rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 1 μg/kg) at gestational day (GD) 15 reduces the pituitary synthesis of luteinizing hormone (LH) during the late fetal and early postnatal period, leading to the imprinting of defects in sexual behaviors at adulthood. However, it remains unclear how the attenuation of pituitary LH is linked to sexual immaturity. To address this issue, we performed a DNA microarray analysis to identify the gene(s) responsible for dioxin-induced sexual immaturity on the pituitary and hypothalamus of male pups, born of TCDD-treated dams, at the age of postnatal day (PND) 70. Among the reduced genes, we focused on gonadotropin-releasing hormone (GnRH) in the hypothalamus because of published evidence that it has a role in sexual behaviors. An attenuation by TCDD of GnRH expression emerged at PND4, and no subsequent return to the control level was seen. A change in neither DNA methylation nor histone acetylation accounted for the reduced expression of GnRH. Intracerebroventricular infusion of GnRH to the TCDD-exposed pups after reaching maturity restored the impairment of sexual behaviors. Supplying equine chorionic gonadotropin, an LH-mimicking hormone, to the TCDD-exposed fetuses at GD15 resulted in a recovery from the reduced expression of GnRH, as well as from the defects in sexual behavior. These results strongly suggest that maternal exposure to TCDD fixes the status of the lowered expression of GnRH in the offspring by reducing the LH-assisted steroidogenesis at the perinatal stage, and this mechanism imprints defects in sexual behaviors at adulthood.

Mutual interaction of kisspeptin, estrogen and bone morphogenetic protein-4 activity in GnRH regulation by GT1-7 cells

Reproduction is integrated by interaction of neural and hormonal signals converging on hypothalamic neurons for controlling gonadotropin-releasing hormone (GnRH). Kisspeptin, the peptide product of the kiss1 gene and the endogenous agonist for the GRP54 receptor, plays a key role in the regulation of GnRH secretion. In the present study, we investigated the interaction between kisspeptin, estrogen and BMPs in the regulation of GnRH production by using mouse hypothalamic GT1-7 cells. Treatment with kisspeptin increased GnRH mRNA expression and GnRH protein production in a concentration-dependent manner. The expression levels of kiss1 and GPR54 were not changed by kisspeptin stimulation. Kisspeptin induction of GnRH was suppressed by co-treatment with BMPs, with BMP-4 action being the most potent for suppressing the kisspeptin effect. The expression of kisspeptin receptor, GPR54, was suppressed by BMPs, and this effect was reversed in the presence of kisspeptin. It was also revealed that BMP-induced Smad1/5/8 phosphorylation and Id-1 expression were suppressed and inhibitory Smad6/7 was induced by kisspeptin. In addition, estrogen induced GPR54 expression, while kisspeptin increased the expression levels of ERα and ERβ, suggesting that the actions of estrogen and kisspeptin are mutually enhanced in GT1-7 cells. Moreover, kisspeptin stimulated MAPKs and AKT signaling, and ERK signaling was functionally involved in the kisspeptin-induced GnRH expression. BMP-4 was found to suppress kisspeptin-induced GnRH expression by reducing ERK signaling activity. Collectively, the results indicate that the axis of kisspeptin-induced GnRH production is bi-directionally controlled, being augmented by an interaction between ERα/β and GPR54 signaling and suppressed by BMP-4 action in GT1-7 neuron cells.

High glucose impairs gonadotropin-releasing hormone neurons in the preoptic area of the hypothalamus

Rabbits with high fat diet (HFD)-induced metabolic syndrome (MetS) developed hypogonadotropic hypogonadism (HH) and showed a reduced gonadotropin-releasing hormone (GnRH) immunopositivity in the hypothalamus (Filippi et al., 2009). This study investigated the relationship between MetS and hypothalamic alterations in HFD-rabbits. Gonadotropins levels decreased as a function of MetS severity, glucose intolerance, hypothalamic gene expression of glucose transporter 4 (GLUT4) and interleukin-6 (IL-6). HFD significantly induced mRNA expression and hypothalamic immunopositivity of GLUT4 and IL-6, as well as a low-grade hypothalamic inflammation (microglial activation with unchanged astroglia morphology), while reduced hypothalamic immunopositivity for KiSS-1 receptor (KiSS-1R), whose mRNA expression negatively correlated with glucose intolerance. Moreover, high glucose exposure down-regulated KiSS-1/KiSS-1R system and GnRH expression in human GnRH-secreting neuroblasts. A subset of HFD rabbits was treated with the farnesoid-X receptor agonist obeticholic acid, able to ameliorate glucose metabolism (Morelli et al., 2012). This treatment significantly increased GnRH mRNA and reduced GLUT4 and IL-6 immunopositivity. Our results suggest that HFD-induced glucose dysregulation negatively affects GnRH neuron function through an inflammatory injury at the hypothalamic level.

