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Endocrinology Vol. 142, No. 11 4729-4739
Copyright © 2001 by The Endocrine Society

ARTICLES

Identification and Characterization of Two Distinct GnRH Receptor Subtypes in a Teleost, the Medaka Oryzias latipes

Kataaki Okubo, Shinji Nagata, Rinkei Ko, Hiroshi Kataoka, Yasutoshi Yoshiura1, Hiroshi Mitani, Mariko Kondo, Kiyoshi Naruse, Akihiro Shima and Katsumi Aida

Department of Aquatic Bioscience (K.O., Y.Y., K.A.), Graduate School of Agricultural and Life Sciences, and Department of Integrated Biosciences (S.N., R.K., H.K.), Graduate School of Frontier Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan; and Department of Integrated Biosciences (H.M., M.K., A.S.), Graduate School of Frontier Sciences, The University of Tokyo, and Department of Biological Sciences (K.N.), Graduate School of Science, The University of Tokyo, Bunkyo, Tokyo 113-0033, Japan

Address all correspondence and requests for reprints to: Dr. K. Aida, Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan. E-mail: aida{at}uf.a.u-tokyo.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We report the identification and characterization of two distinct GnRH receptor (GnRH-R) subtypes, designated GnRH-R1 and GnRH-R2, in a model teleost, the medaka Oryzias latipes. These seven-transmembrane receptors of the medaka contain a cytoplasmic C-terminal tail, which has been found in all other nonmammalian GnRH-Rs cloned to date. The GnRH-R1 gene is composed of three exons separated by two introns, whereas the GnRH-R2 gene has an additional intron and therefore consists of four exons and three introns. The GnRH-R1 and GnRH-R2 genes, both of which exist as single-copy genes in the medaka genome, were mapped to linkage groups 3 and 16, respectively. Inositol phosphate assays using COS-7 cells transfected with GnRH-R1 and GnRH-R2 demonstrated that they had remarkably different ligand sensitivities, although both receptors showed highest preference for chicken-II-type GnRH. Phylogenetic analysis showed the presence of three paralogous lineages for vertebrate GnRH-Rs and indicated that neither GnRH-R1 nor GnRH-R2 is the medaka ortholog to mammalian GnRH-Rs that lack a cytoplasmic tail. This, together with an observation that medaka-type GnRH had low affinity for GnRH-R1 and GnRH-R2, suggests that a third GnRH-R may exist in the medaka.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH IS A decapeptide widely known for its critical role in vertebrate reproduction. GnRH synthesized in the preoptic/hypothalamic area is transported to the pituitary where it stimulates the synthesis and release of gonadotropins (1, 2). In addition to the preoptic/hypothalamic GnRH, one or two other GnRH forms have been found to be expressed in the extrapreoptic/hypothalamic area within the central nervous system of most vertebrates (3, 4, 5). Intriguingly, one of these extrapreoptic/hypothalamic forms, designated chicken-II-type GnRH (cGnRH-II), is conserved among vertebrates from cartilaginous fish to mammals. This conserved form is produced universally in the midbrain tegmentum. The cGnRH-II is suggested to be important in neuromodulation, although its precise function remains to be elucidated (3, 4, 5).

GnRHs exert their actions through interactions with specific receptors that belong to the rhodopsin-like G protein-coupled receptor (GPCR) family (5, 6). GnRH receptors (GnRH-Rs) have been isolated from several mammalian and nonmammalian vertebrate species (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16). Notably, the mammalian GnRH-R has a unique feature in that it is the only GPCR cloned to date that has a truncated cytoplasmic C-terminal tail. It is hypothesized that the cytoplasmic tail was lost on the evolutionary lineage between nonmammalian vertebrates and mammals (10, 12). It has also been suggested that multiple subtypes of GnRH-R are present in individual vertebrate species, based on increasing evidence showing the occurrence of varied GnRH ligand forms in one organism and the distribution of GnRH-binding sites (17). Nevertheless, until quite recently, only a single subtype of GnRH-R had been isolated from each species. Troskie et al. (18), however, have obtained the partial sequences for two candidate GnRH-R genes in the goldfish Carassius auratus, zebrafish Danio rerio, Xenopus laevis, and lizard Agama atra. Subsequently, three full-length GnRH-Rs have been isolated in the bullfrog Rana catesbeiana (19). Neill et al. (20) have cloned second primate GnRH-Rs, which have a cytoplasmic C-terminal tail, unlike other mammalian GnRH-Rs isolated so far. The multiplicity of GnRH-Rs in one organism then raised the following questions: (a) Do other vertebrates besides the bullfrog and primates also have multiple GnRH-Rs; (b) do they have different structural, pharmacological, and/or physiological characteristics; and (c) what are the phylogenetic relationships among multiple GnRH-Rs?

To answer these questions, we have taken one freshwater teleost fish, the medaka Oryzias latipes, as an experimental model system. This fish is a promising model organism for reproductive, developmental, phylogenetic, and genetic studies because of its many useful characteristics such as its well-established reproductive biology and genetics, short generation time, and availability of a large number of inbred strains and mutants (21, 22, 23). Our previous study demonstrated that the medaka possesses three molecular forms of GnRH in its central nervous system; medaka-type GnRH (mdGnRH) expressed in the preoptic area probably as the stimulator of gonadotropin secretion, cGnRH-II in the midbrain tegmentum, and salmon-type GnRH (sGnRH) in the terminal nerve ganglia (24).

In the present study, we report the presence of two distinct GnRH-R subtypes, termed GnRH-R1 and GnRH-R2, in the medaka. We have isolated the cDNAs encoding these two GnRH-Rs and determined the nucleotide sequence, structural organization, copy number, and chromosomal assignment of their genes. The ability of the cloned receptors to couple to the inositol phosphate (IP) second-messenger pathway was investigated using COS-7 cells transiently expressing the GnRH-Rs. In addition, we propose a novel classification of vertebrate GnRH-R subtypes based on phylogenetic analysis in combination with the differences in their protein and gene structures and pharmacological characteristics.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation of the medaka GnRH-R cDNAs
One medaka strain, himedaka (orange-red variety), was purchased from a local dealer. The brains from 50 adult fishes were pooled for RNA extraction. Total RNA was prepared with ISOGEN (Nippongene, Tokyo, Japan), and subsequently poly (A)+ RNA was purified with Oligotex-dT 30 (Takara, Shiga, Japan). Double-strand cDNA was synthesized using a Marathon cDNA amplification kit (CLONTECH Laboratories, Inc., Palo Alto, CA). Degenerate primer pairs, R1-F0 (5'-TA(C/T)AA(C/T)ATGTT(C/T)ACITT(C/T)TCITG-3'; nt 1075–1097)/R1-R0 (5'-A(A/G)IA(A/G)(A/G)TA(A/G)TAIGGIGTCCA(A/G)CA-3'; nt 1288–1310) and R2-F0 (5'-TGGAA(C/T)(A/G)TIACIGTICA(A/G)TG-3'; nucleotide position (nt) 538–557)/R2-R0 (5'-TG(A/G)AACCA(A/G)TACCAIA(C/T)ICC-3'; nt 1087–1106), were used to amplify transmembrane (TM) domain coding regions of the GnRH-R1 and GnRH-R2 cDNAs, respectively. PCR was performed in a thermal cycler PC-701 (Astec, Fukuoka, Japan) in 20 µl containing 1x PCR buffer (Takara), 200 µM of dNTPs (Takara), 0.5 U Taq DNA polymerase (Takara), 0.5 µM each of the primer pair, and an appropriate amount of the brain cDNA template. The PCR cycle protocol was as follows: 94 C for 1 min, 40 cycles of 94 C for 30 sec, 52 C for 30 sec, 72 C for 30 sec, and 72 C for 7 min. The PCR products were analyzed on 2.0% agarose gels and ligated into pBluescript SK (-) (Stratagene, La Jolla, CA). The plasmid DNA was purified, and both strands of the DNA were sequenced using a DNA sequencer SQ-5500 (Hitachi, Tokyo, Japan).

