| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21202
ABSTRACT
Molecular cloning of the channel catfish FSH receptor is reported together with temporal changes in the 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 (RACE) procedures. The cDNA coded for a 662-amino acid protein that was most identical (51%–59%) to salmon gonadotropin receptor I and the FSH receptors of higher vertebrates, and less identical to LH receptors and thyrotropin receptors (45%–49% and 46%–47%, respectively). In addition, PCR analysis of the genomic DNA showed the absence of the LH receptor-specific intron. Expression of the channel catfish FSH receptor gene was highly restricted to the testis and ovary, except for a low-level expression in the spleen. Transfected COS cells expressed an active recombinant receptor as determined by the ligand-specific activation of a cAMP-responsive reporter gene (luciferase). The recombinant receptor was activated by human FSH and, to a small extent, hCG. Seasonal changes in the ovarian expression of the FSH receptor gene, examined by measuring the transcript abundance by quantitative real-time RT-PCR, showed a rise around the time of onset of ovarian recrudescence and a decrease prior to spawning. This pattern of seasonal expression of FSH receptor differs significantly from that of the LH receptor, which we reported recently. The differential expression of the two gonadotropin receptor genes, in addition to the differential secretion of the gonadotropic hormones, seem to be critical for the regulation of steroidogenesis and other gonadal physiological processes.
follicle-stimulating hormone receptor, oocyte development, seasonal reproduction
INTRODUCTION
In vertebrates, gonadotropin-releasing hormone, pituitary gonadotropic hormones (GtH), and gonadal steroids are the vital hormonal elements of the hypothalamo-hypophysial-gonadal axis. The GtHs, FSH and LH, coordinately control gonadal steroidogenesis by binding to their respective receptors (FSHR and LHR) in the gonads [1]. These membrane-bound receptors belong to the superfamily of G-protein-coupled receptors. The G-protein-coupled receptors are anchored to the plasma membrane with seven transmembrane domains, and their tertiary structure is held intact by disulfide bonds between conserved cysteine residues. After binding to the appropriate ligand, the receptors couple with G-proteins and activate dual intracellular signaling: adenylyl cyclase/protein kinase A and phospholipase C/protein kinase C pathways [2].
The gonadotropin receptors (GtHR) and thyroid-stimulating hormone receptor (TSHR), collectively referred to as glycoprotein hormone receptors (GpHR), possess the peculiar feature of a very large extracellular domain, which is made up of nine leucine-rich repeats flanked by cysteine-rich sequences. The transmembrane domain of the three GpHRs is highly conserved, whereas the extracellular domain, which confers ligand specificity, is more variable [3–6]. In higher vertebrates, the genes encoding the FSHR and LHR show distinct patterns of cell-specific and temporal expression. The FSHR is predominantly expressed in the granulosa cells, especially of the developing follicles, and Sertoli cells of the testis. In contrast, the LHR is highly expressed in the granulosa cells of preovulatory follicles and Leydig cells of the testis. In the ovary, pronounced induction of the FSHR and LHR genes thus occur during follicular development, and maturation and ovulation, respectively [3, 7]. The structural organizations of the three genes are highly similar, except that the LHR gene differs from the FSHR and TSHR genes by possessing an additional intron that interrupts the terminal exon [5].
In fishes, the duality of gonadotropic hormones (originally denoted as GtH I and GtH II but now known as FSH and LH, respectively) has been established in numerous teleost species and has been best described in the salmonids [8]. Even though studies have failed to isolate FSH or its cDNA from some teleost species, available data suggest that FSH plays a dominant role during the onset of ovarian recrudescence, which diminishes as the ovarian follicles mature. A pronounced rise in the gene expression of the FSHß subunit at the time of ovulation has also been recorded in a few species [9]. In contrast, LH is poorly expressed until the initiation of final oocyte maturation, at which time elevated LH titers are clearly associated with final oocyte maturation and ovulation [8]. These profiles of FSH and LH expression are essentially similar to those in mammals.
