Molecular cloning and characterization, and prokaryotic expression of the GnRH1 gene obtained from Jinghai yellow chicken.

The gonadotropin-releasing hormone (GnRH) plays an important role in the control of reproductive functions. Recent studies have reported the occurrence of GnRH molecular variants in numerous species. In this study, the GnRH1 gene from Jinghai yellow chicken was cloned by reverse transcriptase-polymerase chain reaction and transformed into BL21 (DE3) competent cells. The GnRH1 gene and amino acid sequences were subjected to bioinformatic analyses. The GnRH1 gene nucleotide sequence was discovered to be 352 bp long, containing a coding, promoter, and section of the 3'-regions. The GnRH1 gene shared 93, 81, 54, 58, 61, 76, 76, 59, 76, and 66% sequence identity with Meleagris gallopavo, Columba livia, Homo sapiens, Bos taurus, swines, Capra hircus, Ovis aries, Pantholops hodgsonii, Equus caballus, and Rattus norvegicus, respectively. The GnRH1 gene showed conserved domains. The GnRH1 protein was a secreted protein comprising 92 amino acids, with a molecular weight of 10205.6 Da and a theoretical pI of 5.67. Most of the amino acid residues were observed to be hydrophilic, indicating water solubility. The predicted secondary structures of proteins included α-helices (h; 23.08%), ß-extensions (e; 10.92%), and random coils (c; 66.0%). The successful construction of prokaryotic expression vector pET32a-GnRH1 was confirmed by restriction and sequence analysis. SDS-PAGE analysis showed the successful expression of recombinant plasmid in Escherichia coli BL21 (molecular weight = 25-28 kDa). Larger quantities of protein were expressed in supernatant, indicating greater expression in soluble form. Western blot analysis confirmed the expression of the target protein.

GnRH receptors and peptides: skating backward.

Gonadotropin-releasing hormone (GnRH) and its receptor are essential for reproduction in vertebrates. Although there are three major types of GnRH peptides and two major types of receptors in vertebrates, the pattern of distribution is unusual. Evidence is presented from genome mining that type I GnRHRs are not restricted to mammals, but can be found in the lobe-finned and cartilaginous fishes. This implies that this tail-less GnRH receptor emerged early in vertebrate evolution, followed by several independent losses in different lineages. Also, we have identified representatives from the three major GnRH peptide types (mammalian GnRH1, vertebrate GnRH2 and dogfish GnRH3) in a single cartilaginous fish, the little skate. Skate and coelacanth are the only examples of animals with both type I and II GnRH receptors and all three peptide types, suggesting this was the ancestral condition in vertebrates. Our analysis of receptor synteny in combination with phylogeny suggests that there were three GnRH receptor types present before the two rounds of whole genome duplication in early vertebrates. To further understand the origin of the GnRH peptide-receptor system, the relationship of vertebrate and invertebrate homologs was examined. Our evidence supports the hypothesis of a GnRH superfamily with a common ancestor for the vertebrate GnRHs, invertebrate (inv)GnRHs, corazonins and adipokinetic hormones. The invertebrate deuterostomes (echinoderms, hemichordates and amphioxus) have derived GnRH-like peptides, although one amphioxus GnRH with a syntenic relationship to human GnRHs has been shown to be functional. Phylogenetic analysis suggests that gene duplications in the ancestral bilaterian produced two receptor types, one of which became adipokinetic hormone receptor/GnRHR and the other corazonin receptor/invGnRHR. It appears that the ancestral deuterostome had both a GnRHR and invGnRHR, and this is still the case in amphioxus. During the transition to vertebrates both the invertebrate-type peptide and receptor were lost, leaving only the vertebrate-type system that presently exists.

Two novel gonadotropin-releasing hormones (GnRHs) from the urochordate ascidian, Halocynthia roretzi: implications for the origin of vertebrate GnRH isoforms.

