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Regular Article |
a Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21202
ABSTRACT
There is little known about the molecular biology of piscine gonadotropin receptors, and information about gene expression during reproductive development is particularly lacking. We have cloned the LH receptor (LHR) in the channel catfish (cc), and examined its gene expression throughout a reproductive cycle. A cDNA encoding the receptor was isolated from the testis using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends procedures. It encoded a 696-amino acid protein that showed the greatest homology (46–50% identity) with the known LHRs and lesser similarity with FSH receptors and thyroid-stimulating hormone receptors (44–47% and 42–44% identity, respectively). In addition, two characteristics unique to the LHRs were conserved in the cloned receptor and the encoding gene: presence of an intron corresponding to intron 10 in mammals and turkey and occurrence of a double cysteine residue in the cytoplasmic tail for potential palmitoylation. The ccLHR gene was well expressed in the gonads and kidney and merely detectable in the gills, muscle, and spleen. The isolated cDNA encoded an active ccLHR protein, as the recombinant receptor expressed in COS7 cells activated a cAMP response element-driven reporter gene (luciferase) upon exposure to hCG in a dose-dependent manner. Seasonal changes in the ovarian expression of the ccLHR gene, as examined by measuring the transcript abundance by quantitative real-time RT-PCR, remained rather low during most of the reproductive cycle but was acutely induced around the time of spawning. This pattern of expression correlates well with the reported expression of its ligand (LH) in fishes and concurs with the notion that LH is a key regulator of the periovulatory maturational events.
seasonal reproduction
INTRODUCTION
The pituitary glycoprotein hormones, FSH, LH, and thyroid-stimulating hormone (TSH) act as high-order controls of the gonads and thyroid in vertebrates. Gonadotropin control of the growth and function of the gonads is mediated by the gonadotropin receptors (GtHR), FSHR and LHR. Genes encoding these receptors are expressed primarily in the accessory cells (follicular and interstitial cells) of the gonads and directly affect gonadal steroidogenesis. The GtHRs bind the respective ligands and activate at least two distinct intracellular signaling pathways: adenylate cyclase and phospholipase C. These signals in turn induce steroidogenesis either via steroidogenic acute regulatory protein or by direct transcriptional activation of the steroidogenic enzymes [1–4]. The GtHR and TSH receptor (TSHR) are membrane receptors belonging to the superfamily of G-protein-coupled receptors as characterized by heptahelical transmembrane domains and conserved cysteine residues. These three receptors constitute the subfamily of glycoprotein hormone receptors and are distinguishable from the rest of the G-protein-coupled receptors by the presence of a long extracellular peptide composed of nine leucine-rich repeat motifs and associated cysteine-rich sequences [5, 6].
Knowledge of the molecular structure, function, and gene-regulation of the GtHRs is imperative to better understand reproductive physiology and can be used to manipulate reproduction. The LHR and FSHR have been cloned in numerous species of the higher vertebrates that are used as research models or valued in animal husbandry. Since the first cloning of these receptors in mammals a decade ago, significant advances have been made in understanding the structure and regulation of gene and signal transduction mechanisms [2, 3].
Fish pituitaries, as in higher vertebrates, secrete two types of GtHs, FSH and LH (previously known as GtH I and GtH II, respectively), that coordinately control ovarian and testicular physiology. The plasma levels of the two hormones exhibit contrasting seasonal (reproductive stage-specific) rhythms: FSH titers rise and remain high during vitellogenesis and spermatogenesis and drop (in most species) during final oocyte maturation and spermiation, whereas LH remains low during vitellogenesis and spermatogenesis but exhibits a periovulatory surge [7]. Complementary DNAs and genes encoding the two hormones have been characterized in several species of fish [7]. There is also limited information available about the gene expression of these hormones during a reproductive cycle. The expression patterns of the two genes are distinct, and the changes in the protein levels correlate with those of the gene transcripts [8].