As our knowledge of the piscine GtHs blossom, information concerning their receptors remains scarce. Although the duality of GtHR was previously demonstrated in a salmonid fish by biochemical methods [10, 11], two types of cDNAs encoding putative FSHR and LHR were reported only recently [12–14]. Efforts in these studies focused on cloning and functional expression of the cDNA. There is yet no information on the temporal changes in the expression of the genes encoding these receptors, but such information will be valuable for definitive identification of the receptors. We have conducted a series of studies on the molecular cloning and temporal gene expression of glycoprotein hormone receptors in two species of fishes, and recently reported on the molecular characteristics of the striped bass TSHR [15] and channel catfish LHR [16]. In this report, we describe the molecular cloning of the FSHR from the channel catfish (Ictalurus punctatus) and the seasonal changes in the gene expression. This is the first seasonal study of any piscine FSHR gene expression and it strengthens the assumption of GtH duality in the catfish.
MATERIALS AND METHODS
Fish and Tissue Collection
Up to seven channel catfish were captured from ponds of a local fish farm (Bowling Catfish Farms, Charles City, MD) at 4-wk intervals for a period of 15 mo. All fish used in this study were pubescent but reproductively inexperienced. The animals were decapitated in accordance with the Animal Care and Use Guidelines, after which dissected tissues were flash-frozen in liquid nitrogen and stored at -80°C.
Molecular Cloning and Sequence Characterization of Channel Catfish FSHR cDNA
The strategy adopted for cloning the channel catfish FSHR (ccFSHR) cDNA was identical to that used for isolating the channel catfish LHR (ccLHR) [16]. The strategy entailed first generating a partial cDNA by reverse transcription-polymerase chain reaction (RT-PCR) using degenerate primers, followed by amplifications of 5'- and 3'-cDNA ends by rapid amplification of cDNA ends (RACE), and finally generation of a cDNA encoding the complete coding region in a single set of RT-PCR reaction. Described briefly, total RNA was extracted from fragments of frozen tissues using Trizol Reagent (Life Technologies, Rockville, MD) and the Fast-Prep (Savant Instruments, Farmingdale, NY) system. Messenger RNA was isolated from total RNA using Straight A's mRNA Isolation System (Novagen, Madison, WI). Messenger RNA (1 µg) isolated from testis or ovary was reverse transcribed using Superscript II reverse transcriptase (Life Technologies) and oligo-dT primer. A PCR was performed using degenerate primers (P1, 5'-TTCAAYCCHTGCGAGGAYATHATGGG, P2, 5'-GTYTGCCAGTCGATDGCGTGGTTGTA) designed from an alignment of the highly conserved segment of the mammalian GpHRs encompassing a portion of the extracellular domain and transmembrane domains I and II (Figs. 1 and 2). One to two microliters of the RT reaction was amplified by PCR using the following cycling conditions: 2-min denaturation at 94°C followed by 30 cycles at 94°C for 30 sec, 52°C for 40 sec, and 72°C for 45 sec. The 5'- and 3'-RACE reactions were carried out on testicular mRNA using the Marathon cDNA Amplification Kit (Clontech, Palo Alto, CA) and primers P3 to P8 (Fig. 1). For each RACE procedure, an initial PCR amplification was followed by a nested amplification.
|
Products of the degenerate PCR and nested RACE reactions were T-A ligated to pCR2.1 (Invitrogen, Carlsbad, CA). The nucleotide sequences of the cloned DNA inserts were determined by dye-terminator automatic sequencing (ABI 373 DNA Sequencer STRETCH or ABI PRISM 310 Genetic Analyzer; PE Applied Biosystems, Foster City, CA). Four to seven clones were sequenced from each nested RACE reaction.
