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a ZymoGenetics, Inc., Seattle, Washington 98102
b School of Molecular Bioscience and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164-4660
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although the activity and function of insulin, IGF-I, IGF-II, and relaxin have been studied extensively, the biological activities of the other family members have yet to be fully elucidated. In this paper, we report the sequence and the pattern of expression of a novel member of this gene family, INSL6, which is highly expressed in the testis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An Expressed Sequence Tag (EST) encoding a novel polypeptide with homology to the B-chain motif of relaxin was identified from a human testis cDNA library EST (dbEST Id: 45235; GenBank accession no. T19007). A nucleic acid probe derived from the EST was used to screen primary testis cDNA libraries, resulting in the isolation of cDNA clones encoding the full-length rat and human polypeptides, termed Zins2 and Zspin2, respectively. In an effort to maintain a consistent nomenclature, the locus symbol adopted by the HUGO nomenclature committee, INSL6, will henceforth be used to refer to the rat Zins2 or human Zspin2 sequence [9].
The human and rat cDNA libraries were made from poly(dT)-selected poly(A)+ testis mRNA purchased from Clontech (Palo Alto, CA). Complementary DNAs were synthesized using Superscript II reverse transcriptase (Life Technologies, Gaithersburg, MD) in accordance with the directions of the vendor. Second strand cDNA syntheses were performed using the method described by Gubler and Hoffman [10]. The resulting cDNAs were directionally cloned into the EcoRI and XhoI sites of 
Zap II phage vector and packaged using Gigapak Gold III extracts (Stratagene, La Jolla, CA). Approximately 130 positive signals were obtained from screening 2 x 106 phage of the rat testis library. Thirty-five positive signals were obtained from screening 1.5 x 106 phage of the human testis library. The rat cDNA sequence was derived from a single clone. The human cDNA were derived from three independent cDNA clones. The sequences of the longest human and rat INSL6-encoding cDNAs were deposited in GenBank under accession nos. AF156094 and AF159506, respectively.
Human Northern Blot Analysis
Human multiple-tissue mRNA blots were purchased from Clontech. The human multiple-tissue blots were prehybridized and hybridized in ExpressHyb solution (Clontech). Twenty nanograms of the cDNAs corresponding to the open reading frame of human INSL6 was labeled with 32P by random priming using the Rediprime DNA Labeling System according to the manufacturer's instructions (Amersham Pharmacia Biotech, Piscataway, NJ). Hybridization with 106 cpm probe/ml hybridization solution was carried out overnight at 65°C. The blots were washed 4 times in double-strength SSC (single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate) and 0.5% SDS at room temperature, followed by 3 washes in 0.1-strength SSC and 0.1% SDS at 55°C. The blots were exposed to film for 16 h at -80°C using a BioMax screen (Eastman Kodak, Rochester, NY). The blot was then hybridized again in ExpressHyb overnight at 65°C with a probe to human clathrin to confirm the integrity of the mRNA on the blot.
Rat Northern Blot Analysis
Total RNA from liver, heart, kidney, lung, brain, skeletal muscle, spleen, pancreas, and testis were isolated from 67-day-old Sprague-Dawley rats. Total RNA from epididymis, prostate, ovary, uterus, and testis were isolated from 90-day-old Sprague-Dawley rats. RNAs were isolated from tissues homogenized in a Trizol solution according to manufacturer's instructions (Life Technologies).
Ten micrograms of each RNA sample was fractionated on a 1.2% agarose gel and transferred to Hybond N+ membrane (Amersham Pharmacia Biotech) via capillary action. The RNA was cross-linked to the membrane by exposure to 120 mJ of UV energy in a Stratalinker 1800 (Stratagene). The membranes were prehybridized for at least 1 h at 60°C in a buffer consisting of 1% BSA, 7% SDS, and 0.5 M sodium phosphate, pH 7.2.
Twenty nanograms of cDNA corresponding to the open reading frame of rat INSL6 was labeled with 32P by random priming using the Rediprime DNA Labeling System according to the manufacturer's instructions (Amersham Pharmacia Biotech). The probe was added to the prehybridized blot and hybridized overnight at 60°C. After hybridization, the blots were washed in 0.1-strength SSC, 0.1% SDS at 65°C. The blots were exposed to a phosphor screen for between 12 and 48 h and analyzed on a phosphorimager (Molecular Dynamics, Sunnyvale, CA). The blot was hybridized to a cyclophilin probe to confirm the integrity of the mRNA on the blot.
