GnRH Variants Research Articles

Distribution and expression of GnRH 1, kiss receptor 2, and estradiol α and ß receptors in the anterior brain of females of Chirostoma humboldtianum.

Reproduction in vertebrates is a complex process regulated by many hormones, and by paracrine factors and their receptors. This study aimed to examine the expression of pjGonadotropin-releasing hormone (GnRH 1), the kisspeptin receptor 2 (kissr2), and estradiol receptors α and β (ER α and ER β) during different stages of the sexual cycle and their distribution within the anterior brain of females of Chirostoma humboldtianum. Among these molecules, the kissr2 showed the maximal variation in expression, while GnRH 1 showed minimal variation of expression, and ERβ and ERα had intermediate variation of expression. The distribution of these molecules in the anterior brain was consistent with their levels of expression; kissr2 was widely distributed throughout the telencephalon and diencephalon, while ER and GnRH 1 showed more restricted distributions. No coexpression of kissr2 and ER in GnRH 1ergic neurons, suggesting that regulation of this GnRH variant is indirectly mediated by kisspeptin and estradiol.

The Gonadotropin-Inhibitory Hormone: What We Know and What We Still Have to Learn From Fish.

Gonadotropin-inhibitory hormone, GnIH, is named because of its function in birds and mammals; however, in other vertebrates this function is not yet clearly established. More than half of the vertebrate species are teleosts. This group is characterized by the 3R whole genome duplication, a fact that could have been responsible for the great phenotypic complexity and great variability in reproductive strategies and sexual behavior. In this context, we revise GnIH cell bodies and fibers distribution in adult brains of teleosts, discuss its relationship with GnRH variants and summarize the few reports available about the ontogeny of the GnIH system. Considering all the information presented in this review, we propose that in teleosts, GnIH could have other functions beyond reproduction or act as an integrative signal in the reproductive process. However, further studies are required in order to clarify the role of GnIH in this group including its involvement in development, a key stage that strongly impacts on adult life.

Effects of heavy metals identified in Chascomús shallow lake on the endocrine-reproductive axis of pejerrey fish (Odontesthes bonariensis)

Some heavy metals related to human activities were measured in the water of Chascomús lake. The maximum concentrations were: 0.23 μg/L for Cd, 4.28 μg/L for Cr, 22.09 μg/L for Cu, 2.49 μg/L for Ni, 3.24 μg/L for Pb and 210.76 μg/L for Zn. The values of Cd, Cr, Cr, Pb and Zn were above the Argentine National Guidelines for the Protection of the Aquatic life. The analysis of gonadal condition of pejerrey fish (Odontesthes bonariensis) from this lake did not revealed any reproductive damages. However, exposures with environmental concentrations of Cd, Cr, Cu and Zn under laboratory conditions of pejerrey males (14 days), caused a significant increase of the expression of the three variants of gnrh in the brain (within Cd exposure) and a decrease in cyp19a1b mRNA (within Cu exposure). Furthermore, at pituitary level, a decrease in fshb transcript levels was observed in the fish exposed to Cd and Cr and a decrease in the expression of both gonadotropin receptors at gonadal level in Zn exposure. Moreover, the gonads of the fish exposed to all the tested metals suffered structural damages showing shortness of the spermatic lobules, fibrosis, testis ova and the presence of piknotic cells. All these findings alert that heavy metals pollution affects the expression of key reproductive genes and gonadal structure of fish species that represent the predominant group of organisms and are considered sentinel species in the aquatic ecosystems.

Seasonal changes and endocrine regulation of pejerrey (Odontesthes bonariensis) spermatogenesis in the wild

