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Endocrinology Vol. 143, No. 12 4673-4682
Copyright © 2002 by The Endocrine Society

ARTICLE

Two Gonadotropin-Releasing Hormone Receptors in the African Catfish: No Differences in Ligand Selectivity, but Differences in Tissue Distribution

Jan Bogerd, W. Bas Diepenbroek, Eveline Hund, Floor van Oosterhout, Ana C. C. Teves, Rob Leurs and Marion Blomenröhr

Research Group Endocrinology (J.B., W.B.D., E.H., F.v.O., A.C.C.T., M.B.), Utrecht University, 3584 CH Utrecht, The Netherlands; and the Department of Pharmacochemistry (R.L.), Free University, 1081 HV Amsterdam, The Netherlands

Address all correspondence and requests for reprints to: Jan Bogerd, Research Group Endocrinology, Utrecht University, Hugo R. Kruytgebouw, Padualaan 8, 3584 CH Utrecht, The Netherlands. E-mail: j.bogerd{at}bio.uu.nl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ligand-binding studies revealed the presence of GnRH-binding sites in African catfish ovary. However, our expression profiling studies failed to detect the previously identified catfish GnRH receptor (cfGnRH-R1) mRNA in this tissue. This negative result instigated us to clone an additional catfish GnRH receptor (cfGnRH-R2) cDNA and study its expression in different tissues in conjunction with the expression of the two catfish GnRH (i.e. cfGnRH and cGnRH-II) genes.

The highest cfGnRH-R1 and cfGnRH-R2 mRNA levels were detected in pituitary for cfGnRH-R1 and in brain and ovary for cfGnRH-R2. cfGnRH mRNA was coexpressed with cfGnRH-R1 mRNA in pituitary and brain and with cfGnRH-R2 mRNA in brain and ovary. Ubiquitous expression of cGnRH-II mRNA was observed in all tissues tested, with the highest expression in brain, heart, pituitary, ovary, and head-kidney.

Binding studies revealed that cfGnRH-R1 had a higher affinity than cfGnRH-R2 for cGnRH-II, cfGnRH, and various other GnRH agonists. However, this was not reflected in the inositol phosphate or cAMP signal transduction properties of both types of cfGnRH-R.

We therefore conclude that in catfish, functional ligand/receptor units evolved by restricted coexpression of a particular receptor in combination with a particular GnRH in particular (nearby) tissue(s).


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE SECRETION OF pituitary gonadotropins is primarily mediated by the hypothalamic decapeptide, GnRH. It is generally accepted that duplications of an ancestral GnRH gene gave rise to three distinct evolutionary GnRH forms (1):a hypothalamic form, which varies in primary structure among species; a completely conserved mesencephalic form, chicken GnRH-II (cGnRH-II; [His5,Trp7,Tyr8]GnRH); and a telencephalic form, salmon GnRH (sGnRH; [Trp7Leu8]GnRH). The sites of GnRH synthesis are not restricted to the brain, and possible functions for GnRHs synthesized at widespread loci have been discussed (2).

In the African catfish, only the species-specific, hypothalamic form of GnRH, catfish GnRH (cfGnRH; [His5,Leu7,Asn8]GnRH) and the conserved mesencephalic cGnRH-II form have been identified by peptide chemistry (3); there are no indications that sGnRH exists in the catfish. The cfGnRH and cGnRH-II cDNAs have been cloned, and their sites of expression in the brain have been determined (4). The cfGnRH-producing neurons have axons projecting directly to the gonadotropes in the pituitary, whereas cGnRH-II-synthesizing neurons do not display such a direct connection to the gonadotropes (5). Nevertheless, both GnRH forms are detected in catfish pituitary and are able to stimulate inositol phosphate (IP) production in gonadotropes and to induce gonadotropin secretion from these cells. cfGnRH is considered to be the physiologically significant GnRH in catfish pituitary and is present at a concentration approximately 700 times higher than cGnRH-II (6), whereas cGnRH-II has a 100–1000 times higher potency to stimulate gonadotrope functions than cfGnRH (7).

GnRH receptors (GnRH-Rs) belong to the G protein-coupled receptor family. A single class of GnRH-R has been detected in catfish pituitary, as revealed by radioligand-binding studies on membranes of this tissue (6, 8), and a single cognate cfGnRH-R has been cloned from catfish pituitary (9). The binding and IP-signaling characteristics of this receptor, transiently expressed in human embryonic kidney (HEK) 293T cells, reflected those of the native receptor present on catfish pituitary gonadotropes (9). Thus, a single molecular entity seems to be capable of mediating GnRH signals in catfish pituitary.

However, King and Millar (10) hypothesized that two or three classes of GnRH-R subtypes may have coevolved with their cognate ligands. Moreover, the presence of multiple structural variants of a particular ligand, in this case GnRH, is often accompanied by the existence of multiple cognate receptor subtypes (11). Indeed, recently two forms of GnRH-R have been characterized in goldfish (12), medaka (13), and primates (14). In bullfrog, we even identified three distinct types of GnRH-R (15).

