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Regular Article |
a Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4660
ABSTRACT
Differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) was used to identify a novel retrovirus, designated SC1, that is expressed at high levels in rat granulosa cells and prepubertal Sertoli cells. The initial DDRT-PCR screen was performed using RNA from cultured prepubertal rat Sertoli cell, liver, and brain samples. SC1 was detected in the prepubertal rat Sertoli cell samples but not in those from liver and brain. SC1 cDNA was 6 kilobases in length and contained regions encoding for the gag, pol, and env retroviral proteins. Northern blot analysis failed to detect expression of the SC1 gene in total RNA isolated from adult brain, heart, spleen, lung, liver, skeletal muscle, kidney, prostate, and epididymis. Similarly, Northern blot analysis of testes from rats at various ages of development showed that high-level expression of the SC1 gene was limited to prepubertal testis samples. In situ hybridization analysis localized the SC1 mRNA to the seminiferous tubules of prepubertal testes and at a much lower level in Sertoli cells of adult testes. Northern blot analysis of total RNA isolated from Sertoli cells from 20-, 27-, and 35-day-old rat Sertoli cells and type A spermatogonia, pachytene spermatocytes, and round spermatids showed expression of the SC1 gene to be restricted to 20- and 27-day-old Sertoli cells, with no expression detected in germ cells. Furthermore, Northern blot analysis also showed expression of the SC1 gene in rat ovaries, and the level of expression was affected during eCG/hCG-induced ovulation. Expression of SC1 mRNA was localized by in situ hybridization of eCG-treated ovaries to the granulosa cell layer in developing follicles. Southern blot analysis showed SC1 to be endogenous in the rat and absent in mouse and human cell genomes. Transient transfection assays using the SC1 promoter region showed high promoter activity in MSC-1 and cultured prepubertal rat Sertoli cells, and no activity in 3T3 or MCF-7 cell lines.
Sertoli cells, spermatogenesis, testes
INTRODUCTION
Retroposons are DNA sequences that are believed to originate from viral RNAs that were inserted into the genomes of eukaryotes and prokaryotes [1]. The integration of retroviral RNA into the mammalian genome has the potential for generating genetic diversity by recombining with the host genome. It has been reported that 0.5% of the mouse genome [2] and 1% of the human genome are retroviral [3].
Most retroviruses are similar in organization. Retroviruses contain 3' and 5' long terminal repeats (LTRs) and generally encode for essential viral proteins such as gag, pol, and env [4]. LTR regions may be as short as a few hundred bases or as long as 2000 bases in length, usually beginning and ending with short, inverted repeats. Many, but not all, proviruses (retroviruses that have integrated into the germ line) have mutations or deletions of one or more of the viral genes and can no longer express functional proteins.
There is some evidence that retroviral sequences are maintained in the mammalian genome because they have a biological function [5]. The retrovirus may affect the host by 1) altering the expression of genes by trans- or cis-acting mechanisms [6]; 2) retrotransposing, leading to insertional mutations in host genes [7]; 3) promoting genomic plasticity [8]; or 4) generating a disease state for the host [3, 9].
Retroviruses may be expressed in a cell-specific, ubiquitous, or limited manner [2, 10, 11]. The expression of a retroviral transcript is controlled by the 5' LTR [10]. In a few cases the 3' LTR has been involved as well [11]. The 5' LTR region of the retroviral gene generally contains CAAT, TATA, and GATA nuclear binding sites that aid in transcriptional activation [10, 11].
There have been several reports of specific retroviral gene expression in the reproductive tissues, including the ovary and epididymis [12], type A spermatogonia [10], and epididymis [13]. In this study we used differential display to identify genes that are expressed specifically in Sertoli cells. We report the expression of a novel retroviral transcript (SC1) that is expressed at high levels in prepubertal rat Sertoli cells in the male and at lower levels in granulosa cells in the female. To date, the study of Sertoli cell function in vivo has been hampered by the absence of an adequate Sertoli cell promoter for transgenic analysis. The finding that this gene is expressed only in dividing Sertoli cells and at high levels in the male suggests that the promoter for this gene could be a valuable tool in the study of Sertoli cell function in vivo.
