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Regular Article |
a Endocrinologie Moléculaire de la Reproduction, UMR CNRS 6026, Campus de Beaulieu, 35042 Rennes cedex, France
b Instituto de Acuicultura de Torre de la Sal, CSIC, Castellón, Spain
c Research Group Comparative Endocrinology University of Utrecht, NL-3584 CH Utrecht, The Netherlands
ABSTRACT
A full-length cDNA encoding a GnRH receptor (GnRH-R) has been obtained from the brain of rainbow trout. This cDNA encodes a protein of 386 amino acids (aa) exhibiting the typical arrangement of the G-protein-coupled receptors in seven transmembrane domains. However, a second ATG could give rise to a receptor with a 30-aa longer extracellular domain. As already shown in other fish and Xenopus, this protein possesses an intracellular domain, in contrast with its mammalian counterparts. In the case of rainbow trout, this intracellular carboxy-terminal tail consists of 58 residues. Northern blotting experiments carried out in the brain, the pituitary, and the liver only resulted in a single band of 1.9–2 kilobases in the pituitary, although reverse transcription-polymerase chain reaction amplification products were found in the brain, the pituitary, the retina, and the ovary. In situ hybridization using a probe corresponding to the full-length coding region of the receptor was performed on vitellogenic or ovulating females and allowed to detect a weak but specific signal in the proximal pars distalis of the pituitary, the preoptic region, the mediobasal hypothalamus, and the optic tectum. However, the strongest signal was consistently detected in a mesencephalic structure, the nucleus lateralis valvulae, the significance of which is presently open to speculation.
anterior pituitary, central nervous system, GnRH, GnRH receptor
INTRODUCTION
Gonadotropin-releasing hormone plays a central role in the neuroendocrine control of the reproductive process in vertebrates, notably by stimulating synthesis and release of gonadotropins. However, GnRH may also deserve other functions centrally. Indeed, a major outcome of the numerous comparative studies based on biochemical, morphological, and physiological approaches in all vertebrate classes is the fact that the brain of many species expresses at least two GnRH variants. One GnRH form is mainly synthesized in the forebrain and varies according to the species, whereas the second GnRH variant, chicken GnRH-II (cGnRH-II), is highly conserved in terms of both structure and site of expression [1]. Indeed, cGnRH-II-expressing neurons have been consistently detected in the synencephalic-mesencephalic area of many vertebrate groups, including primates [2]. If it is now clear that, at least part of the GnRH-expressing neurons of the anterior brain sustain mainly a hypophysiotropic function, the role of the cGnRH-II mesencephalic neurons remains totally unknown. However, the high conservation of this system in the vertebrate lineage suggests important neuromodulatory functions for this peptide. Because of the diversity of GnRH variants identified in teleosts, the GnRH systems of fish have been extensively studied by either immunohistochemistry and in situ hybridization [3]. There is now extensive information about the localization of the GnRH neurons and their respective projections in different species, but surprisingly, little attempt has been made to correlate this information with the expression of GnRH-R. Indeed, most studies on GnRH-R have focused on the pituitary and, more recently, on the gonads.
The first fish GnRH receptor (GnRH-R) has been characterized in the goldfish pituitary, and the presence of two classes of binding sites, one with high affinity and low capacity and one with low affinity and high capacity, was demonstrated [4]. However, in other species, such as the catfish [5], winter flounder [6, 7], sea bream, and stickleback [8], a single class of GnRH binding sites has been described in the pituitary, whereas in the lamprey, two high-affinity binding sites were shown [9]. In addition, similar to what has been shown in mammals [10, 11], extrapituitary sites of GnRH actions have been detected in a number of reproductive and nonreproductive organs such as the ovary, testis, brain, liver, and kidney in the goldfish [12].
