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in Rat Testicular Sertoli Cells1
a Center for Reproductive Biology, Department of Genetics and Cell Biology, Washington State University, Pullman, Washington 99163-4231
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in Sertoli cells. REB and REBß are spliced variants of the REB gene that is implicated in cell-specific gene expression as part of dimeric bHLH complexes acting on E-box response elements. Although both the transcripts of the REB gene are widely expressed, differential expression of the REB gene transcripts REB and REBß has been shown. In the current study, a polymerase chain reaction (PCR)-based approach demonstrated that REB gene transcripts are expressed in the testis. Characterization of the REB transcripts suggested that REB is the major splice variant in Sertoli cells. PCR primers specifically designed to amplify either REB or REBß demonstrated that Sertoli cells express only REB, not REBß. REBß was present in the RNA samples obtained from whole testis, suggesting expression in other testicular cell types. A Northern blot analysis of RNA from Sertoli cells treated with or without FSH or cAMP demonstrated that REB is not hormone responsive. REB was also found to be expressed in germ cells and peritubular cells. An immunocytochemical analysis demonstrated that REB is predominantly expressed by Sertoli cells within the seminiferous tubules. The activity of REB in Sertoli cells was demonstrated with an E-box gel shift with Sertoli cell nuclear extracts. The E-box gel shift was found to contain REB and E47/E12 bHLH proteins. In summary, the Sertoli cell is one of the first cells shown to specifically express the REB isoform of the REB gene. The results are discussed in relation to the possibility that Sertoli cells may express a cell-specific bHLH protein that can preferentially dimerize with REB.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous observations have suggested that the basic helix-loop-helix (bHLH) class of transcription factors may influence Sertoli cell functions. Down-regulation of transferrin [9] and c-fos promoter (unpublished results) activity by overexpression of Id, a dominant negative HLH protein, suggests that bHLH proteins influence Sertoli cell differentiation. The role of bHLH proteins in Sertoli cells is also supported by the observation that several Sertoli cell genes contain highly conserved E-box sequences within their proximal promoter regions as potential targets of bHLH proteins [9].
Basic HLH proteins have previously been shown to regulate cell-specific gene expression. These proteins form heterodimers consisting of ubiquitous class A and tissue-specific class B bHLH proteins [10, 11]. This family is characterized by a common structural motif, the HLH domain, which mediates protein interactions to form homo- and heterodimers [12]. The presence of a highly charged basic region in each of the dimerization partners immediately upstream of the HLH region forms a specific DNA-binding domain that can recognize the consensus DNA sequence, CANNTG, known as an E-box. The activity of these factors is often negatively regulated by another class of HLH proteins that lack the basic domain and are known as inhibitors of differentiation or Id [13]. The class A bHLH proteins HEB [14], E2–2 [15], and the E2A gene products E12 and E47 [16] are capable of forming heterodimers with class B HLH proteins like MyoD [10, 11].
REB is a class A bHLH protein and is the rat homologue of the human E-box-binding protein (HEB, [14]). HEB/REB is expressed in a wide variety of tissues [17]. Expression of REB/HEB in the testis and more specifically in the Sertoli cells has not been reported. In contrast to its human homologue, REB is expressed as two alternatively spliced variants, REB and REBß. REBß is characterized by a 24-amino acid insertion in the leucine heptad repeat near the amino terminal of the bHLH domain and may function as an inhibitory domain. The tissue-specific expression of REB RNA splicing may be involved in determining tissue-specific combinations of heterodimeric complexes between ubiquitous and tissue-restricted bHLH proteins.