MATERIALS AND METHODS
Cell Isolation and RNA Isolation
Sertoli cells and interstitial cells were isolated from 20- and 35-day-old Sprague-Dawley rats as previously described [14] and maintained in serum-free Ham F12 media (Gibco BRL Life Sciences, Grand Island, NY) for 4 days at 32°C. Type A spermatogonia, pachytene spermatocytes, round spermatids, and Sertoli cells from 27- and 55-day-old Sprague-Dawley rats were isolated from rat testes as previously described [15, 16]. Brain, lung, liver, kidney, spleen, heart, skeletal muscle, prostate, and epididymal samples were taken from adult male rats for Northern blot analysis. Brain and liver samples were taken from 20-day-old Sprague-Dawley rats for differential display analysis. Ovary and uterus were taken from adult females. Female rats at 25 days of age were superovulated with a treatment of 10 units of eCG for 48 h, followed by 10 units of hCG for 24 h as described [17]. Ovaries were collected at specific time points during hormonal treatments. Total RNA was isolated from the testes of 3- to 5-day-old, and 40-day-old balb/c mice. A mouse Sertoli cell line (MSC-1), NIH 3T3 cells, and a human breast cancer cell line, MCF-7, were cultured in Dulbecco modified Eagle medium (DMEM; Sigma-Aldrich, St. Louis, MO) supplemented with 10% bovine calf serum at 37°C in 5% CO2 and 95% air. Total RNA was purified from all cell cultures and tissues using the Trizol method (Gibco BRL) according to the manufacturer's specifications.
Differential Display Reverse Transcriptase-Polymerase Chain Reaction
Differential display was performed using the Hieroglyph kit (Genomyx, Foster City, CA). Reverse transcriptase (RT) reactions were performed using 200 ng of total RNA isolated from cultured rat Sertoli cells, and from liver and brain from 20-day-old rats. RT reactions (20 µl) were performed in duplicate at 42°C for 60 min using two different RT enzymes, avian myeloblastosis virus (AMV) RT (Gibco BRL) and Superscript II (Gibco BRL), with an oligo(dT) primer (200 nM); 5'-
(dT12)MN-3' (M = A, G, or C; N = A, T, G, or C) according to the manufacturer's specifications for each RT enzyme. The underlined sequence of the oligo(dT) primer is half of the T7 promoter sequence. Negative controls were included in which RNA or the RT were omitted and replaced with deionized water. Complementary DNA samples (2 µl) were amplified by polymerase chain reaction (PCR) in duplicate 20-µl reactions containing single-strength buffer (1.5 mM MgCl2, 20 µM dNTPs, the 3' oligo(dT) primer [0.2 µm]), and one arbitrary 5' primer (0.2 µm ARP1-4), 0.125 µCi [
-33P]dATP, and 1 unit of AmpliTaqR (Perkin Elmer). Reactions were carried out at 95°C for 2 min, then for 4 cycles at 94°C for 30 sec, 46°C for 30 sec, and 72°C for 2 min; followed by 26 cycles at 94°C for 30 sec, 60°C for 30 sec, and 72°C for 2 min; and a final extension at 72°C for 7 min. In the negative controls, either RNA, RT, or cDNA were replaced with deionized water. Samples were analyzed by denaturing polyacrylamide (4.5% LR-Optimized, HR-1000; Genomyx) gel electrophoresis using a GenomyxLR GX100 DNA sequencer (Genomyx). Complementary DNA fragments were visualized by autoradiography using Biomax film (Kodak). Putative Sertoli cell-specific cDNA fragments were excised from the gel and incubated in 100 µl of deionized water overnight at room temperature. Complementary DNA fragments were reamplified by PCR in 100-µl reactions containing single-strength buffer, 20 µM dNTPs, 1.5 mM MgCl2, 0.2 µM full-length T7 primer, 0.2 µM full-length M13 reverse (M13r) primer, 0.5 units Taq DNA Polymerase (Gibco BRL), and 40 µl of cDNA for 30 cycles at 94°C for 30 sec, 60°C for 30 sec, and 72°C for 2 min, and a final extension at 72°C for 7 min. PCR products were then cloned into p-GemT Easy Vector (Promega, Madison, WI) using standard protocols. The arbitrary primers used for the differential display reverse transcriptase (DDRT)-PCR were as follows:
CGACTCCAAG-3' GCTAGCATGG-3' GACCATTGCA-3' GCTAGCAGAC-3' DNA Sequencing and Synthesis
Oligonucleotide primers SP6END (5'-AGCTATGCATCGAACGCGTT-3') and T7END (5'-TTGGACCCGACGTCGCA-3') were used for sequencing of the DDRT-PCR clones. The amplified differential display fragments contain the T7 and M13r sequences; therefore, primers SP6END and T7END were designed to bind to the polylinker region between the SP6 and T7 promoters, respectively, of the p-GemT Easy Vector. DNA sequencing analysis was performed at the Laboratory of Bioanalysis and Biotechnology at Washington State University, Pullman, Washington. The sequence of each cloned DDRT-PCR fragment was analyzed with the GenBank/European Molecular Biology Laboratory database using the basic local alignment search tool (BLAST) for homology identification. Full-length cDNA was submitted to GenBank using the Bankit internet program at the National Center for Biotechnology Information.