The first functional GnRH-R was cloned using RNA purified from the mouse gonadotrope cell line 
T3-1 [13, 14]. A number of subsequent publications have described the cloning of GnRH receptors in several mammalian species including the rat [15], sheep [16], human [17], cow [18], and pig [19]. Analysis of the primary sequence shows that the GnRH-R has the predicted structure characteristic of a member of the large rhodopsin-like G protein-coupled receptor (GPCR) superfamily, consisting of a single polypeptide chain containing seven hydrophobic transmembrane domains connected by hydrophilic extra- and intracellular loops. Several features conserved among the GPCR family are altered in the mammalian GnRH-R, the most striking one being the complete absence of an intracellular C-terminal domain. These structural differences have been reviewed recently [20]. In addition to a structure-function analysis, the cloning of GnRH-R cDNAs allowed in situ hybridization to localize the GnRH-R mRNA-expressing cells in the rat brain [21] and ovary [22] where it was shown that the GnRH-R had the same primary structure as its pituitary counterpart [23].
Until now, GnRH-R have been cloned and characterized from only two teleost species: the African catfish, Clarias gariepinus [24], and the goldfish, Carassius auratus [25]. This latter species is until now the only one in which two GnRH-R cDNAs, gf A and gf B, sharing 71% identity have been found. These two cDNAs share 71% and 82% identity, respectively, with the catfish GnRH-R (cfGnRH-R) and 43% with the human receptor. The corresponding receptors show marked differences in their ligand selectivity and tissue distribution [25]. In contrast with the mammalian GnRH-R, but in accordance with other GPCRs, the receptors cloned in fish and also in Xenopus [26] contain an intracellular carboxy-terminal domain.
As pointed out before, information on the central distribution of GnRH-R in vertebrates is very limited. Therefore, one of the objectives of this study was to clone one or more GnRH-R in the rainbow trout (Oncorhynchus mykiss), to analyze its (their) tissue distribution(s), and to perform a detailed in situ hybridization study of its (their) sites of expression within the brain.
MATERIALS AND METHODS
Animals
Mature female rainbow trout were obtained from the INRA experimental fish farm (Le Drennec, France) and kept under recycled water at 12–15°C and natural photoperiod (46° north) until use. The developmental stage of the fish was estimated by the time of the year and confirmed by the gonadosomatic index. Animals were treated in agreement with the European Union regulations concerning the protection of experimental animals.
Cloning of a Full-Length cDNA Encoding the Trout GnRH-R
Total RNA was prepared from brain using the TRIzol reagent (Gibco-BRL, Gaithersburg, MD) according to the manufacturer's protocol. A first partial cDNA was obtained after two successive polymerase chain reaction (PCR) amplifications performed with degenerate primers designed from conserved regions of known GnRH-R. The first amplification was carried out using the following primers: For1, 5'-GAYGGVATGTGGAAYATHAC-3'; and Rev1, 5'-ACRTARTADGGNGTCCARCA-3'. A 336-base pair (bp) product spanning from the first to the second extracellular loop was then obtained using primers For1 and a second reverse primer Rev2 (5'-GRAACATGTTRTAKGCYGTTTCC-3').
The 3' extremity of the cDNA was cloned by 3' RACE (rapid amplification of cDNA extremity)-PCR, using a 5'-3' RACE kit according to the manufacturer's conditions (Boehringer Mannheim, Mannheim, Germany). Briefly, a reverse transcription (RT) was performed on 2.5 µg of total RNA using an oligo(dT)-anchor primer (Boehringer Mannheim). A first amplification was then carried out using primer 1 (5'-GGTGCAGTGGTATGGCGGAG-3') and the anchor-primer (Boehringer Mannheim). A nested PCR was conducted using 1 µl of the first reaction as template with primer 2 (5'-GCCACGTGTAAGATGCTATGCTTCC-3') and the same anchor-primer. Primers 1 and 2 were designed from the 336-bp cDNA. The PCR products were purified and cloned in the EcoRV site of Bluescript plasmid for sequencing.
