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Department of Anatomy and Neurobiology,3 Department of Obstetrics and Gynecology,4 Cooperative Reproductive Science Research Center,5 Morehouse School of Medicine, Atlanta, Georgia 30310-1495
Department of Cell Biology and Neuroscience,6 University of South Carolina School of Medicine, Columbia, South Carolina 29208
Department of Biology,7 University of Texas, San Antonio, Texas 78249
Department of Cell Biology and Physiology,8 University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
Department of Pharmaceutical Sciences,9 University of Pittsburgh School of Pharmacology, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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male reproductive tract, testis, developmental biology, sperm motility and transport, spermatogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To identify the cis-regulatory elements present in the Ldh3 promoter, we initially performed in vivo DNase 1 footprinting analysis in isolated pachytene spermatocytes. These molecular studies detected 3 upstream hyposensitive sites and a single footprint associated with the SP-binding GC-box element present in the Ldh3 promoter [6]. Two germ cell-specific trans-acting factors interacting with the GC-box element of the Ldh3 promoter were subsequently identified using electrophoretic mobility gel shift assays. These included a member of the SP family of transcription factors that was demonstrated to bind directly to the Ldh3 consensus GC-box domain, and another uncharacterized germ cell factor that was shown to bind specifically at an overlapping site [7]. DNA sequence analyses have also identified SP-binding GC-box elements in the promoter regions of ldh1 [8, 9]; ldh2 (Thomas and Yang, GenBank accession number AF174288); and in a number of other well characterized testis-specific genes including Pgk2 [10, 11], Pdha2 [12, 13], and histone H1t [14].
The SP family of transcription factors that bind to the consensus GC-box sequence 5'-(G/T)(G/A)GGCG(G/T)(G/ A)(C/T)-3' belongs to a class of glutamine-rich, zinc-finger transactivators. The transcription factor SP1, originally identified by Kadonaga et al. [15], is considered the prototype for these zinc-finger GC-binding factors that include SP2 [16], SP3 [16, 17], SP4 [17], and the Krupple factors [18–24]. Substantial variations in the level of Sp1 transcript expression were shown to occur in mouse tissues and cells during development [25]. In fact, these investigators identified 3 different Sp1 transcripts (8.8, 8.2, and 2.5 kilobases [kb] in size) that were found to be expressed in differentiating germ cells during mouse spermatogenesis [26].
In the present study, we isolated additional members of the SP1 transcription factor family expressed in male germ cells by screening a mouse pachytene spermatocyte cDNA library. The expression patterns of the corresponding Sp1 transcripts expressed in the respective differentiating male germ cell populations were examined using a cell type-specific reverse Northern blot, quantitative real-time polymerase chain reaction (PCR) assays, and in situ hybridization analysis. Immunohistochemcial analysis of histological sections of the mouse seminiferous epithelium were performed to demonstrate that the encoded 60-kDa and 90-kDa SP1 proteins are expressed in differentiating germ cells. The functional activities of these SP1 transcription factors were analyzed in SP-deficient Drosophila SL2 cells stably transfected with Sp1 cDNA expression vectors by transfection/transient expression assays using luciferase reporter vectors driven by Ldh2, Ldh3, and Creb promoters. Collectively, these studies have confirmed that the 60-kDa and 90-kDa SP1 transcription factor isoforms encoded by the Sp1 transcripts of 3.2, 3.7, and 4.1 kb are functional SP1 transcription factors. This suggests that these SP1 transcription factors are able to mediate transactivation of specific target genes such as Creb, Ldh2, and Ldh3 that are expressed in a stage- and cell type-specific manner during spermatogenesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Seminiferous cords and tubules were prepared from Swiss Webster mice (Charles River Breeding Laboratories) by collagenase treatment. Monodispersed suspensions of spermatogenic cells were prepared from the seminiferous cords/tubules with collagenase and trypsin digestion [27, 28]. Primitive type A spermatogonia were isolated from the testes of 6-day-old prepubertal mice, and types A and B spermatogonia were isolated from the testes of 8-day prepubertal mice (300 animals). Preleptotene, leptotene/ zygotene, and early pachytene spermatocytes (P17) were isolated from seminiferous epithelia of Day 17 mice (100 animals) [28]. Pachytene spermatocytes (P60), round spermatids (steps 1–8), and residual cytoplasmic bodies were isolated from testes of >60-day-old mice (60 animals). The germ cells were separated by velocity sedimentation at unit gravity on 2%–4% BSA gradients [27]. Adult pachytene and round spermatids were >95% pure. Populations of the other spermatogenic cell types were >85% pure based on examination of cell morphology under phase contrast optics. Animals used in these studies were maintained and killed according to procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Approval for these studies was received from the Morehouse School of Medicine institutional animal care and use committee.