Northern Blot Analysis
Total RNA (10 µg) from each sample was fractionated in a 1% agarose/formaldehyde gel and transferred to a nylon membrane (Hybond-N, Amersham Pharmacia, Arlington, Heights, IL) and UV cross-linked (UV Stratagene 1800). The DDRT-PCR cDNA fragment, 281 nucleotides from bases 5697–5978, was radiolabeled with [-32P]dATP using the Rad Prime DNA Labeling Kit (Gibco BRL). Northern blots were hybridized overnight at 42°C with labeled cDNA probes in 50% formamide, 50 mM NaH2PO4, 5x Denhardt solution, 5x saline-sodium citrate (SSC), 0.1% SDS, 2% dextran, and 1 mM EDTA. Following hybridization, blots were washed in 2x SSC/0.1% SDS for 10 min at room temperature; 1x SSC/0.1% SDS for 30 min at 65°C, and 0.1% SSC/0.1% SDS for 30 min at 65°C. After blots were washed, they were placed in a phosphor screen cassette (Molecular Dynamics, Sunnyvale, CA) and allowed to expose a phosphor screen for 8–12 h. The signals were detected using a Molecular Dynamics PhosphorImager 445 SI and ImageQuant software (Molecular Dynamics). Ribosomal protein S2 (ChoB) cDNA probe was used as a control for loading of RNA [17].
5' Rapid Amplification of cDNA Ends
5' Rapid amplification of cDNA ends (RACE) reactions were done by using the 5' RACE system version 2.0 (Gibco BRL, 18374-058). Briefly, first-strand cDNA reactions were done in 25-µl reactions with 20 mM Tris (pH 8.4), 50 mM KCl, 2.5 mM MgCl2, 10 mM dithiothreitol (DTT), 100 nM gene-specific primer (GSP, see below), 400 µM dNTPs, 3 µg of total RNA from rat prepubertal Sertoli cells, and 200 units of Superscript II RT at 50°C for 60 min. Following the 60-min incubation, the transcription was stopped by heating at 70°C for 10 min. RNA in the reaction was degraded by adding 1 µl of RNase mix (Gibco BRL) and incubating at 37°C for 30 min. The cDNA was then purified with a GlassMax spin column (Gibco BRL) by adding 120 µl of binding buffer to the cDNA reaction mixtures, transferred to the GlassMax spin column, and centrifuged at 13 000 x g for 20 sec. The cartridge was washed four times with 400 µl of cold wash buffer and twice with cold, 70% ethanol. Complementary DNA was eluted from the cartridge with 50 µl of distilled water preheated to 65°C. Purified cDNA was then tailed with terminal transferase (TdT) in a reaction containing 10 mM Tris pH 8.4, 25 mM KCl, 1.5 mM MgCl2, 200 µM dCTP, 10 µl of purified cDNA, and 1 µl TdT for 10 min at 37°C. The cDNA fragment was amplified by PCR using 20 mM Tris pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 200 µM dNTP, 400 nM nested GSP, 400 nM abridged anchored primer, 5 µl of tailed cDNA, and 0.5 unit of Taq for 35 cycles at 94°C for 30 sec, 55°C for 1 min, 72°C for 4 min, and a final extension at 72°C for 10 min. PCR products were fractionated and gel-purified from a 1% agarose gel, cloned into p-GemT Easy Vector (Promega) using standard protocols, and sequenced. The procedure was repeated with new primers until the full-length cDNA was obtained. Also, reverse primers were generated to verify the sequence following the protocol outlined for the PCR. Antisense primers for 5' RACE were as follows:
Sense primers were as follows:
In Situ Hybridization Analysis