In the ovary of the African catfish, compounds with GnRH-like activity have been detected (16). Moreover, GnRH-binding sites have been characterized in catfish ovary (16). The latter finding suggested that GnRH-Rs are expressed at extrapituitary sites, and that GnRHs, in addition to their gonadotropin-releasing activity, may affect the function of other organs, such as ovary, in catfish. For example, evidence exists for the direct action of GnRH on oocyte meiosis and/or gonadal steroidogenesis in goldfish and carp (17, 18, 19). Moreover, studies performed on mammals demonstrated an inhibitory effect of GnRH upon steroidogenesis in hypophysectomized rats and the mRNAs for GnRH and GnRH-R in rat gonads (20), as well as the presence of endogenous compounds with GnRH-like activity in a number of reproductive and nonreproductive organs, direct extrapituitary actions of GnRH and the presence of GnRH-binding sites in a variety of tissues, including testis and ovary, as well as cancer cells of breast, prostate, and pancreas origin (for review, see Refs. 21 and 22).

To further examine the gonadal GnRH-binding sites in African catfish, we studied whether the previously characterized cfGnRH-R (9) was expressed in ovary. Negative results instigated us to clone an additional catfish GnRH-R (cfGnRH-R2) cDNA, and to study its expression in different tissues in relation to the expression of the two catfish GnRH (i.e. cfGnRH and cGnRH-II) genes. In addition, the binding and IP- and cAMP-signaling characteristics of the two catfish GnRH-Rs were compared.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
African catfish were bred and raised in the laboratory by induced ovulation and artificial fertilization as described previously (23), except that catfish pituitary extract was used instead of human chorionic gonadotropin to induce ovulation. Animal culture and experimentation were consistent with the Dutch national regulations; experimental protocols were submitted to and approved by the respective university committee.

RNA and poly(A)+ RNA isolation, cDNA synthesis, and genomic DNA isolation
Total RNA was isolated from several tissues of three adult catfish as well as from ovaries of three 15-wk-old catfish using the method described by Chirgwin et al. (24). Poly(A)-rich RNA was obtained by Dynabeads-oligo dT25 (Dynal, Oslo, Norway) purification, according to the manufacturer’s instructions. Pituitary, testis, and ovary total RNAs were reverse transcribed with oligo dT12–18 using the SuperScript preamplification system according to the manufacturer’s instructions (Life Technologies, Inc., Breda, The Netherlands). Testis poly(A)-rich RNA was reverse transcribed to either 5'-rapid amplification of cDNA end (RACE) testis cDNA or 3'-RACE testis cDNA using the SMART RACE cDNA amplification kit, according to the manufacturer’s instructions (CLONTECH Laboratories, Inc., Palo Alto, CA). Genomic DNA was extracted from sperm of a single adult male catfish, according to Ausubel et al. (25).

PCR, primers, DNA sequence analysis, and multiple sequence alignment analysis
PCRs were performed in 100-µl volumes containing 50 mM KCl, 10 mM Tris HCl (pH 8.3), 1.5 mM MgCl2, 0.01% gelatin, 200 µM each dNTP, and 20–100 pmol primers in a Perkin-Elmer Cetus cycler (Applied Biosystems, Foster City, CA), using 2 U SuperTaq (HT Biotechnology Ltd., Cambridge, UK). The following primers were used: 104, 5'-CTGGATCCRRTGTGGAAYRTNACWGTKCARTGG-3'; 591, 5'-TACGAATTCGGNATHTGGTAYTGGTT-3'; 592, 5'-ACACTCGAGCCRTADATNTRNGGRTC-3'; 624, 5'-CCAGAGATGTTGAAGGTCACT-3'; 626, 5'-GAGTACGTCCACCACCTGC-3'; 654, 5'-GTGGAATTCGTACTCAG- GAGTGACCTTCAA-3'; 668, 5'-CCCCCGCTTTGTTTTTGTGTTCC-3'; 760, 5'-GGCGCCAACTCGGAACTGTGC-3'; 785, 5'-CGCGAATTCGCCACCATGCCGAGGAACGACTCTCTCTT-3'; 786, 5'-TTTGTTTCTAGATTAGCCCTCAGCTCCTTTAACACT-3', in which N = G, A, T, or C; H = A, T, or C; Y = T or C; R = G or A; D = G, A, or T; W = T or A; and K = T or G. In addition, EcoRI and XbaI restriction endonuclease sites are underlined, and the Kozak consensus translation initiation sequence (26) is shown in italics.

DNA sequence analysis was performed on an automated ABI PRISM 310 or 377 DNA sequencer (Applied Biosystems), using Dye Terminator cycle sequencing chemistry (Applied Biosystems). Multiple sequence alignment analysis was performed using Lasergene software (DNASTAR Inc., Madison, WI).