Isolation of Total RNA and Amplification and Isolationof Sp1 cDNAs
Cytoplasmic RNA was isolated using the Clontech Micro-scale total RNA separator kit according to protocols supplied by the manufacturer (Clontech, Palo Alto, CA). Total RNA isolated from purified pachytene spermatocytes was treated with DNase 1 before the synthesis of double-stranded DNA using the SMART PCR cDNA synthesis kit (Clontech). First-strand synthesis was performed (42°C, 1 h) using a modified oligo dT primer (10 µM) and the SMART 5-oligonucleotide (10 µM) in 1x first-strand buffer (50 mM Tris-HCl pH 8.3, 75 mM KCl, 6 mM MgCl2, and 2 mM dithiothreitol [DTT]), to which 1 mM of deoxynucleotide triphosphates (dNTPs) and 200 U of Maloney murine leukemia virus reverse transcriptase were added. Long-distance PCR (LDPCR) amplification was performed with 2 µl of first-strand reaction containing 1x KlenTaq PCR buffer, 200 µM dNTPs, 10 µM 5' PCR primers (5'-AAGCAGTGGTATCAACGCAGAAGT-3') and 1x Advantage KlenTaq polymerase mix (Clontech). PCR conditions were 27 cycles at 95°C for 15 sec, 65°C for 30 sec, and 68°C for 10 min. The double-stranded cDNAs were subcloned into Invitrogen pCRII TA vectors (Invitrogen, Carlsbad, CA).
After transfection of competent bacteria, the resulting bacterial colonies were blotted onto nitrocellulose filters, and these filters were screened using a 1.5-kb human Sp1-specific hybridization probe [29]. Positive colonies were selected, the plasmid DNA was purified, and the cDNA inserts were characterized by DNA sequence analysis using flanking Sp6 and T7 universal primers.
Reverse Northern Blot Analysis
Total RNA were purified from A/B spermatogonia; from preleptotene, leptotene/zygotene, and pachytene spermatocytes; and from round spermatids as described above. The total RNA samples were treated with DNase 1 and double-stranded cDNAs were synthesized using SMART PCR cDNA amplification techniques as previously described (Clontech). The PCR-amplified LDPCR double-strand DNA samples were resolved on a 1% agarose gel and blotted onto a nylon membrane, prehybridized with Express Hyb solution (Clontech), and probed using the control and Sp1 32P-labeled probes. PCR fragments representing the 0.8-kb 5'-Sp1 probe and 1.5-kb 3'-Sp1 probe derived from the 4.1-kb Sp1 cDNA were subcloned in Bluescript vectors (Stratagene, La Jolla, CA). Labeled hybridization probes were generated from these cloned fragments, which were isolated from the respective vectors using XhoI and XbaI, gel purified, and labeled with 32P-GTP using random-priming reaction kits (Boehringer-Mannheim, Indianapolis, IN). Northern hybridization assays were performed as previously described [5].
Quantitative Real-Time PCR Analysis
The reverse transcription reactions were performed at 37°C for 1 h using SensiScript Reverse Transcriptase (Qiagen, Valencia, CA) with 1 µM Random hexamers (Invitrogen), 1 µM dNTPs, total RNA (200 ng), and RNAsin Ribonucleotide Inhibitor (1 U). Quantitative SYBR green real-time PCR was performed in an iCyclerQ (Bio-Rad Laboratories, Hercules, CA). Gene-specific Sp1 PCR primer sequences were designed using Oligo Primer Analysis Software (National Biomedical Systems, San Diego, CA). For 4.1-, 3.7-, and 3.2-kb Sp1 transcripts, respectively, the transcript-specific sequences were as follows: 5'-CTTGCCTCGTCAGCGTCCGCGTTTTTC-3' and 5'-GAAAGTTGTGTGGCTGTGAGGTCA-3,', 5'-GCTTGCACTCCTTTCCTGACTTAATCC-3' and 5'-GAAAGACTTCTGATTTCTGAT-3,', and 5'-GGCGGAGGAGGGCAGCAGAACCAGACATCA-3' and 5'-CCGAATGATGATGGGACCAGAGTTTT-3'.
The PCR reaction mixture consisted of a cDNA template from 200 ng of total RNA, 0.3 µM of each primer and 1x QuantiTect SYBR green PCR mixture (Qiagen) containing HotStart Taq DNA polymerase, dNTPs (1 µM) and SYBR green amplified with a precycling activation at 95°C for 15 min, followed by 27 cycles of denaturation at 95°C for 15 sec, annealing at 54°C for 30 sec, and extension at 72°C for 30 sec. Internal reference (18S rRNA) and reagent controls (minus RNA or cDNA) were included in each assay. The assays were performed in triplicate to verify the results, and the mean threshold cycle (Ct) number was used to calculate the relative gene expression levels using the comparative 2–
Ct method [30]. Finally, melting curve analyses were performed to demonstrate the absence of any nonspecific PCR products amplified in these PCR reactions. The graphical data reported represent the mean ± SEM from triplicate samples from 2 independent studies. Statistical comparisons were made by using an unpaired t-test or analysis of variance. Significant difference defined as P < 0.05.