Three forms of gonadotropin-releasing hormone (GnRH) are found in vertebrates; these differ in amino acid sequence, localization, distribution, and embryological origin. We used northern blot analysis, and in situ hybridization to detect GnRH transcripts in various tissues in the large ascidian Halocynthia roretzi. We cloned a cDNA encoding two novel GnRHs, termed tGnRH-10 and tGnRH-11, from H. roretzi, with deduced amino acid sequences of QHWSYGFSPG and QHWSYGFLPG, respectively. Both GnRHs are highly similar to those of teleosts and tetrapods. For example, the tGnRH-10 sequence is 90% identical to seabream GnRH1, and tGnRH-11 is 90% identical to salmon GnRH3. The primary structure of the deduced preprotein is similar to that of chordate GnRHs and consists of a signal peptide, two decapeptides, up- and downstream processing sequences (containing lysine and arginine), and a GnRH-associated peptide. The transcripts of the H. roretzi GnRH gene were expressed in all tissues examined. Comparison of the signal peptide of the lamprey GnRH-II precursor with those of three forms from representative vertebrates revealed homology to GnRH2 precursors. These novel ascidian GnRHs offer a new perspective on the origin of vertebrate GnRH subtypes. We hypothesize that gnathostome GnRH2 was derived only from lamprey GnRH-II and that ancestral gnathostome GnRH, which produces neurons that originate in peripheral organs, gave rise to vertebrate GnRH1 and GnRH3 through whole-genome duplication.

Identification and distribution of three gonadotropin-releasing hormone (GnRH) isoforms in the brain of a clupeiform fish, Engraulis japonicus.

To gain a better understanding of the reproductive endocrinology of a primitive order clupeiform fish (Japanese anchovy, Engraulis japonicus), cDNAs encoding three gonadotropin-releasing hormone (GnRH) isoforms were isolated from the brain, and their distribution was analyzed using insitu hybridization (ISH). The three GnRH isoforms include GnRH1 (herring GnRH), GnRH2 (chicken GnRH-ll) and GnRH3 (salmon GnRH), and their full-length cDNAs encode 88, 86, and 89 deduced amino acids (aa), respectively. Alignment analysis of Japanese anchovy GnRH isoforms showed lower identities with other teleost fish. The major population of GnRH1 neurons was localized in the ventral telencephalon (VT) and nucleus preopticus (NPO) of the preoptic area (POA) with minor population in the anterior olfactory bulb (OB). GnRH2 neurons were restricted to the midbrain tegmentum (MT), specific to the nucleus of the medial longitudinal fasciculus (nMLF). GnRH3 neurons were localized in the olfactory nerve (ON), ventral OB, and transitional area between OB and ON. Interestingly, GnRH1 neurons were also localized in the olfactory bulb, in addition to its major population in the preoptic area. These results indicate the differential distribution of three GnRH isoforms expressed in the brain of the Japanese anchovy.

Reproductive development, GnRHa-induced spawning and egg quality of wild meagre (Argyrosomus regius) acclimatised to captivity.

