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Male Reproductive Tract |
Department of Obstetrics and Gynecology,3 Baylor College of Medicine, Houston, Texas 77030
Department of Animal Science,4 Swiss Federal Institute of Technology and Faculty of Veterinary Medicine, University of Zürich, CH-8092 Zürich, Switzerland
Institute of Animal Genetics, Nutrition, and Housing,5 University of Berne, CH-3012 Berne, Switzerland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, gene regulation, male reproductive tract, relaxin, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Genetic targeting of the Insl3 gene in mice revealed its involvement in testicular descent during development [6, 7]. In mutant males deficient for Insl3, gubernaculum ligament failed to differentiate, resulting in a high intra-abdominal cryptorchidism. Transgenic overexpression of Insl3 in female mice causes a male-like differentiation of gubernaculum and descent of the ovaries into inguinal region [8]. Thus, INSL3 controls the first intra-abdominal stage of testicular descent in androgen-independent fashion. It is shown that steroidogenic factor-1 (SF-1) mediates the Insl3 expression in vitro by direct binding to its cognate sites within the Insl3 promoter [9, 10].
Mutations of the INSL3 gene were found also in human cryptorchid patients [11–15]. We established that some of these mutations render peptides unable to stimulate the receptor for INSL3 in vitro (unpublished data). We have isolated a new gene called Great (G protein-protein-coupled receptor affecting testicular descent) [16]. Deficiency of Great in mice caused the same high intra-abdominal cryptorchidism as in Insl3 knockout mutants [17]. Based on the identical phenotypes of the Great and Insl3-deficient mice, we predicted that the Great receptor might be in fact a cognate receptor for Insl3 [16]. This was confirmed later through demonstration of the receptor activation by synthetic INSL3 peptide in vitro [18]. Mutation analysis of human cryptorchid patients revealed a nonfunctional mutant allele of the GREAT gene [17].
Cryptorchidism is one of the most frequent congenital abnormalities in dogs. In some of the purebred dogs, a frequency of the disease was reported as high as 15% [19]. In many cases this abnormality is more prevalent in particular families/breeds, indicating a strong hereditary component in the development of this abnormality. As the first step in analysis of the genetic basis of cryptorchidism in dogs, we describe here an isolation and expression analysis of the canine INSL3 gene. Contrary to the recent report [20], we have established that canine INSL3 gene is well conserved and encodes nondeleted peptide with a typical receptor-binding motif. We have isolated promoter of the canine INSL3 gene and demonstrated a strong upregulation of the promoter by SF-1 transcription factor.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult canine testicular and ovarian tissue samples were collected from dogs killed for reasons unrelated to the current work. All dogs used in this project were cross-bred hound dogs. An anatomical examination during surgery did not reveal any abnormalities in the reproductive organs. Tissues were frozen at -80°C until the DNA/RNA isolation. Total RNA from the canine tissues was extracted with the TRIzol reagent (Life Technologies, Rockville, MD). Genomic DNA was extracted from the tissue samples using conventional phenol-chloroform extraction protocol. Additional DNA samples have been prepared from the buccal swabs using a genomic DNA purification kit (Gentra Systems, Minneapolis, MN). Southern blot analysis of the genomic DNA was performed using conventional techniques. The P32-random labeled DNA fragments (Stratagene, La Jolla, CA) corresponding to the first exon and the 150-base pair (bp) promoter region of canine INSL3 gene were used as the hybridization probes. Hybridization was performed at 65°C overnight in the PerfectHyb Plus hybridization buffer (Sigma, St. Louis, MO). Blots were washed under highly stringent conditions at 68°C three times for 30 min with 0.1 x saline sodium citrate: 0.1% SDS and exposed to X-Omat film (Kodak, Rochester, NY).
Reverse Transcription-Polymerase Chain Reaction and Northern Blot Analysis of the Gene Expression
First-strand cDNA was synthesized using the oligo(dT) primer and RETROscript kit (Ambion, Austin, TX). The following primers have been used for reverse transcription-polymerase chain reaction (RT-PCR) of the canine INSL3 gene: Insl3-dog1F, 5'-GTGTGGCCACCACTTCGT-3'; Insl3-dog2R, 5'-CAGTAGTGTGCGGGATTGGT-3', and Insl3-dogORF/R, 5'-TCAGTGAGGACAGAGGGTCAGC-3'. Cycling conditions were: denaturation at 94°C for 1 min, followed by 35 cycles of 94°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec, with a final extension at 72°C for 5 min. Northern blots were prepared from 10 µg of total RNA isolated from canine testes and ovary using Hybond-N+ membrane (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer protocol. Canine INSL3 cDNA was labeled by random priming and hybridized to the Northern blot as described above.