In Situ Hybridization
Male mice that had been killed were perfused with 4% paraformaldehyde in PBS, and the testes were removed and embedded in paraffin. The embedded tissues were sectioned (10 µm), mounted on poly(L-lysine) coated slides and analyzed by in situ hybridization. Briefly, hybridizations were performed in a humidified chamber for 12 h at 50°C using 4 x 106 cpm per slide of 33P-labeled sense or antisense RNA in a buffer containing 50% formamide, 20 mM DTT, 10% dextran sulfate, and 1x salts (0.3 M NaCl, 10 mM Tris-HCl pH 8.0, 10 mM NaH2PO4 pH 6.8, 5 mM ethylenediamine tetraacetic acid [EDTA], and 1x Denhardt solution). Sense and antisense RNAs were obtained by "in vitro" transcription initiated at the T3 or T7 promoter (or both) of the Bluescript plasmid (Stratagene) into which 5'- and 3'-specific Sp1 cDNA fragments had been subcloned. After hybridization, the preparations were washed for 4 h in rinse buffer (50% formamide, 10 mM DTT, and 1x salts [0.3 M NaCl, 10 mM Tris-HCl pH 8.0, 10 mM NaPO4 pH 6.8, 5 mM EDTA, and 1x Denhardt]) at 50°C, incubated in 20 mg/ml RNase A in 1.5 M NaCl, 10 mM Tris-HCl pH 8.0, and 1 mM EDTA at 37°C, and followed by 16 h in WDTT at 50°C. The slides were dehydrated in ethanol, coated with Kodak NTB-2 emulsion, and developed after exposure for 2 to 3 wk. The slides were counterstained with hematoxylin, and visualized and photographed using an Olympus microscope (Olympus, Melville, NY) under brightfield and darkfield illumination.
Immunohistochemical Analysis
Testes from young adult (2-mo-old) Swiss Webster mice were collected and frozen on dry ice. Frozen sections (10 µm) were cut, treated with 0.3% H2O2 in 50 mM PBS for 15 min, and rinsed extensively with PBS. All antibodies were diluted with 50 mM PBS containing 0.05% Triton X-100 and 5% normal goat serum. Sections stained with the SP1 glutamine-rich (GluR) peptide antibody were incubated with primary antibody (1: 25 000) for 3 days at 4°C. Controls received no primary antibody or were incubated with preimmune serum similarly diluted or with primary antibody plus blocking SP1 peptide at a concentration of 5 µg/ml. These sections were then rinsed with PBS and incubated with biotinylated goat anti-rabbit immunoglobulin G (diluted 1:500; Vector Laboratories, Burlingame, CA) for 3 h at room temperature. Sections were rinsed again with PBS and then incubated with avidin-biotin-horseradish peroxidase (HRP) complex (Vectastain Elite kit; 5 µl reagent A + 5 µl reagent B per milliliter of PBS; Vector Laboratories) for 3 h at room temperature. Sections were then rinsed with 50 mM Tris pH 7.6, placed into a solution of 3-3' diaminobenzidine (DAB; 0.25 mg/ml in 50 mM Tris; Sigma-Aldrich, St. Louis, MO) for 5 min, and then incubated with DAB solution containing 0.0075% NiCl and 0.01% H2O2 for 10 min. Following staining, sections were rinsed with PBS, mounted onto glass slides, coverslipped with DePeX (Gallard-Schlessinger), and viewed with a Leica DMR microscope.
Western Blot Analysis of Nuclear Extracts from Spermatogenic Cells Using SP1 Peptide Antibodies
Testes were dissected from killed 60- to 90-day-old Swiss Webster rats (Charles River Breeding Laboratories) and spermatogenic cells were isolated by collagenase and trypsin digestion as previously described. After trypsin treatment, the germ cells were subjected to centrifugation at 3000 x g for 10 min through enriched Krebs-Ringer bicarbonate medium (EKRB) + 2% BSA, and the pellets were washed twice with fresh EKRB. The final pellet was resuspended in cytoplasmic extraction reagent (CER1) from the NE-PER nuclear extraction kit and processed according to the manufacturer's instructions (Pierce Biotechnology, Rockford, IL). Briefly, the EKRB-washed germ cells were pelleted by centrifugation at 500 x g for 2–3 min. The supernatant was removed and the pellet was resuspended in 200 µl of cytoplasmic extraction reagent (CER1) and vortexed vigorously to lyse the cells. The nuclei were pelleted at 16 000 x g for 5 min, the supernatant was removed, and the nuclear pellet was resuspended in 100 µl of ice-cold nuclear extraction reagent. The resuspended nuclei were incubated on ice and vortexed every 10 min for a total of 40 min. After a 10-min centrifugation (16 000 x g), the supernatant containing the nuclear extract was removed and stored at –80°C. Protein concentrations were determined using the Bradford Assay Kit (Bio-Rad Laboratories, Richmond, CA). For gel electrophoresis, nuclear extracts were diluted in 2x SDS gel loading buffer (100 mM Tris Cl pH 6.8, 20 mM DTT, 4% SDS, 0.2% bromophenol blue, and 20% glycerol). The nuclear extracts (50 µg total protein/lane) were resolved on 10% SDS-PAGE and transferred onto nitrocellulose membranes (Amersham Biosciences, Arlington, CA) with prestained protein markers (Invitrogen). The membrane was probed with peptide antibody (1:200) raised in rabbits to synthetic peptides representing the SP1 GluR domain A region (SP1-GluR; KEQSGNSTNGSNGSESG). The antigen/antibody complexes formed were detected with anti-rabbit HRP antibody diluted in 0.1% TTBS buffer (TBS; 0.1% Tween-20 containing 0.5% nonfat dry milk).
