Pem Homeobox Gene Promoter Sequences that Direct Transcription in a Sertoli Cell-Specific, Stage-Specific, and Androgen-Dependent Manner in the Testis in Vivo
Manjeet K. Rao,
Chad M. Wayne,
Marvin L. Meistrich and
Miles F. Wilkinson
Departments of Immunology (M.K.R., C.M.W., M.F.W.) and Experimental Radiation Oncology (M.L.M.), The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
Address all correspondence and requests for reprints to: Miles F. Wilkinson, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030. E-mail: mwilkins{at}mdanderson.org.
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ABSTRACT
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Although many genes are expressed selectively in Sertoli cells, regulatory sequences sufficient to drive Sertoli cell-specific expression in the postnatal and adult testis in vivo have not been identified. In the present study, we identified promoter sequences from the Pem homeobox gene that direct Sertoli cell-specific expression in an androgen-dependent and stage-specific manner. Immunohistochemical and RNA analysis demonstrated that 0.6-kb 5'-flanking sequence directed transgene expression specifically in the testis and the epididymis but not in any other tissues tested. In the adult testis, this promoter fragment targeted the transgene expression specifically to Sertoli cells during stages IVVIII of the seminiferous epithelial cycle, thereby mimicking the expression pattern of the endogenous Pem gene. This promoter fragment also recapitulated Pems normal postnatal expression pattern, as it directed transcript induction between d 6 and d 9 post partum. Deletion of 0.3 kb from the 5'-end of the transgene had no effect on androgen-dependent Sertoli-specific expression but altered stage-specific expression in adult testes and caused premature postnatal expression. Our results suggest that there are at least two regulatory regions in the Pem proximal promoter: one that directs androgen receptor-dependent expression specifically in Sertoli cells within the testis and another that confers stage-specific expression in neonates and adults by acting as a negative regulator. To our knowledge, this is the first identification of regulatory regions that direct faithful developmentally regulated gene expression in postnatal and adult Sertoli cells in vivo.
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INTRODUCTION
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THE REGULATION OF gene transcription in Sertoli cells and other male reproductive tract somatic cells has been studied primarily in cultured cell lines. These in vitro studies have revealed many transcription factors that control basal transcription, tissue-specific transcription, and hormonally regulated transcription (1, 2, 3, 4). Although informative, these in vitro studies have not provided a complete picture of transcriptional regulation. First, it is currently not possible to recreate the stage-specific events that normally occur during the testicular seminiferous epithelial cycle in cultured cell lines. Thus, to date, no regulatory elements that control stage-specific transcription have been defined. Second, although Sertoli cell lines have been used to identify transcription factors that control cell type-specific transcription (1), it is not clear how closely these cell lines resemble normal Sertoli cells in their normal physiological environment. Third, although hormonal regulation of transcription has been studied in vitro (5, 6, 7), in most cases it is not known how well the results obtained in cultured cell lines apply to physiological hormonal regulation in vivo.
Because of these limitations of in vitro systems, investigators have turned to generating transgenic mice containing 5'-flanking sequences from genes of interest to attempt to identify factors that govern regulated expression in Sertoli cells in vivo (8, 9, 10, 11). Unfortunately, such studies have not yet yielded any sequences that permit specific expression in adult Sertoli cells but not in nonmale reproductive cell types in vivo.