The complete coding region of ccFSHR was amplified from testicular mRNA in a RT-PCR using primers targeting the 5'UTR (P9) and 3'UTR (P10) portions of the cDNA (Fig. 1) and high-fidelity DNA polymerase enzyme (Platinum Pfx DNA polymerase; Life Technologies). The following step-down annealing temperatures were employed in this reaction: two steps of four cycles each at 66°C and 64°C followed by 28 cycles at 62°C. The resulting amplicon was T-A ligated to pBluescript (Stratagene, La Jolla, CA), designated as ccFSHR/pBluescript, and two of the resulting clones were sequenced in both directions.
Potential identity of the protein encoded by the ccFSHR was determined by homology search with the Position Specific Iterated-Basic Local Alignment Search Tool (PSI-BLAST) method [17]. Multiple protein sequences were aligned by the CLUSTAL W method [18] and a phylogenetic tree was constructed by the neighbor-joining method [19]. All of the above analyses were performed with the Internet server of the DNA Data Bank of Japan (DDBJ; http://www.ddbj.nig.ac.jp). Signal peptide and its putative cleavage site were predicted according to Nielsen et al. [20] using SignalP v2 (http://www.cbs.dtu.dk/services/SignalP).
Partial Analysis of ccFSHR Gene
A PCR analysis of catfish genomic DNA was performed to determine whether or not the ccFSHR gene contains an intron at the position corresponding to the unique intron of the LHRs (see below). Catfish genomic DNA was isolated from liver using DNAzol Reagent (Life Technologies), and the genomic DNA and ccFSHR cDNA were PCR amplified with primers P11 and P5, and the size of the two amplicons were compared.
Tissue Expression of the ccFSHR Gene
Presence of the ccFSHR transcript was determined in various tissues by RT-PCR. Single-stranded cDNA was synthesized from 3 µg of oligo(dT)-primed total RNA isolated from each tissue (listed in Results). PCR analysis was performed using primers P12 and P5, and the following cycling conditions: 30 cycles at 92°C for 30 sec, 58°C for 40 sec, and 72°C for 1 min. The analysis was designed to generate specific amplicons that spanned four potential intron-exon boundaries in order to eliminate false-positive amplicons arising from any contaminating genomic DNA. Ten-microliter aliquots of the PCR reactions were analyzed electrophoretically and the amplicons were visualized under ultraviolet light.
Functional Analysis of ccFSHR cDNA
The cDNA insert from ccFHR/pBluescript was first subcloned into a cytomegalovirus (CMV) expression vector (pcDNA3.1/Zeosin; Invitrogen) at Not1-KpnI restriction sites (ccFSHR/pcDNA3.1-Zeo). The sense orientation of cDNA insert in the vector was confirmed by sequencing analysis. The functional integrity of the protein encoded by ccFSHR/pcDNA3.1-Zeo was verified in COS cells that were transiently cotransfected with pCRE-Luc (a plasmid containing the firefly luciferase reporter gene driven by a basic promoter plus multiple cAMP-response elements). COS cells were cultured in Dulbecco modified Eagle medium supplemented with 10% newborn calf serum and cotransfected with ccFSHR/pcDNA3.1-Zeo (50 ng/ml) and pCRE-Luc (1 µg/ml) using Lipofectamine 2000 Reagent (6 µl/ml; Life Technologies). Negative control transfections were performed with "empty" pcDNA3.1-Zeo in place of the ccFSHR/pcDNA3.1-Zeo. Thirty-six h after transfection the cells were serum-starved overnight and then exposed to medium containing BSA (1 mg/ml) and immunopurified human FSH (National Hormone and Pituitary Program) or hCG (Sigma Chemical Co., St. Louis, MO) for 6 h. Ligand activation of the recombinant receptor was indirectly examined by measuring cAMP-mediated induction of luciferase activity using a Luciferase Assay kit (Promega Corporation, Madison, WI) and MicroLumat LB96P Luminometer (EG&G Berthold, Stammwerk, Wilbad, Germany).