In marked contrast to the GtHs, knowledge of the piscine receptors for GtHs is lacking. Based on early studies [9, 10], fishes were thought to express a single type of GtHR until the duality of the receptors was first demonstrated using radioligand binding studies several years ago [11] and later confirmed by ligand autoradiography [12]. However cDNA cloning of these receptors remained elusive in spite of rigorous efforts in many laboratories. Recently, cloning of two types of GtHRs from the ovaries of amago salmon has been reported [13, 14]. However, little is known about the molecular genetics, particularly the seasonal gene expression, of the GtHRs in fishes, a highly diverse group of vertebrates. This communication is the second in our series on the molecular cloning and temporal gene expression of fish gonadal glycoprotein hormone receptors [15]. Here we report the isolation and functional characterization of a cDNA encoding the LHR from the gonads of a nonsalmonid species, the channel catfish (Ictalurus punctatus). In addition, a striking induction of the gene encoding this receptor at the time of final oocyte maturation was demonstrated.
MATERIALS AND METHODS
Fish and Tissue Collection
Mature but reproductively inexperienced channel catfish were captured from a local fish farm (Bowling Catfish Farms, Charles City, MD). Up to seven animals were collected at 4-wk intervals over a period of 15 mo. Few fish could be caught in the winter months. Tissues were collected in the field and flash-frozen in liquid nitrogen. The tissues were stored at -80 °C until use.
Cloning of Channel Catfish LHR cDNA
Total RNA was extracted from fragments of frozen tissues using Trizol Reagent (Life Technologies, Rockville, MD) and a Fast-Prep (Savant Instruments, Farmingdale, NY) system. This method eliminates the use of an homogenizer, which is frequently a source of cross-contamination of samples. Messenger RNA was isolated from total RNA using a Straight A mRNA Isolation System (Novagen, Madison, WI). Two micrograms of mRNA isolated from testis or ovary was reverse transcribed using oligo(dT) primer and Superscript II reverse transcriptase (Life Technologies, Rockville, MD). Polymerase chain reaction (PCR) was performed using degenerate primers (P1, 5'-TTCAAYCCHTGCGAGGAYATHATGGG; P2, 5'-GTYTGCCAGTCGATDGCGTGGTTGTA) designed on the basis of mammalian glycoprotein hormone receptors targeting a portion of the extracellular domain and transmembrane domains I and II (Figs. 1 and 2). The PCR was performed in a 50-µl volume under the following conditions: a 2-min denaturation at 94°C followed by 30 cycles of 94°C for 30 sec, 52°C for 40 sec, and 72°C for 45 sec. The amplicon was T-A cloned in pCR2.1 (Invitrogen, Carlsbad, CA), and the nucleotide sequence of the cloned DNA inserts was determined by dye-terminator automatic sequencing based on gel or capillary electrophoresis (ABI 373 DNA Sequencer Stretch or ABI Prism 310 Genetic Analyzer; PE Applied Biosystems, Foster City, CA).
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Based on the sequence information of the presumptive LHR cDNA amplicon, rapid amplification of cDNA ends (RACE) procedures were performed to isolate the 5' and 3' ends of the cDNA (Marathon cDNA Amplification Kit; Clontech, Palo Alto, CA). In each RACE procedure, the initial PCR amplification was followed by a nested amplification. Gene-specific primer sets (P3–P4 and P5–P6; see Fig. 1 for annealing positions) in combination with adopter primers were used, respectively, for the 5'- and 3'-RACE. The PCR parameters were the same as described above except that the annealing temperatures ranged from 54 to 58°C. The RACE amplicons were subcloned and sequenced as described above. From each RACE reaction 5–10 clones were sequenced.