After determination of the partial cDNA sequences, rapid amplification of cDNA ends (RACE) was carried out to isolate the full-length cDNAs as described before (13). Gene-specific primers, R1-F1 (5'-AAAACGTTTCCTCCGACGAGCCG-3'; nt 1178–1200)/R1-F2 (5'-GCCGCATCTGCGCTGCTCAAAG-3'; nt 1197–1218) and R2-F1 (5'-CAGGTGAATCACATCTTCGTCGCAGC-3'; nt 962–987)/R2-F2 (5'-GTGCTGGACGCCATACTACCTGCTG-3'; nt 1062–1086) were designed to use as primary/nested primers for 3'-RACE of the GnRH-R1 and GnRH-R2, respectively. Primers, R1-R1 (5'-TTTTTTGGTCATCTGCTTGGAGATCTG-3'; nt 1179–1153)/R1-R2 (5'-GTAGCAGATGATCATGATGATGAGC-3'; nt 1113–1137) and R2-R1 (5'-GGCGTGCATGGCAAACAGCTTCAGG-3'; nt 594–618)/R2-R2 (5'-GCAGAGCA-GCTTGCACAGAGCGTC-3'; nt 568–591) were also designed as primary/nested primers for 5'-RACE of the GnRH-R1 and GnRH-R2, respectively. Electrophoresis, subcloning, and sequencing were performed as described above.

Sequence analysis was performed using Sequencher software version 3.1.1 (Hitachi) and SeqEd software version 1.0.3 (Perkin-Elmer Corp., Branchburg, NJ). The nucleotide sequence was determined by analyzing more than four clones from distinct amplifications to avoid PCR errors.

Comparison of the GnRH-Rs of the medaka and other vertebrates
The deduced amino acid sequences of the GnRH-Rs in the medaka and other vertebrates were aligned using CLUSTAL W (25) with default setting. Sequence identity among TM domains I-VII in the GnRH-Rs of the medaka and other vertebrates was calculated using Mac Vector version 6.0 (Oxford Molecular, Beaverton, OR).

Isolation of the medaka GnRH-R genes
Genomic DNA was extracted from the muscle of a medaka strain, himedaka, as described elsewhere (26). The two GnRH-R genes were isolated by means of PCR amplifications of partial sequences and alignment of the contigs by sequence analysis. The following primer pairs were used to amplify GnRH-R1 and GnRH-R2 genes: for the GnRH-R1 gene, R1-F3 (5'-CACAGACCAATCATCTGCACGCGG-3'; nt 1–24)/R1-R3 (5'-CACCGGCATCACGATGAAGGTGAC-3'; nt 724–748), R1-F4 (5'-CCACAAGCGCAAGTCTCACGTCCG-3'; nt 663–686)/R1-R4 (5'-TCCTGCCAGTGGGTCACGAAGCTC-3'; nt 1044–1067), R1-F5 (5'-CAAACTTCACCCAGTGCACCACTAGAG-3'; nt 1016–1042)/R1-R5 (5'-ACGCTCATCTTCAGAGTTCTCATCCG-3'; nt 1237–1262), R1-F6 (5'-GCCGCATCTGCGCTGCTCAAAG-3'; nt 1197–1218)/R1-R6 (5'-CCCGGTGTGAACATAGCATCAGACG-3'; nt 1703–1727), R1-F7 (5'-TCATCTTCGGGCTTTTCAACACCTGC-3'; nt 1382–1407)/R1-R7 (5'-ATTGCAGGAATTGTACGACACCAAGTC-3'; nt 2053–2079), and R1-F8 (5'-TCAGCGGGTTCAAGTCTTGCGAAAC-3'; nt 1984–2008)/R1-R8 (5'-GGCTGGTTCATCTGCAGACACCACT-3'; nt 2712–2736); for the GnRH-R2 gene, R2-F3 (5'-CTACTGGACCGACATGGAGCCGAG-3'; nt 175–198)/R2-R3 (5'-GGCGTGCATGGCAAACAGCTTCAGG-3'; nt 594–618), R2-F4 (5'-GGTGACGCTCTGTGCAAGCTGCTC-3'; nt 565–588)/R2-R4 (5'-CCAGCGATGACTAAAGCTGCCATGAG-3'; nt 815–840), R2-F5 (5'-CTCTTCAGGACCATCAAAGTCGACCG-3'; nt 766–791)/R2-R5 (5'-CGAGCCTTTGGGATGATGTCTGTGC-3'; nt 989-1013), R2-F6 (5'-CAGGTGAATCACATCTTCGTCGCAGC-3'; nt 962–987)/R2-R6 (5'-TAATCCATGGACACCAGTGAAGATCAC-3'; nt 1490–1516), and R2-F7 (5'-CCTTGTTCTATCCTAGGCACTTTAACG-3'; nt 1401–1427)/R2-R7 (5'-AGTGTGTCAGAATAAAAGCTTTAAGCGTC-3'; nt 1863–1891). The PCR products were electrophoresed on 2.0% agarose gels and ligated into pCR-TOPO vector (Invitrogen, Groningen, The Netherlands). The plasmid DNA was purified, and both directions of the DNA were sequenced using two DNA sequencers, Long-Read Tower (Amersham Pharmacia Biotech, Buckinghamshire, Little Chalfont, UK) and SQ-5500 (Hitachi). Sequence analysis was performed as described above. Within introns, several nucleotide sequence differences, which could be owing to interspecific polymorphisms, occurred. Thus, only the consensus sequences were deposited into the database.

