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Molecular forms of GnRH in three model fishes: Rockfish, medaka and zebrafish
Abstract
Three species of fish have become important in the study of reproduction and development. Rockfish are a model for developmental studies of live-bearing perch-like fish, whereas medaka and zebrafish are models for developmental and genetic studies. The forms of GnRH are identified in the brains of each of these fish and in the pituitary of the rockfish to investigate the role of GnRH in reproduction. Here, we report that grass rockfish ( Sebastes rastrelliger ) have three forms of GnRH in brain extracts as determined by HPLC elution position and RIA. These forms are identified as sea bream GnRH, chicken GnRH-II and salmon GnRH. In contrast, only two forms of GnRH were detected in brain extracts of medaka ( Oryzias latipes ) and zebrafish ( Brachydanio rerio ): salmon GnRH and chicken GnRH-II. Rockfish is distinct from medaka and zebrafish in that the most abundant form of GnRH in the rockfish pituitary is sea bream GnRH, whereas this form is absent in the other two fishes. The identification of sea bream GnRH in the rockfish brain and pituitary extracts indicates that the phylogenetic emergence of sea bream GnRH is earlier than the order Perciformes.
Journal of Endocrinology (1996) 150, 17–23
... Research up until now using diagnostic methods like RIA, HPLC, in situ hybridization, etc., has confirmed the occurrence of only two GnRH formes (GnRH2, GnRH3) in the members of the family Cyprinidae, e.g. goldfish (Carassius carassius), (Peter et al., 1991), roach (Rutilus rutilus), (Penlington at al., 1997) and zebra danio (Danio rerio), (Powell et al., 1996;Steven et al., 2003;Palevitch et al., 2007). However, in some species the three GnRH forms were detected simultaneously, e.g., gilthead seabream (Sparus aurata), (Powell et al., 1994). ...
... It seems that it is mainly GnRH3 line, which compensates for the LH inducing role of the missing GnRH1 line in Cyprinidae. The projection of pre-optic GnRH3 neuronal axons into the pituitary (Kobayashi et al., 1997) and the fact that it is the more abundant form in the goldfish pituitary (Powell et al., 1996;Steven et al., 2003) confirm this assumption. Line 3 comprises only the sGnRH form (Sherwood et al., 1983) found only in Teleostei. ...
... After the application of exogenous cGnRH-II, its effect on sexual behaviour (Volkoff and Peter, 1999) and its inhibitory effect on food intake in goldfish (Matsuda et al., 2008) has been demonstrated. In terms of LH secretion stimulation cGnRH-II is more effective as compared to the hypophysiotropic sGnRH form (Illing et al., 1999), but with regard to the low cGnRH-II content in the pituitary (Powell et al., 1996;Steven et al., 2003) its impact on the LH level in plasma is minimal. A wide conservation of cGnRH-II in vertebrate species suggests an important role, although it has not been elucidated clearly until now. ...
Gonadotropin-releasing hormone in Cyprinidae as in other Vertebrates functions as a brain signal which stimulates the secretion of luteinizing hormone from the pituitary gland. Two forms of gonadotropin-releasing hormone have been identified in cyprinids, chicken gonadotropin-releasing hormone II and salmon gonadotropin- releasing hormone. Hypohysiotropic functions are fulfilled mainly by salmon gonadotropin-releasing hormone. The only known factor having an inhibitory effect on LH secretion in the family Cyprinidae is dopamine. Most cyprinids reared under controlled conditions exhibit signs of reproductive dysfunction, which is manifested in the failure to undergo final oocyte maturation and ovulation. In captivity a disruption of endogenous gonadotropin- releasing hormone stimulation occurs and sequentially that of luteinizing hormone, which is indispensible for the final phases of gametogenesis. In addition to methods based on the application of exogenous gonadotropins, the usage of a method functioning on the basis of hypothalamic control of final oocyte maturation and ovulation has become popular recently. The replacement of natural gonadotropin-releasing hormones with chemically synthe- sized gonadotropin-releasing hormone analogues characterized by amino acid substitutions at positions sensitive to enzymatic degradation has resulted in a centuple increase in the effectiveness of luteinizing hormone secretion induction. Combining gonadotropin-releasing hormone analogues with Dopamine inhibitory factors have made it possible to develop an extremely effective agent, which is necessary for the successful artificial reproduction of cyprinids.
... Desde el descubrimiento de la GnRH de mamíferos (mGnRH,), se han identificado 14 isoformas diferentes de GnRH en vertebrados ( King y Millar, 1982a; King y Millar, 1982b; Sherwood et al., 1983; Sower et al., 1993; Powell et al., 1994; Powell et al., 1996a; JimenezLiñan et al., 1997; Carolsfeld et al., 2000; Okubo et al., 2000; Yoo et al., 2000; Montaner et al., 2001), si bien la presencia de moleculas de tipo GnRH se ha puesto también de manifiesto en protocordados e invertebrados (Powell et al., 1996b, Zhang et al., 2000 Iwakoshi et al., 2002 y Adams et al., 2003). Estas formas de GnRH han recibido tradicionalmente el nombre de la especie en la que han sido puestas de manifiesto por primera vez, si bien pueden estar presentes en otras especies distintas. ...
... No obstante, y como en otros perciformes, la forma sbGnRH es la más abundante en la hipófisis de la lubina (González-Martínez et al., 2002a) y parece representar la forma funcional en la regulación de la síntesis y secreción de gonadotrofinas. Hoy en día, existen evidencias de que la expresión de 3 formas distintas de GnRH en el cerebro de teleósteos no está restringida a perciformes y es, probablemente, una característica de todos los teleósteos, ya que también se han detectado 3 isoformas de GnRH distintas en clupeiformes (Clupea harangus, Carosfeld et al., 2000), caraciformes (Piaractus mesopotamicus, Powell et al., 1997), salmoniformes (Coregonus clupeaformis, Adams et al., 2002), ateriniformes (Odonthestes bonariensis, Montaner et al., 2001), sinbranchiformes (Synbranchus marmoratus, Somoza et al., 2002), beloniformes (Oryzias latipes, Okubo et al., 2000), ciprinodontiformes (Xiphophorus maculatus, Xiphophorus helleri, Somoza et al., 2002), escorpeniformes (Sebastes rastrelliger, Powell et al., 1996a), pleuronectiformes (Scophthalmus maximus, Anderson et al., 2001;Verasper moseri, Amano et al., 2002) y tetraodontiformes (Fugu rubripes, Aparicio et al., 2002, Lethimonier et al., 2004). Además, la presencia de una tercera forma de GnRH ha sido sugerida en otros vertebrados, incluidos los humanos (Sherwood et al., 1986; Montaner et al., 1998; Yahalom et al., 1999). ...
... Two GnRH forms, GnRH2 and GnRH3, have been identified in the zebrafish brain using HPLC analysis ( Powell et al., 1996). Several years later, two genes encoding these forms of GnRH were isolated and characterized (Torgersen et al., 2002;Steven et al., 2003). ...
... Additionally, levels of GnRH3 peptide in the adult zebrafish pituitary were shown to be 3-to 4-fold higher than those of GnRH2. Therefore, GnRH3 is considered to be the hypophysiotropic form ( Powell et al., 1996;Steven et al., 2003). ...
Development and function of the forebrain gonadotropin-releasing hormone (GnRH) neuronal system has long been the focus of study in various vertebrate species. This system is crucial for reproduction and an important model for studying tangential neuronal migration. In addition, the finding that multiple forms of GnRH exist in the CNS as well as in non-CNS tissues, coupled with the fact that GnRH fibers project to many CNS regions, implies that GnRH has a variety of functions in addition to its classic reproductive role. The study of the GnRH system and its functions is, however, limited by available model systems and methodologies. The transgenic (Tg) GnRH3:EGFP zebrafish line, in which GnRH3 neurons express EGFP, allows in vivo study of the GnRH3 system in the context of the entire animal. Coupling the use of this line with the attributes and molecular tools available in zebrafish has expanded our ability to study the forebrain GnRH system. Herein, we discuss the use of the Tg(GnRH3:EGFP) zebrafish line as a model for studying forebrain GnRH neurons, both in developing larvae and in sexually mature animals. We also discuss the potential use of this line to study regulation of GnRH3 system development.
... In addition, a form of GnRH are present in the teleost brain. Additionally, in the brains of the most advanced fish, the perchlike fish, three forms of GnRH have been identified by primary structure [7,8], cDNA coding [9,10] or antisera performance liquid chromatography (HPLC) [11,12]. ...
... JFFP sbGnRH is present not only in pacu, but also in the and JC were supported by British Columbia Science brains of several species of perchlike fish: snook [11], sea Council Fellowships. bream [7], sunfish, cichlids [8,10] and rockfish [12]. This suggests that sbGnRH was present in stem line ancestors for both pacu and the perchlike groups before they ...
Perchlike fish are a vast group of advanced teleosts. The species examined to date have three forms of gonadotropin-releasing hormone (GnRH) within a single species, but the origin of the third GnRH peptide is unknown. In this study, the primary structure of three GnRH peptides is determined from the brain of the pacu, Piaractus mesopotamicus, an example of a teleost that is less advanced than the perchlike fish. The GnRH was purified from pacu brain extracts using high performance liquid chromatography (HPLC) and radioimmunoassay (RIA). The three forms identified by chemical sequencing and mass spectrometry are sea bream GnRH (pGlu-His-Trip-Ser-Tyr-Gly-Leu-Ser -Pro-Gly-NH2, 1113.4 Da); chicken GnRH-II (pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2, 1236.6 Da); and salmon GnRH (pGlu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-Gly-NH2, 1212.3 Da). In addition the number of forms of GnRH in the brains of male and female fish was determined separately. The same three forms of GnRH were present in the brains of both sexes as determined by antisera cross-reactivity and elution position from the HPLC column. The results indicate that the pacu brain has the identical forms of GnRH identified in perchlike fish and hence, the origin of three forms occurred earlier in evolution than previously thought.
... At least two GnRH forms are present in the brain of all vertebrates [(commonly cGnRH-II (GnRH-II), plus one of either GnRH-I or GnRH-III)] Karigo and Oka, 2013) [4,32] . Teleost fish of the order Perciformes were the first group of vertebrates in which three GnRH forms were found based on immunological and molecular characterization: salmon GnRH (sGnRH; GnRH-III), chicken GnRH-II (cGnRH-II; GnRH-II), and seabream GnRH (sbGnRH; GnRH-I) (Powell et al., 1994(Powell et al., , 1996Dubois et al., 2002;Lethimonier et al., 2004;Kah et al., 2007;Okubo and Nagahama, 2008;Selvaraj et al., 2013) [67-68, 13, 41, 31, 61, 78] . Particularly, GnRH neurons distributed in the preoptic area (POA) and the hypothalamus (HYP) regions of the brain are shown to be involved in the stimulation of pituitary gonadotrophic hormones (GtHs), folliclestimulating hormone (FSH) and luteinizing hormone (LH) (Yamamoto et al., 1998;Schulz et al., 2001;Swanson et al., 2003;Shahjahan et al., 2010) [103,74,90,81] . ...
... In teleost fish, depending on the species, two or three different variants of GnRH are expressed in the brain (Gothilf et al., 1995;. In zebrafish (Danio rerio), a popular teleost model, two GnRH variants have been identified (Powell et al., 1996) and characterized (Steven et al., 2003): GnRH2 ...
... It appears that with the evolutionary loss of GnRH1, the remaining isoform takes over its function and initiate a compensatory response, which influences the production of gonadotropins, which activated to maintain reproduction (Karigo & Oka, 2013;Muñoz-Cueto et al., 2020). Therefore, it can be suggested that GnRH3 serves as the hypophysiotropic form in this species rather than GnRH1, as has been reported in studies on other species of the order Cypriniformes (Abraham, Palevitch, Gothilf, & Zohar, 2009;Corchuelo et al., 2017;Powell, Krueckl, Collins, & Sherwood, 1996). ...