Real-Time Quantitative RT-PCR
Abundance of ccFSHR transcript was determined as a measure of gene expression throughout an annual ovarian cycle using real-time quantitative RT-PCR analyses described previously [16, 21]. This technique allows for the measurement of transcripts of several genes in a small amount of tissue. Described briefly, 400 ng of total RNA was reverse transcribed in a 20-µl reaction volume using random hexamer primers and Maloney murine leukemia virus reverse transcriptase (Life Technologies). Aliquots of the RT reaction 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). Primers for these PCR analyses, P13 and P14, were designed to generate an amplicon of about 100 base pairs (bp). The PCR amplifications and fluorescence detection were performed with the ABI PRISM Sequence Detector 7700 under the manufacturer's universal thermal cycling conditions. The computations and descriptions of a modification of the procedure, called TaqMan technology, have been published elsewhere [22, 23]. As an internal control, 18S ribosomal RNA was amplified in an identical manner using primers specific for channel catfish 18S (5'-TGGTTAATTCCGATAACGAACGA-3' and 5'-CGCCACTTGTCCCTCTAAGAA-3'). Abundances of ccFSHR transcript were normalized to those of 18S and reported as a fold change in abundance relative to the values obtained in July of the first year of sampling.
Statistics
All numerical data are presented as means ± SEM. Statistical differences between groups were determined by one-way ANOVA followed by the Tukey multiple comparison test in the case of ligand binding studies of the recombinant receptor, and the Kruskal-Wallis test followed by the Dunn multiple comparison test in the case of seasonal gene expression data.
RESULTS
The initial PCR using the degenerate primers amplified a partial cDNA of the expected size (259 bp) from testis and ovary. More than 15 clones of this amplicon were sequenced and the nucleotide sequences of about half of the clones were of presumptive FSHR. The remaining clones represented LHR, which has been previously reported [16]. RT-PCR of ovarian mRNA also generated an amplicon of identical size, however, this amplicon was not characterized further. A single set of nested 3'-RACE reactions using sense primers P3 and P4 in combination with adopter primers amplified the entire 3' terminus of the cDNA (including the poly-A tail). On the other hand, a nested 5'-RACE reaction using the antisense primers P5 and P6 stopped short of the translation start codon. Therefore, a second 5'-RACE using primers P7 and P8 was conducted, and it amplified the remainder of the 5' end and a portion of the untranslated region. One of the 5'-RACE clones was 236 bp longer than the rest, representing transcripts containing an unspliced intron with characteristic GT and AG splice donor and acceptor sites (exon-exon boundary indicated in Fig. 1). The splice junction interrupted the codon between the second and third nucleotide, indicating that it is of phase 2 type (Fig. 1). The RT-PCR using testicular RNA and primers P9 and P10 generated a single amplicon of the expected size of 2.3 kilobase (kb), and it contained the complete coding sequence. The nucleotide sequence of clones from this independent PCR confirmed those derived from the RACE clones.
The isolated cDNA encoded a 662-amino acid protein of which the first 22 amino acids were predicted to constitute the signal peptide (Fig. 2). The deduced protein was most identical to the FSHRs of other species (59% identical to putative salmon FSHR and 51%–53% identical to chicken and mammalian FSHRs, respectively). Its identity to the LHRs, including that of the channel catfish, amago salmon, chicken, and mammals was 45%–49%. It shared only 46%–47% identity with the TSHRs of striped bass and mammals.
|
Characterization of the deduced protein according to the method of Sonnhammer [24] indicated the presence of three major subdivisions: a 341 amino acid extracellular domain, 263 amino acid transmembrane domain, and a 58 amino acid cytoplasmic domain (Fig. 2). In the N-terminal extracellular domain, there were nine leucine-rich repeats flanked on the amino- and carboxy-sides by cysteine-rich sequences [25]. The ccFSHR protein possessed seven potential N-linked glycosylation sites and 11 conserved cysteine residues. The distal cysteine-rich sequence, like in the salmon GtHR I (putative FSHR), was shorter than the piscine and mammalian LHRs and TSHRs. In the cytoplasmic tail, only one conserved cysteine residue was present for potential palmitoylation.