Genomic Southern blot analysis
Genomic DNA of the himedaka was digested with DraI, Eco T14, HindIII, or PstI. Ten micrograms of DNA were electrophoresed on 0.8% agarose gels and transferred to nylon membranes (Hybond-N+; Amersham Pharmacia Biotech). Digoxigenin-labeled cDNA probes for GnRH-R1 (323 bp) and GnRH-R2 (270 bp) were generated by PCR amplification with DIG DNA labeling mix (Roche, Grenzach-Wyhlen, Germany) and primer pairs, R1-F9 (5'-TGCCTGGACCCCATCATCTACG-3'; nt 1405–1426)/R1-R9 (5'-CCCGGTGTGAACATAGCATCAGACG-3'; nt 1703–1727) and R2-F8 (5'-CCAGCCTGACATGCTACGTGTC-3'; nt 1104–1125)/R2-R8 (5'-AATGCGAACGAGCGGTCCAGTG-3'; nt 1352–1373), respectively. The membranes were prehybridized at 68 C for 2 h with hybridization buffer containing 5x SSC, 1% blocking reagent (Roche), 0.1% N-lauroyl-sarcosine, and 0.02% SDS. Hybridization was carried out overnight at 68 C with the hybridization buffer containing the labeled probes. The membranes were subsequently washed with 2x SSC containing 0.1% SDS and with 0.1x SSC containing 0.1% SDS. Signals were visualized with antidigoxigenin antibodies conjugated to alkaline phosphatase and CDP-Star as a substrate (Roche).

Interspecific backcross mapping
Several parts of the GnRH-R genes of two inbred strains of the medaka, HNI and AA2, were sequenced. Insertion/deletion polymorphisms between the two strains were found within intron B of the GnRH-R1 gene and intron A of the GnRH-R2 gene. The following primer pairs were designed to amplify the polymorphism-containing sequences: R1-F10 (5'-AGGACAGTCAAATCTGACCT-3')/R1-R10 (5'-TATGAGAAAT(C/T)ACTCATCCTTACT-3') and R2-F9 (5'-CCCACTGAAAGTTCTGCATG-3')/R2-R9 (5'-ATCAGTCAGGAGATTCTGCT-3') for the GnRH-R1 and GnRH-R2 genes, respectively. PCR amplification of the HNI and AA2 genomic DNAs with R1-F10/R1-R10 yielded DNA fragments of 110 and 122 bp, respectively. Amplification of the HNI and AA2 genomic DNAs with R2-F9/R2-R9 produced fragments of 103 and 81 bp, respectively. Chromosomal assignments of these two genes were determined using reference-typing DNA panels derived from 39 offspring of a backcross between an HNI/AA2 male F1 and an AA2 female parental line (27). Genotypes were analyzed by amplification of the polymorphic DNA regions followed by 10% PAGE. PCR was carried out under a cycle protocol of 94 C for 1 min, 40 cycles of 94 C for 30 sec, 50 (GnRH-R1) or 58 C (GnRH-R2) for 30 sec, and 72 C for 30 sec.

IP assay
The cDNAs containing full-length open reading frames for GnRH-R1 and GnRH-R2 were subcloned into the expression vector pcDNA3 (Invitrogen). The plasmid DNA was transfected into monolayer cultures of COS-7 cells in 100-mm dishes using TransFast transfection reagent (Promega Corp., Madison, WI). After 24 h, cells were trypsinized, transferred to 12-well plates (Corning, Inc., Corning, NY), and grown overnight. Cells were subsequently labeled with 2 µCi/ml myo[2-3H]inositol (Amersham Pharmacia Biotech) in inositol-free medium (Life Technologies, Inc., Rockville, MD) containing 2% heat-inactivated FCS and 50 µg/ml gentamicin. Cells were incubated for 24 h and then washed and preincubated for 15 min at 37 C in IP buffer (1x HBSS, 20 mM HEPES, 20 mM LiCl, and 50 µg/ml gentamicin, pH 7.55), followed by stimulation with various concentrations of GnRH ligands in IP buffer for 1 h at 37 C, with gentle agitation. The GnRH ligands used in the assay were mdGnRH (synthesized by Sawady Technology, Tokyo, Japan), cGnRH-II (Phoenix Pharmaceuticals, Inc., Belmont, CA), sGnRH (Sigma, St. Louis, MO), and mammalian-type GnRH (mGnRH) (Sigma). The production of total IP including IP, IP2, and IP3 was assessed as described by Berg et al. (28). Experiments were performed in triplicate and repeated at least twice. Data obtained were analyzed using PRISM software(GraphPad Software, Inc., San Diego, CA).

Phylogenetic analysis of vertebrate GnRH-Rs
A phylogenetic tree was generated by PHYLIP software (29) using the neighbor-joining method (30). The full-length GnRH-R polypeptide was used to generate the tree. Bootstrap values were calculated by PHYLIP. The Drosophila GnRH-R homolog was used as an outroot (31). Full species names and GenBank accession numbers of the species used to generate the tree are as follows: eel Anguilla japonica, AB041327; trout Oncorhynchus mykiss, OMY272116; goldfish Carassius auratus GfA, AF121845; GfB, AF121846; catfish Clarias gariepinus, CHFNRHR; Xenopus laevis, AF172330; bullfrog Rana catesbeiana GnRH-R1, AF144063; GnRH-R2, AF153913; GnRH-R3, AF144062; chicken Gallus gallus, AJ304414; striped bass Morone saxatilis, AF218841; sheep Ovis aries, SHPGRHR; cow Bos taurus, BTU00934; human Homo sapiens, S60587; dog Canis familiaris, AF206513; mouse Mus musculus, MUSGRHR; rat Rattus norvegicus, S59525; possum Trichosurus vulpecula, AF032379; rhesus monkey Macaca mulatta, AF353987; African green monkey Cercopithecus aethiops, AF353988; and Drosophila melanogaster, AF077299.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation of the medaka GnRH-R cDNAs
Degenerate primers were designed based on relatively conserved TM regions among known GnRH-Rs. Amplification of the medaka brain cDNA with these primers yielded the cDNA fragments for two distinct GnRH-Rs, GnRH-R1 and GnRH-R2. The full-length cDNAs for these two GnRH-Rs were subsequently isolated by 5'- and 3'-RACE. The nucleotide and deduced amino acid sequences for the GnRH-R1 and GnRH-R2 cDNAs are shown in Fig. 1Go. Theoretical translation of the sequences indicated that GnRH-R1 and GnRH-R2 are likely to encode 398- and 373-amino acid polypeptides, respectively. Hydrophobicity analysis showed that both GnRH-R1 and GnRH-R2 contained seven hydrophobic putative TM domains connected by three cytoplasmic and three extracellular loops, and extracellular N-terminal and cytoplasmic C-terminal domains, all of which are widely conserved characteristics of GPCRs (data not shown). 3'-RACE resulted in the isolation of two cDNA clones with different lengths for each GnRH-R, probably owing to alternative usage of two polyadenylation signals (AATAAA and ATTAAA) in the GnRH-R1 and GnRH-R2 genes (Fig. 1Go).



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  		Figure 1. Nucleotide and deduced amino acid sequences of the medaka GnRH-R1 and GnRH-R2 cDNAs. Boxed amino acids indicate putative seven transmembrane domains. Stop codons are denoted by asterisks and polyadenylation signals are underlined. Intron positions are indicated by thick, short lines.