The SC1 cDNA fragment corresponding to nucleotides 5911–5691 (PCR product from primers SC31 and SC51) was cloned into p-GemT Easy Vector. The plasmid was linearized with NdeI and NcoI restriction endonucleases according to the manufacturer's specifications. The linearized plasmids were treated with 1 µl of 20 mg/ml proteinase K for 10 min at room temperature, extracted with 1 volume of phenol, and precipitated in 2.5 volumes of ethanol and 0.1 volume of 3 M sodium acetate. Antisense and sense probes were labeled with 33P in reactions consisting of 10 µl of [-33P]UTP; 1 µl (20 units) RNAsin (Promega); 50 units of T7 or Sp6 RNA polymerase (Gibco BRL); 1x transcription buffer; 10 mM DTT; and 2.5 mM rATP, rGTP, and rCTP according to the manufacturer's specifications. In situ hybridization analyses were performed on testis samples from 8-, 20-, and 90-day-old rats and ovary samples from 25-day-old female rats treated for 48 h with eCG as described by Lok et al. [18]. Tissues were submersion-fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. Sections (10 µm) were cut from each block and placed on poly-L-lysine coated slides. Slides were hybridized overnight at 55°C, stringently washed, dipped in Kodak emulsion NTB-2, and were allowed to expose for 3–21 days at room temperature.
Southern Blot Analysis
Genomic DNA was isolated from cultured rat Sertoli cells and mouse kidneys. Tissue and cell cultures were treated with proteinase K (500 µg/ml) at 50°C for 8 h, followed by extraction with 1 volume of phenol (three times), and precipitated with 2.5 volumes of ethanol and 0.5 volume of 5 M ammonium acetate. Purified Jurkat genomic DNA was a generous gift from Mark Nissen in the laboratory of Dr. Raymond Reeves (Washington State University). Samples of genomic DNA (25–30 µg) were digested overnight with restriction endonucleases EcoRI, HindIII, PstI, and BamHI according to the manufacturer's specifications. Restriction digests were extracted with 1 volume of phenol and precipitated in 2.5 volumes of ethanol and 1 volume of 3 M sodium acetate. Digested DNA was fractionated on a 0.8% agarose gel at 14 V for 16 h. Agarose gels were soaked in 0.25% HCl for 30 min followed by 0.5 N NaOH for 30 min, then transferred overnight to a nylon membrane (Hybond-N, Amersham Pharmacia). Random priming probes were generated from the 281 base pair (bp) DDRT-PCR fragment, and membranes were hybridized and washed as described above.
Transient Transfection Assay
Transient transfection assays were used to measure the promoter activity of the SC1 gene. Transient transfection assays were performed using calcium-phosphate precipitation as previously described [19] with modification by Braun et al. [20]. PCR primer SCK2 (see below) was used with SCP1 primer (see below) to amplify a specific region (bases 5873 to 5331) of the SC1 promoter. This promoter region was cloned into pGL3-basic vector (Promega). The SCK2 primer contained a KpnI cut site on the 5' end for directional cloning into the vector. The promoter sequence was cloned into the KpnI-SmaI site of the pGL3-basic vector according to the manufacturer's specifications. The promoter construct was sequenced using RV2 and GL3 primers (Promega) for insert directional verifications. The SV40 promoter in pGL2 basic vector (Promega) was used as a positive control and pGL3-basic without insert was used as a negative control for the transient transfection assays. PSV40-ß-galactosidase vector (Promega) was used to determine the transfection efficiency of each experiment. NIH 3T3, MFC-7, MSC-1, and rat prepubertal Sertoli cells were used in the transient transfection assays. The cell lines were grown in 24-well plates until they reached approximately 75% confluency. Approximately 1–2 x 105 rat prepubertal Sertoli cells were plated per well and cultured for 4 days prior to transfection. Each well was transfected with 1.5 µg of pGL3 or pGL2 reporter constructs and 1.5 µg of the pSV40-ß-galactosidase reporter construct. Luciferase and ß-galactosidase assays were performed 48 h following transfections. Sense primer used to generate the promoter sequence was SCK2 (5'-GGGGTACCCCTGCTGTCAGCCTTAGCT-3'). Antisense primer used to generate the promoter sequence was SCP1 (5'-CCTCTCTAGGCAGGATCGTAT-3').