The 5' extremity was obtained from the brain by RT-PCR using specific primers designed from the 336-bp cDNA (primer 3, 5'-TGCCATCGCTCTTTGAAGCT-3') and a genomic clone obtained by screening a genomic library with a probe corresponding to the 336-bp sequence (primer 4, 5'-GAGCAATGTAGAATAACTGACGGCA-3'). The PCR products were then cloned in the EcoRV site of Bluescript plasmid.
A cDNA containing the complete reading frame was generated under the same conditions using the following primers (primer 5, 5'-ACTTTGATGTGTATATGATAATAATGTATGTAATC-3' and primer 6, 5'-CTCCAGTCATCTACCGTCACCT-3') and was cloned into the Bluescript plasmid (p-rtGnRH-R).
Northern Blot Analysis
Thirty micrograms of total RNA from brain, optic tectum, pituitary, and liver were heat-denatured (65°C for 15 min) in formaldehyde-formamide, electrophoresed on a 1% agarose gel containing formaldehyde, and transferred onto a nylon membrane (Hybond-N; Amersham, Uppsala, Sweden). The RNA was fixed by UV illumination (254 nm) for 1 min and baking for 1 h at 80°C. The membrane was hybridized under the conditions previously described [27] with a 1372-bp probe corresponding to the coding region of the cDNA, labeled with [32P]dCTP by random priming. The membrane was then washed four times in 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.1% SDS for 5 min at room temperature and three times in 0.2x SSC, 0.1% SDS for 15 min at 50°C, and exposed to a Biomax film (Eastman-Kodak, Rochester, NY) at -80°C.
Analysis of rtGnRH-R Tissue Distribution by RT-PCR and Southern Blotting
For the detection of rtGnRH-R mRNA, tissues were collected and total RNA was prepared and reverse transcribed as described above with random hexamers. A 300-bp rtGnRH-R cDNA, corresponding to transmembrane domains III and IV, was amplified using the following primers: 300F, 5'-GGTGCAGTGGTATGGCGGAG-3' and 300R, 5'-TGCCATCGCTCTTTGAAGCT-3'. The PCR products were transferred onto a nylon membrane (Hybond-N; Amersham) and hybridized as described [27] with the corresponding probe labeled with [32P]dCTP by random priming.
In Situ Hybridization
Sense and antisense rtGnRH-R riboprobes were synthesized using the pBS-rtGnRH-R transcription vector containing the complete coding region, linearized with BamHI or HindIII as a template for T3 and T7 RNA polymerase, respectively.
Mature female rainbow trout, anesthetized with phenoxyethanol (0.3 ml/L), were perfused through the heart with 0.65% NaCl followed by a fixative solution (4% paraformaldehyde, 0.1 M phosphate buffer, pH 7.4). Brains and pituitaries were collected, fixed overnight at room temperature, dehydrated, embedded in paraffin, and cut at 6 µm. Sections were mounted on Tespa-treated (2% Tespa; Sigma, St. Louis, MO) slides for subsequent processing using the in situ hybridization protocol described previously [28].
Following hybridization and washes, sections were dehydrated, air-dried, and exposed to Biomax film (Amersham) for 5 days. Slides were then dipped in an autoradiographic emulsion (Ilford K5 Nuclear Track; Amersham) and stored at 4°C for 4–5 wk before development. Sections were then stained with toluidine blue and photographed with an Olympus Provis photomicroscope under darkfield or brightfield illumination.
The nomenclature used for rainbow trout brain nuclei is from Meek and Nieuwenhuys [29].
RESULTS
Cloning of a Full-Length cDNA for rtGnRH-Receptor
The cloning strategy and the different clones obtained are summarized in Figure 1. Using degenerate primers, a first cDNA of 336 bp (p336), corresponding to a region spanning from the first to the second extracellular loop, was obtained. This cDNA showed about 80% identity with the cfGnRH-R or the two gfGnRH-Rs. Sequence identities were lower with the mammalian GnRH-Rs (about 45%).
