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BOR - Papers in Press, published online ahead of print October 14, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.004515
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BIOLOGY OF REPRODUCTION 68, 262–271 (2003)
DOI: 10.1095/biolreprod.102.004515
© 2003 by the Society for the Study of Reproduction, Inc.

Testis

Cloning and Functional Characterization of a Gonadal Luteinizing Hormone Receptor Complementary DNA from the African Catfish (Clarias gariepinus)

H.F. Vischera, and J. Bogerd1,a

a Faculty of Biology, Research Group Endocrinology, Utrecht University, NL-3584 CH Utrecht, The Netherlands


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A cDNA encoding a putative African catfish (Clarias gariepinus) gonadal LH receptor (cfLH-R) has been cloned. Multiple sequence alignment of the deduced amino acid sequence revealed that the cfLH-R had the highest identity with vertebrate LH receptors (>50%). Overall sequence identity between the cfLH-R and the African catfish FSH receptor (cfFSH-R) is 47%. Sequence analysis of part of the cfLH-R gene revealed the presence of an intron typically found in other vertebrate LH-R genes. Abundant cfLH-R mRNA expression was detected in ovary and testis as well as in head-kidney (the adrenal homologue in fish). Other tissues, such as muscle, brain, cerebellum, stomach, heart, and seminal vesicles, also contained detectable cfLH-R mRNA. Transient expression of the cfLH-R in HEK-T 293 cells resulted in significantly increased basal cAMP levels in the absence of gonadotropic hormone. The cAMP levels could be further elevated in response to catfish LH, salmon LH, human LH, human choriogonadotropin, and human FSH. Salmon FSH and human TSH, however, were inactive. We conclude that we have cloned a cDNA encoding the LH-R of the African catfish. This receptor displays constitutive activity but is still responsive to additional ligand-induced activation.

luteinizing hormone, ovary, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vertebrate reproduction is controlled by the brain-pituitary-gonadal axis [1]. The pituitary-derived gonadotropins, FSH and LH, act on the gonads via their respective cell-surface receptors. The receptors for FSH and LH (FSH-R and LH-R) belong to the superfamily of G protein-coupled receptors and constitute, together with the receptors for thyroid-stimulating hormone (TSH-R), the subfamily of glycoprotein hormone receptors (GpHRs). Members of this subfamily are characterized by their large N-terminal extracellular hormone-binding domains, which are thought to determine the specificity of hormone binding to each type of receptor [2].

The biological activities of FSH and LH in the mammalian testis are directed to two cell types by FSH-R and LH-R being expressed by Sertoli and Leydig cells, respectively [3]; there is little (<0.1%) cross-activation of the FSH-R by LH or of the LH-R by FSH [2, 4]. However, this situation seems to be less clear in fish. For two salmonid species, it has been shown that one type of gonadotropin receptor can respond to both LH and FSH [5–8]. A similar observation was made with the recently cloned testicular cDNA encoding the African catfish (Clarias gariepinus) FSH-R (cfFSH-R) [9] that, when transiently expressed, mediated intracellular cAMP production after stimulation with catfish LH (cfLH). Because testicular steroidogenesis can be stimulated with hCG, which is not mediated by the cfFSH-R, it has been suggested that also a LH-R is present in the catfish testis [9].

We report here the cloning and functional characterization of a catfish LH-R cDNA and demonstrate the expression of a LH-R, next to the FSH-R, in the gonads of the African catfish. In addition, pharmacologic studies revealed that both types of catfish gonadotropin receptors can be activated, with an equal potency, by cfLH.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

African catfish were bred and raised in the laboratory as previously described [10] except that catfish pituitary extract instead of hCG was used to induce ovulation. All tissue and sperm samples used in the present study for RNA and genomic DNA isolation were collected from sexually mature (10- to 12-mo-old) catfish as regards spermatogenesis (spermatozoa in many tubuli). Fish raised in captivity do not differ in testis size or histology from wild fish collected from spawning grounds (compare [11] and [12]). However, the captive fish are in a prespawning condition; in natural habitats, the full spawning condition is triggered by environmental stimuli (i.e., rainy season, resulting in flooded meadows used as spawning grounds).

Gonadotropins

Catfish LH (cfLH) was isolated from pituitaries of mature catfish as described earlier [13]. Salmon LH and salmon FSH (sLH and sFSH, respectively) were a kind gift of Dr. B. Breton (INRA, Rennes, France). Human recombinant LH (hrLH), human recombinant FSH (hrFSH), and hCG were kindly provided by Dr. W.G.E.J. Schoonen (Organon, Oss, The Netherlands). Human TSH (hTSH) was obtained from Sigma (St. Louis, MO).

Total RNA, Poly(A)+ RNA, and Genomic DNA Isolation

Total RNA was isolated from various tissues (for each tissue, n = 3 animals) of mature African catfish using the guandinium isothiocyanate method [14] or using RNAzol B (Campro Scientific, Veenendaal, The Netherlands) according to the manufacturer's instructions. Poly(A)+ RNA was isolated using Dynabeads-oligo dT25 (Dynal A.S., Oslo, Norway) according to the manufacturer's instructions. Genomic DNA was isolated from African catfish sperm according to Ausubel et al. [15].