Full-length cfGnRH-R2 cDNA cloning
To isolate a DNA fragment, coding for part of a second type of catfish GnRH-R (cfGnRH-R2), 100 ng of genomic catfish DNA was PCR amplified using primers 591 and 592, based on conserved amino acid sequences in transmembrane domains (TM) 6 and 7 found in several GnRH-Rs (27). PCR conditions were: denaturation at 94 C for 2 min, followed by 35 cycles at 94 C for 1 min, 55 C for 2 min, and 72 C for 3 min. PCR products of approximately 130 bp were cloned in pGEM-T (Promega Corp., Madison, WI) for sequence analysis. On the basis of the partial genomic cfGnRH-R2 DNA sequence obtained, primer 654 was designed to obtain additional cfGnRH-R2 cDNA sequence information. To this end, we performed RT-PCR amplifications with primer 654 in combination with degenerate primer 104 (based on a conserved amino acid sequence in TM2 of GnRH-Rs) on pituitary, testis, and ovary cDNA. PCR products of approximately 600 bp were cloned in pGEM-T (Promega Corp.) for sequence analysis. Next, 5'- and 3'-RACE amplifications were performed to specifically amplify the 5'- and 3'-ends of the cfGnRH-R2 cDNA, using 5'-RACE testis cDNA and 3'-RACE testis cDNA, respectively, as template. To this end, primers 668 and 760, and primers 624 and 626 were used in combination with the universal primer mix and nested universal primer, respectively, in SMART RACE cDNA amplification (CLONTECH Laboratories, Inc.). The PCR products, obtained by 5'- and 3'-RACE, were cloned in pGEM-T for sequence analysis.

Real-time quantitative PCR
A detailed description of the real-time, quantitative PCR procedure that was used has been described previously (28). Primers and fluorogenic probes (Table 1Go), specific for the cfGnRH-R1, cfGnRH-R2, cfGnRH, and cGnRH-II mRNAs, and specific for the endogenous control [catfish 28S rRNA (cf28S rRNA)], were designed with Primer Express software (Applied Biosystems), according to the manufacturer’s guidelines as described previously (29), and were purchased from Applied Biosystems. The PCR efficiency and whether the relationship between Ct and log starting copy number was linear was tested for all primer/probe sets using defined amounts of pituitary cDNA. For all primer/probe sets, the slope of the standard curves was close to -3.32, and the correlation coefficients were close to unity over four orders of magnitude, indicating maximal PCR amplification. This allowed quantification of the relative cfGnRH-R1, cfGnRH-R2, cfGnRH, and cGnRH-II mRNA levels in cDNA samples from different tissues, using the {Delta}{Delta}Ct method.


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  		Table 1. Primers and fluorogenic probes1

 
Expression of both cfGnRH-Rs and cell culture techniques
We specifically PCR amplified the coding region of the cfGnRH-R2 using primers 785 and 786. The PCR product obtained was cloned into pcDNA3, using the EcoRI and XbaI restriction endonuclease sites present at the 5' ends of the primers, respectively, and checked for the correct DNA sequence. HEK 293T cells were cultured as previously described (30) and transiently transfected with pcDNA3 vector containing either the cfGnRH-R1 (9) or the cfGnRH-R2 coding region using the SuperFect transfection method (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. For binding studies, 5 µg DNA/100-mm2 dish was used for each of the catfish receptor constructs; for IP and cAMP studies, 5 µg DNA/100-mm2 dish and 0.1 µg DNA/100-mm2 dish were used for the cfGnRH-R1 and cfGnRH-R2 constructs, respectively, to yield similar expression levels. To prevent cells from detaching, 24-well and 48-well plates were coated with poly-D-lysine (Sigma, St. Louis, MO) before seeding cells.

Peptides
Mammalian GnRH (mGnRH) was purchased from Sigma. cGnRH-II ([His5,Trp7,Tyr8]GnRH), cfGnRH ([His5,Leu7,Asn8]GnRH), as well as the chimeric GnRH peptides cGnRH-II-L7 ([His5,Leu7,Tyr8]GnRH), cGnRH-II-N8 ([His5,Trp7,Asn8]GnRH), cGnRH-II-R8 ([His5,Trp7,Arg8]GnRH), and cfGnRH-R8 ([His5,Leu7,Arg8]GnRH) were synthesized at the Institute of Molecular Pharmacology (Berlin, Germany), as described previously (31).

Receptor binding assay
cGnRH-II was iodinated using the chloramine-T method and subsequently purified by C18 column chromatography (30). The specific activity of the radioligand was 111 µCi/mmol (32). Ligand-binding assays were performed on cell membranes from cfGnRH-R1- or cfGnRH-R2-expressing HEK 293T cells as described previously (33). Briefly, purified membranes were incubated in 0.5 ml assay buffer [40 mM Tris HCl, 2 mM MgCl2, 0.1% BSA (pH 7.4)] at 4 C for 2 h with increasing concentrations of [125I]-labeled cGnRH-II in the presence or absence of 1 µM unlabeled cGnRH-II. For concentration-displacement studies, purified membranes were incubated with approximately 1 nM [125I]-labeled cGnRH-II in 0.5 ml assay buffer at 4 C for 2 h in the presence of various concentrations of unlabeled native and chimeric GnRH analogs. The concentration of [125I]-labeled cGnRH-II approximated its Kd value at the cfGnRH-R1 (2 nM; see Results). The membranes were then filtered through Whatman GF/B filters using a Brandel cell harvester (Gaithersburg, MD), and the radioactivity retained on the filters was counted. All binding studies were performed in triplicate in three independent experiments. Binding parameters were determined from saturation and dose-displacement curves using the GraphPad PRISM2 (GraphPad Software, Inc., San Diego, CA) software package.