 
Comparison of the GnRH-Rs of the medaka and other vertebrates
Alignment of the GnRH-Rs of the medaka and those of other representative species is shown in Fig. 2Go. The amino acid sequence identity among the species was calculated using relatively conserved TM domains, excluding the highly variable N- and C-terminal domains (Table 1Go). The medaka GnRH-R1 and GnRH-R2 shared 53% amino acid identity. The medaka GnRH-R1 had highest identity (82%) with the striped bass GnRH-R. The medaka GnRH-R1 also showed relatively high identity of 72% and 65% with the bullfrog GnRH-R1 and GnRH-R3, respectively. On the other hand, the medaka GnRH-R2 exhibited high identity (60–78%) with the GnRH-Rs of other teleosts except the striped bass.



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  		Figure 2. Alignment of the deduced amino acid sequences of the GnRH-Rs in the medaka and those in other representative vertebrates. The full-length GnRH-Rs were aligned by CLUSTAL W (25 ) using the default settings. Identical residues are shaded. Lines at the top of the sequences represent putative seven TM domains. Numbers on the right indicate the amino acid positions.

 

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  		Table 1. Amino acid identity (%) between the GnRH-Rs of the medaka and other vertebrates

 
Isolation of the medaka GnRH-R genes
The nucleotide sequences of the transcribed regions in the GnRH-R1 and GnRH-R2 genes including full intron sequences were determined. The transcribed regions in the GnRH-R1 and GnRH-R2 genes were extended over 7 and 11 kb, respectively, on the medaka genome. Sequence analysis revealed that the GnRH-R1 and GnRH-R2 genes had different exon/intron organizations: The GnRH-RI gene was composed of three exons separated by two introns, whereas the GnRH-R2 had an additional intron and therefore consisted of four exons and three introns (Fig. 3Go). The three introns in the GnRH-R2 gene, denoted introns A, B, and C, were located in the extracellular N-terminal domain, TM IV, and the third cytoplasmic loop, respectively (Figs. 1Go and 3Go). On the other hand, the two introns in the GnRH-R1 gene were located in TM IV and the third cytoplasmic loop, and designated introns B and C, because their positions clearly correspond to introns B and C in the GnRH-R2 gene (Figs. 1Go and 3Go). Thus, the GnRH-R1 gene lacked intron A. The lengths of introns B and C in the GnRH-R1 gene were 3.39 and 1.08 kb, respectively. The introns A, B, and C in the GnRH-R2 were 2.69, 2.62, and 4.60 kb, respectively. The nucleotide sequences of all introns adjoining the splicing junctions were consistent with the recognized consensus sequence GT/AG.



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  		Figure 3. Schematic diagram illustrating the organization of the medaka GnRH-R1 and GnRH-R2 genes. Boxes represent exons, and horizontal lines adjacent to exons represent introns A, B, and C. Size in base pairs is indicated. ORF, Open reading frame; UTR, untranslated region.

 
Copy number of the medaka GnRH-R genes
Genomic Southern blot analysis was carried out to determine the copy number of the GnRH-R genes in the medaka genome. Hybridization with both GnRH-R1 and GnRH-R2 cDNA-specific probes yielded single signals for each partial digest of genomic DNA as shown in Fig. 4Go. The size of the signals obtained was different between the hybridizations with the GnRH-R1 and GnRH-R2 probes. Thus, it can be concluded that each of GnRH-R1 and GnRH-R2 is encoded by a distinct, single-copy gene.



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  		Figure 4. Genomic Southern blot of the medaka GnRH-R1 and GnRH-R2 genes. Genomic DNA digested with DraI, Eco T14, HindIII, or PstI was electrophoresed and hybridized with the GnRH-R1- and GnRH-R2-specific probes.

 
Chromosomal assignment of the medaka GnRH-Rs
The two GnRH-R genes in the medaka were mapped using reference typing DNA panels derived from 39 backcross progenies between the two strains, HNI and AA2. Medaka has 24 chromosomes per haploid, and several hundreds of genetic polymorphic markers in this fish have been classified into 24 linkage groups (LGs) (27). Thus, one LG should represent one chromosome. The GnRH-R1 and GnRH-R2 genes were mapped to LGs 3 and 16, respectively (Fig. 5Go). Independent confirmation for this assignment of the two GnRH-R genes was provided by analysis of other backcross panels derived from 52 offspring between an HNI/AA2 female F1 and an AA2 male parental line.



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  		Figure 5. The chromosomal locations of the medaka GnRH-R1 and GnRH-R2 genes. Map distance from the top locus is described in terms of centimorgans (Kosambi mapping function) in parentheses. The expressed sequence tagged sites and phenotypic markers are described with boldface characters. Markers within boxes showed no recombination in 39 meioses. The locations of the markers (except the GnRH-R1 and GnRH-R2 genes) have all been previously reported (27 ).

 
Pharmacological characterization of the medaka GnRH-Rs
Three native GnRH forms in the medaka (mdGnRH, cGnRH-II, and sGnRH) and a mammalian ortholog of the medaka mdGnRH (mGnRH) stimulated IP production in COS-7 cells transfected with either GnRH-R1 or GnRH-R2 (Fig. 6Go). The GnRH-Rs responded in a dose-dependent manner to all four GnRH forms tested. Table 2Go shows the EC50 values of various GnRH ligands pertaining to IP production. GnRH-R1 and GnRH-R2 showed the same preference of sensitivities for GnRH ligands: cGnRH-II > sGnRH > mGnRH > mdGnRH. However, GnRH-R1 exhibited similar preference for all four GnRH forms tested, whereas GnRH-R2 exhibited particular sensitivity for cGnRH-II, compared with the other three GnRH forms.



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  		Figure 6. IP production in COS-7 cells transfected with the medaka GnRH-R1 and GnRH-R2 after stimulation with mdGnRH ({blacksquare}), cGnRH-II (•), sGnRH ({blacktriangleup}), and mGnRH ({square}). The x-axis indicates increasing concentrations of GnRH ligands in log scale. The y-axis represents IP production as a percentage of the maximum IP production.

 

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  		Table 2. Comparison of GnRH ligand potencies in IP assays involving COS-7 cells transfected with the medaka GnRH-Rs

 
Phylogenetic analysis of vertebrate GnRH-Rs
A phylogenetic tree of vertebrate GnRH-Rs was generated using the neighbor-joining method (Fig. 7Go). The tree constructed using the Drosophila GnRH-R homologue as an outroot showed that the vertebrate GnRH-Rs identified to date fell into three distinct lineage groups. One lineage included the medaka GnRH-R1 in addition to the striped bass GnRH-R, bullfrog GnRH-Rs 1 and 3, and second primate GnRH-Rs. The medaka GnRH-R2 was clustered with the GnRH-Rs of the eel, trout, goldfish (both two receptors termed GfA and GfB), catfish, Xenopus, bullfrog (GnRH-R2), and chicken. Mammalian GnRH-Rs that lack a cytoplasmic C-terminal tail solely formed the third lineage group. High bootstrap values of >99% were generated for the nodes at the roots of the three lineages, validating these groupings.