Rat In Situ Hybridization
Rat INSL6 cDNA corresponding to nucleotides 470–790 was cloned into pBluescript SK+ (Stratagene). A clone (5–2R) was chosen and linearized with either SstI (antisense probe) or KpnI (sense probe) to produce template for in vitro transcription. 33P-Labeled antisense or sense probes to rat INSL6 were generated from 20 ng of linearized template in a reaction consisting of 10 µl of [33P]UTP (cat. no. NEG-607H; NEN, Boston, MA), 1 µl (20 units) of RNase inhibitor (Life Technologies), and 1 µl (50 units) of either T3 or T7 RNA polymerase (Stratagene) according to the directions of the vendor of the polymerase.
Testes were removed from adult rats. A small incision was made in the tunica on each end of the testis, and the testes were placed in freshly prepared 4% (v:v) paraformaldehyde for 4 h at 4°C. The testes were bisected with a razor blade and returned to the 4% paraformaldehyde solution for an additional 10 h at 4°C. Sections (10 µm thick) were cut from paraffin-embedded 90-day-old rat testes and mounted onto slides. Immediately prior to hybridization, sections were deparafinized with xylene and rehydrated through a series of graded solutions consisting of 100%, 95%, 85%, 60%, and 30% ethanol (v:v) and rinsed in PBS. The slides were treated with 20 µg/ml proteinase K (Boehringer-Mannheim, Indianapolis, IN) in PBS for 7 min at room temperature, rinsed in PBS twice for 5 min each, and rinsed in diethyl pyrocarbonate (DEPC)-treated water for approximately 30 sec. The slides were acetylated for 10 min at room temperature in 0.25% acetic anhydride in 0.1 M triethanolamine, pH 8.0. The slides were rinsed in DEPC-treated water for approximately 30 sec and rehydrated through a series of graded ethanol solutions.
Sections were hybridized in a humidified chamber overnight at 55°C in a buffer containing 50% formamide, 10% dextran sulfate, 0.3 M NaCl, 20 mM Tris-HCl (pH 7.5), 10 mM dithiothreitol (DTT), 5 mM EDTA (pH 8.0), Denhardt's solution, and 0.4 mg/ml yeast tRNA. The hybridization mixture consisted of 90% of this buffer and 10% probe (2 x 106 cpm/cm2) in DEPC-treated water. Before hybridization, the probe was denatured at 80°C for 2 min. Slides were washed in 5-strength SSC and 1 mM DTT at 55°C for 20 min; 50% formamide, double-strength SSC, and 1 mM DTT at 70°C for 30 min; 10 mM Tris (pH 7.5), 0.5 M NaCl, and 5 mM EDTA (TNE buffer) at 37°C 3 times for 10 min each; TNE buffer containing 20 µg/ml RNase A (Life Technologies) at 37°C for 30 min; TNE buffer at 37°C for 15 min; 50% formamide, double-strength SSC at 70°C for 30 min; double-strength SSC at 37°C for 15 min; and 0.1-strength SSC at 37°C for 15 min. Slides were dehydrated through a series of graded ethanol solutions with 0.3 M ammonium acetate and up to 95% ethanol followed by 2 washes in 100% ethanol. Slides were dipped in Kodak emulsion NTB-2 and exposed for 2 days at room temperature. The sections were counterstained lightly with hematoxylin and eosin and mounted with a xylene-based mounting medium.
Rhesus Macaque In Situ Hybridization
The coding region of human INSL6 cDNA was cloned into the SalI and XbaI sites of pBluescript SK+ to yield the plasmid pSLHIT-7. The plasmid was linearized with XbaI (antisense probe) or XhoI (sense probe) to produce template for in vitro transcription. [35S]UTP sense or antisense probes to human INSL6 were generated from 1 µg of linearized template using 5 µl of [35S]UTP (cat. no. SJ603; Amersham Pharmacia Biotech) and 2 µl (40 units) of either T3 or T7 RNA polymerase (Ambion, Austin, TX) according to the directions of the vendor. The probes were purified over a Sephadex G-50 spin column (Amersham Pharmacia Biotech). The preparation of the Rhesus macaque testis sections and the hybridization and washing conditions were as described for the rat testis sections.
