Cloning and expression of gonadotropin-releasing hormone receptor in the brain and pituitary of the European sea bass: an in situ hybridization study

A full-length cDNA encoding a GnRH receptor (GnRH-R) has been obtained from the pituitary of the European sea bass, Dicentrarchus labrax. The complete cDNA is 1814 base pairs (bp) in length and encodes a protein of 416 amino acids. The 5' UTR and 3' UTR are 239 bp and 324 bp in size, respectively. The expression sites of this GnRH-R were studied in the brain and pituitary of sea bass by means of in situ hybridization. A quantitative analysis of the expression of the GnRH-R gene along the reproductive cycle was also performed. The GnRH-R brain expression was especially relevant in the ventral telencephalon and rostral preoptic area. Some GnRH-R messenger-expressing cells were also evident in the dorsal telencephalon, caudal preoptic area, ventral thalamus, and periventricular hypothalamus. A conspicuous and specific GnRH-R expression was detected in the pineal gland. The highest expression of the GnRH-R gene was observed in the proximal pars distalis of the pituitary. This expression was evident in all LH cells and some FSH cells but not in somatotrophs. In the pituitary, the quantitative analysis revealed a higher expression of GnRH-R gene during late vitellogenesis in comparison with maturation, spawning, and postspawning/resting periods. However, in the brain, the highest GnRH-R expression was evident at spawning or postspawning/ resting periods. These results suggest that the expression of this GnRH-R is regulated in a different manner in the brain and the pituitary of sea bass.     

Evidence for different gonadotropin-releasing hormone response sites in rat ovarian and pituitary cells

The participation of type I GnRH receptor (GnRH-R) on GnRH-II-induced gonadotropin secretion in rat pituitary cells was investigated. Furthermore, we extended the study of GnRH-II action to ovarian cells. The GnRH-II was able to mobilize inositol triphosphate (IP(3)) and to induce LH and FSH release in a dose-dependent manner in pituitary cells and in a GnRH-I-like manner. The GnRH-analog 135-18 (agonist for type II GnRH-R and antagonist for type I GnRH-R) was unable to elicit any cellular response tested in these pituitary cells. The GnRH-II responses were blocked by the type I GnRH-R-antagonists CRX or 135-18, suggesting that these effects were mediated by the type I GnRH-R. In contrast to pituitary cells, GnRH-I, but not GnRH-II, elicited an IP(3) response in superovulated ovarian cells; 135-18 also had no effect. However, GnRH-II as well as GnRH-I presented antiproliferative effects on these cells. Surprisingly, 135-18 had stronger antiproliferative effects than either GnRH peptide. The 135-18 analog, but not GnRH-I or GnRH-II, increased progesterone secretion in superovulated ovarian cells. These results strongly suggest that GnRH-II is able to stimulate rat pituitary cells through the type I GnRH-R, with no evidence for the presence of type II GnRH-R. On the other hand, our results indicate a putative GnRH-R in superovulated ovarian cells with response characteristics that differ from those of the GnRH-R in the pituitary.     

Pituitary gonadotropin-releasing hormone (GnRH) receptor: structure, distribution and regulation of expression

Reproduction in mammals is controlled by interactions between the hypothalamus, anterior pituitary and gonads. Interaction of GnRH with its cognate receptor is essential to regulating reproduction. Characterization of the structure, distribution and expression of GnRH receptors (GnRH-R) has furthered our understanding of the physiological consequences of GnRH stimulation of pituitary gonadotropes. Based on the putative topology of the amino acid sequence of the GnRH-R and point mutation studies, key elements of the GnRH-R have been identified to play a role in ligand recognition and binding, G-protein activation and internalization. Normally, reproductive function is mediated by GnRH-R expressed only on the membranes of pituitary gonadotropes. The density of GnRH-R on gonadotropes determines their ability to respond to GnRH. This density is highest just prior to ovulation and likely is important for complete expression of the pre-ovulatory surge of LH. Therefore, knowledge regarding what regulates the density of GnRH-R is essential to understanding changes in pituitary sensitivity to GnRH and ultimately, to expression of the LH surge. Regulation of GnRH-R gene expression is influenced by a multitude of factors including gonadal steroid hormones, inhibin, activin and perhaps most importantly GnRH itself.     