RESULTS
DDRT-PCR
The goal of the study was to identify genes expressed specifically in rat Sertoli cells. The DDRT-PCR analysis was performed using RNA isolated from cultured Sertoli cells, liver, and brain isolated from 20-day-old rats. By comparing the cDNA fragments produced from the DDRT-PCR analyses of cultured Sertoli cells, liver, and brain, we identified mRNAs that are expressed in Sertoli cells but not in liver and brain. The liver and brain were chosen for DDRT-PCR analysis because they have a wide diversity of expressed genes and many testicular genes are also expressed in one of these tissues.
A 281-bp cDNA fragment was originally identified from the Sertoli cell sample from the DDRT-PCR analyses. The cDNA fragment was not present in samples from liver and brain (Fig. 1) and therefore represented a candidate that was expressed uniquely in Sertoli cells. The cDNA fragment was reamplified by PCR, cloned, sequenced, and analyzed by BLAST analysis in the GenBank nucleotide database. This cDNA fragment contained homology to several expressed sequence tags (ESTs) cloned from pooled rat tissue libraries and the ovary, but had no homology to any known transcripts.
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5' RACE
To determine the full length of the SC1 transcript, 5' RACE PCR analyses were performed using gene-specific primers. The full-length transcript was 5978 bases (GenBank accession number AY009092). Sequence analysis of the full-length clone identified this sequence as a retrovirus (SC1), with 89% nucleotide similarity with the Moloney murine leukemia virus-like 30 (Mo-VL30) [21] and 90% nucleotide similarity with a retroviral-like ovarian-specific transcript 30-1 [22]. The organization of this retroviral transcript is similar to that found in most retroviruses. The sequence contains 5' and 3' LTRs and a classic poly adenylation signal, AATAAA. The open reading frames (ORFs) for the retroviral gag, pol, and env proteins are also present. The 3' LTR sequence of SC1 cDNA contains CAAT and TATA box promoter elements and many putative transcription factor binding site regulatory elements.
Northern Blot Analysis
To determine the expression pattern of SC1, Northern blot analyses were performed on total RNA isolated from prepubertal cultured Sertoli cells and adult heart, liver, brain, spleen, lung, kidney, and skeletal muscle. The 281-bp DDRT-PCR fragment was used as a probe as described in Materials and Methods. SC1 mRNA was detected in cultured Sertoli cells (Fig. 2) but not in heart, liver, brain, spleen, lung, kidney, and skeletal muscle. This suggests that SC1 was expressed only in Sertoli cells.
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There have been several reports of reproductive tissues containing high levels of retroviral expression in mice [12, 13]. Northern blot analyses of total RNA isolated from adult rat ovary, uterus, epididymis, prostate, and whole testis from 90-day-old rats were performed (Fig. 3). SC1 mRNA was detected in the ovary (Fig. 3). The RNA from the uterus appeared, interestingly, to contain a truncated transcript, and SC1 was not detected in the testis sample from a 90-day-old rat.
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To examine the expression of the SC1 gene in testis samples from rats at various ages, total RNA was isolated from rat testes from Days 0, 10, 20, 25, 30, 40, 52, 67, and 90, for Northern blot analyses (Fig. 4). Northern blot analyses comparatively showed that high levels of SC1 mRNA were present in testis samples from 0- and 10-day-old rats, and SC1 expression decreases rapidly in testis samples from 20- through 25-day-old rats, and was absent in the testis samples from the 90-day-old rat. The highest expression was detected in the testis sample of the 10-day-old rat.