Primers and PCR

The following primers were obtained from Life Technologies (Breda, The Netherlands):

  1. 649, 5'-CGCGAATTCGCNTTYAAYCCNTGYGARGAYAT-3';
  2. 651, 5'-CGCGAATTCRTRNGTDATNGTRTGCCAICGYTC-3';
  3. 687, 5'-GGCTATCTGGTTCATCAACATCC-3';
  4. 688, 5'-GTGTAGATGGACAGCTCTCCCC-3';
  5. 737, 5'-CTCTGCCTGGGCATGGCACTA-3';
  6. 738, 5'-CACGTTGCCAAAGCTGGCCTC-3';
  7. 783, 5'-TTCATACTGTGAACACCAAAGTGC-3';
  8. 818, 5'-AATACAGCTGGGCCATGAGCTCCC-3';
  9. 928, 5'-CATCAGCACAGACTGT-3';
  10. 929, 5'-TTTCGAGGGCAGTGGAGGACACATC-3';
  11. 938, 5'-CGCGAATTCATGAGGACATCACTTTTCATCCTG-3';
  12. 939, 5'-CGCGAATTCATGATCCGCTTTGTTCGTAAATTCG-3';
  13. 940, 5'-CGCCGCTCTAGACTCAGACAAGCAGTCAGAATGTG-3';
  14. 944, 5'-TTCGGATCCATGAAGAAGGCCATGCTGCGCTAC-3';
  15. 1006, 5'-CCCAGCAATATCTTC-GCACGGGTTGAAGGCATCTGGCTCAG-3';
  16. 1007, 5'-TTCAACCCGTGC-GAAGATATTGCTGGGTTTGGGTTTCTCCGCGT-3';
in which N = any deoxynucleotide, Y = C or T, R = G or A, D = G, A or T, and I = deoxyinosine. Primers 938, 939, and 944 contained a consensus translation initiation sequence [16], which is underlined.

Polymerase chain reactions (PCRs) were carried out in 50-µl volumes containing 50 mM KCl, 10 mM Tris/HCl (pH 8.3), 1.5 mM MgCl2, 0.01% gelatin, 200 µM each dNTP, 50 pmol primers, and 100 ng genomic DNA or 1 µl of the testis cDNA sublibraries as template (see below) in a Perkin-Elmer Cetus cycler (Applied Biosystems, Foster City, CA), using 1 U SuperTaq (HT Biotechnologies Ltd., Cambridge, U.K.).

Catfish genomic DNA was used as template for PCR amplification with two degenerate primers (649 and 651) corresponding to highly conserved regions unique for the LH-R subfamily. PCR was initiated with denaturation at 94°C for 5 min. Next, SuperTaq polymerase was added at 75°C, followed by 10 cycles at 94°C for 45 sec, at 37°C for 30 sec, at 72°C for 1 min and then by 25 cycles at 94°C for 45 sec, at 55°C for 30 sec, at 72°C for 1 min. A PCR product of approximately 0.3 kilobases (kb) was amplified and subcloned in pGEM-T (Promega, Madison, WI) for sequence analysis.

In addition, catfish genomic DNA was used as template for PCR amplification with primers 738 and 783, positioned in the putative exons 10 and 11. The generated PCR product (~0.8 kb) was subcloned in pGEM-T (Promega) and sequenced.

Isolation of cDNA Clones

A PCR-based screening method of the random-primed ZAP Express catfish testis cDNA sublibraries [9], using primers 687 and 688 (based on the sequence of the cloned 0.3-kb genomic PCR product), revealed that sublibrary 11 contained a cDNA of interest. Next, the partial cfLH-R PCR product was radiolabeled with [{alpha}-32P]dATP (ICN, Costa Mesa, CA) using a DNA labeling kit (ICN) according to the manufacturer's instructions. Approximately 5 x 104 clones of sublibrary 11 were absorbed to two replica Hybond-N filters (Amersham, Roosendaal, The Netherlands) and hybridized with the radiolabeled probe. The filters were prehybridized, hybridized, washed, and autoradiographed according to Ausubel et al. [15]. Positive clones were picked for rescreening at lower plaque densities and excised in vivo as pBK-CMV phagemids.

SMART RACE cDNA Synthesis and 5'-RACE

African catfish testis, ovary, and seminal vesicle poly(A)+ RNA was reverse transcribed to 5'-RACE-ready cDNA with a cfLH-R-specific primer (928) using the SMART RACE cDNA amplification kit (Clontech, Palo Alto, CA) according to the manufacturer's instructions. Next, 5'-RACE amplification was carried out using the Advantage 2 PCR kit (Clontech) with primer 929 in combination with the universal primer mix (UPM) supplied with the SMART RACE cDNA amplification kit. The PCR was diluted 50-fold in Tricine-EDTA buffer (10 mM Tricine-KOH [pH 8.5], 1 mM EDTA), and a nested PCR was performed using primer 818 and the nested universal primer (NUP), supplied with the SMART RACE cDNA amplification kit. All PCRs were carried out according to the instructions of the SMART RACE cDNA amplification kit (Clontech) in a Perkin-Elmer 2400 cycler (Applied Biosystems). PCR products were cloned into pGEM-T (Promega), and at least two clones derived from each nested RACE reaction were sequenced.