The gonadotrophin-releasing hormone receptor: signalling, cycling and desensitisation

Sustained stimulation of G-protein coupled receptors (GPCRs) typically causes receptor desensitisation that is mediated by phosphorylation, often within the C-terminal tail of the receptor. The consequent binding of beta-arrestin not only prevents the receptor from activating its G-protein (causing desensitisation) but can also target it for internalisation via clathrin-coated vesicles and can mediate signalling to proteins regulating endocytosis and mitogen-activated protein kinase (MAPK) cascades. GnRH acts via phospholipase C coupled GPCRs on pituitary gonadotrophs. The type I GnRH-receptors (GnRH-Rs) found only in mammals, are unique in that they lack C-terminal tails and apparently do not undergo agonist-induced phosphorylation or bind beta-arrestin. They are therefore resistant to receptor desensitisation and internalise slowly. In contrast, the type II GnRH-Rs, found in numerous vertebrates, possess such tails and show rapid desensitisation and internalisation with concomitant receptor phosphorylation (within the C-terminal tails) and/or binding of beta-arrestin. The binding to beta-arrestin may also be important for association with dynamin, a GTPase that controls cleavage of endosomes from the plasma membrane. Using recombinant adenovirus to express GnRH-R, we have found that blockade of dynamin-dependent endocytosis inhibits internalisation of type II (Xenopus) GnRH-Rs but not type I (human) GnRH-Rs, revealing the existence of functionally distinct routes through which these receptors are internalised. Although type I GnRH-R do not rapidly desensitise, sustained activation of GnRH receptors does cause desensitisation of gonadotrophin secretion, an effect which must therefore involve adaptive responses distal to the receptor. One such response is the GnRH-induced down regulation of inositol 1, 4, 5 trisphosphate receptors that apparently underlies desensitisation of Ca2+ mobilisation in a gonadotroph-derived cell line. Although activation of other GPCRs can down-regulate inositol 1, 4, 5 trisphosphate receptors, the effect of GnRH is atypically rapid and pronounced, presumably because of the receptor's atypical resistance to desensitisation. GnRH-Rs are also expressed in several extra-pituitary sites and these may mediate direct inhibition of proliferation of hormone-dependent cancer cells. Infection with type I GnRH-R expressing adenovirus facilitated expression of high affinity, PLC-coupled GnRH-R in mammary and prostate cancer cells and these mediated pronounced antiproliferative effects of receptor agonists. No such effect was seen in cells transfected with a type II GnRH-R, implying that it is mediated most efficiently by a non-desensitising receptor. Thus it appears that the GnRH-Rs have undergone a period of rapidly accelerated molecular evolution that is of functional relevance to GnRH-R signalling in pituitary and extra-pituitary sites.     

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.     

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.     

Molecular characterization of the GHRH/GHRH-R and its effect on GH synthesis and release in orange-spotted grouper (Epinephelus coioides)

Growth hormone-releasing hormone (GHRH) is a hypothalamic neuropeptide that stimulates growth hormone (GH) synthesis and secretion in the pituitary gland. In this paper, the full-length cDNAs of orange-spotted grouper GHRH and its receptor (GHRH-R) were cloned. The grouper GHRH cDNA is 713 bp in length and encodes a 141-aa precursor that includes an 18-aa signal peptide, a 27-aa mature GHRH mature peptide and a 47-aa carboxyl terminus. The grouper GHRH-R cDNA sequence is 1495 bp in length, encoding a 422-aa receptor with seven transmembrane domains. Tissue distribution analyses showed that both GHRH and GHRH-R mRNAs were predominantly expressed in the brain, while the GHRH-R mRNA was also abundantly detected in the pituitary gland. Both GHRH and GHRH-R mRNAs were expressed throughout embryonic development from the multi-cell stage to the newly hatched larvae stage, and the highest GHRH and GHRH-R expressions appeared at the brain vesicle stage and the heart stage, respectively. In vitro studies performed on the grouper pituitary primary cells showed that a synthetic grouper GHRH-NH(2) increased both GH mRNA expression and GH protein release in a dose-dependent manner. Together, these results suggest that the newly obtained grouper GHRH was able to stimulate GH synthesis and release, similar to its mammalian counterparts.     