The endocrine mechanisms that regulate spermatogenesis and their interaction with environmental cues have been poorly studied compared with oogenesis in fish. The aim of this work was to study the spermatogenesis in pejerrey under the influence of photoperiod and water temperature fluctuation in the wild, evaluating the transcript levels of brain Gnrh variants and cyp19a1b, pituitary Gth subunits, gonadal Gth receptors, 11β-hsd, and 11-KT plasma levels. Males at spermiogenic stage were observed during spring and autumn, under a photoperiod above 11h of light and a water temperature below 23°C. Most arrested males were observed in summer when water temperatures increased above 23°C. Males at spermatogonial stage were mainly observed in autumn, while most males at spermatocytary stage were caught in winter. An increase of gnrh-I, cyp19a1b, fshb, gpha and 11β-hsd transcripts and 11-KT plasma levels was observed during spermatogonial and/or spermatocytary stage (early spermatogenesis). The spermiogenic stage was associated to the maximum gnrh-I gene expression level and a significant increase of Gth receptors transcripts, being this fact more evident for lhcgr. During this last gonadal stage, cyp19a1b transcript level remained high, while fshb mRNA and 11-KT plasma levels showed a significant decreased compared to that occurred at the spermatocytary stage. Also, gphα and 11β-hsd gene expression levels fell during spermiation up to similar values to those observed in arrested males. A significant correlation between 11-KT and gnrh-I, cyp19a1b, gphα, fshb, 11β-hsd transcripts, and the number of spermatocytes was observed during spermatogenesis. All these findings suggested that in pejerrey, the spermatocyte proliferation occurs mainly during winter under the stimulation of 11-KT induced by FSH through the stimulation of specific enzymes, including the 11β-hsd while spermiation occurs after photoperiod increase and with temperatures of the water below 23°C, through the stimulation of gnrh-I, cyp19a1b and lhcgr.

Differential regulation of GnRH ligand and receptor genes in the brain and pituitary of Atlantic cod exposed to different photoperiod

The onset of puberty and reproduction are tightly controlled by extrinsic and intrinsic inputs combined with genetically determined biological blueprints. Environmental inputs are then mediated by the brain–pituitary–gonad endocrine axis resulting in a unified output. In fish, one of the primary factors controlling the timing of sexual maturation is light, although how these signals are mediated in the brain and pituitary is not well understood. We therefore aimed to elucidate the molecular basis of the control of reproduction during the first spawning season in two year old female Atlantic cod. To this end, we measured GnRH and GnRH-R variant gene expression in brains and pituitaries collected from cod kept under four different photoperiod regimes: natural light (NL), continuous light (LL) and combined treatment of NL–LL and LL–NL. LL inhibited sexual development and spawning and LL–NL delayed sexual development and spawning. LL inhibited the spawning-related increase in brain GnRH3 and pituitary GnRH-R2a gene expression found under NL conditions, and the expression of these genes were delayed in concert with spawning of LL–NL cod. This study indicates that regulation of brain GnRH3 and pituitary GnRH-R2a genes likely mediates photoperiod induced changes in cod gonadal maturation.

The Highly Conserved Gonadotropin-Releasing Hormone-2 Form Acts as a Melatonin-Releasing Factor in the Pineal of a Teleost Fish, the European Sea Bass Dicentrarchus labrax

With the exception of modern mammals, most vertebrate species possess two GnRH genes, GnRH-1 and GnRH-2. In addition, in many teleost fish, there is a third gene called GnRH-3. If the main function of GnRH-1 is unambiguously to stimulate gonadotropin release, the other two GnRH forms still lack clear functions. This is particularly true for the highly conserved GnRH-2 that encodes chicken GnRH-II. This GnRH variant is consistently expressed in neurons of the dorsal synencephalon in most vertebrate groups but still has no clear functions supported by anatomical, pharmacological, and physiological data. In this study performed on a perciform fish, the European sea bass, we show for the first time that the pineal organ receives GnRH-2-immunoreactive fibers originating from the synencephalic GnRH-2 neurons. This was shown through a combination of retrograde tracing and immunohistochemistry, using highly specific antibodies. Supporting the presence of GnRH-2 functional targets, RT-PCR data together with the in situ hybridization studies showed that the sea bass pineal gland strongly expressed a GnRH receptor (dlGnRHR-II-2b) with clear selectivity for GnRH-2 and, to a lesser extent, the dlGnRHR-II-1a subtype. Finally, in vitro and in vivo experiments demonstrate stimulatory effects of GnRH-2 on nocturnal melatonin secretion by the sea bass pineal organ. Altogether, these data provide, for the first time in a vertebrate species, converging evidence supporting a role of GnRH-2 in the modulation of fish pineal functions.