The complete open reading frame (ORF) of the channel catfish (cc)LHR was generated from testicular mRNA in a reverse transcription (RT)-PCR with primers targeting the untranslated portions immediately upstream (P7) and downstream (P8) of the ORF (Fig. 1) and using high-fidelity DNA polymerase enzyme (Platinum Pfx DNA polymerase; Life Technologies). This reaction employed step-down annealing temperatures: 4 cycles each at 66°C and 64°C prior to 28 cycles at 62°C. The resulting product was cloned in pBluescript (Stratagene, La Jolla, CA), designated ccLHR/pBluescript, and one of the resulting clones was sequenced in both directions.
Sequence Computations
Homology search by the position-specific iterated-basic local alignment search tool (PSI-BLAST) method [16], alignment of multiple protein sequences by the CLUSTAL W method [17], and construction of a phylogenetic tree by the neighbor-joining method [18] were performed with the internet server of the DNA Data Bank of Japan (DDBJ; http://www.ddbj.nig.ac.jp). The signal peptide and its putative cleavage site were predicted according to Nielsen et al. [19] using SignalP v2 (http://www.cbs.dtu.dk/services/SignalP). Leucine-rich repeat (LRR) motifs were identified manually.
Genomic Analysis of ccLHR
Catfish genomic DNA was analyzed by PCR to determine if the ccLHR gene contains an intron corresponding to intron 10 of the LHRs. This intron is absent in mammalian FSHR and TSHR genes studied thus far. Catfish genomic DNA was isolated from liver using DNAzol Reagent (Life Technologies). The genomic DNA and ccLHR cDNA were PCR amplified with primers P9 and P4, and the size of the two amplicons was compared. The nucleotide sequence of the genomic amplicon was determined by direct sequencing in both directions.
Tissue-Specific Expression of the ccLHR Gene
Transcriptional expression of the ccLHR gene in various tissues was determined by RT-PCR in which 3 µg of total RNA from various tissues (listed in Results) was reverse transcribed using oligo(dT) as a primer. A cDNA segment spanning four potential intron-exon boundaries (so that false amplicons from genomic contaminants could be identified) was amplified by PCR using primers P10 and P4 in 30 cycles of 94°C for 30 sec, 58°C for 40 sec, and 72°C for 1 min, and the products were analyzed electrophoretically. Ethidium bromide-stained agarose gels were fluorescence-scanned with FluorImager 575 (Molecular Dynamics, Sunnyvale, CA).
Functional Expression of ccLHR cDNA
The cDNA insert from ccLHR/pBluescript was subcloned into a cytomegalovirus expression vector (pcDNA3.1/Zeosin, Invitrogen) at NotI-HindIII restriction sites and designated ccLHR/pcDNA3.1-Zeo. This clone was partially sequenced to ensure that the cDNA was inserted in the sense orientation. The COS7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% newborn calf serum. Cells were cotransfected with ccLHR/pcDNA3.1-Zeo (50 ng/ml) and pCRE-Luc (1 µg/ml) using Lipofectamine 2000 Reagent (6 µl/ml, Life Technologies). The pCRE-Luc plasmid, which encoded the firefly luciferase gene downstream of a basic promoter composed of multiple cAMP response elements, served as a reporter gene for changes in intracellular cAMP. In control transfections, empty vector (pcDNA3.1-Zeo) replaced the ccLHR/pcDNA3.1-Zeo construct. Cells were serum-starved overnight prior to hormone treatments. Two days post-transfection, cells were exposed to medium containing BSA (1 mg/ml) and hCG (Sigma Chemicals, St. Louis, MO) or immunopurified human FSH (National Hormone and Pituitary Program) for 6 h. Incubations were stopped by washing the cells with PBS and adding cell lysis buffer, and luciferase activity was measured using a Luciferase Assay kit (Promega Corporation, Madison, WI) and MicroLumat LB96P luminometer (EG&G Berthold, Stammwerk Wilbad, Germany).