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  		Figure 7. A phylogenetic tree showing three lineages for vertebrate GnRH-Rs. The tree was generated by PHYLIP software (29 ) using the neighbor-joining method (30 ), following alignment by CLUSTAL W (25 ). Unique characteristics of GnRH-Rs within each lineage are summarized on the right side. Bootstrap values are indicated for all nodes on the tree. The scale bar corresponds to estimated evolutionary distance units. For species names and accession numbers, see Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Structural, pharmacological, and physiological characteristics of the two GnRH-Rs in the medaka
In the present study, we have isolated the cDNAs and genes encoding two distinct GnRH-Rs, designated GnRH-R1 and GnRH-R2, from a model fish species, the medaka. IP assays using COS-7 cells transfected with these receptors confirmed that they are indeed functional receptors, and their ligand selectivities were determined. These experiments have demonstrated several differing as well as several common features of the receptors in terms of structure, pharmacology, and physiology.

Both GnRH-R1 and GnRH-R2 contain seven TM domains and an extracellular N terminus. Each of them also has a cytoplasmic C-terminal tail, which is a common characteristic to nonmammalian GnRH-Rs identified so far. The cytoplasmic tails of the two GnRH-Rs in the medaka, however, share no significant homology. In GPCRs, the cytoplasmic tail has been demonstrated to be associated with receptor desensitization and internalization (32, 33, 34). Therefore, it is possible that GnRH-R1 and GnRH-R2 exhibit distinct patterns of desensitization and internalization.

Southern blot analysis revealed that both GnRH-R1 and GnRH-R2 are encoded by single copy genes. These receptors, however, show remarkable differences in gene organization. The GnRH-R1 and GnRH-R2 genes have two introns, termed introns B and C, at identical positions in the genes (TM IV and third cytoplasmic loop, respectively), but there is another intron, denoted intron A, in the extracellular domain of GnRH-R2. All mammalian GnRH-R genes have two introns corresponding to introns B and C in TM IV and the third cytoplasmic loop but lack intron A (6, 20). On the other hand, the Xenopus GnRH-R contains three introns at the same location as the medaka GnRH-R2 (12). The conservation of the location of introns B and C supports the idea that multiple GnRH-Rs arose from a duplication event sometime in the past (5, 18). Although it is unknown whether the ancestral GnRH-R gene was composed of four exons/three introns or three exons/two introns, intron A should be lost or appended in some GnRH-R genes during evolution. In terms of the evolution of the GnRH-R gene, the assignment of the two GnRH-R genes in the medaka to distinct chromosomes eliminates the possibility that these receptor genes have arisen by tandem duplication of their progenitor gene.

When expressed in COS-7 cells, the medaka GnRH-R1 and GnRH-R2 stimulated IP formation in response to GnRH ligands. We used three native GnRH forms in the medaka (mdGnRH, cGnRH-II, and sGnRH) and a mammalian ortholog to the mGnRH as experimental ligands. GnRH-R1 and GnRH-R2 showed the same preference of potencies for GnRH ligands with cGnRH-II > sGnRH > mGnRH > mdGnRH and exhibited the almost same EC50 value for cGnRH-II (0.52 and 1.0 nM, respectively). However, there was a significant difference in the ligand selectivity between GnRH-R1 and GnRH-R2: The sensitivities of GnRH-R1 for sGnRH, mGnRH, and mdGnRH were approximately 100 times greater than those of GnRH-R2.

It is noteworthy that GnRH-R2 exhibited less sensitivity for mGnRH than did GnRH-R1. An acidic residue in the third extracellular loop of mammalian GnRH-Rs (i.e., Glu301 in the mouse and Asp302 in the human GnRH-Rs) is thought to be responsible for high selectivity for mGnRH ligand form, which possesses Arg in position 8 (35). The acidic residue in the third extracellular loop is also conserved as Glu308 in the medaka GnRH-R2 but is replaced by His311 in the medaka GnRH-R1. Therefore, the lower sensitivity of GnRH-R2 for mGnRH was an unexpected observation. It could be that the GnRH-Rs in the medaka have different ligand-binding sites from mammalian GnRH-Rs.

Another intriguing aspect of the pharmacology of the medaka GnRH-Rs is the finding that both receptors exhibited highest selectivities for cGnRH-II, physiological function of which is still unknown. This GnRH form occurs along with one or two other forms in all vertebrates examined so far including humans (36) and the medaka (24). The cGnRH-II-producing neurons are distributed in the midbrain tegmentum and project their axons throughout the brain (3, 4, 5). These lines of evidence suggest the importance of cGnRH-II in the central nervous system. Additional characterization of cGnRH-II and its possible receptor(s) will facilitate the understanding of physiological roles of this GnRH form.

We also examined the expression of the two GnRH-Rs in the brain and pituitary of the medaka. RT-PCR analysis demonstrated the presence of mRNAs for both GnRH-R1 and GnRH-R2 in the pituitary glands of adult medakas, suggesting that both receptors are implicated in the stimulation of gonadotropin secretion from this tissue (data not shown). In the medaka, mdGnRH is considered to be the hormone that stimulates gonadotropin secretion (23). Thus it is surprising that both of the GnRH-Rs expressed in the pituitary exhibited less sensitivity to mdGnRH than the other two native ligand forms in the medaka. One possible explanation for this phenomenon is that an additional GnRH-R that prefers mdGnRH exists in the pituitary besides the GnRH-R1 and GnRH-R2. The possibility of the occurrence of a third GnRH-R will be discussed below.

In addition to expression in the pituitary, GnRH-R1 and GnRH-R2 were found to be expressed throughout the brain (data not shown). This observation predicts physiological roles for these two receptors in the brain, although the precise functions of GnRHs in this organ remain to be elucidated. Several lines of evidence have demonstrated that GnRH, when administered in the brain, promotes the lordosis reflex, a sexual receptive behavior of the female rat (37, 38). GnRH was also shown to modulate reproductive behavior in teleosts including the dwarf gourami Colisa lalia and goldfish (39, 40). One or both of the two GnRH-Rs, therefore, may mediate GnRH function as a neurotransmitter and neuromodulator to regulate reproductive behaviors. We did not observe any difference between the expression patterns of GnRH-R1 and GnRH-R2 in the central nervous system of the adult medaka. Thus, the natures of their physiological roles are still obscure. It is necessary to conduct further studies to determine their spatiotemporal expression and distribution patterns in detail.

A single vertebrate species has at least two distinct GnRH-R subtypes
Within the teleost lineage, the occurrence of two separate genes has been reported for several substances including a homeobox gene (41), cytochrome P450 aromatase (42, 43), rhodopsin (44), and synaptosome-associated protein (45). The duplicity of these genes is thought to result from a genome duplication early in teleost evolution after the divergence of teleosts and tetrapods (46, 47, 48). Thus, one may suspect that the presence of the two GnRH-Rs in the medaka reported in the present study was likewise caused by a duplicative event within the teleost lineage and is therefore a restricted characteristic of this lineage. GnRH-R1 and GnRH-R2 are, however, highly divergent, sharing only 43% overall amino acid identity and 53% identity even in the relatively highly conserved TM domains. Furthermore, phylogenetic analysis revealed that the two GnRH-Rs in the medaka fell into distinct lineages, each of which contains the bullfrog GnRH-R(s). These data indicate that the medaka GnRH-R genes cloned here arose from a gene duplication predating the divergence of teleosts and tetrapods. Hence, the GnRH-R1 and GnRH-R2 should be representatives for different GnRH-R subtypes, and the occurrence of these two GnRH-R subtypes would be a common characteristic in teleosts and tetrapods. In support of this notion, we have also identified a GnRH-R1-like gene, in addition to a previously reported counterpart of the medaka GnRH-R2 (16), in one of the most primitive teleost species, the eel Anguilla japonica (unpublished data).