Catfish LH-R Expression Vector Construct

Catfish testis, ovary, and seminal vesicle cDNAs were generated by reverse transcription of total RNA with random hexamers using the SuperScript preamplification system (Life Technologies) according to the manufacturer's instructions. Subsequently, the cfLH-R open-reading frame was PCR amplified using the Advantage-HF PCR kit (Clontech) in combination with primers 938 or 939 and 940 on the different cDNAs. PCR products were cloned into the pcDNA3.1/V5-His expression vector (Invitrogen, Groningen, The Netherlands) and sequenced.

A chimeric receptor (cfFL-R) construct, containing the N-terminal ectodomain of the cfFSH-R and the transmembrane domain of the cfLH-R, was generated using a fusion-PCR method [17]. Briefly, a PCR fragment was generated using primers 944 and 1006 on a cfFSH-R cDNA template [9] and another PCR fragment using primers 940 and 1007 on a cfLH-R cDNA template (this article). Next, the two PCR fragments obtained were fused in a self-primed PCR reaction, cloned in the pcDNA3.1/V5-His expression vector (Invitrogen), and sequenced. The junction between the cfFSH-R and the cfLH-R was located in a highly conserved region near transmembrane {alpha}-helix (TM) 1 [18] and is represented by a slash: N-terminus ... DAFNPC (cfFSH-R)/EDIAGF ... C-terminus (cfLH-R).

DNA Sequence Analysis and Phylogenetic Analysis

DNA sequence analyses were performed on automated ABI PRISM 310 and 377 DNA sequencers (Applied Biosystems) using Dye Terminator cycle sequencing chemistry (Applied Biosystems). Nucleotide and deduced amino acid sequences were analyzed with Lasergene software (DNASTAR Inc., Madison, WI). Multiple sequence alignment was performed by means of the Megalign program of the Lasergene software using the Clustal method (PAM250). The putative signal peptide cleavage site was predicted using SignalP V1.1 software (http://www.cbs.dtu.dk/services/SignalP/) [19].

Transient Expression of the Putative cfLH-R in HEK-T 293 Cells

Human embryonic kidney (HEK-T) 293 cells [20] were maintained under 5% CO2 in culture medium (Dulbecco modified Eagle medium (DMEM) containing 2 mM glutamine, 10% fetal bovine serum, and 1x antibiotic/antimyotic solution, all from Life Technologies). Transient transfections were performed in a 10-cm dish containing approximately 5 x 106 cells, with 10 ng or 1 µg receptor expression vector construct in the presence of 10 µg of a plasmid containing a ß-galactosidase gene under the control of a human vasoactive intestinal peptide promotor containing five cyclic AMP-response elements (pCRE/ß-gal plasmid) [21], using the modified bovine serum transfection method according to the instructions of the manufacturer (Stratagene, La Jolla, CA). "Empty" pcDNA3.1/V5-His vector was used for mock transfections, while 5 µg of cfLH-R expression construct, in the absence of the pCRE/ß-gal plasmid, was transfected for determining the ligand-induced inositol phosphates production (see below).

Colorimetric Detection of Ligand-Induced cAMP Production

The ß-galactosidase activity was measured according to Chen et al. [21] with minor modifications, as described previously [9]. Briefly, 16–18 h after cotransfection of receptor expression vector construct (10 ng or 1 µg) and pCRE/ß-gal plasmid (10 µg), the cells were collected and split into 96-well plates (~2.5 x 105 cells/well). The next day, the transfected cells were stimulated for 6 h with different concentrations of various gonadotropins in 25 µl Hepes-modified DMEM containing 0.1% BSA (Sigma). Ligand-induced changes in absorbance were related to the forskolin-induced changes (10 µM) on each 96-well plate. Therefore, the results are expressed as arbitrary units, related to the forskolin-induced cAMP-mediated reporter gene activation. The agonist concentrations inducing a half-maximal stimulation (EC50) were calculated using the GraphPad PRISM2 software package (San Diego, CA). All experiments were repeated at least three times using cells from independent transfections.

Detection of Ligand-Induced Inositol Phosphates Production

The ligand-induced hydrolysis of [3H]phosphatidylinositol was assayed as described previously [22]. Briefly, 24 h after transfection with cfLH-R expression vector construct (5 µg) only, the HEK-T 293 cells were seeded into 48-well plates (~2.5 x 105 cells/well) in 0.5 ml inositol-free DMEM supplemented with 10% dialyzed fetal calf serum containing 1 µCi/ml [3H]inositol (Amersham, Little Chalfont, England). The next day, the cells were washed and preincubated for 10 min with assay medium (Hepes-modified DMEM containing 20 mM LiCl). After removing the assay medium, the transfected cells were incubated in 200 µl assay medium containing increasing concentrations of hCG (0.01–20 µg/ml) at 37°C for 45 min. The assay medium was aspirated and cellular lipids were extracted from the cells by 10 mM formic acid at 4°C for 90 min. Total inositol phosphates were separated on Dowex (AG 1X8-200) anion-exchange columns and counted with a scintillation counter. As a positive control for the procedure, HEK-T 293 cells, transiently transfected with a catfish gonadotropin-releasing hormone receptor 1 (cfGnRH-R1), were stimulated with chicken GnRH-II [23].