Chromosomal Mapping
INSL6 was mapped to human chromosome 9 by polymerase chain reaction (PCR) using the human/rodent somatic cell hybrid mapping panel number 2 (National Institute of General Medical Sciences [NIGMS], Camden, NJ). INSL6 was further mapped regionally using the Whitehead Institute/MIT Center for Genome Research's Genebridge 4 radiation hybrid cell panel (Research Genetics, Huntsville, AL). Mapping panel 2 consists of DNA isolated from 24 human/rodent somatic cell hybrid lines, each retaining one specific human chromosome and the parental DNAs. The GeneBridge 4 RH panel contains PCR-amplifiable DNAs from each of 93 radiation hybrid clones, plus 2 control DNAs (the HFL donor and the A23 recipient). A publicly available World Wide Web server (http://carbon.wi.mit.edu:8000/cgi-bin/contig/rhmapper.pl) allowed mapping relative to the Whitehead Institute/MIT Center for Genome Research's (WICGR) radiation hybrid map of the human genome.
For the mapping of INSL6 with the GeneBridge 4 RH panel, 25-µl reactions were set up in a RoboCycler Gradient 96 thermal cycler (Stratagene). Each PCR reaction consisted of KlenTaq reaction buffer (Clontech), 200 µM each dNTP, 125 pM sense primer ZC10684 (5' TCC TCC GCT TGT CCC TGC TGT GG 3'), 125 pM antisense primer ZC10685 (5' CTC CTC GAA ACG GAA CTG GCT CC 3'), 2.5 µl RediLoad (Research Genetics), 0.5 units Advantage KlenTaq Polymerase Mix (Clontech), 25 ng of DNA from an individual hybrid clone or plasmid DNA control, and H2O for a total final volume of 25 µl. The PCR conditions were as follows: an initial 5-min denaturation at 95°C, then 35 cycles of 1 min at 95°C, 1 min at 70°C, and 1 min at 72°C, followed by a final extension of 7 min at 72°C. The reactions were separated by electrophoresis on a 3% NuSieve GTG agarose gel (FMC Bioproducts, Rockland, ME). Similar conditions were used for the initial mapping on the human/rodent somatic cell hybrid mapping panel number 2 (NIGMS, Coriell Institute of Medical Research, Camden, NJ), except that 50-µl reactions were used.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INSL6 was first identified as an EST (dbEST Id: 45235; GenBank accession no. T19007) encoding a polypeptide that exhibits homology to the relaxin B-chain. A 790-base pair (bp) rat and a 717-bp human cDNA encoding full-length INSL6 were isolated from testis cDNA libraries using a probe derived from the EST sequence. The rat INSL6 cDNA encoded an open reading frame of 188 amino acids composed of a putative signal sequence and a mature polypeptide. The tentative signal sequence and the B-, C-, and A-domains that are characteristic of the gene family are as indicated in Figure 1. The human cDNA had a similar sequence with an open reading frame of 213 amino acids. The prohormone of human INSL6 had a slightly longer C-peptide domain and A-domain than rat INSL6. Sequence alignment of human and rat INSL6 is shown in Figure 1.
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As shown in Figure 2, INSL6 was homologous with insulin, the IGFs [2, 3], human relaxin H2 [4], INSL3 [5], INSL4 [6], and INSL5 [7]. Within all members of this family, the cysteine motif with its characteristic spacing was highly conserved within B- and A-domains. The A-domain motif is CC X3 C X8 C, where XN represents the number of residues comprising any amino acids other than cysteine. The B-domain motif is LCG X10 C. Within the B- and A-domains, human INSL6 was 55% identical to rat INSL6, 43% identical to human relaxin H2, 38% identical to human Leydig INSL3, 36% identical to human insulin, 36% identical to human IGF-II, 33% identical to human IGF-I, 28% identical to human INSL5, and 24% identical to human placenta INSL4. Human and rat INSL6 C-domain sequences had no significant sequence similarity with the C-domain sequences of the other family members, but exhibited a significant degree of similarity (43%) with each other.