Total IP assay
Total IPs were extracted and separated as described previously (34). Briefly, 24 h after transfection, cells were transferred to 48-well plates [2.5 x 105 cells per well in 0.5 ml inositol-free DMEM (Life Technologies, Inc.) containing 10% dialyzed fetal calf serum] and incubated for 24 h with [3H]inositol (1 µCi/ml; Amersham Pharmacia Biotech, Little Chalfont, UK). Next, the medium was aspirated, and cells were washed and preincubated for 10 min with assay medium [HEPES-modified DMEM (Sigma) containing 10 mM LiCl]. Various concentrations of different native and chimeric GnRH analogs were added at 37 C for 45 min, after which the assay medium was aspirated. After an extraction with 10 mM formic acid at 4 C for at least 90 min, extracts were transferred to columns containing Dowex (AG 1x8–200) anion-exchange resin (Sigma). Next, total IPs were eluted, and the amount of radioactivity was counted. Assays were performed in duplicate in three separate experiments. EC50 values were determined from dose-response curves using the GraphPad PRISM2 (GraphPad Software, Inc.) software package.

cAMP assay
Twenty-four hours after transfection, cells were transferred to 24-well plates (5 x 105 cells per well in 0.5 ml DMEM containing 10% fetal bovine serum, 2 mM glutamine, and 1x antibiotics/antimycotics; all from Life Technologies, Inc.). After another 24 h, the medium was removed, and cells were washed and preincubated at 37 C for 30 min with HEPES-modified DMEM (Sigma). Thereafter, the medium was aspirated, and cells were incubated with various concentrations of cGnRH-II and cfGnRH at 37 C for 10 min in HEPES-modified DMEM, supplemented with 300 µM of the phosphodiesterase inhibitor isobutylmethylxanthine (Sigma). The reaction was stopped by rapid aspiration of the incubation medium and the addition of 200 µl of 0.1 N cold HCl. After storage at -20 C, cells were disrupted by sonification (2 sec, 40% output) in a Sonifier (Branson, St. Louis, MO). The resulting homogenate was immediately neutralized with 1 N NaOH and assayed for the presence of cAMP using a competitive protein kinase A binding assay according to Norstedt and Fredholm (35), with some minor modifications (36). Briefly, 200 µl of protein kinase A was mixed with 200 µl of cell homogenate or cAMP (Sigma) standards and 30,000 dpm [5,8-3H]cAMP (30–60 Ci/mmol; Amersham Pharmacia Biotech). After incubation at 4 C for 150 min, the mixture was rapidly diluted with 3 ml of ice-cold 50 mM Tris HCl (pH 7.4 at 4 C) and filtered through Whatman GF/B filters using a Brandel cell harvester. The radioactivity retained on the filters was measured by liquid scintillation counting.

Statistical analysis
All data are presented as mean ± SEM of three independent experiments. Statistical analysis was performed using one-way ANOVA and, where the P value was less than 0.05, was followed by the Bonferroni test. A P value less than 0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tissue distribution of the cfGnRH-R1 mRNA
Previously, Habibi et al. (16) demonstrated that GnRH-binding sites are present in African catfish ovary. To confirm this observation, we determined the relative cfGnRH-R mRNA expression levels in various tissues, including preovulatory, postvitellogenic, and postovulatory ovaries of adult catfish as well as ovaries of 15 wk-old catfish, using sensitive real-time, quantitative PCR. Highest cfGnRH-R mRNA levels were detected in pituitary (set at 100%; Fig. 1AGo). Other tissues expressing this mRNA, although at much lower levels (Fig. 1AGo), were brain (~11% of the levels in pituitary), cerebellum (~1%), and testis (~1%).



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  		Figure 1. Relative cfGnRH-R1 and cfGnRH-R2 mRNA levels in different tissues. The data of the cfGnRH-R1 (A) and cfGnRH-R2 (B) mRNA levels in different catfish tissues (for each tissue, three independent RNA isolations were performed from three different catfish) are expressed as the percentage of the cfGnRH-R1 or cfGnRH-R2 mRNA levels in pituitary, normalized to cf28S rRNA levels, respectively, that were each analyzed in duplicate. Te, Testis; Ce, cerebellum; Mu, muscle; St, stomach; In, intestine; HK, head-kidney; SV, seminal vesicles; Li, liver; Br, brain without cerebellum; Pit, pituitary; Ov1, preovulatory, postvitellogenic ovary from adult catfish; Ov2, postovulatory ovary from adult catfish; Ov3, ovary from 15-wk-old catfish; He, heart; CA, conus arteriosus (i.e. the ventral aorta, delivering blood to the gills and surrounded by thyroidal follicles).

 
However, cfGnRH-R mRNA was not detectable in any of the three types of ovary cDNA tested. Because this result suggested that an additional type of GnRH-R, different from the one previously identified (9), was present in catfish ovary, we initiated the cloning of the cDNA coding for this receptor (see Primary structure of cfGnRH-R2).

Primary structure of cfGnRH-R2
Two degenerate primers, designed on amino acid sequences conserved among GnRH-Rs and flanking the third extracellular loop, were used for PCR amplification in combination with catfish genomic DNA as template. PCR products of the expected length were cloned and sequenced. Apart from the known catfish GnRH-R sequence (9), 50% of the cloned PCR products contained a different though related sequence. The latter PCR products most likely coded for part of a second type of catfish GnRH-R, which we designated cfGnRH-R2. As a consequence, the first type of cfGnRH-R (9) was renamed cfGnRH-R1.