Analysis of cfLH-R mRNA Tissue Distribution by Real-Time Quantitative PCR

The relative cfLH-R mRNA levels in different tissues were determined in a similar way as described previously [9]. Specific primers and fluorogenic probes for the cfLH-R mRNA and the endogenous control RNA (i.e., catfish 28S rRNA) are shown in Table 1.


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  		TABLE 1. Primers and TaqMan fluorogenic probes.a

Statistics

All results are shown as the mean ± SEM. Statistical analyses to identify differences in hormone efficacies (EC50) or constitutive activities were performed using analysis of variance (ANOVA) using Statview 4.5 (Abacus Concepts, Berkeley, CA). The ANOVA was followed by a Fisher probable least-squares difference test to identify significant differences (P < 0.05). A t-test was used to confirm significant hormone-induced signaling over basal.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation and Characterization of the cfLH-R cDNA

Two degenerate oligonucleotide primers (649 and 651) based on conserved amino acid sequences of known vertebrate LH-Rs, flanking TM 1 and TM 3, respectively, were used to PCR amplify part of the cfLH-R gene using catfish genomic DNA as a template. An ~0.3-kb fragment was generated, subcloned, and sequenced. Based on the DNA sequence obtained, two specific primers (687 and 688) were designed in order to isolate a plasmid containing a cfLH-R cDNA from a ZAP Express catfish testis cDNA library. To this end, primers 687 and 688 were used for a PCR-based screening of the catfish testis cDNA sublibraries and to synthesize a probe. In this way, screening of sublibrary 11 yielded 16 independent positive clones. Three individual {lambda} clones were selected and excised in vivo as pBK-CMV phagemids. Sequence analysis showed that the three clones were identical. Computer-assisted Blast searches revealed that they were missing coding sequences at the 5' end.

In order to obtain additional cfLH-R cDNA sequence information at the 5'-end, independent 5'-RACE reactions on different cDNA pools were performed. To this end, 150 ng of poly(A)+ RNA, obtained from catfish testis, seminal vesicles (known to contain Leydig cell-like cells [24]), and ovary, was reverse transcribed using the gene-specific primer 928 in combination with the SMART RACE cDNA amplification kit (Clontech). Subsequently, 5'-RACE was carried out, and the generated PCR products were subcloned in pGEM-T and sequenced. The combination of the pBK-CMV phagemid and the 5'-RACE product sequences yielded a 2447-base pair (bp) cfLH-R cDNA sequence (Fig. 1), consisting of an open-reading frame of 2133 nucleotides that was flanked by a leader sequence of 188 nucleotides and a trailer sequence of 126 nucleotides.



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  		FIG. 1. Nucleotide sequence and deduced amino acid sequence of the cfLH-R cDNA. Numbers on the right refer to position of the nucleotides (top) and the amino acid residues (bottom). Amino acid numbering begins with the proposed initial methionine. The signal peptide is indicated in bold and italics. Putative sites for N-linked glycosylation are indicated by grey boxes. The positions of the proposed hydrophobic transmembrane regions of the receptor are underlined. Potential sites for cAMP-/cGMP-dependent protein kinase and PKC phosphorylation are indicated by a white box and filled diamonds, respectively. Putative palmitoylation sites in the intracellular C-terminal domain are indicated by grey boxes. Conserved cysteine clusters in the N-terminal half of the receptor are indicated by black boxes. An arrowhead indicates the position of the LH-R specific intron. The nucleotide sequence data is available in the GenBank database under the accession number AF324540

Two putative translation initiation codons, starting at nucleotide positions 54 (designated extended cfLH-R) and 189 (designated cfLH-R), respectively, could be identified (Fig. 1). The second ATG was considered to be the major translation initiation codon, taking into account the results of the multiple sequence alignment analysis of the deduced amino acid sequence of the cfLH-R and various other known vertebrate LH-Rs (see below) and because it has a more favorable sequence context for translation initiation (CC[A/G]CCATG[G]) [16]. Moreover, functional characterization of the extended cfLH-R and the cfLH-R revealed no significant differences (see below).

Conceptual translation of the cfLH-R coding region following the proposed initiation codon predicted a polypeptide of 710 amino acids (Fig. 1). A putative signal peptide, consisting of the first 17 amino acids, was predicted by SignalP V1.1 [19]. Hence, the mature cfLH-R protein begins with Leu18 and shows the typical characteristics of members of the LH-Rs of the glycoprotein hormone receptor subfamily of the G protein-coupled receptor family. The large extracellular amino-terminal domain consists of 351 amino acids and is predicted to adopt a horseshoe-shaped configuration [25]. The transmembrane domain of 264 amino acid residues includes seven {alpha}-helices, typical for all members of the G protein-coupled receptor family. The intracellular carboxy-terminal domain consists of 78 amino acids and contains two adjacent cysteine residues that are presumably palmitoylated and thereby anchored in the cell membrane [26].

A computer-assisted Blast 2.0.12 search of the protein database [27] and multiple sequence alignment analysis (Fig. 2) showed that the cfLH-R has the highest overall identity to vertebrate LH-Rs (50–65%), followed by FSH-Rs (45–47%) and TSH-Rs (44–45%). Restricting the multiple sequence alignment analysis to separate receptor domains showed that the percentage of identity for the ectodomain of the cfLH-R was 42–50% to vertebrate LH-Rs and 32–34% to FSH- and TSH-Rs while an identity of 57% to all other glycoprotein hormone receptors was found for the TM region of the cfLH-R.