Sequencing
INSL3 cDNA fragments obtained by RT-PCR and genomic PCR fragments were separated on an agarose gel and purified using Ultrafree-DA spin columns (Millipore, Bedford, MA). Both strands of the DNA fragment were directly sequenced by the dye terminator method on an automated 373 DNA sequencing machine using the same primers as for PCR. Plasmid DNA containing a 3.5 kb genomic fragment (see below) with the first exon of INSL3 was isolated using Wizard mini-prep (Promega, Madison, WI), and sequenced using T7, T3, and INSL3 specific primers. Sequences of the canine INSL3 cDNA and promoter region have been deposited into GenBank under accession numbers AY251013, AY251014, and AY251015.
Canine BAC Library Screening
A dog bacterial artificial chromosome (BAC) library was constructed based on the pBeloBAC11 (EMBL U51113) vector and arrayed for PCR-based screening [21]. Details of the library construction and screening approach are available on www.dogmap.ch. Primer pair from the second exon of the canine INSL3 was used to screen the library: Insl3-dogex2F2, 5'-ATCTCCATGGGCAGGTGTC-3', and Insl3-dog2R. After rescreening, one of the isolated clones (S088P10F12) was shown to contain the entire INSL3 gene. The 3.5-kb Xma I BAC fragment containing the first exon of canine INSL3 gene was identified after Southern blot hybridization of the digested BAC DNA with a probe corresponding to the part of the first exon. This fragment was subcloned into the Xma I site of Bluescript KSII plasmid vector (Stratagene)
Analysis of the INSL3 Promoter
Canine INSL3 genomic fragment of 150 bp upstream of the first ATG codon of the open reading frame (ORF) was obtained by PCR using primers dogINSL3promF, 5'-CCCGCTAGCGGGTTTACACTACACTTGCA-3', and dogINSL3promR, 5'-CCCCTCGAGTCATGGTGGCAGGAGGC-3' with the additional Nhe I and Xho I sites on the 5' ends of the primers. The resultant PCR fragment was subcloned into pGEM-T vector, sequenced, and recloned in front of the luciferase reporter gene into the Nhe I and Xho I sites of the pGL3-Basic vector (Promega). All plasmids were purified using the High Purity Midi-prep plasmid preparation kit (Marligen Biosciences, Ijamsville, MD). Sequence of the construct (pGL3-cINSL3) was verified by double-strand sequencing using vector-derived primers. To produce a site-specific mutation of the SF-1 binding site within the INSL3 promoter in pGEM-T vector we used the QuikChange site-directed mutagenesis kit from Stratagene and two primers: 5'-TTCCATCCCCATCC-GAATTCCTCAGCTGGG-3', and 5'- CCCAGCTGAGGAATTCGGATG-GGGATGGAA-3'. Resultant clones were verified by a restriction digest with EcoRI and sequencing, and then the mutated fragments were recloned into pGL3-Basic vector (pGL3-Mut/cINSL3). Plasmid containing cDNA of the human SF-1 in pCEP4 vector was a kind gift from Dr. D. Moore (Baylor College of Medicine, Houston, TX). The KpnI/BamHI SF-1 cDNA fragment was isolated from pCEP4-SF1 and subcloned into the KpnI/BamHI sites of pCR3.1 (pCR3.1-SF1) vector (Invitrogen, San Diego, CA). The 293T cells derived from human embryonic kidney (HEK) fibroblast were maintained in Dulbecco's minimal essential medium supplemented with 10% fetal bovine serum, 1 mM glutamine, and antibiotic/antimycotic mixture (all from Life Technologies). The cells were cotransfected at 80% of confluency on 24-well plates with 100 ng/well of pGL3-cINSL3, pGL3-Mut/cINSL3, or pGL3-Basic plasmid, with either pCR3.1 vector or pCR3.1-SF1 in OptiMEM without antibiotic using FuGENE 6 transfection reagent (Roche, Indianapolis, IN). To assess the efficiency of transfection, beta-galactosidase reporter plasmid was used for cotransfection. After 24 h, cells were washed and treated with the luciferase extraction buffer (Promega). Luciferase and galactosidase activity was measured with the luciferase and galactosidase reagent kits (Promega), respectively. Transfections were performed in triplicate, and experiments were repeated three times.