In contrast, phylogenetic analysis demonstrated that two GnRH-Rs isolated from another teleost species, the goldfish (8), are variant forms within the same subtype. This might result from the genome duplication early in the evolution of teleosts. The bullfrog has been shown to possess three GnRH-Rs in its central nervous system, designated GnRH-Rs 1, 2, and 3 (19). However, the present phylogenetic analysis also placed the bullfrog GnRH-Rs 1 and 3 in the same lineage, suggesting that these two receptors in the bullfrog are also duplicated variant forms within a single subtype. In this amphibian species, several or all genes, probably including GnRH-R genes, may be duplicated, similar to the Xenopus. This idea is consistent with the result of genomic Southern blot analysis performed by Wang et al. (19), which showed two bands for the other GnRH-R in the bullfrog, the GnRH-R2. Therefore the present paper, in combination with the recent studies of the bullfrog and primates, provides evidence that a single vertebrate species has at least two distinct GnRH-R subtypes.

Evolution of vertebrate GnRH-R subtypes: the possible occurrence of three GnRH-R subtypes in one organism
The medaka GnRH-R1 shares relatively high identity with the striped bass GnRH-R, and bullfrog GnRH-Rs 1 and 3. In addition, phylogenetic analysis clustered these genes into one lineage. Thus, it is certain that they form one subtype lineage of vertebrate GnRH-Rs. Phylogenetic analysis also showed that the recently reported second primate GnRH-Rs belong to this subtype group.

According to a widely accepted classification (18), it is believed that the GnRH-Rs of teleosts including the catfish, goldfish, eel, and trout are orthologs to mammalian GnRH-Rs that lack a cytoplasmic tail. In this case, the medaka GnRH-R2 also should be classified into this subtype. However, this does not seem to be a true case because there are several crucial differences on the structural and pharmacological characteristics between the medaka GnRH-R2 and the mammalian GnRH-Rs. First, the medaka GnRH-R2 has a cytoplasmic C-terminal tail, which is absent in these mammalian GnRH-Rs. Second, the medaka GnRH-R2 gene consists of four exons separated by three introns, whereas the mammalian GnRH-R genes have three exons and two introns, resulting from the absence of intron A. Third, although relatively conserved TM domains were used for sequence alignments, the amino acid identities between the medaka GnRH-R2 and the mammalian GnRH-Rs were substantially low. Finally, the pharmacological studies have demonstrated that the medaka GnRH-R2 has over 100 times higher selectivity for cGnRH-II than the other GnRH ligands tested, but the cytoplasmic tail-lacking mammalian GnRH-Rs prefer mGnRH over other forms, including cGnRH-II. Therefore, similar to GnRH-R1, GnRH-R2 is not likely to be the medaka ortholog to the mammalian GnRH-Rs that lack a cytoplasmic tail. The phylogenetic tree constructed using the Drosophila GnRH-R homologue as an outroot indeed assigned the medaka GnRH-R1 and GnRH-R2 and the cytoplasmic tail-lacking mammalian GnRH-Rs to three separate lineages with high bootstrap values for principal nodes. These data led us to the idea that there may be three paralogous lineages in the evolution of vertebrate GnRH-Rs. Accordingly, it is logically possible that one vertebrate species has three distinct GnRH-R subtypes.

Based on the logic mentioned above, we propose a possible classification of vertebrate GnRH-R subtypes. The first subtype contains the medaka GnRH-R1, striped bass GnRH-R, bullfrog GnRH-Rs 1 and 3, and second primate GnRH-Rs. The upper lineage in the phylogenetic tree represents this subtype, possessing a cytoplasmic C-terminal tail but lacking intron A. The second subtype represented by the middle lineage in the tree includes the medaka GnRH-R2 and other GnRH-Rs found in several teleosts, amphibia, and chickens. This subtype also has a cytoplasmic tail, and its gene contains intron A. The third subtype represented by the lower lineage in the tree contains only the GnRH-Rs that have been found in mammals. Unlike the first and second subtypes, the third subtype lacks a cytoplasmic tail and also lacks intron A.

In humans, only one functional GnRH-R has been identified to date. It is uncertain whether other functional GnRH-Rs occur in humans. Recently, Millar et al. (49) have found the human genomic DNA sequence showing high homology of over 40% with the functional human GnRH-R and referred to it as a putative second GnRH-R, but its transcript could not be detected. Instead of the putative second GnRH-R-encoding sense DNA, its antisense DNA is shown to be highly transcribed in various tissues and encodes a ribonucleoprotein (50). This putative second GnRH-R gene contains only two intronic sequences, the locations of which are the same as introns B and C in the medaka GnRH-R genes. Also, this human gene sequence resembles the second GnRH-Rs in primates (20), which belong to the same subtype group as the medaka GnRH-R1 as described above. Therefore, humans would have the second GnRH-R subtype that is orthologous to the medaka GnRH-R1, although it could have become a pseudogene during evolution. It is of interest to note that a GnRH-R gene that has intron A, which the medaka GnRH-R2 possesses, has not been identified in humans. However, the present phylogenetic analysis logically suggests the existence of a human ortholog of the medaka GnRH-R2. Further studies are required to determine whether humans possess another GnRH-R gene that contains intron A.

Conservation of synteny is often seen between the genomes of humans and teleosts including the medaka and zebrafish (27, 46, 47, 48, 51, 52). This phenomenon allows the definition of the relationship between genes of humans and these teleosts. The human GnRH-R gene was mapped to 4q13–21 (53), and the putative second GnRH-R sequences in the human genome were to 1q21.1 (49) and 14q21-q23 (50). In spite of our expectations, the present mapping of the medaka GnRH-R genes failed to find syntenies of these genes owing to the lack of appropriate genetic markers located near the GnRH-R genes. However, when the markers in the medaka linkage map grow denser in the near future, the present mapping would illuminate the evolutionary relationships among the diversified GnRH-R subtypes. Then it also could be determined whether the medaka has the orthologous gene to mammalian GnRH-Rs that probably consists of three exons/two introns and encodes a cytoplasmic tail-lacking receptor.

In conclusion, the present study has identified two distinct GnRH-R subtypes in a model teleost species, the medaka. Their genes have different structures and distinct chromosomal locations. The two receptor subtypes also showed distinct ligand preferences, with each receptor exhibiting particularly high sensitivity for cGnRH-II. Phylogenetic analysis proposed that a third GnRH-R subtype may remain to be found in the medaka.