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To determine the expression of the SC1 gene in Sertoli cells isolated from testes of rats at different ages, Northern blot analyses were performed on samples from Sertoli cells isolated from 20-, 27-, 35-, and 55-day-old rats, and type A spermatogonia, pachytene spermatocytes, round spermatids, and testes from 90-day-old rats (Fig. 5). Expression of SC1 was detected in cultured Sertoli cells from 20-, 27-, and 35-day-old rats, and type A spermatogonia samples, but was not detected in pachytene spermatocytes, round spermatids, or cultured Sertoli cells from 55-day-old rats or in testis sample from 90-day-old rats (Fig. 5). This expression in the type A spermatogonia sample was probably due to Sertoli cell contamination.
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The expression of SC1 was also detected in the adult ovary sample. To determine the expression of SC1 during follicular maturation, prepubertal female rats were treated with eCG for 48 h, followed by treatment with hCG for 48 h. Total RNA was isolated from ovaries at 0, 24, and 48 h after treatment with 10 units of eCG and 14, 24, 48 h after treatment with 10 units of hCG (Fig. 6) as described in Materials and Methods. Northern blot analyses detected SC1 in all ovary samples; however, relative expression was highest at 48 h after treatment with eCG. In contrast, the 48-h posttreatment with hCG had the lowest expression of the retrovirus transcript.
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To determine whether the SC1 gene was expressed in a mouse Sertoli cell-derived cell line and mouse testis samples, Northern blot analyses were performed on mouse testis samples from 3- to 5-day-old and 40-day-old mice, mouse Sertoli cell line MSC-1, and rat prepubertal cultured Sertoli cells (Fig. 7). SC1 mRNA was not detected in the mouse testis samples or MSC-1 cells, suggesting that SC1 is specific to the rat.
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In Situ Hybridization Analysis
To localize the expression of SC1 to specific regions in the testis, in situ hybridization analyses were performed on testis samples from 8-, 20-, and 90-day-old rats (Fig. 8A). SC1 was localized to the seminiferous tubules in testis samples from 8- and 20-day-old rats. SC1 was not detected in the testis of the 90-day-old rat after 3 wk of exposure. The testis samples from an 8-day-old rat showed expression of SC1 after 3 days of exposure, and the testis samples from a 20-day-old rat showed expression after 10 days of exposure. The difference in exposure times needed to detect SC1 between the testis samples from the 8- and 20-day-old rats suggested that the testis sample from a 20-day-old rat contained a much lower level of expression than the testis sample from an 8-day-old rat. The expression of this retroviral transcript appeared to be restricted to the prepubertal Sertoli cells.
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To localize the expression of SC1 in the ovary, in situ hybridization analyses were performed on samples from ovaries treated with eCG for 48 h. The sample from the 48-h treatment with eCG had the highest level of expression of SC1 from the Northern blot analyses (Fig. 6A). SC1 expression was localized to the granulosa cell layer surrounding the developing follicle (Fig. 8B). The large antral follicles had the highest expression of SC1.
Southern Blot Analysis
Retroviral sequences can be present from 1 to 20 copies within the host genome [2]. To determine the genomic copy number of SC1, Southern blot analyses were performed on restriction-digested rat genomic DNA (Fig. 9). Also, to determine whether SC1 was endogenous to the rat or exogenous between different organisms, Southern blot analyses were performed on restriction-digested genomic DNA isolated from balb/c mouse kidneys and a human cell line, Jurkat cells. Southern blot analyses detected SC1 only in the rat genomic DNA. This suggests that SC1 is endogenous to the rat and appears to have multiple copies within the rat genome.