Activation of the GREAT receptor
The expression constructs of two splice variants of canine INSL3 gene were produced by subcloning of the corresponding cDNA fragments into pCR3.1 vector. The INSL3 cDNAs were obtained by RT-PCR with primers Insl3-dogORF/F, 5'-CCACCATGAGCCCCCGCCC-3', and Insl3-dogORF/R. The cINSL3/fl expression construct contained full-length canine INSL3 cDNA and cINSL3/sv contained truncated splice variant of INSL3. Recombinant INSL3 peptides were obtained by transfecting pancreatic HIT cells grown in T-25 flask with approximately 5 µg of the expression constructs. One week after transfection, conditioned media were harvested and used for the receptor activation in cAMP assay. Medium from cells transfected with pCR3.1 vector DNA was used as a control. An efficiency of transfection was assessed by analysis of secreted alkaline phosphatase activity in the media (pAPtag-5 vector from GenHunter, Nashville, TN, was used for cotransfection). The amount of the recombinant product was estimated in relative units according to the alkaline phosphatase activity in the medium. Activation of the GREAT receptor was assayed as described previously [17]. The 293T cells grown in 24 wells were transfected with approximately 0.5 µg/well of the human GREAT construct. One day after transfection, the cells were treated with the conditioned medium containing recombinant peptides, in the presence of 250 µM 3-isobutyl-1-methylxanthine for 30 min. Cells were harvested, washed, and lysed with cAMP extraction buffer (Amersham Pharmacia Biotech). The cAMP level was detected using Amersham enzyme immunoassay system. The cAMP concentrations in each well were measured in duplicate. All experiments were repeated three times using cells from independent transfections.
Phylogenetic Analysis
The alignment of amino acid sequences for genes was done using the ClustalW Service at the European Bioinformatics Institute (http://www2.ebi.ac.uk/clustalw); the final alignment was optimized by hand to maximize homology. The full-length amino acid sequences of different INSL3s have been retrieved from the GenBank.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Northern blot analysis was performed on the RNA isolated from the adult canine ovary and the testis. Equal amounts of the total RNA were loaded on a gel and hybridized with the isolated canine cDNA fragment (Fig. 1B). A single band of 1 kb was detected in both samples. Expression of INSL3 was much stronger in the testis than in the ovary. To determine the missing 5' end of the first exon as well as the promoter sequence of INSL3, we isolated a genomic fragment containing the INSL3 gene. Canine BAC library was screened by PCR with primers derived from the second exon of INSL3. After rescreening, one BAC clone (S088P10F12) was chosen for further analysis. Genomic Xma I fragment of 3.5 kb, containing the first exon of INSL3, was isolated from the BAC DNA and partially sequenced, providing sequence of the missing 5' part of the first exon and the promoter region.
The promoter sequence and combined cDNA sequences of the major transcript and the minor truncated variant of canine INSL3 are shown in Figure 2. In silico translation of the major 399-bp splice variant produced the full-length 132 amino acids canine INSL3 peptide. The amino acid sequence of the peptide was well conserved and, by homology to the other relaxin/insulin-like factors [1, 2], contained 28 amino acid signal peptide, 39 amino acid B-chain, 39 amino acid C-peptide, and 26 amino acid A-chain. Sequencing of the minor splice variant revealed presence of an internal deletion. Comparison of the two cDNAs and the genomic sequence revealed that the smaller cDNA was produced through an alternative splicing utilizing the site within the first exon. The minor transcript potentially encoded truncated 105 amino acid peptide with a deletion of C-terminus of the putative B-chain. Because of a frame shift at the splice site, the C-terminus of the peptide was completely unrelated to the INSL3 peptides. Comparison of the resulted nucleotide sequence with the previously reported canine INSL3 sequence showed several mismatches at the 5' end of the gene and a presence of two Gs instead of 3Gs at positions 215-216 of the ORF at the beginning of the second exon.
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To assess functional properties of the two splice variants, we analyzed their ability to activate the GREAT receptor. Expression constructs corresponding to two splice variants were transiently transfected into human pancreatic cell line HIT. Both constructs produced RNA transcripts of the correct size as was determined by RT-PCR (data not shown). After 1 wk conditioned media from these cells containing recombinant INSL3 peptides were used to stimulate 293T cells expressing the human GREAT receptor. As shown in Fig. 3, the recombinant INSL3, which derived from the full-length INSL3 expression construct, induced a dose-dependent stimulation of the receptor (Fig. 3). The medium from cells transfected with a construct corresponding to the truncated cDNA did not cause any receptor activation. Thus, the minor transcript was nonfunctional in this assay.