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  		FIG. 2. Phylogenetic tree of the leucine-rich repeat-containing, G protein-coupled receptors. The deduced amino acid sequences of 49 LGRs from invertebrate, fish, avian, and mammalian species were analyzed using the Clustal method (PAM250) of the Megalign program of the Lasergene software package (DNASTAR). LGRs used: African catfish LH-R (accession number: AF324540), sheep LH-R (L36329), pig LH-R (M29525), bovine LH-R (U20504), human LH-R (M63108), marmoset monkey LH-R (U80673), rat LH-R (M26199), mouse LH-R (M81310), chicken LH-R (AB009283), quail LH-R (S75716), turkey LH-R (U92082), amago salmon LH-R (AB030005), tilapia LH-R (AB041763), channel catfish LH-R (AF285181), bovine TSH-R (U15570), sheep TSH-R (Y13434), dog TSH-R (P14763), human TSH-R (M32215), rat TSH-R (M34842), mouse TSH-R (U02602), amago salmon TSH-Ra (AB030954), amago salmon TSH-Rb (AB030955), striped bass TSH-R (AF239761), sheep FSH-R (L07302), bovine FSH-R (L22319), pig FSH-R (AF025377), horse FSH-R (S70150), donkey FSH-R (U73659), human FSH-R (M95489), macaque FSH-R (X74454), mouse FSH-R (AF095642), rat FSH-R (L02842), newt FSH-R (AB005587), chicken FSH-R (D87871), amago salmon FSH-R (AB030012), tilapia FSH-R (AB041762), African catfish FSH-R (AJ012647), channel catfish FSH-R (AF285182), Drosophila melanogaster GPHR-I (U47005), Anthopleura elegantissima LGR (Z28332), human LGR5 (AF061444), mouse FEX (AF110818), human LGR6 (AF190501), rat LGR4 (AF061443), Drosophila melanogaster GPHR-II (AF142343), Drosophila melanogaster LGR2 (AF274591), human LGR7 (AF190500), Lymnaea stagnalis GRL101 (Z23104), and Caenorhabditis elegans LGR (AF224743).

PCR amplification on genomic DNA of the African catfish between primers 783 and 738 (corresponding to the homologous exon 10 and 11 sequences, respectively, in mammalian LH-R genes) yielded an ~800-bp PCR product. Sequence analysis of the cloned PCR product revealed that the cfLH-R gene contained a single 696-bp intron located between nucleotides 1115 and 1116 in the cfLH-R cDNA sequence (Fig. 1). In addition, the length of the PCR product obtained with primers 687 and 940, amplifying the region between nucleotides 1295 and 2334 (Fig. 1), on genomic DNA of the catfish indicated that no introns were present in the region of the cfLH-R gene coding for the transmembrane domain and intracellular carboxy-terminal domain.

Functional Characterization of the cfLH-R

In order to functionally characterize the cfLH-R, we generated two cfLH-R cDNA expression vector constructs in pcDNA3.1/V5-His: extended cfLH-R (by PCR amplification between primers 938 and 940) and cfLH-R (by PCR amplification between primers 939 and 940). The functionality of the two types of cloned cfLH-Rs was tested by challenging transiently transfected HEK-T 293 cells with various purified and recombinant glycoprotein hormones, followed by the measurement of intracellular cAMP levels using a colorimetric reporter gene assay [21]. No significant differences, with respect to the ligand-stimulated cAMP production as well as basal signaling, were observed between the extended cfLH-R and cfLH-R expression vector constructs (data not shown). The cfLH-R construct was therefore used for further experimentation.

Expression of cfLH-R significantly increased basal cAMP levels compared with mock or cfFSH-R transfected cells (Fig. 3A); transfections with a higher amount of cfLH-R expression vector construct resulted in a more important increase in basal cAMP levels. The ligand-mediated increase in cAMP production by different glycoprotein hormones is shown in Figure 3B. Except for sFSH and hTSH, all ligands were able to significantly increase the intracellular cAMP levels in a dose-dependent manner (1–10 000 ng/ml). Similar to wild-type cfLH-R, transfection of HEK-T 293 cells with the chimeric cfFL-R expression vector construct resulted in significantly elevated basal adenylate cyclase signaling. However, the chimeric cfFL-R showed the same ligand-activation properties as the wild-type cfFSH-R [9] and was responsive to cfLH and hFSH but was not able to respond to hCG (data not shown).