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Northern Blot Analysis
On human multiple-tissue mRNA blots, a single INSL6 transcript of 1.2 kilobases (kb) was detected only in mRNA derived from testis (Fig. 3a). The cDNAs isolated in this study were approximately 350 bp shorter than the transcript detected by Northern blot analysis. The difference is likely attributable to the poly(A) tail present on the transcript as well as to the fact that our cDNA clones were missing portions of the 5' noncoding sequence. Northern blots containing human mRNAs from heart, brain, placenta, lung, liver, skeletal muscles, kidney, pancreas, spinal cord, lymph nodes, trachea, adrenal gland, and bone marrow were all negative for INSL6 transcripts (data not shown).
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In the rat, INSL6 mRNA was primarily found in testis in the adult (Fig. 3b). In contrast to the case in the human, trace levels of INSL6 transcripts were detected in the rat prostate. INSL6 transcripts were not detected in Sertoli cultures prepared from 20-day-old rat cells or mouse MSC1 Sertoli cells (data not shown). While a single mRNA species was detected in the human testis, there appeared to be an additional species of 1.9 kb in the rat.
In Situ Hybridization Analysis
In situ hybridization studies of both adult rat and Rhesus macaque tissue sections showed that INSL6 mRNA was localized primarily in the seminiferous tubules with the highest expression apparent in spermatocytes and round spermatids (Figs. 4 and 5). INSL6 could not be detected in spermatogonia or peritubular myoid cells, suggesting that its expression in these cells either is very low or is absent. No expression of INSL6 was detected in Leydig cells, suggesting specific and independent roles for INSL3 and INSL6 in testis development and regulation. The signal strength varied considerably among different seminiferous tubules when sections probed with the antisense probe were viewed under low magnification (Fig. 4b), suggesting that INSL6 expression is differentially regulated during the cycle of the seminiferous epithelium.
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Radiation Hybrid Mapping
INSL6 was mapped to chromosome 9 on the NIGMS human/rodent somatic cell hybrid mapping panel number 2. Further regional mapping was done on the GeneBridge 4 RH panel. The WICGR linkage results showed that INSL6 mapped 0 cR_3000 from the chromosome 9 framework marker CHLC.GCT3G05 (lod > 3.0). Proximal and distal framework markers were AFMA113WA5 and D9S178, respectively. CHLC.GCT3G05 is present in YAC clone 951-G-6 contained in the WICGR YAC contig WC9.0. The use of surrounding markers positioned INSL6 in the 9p24 region on the integrated LDB chromosome 9 map (The Genetic Location Database, University of Southhampton, WWW server: http://cedar.genetics.soton.ac.uk/public_html/).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression of INSL6 is testis-specific in humans and is restricted to the testis and prostate in rats. The strong hybridization signal found in testis suggests that INSL6 is an abundant mRNA species. While a single 1.2-kb mRNA species is detected in human testis, there appears to be an additional species of 1.9 kb in the rat. Reverse transcription-PCR, carried out using primers spanning the initiation methionine and the translation stop codon of rat INSL6, consistently produced a single product corresponding to the rat INSL6 open reading frame. This result indicates that the 1.9-kb transcript species in rat testes is not due to alternative splicing involving the coding region of the gene. We have not explored the possibility of other mechanisms such as alternative testis-specific promoters or polyadenylation sites that may give rise to a longer INSL6 transcript. Nor have we been able to isolate a cDNA species corresponding to the 1.9-kb mRNA species detected by the Northern blot analysis. The possibility remains that the longer transcript may be transcribed from a separate locus in rat testes.
The N-terminus of the rat and human INSL6 B-domain was predicted using the method of von Heijne [11] and is indicated in Figure 1. For rat INSL6, the predicted signal sequence cleavage site is after residue Glu22, and in the human sequence this cleavage is predicted after Ser20. The cleavage of the rat sequence at Glu22 will yield an amino terminal Gln residue. The presence of this amino acid at the amino terminus of peptides is known to cyclize to form a pyroglutamic acid [12]. This type of amino terminal modification is known to occur in other members of the relaxin family, such as the amino terminus of the A-chain of human relaxin H2 [4, 12].