Gonadotropin‐releasing hormone (GnRH) is a key molecule in the initiation of the hypothalamic–pituitary–gonadal axis. Thus, knowledge about GnRH may contribute to the effectiveness of species reproduction. Using a Neotropical tetra Astyanax altiparanae as a fish model species, the GnRH forms were characterized at the molecular level and the role of injected GnRHs in vivo was evaluated. The full‐length complementary DNA (cDNA) sequences of preproGnRH2 (612 bp) and preproGnRH3 (407 bp) of A. altiparanae were obtained, and the GnRH1 form was not detected. The cDNA sequences of preproGnRH2 and preproGnRH3 were found to be conserved, but a change in the amino acid at position 8 of the GnRH3 decapeptide of A. altiparanae was observed. All the injected GnRHs stimulated lhβ messenger RNA (mRNA) expression but not fshβ mRNA expression, and only GnRH2 was able to increase maturation‐inducing steroid (MIS) levels and possibly stimulate oocyte release. Furthermore, only GnRH2 was able to start the entire reproductive hormonal cascade and induce spawning. cDNA sequences of preproGnRH2 and preproGnRH3 in A. altiparanae are found to be conserved. All GnRH forms stimulated the lhβ but not the fshβ mRNA expression. Only GnRH2 was able to increase the MIS level and stimulate the release of gametes.
... Zebrafish produce two GnRHs -GnRH2 and GnRH3 [170]. gnrh3 is the hypophysiotropic GnRH in zebrafish [171,172]. ...
Sexual behaviour in teleost fish is highly plastic. It can be attributed to the relatively few sex differences found in adult brain transcriptomes. Environmental and hormonal factors can influence sex-specific behaviour. Androgen treatment stimulates behavioural masculinisation. Sex dimorphic gene expression in developing teleost brains and the molecular basis for androgen-induced behavioural masculinisation are poorly understood. In this study, juvenile zebrafish (Danio rerio) were treated with 100 ng/L of 17 alpha-methyltestosterone during sexual development from 20 days post fertilisation to 40 days and 60 days post fertilisation. We compared brain gene expression patterns in MT-treated zebrafish with control males and females using RNA-Seq to shed light on the dynamic changes in brain gene expression during sexual development and how androgens affect brain gene expression leading to behaviour masculinisation. We found modest differences in gene expression between juvenile male and female zebrafish brains. Brain aromatase (cyp19a1b), prostaglandin 3a synthase (ptges3a) and prostaglandin reductase 1 (ptgr1) were among the genes with sexually dimorphic expression patterns. MT treatment significantly altered gene expression relative to both male and female brains. Fewer differences were found among MT-treated brains and male brains compared to female brains, particularly at 60 dpf. MT treatment upregulated the expression of hydroxysteroid 11-beta dehydrogenase 2 (hsd11b2), deiodinase, iodothyronine, type II (dio2), gonadotrophin releasing hormones (GnRH) 2 and 3 (gnrh2 and gnrh3) suggesting local synthesis of 11-ketotestosterone, triiodothyronine and GnRHs in zebrafish brains which are influenced by androgens. Androgen, estrogen, prostaglandin, thyroid hormone and GnRH signalling pathways likely interact to modulate teleost sexual behaviour.
... With respect to their function, GnRH1 is associated with gonadotropin secretion (Schwanzel-Fukuda and Pfaff, 1990); GnRH2 appears to be involved in the regulation of sexual and feeding behavior (Volkoff and Peter, 1999;Temple et al., 2003;Matsuda et al., 2008) and GnRH3 is associated with neural mechanisms of sexual behavior (Eisthen et al., 2000;Biju et al., 2003Biju et al., , 2005. In cyprinids, such as Danio rerio, two Gnrh forms were described in the brain; Gnrh2 in diencephalon and midbrain, and Gnrh3 in the ventral forebrain (Powell et al., 1996;Abraham et al., 2009). ...
Gonadotropin releasing hormone (GnRH) is one of the key players of brain-pituitary-gonad axis, exerting overall control over vertebrate reproduction. In zebrafish, two variants were characterized and name as Gnrh2 and Gnrh3. In this species, Gnrh3, the hypohysiotropic form, is expressed by neurons of the olfactory-retinal system, where it is related with food detection, intra/interspecific recognition, visual acuity and retinal processing modulation. Previous studies have reported the presence of Gnrh receptors in the zebrafish retina, but not yet in the zebrafish olfactory epithelium. The current study analyzed the presence of gnrh2 and gnrh3, their receptors (gnrhr 1,2,3 and 4) and gnih (gonadotropin inhibitory hormone) transcripts, as well as the Gnrh3 protein in the olfactory epithelium (OE), olfactory bulb (OB), retina and ovary during zebrafish ovarian maturation. We found an increase of gnrh receptors transcripts in the OE at the final stages of ovarian maturation. In the OE, Gnrh3 protein was detected in the olfactory receptor neurons cilia and in the olfactory nerve fibers. Interestingly, in the OB, we found an inverse expression pattern between gnih and gnrh3. In the retina, gnrhr4 mRNA was found in the nuclei of amacrine, bipolar, and ganglion cells next to Gnrh3 positive fibers. In the ovary, gnrh3, gnrhr2 and gnrhr4 transcripts were found in perinucleolar oocytes, while gnih in oocytes at the cortical alveolus stage. Our results suggested that Gnrh/Gnih elements are involved in the neuromodulation of the sensorial system particularly at the final stages of maturation, playing also a paracrine role in the ovary.
... The developmental origins of GnRH neurons in zebrafish as described above, two gnrH forms, gnrh2 and gnrh3, have been identified in the zebrafish brain. 33 The zebrafish gnrh2, similarly to other jawed vertebrates, is localized in the midbrain tegmentum, while gnrh3 is expressed in the olfactory placode-and in the Poa-hypothalamus. 25 Moreover, analysis of the adult zebrafish pituitary reveals levels of gnrh3 peptide 3-to 4-fold higher than those of gnrh2 confirming its hypophysiotropic role. ...
Gonadotropin-releasing hormone (GnRH) neurons have a pivotal role in the physiological functions of hypotahlamic-pituitary-gonadal (HPG) axis. The pulsatile releasing of GnRH hormone into the hypophyseal portal circulation at the median eminence represent the first domino in the HPG cascade of events that regulate the development, fertility and aging in all vertebrates. These neurons principally originate in the olfactory placode and migrate during early embryonal stages into the hypothalamus. Alterations in developmental processes or in the releasing of GnRH hormone lead to a rare and complex disorder of the reproductive axis called congenital hypogonadotropic hypogonadism (CHH). Genetic screening of human patients and the use of model systems have led to the identification of several genes involved in the CHH pathogenesis underlying its oligogenic nature. Nevertheless CHH remains, for a large cohort of patients, idiopathic and GnRH neurogenesis processes not fully understood. This is due to intrinsic difficulties that exist in the analysis of earliest embryonic developmental stages and in the methodologies developed to study the CHH-causing genes. In this regard, zebrafish embryos, on account of its external fertilization and development, allow a real-time analysis that could overcome some of the above mentioned limitations. Moreover, the recent availability of several transgenic zebrafish reporter lines makes it an excellent model for the study of the oligogenic mechanisms leading to CHH.
... Salmon GnRH (sGnRH, or GnRH3 in zebrafish) is localized to the olfactory bulb-terminal nerve as well as to the preoptic area (POA) in the hypothalamus [9,10]. The sGnRH has been shown to elicit GTH-releasing activity and is considered to be the hypophysiotropic form [10,11]. ...
Reproductive homing migration of salmonids requires accurate interaction between the reception of external olfactory cues for navigation to the spawning grounds and the regulation of sexual maturation processes. This study aimed at providing insights into the hypothesized functional link between olfactory sensing of the spawning ground and final sexual maturation. We have therefore assessed the presence and expression levels of olfactory genes by RNA sequencing (RNAseq) of the olfactory rosettes in homing chum salmon Oncorhynchus keta Walbaum from the coastal sea to 75 km upstream the rivers at the pre-spawning ground. The progression of sexual maturation along the brain-pituitary-gonadal axis was assessed through determination of plasma steroid levels by time-resolved fluoroimmunoassays (TR-FIA), pituitary gonadotropin subunit expression and salmon gonadotropin-releasing hormone (sgnrh) expression in the brain by quantitative real-time PCR. RNAseq revealed the expression of 75 known and 27 unknown salmonid olfactory genes of which 13 genes were differentially expressed between fish from the pre-spawning area and from the coastal area, suggesting an important role of these genes in homing. A clear progression towards final maturation was characterised by higher plasma 17α,20β-dihydroxy-4-pregnen-3-one (DHP) levels, increased pituitary luteinizing hormone β subunit (lhβ) expression and sgnrh expression in the post brain, and lower plasma testosterone (T) and 17β-estradiol (E2) levels. Olfactomedins and ependymin are candidates among the differentially expressed genes that may connect olfactory reception to the expression of sgnrh to regulate final maturation.
... It is important to note that there is growing evidence to indicate that multiple forms of GnRH will not be limited to perciform fish. Both HPLC and immunocytochemical studies have demonstrated that multiple forms of GnRH are present in non-perciform fish (Scorpaeniformes (Powell et al. 1996) and Characiformes (Somoza et al. 1994)), amphibians (Conlon et al. 1993, Iela et al. 1996), reptiles (Negrin & Bello 1996), birds (Norgren & Gao 1994) and even mammals (Lescheid et al. 1997). This recent discovery, that primate brains contain three forms of GnRH (mammalian GnRH (mGnRH), cGnRH-II and an unidentified GnRH form), clearly suggests that the presence of multiple forms of GnRH is likely to be prevalent in most, if not all, vertebrate species. ...
GnRH is a neuropeptide which plays an essential role in the control of reproductive fitness for all vertebrates. Increasing evidence suggests that multiple forms of GnRH may exist in most vertebrate brains. Southern blot analysis of the three GnRHs known to be present in perciform fish, the seabream (sb)GnRH, the salmon(s) GnRH and the chicken (c) GnRH-II, demonstrates that each is present as a single gene copy in the genome of the striped bass, Morone saxatilis. In order to investigate the physiological consequences of multiple GnRHs in a single vertebrate species, we have isolated and characterized two of the GnRH genes, those for sbGnRH and cGnRH-II. Computer analysis of 3.5 kb of sequence upstream of the sbGnRH gene reveals a number of consensus DNA binding sites which implicate steroids, such as estrogen and glucocorticoids, and the steroidogenic transcription factor, SF-1, as being involved in the regulation of sbGnRH gene expression. Sequence analysis of the cGnRH-II gene reveals evidence of multiple promoters. Expression studies using (1) solution hybridization-RNAse protection mapping with several RNA probes directed at various regions of the proGnRH gene, (2) primer extension assays using two specific oligonucleotide primers, and (3) reverse transcription PCR with several oligonucleotide primers on cGnRH-II transcripts demonstrate that the cGnRH-II gene initiates transcription at numerous sites using a TATA-less promoter within the brains of sexually mature striped bass. This study is the first to characterize and compare the promoter structures of two GnRH genes present in a single vertebrate species.
... In most ancient teleosts, this form is the mGnRH (eel, sturgeon). In teleosts that evolved later, the prevalent second form is sGnRH (zebrafish, salmon, medaka) (Steven et al., 2003;Powell et al., 1996;Amano et al., 2002). ...
... Only the partial genome of zebrafish (Danio rerio) has been sequenced to date, but it confirms that only two forms of GnRH are present: sGnRH and cGnRH-II. 9 Additionally, the genes for these two GnRHs have been isolated and sequenced. 10,11 To date, only one copy of each zebrafish GnRH gene has been identified in genome analysis or experimentally. ...
Fish, the largest group of vertebrates, use diverse reproductive strategies. Considerable advances have been made in understanding the structure and role of GnRH, the main hormone regulating reproduction. Twenty four distinct peptide structures are known for GnRH from octopus to human. Genes and cDNAs have been cloned and sequenced for both invertebrates and vertebrates. Genomic studies are being used to verify the number and type of GnRH forms and to look for novel forms. Mapping of the evolutionary pathway of GnRH orthologs and paralogs has just begun, but is advancing with the combined use of structures for the peptide, cDNA and gene structures. Phylogenetic analysis, distribution patterns and linkage studies in individual species will help to define the relationships and possibly the orthologs for the GnRH molecule. Expression studies suggest that GnRH is widely expressed outside of the classical brain areas of the olfactory brain, telencephalon, preoptic area and midbrain. An emerging area of interest in fish reproduction is GnRH gene regulation in which upstream and downstream promoters, transcription factors, cell-specific expression and duplicate genes have been studied.
... The zebrafish possesses two molecular forms of GnRH: GnRH2 (pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH 2 ), and GnRH3 (pGlu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-Gly-NH 2 ; Powell et al., 1996;Steven et al., 2003). Therefore, in order to examine the effect of ICV injection of GnRH2 on food intake, GnRH2 was purchased from Bachem AG (Bubendorf, Switzerland) and used. ...