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The 3' extremity of the trout GnRH-R cDNA was obtained by 3' RACE-PCR. Using this method, we have cloned a PCR product of 1250 bp. This clone, pBS-1202, was shown to contain a part of the coding region spanning from transmembrane domain III to the intracellular COOH-terminus of the receptor and 429 bp of the 3' untranslated region. A polyadenylation signal AATAAA was found 35 nucleotides upstream from the poly(A) tail.
The 5' extremity was obtained by RT-PCR using specific primers designed from the cDNA cloned previously and a genomic clone containing the first exon of rtGnRH-R gene (T. Madigou, unpublished data). A 750-bp long cDNA (pATG-750) was obtained. This clone contained part of the 5' untranslated region and an open reading frame encoding the extracellular NH2 terminus of the rtGnRH-R and transmembrane domains I to IV. The two clones (pATG-750 and pBS-rtGnRH-R1202) overlapped and were 100% identical to a region of 300 bp corresponding to transmembrane domains III and IV. The complete coding sequence of the trout GnRH-R was deduced from these two clones, and in order to check if these clones corresponded to the two extremities of the same RNA, a cDNA of about 1.3 kilobases (kb) containing the entire reading frame was generated using two specific primers located in the 5'- and the 3'-untranslated regions, respectively. The sequence of this cDNA has been deposited in the EMBL Nucleotide Sequence Database (accession number AJ272116). Different attempts to obtain a second potential GnRH-R cDNA have failed until now.
Sequence Analysis and Comparison of rtGnRH-R with Other GnRH-R
The nucleotide sequence and the deduced amino acid (aa) sequence of the rtGnRH-R are shown on Figure 2. The potential translation initiation site, as predicted using Netstart 1.0 neural network prediction server (Center for Biological Sequence Analysis, Denmark), corresponds to the ATG present in all other species. If we consider this codon as the initiation site, the extracellular domain consists of 39 aa. However, another ATG is located 90 bp upstream from this first site. This second ATG could give rise to a receptor with a 30-aa longer extracellular domain. However, until the functionality of this first ATG as a translation initiation codon is established, we will consider the second methionine as Met1, giving rise to a protein of 386 aa (Fig. 2). Hydrophobicity analysis of the deduced aa sequence, as determined by the method of Kyte and Doolittle [30], shows that the transmembrane domain is constituted of 289 aa arranged as seven transmembrane segments typical of a GPCR. In contrast to its mammalian counterparts, but like all nonmammalian GnRH-R described at this time, it contains an intracellular domain consisting of 58 aa. In this domain, as in catfish and goldfish receptors, serine residues that are potential phosphorylation sites (SXXS*) are conserved (Ser380 and Ser383). Similarly, a single cysteine (Cys369 in the trout receptor) that may be a site of palmitoylation is conserved in all fish GnRH-Rs.
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Alignment of the predicted rtGnRH-R amino acid sequence with the corresponding sequences described in catfish [24], goldfish [25], mouse [13], rat [15], sheep [16], human [17], bovine [18], Drosophila [31], and Typhlonectes natans (Ebersole et al., Genbank AF174481) was performed using Clustal W (Fig. 3), and a phylogenetic tree was drawn (Fig. 4). These analyses showed that the rtGnRH-R is closely related to the other fish GnRH-R and particularly to the goldfish GnRH-R A (>80% identity). The rtGnRH-R shares about 40% identity with the mammalian or amphibian receptors.
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Tissue Distribution of the rtGNRH-R mRNA
The size of the rtGnRH-R mRNA was determined by Northern blot analysis on total RNA extracted from various tissues of an ovulating female. This analysis revealed the presence of a single mRNA of approximately 1.9–2 kb in[fj[fpthe pituitary, but no signal could be detected in the brain, even on poly(A)+ RNA, or the ovary (Fig. 5A). The absence of signal in these latter tissues might indicate that mRNA levels were below the detection limits of this method. Thus, we used the more sensitive RT-PCR and Southern blotting technique. Products of amplification of the expected size were found in extrapituitary tissues such as the brain, particularly the optic tectum, the retina, and the ovary (Fig. 5B). Surprisingly, a weaker signal was obtained in the pituitary. However, the tissues used for this analysis were collected from vitellogenic females.
