Four gonadotropin releasing hormone receptor genes in Atlantic cod are differentially expressed in the brain and pituitary during puberty.

Gonadotropin releasing hormones (GnRH) are an important part of the brain-pituitary-gonad axis in vertebrates. GnRH binding to its receptors (GnRH-R) stimulates synthesis and release of gonadotropins in the pituitary. GnRH-Rs also mediate other processes in the central nervous system such as reproductive behavior and neuromodulation. As many as five GnRH-R genes have been identified in two teleost fish species, but the function and phylogenetic relationship of these receptors is not fully understood. To gain a better understanding of the functional relationship between multiple GnRH-Rs in an important aquaculture species, the Atlantic cod (Gadus morhua), we identified four GnRH-Rs (gmGnRH-R) by RT-PCR, followed by full-length cloning and sequencing. The deduced amino acid sequences were used for phylogenetic analysis to identify conserved functional motifs and to clarify the relationship of gmGnRH-Rs with other vertebrate GnRH-Rs. The function of GnRH-R variants was investigated by quantitative PCR gene expression analysis in the brain and pituitary of female cod during a full reproductive cycle and in various peripheral tissues in sexually mature fish. Phylogenetic analysis revealed two types of teleost GnRH-Rs: Type I including gmGnRH-R1b and Type II including gmGnRH-R2a, gmGnRH-R2b and gmGnRH-R2c. All four gmGnRH-Rs are expressed in the brain, and gmGnRH-R1b, gmGnRH-R2a and gmGnRH-R2c are expressed in the pituitary. The only GnRH-R differentially expressed in the pituitary during the reproductive cycle is gmGnRH-R2a such that its expression is significantly increased during spawning. These data suggest that gmGnRH-R2a is the most likely candidate to mediate the hypophysiotropic function of GnRH in Atlantic cod.

Alternative splicing of a single precursor mRNA generates two subtypes of Gonadotropin-Releasing Hormone receptor orthologues and their variants in the bivalve mollusc Crassostrea gigas.

Despite their economic importance, only very little information is available regarding (neuro)endocrine regulation of reproduction in bivalve molluscs. To gain insights into the molecular control of gonadic development of these animals, G protein-coupled receptors (GPCR) specifically expressed in the gonad of the pacific oyster Crassostrea gigas were investigated. One such receptor, Cg-GnRH-R, an oyster GPCR orthologue of vertebrate GnRH receptors clearly involved in the control of oyster gametogenesis was first identified [Rodet, F., Lelong, C., Dubos, M.P., Costil, K. and Favrel, P., 2005. Molecular cloning of a molluscan Gonadotropin-Releasing Hormone receptor orthologue specifically expressed in the gonad. Biochim Biophys Acta 1730 187-95.]. We report here the characterization of multiple transcripts encoding GnRH-R orthologues (Cg-GnRH-RII-L/Cg-GnRH-RII-S) including a truncated receptor (Cg-GnRH-R-TF) and demonstrate they are generated by the alternative splicing of a single mRNA precursor. The differential structure of these receptors suggests that Cg-GnRH-R on one hand and Cg-GnRH-RII-L/Cg-GnRH-RII-S on the other hand constitute two receptor subtypes with regard to ligand specificity. Pattern of expression of these transcripts suggests that Cg-GnRH-R cognate ligand is specifically involved in the control of gametogenesis while Cg-GnRH-RII-L and Cg-GnRH-RII-S ones likely do not control reproductive functions specifically. Hypothesis on the involvement of this family of receptors in signalling both GnRH and APGWamide in molluscs is discussed.

Functions of a GnRH receptor heterodimer of the ascidian, Ciona intestinalis.

Gonadotropin-releasing hormone (GnRH) is a ten-amino acid peptide hormone that plays pivotal roles in reproduction in vertebrates and octopus. Recently, six GnRH forms (t-GnRH-3-8) and four GnRH receptor subtypes (Ci-GnRHR-1-4) were identified in the protochordate, Ciona intestinalis. In this study, we show the functional modulation of Ci-GnRHR-1 via heterodimerization with the orphan receptor subtype, Ci-GnRHR-4. The dimerization between Ci-GnRHR-1 and R-4 was detected by co-immunoprecipitation and immunoblot analysis. Binding assays confirmed the binding of t-GnRHs to Ci-GnRHR-1 but not to R-4, and verified no alternation in ligand-binding affinity between Ci-GnRHR-1 homodimer and Ci-GnRHRI&4 heterodimer. The heterodimer was found to stimulate the elevation of intracellular calcium, time-extension of ERK phosphorylation, and up-regulation of cell proliferation, all in a ligand specific manner, compared with the Ci-GnRHR-1 homodimer. In combination, these results indicated that Ci-GnRHR-4 is not an inactive receptor, but a modulatory factor for Ci-GnRHR-1 in C. intestinalis.