Gonadotropin-releasing hormone (GnRH) is an evolutionarily conserved neuropeptide with 10 amino acid residues, of which several structural variants exist. A molecular form known as GnRH2 ([His⁵ Trp⁷ Tyr⁸]GnRH, also known as chicken GnRH II) is widely distributed in vertebrates except for rodents, and has recently been implicated in the regulation of feeding behavior in goldfish. However, the influence of GnRH2 on feeding behavior in other fish has not yet been studied. In the present study, therefore, we investigated the role of GnRH2 in the regulation of feeding behavior in a zebrafish model, and examined its involvement in food intake after intracerebroventricular (ICV) administration. ICV injection of GnRH2 at 0.1 and 1 pmol/g body weight (BW) induced a marked decrease of food consumption in a dose-dependent manner during 30 min after feeding. Cumulative food intake was significantly decreased by ICV injection of GnRH2 at 1 pmol/g BW during the 30-min post-treatment observation period. The anorexigenic action of GnRH2 was completely blocked by treatment with the GnRH type I receptor antagonist Antide at 25 pmol/g BW. We also examined the effect of feeding condition on the expression level of the GnRH2 transcript in the hypothalamus. Levels of GnRH2 mRNA obtained from fish that had been provided excess food for 7 days were higher than those in fish that had been fed normally. These results suggest that, in zebrafish, GnRH2 acts as an anorexigenic factor, as is the case in goldfish.
... The existence of three GnRH forms was confirmed, either by cDNA sequencing or biochemical characterization, in other perciform species including the African cichlid, Haplochromis burtoni (White et al., 1995), the red seabream, Pagrus major, the black seabream, Acanthopagrus schlegeli, the striped knifejaw, Oplegnathus fasciatus, and the Nile tilapia, Oreochromis niloticus (Senthilkumaran et al., 1999). However, recent studies also indicate that sbGnRH is not restricted to perciforms since it was also found in a scorpaeniform, Sebastes rastrelliger (Powell et al., 1996a) and a characiform, Piaractus mesopotamicus (Powell et al., 1997). In the African cichlid (White et al., 1995; and the red seabream (Okuzawa et al., 1997), distribution of cells expressing the different GnRHs was found to be similar to that reported in the gilthead seabream by Gothilf et al. (1996), indicating a neuroanatomical segregation of the different GnRH systems. ...
The expression sites of three prepro-gonadotrophin-releasing hormones (GnRHs), corresponding to seabream GnRH (sbGnRH: Ser8-mGnRH, mammalian GnRH), salmon GnRH (sGnRH: Trp7Leu8-mGnRH), and chicken GnRH-II (cGnRH-II: His5Trp7Tyr8-mGnRH) forms were studied in the brain of a perciform fish, the European sea bass (Dicentrarchus labrax) by means of in situ hybridization. The riboprobes used in this study correspond to the three GnRH-associated peptide (GAP)-coding regions of the prepro-GnRH cDNAs cloned from the same species (salmon GAP: sGAP; seabream GAP: sbGAP; chicken GAP-II: cIIGAP), which show little oligonucleotide sequence identity (sGAP versus sbGAP: 42%; cIIGAP versus sbGAP: 36%; sGAP versus cIIGAP: 41%). Adjacent paraffin sections (6 mm) throughout the entire brain were treated in parallel with each of the three anti-sense probes and the corresponding sense probes, demonstrating the high specificity of the hybridization signal. The results showed that both sGAP and sbGAP mRNAs had a broader expression in the olfactory bulbs, ventral telencephalon, and preoptic region, whereas cIIGAP mRNA expression was confined to large cells of the nucleus of the medial longitudinal fascicle. In the olfactory bulbs, both the signal intensity and the number of positive cells were higher with the sGAP probe, whereas sbGAP mRNA-expressing cells were more numerous and intensely stained in the preoptic region. Additional isolated sbGAP-positive cells were detected in the ventrolateral hypothalamus. These results demonstrate a clear overlapping of sGAP- and sbGAP-expressing cells in the forebrain of the European sea bass, in contrast to previous reports in other perciforms showing a clear segregation of these two cell populations. J. Comp. Neurol. 429:144–155, 2001. © 2000 Wiley-Liss, Inc.
... In teleosts, as in all gnathostomes, chicken GnRH-II (cGnRH-II) is systematically found, while the second form is either mammalian GnRH (mGnRH) (King et al. 1990;Lescheid et al. 1995;O'Neill et al. 1999;Sherwood et al. 1991), catfish GnRH (cfGnRH) (Ngamvongchon et al. 1992), or salmon GnRH (sGnRH) (Powell et al. 1996;Ravaglia et al. 1997;Yu et al. 1988). The ocurrence of a third variant as seabream GnRH (sbGnRH), or herring GnRH (hrGnRH) has also been reported (Carolsfeld et al. 2000;Powell et al. 1994). ...
The brain of the pejerrey (Odontesthes bonariensis) has recently been shown to contain three forms of gonadotropin-releasing hormone (GnRH): salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and pejerrey GnRH (pjGnRH), nevertheless neuroanatomical studies on the distribution of these peptides are lacking. In this study we investigated the distribution of immunoreactive GnRH in the brain of adult pejerrey. Four different policlonal antisera and a monoclonal antibody against different GnRH variants were applied on cryosections and visualized using the ABC method. Three antisera (PBL#49, sGnRH#2 and cII741) revealed three different immunoreactive areas: the terminal nerve ganglion (at the junction between the olfactory bulbs and the anterior telencephalon), the preoptic area just anterior to the hypothalamus and the midbrain tegmentum. Fibers immunoreactive to GnRH were detected in different brain areas: the olfactory bulbs, the ventral thelencephalon, the hypothalamus, the mesencephalic area and an important innervation entering into the pituitary gland. Two other antibodies (LRH13 and s1668) labeled the two nuclei corresponding to the forebrain but not the midbrain tegmentum. As both antibodies have low crossreactivity to cGnRH-II, the data suggest that this group of cells express cGnRH-II. In summary, three different areas with immunoreactivity to GnRH were detected in the pejerrey brain. The distribution of sGnRH, pjGnRH and cGnRH-II expressing neurons, is discussed.
... Two or three unique GnRH variants have been identified in the central nervous system of some teleost fish (Somoza et al., 2002;Dubois et al., 2002;Nabissi et al., 2000;Amano et al., 2002). The Cyprinid fish family, to which the common carp belongs, has two forms of GnRH molecules: salmon GnRH (sGnRH) and chicken type-II GnRH (cGnRH-II; Penlington et al., 1997;Lin and Peter, 1996;Powell et al., 1996;Steven et al., 2003;Li et al., 2004aLi et al., , 2004b, which show regional differences in brain expression (e.g., the anterior parts of the cyprinid brain express more sGnRH than cGnRH-II; Yu et al., 1998). In addition to the brain, sGnRH present peripherally in terminal nerves is believed to be a neuron regulator of the reproductive system (Fernald and White, 1999). ...
Generating transgenic fish with desirable traits (e.g., rapid growth, larger size, etc.) for commercial use has been hampered by concerns for biosafety and competition if these fish are released into the environment. These obstacles may be overcome by producing transgenic fish that are sterile, possibly by inhibiting hormones related to reproduction. In vertebrates, synthesis and release of gonadotropin (GtH) and other reproductive hormones is mediated by gonadotropin-releasing hormone (GnRH). Recently two cDNA sequences encoding salmon-type GnRH (sGnRH) decapeptides were cloned from common carp (Cyprinus carpio). This study analyzed the expression of these two genes using real-time polymerase chain reaction (RT-PCR) in different tissues carp at varying developmental stages. Transcripts of both genes were detected in ovary and testis in mature and regressed, but not in juvenile carp. To evaluate the effects of sGnRH inhibition, the recombinant gene CAsGnRHpc-antisense, expressing antisense sGnRH RNA driven by a carp beta-actin promoter, was constructed. Blocking sGnRH expression using antisense sGnRH significantly decreased GtH in the blood of male transgenic carp. Furthermore, some antisense transgenic fish had no gonadal development and were completely sterile. These data demonstrate that sGnRH is important for GtH synthesis and development of reproductive organs in carp. Also, the antisense sGnRH strategy may prove effective in generating sterile transgenic fish, eliminating environmental concerns these fish may raise.
... Originally considered and named for its important reproductive function, two related GnRH gene-encoding decapeptides (GnRHs), which are not directly involved in HPG axis, have been identified in vertebrates. Within this family of three genes, more than 10 GnRH peptide variants have been identified Sower et al., 1993;Powell et al., 1994Powell et al., , 1996Jimenez-Linan et al., 1997;White & Fernald, 1998a;Carolsfeld et al., 2000;Okubo et al., 2000a;Yoo et al., 2000;Montaner et al., 2001;Adams et al., 2002). All GnRH forms are decapeptides with protein residues 1, 4, 9 and 10 conserved, and all have modified amino and carboxyl termini. ...
Gonadotropin-releasing hormone (GnRH) was originally identified because of its essential role in regulating reproduction in all vertebrates. Since then, three phylogenetically related GnRH decapeptides have been characterized in vertebrates and invertebrates. Almost all tetrapods investigated have at least two GnRH forms (GnRH1 and GnRH2) in the central nervous system. From distributional and functional studies in vertebrates, GnRH1 in the hypothalamus projects predominantly to the pituitary and regulates reproduction via gonadotropin release. GnRH2, which is located in the midbrain, projects to the whole brain and is thought to be involved in sexual behaviour and food intake. GnRH3, located in the forebrain, has only been found in teleost fish and appears to be involved in sexual behaviour, as well as, in some fish species, gonadotropin release. Multiple GnRH receptors (GnRH-Rs), G-protein-coupled receptors regulate endocrine functions and neural transmissions in vertebrates. Phylogenetic and structural analyses of coding sequences show that all vertebrate GnRH-Rs cluster into two main receptor types comprised of four subfamilies. This suggests that at least two rounds of GnRH receptor gene duplications may have occurred in different groups within each lineage. Functional studies suggest that two particular subfamilies of GnRH receptors have independently evolved to act as species-specific endocrine modulators in the pituitary, and these show the greatest variety in regulating neuron networks in the brain. Given the long evolutionary history of the GnRH system, it seems likely that much more remains to be understood about its roles in behaviour and function of vertebrates.
... Investigators have characterized 15 distinct vertebrate GnRH variants that are encoded by distinct genes. The hypophysiotrope-specific GnRH variant expressed in the mammalian hypothalamus is GnRH1, whereas it is GnRH3 in zebrafish (128,129). Rodent GnRH1-positive neurons are scattered throughout the rostral hypothalamus instead of forming distinct expression domains, which reflects mainly the unusual ontogenic origin of GnRH1 cells. GnRH1 neurons in mammals arise from the forebrain placodal region and migrate tangentially through the brain to ultimately reach hypothalamic target regions. ...
Since its introduction as a genetic vertebrate model system approximately 30 years ago, the focus of zebrafish research has increasingly shifted to questions that are also relevant for human development and disease. Here, we review the potential of the zebrafish as a model for human endocrine systems. A recent review compared the functions of the different endocrine systems and glands in zebrafish with those in other vertebrates, including humans, coming to the conclusion that major aspects are conserved. Here, we present an updated overview of this rapidly growing field of zebrafish research, focusing on the hypothalamo-pituitary axis, which links the central nervous system with the endocrine systems, and on major processes that are under (neuro)endocrine control and are the subject of intensive current research in other endocrine model organisms, such as feeding circuits and energy homeostasis, sleep, stress, reproduction, osmoregulation, and calcium homeostasis. Finally, we summarize the strengths and weaknesses of zebrafish as a model for studying endocrine systems.
... In teleost fish, depending on the species, two or three different variants of GnRH are expressed in the brain (Gothilf et al., 1995;Powell et al., 1994). In zebrafish (Danio rerio), a popular teleost model, two GnRH variants have been identified (Powell et al., 1996) and characterized (Steven et al., 2003): GnRH2 (corresponding to the chicken GnRH-II variant) that is synthesized in large neurons of the synencephalon and GnRH3, corresponding to the salmon GnRH variant (Kah et al., 1986;Lethimonier et al., 2004), which is expressed in a continuum of neurons extending from the olfactory bulbs to the anterior hypothalamus (Kah et al., 1986;Lethimonier et al., 2004). The GnRH3 decapeptide corresponds to the hypophysiotrophic variant that controls gonadotropin release (Steven et al., 2003), while the function of GnRH2 is less clear although in fish its roles in sexual behavior (Volkoff and Peter, 1999) and in modulation of pineal function (Servili et al., 2010) are documented. ...