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Localization of rtGnRH-R mRNA in the Brain and Pituitary of the Rainbow Trout
The precise localization of rtGnRH-R mRNA in the brain and pituitary of female rainbow trout was studied by in situ hybridization using the full-length coding region of the rtGnRH-R cDNA as a probe. In general, in situ hybridization showed a very weak expression of rtGnRH-R mRNA throughout the brain and pituitary, confirming our results of Northern blotting and RT-PCR. Nevertheless, specific hybridization signals could be detected in the preoptic region (parvicellular preoptic nucleus; Fig. 6A), the mediobasal hypothalamus (nucleus lateralis tuberis; Fig. 6C), the pituitary (proximal pars distalis; Fig. 6C), and the periventricular layer of the optic tectum (Fig. 6E). Surprisingly, the strongest signal was consistently detected in the nucleus lateralis valvulae, a mesencephalic structure bridging the dorsal tegmentum with the valvula of the cerebellum (Fig. 7). This nucleus contains small cells strongly stained by toluidine blue and larger elements. Figure 7F shows that the labeling preferentially concerns the large pale cells. The specificity of the signal was systematically checked on adjacent sections hybridized with the corresponding sense probe showing only uniform background (Figs. 6, B and D and 7, E and G).
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DISCUSSION
In this study, we have cloned a cDNA encoding a GnRH-R from rainbow trout and used it as a probe to study the localization of the corresponding mRNA in different organs by RT-PCR and in the brain and pituitary by in situ hybridization. This cDNA, containing the full-length coding sequence of the rainbow trout GnRH-R, was obtained by RT-PCR. In this cDNA, a methionine codon is found at the same position as in all the GnRH-Rs. However, another in-frame ATG, 90 bp upstream of this methionine, could give rise to a protein with a putative extracellular domain 30 aa longer than those found in the other GnRH-Rs. Multiple transcription start sites have already been reported in many genes, including GnRH-R as for example in human [32] or sheep [33]. At the present stage, it is not known whether messengers lacking this ATG are generated in rainbow trout. It is interesting to note that such a potential translation initiation site is also present upstream of the predicted first ATG in the gonadotropin receptor of the amago salmon (Oncorhynchus rhodurus) [34]. The sequence identity with the catfish and goldfish GnRH-R were found to be around 80%, whereas it was only about 40% with the mammalian receptors. In the goldfish, recent data have demonstrated the presence of two GnRH-R, Gf A and Gf B, sharing 71% identity [25]. Phylogenetic analyses reveal that the rtGnRH-R is more closely related to the goldfish Gf A than to the Gf B. Until now, there is no indication for the presence of two different receptor subtypes in rainbow trout.
The alignment of the aa sequence of the rtGnRH-R with the other receptors shows that a number of key features are conserved. In most of the GPCR, conserved cysteines in the first and the second extracellular loops form a disulfide bound that is necessary for the correct folding of the receptor [35, 36]. The presence of such a bridge was demonstrated in the rat GnRH-R between Cys114 and Cys195 [37]. In the trout GnRH-R, two cysteines are also found in the corresponding position (Cys119 and Cys186). However, another cysteine is present in the second extracellular loop. In the same way, the putative ligand contact sites Asp98, Asn102, Lys121, and Glu301 described for the mouse [20] are conserved in the rtGnRH-R as Asp103, Asn107, Lys120, and Glu304.