MRNA expression of GnRH variants and receptors in the brain, pituitary and ovaries of pejerrey (Odontesthes bonariensis) in relation to the reproductive status

The present study examined the differential mRNA expression levels of three forms of GnRH (sGnRH, pjGnRH and cGnRH-II) and two forms of GnRH receptor (pjGnRH-R I and pjGnRH-R II) in the brain, pituitary, and ovaries of pejerrey in relation to the reproductive status. The analysis revealed the presence of significant amounts of mRNA of the three GnRH forms while the ovaries showed only two (sGnRH and pjGnRH). The GnRH receptor II was found ubiquitously in the brain, pituitary, and ovaries while the form I was detected only in the brain. The levels of pjGnRH mRNA in the brain and pjGnRH-R II in the pituitary gland varied in correlation with the ovarian condition. However, brain sGnRH and pjGnRH-R I mRNA levels reached a maximum during early stages of ovarian development. In contrast, the brain levels of cGnRH-II mRNA showed no variation. The present study also shows a good correlation of ovarian sGnRH and pjGnRH-R II mRNA levels with the reproductive condition, suggesting that these molecules are may be involved in the regulation of pejerrey ovarian function.

Seasonal Profiles of Brain and Pituitary Gonadotropin-Releasing Hormone and Plasma Luteinizing Hormone in Relation to Sex Change of Protandrous Black Porgy, Acanthopagrus schlegeli1

Three molecular variants of GnRH in the brain (sbGnRH, sGnRH, and cGnRH-II) and two forms in the pituitary (sbGnRH and sGnRH) were detected in protandrous black porgy, Acanthopagrus schlegeli using chromatographic and immunological methods. In juvenile fish, brain sbGnRH, sGnRH, and cGnRH-II levels increased in May and reached their highest levels in July and August (the nonspawning season) and in January through March (the spawning season). In fish aged 1 yr and older, high levels of brain sbGnRH and sGnRH were detected in September, November, and February-March, but the levels of brain cGnRH-II remained constant. A gradual increase in pituitary sbGnRH was detected in juvenile fish from July to March. In fish aged 1+ yr, pituitary sbGnRH levels were high in September and March-May, but low in January-February. A close correlation between pituitary sbGnRH and plasma LH levels was found in juvenile fish and in those aged 1+ yr. In fish aged 2+ yr, significantly lower levels of plasma LH was detected during the nonspawning period in fish that changed sex compared with the fish that remained as males. Higher plasma LH levels were detected in the sex-changing fish from artificially sex-reversed female to male. FSH receptor and LH receptor transcripts were higher in bisexual testicular tissue than in ovarian tissue in 2+-yr-old fish. Direct effects of hCG on sex change were studied and the results show that exogenous hCG did not stimulate gonadal aromatase activity in 2+-yr-old fish. Therefore, it is suggested that high and basal levels of plasma LH during the nonspawning season correlate with the development of male and female gonad, respectively, in black porgy. This important role of the neuroendocrine system in sex change (for male direction) is proposed in hermaphroditic fish.

Single-cell real-time quantitative polymerase chain reaction of immunofluorescently identified neurons of gonadotropin-releasing hormone subtypes in cichlid fish.

The regulatory mechanisms controlling gene expression of GnRH subtypes, particularly of the evolutionarily conserved GnRH2, remain speculative. To address this issue, we have successfully coupled the anatomic specificity of immunofluorescently defined "cell picking" with the sensitivity of real-time quantitative RT-PCR (RT-Q-RT-PCR), which enabled us to examine the presence and quantity of GnRH mRNAs in individual neurons. Here, using RT-Q-RT-PCR, we report change in the levels of transcripts of GnRH subtypes in individual neurons harvested from the brain of mature and immature males of tilapia, Oreochromis niloticus. The levels of GnRH1 mRNA per cell and the percentage of neurons expressing GnRH1 transcripts exceeding 0.05 x 10(2) fg/cell were significantly higher in mature males (44.2%) compared with immature males (4.7%). In contrast, there was no difference in mRNA levels and the percentage of cells expressing GnRH2 and GnRH3 between the two reproductive states. Thus, using a novel approach that enables immunofluorescently labeled single-cell RT-Q-RT-PCR analysis of GnRH neurons, we present evidence that shows preoptic GnRH1 is important for gonadal maturation, whereas GnRH2 and GnRH3 might have supplementary roles in reproductive behaviors or nonreproductive functions. Furthermore, we speculate that the use of this method will allow the identification and quantification of known and unknown genes in single GnRH neurons, which would greatly facilitate our understanding of the complex interactions that govern the physiology of individual cells of GnRH variants in vertebrate species.