Plasmid Constructions, Transient Transfectionsinto Drosophila SL2 Cells, and Western Blot Analysisfor In Vivo SP1 Expression
Sp1 cDNAs of 4.1, 3.7, 3.2, and 2.5 kb were cloned upstream of the V5 tag sequence in pAc5.1V5-His plasmids (Invitrogen). Drosophila Schneider SL2 cells (3 x 106 cells) were cultured in complete Schneider Drosophila medium (Invitrogen) containing 10% heat-inactivated fetal bovine serum (FBS; Gibco-BRL, Rockville, MD) at room temperature for 6–16 h until cells reached concentrations of 2–4 x 106 cell/ml. For transfection, 19 µg of the Sp1 recombinant plasmid DNA in 2 M CaCl2 (300 µl) was mixed with an equal volume of 2x Hepes-buffered saline (300 µl; 50 mM Hepes, 1.5 mM Na2HPO4, and 280 mM NaCl pH 7.1) by a continuous swirling and incubated at room temperature for 30–40 min. The DNA mixture was then added dropwise into Drosophila SL2 cell suspension (1 x 106 cells/ml) and incubated at room temperature for 16– 24 h. The calcium phosphate solution was removed and the cells were washed twice with Schneider Drosophila medium plus 10% FBS (complete medium) by centrifugation at 100 x g for 5–10 min, and incubation was continued in fresh complete medium. The transfected cells were harvested on Days 2, 3, 4, and 5, and assayed by Western blot immunoassay with anti-V5 antibody for the expression of SP1 fusion protein.
Expression of SP1 Transcription Factor Isoforms in Stably Transfected Drosophila Schneider SL2 Cells
Drosophila Schneider SL2 cells were incubated in complete Schneider Drosophila medium (Invitrogen) containing 10% heat-inactivated FBS (Gibco-BRL) at room temperature. Recombinant pAc5.1/V5-His cDNA expression vectors (Invitrogen) were used for expression of the SP1 transcription factors. Briefly, Drosophila SL2 cells (3 x 106) were grown in complete medium (3 ml) at room temperature until they were 60%–80% confluent. For transfection, the plasmid solution A (300 µl) containing Sp1 recombinant pAc5.1/V5-His cDNAs (19 µg) plus pCoHygro (1 µg), the selection vector, in 0.24 M CaC12 was slowly added to an equal volume (300 µl) of solution B containing 2x Hepes-buffered saline (50 mM Hepes, 1.5 mM NaH2PO4, and 280 mM NaCl pH 7.1) with continuous mixing, incubated at room temperature for 30–40 min, and this solution was added dropwise to the Drosophila SL2 cells (1 x 106 cell/ml) with continuous swirling. After incubation for 16–24 h at room temperature, the calcium phosphate solution was removed by centrifugation at 1000 x g for 5 min, and the transfected cells were washed twice with complete medium and incubated in complete medium at room temperature. After incubation for 2 days, the medium was replaced with fresh complete selective medium (3 ml) containing hygromycin B (300 µg/ml). The selective medium was replaced every 4–5 days until resistant colonies appeared. The resistant cells were replated into new plates containing selective medium, passed until cell density reached 6–20 x 106 cells/ml, harvested, and stored in liquid nitrogen.
Transfection of Ldh2, Ldh3, and Creb Promoter/Luciferase Reporter Constructs into Stably Transfected Drosophila SL2 Cells
Drosophila SL2 cells stably transfected with vectors expressing the 3.2- or 4.1-kb germ cell Sp1 transcripts or the 8.8-kb somatic Sp1 transcript were transfected using Lipofectin (Invitrogen) according to the manufacturer's instructions for 1 h with 1 µg of the reporter plasmids Ldh2 Luc, Ldh3 Luc, or –1264 Creb Luc [31]. Whole cell lysates were prepared 48 h after transfection and luciferase assays were performed. Luciferase activity was normalized for protein concentration as determined by the Bradford assay (Bio-Rad Laboratories, Richmond, CA). The luciferase reporter expression data are presented as the mean ± SEM for 3 experiments that were performed in duplicate.