To obtain additional cfGnRH-R2 cDNA sequence, we performed RT-PCR on pituitary, testis, and ovary cDNA using a degenerate primer designed on a highly conserved GnRH-R amino acid sequence in TM2 in combination with a specific primer based on the amplified genomic cfGnRH-R2 DNA sequence. PCR products of the expected length, obtained using pituitary, testis, and ovary cDNA as template, were cloned and sequenced. Next, generating and sequencing 5'- and 3'-RACE cfGnRH-R2 products enabled us to clone the full-length cfGnRH-R2 cDNA (Fig. 2Go).



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  		Figure 2. Alignment of the deduced amino acid sequences of cfGnRH-R1 and cfGnRH-R2. The putative transmembrane domains are underlined. Bold letters indicate amino acid residues that differ between cfGnRH-R1 and cfGnRH-R2. Gaps (indicated by hyphens) were introduced to achieve maximum similarity.

 
The full-length cfGnRH-R2 cDNA encoded a putative GnRH-R, the start codon of which was assigned on the basis of homology to other cloned GnRH-Rs. The cfGnRH-R2 protein (Fig. 2Go) contained seven hydrophobic TMs and an intracellular C-terminal tail, similar to the cfGnRH-R1 (9), the two goldfish GnRH-Rs (12), the Xenopus laevis GnRH-R (37), the two medaka GnRH-Rs (13), and the three bullfrog GnRH-Rs (15). Moreover, potential N-linked glycosylation sites at positions 4, 22, and 104; potential protein kinase C sites at positions 264, 328, 345, 353, and 369 (38); and two cysteine residues at positions 116 and 193, which are possibly linked by a disulfide bridge (39), were identified in the second type of cfGnRH-R. The deduced cfGnRH-R2 amino acid sequence was compared with cfGnRH-R1 (Fig. 2Go) as well as with several other GnRH-Rs (Table 2Go). Highest homology for cfGnRH-R1 and cfGnRH-R2 was obtained with the goldfish GnRH-Rs GfB and GfA, respectively, whereas both cfGnRH-Rs displayed low (<41%) homology to their human and rodent counterparts (Fig. 3Go). The two catfish receptor subtypes shared approximately 69% amino acid identity with each other, with the lowest degree of sequence identity in their extracellular N termini and in their intracellular C termini.


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  		Table 2. Percentage of amino acid identities between cfGnRH-R1 or cfGnRH-R2 and a selection of other GnRH-Rs

 


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  		Figure 3. Phylogenetic tree of various GnRH-Rs. The Clustal method was used to perform multiple sequence alignment. The phylogenetic tree was constructed using the Megalign program of the Lasergene software package (DNASTAR Inc.). The following (deduced) GnRH-R amino acid sequences were used: Anguilla japonica (accession no., AB041327), Oncorhynchus mykiss (AJ272116), goldfish (GfA; AF121845), African catfish (cfGnRH-R2; AF329894), medaka 2 (AB057674), Haplochromis burtoni (AY028476), goldfish (GfB; AF121846), African catfish (cfGnRH-R1; X97497), Rana catesbeiana 2 (AF153913), Xenopus laevis 1 (AF172330), chicken (AJ304414), pig (L29342), sheep (P32237), bovine (U00934), horse (O18821), dog (AF206513), Macaca radiata (AF156930), human (NM_000406), mouse (A44013), rat (JN0463), Trichosurus vulpecula (AF032379), Morone saxatilis (AF218841), Dicentrarchus labrax (AJ419594), Seriola dumerilii (AJ130876), medaka 1 (AB057675), R. catesbeiana 1 (AF144063), R. catesbeiana 3 (AF144062), X. laevis 2 (AF257320), Typhlonectes natans (AF174481), Macaca mulatta 2 (AF353987), Cercopithecus aethiops 2 (AF353988), and Callithrix jacchus 2 (AF368286).

 
Tissue distribution of the cfGnRH-R2 mRNA
To determine the relative cfGnRH-R2 mRNA expression levels in different tissues, we again performed real-time quantitative PCR; however, we were now using primers and a probe specific for cfGnRH-R2. Highest mRNA levels for cfGnRH-R2 were observed in brain (set to 100%; Fig. 1BGo), whereas lower levels were observed in heart (~24% of the levels in brain), testis (~13%), cerebellum (~8%), pituitary (~5%), and seminal vesicles (~1%).

Comparison of the mean, relative cfGnRH-R1, and cfGnRH-R2 mRNA levels revealed that approximately 9-fold higher cfGnRH-R1 mRNA levels were expressed in pituitary than in brain (Fig. 1AGo), whereas approximately 21-fold higher cfGnRH-R2 mRNA levels were expressed in brain than in pituitary (Fig. 1BGo). Moreover, because the amplification efficiencies for both types of cfGnRH-R mRNA as well as cf28S rRNA were approximately equal, these data indicated that approximately 40-fold higher cfGnRH-R1 mRNA levels than cfGnRH-R2 mRNA levels were expressed in pituitary. In contrast, approximately 4-fold higher mRNA levels for cfGnRH-R2 than for cfGnRH-R1 were detected in brain.

