Signal transduction of the gonadotropin releasing hormone (GnRH) receptor: Cross-talk of calcium,protein kinase C (PKC), and arachidonic acid

Summary 1. The decapeptide neurohormone gonadotropin releasing hormone (GnRH) is the first key hormone of the reproductive system. Produced in the hypothalamus, GnRH is released in a pulsatile manner into the hypophysial portal system to reach the anterior pituitary and stimulates the release and synthesis of the gonadotropin hormones LH and FSH. GnRH, a Ca²⁺ mobilizing ligand, binds to its respective binding protein, which is a member of the seven transmembrane domain receptor family and activates a G-protein (Gq).2. The subunit of Gq triggers enhanced phosphoinositide turnover and the elevation of multiple second messengers required for gonadotropin release and biosynthesis.3. The messenger molecules IP₃, diacylglycerol, Ca²⁺, protein kinase C, arachidonic acid and leukotriene C₄ cross-talk in a complex networks of signaling, culminating in gonadotropin release and gene expression.     

Identification of Human GnIH Homologs,RFRP-1 and RFRP-3, and the Cognate Receptor,GPR147 in the Human Hypothalamic Pituitary Axis

The existence of a hypothalamic gonadotropin-inhibiting system has been elusive. A neuropeptide named gonadotropin-inhibitory hormone (GnIH, SIKPSAYLPLRF-NH₂) which directly inhibits gonadotropin synthesis and release from the pituitary was recently identified in quail hypothalamus. Here we identify GnIH homologs in the human hypothalamus and characterize their distribution and biological activity. GnIH homologs were isolated from the human hypothalamus by immunoaffinity purification, and then identified as MPHSFANLPLRF-NH₂ (human RFRP-1) and VPNLPQRF-NH₂ (human RFRP-3) by mass spectrometry. Immunocytochemistry revealed GnIH-immunoreactive neuronal cell bodies in the dorsomedial region of the hypothalamus with axonal projections to GnRH neurons in the preoptic area as well as to the median eminence. RT-PCR and subsequent DNA sequencing of the PCR products identified human GnIH receptor (GPR147) mRNA expression in the hypothalamus as well as in the pituitary. In situ hybridization further identified the expression of GPR147 mRNA in luteinizing hormone producing cells (gonadotropes). Human RFRP-3 has recently been shown to be a potent inhibitor of gonadotropin secretion in cultured sheep pituitary cells by inhibiting Ca²⁺ mobilization. It also directly modulates GnRH neuron firing. The identification of two forms of GnIH (RFRP-1 and RFRP-3) in the human hypothalamus which targets human GnRH neurons and gonadotropes and potently inhibit gonadotropin in sheep models provides a new paradigm for the regulation of hypothalamic-pituitary-gonadal axis in man and a novel means for manipulating reproductive functions.     

A presumptive role for GABA in the stimulatory effects of Des-Gly10, [D-Ala6]-LHRH-ethylamide and pimozide on the gonadotropin release in carp

To investigate the effect of endogenous gamma-aminobutyric acid (GABA) on the blood maturating gonadotropin (GtH) levels, or to study its interaction with pimozide (dopamine antagonist) and a luteinizing hormone-releasing hormone analog (LHRH-a), sexually mature male and female carps were treated with drugs that may either inhibit GABA biosynthesis or GABA degradation. In females the irreversible inhibitor of GABA-transaminase, gamma-vinyl GABA (GVG), which was to increase the endogenous GABA-ergic tone, had no influence on GtH release. On the other hand, the increased GtH response to the combination of pimozide (PIM) and LHRH-a was clearly enhanced by the administration of 3-mercaptopropionic acid (MPA), an inhibitor of the rate limiting enzyme of GABA-biosynthesis. In males the GABA-ergic compound, valproic acid (DPA) decreased LHRH-a stimulated GtH levels. In male carps that received PIM to diminish the dopaminergic inhibition of GtH release, the spermiating response to LHRH-a was increased by administration of MPA. These data suggest that GABA interacts with the action of dopamine and the gonadotropin releasing hormone (GnRH) on the release of GtH.     