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RESULTS |
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To isolate cDNAs corresponding to Sp1 transcripts expressed in germ cells, a mouse pachytene spermatocyte cDNA library was screened with a probe containing the human Sp1 3'-untranslated region (3'-UTR). Four cDNAs were isolated and characterized by DNA sequence analysis. Two of these cDNAs corresponded to 4.1-kb and 3.7-kb Sp1 transcripts that encode full-length SP1 transcription factors. The other two cDNA clones that were isolated represented alternatively spliced 3.2-kb and 5'-truncated 1.4-kb Sp1 transcripts (Fig. 1). A search of the European Molecular Biology Laboratory and GenBank databases using BLASTN and FASTA programs revealed matches with previously submitted Sp1 transcription factor cDNA sequences (Park et al., personal communication, GenBank accession number AF022363; Yajima et al., [32], GenBank accession number AF062566; and Persengiev et al., [25], GenBank accession number S79832). Figure 1 shows a schematic representation and alignment of the cDNAs corresponding to the mouse testis Sp1 transcripts of 4.1, 3.7, 3.2, and 1.4 kb. DNA sequence alignment studies of the full-length cDNAs have indicated that the human, rat, and mouse Sp1 encoded sequences are 98% homologous in their respective open reading frame (ORF) regions. Further, DNA sequence analysis indicates that the 3'-UTR regions of the mouse cDNA clones share 97% identity with the human Sp1 cDNA [33]. The 1.5-kb Sp1-specific hybridization probe used for the reverse Northern blot and in situ hybridization studies represent sequences derived from the 3'-UTR of the 4.1-kb germ cell Sp1 transcript. The 5' Sp1-specific probe corresponds to sequences from a 0.8-kb region of the 4.1-kb transcript that contains the entire transactivation A domain. This region is deleted in the 3.2-kb Sp1 transcript identified in this study (see Fig. 1) and also in the 2.5-kb mouse germ cell Sp1 transcript that was previously isolated and characterized by Persengiev et al. [25].
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Northern Blot Hybridization Analysis of Sp1 Transcript Expression in Germ Cells
Analysis of the expression patterns of the Sp1 transcripts in differentiating germ cells was determined using Northern blot analysis. Due to their low levels of expression in the respective germ cell populations, we expected that transcripts encoding transcription factors such as SP1 would be difficult to detect by traditional hybridization studies on poly(A+) Northern blots. In this study, we used a cell type-specific reverse Northern blot to ensure that all the Sp1 transcripts expressed in differentiating germ cell populations would be detectable in our hybridization assays. This cell type-specific reverse Northern blot was constructed using total RNAs isolated from STAPUT gradient purified germ cell populations. The isolated mRNAs were initially converted to full-length cDNAs using 5'-Cap-specific and 3'-oligo dT primers. These full-length cDNAs were subsequently amplified by PCR, separated by electrophoresis on an agarose gel, and blotted onto nylon membranes. Control hybridization studies were performed using 32P-labeled Ldh3, alpha-tubulin, and beta-actin cDNA probes to validate the respective expression patterns on the reverse Northern blot. Results of these initial hybridization studies confirmed that the transcripts known to be expressed at very low levels in some germ cell populations were detectable using this reverse Northern blot (data not shown).
To analyze the germ cell-specific expression profiles of the Sp1 transcripts represented by the isolated cDNA clones, the reverse Northern blot was hybridized with the 0.8-kb 5'-Sp1 probe that corresponded to the glutamine-rich A domain and flanking regions. Hybridization studies performed using this 5'-probe detected 4 Sp1 transcripts (4.1, 3.7, 3.2, and 2.5 kb) that were expressed in the primary and secondary spermatocytes (Fig. 2A). These hybridization studies also indicated that the 3.7-kb Sp1 transcript was the most abundant Sp1 transcript expressed in preleptotene, leptotene/zygotene, and pachytene spermatocytes. Rehybridization of this blot with the 3'-UTR Sp1 probe also detected Sp1 transcripts of 4.1, 3.7, 3.2, and 2.5 kb in all the germ cell RNA samples. This 3'-UTR-Sp1 probe detected an additional 1.4-kb transcript expressed in preleptotene, leptotene/zygotene, and pachytene spermatocytes (data not shown). No further analysis was performed in this study for the 1.4-kb Sp1 transcript because DNA sequence analysis studies indicated that this 5'-truncated Sp1 transcript contained mainly 3'-UTR sequences (Fig. 1).