The objective of the study was to acclimatise wild-caught meagre (Argyrosomus regius) to captivity to produce viable eggs for aquaculture production. Twelve meagre (3 males and 9 females, mean weight = 20 ± 7 kg) were caught and transported to a land-based facility on 26 October 2006. During, March to June 2007, all three males were spermiating and five of the nine females were in vitellogenesis with mean maximum oocyte diameter ≥550 µm. No spontaneous spawning was observed. Two hormone treatments, either a single injection of gonadotropin-releasing hormone agonist (GnRHa, 20 µg kg(-1) for females and 10 µg kg(-1) for males) or a slow-release implant loaded with the same GnRHa (50 µg kg(-1) for females and 25 µg kg(-1) for males), were used to induce spawning on three different dates on 26 March 2007, 4 May 2007 and 18 April 2008. From each spawning event, the following parameters were determined: fecundity, number of floating eggs, egg size, fertilisation and hatching success, unfed larval survival, and proximal composition and fatty acid profile of the eggs. In 2007, two females that were injected on 26 March and 4 May spawned a total of 5 times producing 9,019,300 floating eggs and a relative fecundity of 198,200 eggs kg(-1) and two different females that were implanted on the same dates spawned 14 times producing 12,430,000 floating eggs and a relative fecundity of 276,200 eggs kg(-1). In 2008, a pair that was implanted spawned five times producing a total of 10,211,900 floating eggs and a relative fecundity of 527,380 eggs kg(-1). The latency period was 48-72 h. Parameters were compared between hormone treatments, date of hormone induction and parents determined by microsatellites. Percentage hatch and egg size were 70 ± 0.3% and 0.99 ± 0.02 mm, respectively, for GnRHa-implanted fish and were significantly higher (P < 0.05) compared to 30 ± 0.3% and 0.95 ± 0.03 mm, respectively, for injected fish. Few differences were observed in proximal composition and fatty acid profile and for all spawns mean (% dry weight) lipid content was 17.3 ± 3.0%, carbohydrate was 4.4 ± 1.9% and protein was 31.5 ± 6.4% and the essential fatty acids: Arachidonic acid (ARA, 20:4n-6) ranged between 0.9 and 1% (of total fatty acids), eicosapentaenoic acid (EPA 20:5n-3) 7.7-10.4% and docosahexaenoic acid (DHA 22:6n-3), 28.6-35.4%. All good quality spawns were obtained in the second and/or third spawn after GnRHa treatment, whereas all bad quality spawns were obtained either on the first spawn or after the fifth spawn. Both spawning protocols gave commercially viable (1,000,000+) numbers of good quality eggs that could form the basis of a hatchery production.

Revisiting the evolution of gonadotropin-releasing hormones and their receptors in vertebrates: secrets hidden in genomes.

Gonadotropin-releasing hormone (GnRH) and its G protein-coupled receptor, GnRHR, play a pivotal role in the control of reproduction in vertebrates. To date, many GnRH and GnRHR genes have been identified in a large variety of vertebrate species using conventional biochemical and molecular biological tools in combination with bioinformatic tools. Phylogenetic approaches, primarily based on amino acid sequence identity, make it possible to classify these multiple GnRHs and GnRHRs into several lineages. Four vertebrate GnRH lineages GnRH1, GnRH2, GnRH3, and GnRH4 (for lamprey) are well established. Four vertebrate GnRHR lineages have also been proposed-three for nonmammalian GnRHRs and mammalian GnRHR2 as well as one for mammalian GnRHR1. However, these phylogenetic analyses cannot fully explain the evolutionary origins of each lineage and the relationships among the lineages. Rapid and vast accumulation of genome sequence information for many vertebrate species, together with advances in bioinformatic tools, has allowed large-scale genome comparison to explore the origin and relationship of gene families of interest. The present review discusses the evolutionary mechanism of vertebrate GnRHs and GnRHRs based on extensive genome comparison. In this article, we focus only on vertebrate genomes because of the difficulty in comparing invertebrate and vertebrate genomes due to their marked divergence.

Evolution of GnRH: diving deeper.

Gonadotropin-releasing hormone (GnRH) plays a central role in vertebrate reproduction. The evolutionary origin of this neuropeptide and its receptor is not obvious, but the advent of genomics makes it possible to examine the roots of GnRH and delve deeper into its ancestral relationships. New peptide sequences identified in invertebrates from annelids to tunicates reveal GnRH-like peptides of 10-12 amino acids. Structural conservation suggests homology between the 15 known invertebrate peptides and the 15 known vertebrate GnRHs. The functions of the invertebrate GnRH-like peptides are not necessarily related to reproduction. We suggest that structurally related families of invertebrate peptides including corazonin and adipokinetic hormone (AKH) form a superfamily of neuropeptides with the GnRH family. GnRH receptors have also been identified in invertebrates from annelids to tunicates suggesting that the origin of GnRH and its receptor extends deep in evolution to the origin of bilaterian animals. To resolve the relationship of invertebrate and vertebrate receptors, we conducted large-scale phylogenetic analysis using maximum likelihood. The data support a superfamily that includes GnRH, AKH and corazonin receptors derived from both published sequences and unpublished gene model predictions. Closely related to the GnRHR superfamily is the vasopressin/oxytocin superfamily of receptors. Phylogenetic analysis suggests a shared ancestry with deep roots. A functional role for GnRH in vertebrates or invertebrates leads to questions about the evolutionary origin of the pituitary. Our analysis suggests a functioning pituitary was the result of genomic duplications in early vertebrates.