Structural elements such as an Asp-Arg-Tyr (DRY) motif in the cytoplasmic end of TMIII or Asn-Pro-X-X-Tyr (NPXXY) in TMVII are highly conserved in the rhodopsin-like GPCR family. The DRY motif was shown to be implicated in receptor activation and the coupling with G-proteins [38]. In mammalian GnRH-Rs, the Tyr is replaced by a Ser except in the marmoset in which a Phe is found [39]. Several studies have shown that mutation of the third aa of this motif may affect receptor functionality. Indeed, substitution of Ser by Tyr or Phe induces an increase in agonist binding affinity and internalization rate [39, 40], whereas replacement by Ala affects neither internalization nor signal transduction [41]. The trout GnRH-R, like all other fish GnRH-Rs, presents a substitution of the Tyr by a His in this DRY motif. Because Phe and Tyr have the same effect on receptor internalization, it has been suggested that aromatic residues at this site could be important in regulating internalization [39]. Although the presence of an aliphatic residue, Ser or Ala, in this motif seems to reduce receptor internalization, the effect of a substitution by a heterocyclic amino acid such as His has not been evaluated. Another conserved domain is the NPXXY motif located in TMVII that has also been shown to be involved in the internalization of some GPCR including the GnRH-R [42]. Although the sequence is modified to DPXXY in all the GnRH-R, the Tyr of this motif, critical for receptor activation and signal transduction, is conserved [42].
The three-dimensional model of the GPCRs and reciprocal mutation of conserved loci in GnRH-R predicts that TMII and TMVII are juxtaposed [43]. Two residues, Asp in TMII and Asn in TMVII, highly conserved among GPCRs but interchanged in all mammalian GnRH-Rs, are likely to be implicated in interhelical interaction [44]. Moreover, the mutation of Asn in TMII by Asp in mouse [43] or rat [45] GnRH-R eliminated detectable ligand binding. However, this seems to be specific to the mammalian receptor because in all other nonmammalian receptors including the rtGnRH-R, two Asp are found at these positions.
Similar to other nonmammalian GnRH-R, the trout receptor contains an intracellular tail that in this case consists of 58 residues. This intracellular tail is important for receptor functioning because the truncation of this domain produces the loss of both the ability for agonist binding and GnRH-stimulated cAMP production [46]. Likewise, addition of cfGnRH-R C-terminal tail to the rat GnRH-R affects its expression, regulation, and the differential coupling with G proteins [47], producing a rapid desensitization of inositol phosphate production as well as increased internalization rate [48].
Northern blot analysis of total RNA revealed the presence of a single GnRH-R mRNA species only in the pituitary of ovulating female trout. This mRNA is shorter than those described in mammals in which a major band of 4–5 kb is detected, together with other shorter bands [13, 16–18]. The structure of the GnRH-R gene has been elucidated in several mammalian species. Unlike many GPCRs, the GnRH-R gene contains introns [32, 33, 49, 50], allowing the existence of alternative splicing, which may explain the presence of multiple bands on Northern blot analysis. In all these species, the gene is constituted of three exons and two introns. In the mouse, several variant transcripts lacking exon 2 or exon 3 have been described [49]. Although only one band is detected by Northern blotting in the trout, we have also found in the brain by RT-PCR an mRNA variant that contains only exons 1 and 2 and may encode a truncated protein corresponding to the NH2 terminus and TM I to V (unpublished data). Unfortunately, brain expression levels of GnRH-R messengers appear too low for allowing detection by Northern blot, even on poly(A)+ RNA. Truncated receptors have already been reported in humans and were shown to be unable to bind GnRH and to activate signal transduction [51]. However, when coexpressed with the wild-type receptor, this truncated protein caused impaired insertion of the wild type into the plasma membrane [51].
Because Northern blotting failed to detect GnRH-R mRNA in other tissues than the pituitary, we used the more sensitive method of RT-PCR followed by Southern blotting and hybridization. The PCR products were found in extrapituitary tissues, namely the brain, the retina and the ovary of vitellogenic females. These results confirm those obtained in the gonads of other teleosts. Indeed, Scatchard analysis revealed the presence of one class of high affinity binding sites in the ovary of common carp [52], African catfish [53], and immature goldfish, whereas two classes of GnRH binding sites with characteristics similar to that of the pituitary were identified in the ovary of mature goldfish [54]. In this latter species, in situ hybridization demonstrated expression of Gf A receptor mRNA in the interstitial tissue and the theca-granulosa cell layers surrounding the oocytes [25]. The precise role of GnRH in the gonads of fish remains unclear, although in vitro studies indicate that it may act as a meiosis-stimulating factor in the oocyte [55]. Similarly, the presence of gonadal GnRH-R has been demonstrated in mammals, and, in the rat, the primary structure of the ovarian receptor was shown to be identical to that of the hypohyseal receptor [23].