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Transient Transfection Analysis
Transient transfection analyses were performed to determine the activity of the SC1 promoter. A reporter construct driving luciferase gene expression was made using a piece of the 3' LTR sequence. This construct was then used in transient transfections of cultured prepubertal rat Sertoli cells, a mouse Sertoli cell line (MSC-1), NIH 3T3 cells, and a human breast cancer cell line, MCF-7. The promoter construct extended from base 5873, containing the core promoter region that consisted of the TATA and CAAT box sequences, and included the 5' promoter sequence out to base 5331. The sequence of SC1 promoter region and a schematic of the construct are shown in Figure 10, A and B. Transient transfection assays were normalized to the luciferase activity of the SV40 promoter used as a positive control. The pSC1 promoter construct did not have luciferase activity in the 3T3 or MCF-7 cell lines (Fig. 11). In contrast, cultured rat prepubertal Sertoli cells displayed high levels of luciferase activity and MSC-1 cells displayed measurable levels of activity relative to SV40 luciferase (Fig. 11). MSC-1 cells treated with 1 mM dibutyryl-cAMP had a slightly decreased luciferase activity of pSC1 construct compared with the nontreated MSC-1 cells.
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DISCUSSION
To date, several tissue-specific retroviral transcripts have been described. Here we report that the expression of a novel retroviral transcript, designated SC1, was highly expressed in prepubertal Sertoli and granulosa cells from the rat. To our knowledge this is the first report of a retrovirus that appears to be specifically expressed at high levels in Sertoli cells and granulosa cells. This 5978-base transcript contained ORFs for the three retroviral proteins, gag, pol, and env. The gag, pol, and env nucleotide sequences within SC1 have 89% nucleotide similarity to the gag, pol, and env sequences of the Mo-VL30 retrovirus reported by Makris and coworkers [21] and 90% nucleotide similarity to the retroviral-like ovarian-specific retrovirus reported by Godwin and coworkers [22].
Southern blot analyses of rat genomic DNA showed that there were multiple copies of this gene in the genome (Fig. 9). This was not surprising because several different sequences were identified during the sequencing of the full-length SC1 transcript. These sequences often differed in blocks of 4–10 base segments, with one or two blocks identified per 700 bases of sequence. These variant sequences were found throughout the full-length sequence and were not specific to any regions, suggesting that through generations of recombination events and cellular divisions the sequences became altered within short, specific segments [2, 11, 23]. The sequence reported here represents a continuous sequence from a clone generated from a PCR using gene-specific primers for the full-length sequence.
The LTR region has been used to classify retroviruses into families [24]. The LTR regions of SC1 had no nucleotide similarities with the Mo-VL30 [21] or the ovarian-specific retroviral transcript [22]. The LTR regions of SC1 have no nucleotide similarities with the LTR regions of other known retroviruses, demonstrating that SC1 is a novel rat retroviral transcript. We have shown that SC1 may be endogenous to the rat by Southern blot analyses of genomic DNA isolated from mouse and human cell lines (Fig. 9).
The transcriptional control of a retroviral gene is found in the LTR region. Because the full-length 3' and 5' LTRs have the same sequence, the transcriptional control elements can be found in the 3' LTR region of the cDNA transcript [11, 25]. The 3' LTR region of SC1 contains CAAT and TATA box promoter elements and many putative transcription factor binding sites such as GATA, SP1, and AP1. Retroviral transcriptional control elements similar to those found in SC1 have been shown to drive the expression of reporter constructions in a tissue-specific manner [3, 10]. Transient transfection assays showed that a reporter construct of the SC1 promoter region was not active in non-Sertoli cell lines such as 3T3 and MCF-7, whereas the mouse Sertoli cell line, MSC-1, had moderate activity, and cultured prepubertal rat Sertoli cells had very high levels of promoter activity (Fig. 11). These data showed that the promoter activity was unique to Sertoli cells, and whereas the retrovirus was not found in mouse cells, the promoter still had activity in mouse Sertoli cells. The promoter activity in cultured rat Sertoli cells was nearly fourfold higher than the activity of the SV40 promoter.
There have also been reports that the methylation state of the transcriptional control elements is important for regulating retroviral expression in specific tissues [26]. However, the SC1 promoter contains few CpG sites, suggesting that methylation may not regulate SC1 promoter activity.