The current study demonstrates that Sertoli cells specifically express REB. This is the first cell type shown to specifically express only one transcript of the REB gene. The demonstration that REB is expressed in Sertoli cells supports previous observations that bHLH proteins appear to act as regulators of Sertoli cell function and differentiation. The hypothesis is discussed that an as-yet-unidentified class B Sertoli cell-specific bHLH protein may be available for dimerization with the class A bHLH protein REB.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sertoli cells were isolated from the testis of 20-day-old rats by a modified procedure described previously [9]. The purified Sertoli cells were more than 98% pure, with 2% contamination of peritubular cells as determined cytochemically [9]. The isolated Sertoli cells were then plated under serum-free conditions in 150 x 20-mm tissue culture dishes (Nunclon; Nunc, Naperville, IL) at a concentration 108 cells per plate. Cells were maintained in a 5% CO2 atmosphere in Ham's F-12 medium (Gibco, Grand Island, NY) with 0.01% BSA at 32°C. Sertoli cells were treated with either FSH (100 ng/ml; ovine FSH-16, National Pituitary Agency, Harbor-UCLA Medical Center, Torrance, CA), dibutryl cAMP (dbcAMP; 100 µM/µl), or vehicle alone (Ham's F-12, control). These optimal concentrations of FSH and cAMP have previously been shown to dramatically stimulate cultured Sertoli cell differentiated functions [18, 19]. The cells were cultured under serum-free conditions for a maximum of 5 days with a change of medium and treatment after 48 h of culture. Cell number and viability did not change during the culture in the absence or presence of treatment.
RNA Preparation
Freshly isolated or cultured Sertoli cells were lysed directly using TRI Reagent (no. T9424; Sigma Chemical Co., St. Louis, MO). The cell lysate was then passed several times through a Pasteur pipette to form homogenous lysate. Tissues (skeletal muscle, brain, liver, kidney, and detunicated testis) were prepared in TRI Reagent (5% w:v) and were homogenized in a tissue homogenizer (Tissue Tearor; BioSpec, Bartlesville, OK). To avoid any possible contamination of extracellular material and high-molecular weight DNA in the final RNA samples, the homogenate was centrifuged at 12 000 x g for 10 min at 4°C. Total RNA was then isolated from the cell lysate and whole tissue homogenate according to the manufacturer's protocol for RNA isolation using TRI Reagent. The final RNA pellet was dissolved in distilled water at a concentration of 1 mg/ml.
Polymerase Chain Reaction (PCR)
Total RNA (2 µg) was reverse-transcribed in a final volume of 20 µl containing 20 U RNasin (Promega, Madison, WI), 200 µM each of dATP, dCTP, dTTP, and dGTP, 1 µg oligo dT (Pharmacia, Piscataway, NJ), 10 µM dithiothreitol, and 200 U of Moloney murine leukemia virus (MMLV) reverse transcriptase (BRL, Gaithersburg, MD) in the MMLV first-strand synthesis buffer supplied by the manufacturer (BRL). The RNA and oligo dT primer in the buffer were first denatured for 5 min at 65°C and then cooled on ice before addition of nucleotides and enzyme. The reverse transcriptase reaction was carried out at 37°C for 1 h. PCR was performed using the GeneAmp 9600 (Perkin Elmer Cetus, Norwalk, CT) with 30 cycles as follows: 94°C, 1 min (denaturation); 58°C, 2 min (primer annealing); 72°C, 1 min (primer extension). Each PCR reaction contained 250 pg reverse-transcribed DNA; 1 µM of each 5' and 3' oligonucleotide primer; 2.5 U Taq polymerase (AmpliTaq; Perkin Elmer); and 200 µM of each dATP, dCTP, dGTP, and dTTP. After amplification, the product of each reaction was subjected to electrophoresis through a 1.5% agarose gel, and bands were visualized by ethidium bromide staining.