In a phylogenetic tree incorporating selected vertebrate GpHRs, the ccFSHR clearly belonged to the FSHR cluster, whereas the ccLHR appropriately belonged to the LHR cluster. The ccFSHR clearly clustered with the other fish FSHRs. The bootstrap support for the branch bearing the three piscine FSHRs was highly significant (100%), suggesting a very close evolutionary relationship (Fig. 3).
|
The PCR analysis using the primers P11 and P5 was performed to test for the presence or absence of the LHR-specific intron in the channel catfish FSHR gene. This intron is present in the LHR genes studied thus far, but is absent in the FSHR and TSHR genes; therefore, it serves as a characteristic structural feature of the LHR genes. The analysis showed that the amplicons from the catfish genomic DNA and the ccFSHR cDNA were identical in size (Fig. 4), indicating the lack of an intron in this portion of the gene.
|
The RT-PCR detected the presence of ccFSHR transcripts in the testis and ovary, and at a very low level in the spleen (Fig. 5). No transcripts were detectable in the brain, pituitary, head kidney (containing interrenal, chromaffin and hematopoetic tissues), kidney, liver, intestine, and gill. In simultaneous reactions, ß-actin was amplified with consistent intensity, indicating the uniformity of the template cDNA among the reactions.
|
In COS cells transiently cotransfected with ccFSHR/pcDNA3.1-Zeo and pCRE-Luc, hFSH increased the reporter gene (luciferase) activity over the basal activity in a dose-dependent manner. There was no significant difference in the luciferase activities of the cells transfected with "empty" expression vector and treated with hFSH (Fig. 6).
|
From the seasonal gene expression study, data collected on the gonadosomatic index of the fish and water temperature have been reported elsewhere [21]. Briefly, the GSI values were lowest in summer and they gradually increased from October. The values increased at a higher rate after April and peaked in June, prior to the time of spawning (early July). Transcripts of ccFSHR were detectable throughout the year but showed a slight elevation in abundance in September, although it was not statistically significant. Transcript abundance declined to a low level in November and fluctuated, but remained low during the winter and spring months. Immediately following spawning, the abundance rose steadily through November, which corresponded to ovarian regression, mitotic follicular multiplication, and onset of recrudescence (Fig. 7).
|
DISCUSSION
In this study we have isolated a cDNA encoding the FSHR of the channel catfish (Ictalurus punctatus) and examined its expression throughout an ovarian cycle. In this series of studies we also cloned a distinct channel catfish LHR [16] and thus the duality of vertebrate gonadotropins appears to be present in the catfishes even though isolation of the catfish FSH has yet to be described.
On the basis of the structural characteristics of the deduced protein and the gene, and the binding characteristics of the recombinant protein, it was evident that the cloned cDNA encoded FSHR. This identification was further authenticated by the temporal and tissue-specific expression patterns of the gene encoding the receptor. The deduced protein showed much higher structural similarity to the known FSHRs than to the LHRs and TSHRs. On the other hand, the sister gonadal cDNA that has been previously identified as ccLHR [16], was most similar to the LHRs. A number of elements of the ccFSHR protein sequence were conserved with the members of the GpHR family. There were 13 conserved cysteine residues throughout the protein, which is consistent with the knowledge that a number of the cysteines are important for tertiary folding to facilitate ligand binding. There are seven potential N-linked glycosylation sites in the extracellular domain, and this compares well with the amago salmon GtHR I (putative FSHR, seven) [13] and African catfish GtHR (putative FSHR; six, [14]), but contrasts with the mammalian FSHRs in which there are only three or four [3]. Of the seven sites, one (N195) is strictly conserved in all FSHRs and two (N49, N288) are unique to piscine FSHRs (Fig. 2; [14]).