Comparative analysis of the pituitary and ovarian GnRH systems in the leopard gecko: signaling crosstalk between multiple receptor subtypes in ovarian follicles.

GnRH regulates reproductive functions through interaction with its pituitary receptor in vertebrates. The present study demonstrated that the leopard gecko possessed two and three genes for GnRH ligands and receptors, respectively, though one of the three receptor subtypes had long been thought not to exist in reptiles. Each receptor subtype showed a distinct pharmacology. All types of ligands and receptors showed different expression patterns, and were widely expressed both inside and outside the brain. This report also shows a comparison of the pituitary and ovarian GnRH systems in the leopard gecko during and after the egg-laying season. All three receptor subtypes were expressed in both the whole pituitary and ovary; however, only one receptor subtype could be detected in the anterior pituitary gland. In situ hybridization showed spatial expression patterns of ovarian receptors, and suggested co-expression of multiple receptor subtypes in granulosa cells of larger follicles. Co-transfection of receptor subtypes showed a distinct pharmacology in COS-7 cells compared with those of single transfections. These results suggest that distinct signaling mechanisms are involved in the pituitary and ovarian GnRH systems. Seasonal and developmental variations in receptor expression in the anterior pituitary gland and ovarian follicles may contribute to the seasonal breeding of this animal.

Terminal nerve gonadotrophin-releasing hormone (GnRH) neurones express multiple GnRH receptors in a teleost, the dwarf gourami (Colisa lalia).

Gonadotophin-releasing hormone (GnRH) peptide released from the terminal nerve (TN)-GnRH neurones of the dwarf gourami primarily modifies the electrical properties of various neurones, including the TN-GnRH neurones themselves. However, our knowledge on the expression of GnRH receptors (GnRHRs) in the TN-GnRH neurones is still limited. Here, we used the single-cell reverse transcriptase-polymerase chain reaction after whole-cell patch-clamp recording to study the distribution of various GnRHR types expressed in the individual TN-GnRH neurones. We found that TN-GnRH neurones express two of the three types of GnRHRs cloned in the dwarf gourami: GnRHR1-2 and -R2, but not -R1-1. Furthermore, in agreement with our previous findings, all TN-GnRH neurones contained mRNAs of salmon GnRH but not chicken GnRH-II.

Differential social regulation of two pituitary gonadotropin-releasing hormone receptors.

In many vertebrates, social interactions regulate reproductive capacity by altering the activity of the hypothalamic-pituitary-gonadal (HPG) axis. To better understand the mechanisms underlying social regulation of reproduction, we investigated the relationship between social status and one main component of the HPG axis: expression levels of gonadotropin-releasing hormone receptor (GnRH-R). Social interactions dictate reproductive capacity in the cichlid fish Astatotilapia burtoni. Reproductively active territory holders suppress the HPG axis of non-territorial males through repeated aggressive encounters. To determine whether the expression of GnRH-R is socially regulated, we quantified mRNA levels of two GnRH-R variants in the pituitaries and brains of territorial (T) and non-territorial (NT) A. burtoni males. We found that T males had significantly higher levels of pituitary GnRH-R1 mRNA than NT males. In contrast, GnRH-R2 mRNA levels in the pituitary did not vary with social status. Pituitaries from both T and NT males expressed significantly higher mRNA levels of GnRH-R1 than GnRH-R2. GnRH mRNA levels in the brain correlated positively with GnRH-R1 mRNA levels in the pituitary but did not correlate with pituitary GnRH-R2. Measurements of GnRH-R1 and GnRH-R2 mRNA levels across the whole brain revealed no social status differences. These results show that, in addition to the known effects of social status on other levels of the HPG axis, GnRH receptor in the pituitary is also a target of social regulation.

Urbilaterian origin of paralogous GnRH and corazonin neuropeptide signalling pathways.