The primer pair sequences used were obtained from published sequences of REB (GenBank acc. no. S53920) and REBß (GenBank acc. no. S53921) and were synthesized from commercial sources. Primer pairs were designed to amplify a region 5' toward the bHLH domain of the REB gene and to selectively amplify either REB or REBß (Fig. 1). To distinguish possible contamination of RNA with DNA, the reverse transcription (RT) reaction was performed without MMLV reverse transcriptase. The absence of any product in the amplification reaction using such a reverse-transcribed preparation indicated the absence of any contaminating DNA in our RNA samples. The sequence and position of the primers were as follows: REB 5' primer (939)—5' CTC ACT TCA CTC GCA GTC TCG AAT GGA; REBß 5' primer (939)—5' TGC AAT GTC CCT CTT AAA GGA TGT TGA GC; REB and REBß 3' primer (REB: 1578; REBß: 1650)—5' GTA AGT TCC TCG AGC CGG CTT ACA GA. As shown in Figure 1, the REB-specific 5' primer was designed to span the junction of 72-base pair (bp) insertion specific to REBß. The REBß-specific 5' primer was designed within the 72-bp insertion absent in REB. Each RT reaction was performed using three different samples. The PCR-based amplification reactions were carried out in duplicate on each reverse-transcribed RNA sample. Simultaneous PCR reactions were also carried out using primers designed to rat cyclophilin to monitor the efficiency of the PCR and to verify that cDNA samples contained equivalent amounts of material [20]. Cyclophilin was faithfully and equally amplified in all the PCR reactions, indicating consistency in the quality of RT and PCR reactions. The data presented are representative of three different RT-PCR reactions carried out in duplicate.
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Subcloning and Sequencing
The PCR fragments were run on 1.5% agarose gel, stained with ethidium bromide, and visualized. The bands were then dissected out, and the DNA was isolated from the gel using Glass MAX DNA isolation system (BRL). The purified DNA fragments were subcloned into pCR 2.1 (Invitrogen, Carlsbad, CA) plasmid. The cloned DNA fragments were sequenced using standard M13 forward and reverse primers in an automated fluorescence-based sequencer (Applied Biosystems, Foster City, CA). Internal sequences were obtained using nested primers that were designed based on previous sequences. All the sequences reported are consensus of two different experiments. The sequence alignments (Genetics Computer Group DNA analysis software, Madison, WI) were carried out using the available sequence of REB and REBß.
Northern Analysis
Total RNA from Sertoli cells, germ cells, and peritubular cells were isolated as described above using TRI Reagent (Sigma). Approximately 10 µg of total RNA was fractionated on a 1% formaldehyde-agarose gel. After fractionation, the RNA in the gel was transferred onto nylon membrane (Hybond N; Amersham, Piscataway, NJ) in 10-strength SSC (single-strength SSC is 0.15 M sodium chloride and 0.015 M sodium citrate) buffer and UV cross-linked as described previously [21]. The membranes were then prehybridized in quick hybridization buffer (Stratagene, La Jolla, CA) for 30 min at 60°C. The hybridization was carried out at 60°C for 1 h with 32P-labeled REB probe obtained by RT-PCR of Sertoli cell RNA using primer set 2. The membrane was subsequently stripped and rehybridized with rat cyclophilin. All the probes were labeled using Prime-it II kit from Stratagene. Kodak X-OMAT AR film (Eastman Kodak, Rochester, NY) was exposed to the membrane overnight at -80°C, and densitometry values were obtained by scanning with Molecular Dynamics ImageQuant Digital Image analysis system (Sunnyvale, CA). The statistical significance of the relative expression between various tissues and treatments was determined by ANOVA.
Immunocytochemistry
Sections (6 mm thick) of paraffin-embedded testes from 20-day-old rats were fixed in buffered formaldehyde (4% in 0.1 M PBS) and incubated either with rabbit anti-human HEB (Santa Cruz Laboratories, Santa Cruz, CA; 1:200) or with normal rabbit serum (negative control) for 2 h at room temperature. Before application of the primary antibody, a microwave antigen retrieval technique was used as described previously [22]. The sections were then rinsed in Tris-buffered saline and incubated with goat anti-rabbit biotinylated antibody (Vectastain Elite kit; Vector Laboratories, Burlingame, CA; dilution 1:100). After rinsing, the sections were incubated in ABC complex (Vector Laboratories; dilution 1:100), and the reaction was visualized with diaminobenzidine (Sigma).