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Quantitative Real-Time PCR Analysis of the Relative Steady State Expression Levels of Sp1 Transcript Variants in Differentiating Germ Cells
The reverse Northern blot assay facilitated utilization of the limited yields of RNA isolated from purified germ cell populations. As shown in Figure 2, the Sp1 transcripts detected matched the sizes of the Sp1 cDNA clones identified in this study. To quantitatively determine the steady state expression levels of these Sp1 transcripts in germ cells, real-time PCR studies were performed using isolated total RNA samples and the data were analyzed by the comparative quantitation method (Ct) described by Livak and Schmittgen [30]. The fluorescent signals produced in these assays accurately represent the amount of PCR product that is amplified in real time as the reaction proceeds. The relative expression levels of the Sp1 transcripts in germ cell RNA samples were calculated by measuring the differences in threshold cycles between the Sp1 transcripts of interest and the endogenous 18S RNA (Ct).
Transcript-specific primers were designed for the Sp1 transcripts of 4.1, 3.7, and 3.2 kb; and the amplification cycle number, represented as the threshold Ct values, was measured during the exponential phase. The threshold Ct value reflects the levels of the respective Sp1 transcript variant in each of the germ cell RNA samples. The real-time PCR data shown in Figure 3 after normalization to the endogenous 18S rRNA indicates that the Sp1 transcript variants of 4.1, 3.7, and 3.2 kb are expressed in a developmentally specific manner in the primary spermatocytes during germ cell differentiation. In fact, these studies revealed that the 3.7-kb Sp1 transcript is the most highly expressed Sp1 transcript in preleptotene, leptotene/zygotene, and pachytene spermatocytes. However, the 4.1- and 3.2-kb Sp1 transcripts also showed increased levels of expression in these differentiating germ cells. The data presented in Figure 3 confirm that the patterns of expression of the individual Sp1 transcripts observed by quantitative real-time PCR analysis are similar to those observed using the reverse Northern blot assay shown in Figure 2.
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In Situ Hybridization Detection of Stage- and CellType-Specific Patterns of Sp1 Transcript Expressionin the Seminiferous Tubules
To verify the cell type-specific expression patterns of the Sp1 transcripts, in situ hybridization analyses were performed on histological sections of the adult mouse seminiferous epithelium. In the adult mouse, the program of germ cell development occurring within the seminiferous epithelium is spatially organized such that individual germ cell types can be identified in histological sections made through the tubules. The spermatogonia are localized along the basement membrane at the periphery of the tubules, whereas the more differentiated germ cells are oriented in a definitive spatial pattern as they develop and migrate toward the lumen. This precise cellular developmental program has been well characterized by histological studies of the mouse seminiferous epithelium [34].
In situ hybridizations performed with the 5'-antisense Sp1 probe detected Sp1 transcripts in all germ cells, but significantly higher levels of expression were observed in preleptotene, leptotene/zygotene, and pachytene spermatocytes (Fig. 4, A–C). Hybridization studies using the 3'-UTR antisense Sp1 probe showed a similar expression profile with higher levels of hybridization signals associated with preleptotene, leptotene/zygotene, and pachytene spermatocytes (Fig. 4, E–G). We observed lower levels of Sp1 transcript expression in spermatogonia and in round and elongated spermatids. No hybridization signals were observed with the 5' and 3' Sp1 sense probes (Fig. 4, D and H). Taken together, the spatial orientation and intensity of the hybridization signals verified that the Sp1 transcript variants detected were most abundantly expressed in primary spermatocytes during the meiotic stages of spermatogenesis. It is possible that detection of Sp1 transcripts observed in spermatogonia may be due to hybridizations of the Sp1 probes with previously identified 8.2 Sp1 transcripts [26]. These in vivo observations confirm the Sp1 transcript expression results derived from both the reverse Northern Blot and quantitative real-time PCR studies.
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Immunohistochemical Analysis of SP1 Transcription Factor Expression Patterns in Differentiating Germ Cells
To determine the patterns of SP1 protein expression in differentiating germ cells using immunohistochemical analysis, antibodies were produced in rabbits from synthetic SP1 peptides. Prior to use on the testicular tissue sections, Western blots containing separated nuclear proteins from purified germ cells were analyzed using these SP1 peptides antibodies. As shown in Figure 5A, SP1 peptide antibodies corresponding to the GluR region recognized both the 90-kDa and 60-kDa SP1 transcription factor isoforms.
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In histological sections through the mouse adult seminiferous epithelium, the differentiating germ cells are spatially and temporally arranged in a definitive pattern that facilitates identification of specific germ cell types. In Figure 5B (I and II) immunohistochemical studies performed using the GluR domain A SP1 peptide antibody demonstrated that this antibody recognized the 60-kDa and 90-kDa SP1 proteins expressed mainly in primary spermatocytes. Nonspecific immunostaining was not observed with the preimmune rabbit antisera (Fig. 5B, III). Further, additional negative control immunohistochemical studies indicated that an excess of the immunizing GluR peptides blocked formation of the SP1 protein-GluR SP1 antibody complexes (data not shown).