Gonadotropin-releasing hormone (GnRH) is a key regulator of reproductive maturation in humans and other vertebrates. Homologs of GnRH and its cognate receptor have been identified in invertebrates-for example, the adipokinetic hormone (AKH) and corazonin (CRZ) neuropeptide pathways in arthropods. However, the precise evolutionary relationships and origins of these signalling systems remain unknown. Here we have addressed this issue with the first identification of both GnRH-type and CRZ-type signalling systems in a deuterostome-the echinoderm (starfish) Asterias rubens. We have identified a GnRH-like neuropeptide (pQIHYKNPGWGPG-NH2) that specifically activates an A. rubens GnRH-type receptor and a novel neuropeptide (HNTFTMGGQNRWKAG-NH2) that specifically activates an A. rubens CRZ-type receptor. With the discovery of these ligand-receptor pairs, we demonstrate that the vertebrate/deuterostomian GnRH-type and the protostomian AKH systems are orthologous and the origin of a paralogous CRZ-type signalling system can be traced to the common ancestor of the Bilateria (Urbilateria).

Five gonadotrophin-releasing hormone receptors in a teleost fish: isolation, tissue distribution and phylogenetic relationships.

Gonadotrophin-releasing hormone (GnRH) is the main neurohormone controlling gonadotrophin release in all vertebrates, and in teleost fish also of growth hormone and possibly of other adenohypophyseal hormones. Over 20 GnRHs have been identified in vertebrates and protochoordates and shown to bind cognate G-protein couple receptors (GnRHR). We have searched the puffer fish, Fugu rubripes, genome sequencing database, identified five GnRHR genes and proceeded to isolate the corresponding complementary DNAs in European sea bass, Dicentrachus labrax. Phylogenetic analysis clusters the European sea bass, puffer fish and all other vertebrate receptors into two main lineages corresponding to the mammalian type I and II receptors. The fish receptors could be subdivided in two GnRHR1 (A and B) and three GnRHR2 (A, B and C) subtypes. Amino acid sequence identity within receptor subtypes varies between 70 and 90% but only 50-55% among the two main lineages in fish. All European sea bass receptor mRNAs are expressed in the anterior and mid brain, and all but one are expressed in the pituitary gland. There is differential expression of the receptors in peripheral tissues related to reproduction (gonads), chemical senses (eye and olfactory epithelium) and osmoregulation (kidney and gill). This is the first report showing five GnRH receptors in a vertebrate species and the gene expression patterns support the concept that GnRH and GnRHRs play highly diverse functional roles in the regulation of cellular functions, besides the "classical" role of pituitary function regulation.

Evidence that the type-2 gonadotrophin-releasing hormone (GnRH) receptor mediates the behavioural effects of GnRH-II on feeding and reproduction in musk shrews.

Gonadotrophin-releasing hormone (GnRH) is a regulatory neuropeptide of which there are multiple structural variants. In mammals, a hypothalamic form (GnRH-I) controls gonadotrophin secretion whereas a midbrain form (GnRH-II) appears to have a neuromodulatory role affecting feeding and reproduction. In female musk shrews and mice, central administration of GnRH-II reinstates mating behaviour previously inhibited by food restriction. In addition, GnRH-II treatment also decreases short-term food intake in musk shrews. GnRH-II can bind two different mammalian GnRH receptors (type-1 and type-2), and thus it is unclear which receptor subtype mediates the behavioural effects of this peptide. Adult female musk shrews implanted with i.c.v. cannula were food restricted or fed ad lib and then tested for sexual behaviour or food intake. One hour before testing, animals were pretreated with vehicle or Antide, a potent type-1 GnRH receptor antagonist (at a dose that blocks GnRH-I or -II mediated ovulation). Twenty minutes before testing, females were infused a second time with either GnRH-II or vehicle. Additional females were tested after an infusion of 135-18, a type-1 receptor antagonist that displays agonist actions at the primate type-2 receptor. GnRH-II treatment increased sexual behaviour in underfed female shrews; pretreatment with Antide did not block this action, suggesting that the effects of GnRH-II are not mediated via the type-1 receptor. Similarly, the inhibitory effects of GnRH-II on short-term food intake were not prevented by pretreatment with Antide. The behavioural effects of the type-2 receptor agonist 135-18 were similar to those seen in GnRH-II-treated females, with 135-18 promoting sexual behaviour and decreasing food intake. Collectively, these results indicate that GnRH-II does not act via the type-1 GnRH receptor to regulate mammalian behaviour but likely activates the type-2 GnRH receptor.