Gonadotropin-releasing hormone receptors: neuroendocrine regulators and neuromodulators

Several lines of evidence support the idea that more than one gonadotropin-releasing hormone receptor (GnRH-R) subtype exists to accommodate the presence of multiple GnRH forms seen in vertebrate species. The development of pituitary endocrine cells, GnRH-Rs type IA (GfA), IB (GfB) and type III in the pituitary and their synchronization with the establishment of GnRH1-3 regulatory pathways reflect a neuroendocrine function for the GnRH-Rs. Based on developmental, morphological and biochemical evidence it can be hypothesized that GnRH1 regulates gonadotrophs through GnRH-R type IA, GnRH2 regulates prolactin secretion through GnRH-R type IB and GnRH3 regulates growth hormone secretion through GnRH-R type III. Therefore, the three native GnRH variants in advance teleost like tilapia might have their respective cognate receptors, each being capable of regulating different and, to some degree the same pituitary endocrine cells. In the brain, the expression pattern of GnRH-R type IB and type III parallels the distribution of GnRH2 and GnRH3 fiber network, which suggests their role in neuromodulation and reproductive behavior.

Expression of pejerrey gonadotropin-releasing hormone in three orders of fish.

Molecular variants of GnRH were characterized by reverse-phase, high-performance liquid chromatography from brain extracts of fish in three different orders: Synbranchiformes (swamp eel [Synbranchus marmoratus]), Cyprinidontiformes (platyfish [Xiphophorus maculatus] and green swordtail [X. helleri]), and Atheriniformes (Patagonia pejerrey [Odontesthes hatchery]). Also, pituitary gland extracts from the pejerrey O. bonariensis (Atheriniformes) were characterized. Eluted fractions were tested in radioimmunoassays with antisera specific to GnRH, including both antisera that detected only one form of GnRH and those that detected several forms. The results show that brain extracts obtained from all species contained the same three molecular forms of GnRH, which were immunologically and chromatographically undistinguishable from chicken GnRH-II, pejerrey GnRH (pjGnRH), and salmon GnRH. This study supports the hypothesis that expression of these three forms is common in different fish orders and that pjGnRH is the main regulator of pituitary function in these fish.

In silico and in situ characterization of the zebrafish (Danio rerio) gnrh3 (sGnRH) gene.

BackgroundGonadotropin releasing hormone (GnRH) is responsible for stimulation of gonadotropic hormone (GtH) in the hypothalamus-pituitary-gonadal axis (HPG). The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio).ResultsWe have characterized a zebrafish [Trp7, Leu8] or salmon (s) GnRH variant, gnrh3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH), was shown capable of driving cell specific reporter gene expression in transgenic zebrafish.ConclusionsThe characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar) GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.

Molecular cloning, distribution and pharmacological characterization of a novel gonadotropin-releasing hormone ([Trp 8] GnRH) in frog brain