Real-Time Quantitative RT-PCR
Real-time quantitative (rtq) RT-PCR was employed to determine the transcript abundance as a measure of expression of the ccLHR gene through a complete reproductive cycle. Unlike conventional quantitative RT-PCR, transcript quantification by rtqRT-PCR is based on a parameter (threshold cycle) that occurs early in the PCR process, and therefore the measurements are more accurate and highly reproducible. Described briefly, 400 ng of total RNA was reverse transcribed in a 20-µl reaction volume using random hexamer primers and Moloney murine leukemia virus reverse transcriptase (Life Technologies). Primers P11 and P12 were designed according to the requirements set forth by Primer Express software and the developer of the ABI Prism Sequence Detector (see below). In short, the primer sets were designed to generate amplicons of 50–150 base pairs (bp) in length and the 3' ends of the primers were A/T-rich. The cDNA corresponding to 5 ng of reverse-transcribed RNA served as templates for each of duplicate 25-µl PCR reactions using SYBR Green Core Reagents (PE Applied Biosystems). The PCR amplifications and fluorescence detection were performed with the ABI Prism Sequence Detector 7700 using the manufacturer's universal thermal cycling conditions. The computations and descriptions of a modification of the procedure, called TaqMan technology, have been published elsewhere [20, 21]. As an internal control, 18S rRNA was amplified in an identical manner using primers specific for channel catfish 18S (5'-TGGTTAATTCCGATAACGAACGA and 5'-CGCCACTTGTCCCTCTAAGAA). Abundance of the ccLHR transcript was normalized to 18S and reported as a fold change in abundance relative to the values obtained in July of the first year of sampling (gonadal regression).
The rtqRT-PCR procedure was validated according to the manufacturer: the efficiencies of amplification of ccLHR and 18S were verified to be equal statistically; gel electrophoresis of randomly selected PCR reactions confirmed the presence of a single amplicon of expected size; control reactions in which RNA replaced the cDNA template produced no significant signals, ensuring the nonamplification of any contaminating genomic DNA.
Statistics
All numerical data (functional expression studies) are presented as mean ± SEM. Statistical differences between groups of seasonal gene expression data were determined by ANOVA followed by Tukey's multiple comparison test.
RESULTS
Cloning of ccLHR cDNA
The degenerate PCR generated an amplicon of the expected size (259 bp) from the testis and ovary. About 15 clones of this amplicon were sequenced, and the nucleotide sequences of the clones belonged to two types: those of presumptive LHR and FSHR. A description of the ccFSHR will be reported separately. The RACE procedures led to the isolation of the 5' and 3' ends of the cDNA covering the ORF and untranslated regions (GenBank accession no. AF285181). The specific RT-PCR to generate a full-length clone using primers P7 and P8 generated a single amplicon of 2.2 kilobases as expected. The nucleotide sequence of this clone was identical to those of the RACE clones.
Primary Structure of the Deduced ccLHR Protein
The isolated cDNA encoded a 696-amino acid protein including a predicted 23-amino acid signal peptide (Fig. 2). The deduced protein showed the greatest homology with the LHRs of other species: 50%, 47%, and 46–47% identities with chicken (GenBank accession no. AB009283), salmon (AB030005), and mammalian (cow, U20504; human, S57793; mouse, M81310; pig, M29525) LHRs, respectively. It shared 44–47% identity with the FSHRs of salmon (AB030012), chicken (U51097), and mammals (human, S59900; mouse, AF095642; cow, L22319; pig, AF025377), and 42–44% identity with the TSHRs of striped bass [15] and mammals (human, M32215; rat, M34842; cow, U15570).
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The C-terminal half of the deduced protein was composed of seven transmembrane domains and a 67-residue intracellular tail. The N-terminal half constituted a large (370-residue) extracellular domain in which nine leucine-rich repeats together with associated cysteine-rich flanking regions [6] and three potential N-linked glycosylation sites were identified. The TSHR-specific insertion [15] was absent in the distal cysteine-rich sequence. There were 18 cysteine residues and two of them (646CC) occur consecutively in the cytoplasmic tail for potential palmitoylation [2]. Occurrence of double cysteine residues in this position is characteristic of the LHRs and is present in all species characterized to date (partially shown in Fig. 2).