Seasonal variation in the expression of five subtypes of gonadotropin-releasing hormone receptor genes in the brain of masu salmon from immaturity to spawning.

Seasonal variation in the expression of five subtypes of gonadotropin-releasing hormone receptor (GnRH-R) genes, designated as msGnRH-R1, -R2, -R3, -R4, and -R5, was examined in the brain of masu salmon (Oncorhynchus masou). In addition, responses of these genes to GnRH were examined in a GnRH analog (GnRHa) implantation experiment. Brain samples were collected one week after the implantation every month from immaturity through spawning. The absolute amount of GnRH-R mRNA in single forebrains was determined by real-time PCR assays. Among the five genes, R4 and R5 were dominantly expressed in both sexes. R1, R4, and R5 mRNAs showed similar changes throughout the experimental period in both sexes. Levels tended to be high in winter and low in the pre-spawning season, followed by elevations in the spawning period. The mRNA levels had weak to moderate negative correlations with the plasma level of estradiol-17beta (E2) in females. The effects of GnRHa on msGnRH-R mRNAs were not apparent for all the subtypes. These results indicate that the msGnRH-R1, -R4, and -R5 genes are synchronously expressed during sexual maturation. There was a trend toward decreased levels of their expression prior to the spawning period and then increased levels at spawning, possibly causing GnRH target neurons to sensitize to a GnRH stimulus. Furthermore, E2 may be involved in msGnRH-R gene expression in the brain of female masu salmon during sexual maturation.

Differential desensitization and internalization of three different bullfrog gonadotropin-releasing hormone receptors.

We previously demonstrated the presence of three distinct types of the gonadotropin-releasing hormone receptor (GnRHR) in a bullfrog (denoted bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3). The bfGnRHRs exhibited differential tissue distribution and ligand selectivity. In the present study, we demonstrated the desensitization and internalization kinetics of these receptors in both transiently-transfected HEK293 cells and retrovirus-mediated stable cells. The time-course accumulation of the inositol phosphate in response to GnRH revealed that bfGnRHR-1 and -2 were rapidly desensitized, whereas bfGnRHR-3 was slowly desensitized. A comparison of the internalization kinetics revealed the most rapid rate and highest extent of internalization of bfGnRHR-2 among the three receptors. Interestingly, the mechanisms that underlie the receptor internalization appear to differ from each other. Internalization of bfGnRHR-1 was dependent on both dynamin and beta-arrestin, whereas those of bfGnRHR-2 and -3 were dependent on dynamin, but not on arrestin. These results, therefore, suggest that differential regulatory mechanisms for desensitization and internalization of the GnRHR are involved in diverse cellular and physiological responses to GnRH stimulation.

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.

Bisphenol A affects gene expression of gonadotropin-releasing hormones and type I GnRH receptors in brains of adult rare minnow Gobiocypris rarus.

Recent studies support the notion that endocrine disrupting chemicals (EDCs) could affect the reproductive regulations of the neuroendocrine system. The objectives of the present study were to determine whether the weak estrogenic chemical, bisphenol A (BPA), disrupts gonadotropin-releasing hormone (GnRH) system by altering the transcription of GnRHs and GnRH receptor (GnRHR) genes in adult rare minnow Gobiocypris rarus. In the present study, the histological examination of the ovary after 35-day BPA exposure at 15 µg/L demonstrated the perturbing effects of environmentally relevant BPA on the ovarian development in G. rarus. In addition mRNA expression of ovarian P450 aromatase in both ovaries and testes were significantly down-regulated by 15 µg/L BPA. GnRH2, GnRH3, GnRHR1A and GnRHR1B gene were identified in G. rarus. The expression patterns of GnRHs and GnRHR1s were analyzed in various tissues of G. rarus by quantitative real-time PCR. GnRHs and GnRHR1s were all predominantly expressed in the brains. Both GnRH3 and GnRHR1A were significantly upregulated in the brains of female exposed to 15 µg/L BPA for 35 days. It would suggest a potential negative feedback in the GnRH system in response to the disturbance of downstream of the brain-pituitary-gonadal axis. Collectively, the present findings suggest that the transcripts of some key genes in the neuroendocrine system can be used as critical biomarkers in endocrine disruption assays of teleost fish.