Development of a synaptosomal model to determine drug-induced in vivo changes in GABA-levels of nerve endings in 11 brain regions of the rat

An experimental procedure was developed which allowed the simultaneous measurement of GABA in synaptosomes from 11 regions of one rat brain. Synaptosomal fractions were prepared by conventional subcellular fractionation procedures and characterized by electron microscopy. Post-mortem increases of GABA during removal and dissection of brain tissue, homogenization and fractionation procedures could be sufficiently minimized by rapid processing of the tissue at low temperatures and inclusion of 3-mercaptopropionic acid (1 mM) in the homogenizing medium. Experiments with addition of aminooxyacetic acid (AOOA, 1 mM) to the homogenizing medium indicated that GABA was not being degraded during synaptosome preparation. The presence of exogenous GABA (1 mM) did not alter the GABA levels in the organelles, indicating that no significant redistribution of GABA occurred during subcellular fractionation. On the basis of these findings, it was suggested that synaptosomal fractions could be used as a model to monitor indirectly the drug-induced changes in GABA levels of nerve endings in discrete brain areas of the intact animal. In vivo experiments with AOAA (30 mg/kg i.p.) and valproic acid (VPA, 200 mg/kg i.p.) showed that both drugs caused differential effects on synaptosmal GABA levels in different brain regions. Although AOAA was more potent than VPA in increasing GABA in whole tissue of most brain regions, significant increases of synaptosomal GABA levels after AOAA were only determined in olfactory bulbs and frontal cerebral cortex. In contrast, VPA induced significant synaptosomal GABA increases in olfactory bulbs, hypothalamus, superior and inferior colliculus, substantia nigra, and cerebellum. The data indicate that the synaptosomal model can provide useful information on the in vivo effects of drugs on GABA levels in nerve terminals and their ability to exert this effect in specific brain areas.     

Enhanced Glutamatergic and Decreased Gabaergic Synaptic Appositions to GnRH Neurons on Proestrus in the Rat: Modulatory Effect of Aging

Background

Previous work by our lab and others has implicated glutamate as a major excitatory signal to gonadotropin hormone releasing hormone (GnRH) neurons, with gamma amino butyric acid (GABA) serving as a potential major inhibitory signal. However, it is unknown whether GABAergic and/or glutamatergic synaptic appositions to GnRH neurons changes on the day of the proestrous LH surge or is affected by aging.

Methodology/Principal Findings

To examine this question, synaptic terminal appositions on GnRH neurons for VGAT (vesicular GABA transporter) and VGLUT2 (vesicular glutamate transporter-2), markers of GABAergic and glutamatergic synaptic terminals, respectively, was examined by immunohistochemistry and confocal microscopic analysis in young and middle-aged diestrous and proestrous rats. The results show that in young proestrous rats at the time of LH surge, we observed reciprocal changes in the VGAT and VGLUT2 positive terminals apposing GnRH neurons, where VGAT terminal appositions were decreased and VGLUT2 terminal appositions were significantly increased, as compared to young diestrus control animals. Interestingly, in middle-aged cycling animals this divergent modulation of VGAT and VGLUT2 terminal apposition was greatly impaired, as no significant differences were observed between VGAT and VGLUT2 terminals apposing GnRH neurons at proestrous. However, the density of VGAT and VGLUT2 terminals apposing GnRH neurons were both significantly increased in the middle-aged animals.

Conclusions/Significance

In conclusion, there is an increase in glutamatergic and decrease in GABAergic synaptic terminal appositions on GnRH neurons on proestrus in young animals, which may serve to facilitate activation of GnRH neurons. In contrast, middle-aged diestrous and proestrous animals show a significant increase in both VGAT and VGLUT synaptic terminal appositions on GnRH neurons as compared to young animals, and the cycle-related change in these appositions between diestrus and proestrus that is observed in young animals is lost.     