Until now, studies dedicated to the actions of endocrine disrupting chemicals (EDCs) on the reproductive axis have been concerned with their effects at the gonadal level leaving their actions on neuroendocrine circuits controlling reproduction virtually unexplored. In vertebrates, gonadotropin-releasing hormone (GnRH) is the key factor controlling the activity of the reproductive axis. The development and functioning of GnRH neurons are finely tuned by a series of factors, notably sex steroids, making these neurons potential targets of EDCs, notably in aquatic species. By means of immunohistochemistry, we examined the effects of low levels of ethinylestradiol (EE2 0.02 nM, 0.1 nM, 0.5 nM), a potent synthetic estrogen, on early development (at 5, 10, 20, 30 days post-fertilization) of the forebrain GnRH neurons in a model fish species, the zebrafish (Danio rerio). In parallel, the ER-regulated expression of cytochrome P450 aromatase B (AroB) protein, which is encoded by the cyp19a1b gene, was precisely mapped at the brain and pituitary levels in developing control and EE2-exposed zebrafish. We show that EE2 disrupts the ontogeny of GnRH system by inducing an increase in the number of GnRH-ir neurons and GnRH fibers based on their immunoreactivity as well as a decrease in the size of the GnRH-ir soma and a modification of the migration profile of GnRH-ir neurons. Furthermore, we report a spectacular dose and time-dependent induction of AroB expression in radial glial cells of the developing brain further illustrating the extreme sensitivity of AroB to xenoestrogen and the relevance of AroB as biomarker of xenoestrogen effects on the central nervous system. Collectively, these original and relevant observations highlight the sensitivity of GnRH and AroB to a synthetic estrogen during embryogenesis. These data reinforce the need to further study the mechanisms underlying EDC effects on key neuroendocrine circuits involved in reproduction and brain development of vertebrates.
... This situation may be similar to that in GnRH systems: in most vertebrates, only one of the GnRH paralogous systems that synthesizes one of the three GnRH paralogs, GnRH1, is hypophysiotropic, and the other systems (GnRH2 and GnRH3) are nonhypophysiotropic and assume neuromodulatory functions (24). Interestingly, in cyprinid fish, the GnRH1 gene is lost and the GnRH2 and GnRH3 systems are hypophysiotropic (25,26). This situation may be similar to that of fish species that have lost kiss1 during evolution. ...
Kiss2, a paralogous gene for kiss1, has recently been identified in several vertebrates. However, their relative potencies for the regulation of reproductive functions appear to differ among species. Here we used medaka as a model animal to examine the kiss1 and kiss2 expression dynamics by in situ hybridization under different conditions: breeding or nonbreeding and ovariectomized or sham operated. Medaka kiss1-expressing neurons and kiss2-expressing neurons were mainly localized in two hypothalamic nuclei, nucleus ventralis tuberis (NVT) and nucleus recessus lateralis (NRL), respectively. NRL kiss2 expression did not change according to differences in breeding condition, whereas NVT kiss1 expression was strongly correlated with breeding condition. In addition, ovariectomy did not change kiss2 expression but significantly decreased the kiss1 expression. Moreover, double-label in situ hybridization revealed that NVT Kiss1 neurons coexpress estrogen receptor-alpha, whereas NRL Kiss2 neurons do not. From these results, we conclude that the NVT Kiss1 neurons are positively regulated by ovarian estrogen via their coexpressed estrogen receptor-alpha and are directly involved in the central regulation of reproduction in medaka. In contrast, we argue that the NRL Kiss2 neurons in medaka may serve nonreproductive functions. These functional differences between Kiss1 and Kiss2 neurons are discussed from a phylogenetic viewpoint.
... The GnRH3 axons project to various parts of the brain including the pituitary gland (Abraham et al., 2008). Furthermore, combined high performance liquid chromatography/radioimmunoassay analyses have indicated that the GnRH3 peptide is the predominant GnRH peptide in the adult zebrafish pituitary (Powell et al., 1996). Therefore, GnRH3 is considered the hypophysiotropic form of GnRH in zebrafish. ...
The initiation of puberty and the functioning of the reproductive system depend on proper development of the hypophysiotropic gonadotropin-releasing hormone (GnRH) system. One critical step in this process is the embryonic migration of GnRH neurons from the olfactory area to the hypothalamus. Using a transgenic zebrafish model, Tg(gnrh3:EGFP), in which GnRH3 neurons and axons are fluorescently labeled, we investigated whether zebrafish NELF is essential for the development of GnRH3 neurons. The zebrafish nelf cDNA was cloned and characterized. During embryonic development, nelf is expressed in GnRH3 neurons and in target sites of GnRH3 projections and perikarya, before the initiation of their migration. Nelf knockdown resulted in a disruption of the GnRH3 system which included absence or misguiding of GnRH3 axonal outgrowth and incorrect or arrested migration of GnRH3 perikarya. These results suggest that Nelf is an important factor in the developmental migration and projection of GnRH3 neurons in zebrafish.
... Furthermore, the preoptic specific seabream-GnRH antisense oligonucleotide probes did not form hybridization signals in the medaka brains. Similarly, by using high pressure liquid chromatography, Powell et al. (1996) failed to detect seabream-GnRH in the medaka brains. Thus, the medaka brains either lack a preoptic form of GnRH or might synthesize a preoptic molecular form different from the known forms of GnRH. ...
The origins of the different populations of gonadotropin-releasing hormone (GnRH)-containing neurons in the brains of two genotypes (HO4C; HNI-II) of medaka Oryzias latipes were analyzed at different stages of development (day 1 after fertilization through adulthood), by using oligonucleotide probes specific to salmon-, seabream-, and chicken II-GnRH mRNA and antisera against specific GnRH peptides. Between the two genotypes, there was no difference in the site and time of GnRH expression or the final pattern of GnRH neuronal organization. In the adult fish of both sexes, salmon GnRH mRNA and peptide-containing neurons were seen in the terminal nerve ganglia (nucleus olfactoretinalis; NOR) and chicken II-GnRH mRNA and peptide-containing neurons in the midbrain tegmentum. GnRH cells at the base of the olfactory placode (1-2 cells) and in the midbrain tegmentum were first seen in 1-day-old fish of both genotypes. On day 15, lightly immunoreactive GnRH cells were seen in the NOR of only HNI genotype. By day 30, GnRH expression in the NOR and in the midbrain was prominent. GnRH cells along the basal olfactory bulb and basal telencephalon were occasionally seen in animals 30 days or older. This developmental study shows differential distribution of salmon and chicken II-GnRH mRNA subtypes and emphasizes their separate embryonic origins from the olfactory apparatus (salmon-GnRH) and the ependymal cells of the third ventricle (chicken II-GnRH). The absence of preoptic GnRH hybridization signals, immunoreactivity and the lack of GnRH fibers in the pituitary suggests that the preoptic GnRH neurons are distinct from the olfactory derived-terminal nerve GnRH neurons, and that the GnRH neurites reported in the pituitary of teleost must be of preoptic origin.
... It is important to note that there is growing evidence to indicate that multiple forms of GnRH will not be limited to perciform fish. Both HPLC and immunocytochemical studies have demonstrated that multiple forms of GnRH are present in non-perciform fish (Scorpaeniformes (Powell et al. 1996) and Characiformes (Somoza et al. 1994)), amphibians (Conlon et al. 1993, Iela et al. 1996), reptiles (Negrin & Bello 1996), birds (Norgren & Gao 1994) and even mammals (Lescheid et al. 1997). This recent discovery, that primate brains contain three forms of GnRH (mammalian GnRH (mGnRH), cGnRH-II and an unidentified GnRH form), clearly suggests that the presence of multiple forms of GnRH is likely to be prevalent in most, if not all, vertebrate species. ...
GnRH is a neuropeptide which plays an essential role in the control of reproductive fitness for all vertebrates. Increasing evidence suggests that multiple forms of GnRH may exist in most vertebrate brains. Southern blot analysis of the three GnRHs known to be present in perciform fish, the seabream (sb)GnRH, the salmon(s) GnRH and the chicken (c) GnRH-II, demonstrates that each is present as a single gene copy in the genome of the striped bass, Morone saxatilis. In order to investigate the physiological consequences of multiple GnRHs in a single vertebrate species, we have isolated and characterized two of the GnRH genes, those for sbGnRH and cGnRH-II. Computer analysis of 3.5 kb of sequence upstream of the sbGnRH gene reveals a number of consensus DNA binding sites which implicate steroids, such as estrogen and glucocorticoids, and the steroidogenic transcription factor, SF-1, as being involved in the regulation of sbGnRH gene expression. Sequence analysis of the cGnRH-II gene reveals evidence of multiple promoters. Expression studies using (1) solution hybridization-RNAse protection mapping with several RNA probes directed at various regions of the proGnRH gene, (2) primer extension assays using two specific oligonucleotide primers, and (3) reverse transcription PCR with several oligonucleotide primers on cGnRH-II transcripts demonstrate that the cGnRH-II gene initiates transcription at numerous sites using a TATA-less promoter within the brains of sexually mature striped bass. This study is the first to characterize and compare the promoter structures of two GnRH genes present in a single vertebrate species.
... No negatively charged group is found in the sequence of mammalian GnRH, excluding an interaction of GnRH with its receptor similar to that suggested for peptide 1. Nevertheless, two other forms of GnRH found in lower vertebrates have a negatively charged side chain: the Lampry GnRH I form contains a Glu 6 residue (Sherwood et al., 1986), and the newly identified Tunicate GnRH I form contains an Asp 5 residue, supposedly connected via a salt bridge to a Lys 8 residue (Powell et al., 1996). ...
In the course of our studies toward the development of novel analogs of the decapeptide gonadotropin releasing hormone (GnRH), we have examined a hexapeptide that is an antagonist of endothelin (ET). It was found that this peptide, Ac-D-Trp-Leu-Asp-Ile-Ile-Trp (peptide 1), binds specifically to the pituitary GnRH receptor. Moreover, peptide 1 exhibits a GnRH agonistic activity (i.e., it induces luteinizing hormone release from rat pituitary). This activity is mediated directly by the GnRH receptor and is suppressed by a GnRH antagonist. Removal of the acetyl group of peptide 1 results in a hexapeptide (peptide 2) with binding properties similar to those of GnRH but with a diminished affinity toward the ET receptor. Several other ET antagonists were screened for a potential interaction with the GnRH receptor. Two of these, the hexapeptide PD145065 and the cyclic pentapeptide BQ-123, expressed GnRH agonistic activity at micromolar concentrations in vitro. BQ-123, previously approved for trials on humans as an ET antagonist, is demonstrated to act in vivo as a GnRH agonist, in a dose that was demonstrated previously as the minimal required dose for significant ET antagonism. The GnRH agonistic activity of ET antagonists may therefore result in interference with the physiological control of the reproductive system. Such effects may be most severe when ET antagonists are used chronically. Thus, the major practical message of this study is the need to circumvent the potential side effects of ET antagonist-based drugs.
... The existence of three GnRH forms was confirmed, either by cDNA sequencing or biochemical characterization, in other perciform species including the African cichlid, Haplochromis burtoni (White et al., 1995), the red seabream , Pagrus major, the black seabream, Acanthopagrus schlegeli, the striped knifejaw, Oplegnathus fasciatus, and the Nile tilapia, Oreochromis niloticus (Senthilkumaran et al., 1999). However, recent studies also indicate that sbGnRH is not restricted to perciforms since it was also found in a scorpaeniform, Sebastes rastrelliger (Powell et al., 1996a) and a characiform, Piaractus mesopotamicus (Powell et al., 1997). In the African cichlid (White et al., 1995; White and Fernald, 1998) and the red seabream (Okuzawa et al., 1997), distribution of cells expressing the different GnRHs was found to be similar to that reported in the gilthead seabream by Gothilf et al. (1996), indicating a neuroanatomical segregation of the different GnRH systems. ...