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  		FIG. 3. Cyclic AMP production was measured in HEK-T 293 cells transiently cotransfected with various receptor constructs and a plasmid containing a ß-galactosidase gene under control of a promotor containing five cAMP-response elements (pCRE/ß-gal). A) Basal cAMP production was measured in HEK-T 293 cells transfected with an empty vector (10 ng/transfection) or plasmids encoding the cDNA for cfLH-R (10 and 1000 ng/transfection), cfFSH-R (10 and 1000 ng/transfection), or the chimeric receptor cfFL-R (10 ng/transfection). The basal cAMP production levels, mediated by a 1000-ng cfLH-R per transfection, were (at least) as high as the forskolin (reporter gene assay) control (set to 1). Values sharing the same letter do not differ significantly (P < 0.05; ANOVA, followed by Fisher probable least-squares difference test). B) Effects of different gonadotropins on cAMP production in HEK-T 293 cells transfected with 10 ng cfLH-R. For clarity, the data for fish gonadotropins (top) and human glycoprotein hormones (bottom) are shown in separate graphs and are expressed as the mean ± SEM of triplicate observations from a single representative experiment. Numbers in brackets are the EC50 concentrations and represent an average of at least three independent assays with triplicate observations each and are given as mean ± SEM. Values sharing the same letter do not differ significantly (P < 0.05; ANOVA, followed by Fisher probable least-squares difference test)

We also tested hCG for its capacity to increase the production of intracellular inositol phosphates in HEK-T 293 cells transiently transfected with the cfLH-R expression vector construct. The cfLH-R responded dose dependently to increasing doses of hCG with elevated levels of inositol phosphates (Fig. 4). Maximum stimulation of inositol phosphates formation was still not reached with a dosis of 20 µg/ml hCG, and at least 100-fold higher hCG concentrations are estimated to be required to elicit 50% inositol phosphates production compared with those giving half-maximal stimulation of the cAMP signal transduction pathway. Basal phosphoinositide breakdown in the absence of hCG was not significantly different between cfLH-R and mock-transfected cells.



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  		FIG. 4. Human CG-mediated inositol phosphates (IPx) accumulation in HEK-T 293 cells transfected with 5 µg of the cfLH-R expression vector construct or empty vector. Results are shown as the mean ± SEM of triplicate observations from a single representative experiment

Tissue Distribution of the cfLH-R mRNA

Sensitive, real-time quantitative PCR allowed specific detection of the relative cfLH-R mRNA levels in different tissues and revealed that the cfLH-R transcript is most abundantly expressed in testis, ovary, and head-kidney (Fig. 5). In addition, muscle and brain were positive, while relatively low cfLH-R mRNA levels were detected in seminal vesicles, cerebellum, heart, liver, and stomach.



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  		FIG. 5. Relative cfLH-R mRNA levels in different tissues. Data are expressed as percentage of the cfLH-R mRNA levels (mean ± SEM) in head-kidney, normalized to cf28S rRNA levels, from three individual tissues that were analyzed in duplicate. Te, Testis; Ce, cerebellum; Mu, muscle, St, stomach; In, intestine; HK, head-kidney; SV, seminal vesicles; Li, liver; Ki, kidney. Br, brain without cerebellum; Pit, pituitary; Ov, ovary; He, heart; CA, conus arteriosus (i.e., the ventral aorta, delivering blood to the gills and surrounded by thyroidal follicles)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, the cDNA coding for the cfLH-R was cloned from gonadal tissues of the African catfish. Analysis of the overall deduced amino acid sequence of the cfLH-R revealed that this receptor was highly homologous to vertebrate LH receptors, especially in the amino-terminal ectodomain. The TM domain of the cfLH-R was equally homologous to all GpHRs.

Additional evidence that the cloned receptor cDNA encoded a LH-R was obtained by partial analysis of the cfLH-R gene. A single intron, intervening the regions corresponding to exons 10 and 11 in mammalian LH-Rs, was found, while other glycoprotein hormone receptor (FSH-R and TSH-R) genes, including the cfFSH-R gene [9] lacked such an intron at the homologous position. In tetrapod glycoprotein hormone receptor genes, the region for the TM and intracellular domains is encoded by a single exon, namely by exon 10 in the FSH-R and TSH-R subfamilies and by exon 11 in the LH-R subfamily [28]. Like tetrapod glycoprotein hormone receptor genes, both the cfFSH-R gene and the cfLH-R gene are intronless in this region. However, introns have been observed in the TM coding region of salmon gonadotropin receptor and other related invertebrate leucine-rich repeat-containing G protein-coupled receptor (LGR) genes [7, 8, 29, 30].

The cfLH-R shows the hallmarks of the GpHR subfamily: a relatively large amino-terminal extracellular domain (351 amino acid residues) connected to a rhodopsin-like seven TM module. The N-terminal ectodomain of the cfLH-R contains nine putative leucine-rich repeats (LRRs) encompassing amino acid residues 49–265 and is predicted to be arranged in a horseshoe-shaped configuration, shown to be involved in hormone binding [25, 31]. The concave amphipathic inner surface of this domain, formed by parallel ß strands, interacts with the large glycoprotein hormone ligands [32–34]. Particular ionizable amino acid residues on this surface that have been identified to be involved in ligand binding [31, 35] are conserved in the cfLH-R or substituted by amino acids with a similar chemical or physical character (e.g., D152, N178, and N204 in the cfLH-R are E132, K158, and E184, respectively, in the rat LH-R). The LRR domain of the cfLH-R is flanked by conserved N- and C-terminal cysteine clusters (Cys residues on positions 22, 26, 28, and 35 and on positions 277, 278, 348, and 358), which are thought to be important for ligand binding and receptor expression at the cell surface, respectively [36]. In addition, the latter four Cys residues may position the ectodomain in relation to the transmembrane domain by forming disulfide bridges with Cys residues in the first and second extracellular loops (Cys444 and Cys519, respectively [31]). Cys358 is part of a 10 amino acid sequence (residues 355–364, FNPCEDIAGF), located just upstream of TM 1, that is important for receptor cell-surface expression as well as for ligand-mediated signaling and is almost invariably recognized in all members of the glycoprotein hormone receptor family (consensus sequence: FNPCEDIMGY). Residues P357, E359, and D360 of the cfLH-R have been shown to be essential for signaling and are conserved in all GpHRs, while the less conserved A362 and F364 appear to have no significant function in receptor expression or signal transduction [18]. In the rat LH-R, Cys109 and Cys134 are important residues for receptor cell-surface expression as well as for hormone binding [36]. These cysteine residues, conserved in the LH-Rs, are localized in two adjacent ß strands and are predicted to form a disulfide bridge [31]. The amino acid residue on the similar position as Cys109 in the rat LH-R, however, is not conserved in the cfLH-R (i.e., Ser129), suggesting that a disulfide bond within this LRR is not strictly necessary to maintain the horseshoe-shaped conformation. In mammalian LH-Rs, six potential N-linked glycosylation sites are present in the N-terminal ectodomain [26], while only three of these sites were recognized in the cfLH-R: Asn97 and Asn193 are conserved in all members of the glycoprotein hormone receptor family, while Asn296 is conserved in mammalian LH-Rs.