Two gonadotropin-releasing hormone receptor subtypes with distinct ligand selectivity and differential distribution in brain and pituitary in the goldfish (Carassius auratus).

In the goldfish (Carassius auratus) the two endogenous forms of gonadotropin-releasing hormone (GnRH), namely chicken GnRH II ([His5, Trp7,Tyr8]GnRH) and salmon GnRH ([Trp7,Leu8]GnRH), stimulate the release of both gonadotropins and growth hormone from the pituitary. This control is thought to occur by means of the stimulation of distinct GnRH receptors. These receptors can be distinguished on the basis of differential gonadotropin and growth hormone releasing activities of naturally occurring GnRHs and GnRHs with variant amino acids in position 8. We have cloned the cDNAs of two GnRH receptors, GfA and GfB, from goldfish brain and pituitary. Although the receptors share 71% identity, there are marked differences in their ligand selectivity. Both receptors are expressed in the pituitary but are differentially expressed in the brain, ovary, and liver. Thus we have found and cloned two full-length cDNAs that appear to correspond to different forms of GnRH receptor, with distinct pharmacological characteristics and tissue distribution, in a single species.

Differential brain distribution of gonadotropin-releasing hormone receptors in the goldfish.

The present study describes the differential distributions in the brain of the two goldfish gonadotropin-releasing hormone (GnRH) receptors, using both immunohistochemistry and in situ hybridization approaches. The goldfish GnRH GfA and GfB receptors are variant forms of the same receptor subtype, although with distinct differences in ligand binding characteristics, and differential distributions in the pituitary and body tissues [Proc. Natl. Acad. Sci. USA 96 (1999) 2526]. The goldfish GnRH GfA receptor was found to be widespread throughout the brain, with neurons showing immunoreactivity in the olfactory bulbs, telencephalon, preoptic region, ventro-basal hypothalamus, thalamus, midbrain, motor neurons of the fifth, seventh, and tenth cranial nerves, reticular formation, cerebellum, and motor zone of the vagal lobes. The tracts in the posterior commissure, optic tectum, and motor zone of the vagal lobes also demonstrated immunoreactivity. While the brain was not systematically surveyed for in situ hybridization, hybridization was found in similar locations in the telencephalon, preoptic region, ventro-basal hypothalamus, cerebellum, and optic tectum. Hybridization was additionally found in the medial hypothalamus. The goldfish GnRH GfB receptor was found to have a more restricted distribution in the brain, with neurons showing immunoreactivity in the telencephalon, preoptic region, and ventro-basal hypothalamus. In situ hybridization demonstrated a somewhat wider distribution of expression of the receptor, with hybridization occurring in the preoptic region, ventro-basal and medial hypothalamus, as well as in the thalamus, epithalamus, and optic tectum. The widespread distribution of GnRH GfA receptor, and in particular its localization in the midbrain tegmentum in the region of the GnRH-II neurons, suggests that this receptor may be involved in the behavioral actions of GnRH peptides in the goldfish.

Three distinct types of GnRH receptor characterized in the bullfrog.

It has been proposed recently that two types of GnRH receptors (GnRHR) exist in a particular species. Here we present data demonstrating that at least three types of GnRHR are expressed in a single diploid species, the bullfrog. Three different cDNAs, encoding distinct types of bullfrog GnRHR (bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3), were isolated from pituitary and hindbrain of the bullfrog. BfGnRHR-1 mRNA was expressed predominantly in pituitary, whereas bfGnRHR-2 and -3 mRNAs were expressed in brain. The bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3 proteins have an amino acid identity of approximately 30% to approximately 35% with mammalian GnRHRs and approximately 40% to approximately 50% with nonmammalian GnRHRs. Interestingly, bfGnRHR-2 has an 85% amino acid homology with Xenopus GnRHR. Less than 53% amino acid identity was observed among the three bfGnRHRs. All isolated cDNAs encode functional receptors because their transient expression in COS-7 cells resulted in a ligand-dependent increase in inositol phosphate production. Notably, all three receptors exhibited a differential ligand selectivity. For all receptors, cGnRH-II has a higher potency than mGnRH. In addition, salmon GnRH also has a strikingly high potency to stimulate all three receptors. In conclusion, we demonstrated the presence of three GnRHRs in the bullfrog. Their expression in pituitary and brain suggests that bfGnRHRs play an important role in the regulation of reproductive functions in the bullfrog.