Gel Mobility Shift and Blot Assay
Gel-shift assays were performed with nuclear extracts of cultured Sertoli cells. The Sertoli cells were isolated as described above and cultured in 150 x 20-mm tissue culture dishes (Nunclon). The cells either were treated after 48 h in culture with FSH or were not treated (for controls). After 72 h, the cells were scraped off the tissue culture dishes and washed once with PBS. The nuclear extracts of these cells were then prepared as described by Guillou et al. [23]. Typically 70–100 µg of protein was obtained from 108 plated cells. The double-stranded DNA probe used in the gel retardation assay was a 20-bp mouse transferrin promoter proximal E-box (-327CAGCTG) containing flanking sequence. The single-stranded oligonucleotides were 5' 32P end-labeled with [
-32P]ATP (150 µCi/µl; NEN, Boston, MA) and T4 polynucleotide kinase (Boehringer-Mannheim, Indianapolis, IN). The complementary oligonucleotides were annealed, electrophoretically purified, and then used as probes in gel-shift assays [9].
For immunoblot analysis, a gel-shift assay was performed as described above in duplicate on the same gel, using radioactive and nonradioactive E-box oligonucleotides. A gel-shift assay using nonradioactive E-box probe on the polyacrylamide gel was electrophoretically transferred to a nitrocellulose membrane (BA85; Schleicher & Schuell, Keene, NH) by electrophoresis in Tris-glycine buffer containing 12% methanol. The blot was then blocked in 5% nonfat milk in TBSN (TBSN: 50 mM Tris [pH 7.4], 150 mM NaCl, and 0.05% Nonidet P-40) and incubated with 1:3000 dilution of antibodies to either E12/E47 or HEB (Santa Cruz Biotechnology, Santa Cruz, CA) for 3 h. After three washes of 15 min each, the blot was hybridized with a secondary antibody (1:3000 dilution; directed against rabbit IgG) conjugated to alkaline phosphatase for 1 h at room temperature. After five washes in TBSN, the immune complex was detected using an Immun-Star chemiluminescent protein detection kit (Bio-Rad, Hercules, CA). The nonimmune serum was included to determine the specificity of the blot. As a positive control, a gel shift was carried out using AP-1 oligo and blotted with c-fos, E12, USF, and SRF antibody. When AP-1 was used in the gel shift, a band in the blot was observed only with the c-fos antibody (data not shown).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and REBß, whereas the two 5' primers were designed to selectively amplify either REB or REBß transcripts (Fig. 1A). As shown in Figure 1B, when REB-specific 5' primer was used in the PCR reaction, a single predominant band of approximately 640 bp was observed in the RNA from whole testis (lane 1), freshly isolated Sertoli cells (lane 2), cultured Sertoli cells (lane 3), cultured Sertoli cells treated with either FSH (lane 4) or dbcAMP (lane 5), and kidney (lane 6, positive control). The 640-bp PCR fragment was isolated from the gel, purified, and subcloned for sequencing. Alignment of the sequence obtained to the available nucleotide sequence of REB confirmed that the 640-bp band was REB. The expression of REB appears constitutive because of its presence in untreated and FSH- or dbcAMP-treated Sertoli cells. The identity of the 450-bp testis-specific band seen in Figure 1B (lanes 1, 2, 3, 4, and 5) is under investigation. A PCR analysis for a constitutively expressed cyclophilin gene demonstrated an equivalent amount of cyclophilin PCR product in all RNA samples (data not shown).