Application of cryosubstitution in neurohormone-and neurotransmitter-immunocytochemistry

Summary Pituitaries of the African catfish, Clarias gariepinus, were prefixed in aldehyde fixatives, frozen in liquid propane and submitted to a cryosubstitution procedure. Ultrathin sections of the Lowicryl HM20-embedded tissue were treated with primary antisera raised in rabbits to gonadotropin releasing hormone (GnRH), vasopressin or gamma amino butyric acid (GABA) respectively. Binding of the primary antisera was visualized with goat anti-rabbit (GAR) labeled with gold. The general morphology of the tissue components in the cryosubstituted pituitaries matches with that obtained after routine embedding procedures. In addition, a strong labeling intensity of the neuropeptides/neurotransmitters investigated in the present study was demonstrated. Due to these qualities cryosubstitution provides optimal conditions for studying co-localization of neurosecretory products, using double-immunostaining procedures. In the pars distalis of the catfish pituitary several types of hypothalamus-derived nerve fibers are present between or synapting on the secretory cells. It is demonstrated that the two known catfish GnRHs are co-localized in the same nerve fiber and within these nerve fibers even co-exist in the same neurosecretory granules. GABA and vasopressin-immunolabeling each occurred in different nerve fibers. The present data demonstrate that cryosubstitution and low temperature-embedding results in an excellent morphological preservation compared to ultracryotomy and a better preserved immunoreactivity of small antigenic molecules in comparison to conventional fixation and embedding techniques.     

Molecular dissection of valproic acid effects in acute myeloid leukemia identifies predictive networks

Histone deacetylase inhibitors (HDACIs) like valproic acid (VPA) display activity in leukemia models and induce tumor-selective cytotoxicity against acute myeloid leukemia (AML) blasts. As there are limited data on HDACIs effects, we aimed to dissect VPA effects in vitro using myeloid cell lines with the idea to integrate findings with in vivo data from AML patients treated with VPA additionally to intensive chemotherapy (n = 12). By gene expression profiling we identified an in vitro VPA response signature enriched for genes/pathways known to be implicated in cell cycle arrest, apoptosis, and DNA repair. Following VPA treatment in vivo, gene expression changes in AML patients showed concordant results with the in vitro VPA response despite concomitant intensive chemotherapy. Comparative miRNA profiling revealed VPA-associated miRNA expression changes likely contributing to a VPA-induced reversion of deregulated gene expression. In addition, we were able to define markers predicting VPA response in vivo such as CXCR4 and LBH. These could be validated in an independent cohort of VPA and intensive chemotherapy treated AML patients (n = 114) in which they were inversely correlated with relapse-free survival. In summary, our data provide new insights into the molecular mechanisms of VPA in myeloid blasts, which might be useful in further advancing HDAC inhibition based treatment approaches in AML.     

Regulation of gonadotropin-releasing hormone (GnRH) gene expression in hypothalamic neuronal cells

Summary 1. Gonadotropin-releasing hormone (GnRH) is the hypothalamic releasing factor that controls pituitary gonadotropin subunit gene expression and indirectly gametogenesis and steroidogenesis from the gonad, which results in reproductive competence.2. GnRH is synthesized in only about 1000 neurons in the hypothalamus and released in an episodic fashion down the median eminence to regulate gonadotropin biosynthesis.3. Although much is known about the secretory dynamics of GnRH release, little is known about the pretranslational control of GnRH biosynthesis due to lack of appropriate model systems. The recent availability of immortalized neuronal cell lines that produce GnRH allows investigators for the first time to begin to dissect the factors that directly regulate GnRH gene expression.4. This article reviews the current state of knowledge concerning the mechanisms that direct tissue-specific and peptide hormone control of GnRH biosynthesis.     

The hypogonadotropic state of the prepubertal male rhesus monkey (Macaca mulatta) is not associated with a decrease in hypothalamic gonadotropin-releasing hormone content

Hypothalamic contents of gonadotropin-releasing hormone (GnRH) in neonatally orchidectomized infant, juvenile, and adult monkeys were measured by a radioimmunoassay (RIA) and by an in vivo bioassay that utilized luteinizing hormone (LH) secretion in estrogen- and progesterone-treated ovariectomized rats. The results of the bioassay provided no evidence to suggest that hypothalamic GnRH content in juvenile monkeys (mean = 83 ng/hypothalamus; n = 3) was less than that in infants (mean = 54 ng/hypothalamus; n = 4) and adults (mean = 36 ng/hypothalamus; n = 3). A similar developmental pattern in hypothalamic GnRH content was also observed when the decapeptide was measured by RIA. In striking contrast to the maintenance of hypothalamic GnRH content throughout postnatal development, pituitary gonadotropin contents and serum gonadotropin concentrations were markedly reduced in juvenile monkeys.     