Phylogenetic analysis (Fig. 3) clearly placed the cloned receptor within the LHR cluster with a high bootstrap support of 83%. Further, the ccLHR branched out with the salmon GtHR II (homologous to LHR) with highly significant bootstrap support (98%), suggesting a cognate relationship. The protein deduced from the other amplicon amplified by the degenerate primer was classified within the FSHR cluster (unpublished results).
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Presence of LHR-Specific Intron in the ccLHR Gene
The PCR with primers P9 and P4 amplified an expected 350-bp product from the ccLHR/pcDNA3.1-Zeo, whereas a larger product was amplified from the catfish genomic DNA, suggesting the presence of an intron in this region (Fig. 4A). In order to confirm this and to determine the precise position of the intron, the nucleotide sequence of the PCR product was directly determined. A 102-bp intron was located at a position that is conserved in other known LHR genes (Figs. 2 and 4B) but absent in the FSHR and TSHR genes. The location of this intron followed phase 2 (Fig. 4B), similar to the genes encoding other glycoprotein hormone receptors [2, 22].
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Tissue-Specific Expression of the ccLHR Gene
The RT-PCR examination revealed that the ccLHR gene was expressed in the gonads, kidney, and, at low levels, in the gills and muscle (Fig. 5). Expressions in the muscle and spleen were barely detectable. Renal expression of the gene was confirmed in a second male animal (Fig. 5, kidney2).
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Functional Expression of ccLHR cDNA
In COS cells transiently cotransfected with ccLHR/pcDNA3.1-Zeo and pCRE-Luc, hCG increased reporter gene (luciferase) activity over the basal activity in a dose-dependent manner. Human FSH seemed to produce a small but statistically nonsignificant increase in the activity. Basal luciferase activity found in cells transfected with the empty expression plasmid was much lower than in cells expressing the LHR and remained unchanged with hormonal treatments (Fig. 6).
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Seasonal Expression of the ccLHR Gene
The data on the gonadosomatic index (GSI) of the fish and water temperature were presented elsewhere [23]. Briefly, from the lowest summer values, the GSI slowly increased from October, increased rapidly after April, and peaked in June, antecedent to the time of spawning (early July). The abundance of ccLHR transcript increased slightly (August–September) just before the onset of ovarian recrudescence and remained low throughout the period of vitellogenic growth (through March). At the anticipated time of final spawning, the abundance increased nearly fivefold and thereafter quickly returned to the basal level (Fig. 7).
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DISCUSSION
The LH-mediated control of ovarian physiology in fish, especially final oocyte maturation, requires the timely and quantitative expression of the LHR in the ovary [7, 24]. While there is abundant literature on the temporal ovarian expression of LHR gene in mammals [1, 25, 26], there is little information about birds [27, 28] and none for fishes. We have addressed this deficiency by first isolating the LHR cDNA in this species and then systematically determining the expression of LHR throughout an annual reproductive season in the channel catfish, a species that offers the advantage of synchronous development of oocytes. In addition to determining the primary structure of the ccLHR, the present study has revealed the evolutionary conservation of an intronic site unique to the LHRs.
The identity of the receptor was established on the basis of protein structure, gene organization, and ligand specificity of the recombinant receptor and confirmed by the seasonal gene expression. The cloned glycoprotein hormone receptor showed the highest homology to LHRs of other species including a species of fish, the amago salmon. It is interesting that it shows greater identity to the chicken LHR [28] than the salmon GtHR II (putative LHR; [13]). Nevertheless, it has a closer evolutionary relationship to the salmon LHR as indicated by the phylogenetic analysis (Fig. 3).