To date nine structural variants of GnRH have been identified in vertebrates and two additional forms have been isolated from a tunicate. In amphibians only mammalian GnRH ([Arg 8] GnRH) and type II GnRH (chicken GnRH II, [His 5, Trp 7, Tyr 8] GnRH) have been identified. In the present study, a full-length cDNA encoding a novel type of GnRH was isolated from pituitary of Rana dybowskii. The GnRH gene encodes a GnRH peptide ([Trp 8] GnRH) in which tryptophan is substituted for arginine of mammalian GnRH Northern blot analysis revealed the presence of a single 500 bp transcript for the [Trp 8] GnRH precursor in forebrain but its absence in testis, ovary, kidney and liver. Restriction digests of genomic DNA demonstrated a single copy of the gene. The [Trp 8] GnRH immunoreactive cells were identified in the preoptic area of the frog brain. Synthetic [Trp 8] GnRH was tested for its ability to stimulate inositol phosphate production by COS-1 cells transfected with the cloned Xenopus pituitary GnRH receptor and the cloned human GnRH receptor. [Trp 8] GnRH had a potency of about 60% compared with mammalian GnRH ([Arg 8] GnRH) for the Xenopus receptor, whereas the potency of [Trp 8] GnRH was ≈5% compared with mammalian GnRH for the human receptor. Both mammalian GnRH and [Trp 8] GnRH were 1000-fold less potent than type II GnRH for the Xenopus GnRH receptor. The similar potency of [Arg 8] GnRH and the novel [Trp 8] GnRH for the Xenopus pituitary receptor indicates that, unlike the human receptor, the Xenopus receptor does not discriminate between these amino acids in position eight thereby allowing substitution of the arginine in the mammalian GnRH.

Colocalization of GnRH Binding Sites with Gonadotropin-, Somatotropin-, Somatolactin-, and Prolactin-Expressing Pituitary Cells of the Pejerrey, Odontesthes bonariensis, in Vitro

Previous studies in the pejerrey, Odontesthes bonariensis, have demonstrated that fibers with immunoreactivity to gonadotropin-releasing hormone (ir-GnRH) reach all areas of the pituitary gland, the rostral pars distalis (RPD), the proximal pars distalis (PPD), and the pars intemedia (PI). A close association was shown between ir-GnRH fibers and gonadotropin (GtH)-, growth hormone (GH)-, somatolactin (SL)-, and prolactin (PRL)-expressing cells. The presence of only one GnRH variant, suspected to be a novel form, has been shown in pituitary extracts of this fish. In addition, GnRH may stimulate GtHs, GH, SL, and PRL levels in different fish species. The objective of the present study was to seek GnRH receptors and therefore colocalization with GtHs, GH, SL, and PRL cells in O. bonariensis using a pituitary primary cell culture system. GnRH binding sites were revealed by autoradiography of an iodinated superactive GnRH agonist ([125I]GnRH-A) and pituitary cells were identified by immunocytochemistry using piscine antisera. Following autoradiography, silver grains representing specific [125I]GnRH-A binding were associated with anti GtH, GH, SL, and PRL positive cells. These results demonstrate the presence of GnRH binding sites on these cells. It is suggested that GnRH may play a wide role in the neuroendocrine control of different pituitary hormones in addition to the GtHs.

Differential distribution of gonadotropin-releasing hormone variants in the brain of Hydrochaeris hydrochaeris (Mammalia, Rodentia).

1. In a previous paper we reported evidence for the presence of mGnRH- and sGnRH-like peptides in the preoptic-hypothalamic region of the capybara Hydrochaeris hydrochaeris (Montaner et al., 1998). In that study, the presence of a cGnRH-II like molecule in olfactory bulb extracts was suggested. 2. The capybara, the largest living rodent in the world, belongs to the order Hystricomorpha, which is considered to be one of the oldest groups of rodents. Some authors consider that this group is the ancestor of all remaining rodents. 3. In this study we have characterized GnRH molecular variants found in extracts from the olfactory bulbs and the mesencephalic region of capybara. These regions represent the two GnRH neuronal systems: the terminal nerve-septopreoptic and the midbrain systems. 4. An indirect method combining reverse-phase high-performance liquid chromatography (RP-HPLC) and radioimmunoassay (RIA) was used to characterize GnRH variants. The analysis of both extracts with two different RIA systems revealed three immunoreactive GnRH peaks, coeluting with mGnRH, cIIGnRH, and sGnRH synthetic standards. These results were additionally supported by serial dilution studies with specific antisera. 5. To our knowledge this the first report on the presence of three GnRH variants in the brain of an eutherian mammal. These results suggest that, similarly to other vertebrates, the expression of multiple GnRH variants may also be a common pattern in mammals.