As expected, the three types of ovary were also positive (Fig. 1BGo); highest cfGnRH-R2 mRNA levels were found in ovaries of 15-wk-old catfish (~50% of the levels in brain). Relatively lower levels were detected in preovulatory, postvitellogenic (~16% of the levels in brain), or postovulatory (~12% of the levels in brain) ovaries of adult catfish.

Tissue distribution of the cfGnRH and cGnRH-II mRNAs
Highest cfGnRH mRNA levels were detected in brain (set at 100%; Fig. 4AGo) and pituitary (~53% of the levels in brain). Much lower levels were observed in the ovary of 15-wk-old catfish (~3%) and in preovulatory, postvitellogenic (~1%), and postovulatory ovaries (~0.5%) of adult catfish, whereas very low levels were detected in cerebellum (~0.5%), intestine (~0.3%), stomach (~0.2%), and testis (~0.1%).



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  		Figure 4. Relative cfGnRH mRNA and cGnRH-II mRNA levels in different tissues. The data of the cfGnRH (A) and cGnRH-II (B) mRNA levels in different catfish tissues (for each tissue, three independent RNA isolations were performed from three different catfish) are expressed as percentage of the cfGnRH and cGnRH-II mRNA levels in brain, normalized to cf28S rRNA levels, respectively, that were each analyzed in duplicate. Te, Testis; Ce, cerebellum; Mu, muscle; St, stomach; In, intestine; HK, head-kidney; SV, seminal vesicles; Li, liver; Br, brain without cerebellum; Pit, pituitary; Ov1, preovulatory, postvitellogenic ovary from adult catfish; Ov2, postovulatory ovary from adult catfish; Ov3, ovary from 15-wk-old catfish; He, heart; CA, conus arteriosus.

 
Highest cGnRH-II mRNA levels were observed in brain (set at 100%; Fig. 4BGo) and heart (~99% of the levels in brain), whereas intermediate levels were detected in head-kidney (~60%), cerebellum (~47%), pituitary (~43%), and the ovary of 15-wk-old catfish (~27%). Relatively low levels of cGnRH-II mRNA were found in seminal vesicles, intestine, liver, stomach, conus arteriosus, testis, preovulatory, postvitellogenic ovary, muscle, and postovulatory ovary, in this order (between ~15% and ~3% of the levels in brain).

Pharmacological characterization of the cfGnRH-Rs
Binding of [125I]-cGnRH-II to membranes expressing either cfGnRH-R1 or cfGnRH-R2 was saturable with a Bmax value of 9.11 ± 0.17 pmol/mg protein and a pKd value of 8.66 ± 0.03 (2.18 nM; n = 3) for receptor 1 and a Bmax value of 63.7 ± 3.11 pmol/mg protein and a pKd value of 8.34 ± 0.02 (4.57 nM; n = 3) for receptor 2. Thus, receptor 2 showed a slightly lower affinity for [125I]cGnRH-II compared with receptor 1 (P < 0.01). Furthermore, both receptor subtypes had the same order of affinities for various GnRH agonists, such that cGnRH-II = cGnRH-II-R8 > cGnRH-II-N8 > cGnRH-II-L7 > cfGnRH-R8 > cfGnRH = mGnRH (Table 3Go). However, slight (cGnRH-II, cGnRH-II-R8, and cGnRH-II-L7) to substantial (cfGnRH-R8, cfGnRH, and mGnRH) differences in Ki values were detected for the two catfish GnRH-R subtypes using various GnRH agonists, with receptor 2 generally having a lower affinity for these agonists compared with receptor 1 (Fig. 5Go and Table 3Go; n = 3; P < 0.001). Only cGnRH-II-N8 had no preference for any receptor subtype (Table 3Go; n = 3; P > 0.05). Because the Bmax value for receptor 2 was higher than that of receptor 1, the amount of DNA for transfection was decreased for receptor 2 from 5 µg DNA/100-mm2 dish to 0.1 µg DNA/100-mm2 dish. In this way, similar expression levels of receptor 1 and receptor 2 were obtained for IP and cAMP studies (data not shown).


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  		Table 3. Comparison of the binding parameters of cfGnRH-R1 and cfGnRH-R2

 


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  		Figure 5. Ligand binding of cGnRH-II ({bullet}/{circ}) and cfGnRH ({blacksquare}/{square}) in HEK 293T cells transiently expressing cfGnRH-R1 ({bullet}/{blacksquare}) or cfGnRH-R2 ({circ}/{square}). Displacement of [125I]cGnRH-II binding to membranes prepared from HEK 293T cells transiently expressing cfGnRH-R1 or cfGnRH-R2 by various concentrations of the two native GnRH forms was measured as described in Materials and Methods. The results shown are the mean ± SEM of triplicate observations from a single representative competition binding experiment.