The site of the LHR-specific intron is conserved in the catfish ccLHR gene. Following the characterization of glycoprotein hormone receptor genes in some mammals, it was found that the organization of these genes was very similar except that the LHR genes possessed an additional intron (intron 10) compared to the FSHR and TSHR genes (nine introns each) [2, 3]. The presence of this intron has been recently shown in an avian (turkey) LHR gene [29]. The presence of this intron in the channel catfish LHR gene, but not the channel catfish FSHR gene (unpublished results), and the conserved splice junction suggest that the receptor-specific pattern of genomic organization was established in the common ancestor of the teleost fishes and tetrapods.
Another similarly LHR-specific pattern was evident when the peptide sequences of most available glycoprtein hormone receptors were aligned (partly shown in Fig. 2). For potential palmitoylation and consequent membrane anchoring [2, 5], two adjacent cysteine residues are present in the intracellular domain of the LHR (but only one in the FSHRs and TSHRs) in mammals (including marmoset monkey [30]), turkey, chicken, and salmon. This structural characteristic also holds true for the ccGtHRs (LHR, Fig. 2; FSHR, unpublished results); however, the chicken FSHR [31] with double cysteines at this site is an exception.
The distal cysteine-rich sequence that lies between the last leucine-rich repeat and the first transmembrane segment is the most variable region in the glycoprotein hormone receptor proteins and is believed to be critical for ligand specificity [2, 32]. This sequence in the ccLHR (Fig. 2), salmon GtHRs [13, 14], and all other vertebrate gonadotropin receptors is shorter than the corresponding region in the TSHRs, including the sole reported nonmammalian form (striped bass [15]). This segment is shortest in the piscine FSHRs, as consistently observed in amago salmon [14], African catfish (GenBank accession no. AJ012647), and channel catfish (unpublished results).
The ligand preference of the recombinant receptor also lends support to the identification of the ccLHR. The ligand-binding property has not always been a useful criterion to identify a glycoprotein hormone receptor because significant cross-activation by other members of the glycoprotein hormone family have been reported [33, 34]. However, of the two heterologous glycoprotein hormones tested, only hCG activated the receptor in a dose-dependent manner. In the absence of homologous LH, hCG was tested because it has often been effectively utilized as a heterologous GtH preparation for the study of fish reproductive endocrinology [35] and for induced spawning. The low level of activation of the receptor by hFSH indicated limited interaction of the receptor with this hormone. This was anticipated because minimal cross-activation was reported for the putative coho salmon GtHR II [11] and amago salmon GtHR II [14] even with the use of homologous FSH (GtH I).
The activation of the pCRE-Luc reporter in the present study demonstrated that the ccLHR, as in other species, acts via a cAMP-mediated signal transduction pathway. Whether the receptor uses other signaling pathways, specifically the phospholipase C pathway, is unknown; however, it has been shown that glycoprotein hormone receptors in many species use multiple signaling pathways [2, 36]. In addition, the background luciferase activity (i.e., no hormones; Fig. 6) in cells transfected with the LHR construct is almost three times higher than cells transfected with the empty pcDNA vector. These data suggest that the recombinant catfish receptor be partially but constitutively activated in this heterologous expression system. It is unknown if constitutively activated LHR are evident in the catfish ovarian follicle, but it could partially explain the continuous presence and relatively high titers of sex steroid titers in this species [23]. Further studies in a homologous expression system (catfish host cells) using homologous hormone are required to understand the binding kinetics and intracellular signaling mechanisms of the piscine LHR, FSHR, and TSHR.
As expected, expression of the ccLHR gene was clearly evident in the testis and ovary (Fig. 5). The renal expression of the gene in male catfish at levels comparable to the testis was, however, unexpected. Transcript abundance of ccLHR in the kidney of female catfish has not been examined nor has the potential changes in expression associated with the reproductive stage been described. In the human, in which the tissue expression of this gene has been studied extensively, LHR transcripts or protein have been reported in a variety of extragonadal tissues including brain [37], adrenal glands [38], lymphocytes [2], urinary bladder [39], and skin [40], but not in the kidney. While the catfish skin was not examined in this study, the head kidney (containing the piscine homologue of the adrenal gland along with a variety of other tissues) did not express the gene.