The identification and distribution of gonadotropin-releasing hormone-like peptides in the central nervous system and ovary of the giant freshwater prawn, Macrobrachium rosenbergii.

In the present study, we demonstrated the existence of GnRH-like peptides in the central nervous system (CNS) and ovary of the giant freshwater prawn, Macrobrachium rosenbergii using immunocytochemistry. The immunoreactivity (ir) of lamprey (l) GnRH-III was detected in the soma of medium-sized neurons located in neuronal cluster number 11 in the middle part of supraesophageal ganglion (deutocerebrum), whereas ir-octopus (oct) GnRH was observed in the soma of both medium-sized and large-sized neurons in thoracic ganglia, as well as in the fibers innervating the other medium-sized and large-sized neuronal cell bodies in the thoracic ganglia. In addition, ir-lGnRH-I was observed in the cytoplasm of late previtellogenic oocyte and early vitellogenic oocyte. These data suggest that M. rosenbergii contain at least three isoforms of GnRH: two GnRH isoforms closely related to lGnRH-III and octGnRH in the CNS, whereas another isoform, closely related to lGnRH-I, was localized in the ovary. This finding provides supporting data that ir-GnRH-like peptide(s) may exist in this decapod crustacean.

Ligand-selective signal transduction by two endogenous GnRH isoforms involves biased activation of the class I PI3K catalytic subunits p110ß, p110γ, and p110δ in pituitary gonadotropes and somatotropes.

In goldfish, 2 endogenous GnRH isoforms, GnRH2 and GnRH3, are released at the pituitary and directly stimulate LH and GH release using the same population of GnRH receptors (GnRHRs) but with GnRH-specific transduction mechanisms. Previously, we have shown that phosphoinositide 3-kinases (PI3Ks) mediate GnRH2- and GnRH3-stimulated LH and GH release. Among the 3 classes of PI3Ks, class I PI3Ks are the best characterized and consist of 4 110-kDa catalytic isoforms (p110α, p110ß, p110γ, and p110δ). Importantly, p110ß and p110γ, but not p110α or p110δ, can be directly activated by the Gßγ heterodimer of Gαßγ protein complexes. In the present study, we examined the expression of class I PI3K isoforms and the effects of selective inhibitors of p110α, p110ß, p110γ, and p110δ catalytic activity on basal, as well as acute, GnRH2- and GnRH3-stimulated LH and GH release responses using primary cultures of dispersed goldfish pituitary cells in column perifusion. Results demonstrate that p110γ and p110δ are involved in the control of basal LH and GH release, whereas p110α and p110ß only regulate basal LH secretion. However, p110ß and p110γ both participated in GnRH3- and GnRH2-stimulated GH release, whereas p110ß and p110γ mediated GnRH2- and GnRH3-induced LH release responses, respectively. GnRH2- and GnRH3-stimulated LH release, as well as GnRH3-elicited GH release, also required p110δ. These results constitute the first evidence for the differential involvement of class I PI3K catalytic subunits in GnRH actions, in general, and suggest that GnRH2 and GnRH3 binding to GnRHRs can bias the activation of class I PI3K signaling to mediate hormone release responses in 2 distinct pituitary cell types. The involvement of both class IA and IB PI3Ks implicates Gßγ subunits, as well as other known regulators of class I PI3Ks, as important components of GnRHR-mediated responses that could influence GnRH-selective signaling in other cell types.