The expression sites of three prepro-gonadotrophin-releasing hormones (GnRHs), corresponding to seabream GnRH (sbGnRH: Ser(8)-mGnRH, mammalian GnRH), salmon GnRH (sGnRH: Trp(7)Leu(8)-mGnRH), and chicken GnRH-II (cGnRH-II: His(5)Trp(7)Tyr(8)-mGnRH) forms were studied in the brain of a perciform fish, the European sea bass (Dicentrarchus labrax) by means of in situ hybridization. The riboprobes used in this study correspond to the three GnRH-associated peptide (GAP)-coding regions of the prepro-GnRH cDNAs cloned from the same species (salmon GAP: sGAP; seabream GAP: sbGAP; chicken GAP-II: cIIGAP), which show little oligonucleotide sequence identity (sGAP versus sbGAP: 42%; cIIGAP versus sbGAP: 36%; sGAP versus cIIGAP: 41%). Adjacent paraffin sections (6 mm) throughout the entire brain were treated in parallel with each of the three anti-sense probes and the corresponding sense probes, demonstrating the high specificity of the hybridization signal. The results showed that both sGAP and sbGAP mRNAs had a broader expression in the olfactory bulbs, ventral telencephalon, and preoptic region, whereas cIIGAP mRNA expression was confined to large cells of the nucleus of the medial longitudinal fascicle. In the olfactory bulbs, both the signal intensity and the number of positive cells were higher with the sGAP probe, whereas sbGAP mRNA-expressing cells were more numerous and intensely stained in the preoptic region. Additional isolated sbGAP-positive cells were detected in the ventrolateral hypothalamus. These results demonstrate a clear overlapping of sGAP- and sbGAP-expressing cells in the forebrain of the European sea bass, in contrast to previous reports in other perciforms showing a clear segregation of these two cell populations.
In seasonally breeding mammals and birds, the production of the hormones that regulate reproduction (gonadotropins) is controlled by a complex pituitary-brain-pituitary pathway. Indeed, the pituitary thyroid-stimulating hormone (TSH) regulates gonadotropin expression in pituitary gonadotropes, via dio2-expressing tanycytes, hypothalamic Kisspeptin, RFamide-related peptide, and gonadotropin-releasing hormone neurons. However, in fish, how seasonal environmental signals influence gonadotropins remains unclear. In addition, the seasonal regulation of gonadotrope (gonadotropin-producing cell) proliferation in the pituitary is, to the best of our knowledge, not elucidated in any vertebrate group. Here, we show that in the vertebrate model Japanese medaka (Oryzias latipes), a long day seasonally breeding fish, photoperiod (daylength) not only regulates hormone production by the gonadotropes but also their proliferation. We also reveal an intra-pituitary pathway that regulates gonadotrope cell number and hormone production. In this pathway, Tsh regulates gonadotropes via folliculostellate cells within the pituitary. This study suggests the existence of an alternative regulatory mechanism of seasonal gonadotropin production in fish.
In vertebrates, pituitary gonadotropins (follicle-stimulating and luteinizing hormones: FSH and LH) regulate gonadal development and maturation, therefore playing an essential role in reproduction. The seasonal regulation of gonadotropins has been widely studied in mammals and birds, and in these taxa thyroid-stimulating hormone (TSH) was found to play a critical role. By contrast, the seasonal regulation of gonadotropins remains unclear in teleost fish. In addition, the seasonal regulation of gonadotrope (gonadotropin-producing cell) proliferation has not been elucidated in any vertebrate group. Using the teleost fish medaka as a model, we show for the first time that long photoperiod enables reproduction by stimulating gonadotropin mRNA synthesis and gonadotrope cell proliferation. In female medaka, this proliferation is achieved by gonadotrope mitosis. We then demonstrate that in female medaka, photoperiod stimulates gonadotropin mRNA production and mitosis via an indirect intra-pituitary pathway, involving pituitary Tsh cells. We show that non-endocrine folliculostellate cells in the pituitary mediate the Tsh signal regulating gonadotrope activity and proliferation, as they are the only pituitary cells to express Tsh receptors and send projections to gonadotropes. Finally, we show that melatonin suppresses pituitary tshba expression in fish exposed to long photoperiod, suggesting that short photoperiod inhibits gonadotropin synthesis via melatonin in both fish and mammals. This study therefore demonstrates that in fish, photoperiod regulates gonadotrope cell activity and mitosis via a melatonin-Tsh pathway. It also reveals the existence of a novel intra-pituitary pathway for seasonal regulation of gonadotropes, involving folliculostellate cells, which we propose might also exist in other vertebrates.
SIGNIFICANCE
In seasonally breeding mammals and birds, the production of the hormones that regulate reproduction (gonadotropins) by gonadotropes is controlled by the pituitary thyroid-stimulating hormone (TSH) through an indirect pathway via the brain. However, in fish, how seasonal environmental signals influence gonadotropins remains unclear. Here, we show that in a long day seasonally breeding fish, medaka, photoperiod not only regulates the activity (hormone production) of the gonadotropes but also their proliferation. We also reveal a novel intra-pituitary pathway that regulates gonadotrope cell activity and number. This pathway involves melatonin, Tsh, and folliculostellate cells. Interestingly, as all these components are also found in the mammalian pituitary, this study suggests the existence of an alternative regulatory mechanism of seasonal gonadotropin production across vertebrates.
The two pituitary gonadotropins, FSH and LH, regulate the reproductive function in all vertebrates. While many studies have investigated the regulation of gonadotropin production and release by the sex steroid feedback, its role on the regulation of gonadotrope cell number remains unclear. Using medaka as a model and an optimized protocol to restore physiological sex steroids levels following gonadectomy, we show that gonadal sex steroids not only decrease fshb transcript levels, but also Fsh cell number in both sexes. We then investigated the origin of the Fsh cell hyperplasia induced by gonadectomy. In both sexes, BrdU incubation shows that this is achieved via Fsh cell mitosis. In situ hybridization reveals that new Fsh cells also originate from transdifferentiating Tsh cells in females, but not in males. Both phenomena are inhibited by sex steroid supplementation via feeding. In males (but not females), gonadectomy (without recovery with sex steroid supplementation) also reduces sox2 transcript levels and Sox2-immunopositive population size, suggesting that sox2-progenitors may be recruited to produce new Fsh cells. Opposite to Fsh cells, gonadectomy decreases lhb levels in both sexes, and levels are not restored by sex steroid supplementation. In addition, the regulation of Lh cell number also seems to be sex dependent. Removal of gonadal sex steroids stimulates Lh cell mitosis in male (like Fsh cells), but not in females. To conclude, our study provides the first evidence on sexually dimorphic mechanisms used in the fish pituitary to remodel gonadotrope populations in response to sex steroids.
FA161, an 8-page illustrated fact sheet by Roy P. E. Yanong, Carlos Martinez, and Craig A. Watson, explains for ornamental fish producers the mode of action and considerations for the use of ovaprim, a hormone product used as a spawning aid. Includes references. Published by the UF Program in Fisheries and Aquatic Sciences, School of Forest Resources and Conservation, December 2009. FA161/FA161: Use of Ovaprim in Ornamental Fish Aquaculture (ufl.edu)
GnRH-I serves as the neuropeptide that regulates mammalian reproduction. Recently, several groups have identified in the brain of rodents, monkeys, and humans a second isoform of GnRH (GnRH-II) whose structure is 70% identical to that of GnRH-I. In this study we demonstrate for the first time human and mouse neuronal cell lines that express both GnRH-I and GnRH-II. Following the screening of several human neuronal cell lines by RT-PCR and Southern hybridization, we demonstrated that two cell lines, TE-671 medulloblastoma and LAN-1 neuroblastoma cells, coexpress messenger RNA encoding the two isoforms of GnRH. Nucleotide sequencing indicated that the complementary DNA fragments are identical to those of the known human GnRH-I and GnRH-II sequences. Extracts obtained from the TE-671 and LAN-1 cell lines as well as from the immortalized mouse hypothalamic GT1–7 neuronal cell line were found to contain the two isoforms of GnRH, which exhibited identical chromatographic properties as synthetic GnRH-I and GnRH-II, in HPLC followed by specific RIAs. Furthermore, double immunofluorescence studies demonstrated the two GnRH isoforms in LAN-1, TE-671, and GT1–7 cells. The identification of neuronal cell lines expressing both GnRH-I and GnRH-II provides tools for studying the differential regulation of gene expression and secretion and for studying the interaction between the two isoforms. Such studies may contribute to elucidation of the physiological functions of GnRH-II, which are still unknown.
GnRH-I (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), originally isolated from the mammalian hypothalamus, plays a pivotal role as the physiological regulator of reproduction (1, 2). This peptide is synthesized and released by hypothalamic neurosecretory cells and reaches the pituitary gland by way of a specialized portal system to induce the synthesis and secretion of the gonadotropic hormones, which regulate gonadal function (3). Recently, several groups have identified a second isoform of GnRH (GnRH-II; His⁵,Trp⁷,Tyr⁸-GnRH-I), in the brain of mammalian species (4, 5, 6). The GnRH-II gene was cloned from human (7) and monkey (8) brains. Originally, GnRH-II was isolated as a second form of GnRH from the chicken brain (9) and was termed chicken GnRH-II. Since then, it was found to be expressed in cartilaginous and bony fish (10, 11, 12, 13), amphibians (14, 15, 16), reptiles (17, 18), birds (9, 19), and metatherian mammals (20, 21) and recently in rodents (5, 6), monkeys (4, 8), and humans (5, 7). The wide distribution of this neuropeptide over all vertebrate classes demonstrates its conservation over the years of evolution and may imply that its physiological functions are very important. In the vertebrate brain the localization of GnRH-II neurons is restricted mainly to the brainstem and hypothalamic structures. The cells are scattered in the periaqueductal and central regions of the midbrain and in the paraventricular, supraoptic, and medial-basal nuclei of the hypothalamus (4, 5, 6, 8). Recently, a third isoform of GnRH in the human, calf, and mouse brain has been demonstrated (22).
The decapeptide gonadotropin-releasing hormone (GnRH) was isolated originally from the mammalian hypothalamus and was named for its role as a stimulator of gonadotropin release from the pituitary gland. Multiple molecular forms of GnRH have been found in the brain of individual teleost species. However, the exact number of forms and their respective physiological roles are still uncertain. Our isolation and comparison of GnRH cDNAs in three teleosts, the eel, arowana, and medaka, led to the idea that duplications of an ancestral GnRH gene had occurred to give rise to three paralogous forms predating the emergence of teleosts, and thus all teleosts may possess three GnRH forms. Also, three GnRHs were shown to be expressed from different loci within the medaka brain, supporting the concept that each GnRH has a specific physiological role. In addition, we demonstrated the occurrence of two distinct GnRH receptor (GnRH-R) subtypes in the medaka; these subtypes exhibited remarkably different primary structures, gene organizations, and ligand selectivities. As both these receptors were expressed in the brain and pituitary, however, the difference in their respective physiological function remains uncertain. Intriguingly, although only two GnRH-R subtypes have been found in the teleosts to date, structural and pharmacological analysis does not rule out the possibility that there may be a third subtype of GnRH-R in teleost.
The decapeptide gonadotropin-releasing hormone (GnRH) has a critical role in the neural control of vertebrate reproduction. To facilitate the establishment of reproductive biology in teleost, we have identified and characterized GnRHs and their receptors in an experimental model species, the medaka Oryzias latipes. Three GnRHs and two GnRH receptors (GnRH-Rs) were cloned from the medaka. Three GnRHs, designated medaka-type GnRH (mdGnRH), chicken-II-type GnRH (cGnRH-II), and salmon-type GnRH (sGnRH), were expressed in separate brain loci (the preoptic area, midbrain tegmentum, and terminal nerve ganglia, respectively), supporting the idea that each form has a specific physiological role. On the other hand, the expressions of two GnRH-Rs, designated GnRH-R1 and GnRH-R2, overlapped at the tissue level; they were expressed both in the brain and in the pituitary. Despite such similar expression patterns, these two GnRH-Rs exhibited remarkably different ligand selectivities; GnRH-R1 showed relatively high selectivity to all three native GnRHs in the medaka, whereas GnRH-R2 had high selectivity only to cGnRH-II. Intriguingly, several lines of evidence obtained from structural, phylogenetical, and pharmacological studies suggested that a third subtype of GnRH-R remains to be found.