 
All GnRH agonists stimulated IP production in HEK 293T cells transiently expressing either cfGnRH-R1 or cfGnRH-R2. Both receptor 1 and receptor 2 showed the same order of potencies for the endogenous GnRHs, such that cGnRH-II has a greater potency than cfGnRH (Table 4Go and Fig. 6Go; n = 3; P < 0.001). There are no significant differences in EC50 values between receptor 1 and receptor 2 for the GnRH agonists, except for cfGnRH-R8 and mGnRH, that both have slightly higher potencies to stimulate IP production in cells expressing receptor 1 compared with receptor 2 (Table 4Go; n = 3; P > 0.05 and P < 0.05, respectively). The maximal IP response after stimulation with either cGnRH-II or cfGnRH was similar for both receptor subtypes [Emax values (% of basal), 475 ± 73 for receptor 1 after stimulation with cGnRH-II; 644 ± 86 for receptor 2 after stimulation with cGnRH-II; 570 ± 76 for receptor 1 after stimulation with cfGnRH; and 539 ± 59 for receptor 2 after stimulation with cfGnRH; n = 3; P > 0.05].


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  		Table 4. Comparison of the IP-production characteristics of cfGnRH-R1 and cfGnRH-R2

 


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  		Figure 6. Agonist-mediated [3H]IP ({bullet}/{blacksquare}) and cAMP ({circ}/{square}) production of cfGnRH-R2 transiently expressed in HEK 293T cells. [3H]IP and cAMP production were measured after stimulation for 45 min and 10 min, respectively, with various concentrations of cGnRH-II ({bullet}/{circ}) and cfGnRH ({blacksquare}/{square}) as described in Materials and Methods. Data obtained are expressed as percentage of basal [3H]IP production and of basal cAMP production, respectively, in the absence of agonist. Results shown are the mean ± SEM of three independent experiments.

 
Moreover, both cGnRH-II and cfGnRH stimulated cAMP production in HEK 293T cells expressing either receptor 1 or receptor 2 (Fig. 6Go and Table 5Go). For both receptor subtypes, cGnRH-II had a higher potency to stimulate cAMP production compared with cfGnRH (n = 3; P < 0.001), but neither cGnRH-II nor cfGnRH differentiated between receptor 1 or 2 with regard to cAMP-stimulating potency (Table 5Go; n = 3; P > 0.05). The maximal cAMP production after stimulation with either cGnRH-II or cfGnRH was similar for both receptor subtypes (Table 5Go; n = 3; P > 0.05), but in terms of stimulation over basal, it was significantly lower than the maximal IP response (P < 0.01).


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  		Table 5. cAMP production characteristics of cfGnRH-R2 in comparison with cfGnRH-R1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The discrepancy between the presence of GnRH-binding sites and the absence of detectable cfGnRH-R1 expression in ovary instigated us to identify an additional GnRH-R type in the catfish. Here, we report on the cDNA cloning of this second GnRH-R subtype, cfGnRH-R2, from the African catfish that shares highest sequence identity with one of the two goldfish GnRH-Rs, GfA. Native as well as chimeric GnRH agonists bound preferentially to cfGnRH-R1. However, in contrast to the two goldfish GnRH-Rs that display distinct IP responses upon stimulation with native GnRHs (12), the two catfish GnRH-Rs do not distinguish between the endogenous GnRHs in the catfish with respect to IP and cAMP signaling.

Yet, assuming that the observed differences in mRNA expression profiles correlate well with the protein (i.e. each GnRH-R) and peptide (i.e. each GnRH) expression profiles, differential functions for the two types of cfGnRH-Rs seem to be determined by their distinct tissue-specific expression patterns in relation to the expression profiles of the two endogenous GnRHs.

In addition to the distinct expression patterns in the ovary, cfGnRH-R1 and cfGnRH-R2 mRNAs showed marked expression differences in various other catfish tissues. CfGnRH-R2 mRNA was most abundantly expressed in brain (~4-fold higher cfGnRH-R2 than cfGnRH-R1 mRNA levels), whereas the cfGnRH-R1 mRNA is predominantly expressed in pituitary (~40-fold higher cfGnRH-R1 than the cfGnRH-R2 mRNA levels). A similar differential expression of the two goldfish GnRH-R subtype mRNAs has been reported in goldfish brain (12); only probes specific for goldfish GnRH-R GfA, which is highly homologous to cfGnRH-R2, hybridized to a small group of neuronal perikarya in the area ventralis telencephali, whereas probes specific for goldfish GnRH-R GfB, which is highly homologous to cfGnRH-R1, gave no detectable in situ hybridization signals in this region. Differential mRNA expression patterns have also been described for the three bullfrog GnRH-Rs (bfGnRH-Rs): bfGnRH-R1 mRNA was most abundantly expressed in pituitary, whereas the bfGnRH-R2 and bfGnRH-R3 mRNAs were mainly expressed in brain (15). The mRNA expression patterns for the two catfish GnRH-Rs as well as their functional characterization suggest that cfGnRH-R2 has important functions in the brain and might be stimulated predominantly by cGnRH-II, whereas cfGnRH-R1 would mainly function in the regulation of pituitary gonadotroph activity. Although cfGnRH-R1 has a more than 1000-fold higher selectivity for cGnRH-II than for cfGnRH, the latter peptide is thought to be the main regulator of gonadotropin release in catfish (6). The main reasons are the direct innervation of the pituitary gonadotrophs by the cfGnRH-producing neurons in the ventral hypothalamus and by the more than 700-fold excess of cfGnRH over cGnRH-II in the African catfish pituitary. The much higher cfGnRH-R1 than cfGnRH-R2 mRNA expression levels in pituitary might explain why, in previous radioligand-binding studies on catfish pituitary membranes, only a single class of GnRH-R was detected (6, 8).