The function and the expression of forebrain GABA(A) receptors change with hormonal state in the adult mouse.

Neurotransmission mediated by gamma-aminobutyric acid type A (GABA(A)) receptors in the mammalian medial preoptic area (mPOA) plays a pivotal role in the expression of hormone-sensitive behaviors. Hand in hand with GABAergic control of reproduction, hormone treatments that activate gonadal steroid signaling pathways in gonadectomized rats are known to regulate the expression of specific GABA(A) receptor subunit mRNAs. While the effects of exogenous hormone treatments have been well documented, little information is available as to how GABA(A) receptor-mediated transmission in the mPOA is altered by endogenous changes in hormonal state in gonadally-intact adult animals or if those changes can be ascribed to hormone-dependent changes in receptor subunit composition. In the present study, we found that both the peak amplitudes of GABA(A) receptor-mediated synaptic currents in the mPOA, as well as the ability of the endogenous neurosteroids to modulate those currents, varied as a function of the estrous cycle. Moreover, we found that the degree of neurosteroid modulation was also significantly different between wild-type and the androgen-insensitive testicular feminization (Tfm) mutant male mice. Semiquantitative RT-PCR analysis performed to assess levels of GABA(A) receptor subunit mRNAs indicated that levels of specific subunits varied over the course of the estrous cycle and between wild-type and Tfm male mice. The variations in GABA(A) receptor expression and function in the mPOA that are associated with differences in gonadal steroid signaling may contribute to the dynamic nature of GABAergic control of neuroendocrine pathways.     

蒙药乌力吉-18对大鼠下丘脑-垂体-卵巢轴相关激素及受体表达的影响

目的:探讨蒙药乌力吉-18对大鼠下丘脑-垂体-卵巢轴相关激素及受体的影响。方法:选取40只健康雌性未孕SD大鼠,随机分为空白组、对照组、乌力吉-18高、低2个剂量组,每组10只。空白组灌胃等体积蒸馏水,对照组灌胃逍遥丸,高、低剂量组分别灌胃2.0 g·kg^-1·d^-1、1.0 g·kg^-1·d^-1乌力吉-18,连续给药31学艺术d。采用酶联免疫吸附法测定血清促性腺激素释放激素(GnRH)、促卵泡生成素(FSH)、黄体生成素(LH)、雌二醇(E₂)及孕酮(PROG)的含量;免疫组化法检测下丘脑组织促性腺激素释放激素(GnRH)、垂体组织促性腺激素释放激素受体(GnRHR)的表达;以蛋白免疫印迹技术检测卵巢组织促卵泡生成素受体(FSHR)、黄体生成素受体(LHR)蛋白表达量。以实时荧光定量PCR检测卵巢组织中FSHR、LHR基因表达量。结果:与空白组比较,乌力吉-18低剂量组可明显升高血清LH含量(P<0.05),上调下丘脑组织GnRH、垂体组织GnRHR表达及卵巢组织FSHR、LHR蛋白表达(P<0.05);乌力吉-18高剂量组可显著升高血清FSH、LH、E₂含量(P<0.05),上调下丘脑组织GnRH表达及卵巢组织FSHR表达量(P<0.05),并可显著升高卵巢组织中FSHR、LHR基因表达量(P<0.05);对照组可明显升高血清E₂含量(P<0.05)。结论:蒙药乌力吉-18可明显升高血清FSH、LH及E₂的含量,促进下丘脑组织GnRH、垂体组织GnRHR及卵巢组织中FSHR、LHR的表达,表明乌力吉-18能够对下丘脑-垂体-卵巢轴相关激素及受体表达产生影响。     