Gonadotropin-releasing hormone (GnRH) is well known as the most important regulator of gonadotropin (GtH) secretion. An understanding of morphological and molecular characteristics of the GnRH neuronal system, its regulatory mechanisms and of its role in teleost reproduction are very important issues not only for comparative biology but also for aquaculture. However, the information generated on GnRH in teleosts is not similar to that in mammalian species because of some major differences which are unique to teleosts. A few major differences are listed below:
This chapter discusses the endocrinology of zebrafish. The endocrinology of zebrafish comprising the reproductive, stress, growth, and thyroidal systems is supported by a full cascade of neurohormones, pituitary hormones, and peripheral hormones including steroids comparable to human forms with duplicate genes in some cases. Zebrafish possess two forms of gonadotropin-releasing hormone (Gnrh) compared to most other teleosts with three forms. However, zebrafish have Gnrh2, which is identical to one of the human forms (GNRH2), and Gnrh3, which is unique to teleost fishes, but 80% identical to the other human form (GNRH1). The vertebrate stress axis involves a signaling chain of several hormones and receptors. The primary stress response is initiated when corticotropin-releasing hormone (CRH) is secreted by hypothalamic neurons to bind its receptor in the anterior pituitary on the surface of corticotrope cells. This is followed by the release of adrenocorticotropic hormone (ACTH), a post-translational product of pro-opiomelanocortin (POMC). Zebrafish, like other teleosts, have a thyroid axis similar to that of mammals and amphibians, with a few unique variations. Thyrotropin releasing hormone (Trh) is detected throughout the zebrafish brain with a wider distribution than in other teleosts or mammals, suggesting possible additional functions in zebrafish. Zebrafish possess all of the major hormonal components found in mammals necessary to maintain osmotic balance. The best characterized zebrafish osmoregulatory neuropeptides are isotocin (It) and arginine vasotocin (Avt), the fish orthologs of mammalian oxytocin and arginine vasopressin, respectively.
Gonadotropin-releasing hormone (GnRH) is the hypothalamic hormone that regulates the reproductive system by stimulating release
of gonadotropins from the anterior pituitary gland. The molecular variants of the reproductive neuropeptide GnRH were characterized
from brain tissue of three perciform species from Antarctic waters: Pseudochaenichthys georgianus, Chaenocephalus aceratus, and Notothenia rossi. The study involved reverse phase high-performance liquid chromatography (RP-HPLC) followed by radioimmunoassay (RIA) with
two antisera that recognize all GnRH variants already identified: PBL 45 and PBL 49. The results showed that brain extracts
of P. georgianus, C. aceratus, and N. rossi contain, like those of other perciform fish, three forms of GnRH likely to be: sbGnRH (seabream GnRH), cGnRH-II (chicken
GnRH II) and sGnRH (salmon GnRH). They also showed evidence for the presence of a fourth GnRH variant, chromatographically
and immunologically different from the other known forms of the vertebrate hormone. Although final conclusions will require
isolation, purification, and sequencing of these molecules, these results offer encouraging possibilities of further advances
in the characterization of a multiplicity of GnRH molecular variants.
This paper studies the molecular variants of gonadotropin-releasing hormone (GnRH) present in the brain of the protogynous
swamp eel, Synbranchus marmoratus, and the effects of the administration of salmon GnRH analogue (sGnRH-A) and the dopamine
receptor antagonist, domperidone (DOM) on final maturation and gamete release in this species. Evidence for the presence of
two GnRH variants, sGnRH and cIIGnRH were obtained by reverse phase high-pressure liquid chromatography (RP-HPLC) and radioimmunoassay
with different antisera. The effects of treatment with sGnRH-A+DOM were checked by three ways: oocyte and milt release by
stripping, histological analysis of the gonadal tissue, and androgen serum levels at different times throughout the experiment.
In males, spermiation was induced after three weeks of treatment. In the female group, sGnRH-A+DOM did not induce ovulation
at the end of the experiment. Histological analysis of the gonads from the female group showed evidence of sex reversal. All
the treated fish had elevated androgen serum levels from the third week, with respect to control fish. In all cases, serum
estradiol levels were undetectable. These results suggest that treatment with sGnRH analog and DOM induce sex reversal in
female and spermiation in males.
The mammalian gonadotropin-releasing hormone (GnRH-I), which regulates reproduction, was the first isoform of GnRH that was identified in mammals. Recently, we and others have demonstrated the existence of a second isoform of GnRH in the brain of mammals. The presence of a third isoform of GnRH, GnRH-III, in the brain of mammals is reported herein. GnRH-III, extracted from the brain of bovine and human, was purified by high performance liquid chromatography, using two distinct elution programs. In both, GnRH-III was eluted at the same positions as synthetic salmon GnRH, as demonstrated by radioimmunoassay. The luteinizing hormone-releasing activity of purified GnRH-III, using dispersed rat pituitary cells, was found to be similar to that of synthetic salmon GnRH. The total amount of GnRH-III, determined by radioimmunoassay, in the hypothalamus and midbrain of humans and calves is similar to that of GnRH-I. Immunohistochemical studies demonstrated GnRH-III-containing neurons in the hypothalamus and midbrain of human and GnRH-III fibers in the median eminence of rats. The distribution of GnRH-III in the brain suggests that in addition to a putative function as a neurohormone at the hypothalamic-pituitary axis, GnRH-III may have other functions. Our present results suggest that multiple isoforms of GnRH are present in the brain of mammals, and further studies are required in order to elucidate their biological functions.
Hypophysiotropic GnRH neurons are located in the preoptic area and ventral hypothalamus of sexually mature vertebrates. In several species, the embryonic origin of hypophysiotropic GnRH neurons remains unclear. Using the Tg(GnRH3:EGFP) zebrafish line, in which GnRH3 neurons express EGFP, GnRH3 neurons in the olfactory region were specifically and individually ablated during early development using laser pulses. After ablation, the olfactory region maintained the capacity to regenerate GnRH3 neurons. However, this capacity was time-limited. When ablation of GnRH3 cells was conducted at 2 d after fertilization, high regeneration rates were observed, but regeneration capacity significantly decreased when ablation was performed at 4 or 6 d after fertilization. Unilateral GnRH3 neuron ablation results in unilateral soma presence. These unilateral somata are capable of projecting fiber extensions bilaterally. Successful bilateral GnRH3 soma ablation during development resulted in complete lack of olfactory, terminal nerve, preoptic area, and hypothalamic GnRH3 neurons and fibers in 12-wk-old animals. Mature animals lacking GnRH3 neurons exhibited arrested oocyte development and reduced average oocyte diameter. Animals in which GnRH3 neurons were partially ablated exhibited normal oocyte development; however, their fecundity was significantly reduced. These findings demonstrate that the hypophysiotropic GnRH3 populations in zebrafish consist of neurons that originate in the olfactory region during early development. The presence of GnRH3 neurons of olfactory region origin in reproductively mature zebrafish is a prerequisite for normal oocyte development and reproduction.
Reproductive processes are mainly regulated by the brain-pituitary-gonad axis (BPG-axis). Gonadotropin-releasing hormone (GnRH) neurons localized in the brain release their hormone GnRH, which allows the release of gonadotropic hormone by gonadotropic cells in the pituitary. Gonadotropic hormone, in turn, regulates the production of sex steroids and germ cells in the gonads. The steroids complete the dynamic BPG-axis by exerting feedback effects at the level of the brain, pituitary and gonads. The transition period of a juvenile, inactive axis towards a mature, functional system is known as puberty. The prevailing "missing-link" concept assumes that one or more components of the axis are absent or not functional before puberty. For our animal model, the African catfish, it has been proposed that sex steroids initiate and/or accelerate pubertal development. However, based on earlier findings, it was suggested that the "missing link" could also be localized at a higher level of the BPG-axis, i.e. at the GnRH system in the brain. In order to test the hypothesis that sex steroids and/or a functional GnRH system are important determinants in the onset of puberty in the African catfish, we studied the normal development of the GnRH system in the brain and the effects of certain steroids on this development. The present thesis shows that specific sex steroids are important for the development of GnRH neurons. Thus, in the African catfish, steroids are a serious candidate for the "missing-link", since it is required for activating the GnRH neurons in the preparation for the onset of puberty. Another candidate for the "missing-link" is the actual released amount of GnRH that is available for the gonadotropic cells at the onset of puberty. We hypothesize that functional contact between the first GnRH terminals in the pituitary and the gonadotropic cells is required for the initiation of puberty. Once this switch is turned on, the three levels of the BPG-axis simultaneously display their maturational processes: innervation of the pituitary by GnRH fibers, development of gonadotropic cells and the first wave of spermatogenesis.
Hypothalamic gonadotropin-releasing hormone (GnRH) neurons control pituitary gonadotropin secretion and gametogenesis. In the course of development, these neurons migrate from the olfactory placode to the hypothalamus. The precise molecular mechanism of this neuronal migration is unclear. Here, we investigated whether the chemokine receptor, Cxcr4b, and its cognate ligand, Cxcl12a, are required for proper migration of GnRH3 neurons in zebrafish. Deviated GnRH3 axonal projections and neuronal migration were detected in larvae that carry a homozygote cxcr4b mutation. Similarly, knockdown of Cxcr4b or Cxcl12a led to the appearance of abnormal GnRH3 axonal projections and cell migration, including absence of the characteristic lateral crossing of GnRH3 axons at the anterior commissure and optic chiasm. Double-labeling analysis has shown that cxcr4b and cxcl12a are expressed along the GnRH3 migration pathway (i.e. olfactory placode, terminal nerve and the optic chiasm). The results of this study suggest that the Cxcl12a-Cxcr4b ligand-receptor pair are involved in the migration of GnRH3 neurons in zebrafish, and are therefore crucial for the development of this system.
Gonadotropin-releasing hormone (GnRH) molecular variants in the brain and pituitary gland of pejerrey, Odontesthes bonariensis (Atheriniformes), were characterized by gradient reverse phase high performance liquid chromatography (RP-HPLC). Eluted fractions were tested in radioimmunoassays with different antisera. The results show that the brain extract contains three forms of GnRH: one is immunologically and chromatographically similar to cIIGnRH (chicken II), and another is similar to sGnRH (salmon). A third GnRH appears to be chromatographic and immunologically different from the nine other known forms of the vertebrate hormone. This is the only variant present in the pituitary gland.
Gonadotropin-releasing hormone-I (GnRH-I), present in the mammalian hypothalamus, regulates reproduction. In this study we demonstrate, for the first time, that an additional isoform of GnRH, [His5, Trp7, Tyr8] GnRH-I (GnRH-II) is present in the brain of the mouse, rat and human. Human and rat brain extracts contain two isoforms of GnRH, GnRH-I and GnRH-II, which exhibited identical chromatographic properties to the respective synthetic peptides, in high performance liquid chromatography. Using immunohistochemical techniques we have found that GnRH-II is present in neuronal cells that are localized mainly in the periaqueductal area as well as in the oculomotor and red nuclei of the midbrain. It is of interest to note that in the hypogonadal mouse, although the GnRH-I gene is deleted, GnRH-II is present. Substantial concentrations of GnRH-II are also present in the hypothalamus and stored in the human pituitary stalk or in the mouse median eminence. By using reverse transcription (RT)-PCR we have also found that while GnRH-II is not expressed in the cerebellum, it is expressed in all three structures of the brain stem: midbrain, pons and medulla oblongata.
Specific and sensitive radioimmunoassays (RIAs) were newly developed for two types of gonadotropin-releasing hormone (GnRH), namely, seabream (sb) GnRH and chicken (c) GnRH-II. We employed these two RIAs together with a previously reported RIA for salmon (s) GnRH to study the presence and regional distribution of these three GnRHs in the brains and pituitaries of four perciform fishes (red seabream, Pagrus major; black seabream, Acanthopagrus schlegeli; striped knifejaw, Oplegnathus fasciatus; and Nile tilapia, Oreochromis niloticus), as well as clarify seasonal changes in levels of these GnRHs in the brain and pituitary of red seabream. All three GnRHs were found in brains of all fishes examined, with regional distributions in the brains of the three GnRHs being rather similar. sbGnRH was abundant in telencephalon and hypothalamus. cGnRH-II was concentrated from the middle to posterior part of the brain and distributed throughout the brain. sGnRH was concentrated in the olfactory bulb and distributed all over the brain, as was cGnRH-II. The dominant form of GnRH in the pituitary was sbGnRH, with levels 500- to 2400-fold higher than those of sGnRH, while cGnRH-II was undetectable in all four species. In the brain and pituitary of female red seabream, levels of both brain and pituitary sbGnRH increased from October (immature phase) and reached a peak in April (spawning phase), reflecting the increase in gonadosomatic index and vitellogenesis. However, levels of sbGnRH remained high only in the pituitary of completely regressed fish in June. Levels of both sGnRH and cGnRH-II in the brain were higher in the regressed phase and remained lower during the spawning phase. From these and previous results, it appears that sbGnRH is physiologically the most important form of GnRH in reproduction in red seabream and, probably, in other perciforms also.