Isolation and characterisation of mRNA encoding the salmon- and chicken-II type gonadotrophin-releasing hormones in the teleost fish Rutilus rutilus (Cyprinidae).

The complementary DNAs (cDNA) encoding the [Trp7,Leu8]-gonadotrophin-releasing hormone (salmon-type GnRH; sGnRH:GeneBank accession no. u60667) and the [His5,Trp7,Tyr8]-GnRH (chicken-II-type GnRH; cGnRH-II: GeneBank accession no. u60668) precursor in the roach (Rutilus rutilus) were isolated and sequenced following reverse transcription and rapid amplification of cDNA ends (RACE). The sGnRH and cGnRH-II precursor cDNAs consisted of 439 and 628 bp, and included open reading frames of 282 and 255 bp respectively. The structures of the encoded peptides were the same as GnRHs previously identified in other vertebrates. The sGnRH and cGnRH-II precursor cDNAs, including the non-coding regions, had 88.6 and 79.9% identity respectively, to those identified in goldfish (Carassius auratus). However, significant similarity was not observed between the non-coding regions of the GnRH cDNAs of Cyprinidae and other fish. The presumed third exon, encoding partial sGnRH associated peptide (GAP) of roach, demonstrated significant nucleotide and amino acid similarity with the appropriate regions in the goldfish, but not with other species, and this may indicate functional differences of GAP between different families of fish. cGnRH-II precursor cDNAs from roach had relatively high nucleotide similarity across this GnRH variant. Cladistic analysis classified the sGnRH and cGnRH-II precursor cDNAs into three and two groups respectively. However, the divergence between nucleotide sequences within the sGnRH variant was greater than those encoding the cGnRH-II precursors. Consistent with the consensus developed from previous studies, Northern blot analysis demonstrated that expression of sGnRH and cGnRH-II was restricted to the olfactory bulbs and midbrain of roach respectively. This work forms the basis for further study on the mechanisms by which the tapeworm, Ligula intestinalis, interacts with the pituitary-gonadal axis of its fish host.

The Atlantic salmon prepro-gonadotropin releasing hormone gene and mRNA

Screening for the gene encoding salmon gonadotropin releasing hormone (sGnRH) in an Atlantic salmon ( Salmo salar) genomic library resulted in isolation of a positive clone designated λsGnRH-1. An anchor polymerase chain reaction (PCR) technique was used to amplify GnRH cDNA derived from salmon hypothalamic mRNA. The cDNA sequence was aligned to the 7607 base pair genomic sequence which was shown to encode the entire prepro-GnRH gene. The cDNA proved that the cloned gene is expressed in the hypothalamus of mature salmon. The coding domain of sGnRH differs from the mammalian GnRH by six nucleotide changes which allow the two amino acid differences between the two GnRH variants. Salmon GnRH associated peptide (GAP) differs extensively in sequence and size from the mammalian counterpart. Compared to the GnRH cDNA of a cichlid species the similarity is 69.3% in the protein coding sequence.

Distribution of gonadotropin-releasing hormone-like peptides in the brain during development of juvenile male Rana esculenta

The distribution and density of cell bodies and fibers immunoreactive to GnRH-like peptides were investigated in the brain of male juvenile frogs (Rana esculenta) during postmetamorphic development. An immunohistochemical technique was used, involving antisera raised against 4 variants of GnRH: mammalian GnRH, chicken GnRH-I, chicken GnRH-II and salmon GnRH. A comparison of the immunohistochemical distribution at 8 different developmental stages shows that the maximum density of immunoreactive-GnRH elements, and the full distributional complexity of this system, is attained at the completion of spermatogenesis. Immunoreactive-GnRH cell bodies first appear in the anterior preoptic area during the metamorphic climax, and then appear sequentially in the medial septal area, tegmentum and, lastly, in the retrochiasmatic area and olfactory bulb when immunoreactive-fibers also reach the cerebellum. The GnRH system reacts positively to antisera for all 4 GnRH variants, but immunoreactivity for chicken GnRH-I is the weakest.