The seasonal changes in gene expression, as determined by measuring transcript abundance, revealed that the expression of the ccLHR gene follows a highly defined rhythm of expression (Fig. 7). While expression may be extremely low during most of the reproductive cycle, transcript abundance increased slightly prior to the onset of ovarian recrudescence (although not significant) and increased dramatically immediately preceding spawning (final oocyte maturation). The preovulatory induction of the ccLHR gene corresponds well with the known seasonal dynamics of its ligand in fishes. In a typical fish, the plasma titers of LH remain undetectable during the phase of gonadal growth but increase sharply during final oocyte maturation, with peak titers coinciding with ovulation [7, 41, 42]. It should be noted that the quantitative RT-PCR procedure was not designed to detect any potential differences in transcript complexity of the LHR or any seasonal changes in this complexity. Mammalian follicles express multiple LHR transcripts; however, we have no data by Northern blot analysis to indicate if this is also true for the catfish follicle.
There is growing evidence that LH is important in the final oocyte maturation in fishes [43]. Specifically, it is thought to bring about an abrupt switch in steroidogenesis from estradiol to the maturation-inducing steroid (MIS) by inhibiting aromatase activity [44] and stimulating MIS synthesis [43, 45]. The marked preovulatory induction of the ccLHR gene observed in this study is consistent with the above conclusion. Despite major differences in reproductive physiology, the changes in transcript abundance of the ccLHR closely reflect those in rat [1, 26] and chicken [31] in which LHR mRNA was barely detectable in granulosa cells from immature follicles and postovulatory follicles but was highly elevated in large preovulatory follicles.
The binding properties, cellular localization, or molecular characteristics of a similar receptor in two fish species (coho salmon and amago salmon) have been previously reported. In the studies in coho salmon [11, 12], the type II receptor was restricted to the granulosa cells of the preovulatory stage, and it specifically bound LH (GtH II). The GtHR cDNA first cloned in amago salmon not only showed the greatest structural similarity with the LHR of higher vertebrates, but its recombinant receptor was also most responsive to LH [13]. Thus these receptors characterized in coho and amago salmons are clearly the homologs of the ccLHR described in this study.
In summary this report describes the isolation and functional expression of the LHR in the channel catfish. In addition to describing only the second piscine LHR to date, this study shows 1) the evolutionary conservation of an intronic site unique to the LHRs, 2) dramatic induction of the gene in preovulatory follicles, and 3) novel expression of the gene in the kidney. The data presented here will facilitate further molecular studies to understand the LH-mediated regulation of final oocyte maturation and the contribution of receptor modulation to this process.
ACKNOWLEDGMENTS
We thank Dr. A.F. Parlow (National Hormone and Pituitary Program, Torrance, CA) for providing the human FSH and Dr. A. Place for useful discussion. This work was initiated with oligonucleotide primers originally designed by Penny Swanson of the Northwest Fisheries Science Center, National Marine Fisheries Service. This is contribution number 536 from the Center of Marine Biotechnology.
FOOTNOTES
First decision: 17 October 2000.
1 This research was supported by grants to J.M.T. from the United States Department of Agriculture (Enhancing Reproductive Efficiency, grant 00-35203-9105) and the Wallenburg Foundation. The nucleotide sequence reported in this paper has been deposited in GenBank under the accession number AF285181. 
2 Correspondence: John M. Trant, Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 E. Pratt Street, Baltimore, MD 21202. FAX: 410 234 8896; trant{at}umbi.umd.edu
Accepted: November 3, 2000.
Received: August 31, 2000.
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