The Sp1 Transcript Variants Expressed in Mouse Germ Cells Encode 90-kDa and 60-kDa SP1 Proteins
DNA sequence analysis of cDNA clones corresponding to the Sp1 transcripts of 4.1 and 3.7 kb expressed in germ cells indicated that they have similar ORFs and would give rise to identical 90-kDa SP1 proteins. An examination of the ORFs of the 3.2- and 2.5-kb Sp1 transcripts expressed in germ cells predicted that these transcripts encode identical 60-kDa SP1 proteins due to deletion of one of the two GluR transactivation domains present in the 90-kDa SP1 protein. To confirm that the respective cDNAs produced SP1 proteins of the expected sizes, the 4.1- and 3.2-kb Sp1 cDNAs were sublconed in all 3 reading frames in Drosophila expression vectors and transfected into SP-deficient Drosophila SL2 cells. Figure 6 shows the result of a Western Blot analysis of extracts from the transfected Drosophila SL2 cells. Figure 6A shows that the 4.1-kb Sp1 cDNA produced a 90-kDa SP1 protein in reading frame A with maximum expression of this SP1 protein occurring 3 days after transfection. Results in Figure 6B show that the 3.2-kb Sp1 cDNA produced a 60-kDa SP1 protein in reading frame A with maximum expression of this SP protein occurring at 2 days after transfection. In Figure 6C, the control expression vector produced a 118-kDa ß GAL (beta galactosidase) protein throughout the 5-day period of the experiment with the optimum expression levels of this protein occurring at 2 days after transfection.
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Transactivation of the Creb, Ldh2, and Ldh3 Promoters by the 60-kDa and 90-kDa SP1 TranscriptionFactor Isoforms
The transactivation capabilities of the 60-kDa and 90-kDa SP1 isoforms were first investigated in transient transfection studies employing a reporter gene driven by the promoter of the Creb transcription factor gene. The Creb promoter contains 3 SP1 binding sites of which 1 is essential for promoter activity [35]. The transfections were performed in Drosophila SL2 cells that do not normally express endogenous SP1 factors but were stably transfected with 4.1-, 3.2-, or 8.8-kb Sp1 cDNAs. The results shown in Figure 7 indicated that all 3 of the Sp1 transcripts encode active SP1 transcription factors that induce Creb promoter activity over basal levels. The 4.1-kb Sp1 transcript encoding the 90-kDa SP1 factor resulted in the highest transactivation activity. The 8.8-kb Sp1 transcript encoding the 110-kDa SP1 factor and the 3.2-kb Sp1 transcript encoding the 60-kDa SP1 factor provided lower relative transactivation activities of 43% and 30% in comparison to the 4.1-kb Sp1 transcript, respectively.
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Studies were extended to include the germ cell-specific Ldh3 promoter that contains a single SP-binding GC-box and Ldh2 promoter that contains 3 SP-binding GC-box domains. Previous studies demonstrated that the GC-box domain present in the Ldh3 promoter is required for transactivation of the gene [6, 36]. Figure 8 shows the relative activities of the Ldh2 and Ldh3 promoters in Drosophila SL2 cells stably transfected with the respective Sp1 DNA expression vectors. These studies indicated that activation of both promoters were mediated by the stably expressed SP1 factors. The 8.8-kb Sp1 cDNA encoding a 110-kDa SP1 factor induced the highest levels of activity directed by the Ldh2 promoter. In comparison, the 4.1-kb Sp1 cDNA encoding a 90-kDa SP1 factor showed 90% relative transactivation activity, whereas the 3.2-kb Sp1 cDNA encoding the 60-kDa SP1 factor showed 49% relative activity for Ldh2 promoter-driven luciferase expression. In contrast, for studies performed with the Ldh3 promoter-Luc reporter construct, the 4.1-kb Sp1 transcript encoding the 90-kDa SP1 factor resulted in the greatest transactivation activity measured at 40% relative to that observed for Ldh2 promoter-driven luciferase expression in the presence of the 8.8-kb Sp1 transcript. The 8.8- and 3.2-kb Sp1 transcripts induced the Ldh3 promoter 27% and 12%, respectively, relative to the 4.1-kb Sp1 transcript. Induction of Creb promoter activity by the Sp1 transcripts was more similar to that of Ldh3 than Ldh2.