Expression of the GnRH and GnRH receptor (GnRH-R) genes in the hypothalamus and of the GnRH-R gene in the anterior pituitary gland of anestrous and luteal phase ewes

Data exists showing that seasonal changes in the innervations of GnRH cells in the hypothalamus and functions of some neural systems affecting GnRH neurons are associated with GnRH release in ewes. Consequently, we put the question as to how the expression of GnRH gene and GnRH-R gene in the hypothalamus and GnRH-R gene in the anterior pituitary gland is reflected with LH secretion in anestrous and luteal phase ewes. Analysis of GnRH gene expression by RT-PCR in anestrous ewes indicated comparable levels of GnRH mRNA in the preoptic area, anterior and ventromedial hypothalamus. GnRH-R mRNA at different concentrations was found throughout the preoptic area, anterior and ventromedial hypothalamus, stalk/median eminence and in the anterior pituitary gland. The highest GnRH-R mRNA levels were detected in the stalk/median eminence and in the anterior pituitary gland.During the luteal phase of the estrous cycle in ewes, the levels of GnRH mRNA and GnRH-R mRNA in all structures were significantly higher than in anestrous ewes. Also LH concentrations in blood plasma of luteal phase ewes were significantly higher than those of anestrous ewes.In conclusion, results from this study suggest that low expression of the GnRH and GnRH-R genes in the hypothalamus and of the GnRH-R gene in the anterior pituitary gland, amongst others, may be responsible for a decrease in LH secretion and the anovulatory state in ewes during the long photoperiod.     

Evidence that gonadotropin-releasing hormone (GnRH) II stimulates luteinizing hormone and follicle-stimulating hormone secretion from monkey pituitary cultures by activating the GnRH I receptor

Mammalian gonadotropin-releasing hormone (GnRH) I is the neuropeptide that regulates reproduction. In recent years, a second isoform of GnRH, GnRH II, and its highly selective type II GnRH receptor were cloned and identified in monkey brain, but its physiological function remains unknown. We sought to determine whether GnRH II stimulates LH and FSH secretion by activating specific receptors in primary pituitary cultures from male monkeys. Dispersed pituitary cells were maintained in steroid-depleted media and stimulated with GnRH I and/or GnRH II for 6 h. Cells were also treated with Antide (Bachem, King of Prussia, PA), a GnRH I antagonist, to block gonadotropin secretion. In monkey as well as rat pituitary cultures, GnRH II was a less effective stimulator of LH and FSH secretion than was GnRH I. In both cell preparations, Antide completely blocked LH and FSH release provoked by GnRH II as well as GnRH I. Furthermore, the combination of GnRH I and GnRH II was no more effective than either agonist alone. These results indicate that GnRH II stimulates FSH and LH secretion, but they also imply that this action occurs through the GnRH I receptor. The GnRH II receptors may have a unique function in the monkey brain and pituitary other than regulation of gonadotropin secretion.     

A novel IL-1 receptor, cloned from B cells by mammalian expression, is expressed in many cell types.

cDNA clones corresponding to an Mr approximately 80,000 receptor (type I receptor) for interleukin-1 (IL-1) have been isolated previously by mammalian expression. Here, we report the use of an improved expression cloning method to isolate human and murine cDNA clones encoding a second type (Mr approximately 60,000) of IL-1 receptor (type II receptor). The mature type II IL-1 receptor consists of (i) a ligand binding portion comprised of three immunoglobulin-like domains; (ii) a single transmembrane region; and (iii) a short cytoplasmic domain of 29 amino acids. This last contrasts with the approximately 215 amino acid cytoplasmic domain of the type I receptor, and suggests that the two IL-1 receptors may interact with different signal transduction pathways. The type II receptor is expressed in a number of different tissues, including both B and T lymphocytes, and can be induced in several cell types by treatment with phorbol ester. Both IL-1 receptors appear to be well conserved in evolution, and map to the same chromosomal location. Like the type I receptor, the human type II IL-1 receptor can bind all three forms of IL-1 (IL-1 alpha, IL-1 beta and IL-1ra). Vaccinia virus contains an open reading frame bearing strong resemblance to the type II IL-1 receptor.     