Antibodies against different forms of GnRH distinguish different populations of cells and axonal pathways in a urodele amphibian, Taricha granulosa.

Neurons immunoreactive to the peptide hormone gonadotropin-releasing hormone (GnRH) have been identified in the posterior diencephalon or anterior midbrain of diverse vertebrates. These cells are located caudal to the more well-characterized GnRH neurons in the nervus terminalis and septo-preoptic area, and are believed to express one or more of the nonmammalian forms of the GnRH. The present study utilized immunocytochemical techniques to determine whether the posterior GnRH group is present in a urodele amphibian, the newt Taricha granulosa. Antibodies directed against different molecular forms of GnRH were used to evaluate the immunological properties of GnRH-containing neurons in amphibians. An antibody selective for mammalian GnRH labeled perikarya in the nervus terminalis (terminal nerve) and septo-preoptic region, as described previously. Thick fibers that arise from terminal nerve and septo-preoptic neurons project mainly to the median eminence, medial pallium and habenula. An antibody selective for chicken GnRH II labeled cell bodies in the paraventricular organ and posterior tubercle of the caudal diencephalon, and thin fibers that project widely throughout the central nervous system. Region-specific staining with different GnRH antibodies supports the interpretation that different molecular forms of GnRH are expressed by neuroanatomically distinguishable systems.

Cloning and expression analysis in mature individuals of two chicken type-II GnRH (cGnRH-II) genes in common carp (Cyprinus carpio).

Gonadotropin-releasing hormone (GnRH) is a conservative neurodecapeptide family, which plays a crucial role in regulating the gonad development and in controlling the final sexual maturation in vertebrate. Two differing cGnRH-II cDNAs of common carp, namely cGnRH-II cDNA1 and cDNA2, were firstly cloned from the brain by rapid amplification of cDNA end (RACE) and reverse transcription- polymerase chain reaction (RT-PCR). The length of cGnRH-II cDNA1 and cDNA2 was 622 and 578 base pairs (bp), respectively. The cGnRH-II precursors encoded by two cDNAs consisted of 86 amino acids, including a signal peptide, cGnRH-II decapeptide and a GnRH-associated peptide (GAP) linked by a Gly-Lys-Arg proteolytic site. The results of intron trapping and Southern blot showed that two differing cGnRH-II genes in common carp genome were further identified, and that two genes might exist as a single copy. The multi-gene coding of common carp cGnRH-II gene offered novel evidence for gene duplication hypothesis. Using semi-quantitative RT-PCR, expression and relative expression levels of cGnRH-II genes were detected in five dissected brain regions, pituitary and gonad of common carp. With the exception of no mRNA2 in ovary, two cGnRH-II genes could be expressed in all the detected tissues. However, expression levels showed an apparent difference in different brain regions, pituitary and gonad. According to the expression characterization of cGnRH-II genes in brain areas, it was presumed that cGnRH-II might mainly work as the neurotransmitter and neuromodulator and also operate in the regulation for the GnRH releasing. Then, the expression of cGnRH-II genes in pituitary and gonad suggested that cGnRH-II might act as the autocrine or paracrine regulator.

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.

Molecular cloning, tissue distribution, and ontogeny of gonadotropin-releasing hormone III gene (GnRH-III) in half-smooth tongue sole (Cynoglossus semilaevis).