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DISCUSSION |
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In the current study, we have demonstrated that the Sp1 transcripts of 4.1, 3.7, and 3.2 kb are expressed in a stage- and cell type-specific manner in differentiating male germ cells using a cell type-specific reverse Northern blot, quantitative real-time PCR and in situ hybridization assays. These studies suggest that 2 other functionally distinct populations of Sp1 transcript variants exist in differentiating mouse germ cells in addition to the 8.8/8.2-kb Sp1 transcripts encoding 110-kDa SP1 factors. The larger Sp1 transcripts (4.1 and 3.7 kb) encode 90-kDa SP1 transcription factors that contain 2 GluR transactivation domains (A and B). In contrast, the smaller Sp1 transcripts (3.2 and 2.5 kb) encode 60-kDa SP1 transcription factors that contain only a single GluR B transactivation domain. Identification of the 1.4-kb 5'-truncated Sp1 transcript expressed in primary spermatocytes that contained only the 3'-UTR was interesting, and its functional significance is currently under investigation. Western blot analysis of germ cell nuclear extracts using SP1 GluR peptide antibodies indicated that 60-kDa and 90-kDa SP1 proteins are expressed in differentiating germ cells during spermatogenesis. Immunohistochemical analysis of histological sections through the mouse seminiferous epithelium using this SP1 GluR peptide antibody confirmed that these SP1 proteins were expressed mainly in the primary spermatocytes. The specificity of this antibody was demonstrated by 2 independent negative controls that included use of preimmune sera and treatment of the SP1 GluR antibody with the corresponding immunizing peptide.
The functional activities of these SP1 transcription factor isoforms were investigated using Ldh and Creb promoter-directed luciferase reporter expression studies. Due to the lack of appropriate germ cell tissue culture lines, these studies were performed in SP-deficient Drosophila SL2 cells that were stably transfected with cDNA expression vectors for the respective SP1 transcription factor isoforms. These transfection/transient expression studies verified that both the 60-kDa and 90-kDa SP1 proteins expressed in mouse germ cells are able to potently transactivate the Ldh2, Ldh3, and Creb promoters. Further insights into the specific roles that these SP1 transcription factors play during spermatogenesis, however, will require technically challenging in vivo RNA knockdown and overexpression studies using recently developed lentiviral gene transfer techniques [37– 39]. These SP1 knockdown and overexpression lentiviral vectors are currently being developed in the laboratory and will be used in combination with Serial Analysis of Gene Expression (SAGE), microarray analysis, or both, to identify the SP1 target genes activated in stage- and cell type-specific manners in differentiating mouse germ cells.
Multiple members of the SP-family of transcription factors are coexpressed in a number of cell types including differentiating mouse germ cells. Despite their ubiquitous distribution, several published studies have demonstrated that SP factors are able to mediate restricted developmental and cell type-specific target gene expression. Studies by Park (personal communication), Yajima et al. [32], and Persengiev et al. [26] observed that Sp1 transcript variants of 8.8, 8.2, 4.1, 3.7, and 2.5 kb were expressed in rat and mouse somatic tissue and germ cells. However, none of these studies addressed the functional role or the significance of the multiple SP1 transcription factors encoded by these Sp1 transcripts in mediating tissue- and cell type-specific gene expression. The complex mechanisms involved in SP-mediated gene activation/repression have only recently been appreciated, because these regulatory mechanisms are currently being analyzed in a number of somatic cells [40, 41]. The major conclusion of these studies is that SP1 transcription factor activity is likely to depend on cellular context and promoter architecture. Furthermore, coexpressed SP1 factors are able to act synergistically to transactivate promoters containing multiple GC-box sites such as in the Ldh2 promoter. This is due to formation of oligomers and higher-order complexes that are mediated through interactions of the A, B, and D domains present in the SP1 proteins [42, 43]. It is also possible that the presence or absence of these domains in the respective SP1 isoforms can have functional consequences due to alterations in SP1 protein/coregulator interactions.
To our knowledge, neither the expression patterns, functional roles, nor significance of the Sp1 spliced variants of 4.1, 3.7, and 3.2 kb have been thoroughly investigated previously in either somatic or germ cells. SP-binding GC-box regulatory domains are present in the promoter regions of several genes expressed in a developmentally specific manner in germ cells, including Creb, Pgk2, Pdha, Ldh1, Ldh2, Ldh3, and Histone Hlt genes. In addition, a number of growth factor receptor genes that are developmentally expressed in germ cells including retinoic acid receptor [44], Oct 3/4 [45, 46], and c-kit [47] also contain SP-binding GC-box domains in their promoter regions. Furthermore, recent transgenic studies have indicated that the Cdk-2 gene that is essential for meiotic phase of spermatogenesis contains SP-binding GC-box elements in the promoter region [48]. The selective program of transactivation of these genes observed during germ cell differentiation is most likely mediated by SP1 factor/coregulator interactions. In our laboratory, yeast 2-hybrid and chromatin immunoprecipitation studies are now in progress to identify the stage- and cell type-specific coregulators that interact with the SP1 transcription factors identified in study.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Kelwyn Thomas, Department of Anatomy and Neurobiology, Morehouse School of Medicine, 720 Westview Drive SW, Atlanta, Georgia 30310-1495. FAX: 404 752 1028; kethomas{at}msm.edu
Received: 7 April 2004.
First decision: 6 May 2004.
Accepted: 8 December 2004.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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