The present study has identified three molecular forms of gonadotropin-releasing hormone (GnRH) in the brain of a teleost, the medaka, by isolation of their cDNAs. This species has a novel GnRH, which is here named medaka-type GnRH (mdGnRH), in addition to two characterized forms, chicken-II-type GnRH (cGnRH-II) and salmon-type GnRH (sGnRH). Phylogenetic analysis showed that mdGnRH is a medaka homolog of and seabream-type GnRH (sbGnRH) and mammalian-type GnRH (mGnRH) in other species, and suggested that all vertebrates have three distinct GnRHs. Furthermore, in situ hybridization revealed that the mdGnRH gene is expressed only in neurons clustered within the preoptic area as sbGnRH and mGnRH genes in other species are, while the genes for cGnRH-II and sGnRH are only in the midbrain tegmentum and nucleus olfactoretinalis, respectively. This result suggested that mdGnRH is a hypophysiotropic factor and the other two forms are involved in other physiological events as neuromodulators or neurotransmitters.
GnRH-I serves as the neuropeptide that regulates mammalian reproduction. Recently, several groups have identified in the brain of rodents, monkeys, and humans a second isoform of GnRH (GnRH-II) whose structure is 70% identical to that of GnRH-I. In this study we demonstrate for the first time human and mouse neuronal cell lines that express both GnRH-I and GnRH-II. Following the screening of several human neuronal cell lines by RT-PCR and Southern hybridization, we demonstrated that two cell lines, TE-671 medulloblastoma and LAN-1 neuroblastoma cells, coexpress messenger RNA encoding the two isoforms of GnRH. Nucleotide sequencing indicated that the complementary DNA fragments are identical to those of the known human GnRH-I and GnRH-II sequences. Extracts obtained from the TE-671 and LAN-1 cell lines as well as from the immortalized mouse hypothalamic GT1-7 neuronal cell line were found to contain the two isoforms of GnRH, which exhibited identical chromatographic properties as synthetic GnRH-I and GnRH-II, in HPLC followed by specific RIAs. Furthermore, double immunofluorescence studies demonstrated the two GnRH isoforms in LAN-1, TE-671, and GT1-7 cells. The identification of neuronal cell lines expressing both GnRH-I and GnRH-II provides tools for studying the differential regulation of gene expression and secretion and for studying the interaction between the two isoforms. Such studies may contribute to elucidation of the physiological functions of GnRH-II, which are still unknown.
The effects of serotonin (5-HT), GABA and neuropeptide Y (NPY) on in vitro release of seabream (sb) gonadotropin releasing hormone (GnRH) from slices of the preoptic-anterior hypothalamus (P-AH) and pituitary of red seabream were studied. 5-HT, GABA and NPY all stimulated the release of sbGnRH from the P-AH but not from the pituitary of immature red seabream. They also stimulated sbGnRH release from the P-AH with a similar potency during the course of gonadal development. Specific agonists and/or antagonists of 5-HT, GABA and NPY showed that 5-HT and GABA utilize 5-HT(2) and GABAA receptor subtypes, respectively, to mediate their action, and that NPY employs at least NPY(Y1) and NPY(Y2) receptor subtypes to stimulate sbGnRH release. Combinations of different antagonists for 5-HT, GABA and noradrenaline/adrenaline did not block the stimulatory influence of NPY on release of sbGnRH, indicating that the action of NPY on the sbGnRH neuronal system is probably direct.
The primary structure of gonadotropin-releasing hormone (GnRH) isolated from whole brains of lamprey is pGlu-His-Tyr-Ser-Leu-Glu-Trp-Lys-Pro-Gly-NH2. This unique decapeptide was isolated and purified from brain extracts by reverse-phase high performance liquid chromatography. The structure of the peptide was established from chymotryptic fragments that were identified by protein sequence analysis and fast atom bombardment mass spectrometry. The peptide reacts with an antiserum raised against mammalian GnRH and is structurally identified as a member of the GnRH family by the amino and carboxyl termini of pGlu1-His2 and Pro9-Gly10NH2, the conservation of Ser4 in the internal segment of the molecule and its length of 10 amino acids. For the first time, amino acid substitutions are found in positions 3 and 6, critical for biological potency and conformation, respectively. Additionally, a second form of GnRH (lamprey II GnRH), representing about 10% of the total GnRH immunoreactive material in the brain, was isolated; its amino acid composition differs by 3 residues from lamprey I GnRH. Synthetic lamprey I GnRH elevates plasma estradiol in adult female lampreys.
A peptide that is recognized by certain antibodies raised against mammalian gonadotropin-releasing hormone has been purified from extracts of salmon brains by gel filtration and high-performance liquid chromatography. The primary structure of this 10-residue peptide is less than Glu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-Gly-NH2. This represents a difference of two amino acids between salmon and mammalian gonadotropin-releasing hormone and demonstrates that most of the molecule has been conserved during evolution. The synthetic form of salmon gonadotropin-releasing hormone is less potent than is mammalian gonadotropin-releasing hormone on mammalian cells and is biologically active in salmon.
Most vertebrate species have more than one form of gonadotropin-releasing hormone (GnRH) in their brains, but it is not clear whether each form has a distinct function. We report that sea bream (Sparus aurata) brains have three forms of GnRH, one of which is described herein and is called sea bream GnRH (sbGnRH). The primary structures of two forms were determined by Edman degradation and mass spectral analysis. The amino acid sequence of sbGnRH is pGlu-His-Trp-Ser-Tyr-Gly-Leu-Ser-Pro-Gly-NH2. The second peptide is identical to a form originally isolated from chicken brains (cGnRH-II): pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2. cGnRH-II is the most ancient form of GnRH identified to date in jawed fish and the most prevalent form throughout the vertebrates. The third form of GnRH has previously been identified as salmon GnRH by cDNA studies and is confirmed here by chromatographic and immunological studies. Phylogenetic distribution of GnRH peptides suggests sbGnRH arose in the perch-like fish as a gene duplication of the existing cGnRH-II or salmon GnRH genes. All three identified GnRH peptides were synthesized and shown to release gonadotropin in vivo in the sea bream. The dominant form of GnRH stored in the pituitary was sbGnRH. Not only was the content of sbGnRH 500-fold greater than that of salmon GnRH but also cGnRH-II was not detected in the pituitary. The latter evidence suggests that sbGnRH is the endogenous releaser of gonadotropin II.
Selected aspects of the reproduction and development ofSebastes and other rockfishes are reviewed in the context of piscine viviparity. Among the eight subfamilies of the Scorpaenidae,
viviparity is confined to the subfamily Sebastinae; gestation is lumenal and the embryos usually develop to term within the
egg envelope. Transitional states from oviparity to viviparity are evident in different species within the family. A scenario
for the evolutionary origin of viviparity in rockfishes is derived from an analysis of scorpaeniform reproductive biology.
Although viviparity is best developed in the genusSebastes, it is still in a primitive, unspecialized state. Rockfish viviparity is essentially lecithotrophic, i.e. embryonic nutrition
is dependent on the energy reserves laid down during oogenesis. In other groups of viviparous fishes, lecithotrophy has been
shown to be better suited energetically to seasonally unpredictable habitats, whereas matrotrophy requires a predictable food
supply. During the evolution of an essentially primitive form of lecithotrophic viviparity in rockfishes, the advantages of
high fecundity associated with oviparity were retained while an enormous increase in the survival rate of the developing embryos
was acquired. The basic lecithotrophic pattern of oviparous development was not changed since it offered selective advantages
both in terms of energetics and as a basis for retaining a large brood size.
We have developed sensitive and specific radioimmunoassays (RIA) for salmon gonadotropin-releasing hormone (sGnRH) and chicken GnRH-II (cGnRH-II). Synthetic sGnRH and cGnRH-II(2–10) were conjugated to bovine serum albumin and injected into rabbits to raise specific antisera. The antiserum against sGnRH showed cross-reactivities of 1.58 and 0.08% for cGnRH-II and lamprey GnRH, respectively. The antiserum against cGnRH-II showed cross-reactivities of 0.05 and 0.01% for sGnRH and lamprey GnRH, respectively. Both antisera were observed not to cross-react with mammalian GnRH and cGnRH-I or other peptide hormones. Synthetic sGnRH and cGnRH-II were iodinated using the chloramine-T method. The iodinated GnRH was purified by HPLC using a reverse-phase C18 column. The RIA system was developed as a double antibody method. Brain extracts of rainbow trout showed displacement curves which were parallel to the sGnRH and cGnRH-II standards in each RIA. HPLC analysis followed by RIA has revealed that rainbow trout brain contains two types of GnRH: sGnRH and cGnRH-II. Total sGnRH content in the brain was about three-fold higher than that of cGnRH-II. In the olfactory bulbs, telencephalon, optic tectum-thalamus, hypothalamus, and pituitary, sGnRH content (per region) was higher than cGnRH-II content, whereas cerebellum and medulla oblongata contained much more cGnRH-II than sGnRH. sGnRH content in the optic tectum-thalamus and pituitary was the highest in 1-year-old immature fish and 3-year-old mature fish, respectively. Medulla oblongata showed the highest cGnRH-II content in all groups. sGnRH concentrations (per milligram of protein) were high in the pituitary and intermediate in the olfactory bulbs, hypothalamus, and telencephalon. In all groups, the cGnRH-II concentration was high in the medulla oblongata, whereas the concentration in the olfactory bulbs and pituitary gland was below the detectable limit in most individuals.
Two forms of gonadotropin-releasing hormone (GnRH) have been purified from brain extracts of the Thai catfish, Clarias macrocephalus, using reverse-phase high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA). The amino acid sequences of both forms of catfish GnRH (catfish GnRH-I and -II) were determined using Edman degradation. The presence of the N-terminal pGlu residue in both peptides was established by digestion with pyroglutamyl aminopeptidase. In addition, catfish GnRH-I was studied by mass spectrometry. The primary structure of catfish GnRHI is pGluHisTrpSerHisGlyLeuAsnProGlyNH2 and catfish GnRH-II is identical to chicken GnRH-II, pGluHisTrpSerHisGlyTrpTyrProGlyNH2. Functional studies showed that synthetic catfish GnRH-I released not only gonadotropin but also growth hormone from an in vitro preparation of goldfish pituitaries. Catfish GnRH-II is identical to the widely distributed and highly conserved chicken GnRH-II. Indirect evidence has suggested its presence in bony fish, but this is the first report of its primary sequence. The distinct structure of catfish GnRH-I increases the number of GnRH family members to six.
Two forms of immunoreactive gonadotropin-releasing hormone (GnRH) were extracted from brain-pituitary tissues of Thai catfish, Clarias macrocephalus and C. batrachus. The peptides were detected using high performance liquid chromatography (HPLC) and radioimmunoassay (RIA). In both the HPLC systems, catfish GnRH-I eluted earlier than catfish GnRH-II and also eluted before the synthetic standards of mammalian, lamprey, chicken I, chicken II, and salmon GnRH. Hence, catfish GnRH-I appears to be the most hydrophilic GnRH family member because of this early elution from the HPLC. Catfish GnRH-II eluted in a position similar to that of chicken GnRH-II. This study suggests that catfish GnRH-I is a novel form of GnRH, whereas catfish GnRH-II is the same as chicken GnRH-II. Indirect evidence suggests that the catfish molecule is 10 amino acids in length and has an amide at the C-terminus. Moreover, the novel catfish GnRH appears to be different within the domain of amino acids 5 to 10 compared with mammalian GnRH because it is not recognized by antiserum B-6. An injection of native chicken GnRH-II was more effective than salmon or mammalian GnRH for induced ovulation in C. macrocephalus.
Two forms of gonadotropin-releasing hormone (GnRH) have been purified from brain extracts of the African catfish, Clarias gariepinus, using reverse-phase high performance liquid chromatography (HPLC) and radioimmunoassay (RIA). The amino acid sequences of both forms of African catfish GnRH were determined using Edman degradation after digestion with pyroglutamyl aminopeptidase. In addition, both GnRHs were studied by mass spectrometry. The primary structure of African catfish GnRH I is identical to Thai catfish GnRH I, pGlu-His-Trp-Ser-His-Gly-Leu-Asn-Pro-Gly-NH2, and the primary structure of African catfish GnRH II is identical to the widely distributed and highly conserved chicken GnRH II, pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2.