The TM domain includes seven {alpha}-helices, connected by three extracellular and three intracellular loops, and consists of 264 amino acid residues. In addition, amino acid residues totally conserved in this receptor family were found in this region in the cfLH-R, e.g., Arg469 located just downstream of TM 3 (in the so-called ERW-motif), which is believed to be involved in signal transduction, as well as two cysteine residues (Cys444 and Cys519) located in extracellular loops I and II, which are believed to connect both loops via a disulfide bridge. The intracellular carboxy-terminal domain consists of 78 amino acids and contains two adjacent cysteine residues that are presumed to be palmitoylated, fixing this domain in the cell membrane [26]. Two potential phosphorylation sites [37] were recognized in the carboxy-terminal domain of the cfLH-R: Thr639, a potential site for phosphorylation by cAMP- and cGMP-dependent protein kinase, and Thr666, presumably phosphorylated by protein kinase C. In addition, intracellular loop 2 contained a potential cAMP- and cGMP-dependent protein kinase phosphorylation site: T484.

The cfLH-R was found to increase cAMP production in transfected HEK-T 293 cells in the absence of hormones. This was also observed after transient transfection of COS-7 cells with the cfLH-R (data not shown). Constitutive activity in the GpHR-family has also been observed in the wild-type channel catfish LH-R and FSH-R [38, 39], the nematode LGR [30], and multiple mutant human TSH-Rs [40], human FSH-Rs, and human LH-Rs [1]. Ligand-independent activation of GpHRs is almost exclusively associated with mutations in the intracellular halves of the transmembrane helices or in the third intracellular loop [1]. The chimeric receptor cfFL-R, containing the N-terminal ectodomain of the cfFSH-R and the transmembrane domain of the cfLH-R, was also constitutively active but behaved like the cfFSH-R with respect to ligand selectivity. This indicates that the property causing the constitutive activity is located in the transmembrane domain of the cfLH-R. However, all identified activational sensitive amino acid residues of mammalian GpHRs [1] were conserved in the cfLH-R. Five highly conserved amino acids (T-K/R-I-A-K) located in the C-terminal region of the third intracellular loop were identified as being important to stabilize the inactive receptor conformation, resulting in the absence of ligand-independent signaling [30, 41]. This five-amino acid sequence was conserved in the cfLH-R except that the Thr residue is substituted by an Ala residue. An Ala residue at this position is also present in the constitutively active channel catfish FSH-R [39] and also in the FSH-R of the African catfish. Because the latter is not constitutively active, the potency of this Ala residue in disrupting the constrained state of the cfLH-R is questionable.

Fish LHs (cfLH and sLH) and human gonadotropins (hrFSH, hrLH, and hCG) were all able to stimulate cfLH-R-mediated intracellular cAMP signaling with EC50 values varying between ~45–565 ng/ml. From all ligands tested, catfish LH (cfLH) had the highest potency (EC50 = 47 ± 19 ng/ml) in activating the cfLH-R. Apparently the cfLH-R is able to discriminate between fish FSHs and LHs since sFSH was inactive, but the receptor is unable to distinguish between the human gonadotropins. However, the cfLH-R was able to distinguish between hTSH and the human gonadotropins. A different situation was encountered with the cfFSH-R [42]: hrFSH and the fish gonadotropins (cfLH, sLH, and sFSH) were able to activate the receptor, whereas hTSH and hCG were inactive. Definitive conclusions regarding hormone-binding specificity of both cfLH-R and cfFSH-R await the availability of native or recombinant African catfish FSH and TSH for experimentation, as phylogenetically related gonadotropins can exert unpredictable heterologous receptor specificity [43, 44]. Nevertheless, the present data suggest that the cfLH-R has a better ability to discriminate between fish gonadotropins (being activated by cfLH and sLH but not by sFSH) than the cfFSH-R (being activated by cfLH, sLH, and sFSH). Binding studies on membranes obtained from coho salmon ovaria showed the same tendency [5, 6]: the salmon gonadotropin receptor (GTH-R) type I (i.e., the putative FSH-R) showed affinity to both salmon LH and FSH, whereas the salmon GTH-R type II (i.e., the putative LH-R) only bound salmon LH. Reduced ligand specificity was also observed with transiently expressed amago salmon gonadotropin receptors (FSH-R and LH-R), which were both activated by sFSH as well as by sLH, although the receptors appeared to prefer their respective homologous ligands [7, 8].