Endogenous retroviral transcripts can be expressed in both tissue-specific and developmentally specific manners [1]. Using Northern blot and in situ hybridization analyses we have shown in the male rat that the expression of the SC1 gene was restricted to the prepubertal rat Sertoli cells. The expression of SC1 was not detected in adult Sertoli cells (Figs. 5 and 8A). Similarly, Martin and coworkers [11] reported the expression of a retroviral gene in the prepubertal rat heart and liver. Martin and coworkers [11] found that the expression of a retroviral gene was down-regulated 10-fold from heart tissue of an adult compared with that from rapidly growing tissue. The expression of retroviral transcripts is enhanced in tumorigenesis and mitotically active cells [27, 28]. Prepubertal Sertoli cells in 0- and 10-day-old rats are mitotically active [29, 30], and the expression of SC1 correlates well with the mitotic activity of the Sertoli cells. Nakamuta and coworkers [31] demonstrated that the core promoter region of the tissue-specific RATRLTR family of retroviruses are capable of becoming active in nonexpressing cell lines during transient transfections assays, suggesting that mitotic or tissue-specific signals may be regulating the expression of some retroviral genes.
The expression of retroviral genes has been shown to be altered by external signals in reproductive tissues. The expression of a novel retrovirus in the epididymis was decreased in castrated mice, and this decrease was partially recovered with treatments of testosterone [12]. Prepubertal Sertoli cells are responsive to FSH [29]. FSH can stimulate Sertoli cell mitosis and final Sertoli cell maturation (reviewed by [32]). However, the gene expression of SC1 was not responsive to ovine FSH or testosterone treatment of prepubertal cultured rat Sertoli cells (data not shown).
We have also identified and localized the gene expression of SC1 in rat granulosa cells by Northern blot and in situ hybridization analyses (Figs. 6 and 8B). Ovaries treated with eCG for 48 h had the highest level of SC1 expression. Equine chorionic gonadotropin mimics the action of FSH on the ovary. We cannot rule out the possibility that the increase observed in SC1 expression was due to the apparent increase in granulosa cells in the large growing follicles during eCG treatment.
In summary, we report the tissue-specific expression of a novel endogenous retroviral transcript, SC1. In the male rat, SC1 was expressed only in prepubertal Sertoli cells, as shown by Northern blot and in situ hybridization analyses. In the female rat, SC1 was expressed in granulosa cells. Further characterization of the 5' LTR region of this novel retroviral transcript will be necessary to characterize how and why this transcript is expressed and whether it plays any role in spermatogenesis.
ACKNOWLEDGMENTS
We thank Alice Karl and Debra Mitchell for the Sertoli cell and interstitial cell preparation and cultures, and Derek Pouchnik and Gerhart Munske for the DNA sequences and oligonucleotide synthesis. We give special thanks to William Wright at The Johns Hopkins University for total RNA isolated from adult rat Sertoli cells and purified germ cell populations.
FOOTNOTES
First decision: 1 February 2001.
1 This work was supported by the National Institutes of Health grants R37 HD10808 to M.D.G. and HD 08437 to D.S.J. 
2 Correspondence. FAX: 509 335 9688; griswold{at}mail.wsu.edu
Accepted: May 31, 2001.
Received: December 29, 2000.
REFERENCES
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 W. Xia, D. D. Mruk, W. M. Lee, and C. Y. Cheng Differential Interactions between Transforming Growth Factor-beta3/TbetaR1, TAB1, and CD2AP Disrupt Blood-Testis Barrier and Sertoli-Germ Cell Adhesion J. Biol. Chem., June 16, 2006; 281(24): 16799 - 16813. [Abstract] [Full Text] [PDF]
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 L. J. Cheng, J. M. Li, J. Chen, Y. H. Ge, Z. R. Yu, D. S. Han, Z. M. Zhou, and J. H. Sha NYD-SP16, a Novel Gene Associated with Spermatogenesis of Human Testis Biol Reprod, January 1, 2003; 68(1): 190 - 198. [Abstract] [Full Text] [PDF]
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 D. J. McLean, P. J. Friel, D. Pouchnik, and M. D. Griswold Oligonucleotide Microarray Analysis of Gene Expression in Follicle-Stimulating Hormone-Treated Rat Sertoli Cells Mol. Endocrinol., December 1, 2002; 16(12): 2780 - 2792. [Abstract] [Full Text] [PDF]
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