As yet, we have not been able to detect or isolate the native protein from the testis. It is not known whether native INSL6 is processed as is the case for human insulin and relaxin H2, where the C-peptide is excised by prohormone convertases to produce the biologically active form of the hormone. Possible processing of the prohormone B- and C-domains of INSL6 is predicted to occur after Arg52 and Arg54 of the rat and human sequences, respectively. This cleavage at basic residues by a proprotein convertase-like enzyme is frequently followed by a carboxypeptidase cleavage, which in this case would leave Ser51 or Phe53 as the carboxyl terminal residue of the B-domain. Cleavage between the C- and A-domains is predicted to be after Arg162 of the rat sequence and Arg172 of the human sequence. The Arg-XX-Arg motif at this site of both the rat and human sequences is known to be the consensus site for cleavage by the furin family of convertases. Proteolytic cleavage at this site in the rat hormone will leave Gly163 as the N-terminus of the mature A-domain, while the corresponding processing in the human INSL6 sequence will yield Gly173 as the amino terminus of the A-domain.
As illustrated in Figure 2, the sequence at the carboxyl terminus of rat and human INSL6 diverges after rat Phe188 and human Phe198, respectively. The rat cDNA sequence contains a stop codon following this amino acid (Phe188), while translation of the human protein continues for another 15 amino acids. Although the presence of additional amino acids coded for by the human cDNA will generate a larger prohormone polypeptide in humans, the presence of a dibasic (Lys-Arg) sequence exactly at the position where the rat sequence terminates suggests that additional prohormone convertase processing is required at this site. The cleavage of this dibasic site by a prohormone convertase and subsequent cleavage of the basic residues by a carboxypeptidase will yield a carboxyl terminus in human INSL6 that is identical to rat INSL6.
The site of INSL6 expression is similar to that of proprotein convertase 4 (PC4) [13], a testis-specific, furin-like protease believed to be involved in protein maturation. PC4 is a member of a large family of proprotein convertases that includes PC 1/3 and PC2 [1]. The latter enzymes are those believed responsible for the maturation of other members of the insulin family. The coexpression of INSL6 and PC4 within the same cell population lends support for a hypothesis that PC4 is involved in the maturation of INSL6; however, further analysis is required to determine the validity of this hypothesis.
INSL6 is mapped to chromosome 9p24. The gene encoding early placenta insulin-like factor (INSL4), another family member associated with reproductive tissue, also maps to 9p24 [6]. It has been reported that the 9p24 region harbors an autosomal testis-determining factor locus (TDFA) [14]. Failure of testis development has been associated with rearrangement of the 9p24.1 region [15]. Several cases of sex reversal with gonadal dysgenesis have also been reported in translocation or terminal deletion of the distal chromosome 9p region [16], demonstrating that this chromosome region containing INSL6 is important for reproductive function.
In situ hybridization analysis of rat and rhesus macaque testis revealed that INSL6 expression is localized to the seminiferous epithelium. Upon close inspection, the expression appears to be localized in the spermatocytes and round spermatids. At present the role of INSL6 in sperm development and fertilization is under investigation. However, a function for another testis-specific member of the insulin family has been recently reported. Targeted disruption of INSL3, which is expressed in Leydig cells, was shown to exhibit bilateral cryptorchidism with free-moving testes and genital ducts due to failure of gubernaculum development during murine embryogenesis [17].
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ACKNOWLEDGMENTS |
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While our manuscript was in review, the characterization of human [18] and murine [18, 19] INSL6 was reported by two other laboratories.
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FOOTNOTES |
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1 GenBank accession numbers: human INSL6: AF156094; rat INSL6: AF159506. The gene symbol INSL6 has been approved by the HUGO nomenclature committee. 
2 Correspondence: Si Lok, ZymoGenetics, 1201 Eastlake Ave. E., Seattle, WA 98102. FAX: 206 442 6608; loks{at}zgi.com
Accepted: January 5, 2000.
Received: November 1, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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