Gonadotropin-releasing hormone (GnRH) is a neuropeptide that plays a vital role in hypothalamus-pituitary-gonad (HPG) axis. In the present study, the GnRH-III gene was isolated from half-smooth tongue sole (Cynoglossus semilaevis). In the 1160 bp genomic sequence, four exons, three introns, and 5'-/3'-flanking sequences were identified. The putative peptide was 92 residues long, including a putative signal peptide containing 23 amino acids, the GnRH decapeptide, a proteolytic cleavage site of three amino acids and a GnRH associated peptide of 56 amino acids. The overall amino acid sequence of C. semilaevis GnRH-III (csGnRH-III) was highly conserved with other teleost GnRH-III genes. Phylogenetic analysis showed the evolutionary relationships of csGnRH-III with other known GnRH genes. A 320 bp promoter sequence of the csGnRH-III was also analyzed, and several potential regulatory motifs were identified which were conserved in the GnRH promoters of other teleosts. Quantitative real-time PCR analysis indicated csGnRH-III was expressed only in brain and gonads. In C. semilaevis, the csGnRH-III transcript was maternally deposited and appeared to be developmentally regulated during embryogenesis and early larval development. Comparing sequence and expression patterns of csGnRH-III with other teleosts GnRH-IIIs suggested that the main function of GnRH-III might be conserved in teleosts.

[Does the pharmacological class effect between the different luteinizing hormone releasing hormone analogues used in the treatment of prostate cancer have to be assumed?]. / ¿Se ha de asumir el efecto de clase farmacológica entre los diferentes análogos de la hormona liberadora de la hormona luteinizante usados en el tratamiento del carcinoma de próstata?

INTRODUCTION: Evidence-based medicine is transforming clinical practice because of its progressive implantation. OBJECTIVES: We considered studying whether LHRH analogues are agents of the same pharmacological class, i.e., whether they have the same clinical effect, using the approach to evidence-based medicine. MATERIAL AND METHODS: PubMed was used as the main source of search. We have reviewed the evidence on the alleged «drug class effect¼ between analogues and the existing bibliographic support for their use in various medical indications. An evidence level and degree of recommendation have been assigned to each conclusion based on the «Scottish Intercollegiate Guidelines Network¼. RESULTS: There are no studies designed to answer the question of a class effect between LHRH analogues or agonists. Reviews and meta-analyses have been performed on many other issues related to therapeutic management either with analogues, alone or in combination with surgery or radiation therapy. Direct comparisons do not allow for obtain definitive conclusions: Indirect evidence is obtained from randomized studies comparing the different LHRH analogues to other treatments used to obtain androgen deprivation. Other issues related to pharmacokinetics and pharmacodynamics supporting either the existence or non-existence of class effect were evaluated. CONCLUSIONS: The current available evidence is not enough to support a presumed «drug class effect¼ among the various analogues in the treatment of prostate carcinoma.

Evolution of GnRH ligands and receptors in gnathostomata.

Gonadotropin-releasing hormone (GnRH) is the final common signaling molecule used by the brain to regulate reproduction in all vertebrates. Until now, a total of 24 GnRH structural variants have been characterized from vertebrate, protochordate and invertebrate nervous tissue. Almost all vertebrates already investigated have at least two GnRH forms coexisting in the central nervous system. Furthermore, it is now well accepted that three GnRH forms are present both in early and late evolved teleostean fishes. The number and taxonomic distribution of the different GnRH variants also raise questions about the phylogenetic relationships between them. Most of the GnRH phylogenetic analyses are in agreement with the widely accepted idea that the GnRH family can be divided into three main groups. However, the examination of the gnathostome GnRH phylogenetic relationships clearly shows the existence of two main paralogous GnRH lineages: the ''midbrain GnRH" group and the "forebrain GnRH" group. The first one, represented by chicken GnRH-II forms, and the second one composed of two paralogous lineages, the salmon GnRH cluster (only represented in teleostean fish species) and the hypophysotropic GnRH cluster, also present in tetrapods. This analysis suggests that the two forebrain clades share a common precursor and reinforces the idea that the salmon GnRH branch has originated from a duplication of the hypophysotropic lineage. GnRH ligands exert their activity through G protein-coupled receptors of the rhodopsin-like family. As with the ligands, multiple GnRHRs are expressed in individual vertebrate species and phylogenetic analyses have revealed that all vertebrate GnRHRs cluster into three main receptor types. However, new data and a new phylogenetic analysis propose a two GnRHR type model, in which different rounds of gene duplications may have occurred in different groups within each lineage.