Evidences for the regulation of GnRH and GTH expression by GnIH in the goldfish, Carassius auratus

Gonadotrophin-inhibitory hormone (GnIH) plays an important role in regulating of reproduction in teleosts. To clarify the mode of action of GnIH on the synthesis of gonadotropin releasing hormone (GnRH) and gonadotrophin (GtH), three GnIHR cDNAs were cloned from the goldfish brain. In situ hybridization results showed that GnIHRs were localized to the hypothalamus and pituitary. In the hypothalamus, GnIHRs were found in the NPP, NPO and NLT, whereas sGnRH neurons were reported to be located, and potentially regulated by GnIH. In the pituitary, only two GnIHRs were observed and they were localized to the PI instead of the adenohypophysis where GtH-expressing cells are localized, suggesting indirect regulation of GtH by GnIH. In vivo, intraperitoneal (i.p.) injections of synthetic goldfish GnIH-II peptide and GnIH-III peptide significantly decreased sGnRH and FSHβ mRNA levels. Only GnIH-II decreased LHβ mRNA levels significantly. In vitro, both GnIH-II and GnIH-III showed no effect on GtH synthesis, but an inhibition of GnRH-stimulated LHβ and FSHβ synthesis was observed when GnIH-III was applied to primary pituitary cells in culture. Thus, GnIH could contribute to the regulation of gonadotropin in the brain and pituitary in teleosts.

Androgen receptor repression of gonadotropin-releasing hormone gene transcription via enhancer 1

Gonadotropin-releasing hormone (GnRH) plays a major role in the hypothalamic-pituitary–gonadal (HPG) axis, and synthesis and secretion of GnRH are regulated by gonadal steroid hormones. Disruptions in androgen levels are involved in a number of reproductive defects, including hypogonadotropic hypogonadism and polycystic ovarian syndrome. Androgens down-regulate GnRH mRNA synthesis in vivo and in vitro via an androgen receptor (AR)-dependent mechanism. Methyltrienolone (R1881), a synthetic AR agonist, represses GnRH expression through multiple sites in the proximal promoter. In this study, we show AR also represses GnRH transcription via the major enhancer (GnRH-E1). A multimer of the −1800/−1766 region was repressed by R1881 treatment. Mutation of two bases, −1792 and −1791, resulted in decreased basal activity and a loss of AR-mediated repression. AR bound to the −1796/−1791 sequence in electrophoretic mobility shift assays, indicating a direct interaction with DNA or other transcription factors in this region. We conclude that AR repression of GnRH-E1 acts via multiple AR-responsive regions, including the site at −1792/−1791.

Depletion of enteric gonadotropin-releasing hormone is found in a few patients suffering from severe gastrointestinal dysmotility

Objective. Many patients, especially women, suffer from severe gastrointestinal pain and dysmotility for several years without being diagnosed. Depletion of gonadotropin-releasing hormone (GnRH) in the enteric nervous system (ENS) has been described in some patients. The aim of this study was to examine the expression of GnRH in ENS and antibodies against GnRH in serum, in a dysmotility patient cohort of southern Sweden. Materials and methods.All consecutive patients (n = 35) referred for laparoscopic full-thickness biopsy because of symptoms or signs of severe dysmotility between 1998 and 2009, or patients with a severe dysmotility disorder having had a bowel resection within the time frame, were considered for inclusion. In 22 cases, representative biopsy material containing ganglia was available, and these patients were included. Medical records were scrutinized. The expression of GnRH was determined by immunohistochemistry in bowel biopsies from these patients and in patients with carcinoma or diverticulosis without ENS histopathology. Antibodies against GnRH in serum were determined by ELISA in patients and controls. Results. 14 patients were diagnosed with enteric dysmotility (ED) and 8 with chronic intestinal pseudo-obstruction due to varying etiology. Immunostained biopsies showed expression of GnRH in the ENS. A reduced expression of GnRH-containing neurons was found in 5 patients, as well as antibodies against GnRH in serum. 3 of these patients had a history of in vitro fertilization (IVF) using GnRH analogs. Conclusions.A subgroup of patients with severe dysmotility had a reduced expression of GnRH-containing neurons in the ENS and expressed antibodies against GnRH in serum.