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Next we determined an exon/intron structure of the gene. The genomic DNA from four unrelated animals have been used in this analysis. Amplification of the canine genomic DNA with primers designed from the first and the second exons produced a 1.2-kb band in all four samples (two samples are shown in Fig. 4A). Sequencing of this fragment demonstrated that canine INSL3 gene is composed of the two exons separated by an intron of about 950 bp. Surprisingly, in one DNA sample (Fig. 4A, lane 2), we have detected an additional 180 bp band. This fragment was reproducibly amplified from several DNA samples independently prepared from the same animal. The 180-bp DNA fragment was isolated from the gel and directly sequenced. The nucleotide sequence corresponded to the sequence of the minor truncated splice variant of the INSL3 transcript, indicating that an additional genomic copy of INSL3 was originated through retroposition of the cDNA into genomic DNA. The retrogene did not contain the INSL3 promoter. Multiple attempts to PCR a fragment with primers based on the promoter sequence and sequence of the second exon failed to amplify a fragment of the expected size (data not shown). To verify this finding, we prepared a Southern blot of the EcoRI-digested genomic DNA from animals with and without retroposon (Fig. 4B). Hybridization with a probe corresponding to the first exon revealed a presence of the two positive bands (8.5 kb and 18 kb) in the DNA isolated from the dog with retroposon and only one band (8.5 kb) in the DNA from the retroposon-negative animal. Hybridization with the probe corresponding to the 150-bp promoter region adjacent to the first exon revealed only one band in both DNA samples. An EcoRI fragment of the same size (8.5 kb) was detected in the INSL3-containing BAC (data not shown). Thus, the additional genomic copy of INSL3 does not contain promoter sequence of the gene.
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A single base pair difference was found between the INSL3 cDNA and the retrogene. The nucleotide substitution created an additional AatII site in the latter sequence (Fig. 4C). Digestion of the PCR fragment corresponding to the pseudogene with AatII indeed resulted in two bands of the expected 57 bp and 123 bp size (Fig. 4C). The presence of a restriction site polymorphism between two copies of the gene allowed us to analyze expression of the retrogene. The INSL3 RT-PCR products, obtained from the testis RNA, isolated from the dog with a retrogene, have been digested overnight with AatII. If the retrogene was expressed, the restriction of the INSL3 cDNA would lead to the digest of the lower band. However, analysis of the digested DNA did not reveal presence of the INSL3 retrogene transcripts in the RNA pool (Fig. 4C). Thus, the INSL3 retrogene was not expressed in testis.
Previously it has been shown that mouse Insl3 promoter is regulated by SF-1 [9, 10]. We analyzed the effect of the SF-1 on the canine INSL3 promoter. A genomic fragment of 150 bp upstream of the first ATG codon in the ORF was subcloned into pGL3 luciferase reporter vector, and this construct was transfected into HEK 293T cells. No promoter activity was observed 24 h after transfection (Fig. 5A). To examine effect of SF-1 on INSL3 promoter activity, we performed transient cotransfection of the canine INSL3 promoter construct and the human SF-1 expression vector. As shown in Figure 4A, presence of SF-1 had a dramatic effect on the canine INSL3 promoter, with almost 40-fold activation in comparison with the baseline activity.
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Comparison of the canine promoter sequence to four other available sequences of INSL3 promoters from other mammalian species revealed a putative conserved SF-1-binding site (Fig. 5B). We produced a mutant variant of the canine promoter with site-specific substitution of two nucleotides within the identified putative SF-1 binding site. Results of cotransfection of the mutant canine INSL3 promoter construct with the SF-1 expression vector demonstrated complete loss of the promoter response to SF-1 (Fig. 5A). Thus, a single site primarily mediates the SF-1 activation within canine INSL3 promoter.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The genomic structure of the canine INSL3 gene is similar to the one established for the human, mouse, and rat genes. It is composed of two exons separated by an intron of about 1 kb. Analysis of the INSL3 expression in testis and ovary by Northern blot hybridization shows a presence of a single transcript. The INSL3 expression level is much higher in the canine testis than in the ovary, similar to what was reported previously for human [23] and mouse [24].