The presence of GnRH-Rs in the retina correlates with previous studies demonstrating an innervation of the retina by GnRH-immunoreactive fibers [56]. In cichlid, poecilid, and centrarchid fishes, GnRH-immunoreactive neurons are found in the nucleus olfactoretinalis and processes of these neurons project to the contralateral retina [57]. A more recent study in Atlantic salmon showed that some GnRH-positive cells clustered within the medial component of the olfactory nerve project to the retina where the GnRH fibers synapse on the dopaminergic interplexiform cells [58]. It has been shown that the effects of GnRH on horizontal cell activity mimicked the effects of dopamine on these cells, suggesting that GnRH acts by stimulating the release of dopamine from interplexiform cells [59, 60].
In this study, we have also analyzed the distribution of GnRH-R mRNA in the pituitary by in situ hybridization using a probe corresponding to the full-length coding region. The signal was restricted to the proximal pars distalis but remained weak in mature females or ovulating females. This is in agreement with the weak signal detected by RT-PCR (this study) and with the fact that binding studies using different GnRH analogs failed to evidence a strong specific binding in the trout pituitary (C. Weil and L.W. Crim, personal communication) in contrast with other species [5–8]. This is surprising because GnRH is well known to be active in vivo and in vitro in stimulating LH secretion in the trout [61]. The weak but significant signal observed in the pituitary by Northern blotting tends to indicate that the number of GnRH-R mRNA copies per cell is low but still sufficient to be detected by Northern blot. In addition, parallel studies performed in sea bass using the same technique allowed detection of a very strong GnRH-R hybridization signal in the pituitary of mature, and even immature, males [62].
To date, most of the information concerning the localization of GnRH-R expressing cells in the brain has been obtained by autoradiography of binding of 125I-labeled GnRH analogs and in situ hybridization in mammals. In the rat brain, the mRNA encoding the GnRH-R is present in selected regions associated with the regulation of GnRH release from the median eminence and with the generation of reproductive behaviors [21].
In fish, apart from a possible neuromodulatory function, GnRH has been shown to stimulate sexual behavior [63]; however, the precise central sites of GnRH actions are unknown. This paper reports for the first time the precise distribution of GnRH-R mRNA in the brain of a teleost fish. In the brain of trout, a specific signal could be consistently detected in two regions clearly implicated in the neuroendocrine control of the pituitary, the preoptic area, and the mediobasal hypothalamus [64]. In teleosts, the main GnRH-positive fiber tracts cross the preoptic region and the ventral hypothalamus on their way to the pituitary [56, 65] and one may speculate that these varicose fibers establish contacts of passage with some cell populations and influence their activities.
A significant GnRH-R hybridization signal has also consistently been detected in the optic tectum in agreement with the RT-PCR results. The optic tectum of teleosts is a highly laminar retino-recipient structure in which GnRH-R mRNAs were restricted to the piriform neurons of the periventricular layer shown recently to strongly express melatonin receptor in trout [28], and to be the site of a circadian clock in zebrafish [66]. The potential functions of GnRH in the tectum remain to be established. However, the expression of GnRH-R in the tectum is consistent with the significant GnRH innervation of this structure [56, 65].