Four forms of immunoreactive GnRH have been detected in tissue extracts of both whole brains and terminal nerves from the spiny dogfish (Squalus acanthias). The GnRH forms were characterized using reverse-phase high pressure liquid chromatography (HPLC) and immunological recognition with four different antisera. Three of these forms possess immunological and chromatographic properties consistent with known forms of GnRH: mammalian GnRH, chicken GnRH-II and salmon GnRH. An additional form, with an HPLC elution position intermediate between chicken GnRH-II and salmon GnRH appears to be a new structure of GnRH. The presence of all four GnRH forms in the terminal nerve suggests a lack of regional specificity of the expressed forms of GnRH in the brain.
Three species of fish that are phylogenetically older than other members of the bony fish lineage were selected to determine if gonadotropin-releasing hormone (GnRH) is present in their brains. Brain extracts were prepared from each species and found to contain immunoreactive (ir) GnRH. To further characterize the molecular forms of GnRH in each species, the extracts were injected into a high pressure liquid chromatograph (HPLC). The elution time of each GnRH-like form was compared to those of the synthetic forms of the five known GnRHs. Several antisera were used to detect both the synthetic and unknown GnRHs in the HPLC fractions. All three species of fish had two forms of GnRH: a dominant form that is mammalian GnRH-like (mGnRH), and a minor form of irGnRH material that is similar to chicken GnRH-II (cGnRH-II). The other known forms of GnRH (salmon, lamprey, and chicken-I) were not detected. The appearance in these ancient bony fish of a mammalian-like form of GnRH, which has not been found in the jawless or cartilaginous fish studied to date, suggests that mGnRH arose in a common phylogenetic ancestor of the bony fish and tetrapods. This mGnRH-like molecule is known to have been conserved in the amphibian and mammalian lineage, but not in the reptilian or avian line. In addition, the presence of a cGnRH-II-like molecule in the bony fish examined here, and in the cartilaginous fish studied earlier, implies that this form of GnRH may have been present in an ancestor common to both of these classes of fish.
We have recently developed sensitive and specific radioimmunoassays (RIAs) for salmon gonadotropin‐releasing hormone (sGnRH) and chicken GnRH‐II (cGnRH‐II) and have measured the contents of both GnRHs in the rainbow trout brain. Our results showed that contents of the two GnRHs are variable among different brain regions. Therefore, in order to confirm the differential distribution of the two GnRHs by a different technique, we examined the distribution of immunoreactive sGnRH and cGnRH‐II in the brain of masu salmon by using immunocytochemical techniques.
sGnRH immunoreactive (ir) cell bodies were scattered in the transitional areas between the olfactory nerve and the olfactory bulb, the ventral olfactory bulb, between the olfactory bulb and the telencephalon, the ventral telencephalon, and the preoptic area. These sGnRH‐ir cell bodies were dispersed in a strip‐like region running rostrocaudally in the most ventral part of the ventral telencephalon. sGnRH‐ir fibers were distributed in the various brain regions from the olfactory bulb to the spinal cord. They were especially abundant in the olfactory bulb, ventral telencephalon, preoptic area, hypothalamus, deep layers of the optic tectum, and thalamus. sGnRH‐ir fibers also innervated the pituitary directly.
cGnRH‐II‐ir cell bodies were found in the nucleus of the medial longitudinal fasciculus (nMLF). The distribution of cGnRH‐II‐ir fibers was similar to that of sGnRH‐ir fibers, except that cGnRH‐II‐ir fibers were absent in the pituitary. The number of cGnRH‐II‐ir fibers was much fewer than that of sGnRH‐ir fibers.
The results of the present immunocytochemical study are in basic agreement with those of our previous RIA study. Thus, we suggest that in masu salmon, sGnRH not only regulates gonadotropin (GTH) release from the pituitary but also functions as a neuromodulator in the brain, whereas cGnRH‐II functions only as a neuromodulator.
Immunoreactive gonadotropin-releasing hormone (ir-GnRH) was detected in brain extracts of newborn and 10-day-old rats and in adult guinea pigs; it was also present in extracts of the neural ganglion and gland of a protochordate. Radioimmunoassay (RIA) using different GnRH antisera after high-performance liquid chromatography (HPLC) revealed that the dominant form of GnRH is the mammalian form (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) both during ontogenesis in the rat and in the adult guinea pig known to have variant forms of other peptide hormones. None of the other forms of GnRH identified in nonmammalian species to date appear to be present in the rat or guinea pig. A small amount of an unidentified HPLC early eluting form of GnRH is present, but detection by antiserum B-6 implies that it is also mammalian GnRH, with the possibility of changes in positions 2-4. The molecular form of GnRH in a protochordate, the sea squirt Chelyosoma productum, is distinct from salmon and mammalian GnRHs. Cross-reactivity with the sea squirt GnRH-like molecule was highest with an antiserum made against lamprey GnRH; the same antiserum was used to stain nerve fibers in the neural ganglion and some of its roots. This is the first report using RIA, HPLC, and immunocytochemistry to show that protochordates have GnRH-like material. The results suggest that GnRH may have been present at the transition between the invertebrates and vertebrates.
Two molecular forms of gonadotropin-releasing hormone (GnRH) were identified in the extracts of various brain areas, spinal cord and pituitary in female and male goldfish and had chromatographic and immunological properties similar to [His5, Trp7, Tyr8]-GnRH (cGnRH-II) and [Trp7,Leu8]-GnRH (sGnRH). Radioimmunoassay using different GnRH antisera after high pressure liquid chromatography did not reveal significant peaks of mammalian GnRH, [Gln8]-GnRH and [Tyr3,Leu5,Glu6,Trp7,Lys8]-GnRH in the brain extracts. The proportion of cGnRH-II-like immunoactivity to sGnRH-like immunoactivity was higher in the caudal brain areas compared to the rostral areas. The differential distribution of two GnRH forms suggest that the different GnRH forms may have different physiological functions.
Several vertebrate gonadotropin-releasing hormones (GnRH) and analogs were tested for activity in vivo in goldfish. Each peptide was administered intraperitoneally to goldfish, pretreated with pimozide or vehicle for pimozide, and changes in serum levels of gonadotropin were determined. Pimozide potentiates the activity of GnRH in vivo in goldfish by blocking the endogenous gonadotropin release-inhibitory activity of dopamine; relative potencies of GnRH peptides become evident in vivo in goldfish pretreated with pimozide (R. Peter et al. (1985), Gen. Comp. Endocrinol. 58, 231-242). Mammalian GnRH (mGnRH) was used as reference standard. [Try3, Leu5, Glu6, Trp7, Lys8]-GnRH (lamprey GnRH), [Gln8]-GnRH (chicken GnRH-I), and [His5, Trp7, Try8]-GnRH (chicken GnRH-II) caused increases in serum gonadotropin level similar in magnitude to mGnRH. [His5, D-Arg6, Trp7, Tyr8]-GnRH is superactive in the goldfish. [Asn8]-, [Met8]-, [Phe8]-, and [Ser8]-GnRH had activity similar to mGnRH in goldfish; [His8]-, [Ile8]-, and [Leu8]-GnRH had a lower level of activity; [Glu8]-GnRH had no apparent activity. The results indicate that there is no particular requirement for a hydrophobic or hydrophilic amino acid, or for a positively charged amino acid in position 8 of mGnRH for activity in vivo in the goldfish; a negatively charged amino acid in position 8 is detrimental for activity.
Mammalian, chicken, and salmon gonadotropin releasing hormones (GnRHs), and anlogs of each peptide, were injected either alone or in combination with pimozide into goldfish, and the changes in serum gonadotropin (GtH) levels determined. The native peptides had similar potencies in terms of magnitude and duration of the GtH response. Analogs of LHRH that are superactive in mammals are also superactive in goldfish; although [(imBzl)-D-His6, Pro9-NEt]-LHRH is very highly superactive in mammals it has activity similar to [D-Ala6, Pro9-NEt]-LHRH in goldfish. D-Ala6 or (imBzl)-D-His6 substitutions of [Trp7, Leu8, Pro9-NEt]-LHRH are not superactive in goldfish, whereas the D-Arg6 substitution is highly superactive, indicating that there are differences in the factors that make salmon and mammalian GnRH superactive. These results also indicate that the structural modifications that determine superactivity of GnRHs in goldfish differ from what is known for mammals.
GnRH is a decapeptide family with at least nine distinct structures. Vertebrates, except for most placental mammals, have more than one of these GnRH forms within the brain. We report chromatographical and immunological evidence that three forms of GnRH are in the brains of both cichlid (Haplochromis burtoni) and pumpkinseed (Lepomis gibbosus) fishes. We argue that the three forms correspond to those previously described as sea bream GnRH (sbGnRH), chicken GnRH-II and salmon GnRH. In contrast, only one GnRH form was present in the pituitary of the cichlid and is identified as sbGnRH by amino acid sequence. This is the first report in which the primary structure of GnRH is determined from pituitary tissue. The N-terminus was identified by monitoring the digestion of the peptide by pyroglutamate aminopeptidase with matrix assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The amidation of the C-terminus was established using an esterification procedure for monitoring with MALDI-MS. This report supports the idea that three forms of GnRH within one species is widespread in the order Perciformes. The present study establishes sbGnRH as the third GnRH form in H. burtoni and predicts that sbGnRH is synthesized in preoptic neurons, then transported to the pituitary in the preoptic-hypophyseal axons for the release of one or both gonadotropins.
Molecular variants of GnRH (gonadotropin-releasing hormone) in brain and pituitary extracts of the South American characiforme Prochilodus lineatus were studied using a combination of reverse-phase high-performance liquid chromatography and radioimmunoassay with different antisera. In brain extracts our study revealed that this fish has at least two different types of GnRH: cIIGnRH (chicken II) and sGnRH (salmon), and possibly a third variant of this molecule. In pituitary extracts we could find only two immunoreactive peaks corresponding to sGnRH and the possible third form.
The molecular forms of gonadotropin-releasing hormone (GnRH) in brain-pituitary extracts were determined for snook Centropomus undecimalis and black sea bass Centropristis striata. The extracts were analyzed in both isocratic and gradient high performance liquid chromatography (HPLC) programs. Eluted fractions were tested in radioimmunoassays with 4 different antisera made against 3 distinct GnRH peptides. Results show that snook contain 3 forms of GnRH, all of which are present in males and females irrespective of the stage of the reproductive cycle. Larger quantities of these GnRH peptides are present in snook in the nonreproductive phase than in snook in the reproductive phase. One form of snook GnRH is immunologically and chromatographically similar to salmon GnRH, and a second form is similar to chicken GnRH-II. However, the third snook GnRH appears to be distinct from the 7 known forms of the vertebrate hormone. In contrast, sea bass contain only the salmon GnRH-like and chicken GnRH-II-like forms of GnRH and, hence, appear to match the more usual pattern of GnRH peptides in teleosts. We speculate that one of the GnRH genes was duplicated and then altered in a fish ancestral to snook but not sea bass, even though both species of fish are in the recently evolved Perciformes order.
I. Introduction GONADOTROPIN-releasing hormone (GnRH) plays a pivotal role in reproduction. The existence of this neuropeptide, also called LHRH, was predicted in the 1950s (1), but the structure of the 10 amino acid peptide for porcine and ovine brains was not reported until the 1970s (2, 3). In the 20 yr since then, only one form of GnRH has been identified in most placental mammals, and this is still considered to be the sole neuropeptide causing the release of LH and FSH. The structure and functions of the mammalian form of GnRH have been reviewed a number of times (4s–8). Meanwhile, both mammalian GnRH and related forms of GnRH are now known to be present in primitive placental mammals, nonplacental mammals, and other vertebrates. Novel functions, in addition to the release of gonadotropins, exist. Studies of GnRH peptides and their genes have altered our views on the origin, function, and regulation of this neuropeptide.
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Immunocytochemical demonstration of salmon GnRH and chicken GnRH-II in the brain of masu salmon, Oncorhynchus masou An evaluation of rockfish (Sebastes spp) as models for the study of reproduction and development in viviparous marine fish
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WH Freeman & Company. Echeverría TW 1987 Thirty-four species of California rockfishes: Maturity and seasonality of reproduction
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