In most of the tissues examined, REB and REBß were coexpressed [17]. The relative abundance of the expression of REB and REBß was, however, differentially regulated. In the rat head, for example, a distinct difference in the relative levels of REB and REBß was observed [17]. The REBß transcript predominated over REB, and the levels of both the species exhibited a general decrease with continuing embryonic development. With the exception of the lung, where REB is predominantly overexpressed, most other tissues express similar levels of REB and REBß transcripts [17]. To investigate the presence of REBß in Sertoli cells we examined the expression of REBß transcripts in Sertoli cells and whole testis by a PCR-based protocol that distinguishes between the two REB gene transcripts. The total RNA from cultured Sertoli cells treated with FSH and from whole testis were reverse-transcribed using oligo dT primer. To specifically amplify the REBß transcript, a 5' primer in the 72-bp insertion of REBß was used in the PCR reaction. With this 5'-REBß-specific primer, a 655-bp band corresponding to the predicted length between the two primers was observed in the whole testis (lane 5) and lung (lane 6), but not in Sertoli cells (Fig. 1C, lanes 1–4). The REBß transcript in Sertoli cells was also not observed in a PCR reaction carried out for 45 cycles (data not shown). Subsequent subcloning and sequencing of the 655-bp band confirmed its identity as REBß. The differential expression of REB and not REBß in Sertoli cells is a unique observation. To our knowledge, no other system has been reported in which one splice variant of the REB gene is expressed exclusively.
A Northern blot analysis was performed to investigate the potential hormonal regulation of REB gene expression. A [32P]dCTP-labeled random-primed probe was used from a 640-bp REB-specific PCR product from Sertoli cells as described above. As shown in Figure 2A, the predicted size of the RNA species migrating at approximately 4.2 kilobases (kb) corresponding to an REB transcript was detected. The abundance of this message was similar in untreated and FSH-treated Sertoli cells (Fig. 2B). Cyclophilin expression was used as a constitutively expressed gene to normalize for the integrity and amount of RNA (Fig. 2B). Although hormones did not appear to influence REB expression, higher levels of expression were observed in the cultured Sertoli cells in comparison to freshly isolated cells (Fig. 2).
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The RT-PCR data presented in Figure 1 were obtained from RNA collected from Sertoli cells and whole testis from 20-day-old rats. This represents a stage when Sertoli cells are functionally differentiated and have formed a blood-testis barrier. The transcripts of the REB gene were also detected in the whole testis by RT-PCR of RNA obtained from embryonic Day 18 and postnatal Day 0 and 10 testis (Fig. 3). Similar results were obtained for REBß (data not shown). An RT-PCR for cyclophilin demonstrated a PCR product in all the RNA samples (data not shown).
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Immunohistochemistry was performed to complement the PCR and Northern blot data obtained and to show that the REB protein is also expressed in Sertoli cells. As shown in Figure 4A, REB was localized in the nucleus of the Sertoli cells, suggesting that the REB protein is expressed by Sertoli cells. The HEB antibody used in the present study cross-reacts with its rat homologue REB. This antibody does not distinguishes between REB and REBß proteins. Since the REBß transcript was not expressed in Sertoli cells (Fig. 3), it is concluded that the antibody detected REB protein in Sertoli cells. REB staining was also observed in the interstitial and peritubular cells surrounding the seminiferous tubules (Fig. 4A).
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The presence of REB in Sertoli cells suggests that it can potentially bind to E-box sequences present in the promoters of a number of Sertoli cell genes. To test this hypothesis, both gel-shift and gel-shift immunoblot experiments were performed. Nuclear extracts from cultured Sertoli cells treated with FSH were incubated with the proximal transferrin promoter E-box sequence and resolved in a native polyacrylamide gel. The presence of a retarded band (Fig. 5, gel shift) suggested that E-box binding proteins are present in the Sertoli cells. To determine the identity of these E-box binding proteins, the binding reaction with unlabeled E-box was electrophoresed on a native polyacrylamide gel and blotted onto a nitrocellulose membrane. The membrane was subsequently probed with HEB antibody. A similar gel-shift blot was also probed with E12/E47 antibody. The presence of HEB immunoreactive band similar in migration to the gel shift retarded band suggested that REB is part of the complex binding to the E-box oligonucleotide (Fig. 5, blot). Interestingly, a band similar in migration to REB was also observed when E12/E47 antibody was used to probe the blotted E-box gel shift. A control Oct1 oligonucleotide gel shift had no immunoreactive band (Fig. 5). A control with nonimmune serum also had no immunoreactive band.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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is also present in Sertoli cells.