The gene that presumptively encoded the ccFSHR cDNA lacked the LHR-specific intron (Fig. 4). It was first observed in mammals that among the GpHR genes, the LHR genes are unique in possessing an extra intron (intron 10; [5]). Studies in our laboratory and others have shown that this phenomenon holds true for those lower vertebrates in which the LHR gene have been analyzed to date (turkey, [26]; catfish, [16]; and tilapia, [27]). Even though the absence of the LHR-specific intron is shared by TSHR genes, its sequence identity to the TSHRs was the lowest of the three GpHRs. In addition, the ccFSHR lacked the characteristic TSHR-signature sequences and TSHR-specific extra sequence in the distal part of the extracellular domain [15].
The presence of an intron interrupting the codon for L127 is characteristic of GpHRs because its site exactly corresponds to introns in FSHR, LHR, and TSHR genes, and the homologous genes of nematode [28] and fruit fly [29]. Further, it shows phase-2 splicing, like most introns of the GpHR genes and their invertebrate homologues [5, 29].
The expression of the gene encoding the isolated cDNA was restricted to the gonads as demonstrated in mammals [3] and amago salmon [13]. Even though transcripts were detectable in the spleen, the physiological significance of this low expression is unknown. In mammals, the expression of FSHR gene is restricted to the gonads, whereas the LHR and TSHR genes are also expressed in a variety of extragonadal and extrathyroidal tissues [4–6]. Likewise, ccLHR was also expressed in several extragonadal tissues (including the kidney [16]) and the striped bass TSHR is expressed in many extrathyroidal tissues [15].
To verify whether the cloned cDNA encoded a functional protein and to examine the ligand specificity of the recombinant protein, we transiently expressed the cDNA in a mammalian cell line (COS cells). The results demonstrated that the cDNA indeed encoded a functional protein and the recombinant protein showed a clear binding preference for the hFSH over hCG (Fig. 6), which is consistent with the binding properties of the putative salmon FSHR [10, 13]. However, it should be noted that there was an approximately 2.3-fold increase in luciferase activity in both the COS cells transfected with "empty" vector (pcDNA3.1-Zeo + pCRE-Luc) and those transfected with the FSHR constructs (ccFSHR/pcDNA3.1-Zeo + pCRE-Luc) when incubated with 96 IU FSH/ml (Fig. 6). Although the change in luciferase activity in the cells transfected with the "empty" vector was not statistically significant, one could interpret this trend as an FSH-mediated increase in cellular cAMP independent of the ligand binding abilities of the recombinant protein. This trend is not believed to be real because an examination of the data reveals that the elevated mean at a dose of 96 IU/ml was predominately the result of a single well of the triplicate transfections with empty vector. However, even with this potential "outlyer" removed, there remains an apparent 1.6-fold increase (albeit, statistically insignificant) in the luciferase activity. There was no "trend" evident in the COS cells transfected with empty vector and treated with hCG (Fig. 6).
The activation by the ligand-bound ccFSHR of a reporter gene driven by cyclic AMP response elements demonstrated that the adenylate cyclase/protein kinase A pathway is at least one of the pathways by which the receptor transduces its signal. This pathway was considered to be the predominant one for the three GpHRs (e.g., [30]). However, a reconsideration of this notion has been prompted by the report that monkey and human LHRs generate cAMP and IP3 with similar sensitivities [31]. Whether the ccFSHR acts via additional pathways, particularly the phospholipase C/calcium pathway, needs to be examined.
The expression of recombinant ccFSHR in COS cells resulted in significant constitutive activation of the receptor, as was the case with the ccLHR [16] and amago salmon GtHRs I and II (putative FSHR and LHR, respectively) [12, 13]. In a similar situation, an invertebrate (nematode) GpHR homologue showed a high level of constitutive activity when expressed heterologously [28]. Constitutive activation of GpHRs has been frequently demonstrated before, and in mammals this phenomenon has been attributed to "activating" mutations [3, 5]. Kudo and colleagues [28] proposed that the highly conserved pentaplet (T-K/R-I-A-K) present in the extracellular loop 3 of the GpHRs cause a constrained state that results in the absence of ligand-independent activity, and the absence of these residues likely gives rise to the active conformation causing ligand-independent activation, as in the case of the nematode receptor. However, with the pentaplet fairly conserved in the ccFSHR and ccLHR, this theory warrants reconsideration.