Molecular polymorphism of native gonadotropin-releasing hormone (GnRH) is restricted to mammalian GnRH and [hydroxyproline9] GnRH in the developing rat brain.

Although chicken gonadotropin-releasing hormone (GnRH)-II is thought to occur in most animal species, its presence and that of two other variants (lamprey GnRH-III, salmon GnRH) is questionable in rodents. Here we report on the GnRH peptides present in the hypothalamus and the remaining brain of rat of both sexes during development. No immunoreactivity was detected in the elution zone of either native or hydroxylated forms of the above three variants in any of brain extracts chromatographed. The main peptides detected were mammalian GnRH (mGnRH) and m[hydroxyproline9]GnRH (mHypGnRH). In the hypothalamus, these peptides were associated with their free acid and precursor forms. N-terminal fragments from both native decapeptides (GnRH) and mGnRH (GnRH) were observed only in the hypothalamus. C-terminal fragments were detected in both tissues. The relative proportions of mGnRH and mHypGnRH showed no developmental changes in the remaining brain. The hypothalamic proportions of mHypGnRH were high on day 5, and decreased from day 15 onwards. The [Gly11]-precursor to mHypGnRH molar ratio was twofold lower than with the non-hydroxylated peptides. The mGnRH to GnRH molar ratio increased in males but decreased in females during development. No sex-related differences were observed in the native decapeptide to GnRH molar ratio. It was concluded that (1) chicken GnRH-II is not present in all mammals, (2) mGnRH and mHypGnRH are the main GnRH isoforms present in the rat brain, (3) the processing of [Gly11]-precursor into mHypGnRH occurs at a higher rate than that of mGnRH, and (4) the catabolism does not interfere with the developmental changes undergone by the mGnRH and mHypGnRH brain contents.

The olfactory organ modulates gonadotropin-releasing hormone types and nest-building behavior in the tilapia Oreochromis niloticus.

Direct olfactory inputs to any of the known gonadotropin-releasing hormone (GnRH) containing neurons have not been demonstrated. Therefore, the rationale of this study was to examine whether olfactory inputs might in some way interact with the GnRH system(s) to synchronize reproductive behaviors. In order to establish this, we used anosmic mature male tilapia to investigate changes in reproductive behaviors, gonadal morphology, and GnRH1, GnRH2, and GnRH3 cellular morphology and change in GnRH mRNA levels by real-time polymerase chain reaction. Bilateral removal of the olfactory rosettes followed by occlusion of the nasal cavity (ORX) inhibited nest-building behavior, but had no effect on aggressive and sexual behaviors or gonadal morphology. ORX failed to alter the morphological features of GnRH1, GnRH2, and GnRH3 (cell number, size, GnRH optical density), but significantly decreased copies of GnRH1 and GnRH2 mRNAs. GnRH immunoreactive fibers were not evident in the olfactory nerve and rosettes. DiI application to the olfactory nerve labeled inputs primarily to the glomerular layer of the olfactory bulbs and extrabulbar inputs to the forebrain but not to GnRH neurons. These results provide evidence that the olfactory rosette is crucial for modulating nest-building behavior through second-order olfactory pathways interacting with GnRH1 and GnRH2 neuronal systems.

Evolutionary aspects of GnRHs, GnRH neuronal systems and GnRH receptors in teleost fish.

Gonadotrophin-releasing hormone (GnRH) was originally believed to be released by a unique set of hypophysiotrophic neurons to stimulate the release of gonadotrophins from the pituitary, therefore acting as a major initiator of the hormonal cascade controlling the reproductive axis. However, it now appears that each vertebrate species expresses two or three GnRH forms in multiple tissues and that GnRHs exert pleiotropic actions via several classes of receptors. This new vision of the GnRH systems arose progressively from numerous comparative studies in all vertebrate classes, but fish in general, and teleosts in particular, have often plaid a leading part in changing established concepts. To date fish still appear as attractive models to decipher the evolutionary mechanisms that led to the diversification of GnRH functions. Not only do teleosts exhibit the highest variety of GnRH variants, but recent data and whole genome analyses indicate that they may also possess multiple GnRH receptors. This paper intends to summarize the current situation with special emphasis on interspecies comparisons which provide insights into the possible evolutionary mechanisms leading to the diversification of GnRH functions.