Role of LPXRFamide peptide in the neuroendocrine regulation of reproduction in fish

The decapeptide gonadotropin-releasing hormone (GnRH) is the primary factor responsible for the hypothalamic control of gonadotropin (GTH) secretion. This review focuses on a family of neuropeptides, LPXRFamide (LPXRFa) peptides, which have been implicated in the regulation of GTH secretion. LPXRFa acts on the pituitary via a G protein-coupled receptor, LPXRFa-R, to enhance gonadal development and maintenance by increasing gonadotropin release and synthesis. Because LPXRFa exists and functions in several fish species, LPXRFa is considered to be a key neurohormone in fish reproduction control. The precursors to LPXRFamide peptides encoded plural LPXRFamide peptides and were highly divergent in vertebrates, particularly in lower vertebrates. Tissue distribution analyses indicated that LPXRFamide peptides were highly concentrated in the hypothalamus and other brainstem regions. In view of the localization and expression of LPXRFamide peptides in the hypothalamo-hypophysial system, LPXRFamide peptide in fish increase GTH release in vitro and in vivo. This review summarizes the advances made in our understanding of the biosynthesis, mode of action and functional significance of LPXRFa, a newly discovered key neurohormone.     

Effects of GABA(A) receptor modulation on the expression of GnRH gene and GnRH receptor (GnRH-R) gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland of follicular-phase ewes

The effect of prolonged, intermittent infusion of GABA(A) receptor agonist (muscimol) or GABA(A) receptor antagonist (bicuculline) into the third cerebral ventricle on the expression of GnRH gene and GnRH-R gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland was examined in follicular-phase ewes by real-time PCR. The activation or inhibition of GABA(A) receptors in the hypothalamus decreased or increased the expression of GnRH and GnRH-R genes and LH secretion, respectively. The present results indicate that the GABAergic system in the hypothalamus of follicular-phase ewes may suppress, via hypothalamic GABA(A) receptors, the expression of GnRH and GnRH-R genes in this structure. The decrease or increase of GnRH-R mRNA in the anterior pituitary gland and LH secretion in the muscimol- or bicuculline-treated ewes, respectively, is probably a consequence of parallel changes in the release of GnRH from the hypothalamus activating GnRH-R gene expression. It is suggested that GABA acting through the GABA(A) receptor mechanism on the expression of GnRH gene and GnRH-R gene in the hypothalamus may be involved in two processes: the biosynthesis of GnRH and the release of this neurohormone in the hypothalamus.     

Inhibition of tumor-stromal interaction through HGF/Met signaling by valproic acid

Hepatocyte growth factor (HGF), which is produced by surrounding stromal cells, including fibroblasts and endothelial cells, has been shown to be a significant factor responsible for cancer cell invasion mediated by tumor-stromal interactions. We found in this study that the anti-tumor agent valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, strongly inhibited tumor-stromal interaction. VPA inhibited HGF production in fibroblasts induced by epidermal growth factor (EGF), platelet-derived growth factor, basic fibroblast growth factor, phorbol 12-myristate 13-acetate (PMA) and prostaglandin E₂ without any appreciable cytotoxic effect. Other HDAC inhibitors, including butyric acid and trichostatin A (TSA), showed similar inhibitory effects on HGF production stimulated by various inducers. Up-regulations of HGF gene expression induced by PMA and EGF were also suppressed by VPA and TSA. Furthermore, VPA significantly inhibited HGF-induced invasion of HepG2 hepatocellular carcinoma cells. VPA, however, did not affect the increases in phosphorylation of MAPK and Akt in HGF-treated HepG2 cells. These results demonstrated that VPA inhibited two critical processes of tumor-stromal interaction, induction of fibroblastic HGF production and HGF-induced invasion of HepG2 cells, and suggest that those activities serve for other anti-tumor mechanisms of VPA besides causing proliferation arrest, differentiation, and/or apoptosis of tumor cells.     