The presence of REB and not REBß in Sertoli cells is a novel observation and suggests a cell-specific splicing event. The alternatively spliced 24-amino acid insert specific to the REBß transcript is predicted to interact with the bHLH domain. The effect of such an interaction may mask the HLH domain from both homo- and heterodimeric interactions with other bHLH domains [17]. The leucine zipper domain common to both REB and REBß is also present in other bHLH proteins like AP-4 and USF. This domain is required for efficient homodimerization. Unlike these bHLH-ZIP molecules, REB does not require its leucine repeat sequence for efficient dimerization and DNA binding [17]. REB mutants lacking the leucine repeat can efficiently bind E-box sequences both as homodimers and heterodimers with native REB and E47 [17]. This observation helps explain the gel-shift data of Figure 5. The gel-shift data presented in this figure suggest the potential of REB forming heterodimers with E47. Alternatively, REB may form homodimers similar to E47 homodimers and colocalize on the gel shift. Currently being investigated is the potential that yet-unknown Sertoli cell-specific bHLH protein may dimerize with either REB and/or E47.
The tissue-specific alternative splicing of the REB gene observed in different tissues [17] suggests a potential distinct functional importance of the two gene products. The absence of REBß suggests that the REBß actions are not required or are detrimental to Sertoli cells. Given the apparent similarity of their heterodimerization capabilities and the presence of at least two class A bHLH proteins in Sertoli cells, REB and E47, these two bHLH proteins may have a functional redundancy in the cells. This concept is supported by recent E2A and REB gene knockout studies. The E2A deficiency is lethal postnatally, and the surviving F2 progeny are severely growth retarded [25]. In the absence of E2A activity, the earliest committed B-cell precursors are undetectable in the bone marrow. This phenotype is consistent with role of the E2A gene in B-cell development. The females were significantly underrepresented in comparison to males, and the growth defect was more pronounced in the females than in the males. The phenotype of REB knockout [26] is less pronounced than that of the E2A knockout. Although the deficiency is lethal postnatally, the F2 progeny of REB (-/-) were born with close-to-expected frequency, but they died within the first 1 or 2 wk after birth. Although a detailed study of the E2A and REB knockout with respect to reproductive organs and fertility was not reported, it is clear that male sex determination was normal. During rat embryonic development, male sex determination is associated with the differentiation of Sertoli cells and their ability to secrete Müllerian inhibiting substance [2, 3]. It is possible that the expression of Sertoli cell E47 and REB shows a high degree of functional compensation. This idea is supported by the recent report that human HEB cDNA under the control of the endogenous E2A promoter, when substituted for the mouse E2A gene, can functionally replace E2A in supporting B-cell commitment and differentiation and can rescue postnatal lethality associated with E2A disruption [27].
Expression of the bHLH E2A transcription factors has been shown to cause growth arrest in NIH 3T3 cells [28]. This function of the E2A gene products is consistent with its role in cell differentiation. Based on structural homology and functional compensation, it is speculated that REB may also cause growth arrest followed by cellular differentiation. The postpubertal Sertoli cells represent a terminally differentiated nondividing cell population [1]. To ensure that these cells do not undergo dedifferentiation and enter the cell cycle, they may express high levels of REB and E47. Previous observations have shown that overexpression of the dominant negative HLH protein Id in Sertoli cells is able to down-regulate the activation of transferrin [9] and c-fos promoters (unpublished results). Both these proteins are associated with differentiation of Sertoli cells. The hypothesis is being examined that bHLH proteins such as REB have a role in regulating Sertoli cell differentiation.
Given the widespread expression pattern and sequence homology to other class A bHLH proteins, such as the E2A gene products E12/E47, REB is likely to serve a similar function [17]. Analysis of in vivo dimerization between cell-specific class B bHLH proteins and ubiquitously expressed class A bHLH proteins has indicated the necessity for heterodimeric interactions [11]. The presence of REB suggests that Sertoli cells may also express a cell-specific bHLH protein. The Sertoli cell-specific bHLH protein remains to be elucidated.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 509 335 2176; skinner{at}mail.wsu.edu
Accepted: December 21, 1998.
Received: August 7, 1998.
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