Recombinant perciform GnRH-R activates different signaling pathways in fish and mammalian heterologous cell lines

Perciforms have three forms of gonadotropin-releasing hormone (GnRH) in their brain. All three GnRHs are potent secretogogues for luteinizing hormone (LH) from the pituitary. The pivotal role of GnRH-R-GnRH interactions in reproductive homeostasis is well established; however, there is a paucity of information on how a GnRH-R responds to the three endogenous GnRH forms in a perciform species. In this study, a recombinant pituitary GnRH-R from striped bass (stb) was expressed in a mammalian cell line (COS-7) and a fish cell line (CHSE-214). Activation of the signaling pathways was monitored by reporter gene (luciferase) based assays, which were specific for cAMP-PKA or Ca 2+/calmodulin kinase (activated via c-fos promoter) signaling pathways. The stbGnRH-R expressed in two different cell lines triggered different downstream signaling in response to the treatments with chicken (c) GnRH II. Interestingly, when endogenous GnRHs were used in combinations, the luciferase activity was significantly attenuated in transfected CHSE-214 cells.     

赤点石斑鱼两种芳香化酶cDNA的克隆及其表达的组织特异性

以赤点石斑鱼 (Epinephelusakaara)脑垂体中提取的RNA为模板 ,根据芳香化酶的保守序列设计引物 ,利用GeneRacerTM 技术 ,克隆出两种芳香化酶即脑芳香化酶 (P4 5 0aromB)和性腺芳香化酶 (P4 5 0aromA)的cDNA ,其全长分别为 190 1bp (编码 5 0 9aa)和 1833bp (编码 5 18aa)。序列分析结果表明 ,赤点石斑鱼两种芳香化酶cDNA序列的同源性为 5 1 6 % ,氨基酸序列之间同源性为 6 2 5 % ,与斜带石斑鱼两种芳香化酶氨基酸同源性分别为 94 7%和 97 9%。对 8个科的 10种鱼进行了分子系统进化树分析 ,结果与根据传统的形态学和生化特征分类进化地位基本一致。以特异性引物扩增雌、雄赤点石斑鱼各种组织 (垂体、嗅球、端脑、下丘脑、中脑、后脑、延脑、心脏、肾脏、肝脏、脾脏、性腺、鳃、胃、肠、皮肤、脂肪、肌肉、头肾、胸腺、鳔 ) ,以β actin作内标比较各组织芳香化酶基因表达量的差异 ,结果表明 ,赤点石斑鱼脑芳香化酶 (P4 5 0aromB)有广泛的组织分布 ,脑和垂体的表达量很高 ,各组织表达量有明显的雌、雄差异 ;而性腺芳香化酶 (P4 5 0aromA)表达主要集中于垂体和性腺 ,且不论雌雄 ,其性腺表达量均高于脑垂体 ,和P4 5 0aromB的表达模式明显不同 ,表现为在脑部 ,P4 5 0aromB表达量高于P4 5 0aromA ,而在性腺 ,     

cDNA cloning and mRNA expression of neuropeptide Y in orange spotted grouper, Epinephelus coioides

A full-length cDNA encoding the neuropeptide Y (NPY) was cloned from the hypothalamus of orange spotted grouper (Epinephelus coioides) by rapid amplification of cDNA ends approaches. The NPY cDNA sequence is 688 bp long and has an open reading frame of 300 bp encoding prepro-NPY with 99 amino acids. The deduced amino acid sequences contain a 28-amino-acids signal peptide followed by a 36-amino-acids mature NPY peptide. mRNA expression of NPY was determined using semi-quantitative RT-PCR followed by Southern blot analysis. NPY mRNA was expressed in olfactory bulb, telencephalon, pituitary, hypothalamus, optic tectum-thalamus, medulla oblongata, cerebellum and spinal cord. Low levels of NPY mRNA expression were found in retina, ovary and stomach, while much lower levels of expression were detected in liver, heart, gill, skin, anterior intestine, thymus and blood. No NPY mRNA expression was observed in unfertilized eggs, newly fertilized eggs, 16-cells stage and morula stage of the embryo and lower levels of expression were detected in the blastula, gastrula and neurula stages. It was highly expressed from lens formation stage to 52-day-old larval stage. NPY might be involved in the late embryonic and larval development of the orange spotted grouper.