Stimulation of cfLH-R expressing HEK-T 293 cells with hCG concentrations higher than 3 µg/ml leads to significant increases in inositol phosphate accumulation. Coupling to both the adenylyl cyclase and phospholipase C second messenger systems was also observed for some other GpHRs, e.g., the rat [45] and human LH-R [46]. While cAMP responses were elicited by relatively low physiologic levels of hCG, high levels of hCG were required to activate phosphoinositide hydrolysis. Therefore, coupling of the LH-R to the phospholipase C pathway may only be of importance during the preovulatory period and pregnancy in humans, when circulating levels of LH and hCG are elevated. However, the importance of dual coupling in males, in the absence of LH surges or high CG levels, is generally questioned [47]. The duality in signaling may also be relevant in female African catfish because relatively high plasma cfLH levels (comparable with ~100 ng hCG/ml) were required to induce oocyte maturation and ovulation in the African catfish [48].

Catfish LH-R mRNA was abundantly expressed in tissues where expression was expected (i.e., testis and ovary). Surprisingly, however, high cfLH-R mRNA levels were also detected in the head-kidney, which includes the steroidogenic interrenal tissue, the homologue to the adrenals in amniotes [49]. Corticotropic effects of gonadotropins have been reported in teleosts [50]. The observed expression of cfLH-R mRNA in seminal vesicles (a structure developing from the caudal part of the testis anlage and hence not homologous to mammalian seminal vesicles) may seem surprising. However, catfish seminal vesicles contain interstitial cells, which are homologous to Leydig cells [24]. In addition, cfLH-R mRNA was also detected in muscle, stomach, heart, liver, cerebellum, and brain. Expression of LH-R mRNA in nongonadal tissues of fish as well as in mammals has also been reported by other groups [7, 38, 51–54].

In mammals, testis function is regulated by two distinct gonadotropins (FSH and LH) via their specific receptors; there is no significant (<0.1%) cross-activation of the FSH-R by LH or of the LH-R by FSH [2, 4]. LH acts on the Leydig cells and stimulates androgen production, while FSH increases the efficiency of spermatogenesis by stimulating Sertoli cell functions. In contrast with the situation in mammals, a less apparent distinction between the functions of gonadotropins and their receptors is found in teleost fish. While spatiotemporal expression data are not yet available for fish gonadotropin receptors, pharmacologic analysis on transiently expressed receptors revealed that some of these receptors are promiscuous with respect to ligand selectivity [7–9]. The cfFSH-R can be activated by fish LH as well as fish FSH [42] but has a clear preference for mammalian FSH over mammalian LH [9]. On the contrary, the catfish LH-R can be stimulated by all mammalian gonadotropins but has a clear preference for fish LH over fish FSH (this study). It therefore seems reasonable to assume that stimulation of testicular steroidogenesis in African catfish can be achieved via the cfLH-R. The duality of gonadotropins is likely but not yet clearly established in the African catfish. The cDNA for the cfFSH-ß subunit has been cloned recently (unpublished results), but expression and release of the FSH protein still remains to be shown. A potential role for cfFSH in regulating testicular function may be redundant because cfLH is able to stimulate both the cfFSH-R and the cfLH-R with the same potency.

In conclusion, we cloned a LH-R cDNA from African catfish. Cloning of both gonadotropin receptors permits investigations into their specific roles in the regulation of gonadal functions. However, with a promiscuous cfFSH-R and a constitutively active cfLH-R, their spatiotemporal expression patterns as well as ligand availability require further research. Because both cfFSH-R and cfLH-R respond to cfLH with nearly similar EC50's, it will also be interesting to elucidate the molecular basis for the limited specificity of the interaction of cfLH with both catfish gonadotropin receptors. This setting renders African catfish an interesting model for studies on the gonadotropic regulation of gonadal functions.


   ACKNOWLEDGMENTS
 
Dr. B. Breton and Dr. W.G.E.J. Schoonen are gratefully acknowledged for providing salmon and human gonadotropin hormones, respectively. The authors thank Dr. R.W. Schulz (Utrecht University) for critically reading the manuscript.


   FOOTNOTES
 
1 Correspondence: J. Bogerd, Faculty of Biology, Research Group Endocrinology, Utrecht University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands. FAX: 31 30 253 2837; j.bogerd{at}bio.uu.nl Back

Received: 12 February 2002.

First decision: 12 March 2002.

Accepted: 3 July 2002.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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H.F. Vischer, A.C.C. Teves, J.C.M. Ackermans, W. van Dijk, R.W. Schulz, and J. Bogerd
Cloning and Spatiotemporal Expression of the Follicle-Stimulating Hormone {beta} Subunit Complementary DNA in the African Catfish (Clarias gariepinus)
Biol Reprod, April 1, 2003; 68(4): 1324 - 1332.
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biolreprod.102.004515v1
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