Molecular Cloning and Differential Expression of Three GnRH Genes during Ovarian Maturation of Spotted Halibut, Verasper variegatus

In this study, the gonadotropin-releasing hormone (GnRH) genes in spotted halibut were cloned and sequenced by isolating their cDNAs. The species expressed three molecular forms of GnRH in the brain: chicken-type GnRH-II (cGnRH-II), seabream-type GnRH (sbGnRH), and salmon-type GnRH (sGnRH). Phylogenetic analysis divided the molecular forms of GnRHs into three branches: cGnRH-II branch, sGnRH branch, and fish-specific GnRH branch. The spatial expression showed that they had the highest expression levels in the brain. cGnRH-II was exclusively detected in the brain, while sbGnRH had a global expression pattern in all examined organs. sGnRH was detected in the brain, pituitary, and ovary. The temporal changes of brain GnRH mRNA expression levels were examined during ovarian maturation and postspawning, and the serum steroid hormones and gonadosomatic index (GSI) were recorded. Amounts of sbGnRH mRNA substantially elevated (P < 0.05) during ovarian maturation, which concomitant with considerable elevation of GSI and serum steroids levels. On the contrary, neither sGnRH nor cGnRH-II mRNA levels showed significant changes during ovarian maturation in this study. These results suggested that these three GnRH genes are the important regulators for the differential expression of GnRH in spotted halibut, and would help us better understand the reproductive endocrine mechanism of spotted halibut.

Neonatal dexamethasone exposure down-regulates GnRH expression through the GnIH pathway in female mice

Synthetic glucocorticoid (dexamethasone; DEX) treatment during the neonatal stage is known to affect reproductive activity. However, it is still unknown whether neonatal stress activates gonadotropin-inhibitory hormone (GnIH) synthesizing cells in the dorsomedial hypothalamus (DMH), which could have pronounced suppressive action on gonadotropin-releasing hormone (GnRH) neurons, leading to delayed pubertal onset. This study was designed to determine the effect of neonatal DEX (1.0mg/kg) exposure on reproductive maturation. Therefore, GnRH, GnIH and GnIH receptors, G-protein coupled receptors (GPR) 147 and GPR74 mRNA levels were measured using quantitative real-time PCR in female mice at postnatal (P) days 21, 30 and in estrus stage mice, aged between P45–50. DEX-treated females of P45–50 had delayed vaginal opening, and irregular estrus cycles and lower GnRH expression in the preoptic area (POA) when compared with age-matched controls. The expression levels of GPR147 and GPR74 mRNA in the POA increased significantly in DEX-treated female mice of P21 and P45–50 compared to controls. In addition, GPR147 and GPR74 mRNA expression was observed in laser captured single GnRH neurons in the POA. Although there was no difference in GnIH mRNA expression in the DMH, immunostained GnIH cell numbers in the DMH increased in DEX-treated females of P45–50 compared to controls. Taken together, the results show that the delayed pubertal onset could be due to the inhibition of GnRH gene expression after neonatal DEX treatment, which may be accounted for in part by the inhibitory signals from the up-regulated GnIH–GnIH receptor pathway to the POA.

Role of neuropeptide Y in regulating hypothalamus–pituitary–gonad axis in the rats treated with electro-acupuncture