As regards the cfGnRH-R1 and cfGnRH-R2 mRNA levels in gonadal tissues, both mRNAs are expressed in testis, whereas only cfGnRH-R2 mRNA was detected in ovary. Catfish seminal vesicles were also positive for cfGnRH-R2 mRNA expression. The latter tissue contains epithelial cells, which are thought to be homologous to Sertoli cells (40) and to express the catfish FSH-R mRNA (28), and interstitial cells, which are thought to be homologous to Leydig cells (41). In mammals, Leydig cells express GnRH-Rs, and it is thought that the GnRH produced locally in the testis would act as a paracrine hormone to the receptor on Leydig cells in both mature rats and adult humans (42), possibly exerting an inhibitory effect upon steroidogenesis (20). Therefore, the Leydig cell-like cell type in catfish seminal vesicles is the most likely candidate cell-type that expresses cfGnRH-R2. However, this has to be verified by in situ hybridization studies.

African catfish ovary has been shown to contain GnRH-binding sites (16), which therefore are most likely related to the observed cfGnRH-R2 mRNA expression in this tissue. In particular, relatively high cfGnRH-R2 mRNA levels were found in the ovary of 15-wk-old catfish, suggesting an important function during early (previtellogenic) stages of oogenesis. Similar results were obtained in goldfish (12); only goldfish GnRH-R GfA, which is highly homologous to cfGnRH-R2, is expressed in goldfish ovary. Moreover, goldfish ovary also expresses sGnRH ([Trp7Leu8]GnRH; Ref. 43), which is more potent in stimulating goldfish GnRH-R GfA than goldfish GnRH-R GfB. Furthermore, the rainbow trout GnRH-R has been found in the ovary (44), and mRNA of the GnRH-R as well as the mRNA of cGnRH-II have been detected in ovarian surface epithelial cells, primary cultures of ovarian tumors, and ovarian cancer cell lines (45).

In addition to GnRH-binding sites, African catfish ovary has been shown to also contain a GnRH-like compound (16). Coexpression in the same (or nearby) tissue of a particular receptor subtype with a specific ligand indicates the evolution of a coordinated functional unit. We therefore also determined in which tissues the cfGnRH and cGnRH-II mRNAs were expressed.

Relatively high cfGnRH mRNA levels were detected in brain. Also, relatively high mRNA levels were observed in pituitary, indicating either the presence of this mRNA type in axons of the hypothalamic cfGnRH neurons that innervate the catfish pituitary or the presence of (misdirected) cfGnRH neurons in this tissue. Most other tissues were negative or expressing relatively low cfGnRH mRNA levels (e.g. in the ovary of 15-wk-old catfish). Highest cGnRH-II mRNA levels were found in brain and heart. Intermediate cGnRH-II mRNA levels were observed in cerebellum, head-kidney, and pituitary, and in the ovary of 15-wk-old catfish. All other tissues appear to contain very low levels of cGnRH-II mRNA, suggesting a somewhat leaky transcriptional regulation of this gene. Alternatively, the very low levels of cGnRH-II mRNA in most tissues may be related to GnRH expression in cells that has been previously described as GnRH-like immunoreactivity in mast cells (46). These cells circulate in precursor form and enter tissues in which they complete their differentiation. The observed coexpression of the relatively high and intermediate levels of cGnRH-II and the intermediate levels of cfGnRH-R2 in heart and in the ovary of 15-wk-old catfish, respectively, suggests that both may function as a coordinated functional unit in these tissues. This, however, requires further functional studies.

In conclusion, this is the first detailed study on (nearby) colocalization of specific forms of GnRH with specific types of GnRH-R. We demonstrated that next to the GnRH system regulating the activity of pituitary gonadotropes (the functional unit of cfGnRH and the cfGnRH-R1; Ref. 6), other GnRH systems involving selected pairs of receptor and ligand subtypes are present in catfish (for example, coexpression of cfGnRH-R2 and cGnRH-II in the heart). The fact that the two GnRH-Rs in catfish do not differ in their IP- and cAMP-signaling properties suggests that both receptors, when expressed in the same tissue, are redundant. Alternatively, the two receptors show differences in regulation of receptor expression or differ in other signaling routes like, for example, the MAPK pathway.


   Acknowledgments
 
We thank Dr. R. W. Schulz (Utrecht University) for critically reading this manuscript.


   Footnotes
 
Abbreviations: bfGnRH-R, Bull frog GnRH-R; cfGnRH, catfish GnRH; cf28S rRNA, catfish 28S rRNA; cGnRH-II, chicken GnRH-II; GfA and GfB, goldfish GnRH-Rs; GnRH-R, GnRH receptor; HEK, human embryonic kidney; IP, inositol phosphate; mGnRH, mammalian GnRH; RACE, rapid amplification of cDNA end; sGnRH, salmon GnRH; TM, transmembrane domain.

Received June 3, 2002.

Accepted for publication August 26, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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