Notably, the RT-PCR analysis also reveals the presence of a minor truncated transcript as a result of alternative splicing event within the first exon of INSL3. The truncated transcript of the mouse Insl3 gene was reported previously, with an alternative splice site also located in the first exon [25]. Comparison of the mouse and canine transcripts shows that in mouse alternative splicing occurs in a more proximal position to the one identified in the canine INSL3 [25]. Mouse splice variant encodes a truncated INSL3 whereby the beginning of the B-domain region is linked to a downstream region of the C-domain. However, in the canine INSL3, because of a frame shift at the alternative splice site, a second half of the peptide is not related to INSL3. An expression of the truncated transcript is detectable only at the RT-PCR level but not in the Northern blot analysis. The INSL3 peptides belong to the insulin/relaxin superfamily [1, 2]. All peptides of this group are produced as preprohormones consisting of a signal peptide, a B-chain, a connecting C-peptide, and an A-chain [26]. A mature hormone has a conserved heterodimeric structure held together by two interchain disulfide bonds with another intrachain bridge located in the A-chain. Alignment of the INSL3 peptides isolated from several mammalian species is shown in Figure 6. Overall, canine INSL3 is 78% identical to the pig INSL3, 75% to the mouse Insl3, and 71% to the human INSL3. It is important to note that putative INSL3 receptor-binding domain (GGPRW), identified previously in human INSL3 peptide [22], is highly conserved in all species, including dogs (Fig. 6, underlined sequence).
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We have shown that recombinant canine INSL3 peptide corresponding to the full-length transcript is functionally active and capable to stimulate human GREAT receptor. Alternatively, medium from the cells, transfected with the expression construct corresponding to the truncated transcript, failed to stimulate GREAT. Because in the experiments described in this study, we did not monitor protein translation, its stability, or extracellular secretion of the produced peptide, we cannot identify the exact reason for such a failure. It is also possible that the truncated transcript discovered in the testis RNA cannot produce a peptide in the pancreatic cells used in our experiments. Nevertheless, the fact that the alternative splice site renders a frame-shift mutation, truncation of the INSL3 peptide, deletion of the receptor-binding domain, and overall unstable secondary structure indicates that the alternative transcript is not functional. Taking into account its low level of expression detectable only at the RT-PCR level, a significance of this splice variant is presently unclear.
Surprisingly, in one of the animals, we detected an additional genomic copy of INSL3. Sequencing data indicate that this copy of the gene originated through retroposition of the minor truncated cDNA into the canine genome. The retrogene does not contain the INSL3 promoter sequence. Using a single bp difference between the INSL3 gene and its retrocopy, we have shown that the latter one is not expressed at the RNA level. To date no pseudogenes of INSL3 have been described in other species. It remains to be elucidated when such retroposition occurred in canine evolutionary lineage and whether this pseudogene is breed specific. Previously it has been shown that SF-1 orphan nuclear receptor mediates expression of the mouse Insl3 gene in vitro [9, 10]. It is interesting to note that the expression of Sf-1 during male development coincides with expression of Insl3 and testicular descent. During mouse embryogenesis, SF-1 is expressed in the developing urogenital system in both sexes until about day 9 after mating [27]. From day 12.5, expression in male gonads persists in both Sertoli and Leidig cells, but its expression is downregulated in females from day 13.5 [27]. Right at this time a first, transabdominal stage of testicular descent begins in male mice [28] and the Insl3 transcripts are detected in the embryonic testis [4]. The results presented here demonstrate that the canine INSL3 promoter is robustly activated by SF-1 in vitro. In mouse three functional SF-1-binding sites have been identified within 188-bp Insl3 promoter. The most distal SF-1-binding site shows the highest affinity toward SF-1 [10] and is close to a putative consensus sequence of the binding element for SF-1, PyCAAGGPyPyPur [29]. Comparison of the mouse promoter sequence to that from the human, rat, pig, and dog INSL3 genes shows that this most distal site is the only one conserved in all five species (Fig. 5B). We have shown that mutation of this conserved domain drastically reduces response to the SF-1 activation in canine INSL3 promoter. In summary, we have isolated and characterized canine INSL3 gene. INSL3 and its receptor GREAT/LGR8 are responsible for descent of the testes during mammalian development. Cloning of canine INSL3 gene and identification of its promoter provides an opportunity to study its role in etiology of cryptorchidism, one of the most frequent congenital abnormalities in dogs.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Alexander Agoulnik, Department of Obstetrics and Gynecology, Baylor College of Medicine, 6550 Fannin St., Suite 861, Houston, TX 77030. FAX: 713 798 5074; agoulnik{at}bcm.tmc.edu
Received: 7 May 2003.
First decision: 28 May 2003.
Accepted: 11 June 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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