Neuropeptide Y (NPY) is an important regulator of reproductive axis, which mainly plays some roles in regulating secretion of gonadotropin-releasing hormone (GnRH) in hypothalamus. In previous studies, we found that the repeated low frequency electro-acupuncture (EA) down-regulated hypothalamus-pituitary-gonad (HPG) axis of common rats and rabbits during puberty. In this study, we investigated the role of NPY in regulating the reproductive axis of common rats at different developmental stages and rats treated with the repeated EA. Low frequency EA (3 Hz) was performed at acupoints (treatment groups) or non-acupoints (control groups) for 20 min daily for 10 days in Sprague-Dawley (SD) rats at four developmental stages, which were juvenile stage, early puberty stage, later puberty stage and adult stage. NPY expression in the hypothalamus were determined using RT-PCR and real-time quantitative PCR (qPCR) after 10 days-treatments. The results showed that NPY expression in the early pubertal group (EPG) was significantly depressed after repeated EA (P<0.05). Compared with the results of GnRH expression and body weights, the change of NPY expression was similar with the fluctuation of GnRH expression after EA and the increase of body weights of rats was not influenced by the depression of NPY expression after EA during early puberty. The results demonstrated that repeated low frequency EA was an effective method on down-regulating not only the GnRH expression but also the NPY expression in the hypothalamus without reducing body weights of rats during early puberty.

Excess of leptin inhibits hypothalamicKiSS-1expression in pubertal mice

PurposeLeptin has been considered a link between metabolic state and reproductive activity. Defective reproductive function can occur in leptin-deficient and leptin-excessive conditions. The aim of this study was to examine the effects of centrally injected leptin on the hypothalamic KiSS-1 system in relation to gonadotropin-releasing hormone (GnRH) action in the initial stage of puberty.MethodsLeptin (1 µg) was injected directly into the ventricle of pubertal female mice. The resultant gene expressions of hypothalamic GnRH and KiSS-1 and pituitary LH, 2 and 4 hours after injection, were compared with those of saline-injected control mice. The changes in the gene expressions after blocking the GnRH action were also analyzed.ResultsThe basal expression levels of KiSS-1, GnRH, and LH were significantly higher in the pubertal mice than in the prepubertal mice. The 1-µg leptin dose significantly decreased the mRNA expression levels of KiSS-1, GnRH, and LH in the pubertal mice. A GnRH antagonist significantly increased the KiSS-1 and GnRH mRNA expression levels, and the additional leptin injection decreased the gene expression levels compared with those in the control group.ConclusionThe excess leptin might have suppressed the central reproductive axis in the pubertal mice by inhibiting the KiSS-1 expression, and this mechanism is independent of the GnRH-LH-estradiol feedback loop.

Signaling of Cytokines is Important in Regulation of GnRH Neurons

Cytokines encompass a broad class of peptides that mediate signals in a broad range of physiological situations including inflammation, infection, and obesity. The cytokine receptor-associated tyrosine kinase, Jak2, is one of the most important proteins mediating cytokine signaling pathway activation. Recently, our group has demonstrated that Jak2 signaling in the gonadotropin-releasing hormone (GnRH) neuron plays a critical role in fertility in males and females, implicating cytokine activation of the neuron in GnRH neuronal development and function. To date, the specific cytokine(s) essential for activating Jak2 during neuroendocrine development are not known. In this article, we review the evidence for the role of several class 1 cytokines in regulating GnRH neuronal development, GnRH secretion, and GnRH expression.

Hypothalamic Dysregulation and Infertility in Mice Lacking the Homeodomain Protein Six6

The hypothalamus, pituitary, and gonads coordinate to direct the development and regulation of reproductive function in mammals. Control of the hypothalamic-pituitary-gonadal axis is dependent on correct migration of gonadotropin-releasing hormone (GnRH) neurons from the nasal placode to the hypothalamus, followed by proper synthesis and pulsatile secretion of GnRH, functions absent in patients with hypogonadal hypogonadism. In this study, we identify sine oculis-related homeobox 6 (Six6) as a novel factor necessary for proper targeting of GnRH expression to the limited population of GnRH neurons within the adult mouse hypothalamus and demonstrate that it is required for proper reproductive function in both male and female mice. Female Six6-null mice exhibit a striking decrease in fertility, failing to progress through the estrous cycle normally, show any signs of successful ovulation, or produce litters. Although basal gonadotropin production in these mice is relatively normal, analysis of GnRH expression reveals a dramatic decrease in total GnRH neuron numbers. We show that expression of Six6 is dramatically increased during GnRH neuronal maturation and that overexpression of Six6 induces GnRH transcription in neuronal cells. Finally, we demonstrate that this induction in GnRH expression is mediated via binding of Six6 to evolutionarily conserved ATTA sites located within the GnRH proximal promoter. Together, these data indicate that Six6 plays an important role in the regulation of GnRH expression and hypothalamic control of fertility.