Induction of PER1 mRNA expression in immortalized gonadotropes by gonadotropin‐releasing hormone (GnRH): Involvement of protein kinase C and MAP kinase signaling

The initiation and maintenance of reproductive function in mammals is critically dependent on the pulsatile secretion of gonadotropin‐releasing hormone (GnRH). This peptide drives the pulsatile release of FSH and LH from the pituitary pars distalis via signaling pathways that are activated by the type I GnRH receptor (GnRH‐R). Recently, a microarray analysis study reported that a number of genes, including mPer1, are induced by GnRH in immortalized gonadotrope cells. In view of these data, we have begun to analyze in detail the signaling pathways mediating the action of GnRH on mPer1 expression in these cells. Using quantitative real‐time polymprose cho read (PCR), we could confirm that exposure of immortalized gonadotropes (LβT2 cells) to the GnRH analog, buserelin, markedly induces mPer1 (but not mPer2) expression. Consistent with GnRH receptor signaling via the protein kinase (PK)‐C pathway, exposure of the cells to phorbol 12,13‐dibutyrate rapidly elevates both mPer1 and LHβ subunit mRNA levels, while pharmacological inhibition of PKC prevents the mPer1 and LHβ response to buserelin. As GnRH is known to regulate gonadotropin synthesis via activation of p42/44 mitogen‐activated protein kinase (MAPK) signaling pathways, we then examined the involvement of this pathway in regulating mPer1 expression in gonadotropes. Our data reveal that GnRH‐induced mPer1 expression is blocked following acute exposure to a MAPK kinase inhibitor. Although the involvement of this signaling mechanism in the regulation of mPer1 is known in neurons, e.g., in the suprachiasmatic nuclei, the induction of mPer1 in gonadotropes represents a novel mechanism of GnRH signaling, whose functional significance is still under investigation.     

Mammalian gonadotropin-releasing hormone (GnRH) receptor subtypes

Mammalian gonadotropin-releasing hormone (GnRH I) is a hypothalamic decapeptide that governs gonadotropin secretion through interaction with its seven transmembrane (7TM), G protein-coupled receptor (GPCR) expressed by anterior pituitary cells. A second decapeptide, GnRH II, originally discovered in the chicken hypothalamus was recently reported to be expressed in the mammalian hypothalamus as well. A search of the recently-sequenced human genome identified a 7TM/GPCR on chromosome 1 that exhibited a higher identity with non-mammalian vertebrate GnRH II receptors (55%) than with the human GnRH I receptor (39%). Molecular cloning and nucleotide sequencing of this putative GnRH II receptor cDNA from monkey pituitary gland revealed a 379 amino acid receptor that, unlike the GnRH I receptor, possessed a C-terminal tail. Heterologous expression and functional testing of the receptor in COS-1 cells confirmed its identity as a GnRH II receptor: measurement of 3H-inositol phosphate accumulation revealed EC(50)s for GnRH II of 0.86 nM and for GnRH I of 337 nM. Ubiquitous tissue expression of GnRH II receptor mRNA was observed using a human tissue RNA expression array and a 32P-labeled antisense riboprobe representing the 7TM region of human GnRH II receptor cDNA. As predicted by the presence of its C-terminal tail, the GnRH II receptor was desensitized by GnRH II treatment whereas the naturally tail-less GnRH I receptor was not desensitized by GnRH I. Pharmacological analysis of the GnRH II receptor revealed that GnRH I 'superagonists' were more potent than GnRH I but less potent than GnRH II. Numerous GnRH I antagonists showed neither antagonistic nor agonistic activity with the GnRH II receptor. The functions of the GnRH II receptor are unknown but may include regulation of gonadotropin secretion, female sexual behavior, or tumor cell growth.     

GnRH在性成熟高白鲑神经系统及性腺中的分布定位

摘要：应用免疫组织化学方法,系统观察性成熟期高白鲑(Coregonus peled)神经系统及性腺中的促性腺激素释放激素( GnRH)的分布情况。结果表明,GnRH在大脑、小脑、中脑、脊髓、延髓中免疫阳性反应明显,且主要分布在神经元内。GnRH免疫阳性细胞在卵巢和精巢中均有分布,而且其阳性部位在卵巢主要分布于小生长期卵...