   Acknowledgments
 
The authors are grateful to Dr. Kazushige Touhara at the University of Tokyo and Dr. Koichi Okuzawa at National Research Institute of Aquaculture for helpful technical assistance with the IP assay and useful discussions. We would also like to thank Jennifer Ito for critical reading of the manuscript.


   Footnotes
 
The sequences reported in this paper have been deposited in the DDBJ/EMBL/GenBank (accession nos. AB057675, AB057674, AB057677, and AB057676 for the medaka GnRH-R1 cDNA, GnRH-R2 cDNA, GnRH-R1 gene, and GnRH-R2 gene, respectively).

1 Present address: National Research Institute of Aquaculture, Inland Station, Hiruta, Tamaki, Mie 519-0423, Japan. Back

Abbreviations: cGnRH-II, Chicken-II-type GnRH; GnRH-R, GnRH receptor; GPCR, G protein-coupled receptor; IP, inositol phosphate; LG, linkage group; mdGnRH, medaka-type GnRH; mGnRH, mammalian-type GnRH; RACE, rapid amplification of cDNA ends; sGnRH, salmon-type GnRH; TM, transmembrane.

Received April 23, 2001.

Accepted for publication July 16, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Matsuo H, Baba Y, Nair RM, Arimura A, Schally AV 1971 Structure of the porcine LH- and FSH-releasing hormone. I. The proposed amino acid sequence. Biochem Biophys Res Commun 43:1334–1339[CrossRef][Medline]
  2. Amoss M, Burgus R, Blackwell R, Vale W, Fellows R, Guillemin R 1971 Purification, amino acid composition and N-terminus of the hypothalamic luteinizing hormone releasing factor (LRF) of ovine origin. Biochem Biophys Res Commun 44:205–210[CrossRef][Medline]
  3. Fernald RD, White RB 1999 Gonadotropin-releasing hormone genes: phylogeny, structure, and functions. Front Neuroendocrinol 20:224–240[CrossRef][Medline]
  4. Sherwood NM, von Schalburg K, Lescheid DW 1997 Origin and evolution of GnRH in vertebrates and invertebrates. In: Parhar IS, Sakuma Y, eds. GnRH neurons: gene to behavior. Tokyo: Brain Shuppan; 3–25
  5. King JA, Millar RP 1997 Coordinated evolution of GnRHs and their receptors. In: Parhar IS, Sakuma Y, eds. GnRH neurons: gene to behavior. Tokyo: Brain Shuppan; 51–77
  6. Leung PCK, Peng C 1996 Gonadotropin-releasing hormone receptor: gene structure, expression and regulation. Biol Signals 5:63–69[Medline]
  7. Tensen C, Okuzawa K, Blomenrohr M, et al. 1997 Distinct efficacies for two endogenous ligands on a single cognate gonadoliberin receptor. Eur J Biochem 243:134–140[Medline]
  8. Illing N, Troskie BE, Nahorniak CS, Hapgood JP, Peter RE, Millar RP 1999 Two gonadotropin-releasing hormone receptor subtypes with distinct ligand selectivity and differential distribution in brain and pituitary in the goldfish (Carassius auratus). Proc Natl Acad Sci USA 96:2526–2531[Abstract/Free Full Text]
  9. Byrne B, McGregor A, Taylor PL, et al. 1999 Isolation and characterization of the marmoset gonadotropin releasing hormone receptor: Ser(140) of the DRS motif is substituted by Phe. J Endocrinol 163:447–456[Abstract]
  10. King JA, Fidler A, Lawrence S, Adam T, Millar RP, Katz A 2000 Cloning and expression, pharmacological characterization, and internalization kinetics of the pituitary GnRH receptor in a metatherian species of mammal. Gen Comp Endocrinol 117:439–448[CrossRef][Medline]
  11. Cui J, Smith RG, Mount GR, et al. 2000 Identification of Phe313 of the gonadotropin-releasing hormone (GnRH) receptor as a site critical for the binding of nonpeptide GnRH antagonists. Mol Endocrinol 14:671–681[Abstract/Free Full Text]
  12. Troskie BE, Hapgood JP, Millar RP, Illing N 2000 Complementary deoxyribonucleic acid cloning, gene expression, and ligand selectivity of a novel gonadotropin-releasing hormone receptor expressed in the pituitary and midbrain of Xenopus laevis. Endocrinology 141:1764–1771[Abstract/Free Full Text]
  13. Okubo K, Suetake H, Usami T, Aida K 2000 Molecular cloning and tissue-specific expression of a gonadotropin-releasing hormone receptor in the Japanese eel. Gen Comp Endocrinol 119:181–192[CrossRef][Medline]
  14. Alok D, Hassin S, Sampath Kumar R, Trant JM, Yu K, Zohar Y 2000 Characterization of a pituitary GnRH-receptor from a perciform fish, morone saxatilis: functional expression in a fish cell line. Mol Cell Endocrinol 168:65–75[CrossRef][Medline]
  15. Madigou T, Mananos-Sanchez E, Hulshof S, Anglade I, Zanuy S, Kah O 2000 Cloning, tissue distribution, and central expression of the gonadotropin-releasing hormone receptor in the rainbow trout (Oncorhynchus mykiss). Biol Reprod 63:1857–1866[Abstract/Free Full Text]
  16. Sun YM, Flanagan CA, Illing N, et al. 2001 A chicken gonadotropin-releasing hormone receptor that confers agonist activity to mammalian antagonists: identification of D-Lys6 in the ligand and extracellular loop two of the receptor as determinants. J Biol Chem 276:7754–7761[Abstract/Free Full Text]
  17. Jennes L, Centers A, Eyigor O 1997 GnRH receptors in the rat central nervous system. In: Parhar IS, Sakuma Y, eds. GnRH neurons: gene to behavior. Tokyo: Brain Shuppan; 79–95
  18. Troskie B, Illing N, Rumbak E, et al. 1998 Identification of three putative GnRH receptor subtypes in vertebrates. Gen Comp Endocrinol 112:296–302[CrossRef][Medline]
  19. Wang L, Bogerd J, Choi HS, et al. 2001 Three distinct types of GnRH receptor characterized in the bullfrog. Proc Natl Acad Sci USA 98:361–366[Abstract/Free Full Text]
  20. Neill JD, Duck LW, Sellers JC, Musgrove LC 2001 A gonadotropin-releasing hormone (GnRH) receptor specific for GnRH II in primates. Biochem Biophys Res Commun 282:1012–1018[CrossRef][Medline]
  21. Yamamoto T 1975 Medaka (killifish) biology and strains. Tokyo: Keigaku Publishing
  22. Ozato K, Wakamatsu Y 1994 Developmental genetics of medaka. Dev Growth Differ 36:437–443[CrossRef]
  23. Ishikawa Y 2000 Medakafish as a model system for vertebrate developmental genetics. BioEssays 22:487–495[CrossRef][Medline]
  24. Okubo K, Amano M, Yoshiura Y, Suetake H, Aida K 2000 A novel form of gonadotropin-releasing hormone in the medaka, Oryzias latipes. Biochem Biophys Res Commun 276:298–303[CrossRef][Medline]