Very surprisingly, the highest GnRH-R hybridization signal was consistently found in a mesencephalic structure, the nucleus lateralis valvulae, that has been well studied in the carp [67]. In this species, this nucleus consists of small adendritic cells, projecting to both the corpus and the valvula of the cerebellum, and larger dendritic elements [29, 67]. Our study tends to indicate that expression of the GnRH-R is restricted to these larger cells. In the cyprinid Phoxinus phoxinus, part of the cell bodies in the nucleus lateralis valvulae have been shown to be cholinergic on the basis of acetylcholinesterase histochemistry and choline acetyltransferase immunohistochemistry [68]. There is no precise information on the functions of the nucleus lateralis valvulae, but it could be implicated in the processing of sensory and motor information [29]. The nucleus lateralis valvulae receives afferents from the telencephalon and possibly from the nucleus of the medial longitudinal fascicle [67], whose large neurons are well known for expressing cGnRH-II in salmonids [69] and other species. Although no particular cGnRH-II innervation has ever been reported in the nucleus lateralis valvulae, a potential cGnRH-II projection from the nucleus of the medial longitudinal fascicle to the nucleus lateralis valvulae deserves considerable interest, as there is no precise information on the potential role of cGnRH-II nor on its sites of action in fish and other vertebrates. It is important to point out that although initially discovered in fish [56, 70], cGnRH-II neurons in the mesencephalon have now been identified in all vertebrate groups, including mammals [2]. Because the contribution of these cells to the pituitary in fish or median eminence in tetrapods seems to be low or nonexistant, cGnRH-II certainly plays some still highly enigmatic but crucial role within the brain in view of the high conservation of the cGnRH-II system. Interestingly, a recent study in Xenopus showed that the central expression of a GnRH-R is mainly associated with the midbrain in this species [26].
In conclusion, a GnRH-R cDNA has been cloned from the rainbow trout and may encode a protein with a 30-aa longer extracellular domain. As already reported in catfish and goldfish, this receptor contains an intracellular domain, in contrast with its mammalian counterparts. The corresponding mRNAs are expressed in the retina, the brain, the pituitary, and the ovaries. In the brain the highest expression is clearly associated with a mesencephalic structure, the nucleus lateralis valvulae, whose precise function is unknown but may concern the processing of sensory or motor information.
FOOTNOTES
1 Supported by the CNRS, INRA, Ministère de la Recherche et de la Technologie, Fondation Langlois, and European Union (Fair CT97-3785). The first two authors have contributed equally to this study. 
2 Correspondence: O. Kah, Endocrinologie Moléculaire de la Reproduction, UMR CNRS 6026, Bat 13, Campus de Beaulieu, 35042 Rennes cedex, France. FAX: 33 2 99 28 67 94; olivier.kah{at}univ-rennes1.fr
Accepted: August 7, 2000.
Received: April 4, 2000.
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D. Gonzalez-Martinez, T. Madigou, E. Mananos, J. M. Cerda-Reverter, S. Zanuy, O. Kah, and J. A. Munoz-Cueto Cloning and Expression of Gonadotropin-Releasing Hormone Receptor in the Brain and Pituitary of the European Sea Bass: An In Situ Hybridization Study Biol Reprod, May 1, 2004; 70(5): 1380 - 1391. [Abstract] [Full Text] [PDF]
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 R. P. Millar, Z.-L. Lu, A. J. Pawson, C. A. Flanagan, K. Morgan, and S. R. Maudsley Gonadotropin-Releasing Hormone Receptors Endocr. Rev., April 1, 2004; 25(2): 235 - 275. [Abstract] [Full Text] [PDF]
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J. Bogerd, W. B. Diepenbroek, E. Hund, F. van Oosterhout, A. C. C. Teves, R. Leurs, and M. Blomenrohr Two Gonadotropin-Releasing Hormone Receptors in the African Catfish: No Differences in Ligand Selectivity, but Differences in Tissue Distribution Endocrinology, December 1, 2002; 143(12): 4673 - 4682. [Abstract] [Full Text] [PDF]
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K. Okubo, S. Nagata, R. Ko, H. Kataoka, Y. Yoshiura, H. Mitani, M. Kondo, K. Naruse, A. Shima, and K. Aida Identification and Characterization of Two Distinct GnRH Receptor Subtypes in a Teleost, the Medaka Oryzias latipes Endocrinology, November 1, 2001; 142(11): 4729 - 4739. [Abstract] [Full Text] [PDF]
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