Seasonal variation of the three native gonadotropin-releasing hormone messenger ribonucleic acids levels in the brain of female red seabream.

We studied the seasonal variation of the expression of genes encoding the three native gonadotropin-releasing hormones (GnRHs), namely salmon(s) GnRH, chicken(c) GnRH-II, and seabream(sb) GnRH in red seabream, Pagrus (Chrysophrys) major, in order to better understand the regulatory mechanisms of GnRH gene expression by environmental and endocrine factors. Female red seabream, reared under natural conditions, were collected monthly or bimonthly from October to June, and the levels of the three distinct GnRH messenger ribonucleic acids (mRNAs) in the brains of those fish (n = 4-6) were determined by ribonuclease (RNase) protection analysis. The levels of sbGnRH mRNA correlated well with the observed ovarian histology; the levels of sbGnRH mRNA of immature fish in October and December were low, and increased in February and March in conjunction with active vitellogenesis. The sbGnRH mRNA levels reached a maximum level in April (spawning season), after which they rapidly decreased together with the observed ovarian regression in June. In contrast, the levels of sGnRH mRNA showed no variation, while those of cGnRH-II mRNA were elevated only slightly in March and April. The increase in sbGnRH mRNA levels correlates with the increase in day length, water temperature and serum steroids levels, suggesting that these factors are candidates for regulators of sbGnRH synthesis.

Cloning and characterization of cDNAs encoding the GnRH1 and GnRH2 precursors from bullfrog (Rana catesbeiana).

We have isolated the cDNAs encoding the GnRH1 and GnRH2 precursors, respectively, from bullfrog (Rana catesbeiana) brain. The first cDNA consists of 648 bp and contains an open-reading frame of 270 nucleotides, encoding the bullfrog GnRH1 precursor. The second cDNA consists of 1053 bp and contains an open-reading frame of 255 nucleotides, encoding the bullfrog GnRH2 precursor. Both types of bullfrog GnRH precursor have a similar molecular architecture as observed in other GnRH precursors, consisting of a signal peptide, followed by the GnRH decapeptide, a conserved carboxy-terminal amidation and proteolytical processing site, and a GnRH-associated peptide (GAP). In addition, we have identified a third cDNA, containing 24 additional nucleotides in its GAP-coding region. Genomic PCR and sequence analysis confirmed that this cDNA represents an alternative splice variant of the bullfrog GnRH2-precursor pre-mRNA. The bullfrog GnRH1 precursor exhibits 60% and less than 40% amino acid identity to its Xenopus and mammalian counterparts, respectively, whereas the bullfrog GnRH2 precursor displays 50% to 60% amino acid identity to that of its nonmammalian counterparts, but shares only 25% amino acid identity with its mammalian counterparts. Northern blot analysis revealed a single GnRH1-precursor mRNA species of approximately 0.75 kilobases, expressed in bullfrog forebrain, and a single GnRH2-precursor mRNA species of approximately 1.1 kilobases, expressed in bullfrog midbrain/hindbrain. Furthermore, both bullfrog GnRH-precursor mRNAs exhibited a differential spatiotemporal expression pattern. Genomic Southern blot analysis indicated that both bullfrog GnRH genes are present as single copy genes. This is the first report on the molecular cloning of a GnRH2-precursor cDNA from an amphibian species. In addition, we present data showing that alternative splicing is utilized to generate different GnRH2-precursor mRNAs. J. Exp. Zool. 289:190-201, 2001.