  25. Thompson JD, Higgins DG, Gibson TJ 1994 CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680[Abstract/Free Full Text]
  26. Yoshizaki G, Oshiro T, Takashima F 1991 Introduction of carp {alpha}-globin gene into rainbow trout. Nippon Suisan Gakkaishi 57:819–824
  27. Naruse K, Fukamachi S, Mitani H, et al. 2000 A detailed linkage map of medaka, Oryzias latipes: comparative genomics and genome evolution. Genetics 154:1773–1784[Abstract/Free Full Text]
  28. Berg KA, Clarke WP, Chen Y, Ebersole BJ, McKay RD, Maayani S 1994 5-Hydroxytryptamine type 2A receptors regulate cyclic AMP accumulation in a neuronal cell line by protein kinase C-dependent and calcium/calmodulin-dependent mechanisms. Mol Pharmacol 45:826–836[Abstract]
  29. Felsenstein J 1989 PHYLIP (Phylogeny inference package) version 3.5c (distributed by the author) Seattle, WA: University of Washington, Department of Genetics
  30. Saito N, Nei W 1987 The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425[Abstract]
  31. Hauser F, Sondergaard L, Grimmelikhuijzen CJ 1998 Molecular cloning, genomic organization and developmental regulation of a novel receptor from Drosophila melanogaster structurally related to gonadotropin-releasing hormone receptors for vertebrates. Biochem Biophys Res Commun 249:822–828[CrossRef][Medline]
  32. Pawson AJ, Katz A, Sun YM, et al. 1998 Contrasting internalization kinetics of human and chicken gonadotropin-releasing hormone receptors mediated by C-terminal tail. J Endocrinol 156:R9–R12
  33. Heding A, Vrecl M, Bogerd J, et al. 1998 Gonadotropin-releasing hormone receptors with intracellular carboxyl-terminal tails undergo acute desensitization of total inositol phosphate production and exhibit accelerated internalization kinetics. J Biol Chem 273:11472–11477[Abstract/Free Full Text]
  34. Willars GB, Heding A, Vrecl M, et al. 1999 Lack of a C-terminal tail in the mammalian gonadotropin-releasing hormone receptor confers resistance to agonist-dependent phosphorylation and rapid desensitization. J Biol Chem 274:30146–30153[Abstract/Free Full Text]
  35. Flanagan CA, Becker II, Davidson JS, et al. 1994 Glutamate 301 of the mouse gonadotropin-releasing hormone receptor confers specificity for arginine 8 of mammalian gonadotropin-releasing hormone. J Biol Chem 269:22636–22641[Abstract/Free Full Text]
  36. White RB, Eisen JA, Kasten TL, Fernald RD 1998 Second gene for gonadotropin-releasing hormone in humans. Proc Natl Acad Sci USA 95:305–309[Abstract/Free Full Text]
  37. Moss RL, McCann SM 1973 Induction of mating behavior in rats by luteinizing hormone releasing factor. Science 181:177–179[Abstract/Free Full Text]
  38. Pfaff DW 1973 Luteinizing hormone-releasing factor potentiates lordosis behavior in hypophysectomized ovariectomized female rats. Science 182:1148–1149[Abstract/Free Full Text]
  39. Yamamoto N, Oka Y, Kawashima S 1997 Lesions of gonadotropin-releasing hormone-immunoreactive terminal nerve cells: effects on the reproductive behavior of male dwarf gouramis. Neuroendocrinology 65:403–412[Medline]
  40. Volkoff H, Peter RE 1999 Actions of two forms of gonadotropin releasing hormone and a GnRH antagonist on spawning behavior of the goldfish Carassius auratus. Gen Comp Endocrinol 116:347–355[CrossRef][Medline]
  41. Amores A, Force A, Yan YL, et al. 1998 Zebrafish hox clusters and vertebrate genome evolution. Science 282:1711–1714[Abstract/Free Full Text]
  42. Tchoudakova A, Callard GV 1998 Identification of multiple CYP19 genes encoding different cytochrome P450 aromatase isozymes in brain and ovary. Endocrinology 139:2179–2189[Abstract/Free Full Text]
  43. Kishida M, Callard GV 2001 Distinct cytochrome P450 aromatase isoforms in zebrafish (Danio rerio) brain and ovary are differentially programmed and estrogen regulated during early development. Endocrinology 142:740–750[Abstract/Free Full Text]
  44. Mano H, Kojima D, Fukada Y 1999 Exo-rhodopsin: a novel rhodopsin expressed in the zebrafish pineal gland. Brain Res Mol Brain Res 73:110–118[Medline]
  45. Risinger C, Larhammar D 1993 Multiple loci for synapse protein SNAP-25 in the tetraploid goldfish. Proc Natl Acad Sci USA 90:10598–10602[Abstract/Free Full Text]
  46. Barbazuk WB, Korf I, Kadavi C, et al. 2000 The syntenic relationship of the zebrafish and human genomes. Genome Res 10:1351–1358[Abstract/Free Full Text]
  47. Postlethwait JH, Woods IG, Ngo-Hazelett P, et al. 2000 Zebrafish comparative genomics and the origins of vertebrate chromosomes. Genome Res 10:1890–1902[Abstract/Free Full Text]
  48. Woods IG, Kelly PD, Chu F, et al. 2000 A comparative map of the zebrafish genome. Genome Res 10:1903–1914[Abstract/Free Full Text]
  49. Millar R, Conklin D, Lofton-Day C, et al. 1999 A novel human GnRH receptor homolog gene: abundant and wide tissue distribution of the antisense transcript. J Endocrinol 162:117–126[Abstract]
  50. Conklin DC, Rixon MW, Kuestner RE, Maurer MF, Whitmore TE, Millar RP 2000 Cloning and gene expression of a novel human ribonucleoprotein. Biochem Biophys Acta 1492:465–469[Medline]
  51. Postlethwait JH, Yan YL, Gates MA, et al. 1998 Vertebrate genome evolution and the zebrafish gene map. Nat Genet 18:345–349[CrossRef][Medline]
  52. Gates MA, Kim L, Egan ES, et al. 2000 A genetic linkage map for zebrafish: comparative analysis and localization of genes and expressed sequences. Genome Res 9:334–347[Abstract/Free Full Text]
  53. Kaiser UB, Dushkin H, Altherr MR, Beier DR, Chin WW 1994 Chromosomal localization of the gonadotropin-releasing hormone receptor gene to human chromosome 4q13.1-q21.1 and mouse chromosome 5. Genomics 20:506–508[CrossRef][Medline]



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C. Wang, O. Yun, K. Maiti, D. Y. Oh, K. K. Kim, C. H. Chae, C. J. Lee, J. Y. Seong, and H. B. Kwon
Position of Pro and Ser near Glu7.32 in the Extracellular Loop 3 of Mammalian and Nonmammalian Gonadotropin-Releasing Hormone (GnRH) Receptors Is a Critical Determinant for Differential Ligand Selectivity for Mammalian GnRH and Chicken GnRH-II
Mol. Endocrinol., January 1, 2004; 18(1): 105 - 116.
[Abstract]