The success in molecular cloning of a cDNA is frequently followed by advances in the understanding of the gene such as cellular expression, temporal expression, pathophysiology of potential mutations, and gene regulation. In the present study, the changes in the ovarian expression of the FSHR gene throughout an annual reproductive cycle of the channel catfish were determined. The elevated levels of the transcripts in September–October, especially in the second year of study, suggest a heightened sensitivity for FSH preceding and during the onset of ovarian recrudescence. The low but constitutive expression of the FSHR throughout ovarian growth suggests an FSH-mediated negative feedback of FSHR expression on the ovarian follicles. These studies can be best explored after the catfish FSH becomes available.
The steady decrease in the transcript levels culminated in the lowest levels just prior to spawning, presumably associated with the attainment of competence of the oocytes to undergo final maturation. This is consistent with the FSH profile in most teleosts, in which there is a decrease in FSH levels at this time [8]. The drop in FSH is followed by a surge in LH that is known to be important for the processes of final oocyte maturation and the associated shift in the steroidogenic pathway [8, 32]. Our previous report of a dramatic increase in the LHR around the time of ovulation in this species fit well with this model [16].
The oocyte growth dynamics of the channel catfish follow the "group synchronous" type, in which a vast majority of the ovarian mass is constituted by a clutch of large oocytes, and the remainder by a heterogeneous population of small oocytes from which future clutches are recruited. The present data represent the overall FSHR gene expression pattern of all the oocyte subpopulations (and associated follicles) combined. Only a qualitative procedure such as in situ hybridization would reveal the potential difference between the subpopulations: clutch and small oocytes.
The ccFSHR cloned in the present study has been positively identified using multiple criteria that included ligand specificity, genomic organization, and temporal gene expression. Characteristics of dual forms of GtHR from two salmonid fish have been reported previously: binding properties and cellular localization of GtHRs I and II in coho salmon [10, 11], and the molecular structure of GtHRs I and II (putative FSHR and LHR, respectively) in amago salmon [12, 13]. The coho salmon GtHR I, unlike the GtHR II, preferentially bound FSH (GtH I) and was localized in the thecal and granulosa cell layers of vitellogenic follicles, and in the thecal layer and interstitial connective tissue, but not the granulosa layer, of preovulatory follicles. Oba and coworkers [12, 13] have cloned amago salmon GtHRs I and II, of which the GtHR I was most activated by FSH and structurally more similar to the ccFSHR. Isolation of a GtHR from another catfish, the African catfish, has been briefly communicated, although its identity was not established due to a discrepancy between its structural features and ligand specificity [14]. There is a very high level of sequence homology (92%) between the African catfish GtHR and ccFSHR, and both genes lacked the LHR-specific intron (Fig. 4; [14]). Thus, it is reasonable to conclude that the reported African catfish GtHR is also an FSHR, not an LHR.
In summary, we have cloned the channel catfish FSHR and successfully produced recombinant receptor that is specifically activated by FSH. The present study has further provided a profile of ccFSHR gene expression with respect to reproductive stages, for the first time for any piscine FSHR. The molecular cloning would facilitate further advances in understanding the molecular physiology of fish FSHR.
ACKNOWLEDGMENTS
This work was initiated with oligonucleotide primers originally designed by Dr. Penny Swanson of the Northwest Fisheries Science Center, National Marine Fisheries Service, Seattle, Washington. We thank Dr. A.F. Parlow (National Hormone and Pituitary Program, Torrance, CA) for providing the human FSH.
FOOTNOTES
First decision: 22 January 2001.
1 This research was supported by the U.S. Department of Agriculture (Enhancing Reproductive Efficiency grant 00-5203-9105) and by a grant from the Wallenburg Foundation to J.M.T. The nucleotide sequence reported in this paper is accessible from GenBank under accession number AF285182. This is contribution 546 from the Center of Marine Biotechnology. This paper is in sequence to a previously published paper (Biol Reprod 2001; 64:1010–1018). 
2 Correspondence: John M. Trant, Center of Marine Biotechnology, University
of Maryland Biotechnology Institute, 701 East Pratt Street, Baltimore,
MD 21202. FAX: 410 234 8896; trant{at}umbi.umd.edu
Accepted: April 11, 2001.
Received: December 18, 2000.
REFERENCES
This article has been cited by other articles:
|
|
 |
|
  D. L. Villeneuve, L. S. Blake, J. D. Brodin, J. E. Cavallin, E. J. Durhan, K. M. Jensen, M. D. Kahl, E. A. Makynen, D. Martinovic, N. D. Mueller, et al. Effects of a 3{beta}-Hydroxysteroid Dehydrogenase Inhibitor, Trilostane, on the Fathead Minnow Reproductive Axis Toxicol. Sci., July 1, 2008; 104(1): 113 - 123. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Sambroni, F. Le Gac, B. Breton, and J.-J. Lareyre Functional specificity of the rainbow trout (Oncorhynchus mykiss) gonadotropin receptors as assayed in a mammalian cell line J. Endocrinol., November 1, 2007; 195(2): 213 - 228. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Zmora, Y. Kazeto, R S. Kumar, R. W Schulz, and J. M Trant Production of recombinant channel catfish (Ictalurus punctatus) FSH and LH in S2 Drosophila cell line and an indication of their different actions J. Endocrinol., August 1, 2007; 194(2): 407 - 416. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Ko, W. Park, D.-J. Kim, M. Kobayashi, and Y. C. Sohn Biological activities of recombinant Manchurian trout FSH and LH: their receptor specificity, steroidogenic and vitellogenic potencies J. Mol. Endocrinol., January 1, 2007; 38(1): 99 - 111. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Freamat, H. Kawauchi, M. Nozaki, and S. A Sower Identification and cloning of a glycoprotein hormone receptor from sea lamprey, Petromyzon marinus. J. Mol. Endocrinol., August 1, 2006; 37(1): 135 - 146. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Campbell, J. Dickey, B. Beckman, G. Young, A. Pierce, H. Fukada, and P. Swanson Previtellogenic Oocyte Growth in Salmon: Relationships among Body Growth, Plasma Insulin-Like Growth Factor-1, Estradiol-17beta, Follicle-Stimulating Hormone and Expression of Ovarian Genes for Insulin-Like Growth Factors, Steroidogenic-Acute Regulatory Protein and Receptors for Gonadotropins, Growth Hormone, and Somatolactin Biol Reprod, July 1, 2006; 75(1): 34 - 44. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y Kazeto and J M Trant Molecular biology of channel catfish brain cytochrome P450 aromatase (CYP19A2): cloning, preovulatory induction of gene expression, hormonal gene regulation and analysis of promoter region J. Mol. Endocrinol., December 1, 2005; 35(3): 571 - 583. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-F. Kwok, W.-K. So, Y. Wang, and W. Ge Zebrafish Gonadotropins and Their Receptors: I. Cloning and Characterization of Zebrafish Follicle-Stimulating Hormone and Luteinizing Hormone Receptors-- Evidence for Their Distinct Functions in Follicle Development Biol Reprod, June 1, 2005; 72(6): 1370 - 1381. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.F. Vischer and J. Bogerd Cloning and Functional Characterization of a Gonadal Luteinizing Hormone Receptor Complementary DNA from the African Catfish (Clarias gariepinus) Biol Reprod, January 1, 2003; 68(1): 262 - 271. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