Here, we identify, for the first time, regulatory sequences sufficient for Sertoli cell-specific expression in postnatal and adult testes in vivo. These regulatory sequences are from the homeobox gene Pem, which is the founding member of the recently defined PEPP homeobox transcription factor subfamily (12). We previously showed that Pem transcripts are derived from two promoters independently regulated in a tissue-specific manner (13, 14, 15, 16). The proximal promoter (Pem Pp), the subject of this report, is expressed exclusively in male reproductive tissues (testis and epididymis). In the testis, the Pem Pp is expressed in Sertoli cells, where it is dramatically induced when the adjacent germ cells are initiating meiosis between d 8 and d 9 post partum (13, 17, 18, 19). In the adult testis, Pem mRNA and protein are expressed in Sertoli cells during stages IVVIII of the seminiferous epithelial cycle (17, 18). In addition to being developmentally regulated, Pem expression is androgen dependent (13, 16, 20). We report here the first analysis of the regulatory regions that control transcription from the Pem Pp. Our results allowed us to define two regions in the Pem Pp that each conferred unique regulatory control in vivo. Our analysis suggests that the Pem Pp will be a good model system in which to identify and study factors that mediate stage-specific, hormonally regulated gene expression specifically in Sertoli cells. In addition, the Pem promoter sequences that we have identified in this report may be a useful tool for expressing high levels of foreign genes and conditionally knocking out genes specifically within Sertoli cells in vivo.
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RESULTS
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Developmentally Regulated Expression in Sertoli Cells in Vivo Directed by 0.6-kb Pem Pp 5'-Flanking Sequence
We previously reported that the Pem Pp is expressed exclusively in testis and epididymis (13, 19). Here, we examined whether a gene construct containing 0.6 kb of the Pem Pp 5'-flanking sequence (Pem-213) was sufficient to direct this tissue-specific expression pattern (Fig. 1
, A and B). Ribonuclease protection analysis (RPA) of tissues from two independent transgenic lines containing this construct (Pem-213.6 and 213.10) demonstrated that the transgene was only detectably expressed in testes and epididymides but not in any other tissues tested (Fig. 1C
). To determine the level of expression of the transgene relative to that of the endogenous Pem gene, we took two approaches. First we assessed expression at the RNA level by performing RPA with a probe that distinguishes between transcripts from these two genes. This analysis showed that the transgene was expressed at 3.6- and 11.6-fold higher levels than was the endogenous gene in testes from the Pem-213.6 and 213.10 transgenic mice, respectively (Fig. 1D
). Because the level of endogenous Pem Pp transcripts in the transgenic mice did not significantly differ from that of control littermates, there was no evidence for Pem autoregulation. Second, we performed Western blot analysis with a polyclonal antiserum that we and others have previously demonstrated specifically recognizes mouse Pem (18, 19, 21) to determine the level of Pem protein. We found that Pem protein levels were dramatically higher in Pem-213.10 transgenic mice than in nontransgenic mice (Fig. 1E
).

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Figure 1. A Transgene Harboring 0.6-kb Pem Pp 5'-Flanking Sequence Is Expressed in a Normal Developmentally Regulated Manner Specifically in Male Reproductive Tissue in Vivo
A, Schematic diagram of the Pem gene. B, Schematic diagram of the Pem-213 transgene. Probe A corresponds to the region of the transgene shown; probe B corresponds to the endogenous Pem gene and thus is complementary with transgene mRNA over only a short region. C, RPA of total cellular RNA (10 µg) from adult tissues from Pem-213.6 mice using probe A. The protected RNA fragment was approximately 200 nt. D, RPA of adult testes RNA (10 µg) from two Pem-213 founder lines using probe B to distinguish between endogenous and Pem transgene mRNA. The protected band sizes were approximately 180 nt and approximately 130 nt for endogenous and transgene mRNA, respectively. E, Western blot analysis of Pem protein expression in Pem transgenic mice and control (nontransgenic) mice. The Pem band comigrated with that of recombinant Pem (data not shown). F, RPA of testes RNA (10 µg) from 6- and 9-d-old Pem-213.6 mice using probe A. A ß-actin probe was included in all annealing reactions as a loading control (the protected band was 35 nt).
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We next determined whether the Pem-213 transgene was expressed specifically in Sertoli cells within the testis like the endogenous Pem gene. To do this, we performed immunohistochemical analysis on serial cross-sections of testes from Pem-213 transgenic and control littermate mice. This analysis showed that Pem-213.10 transgenic mice expressed much higher levels of Pem protein than that of control nontransgenic littermates (Fig. 2
, AF), which is consistent with the elevated expression of Pem mRNA and protein from these transgenic mice (Fig. 1
, D and E). Pem-213.6 and 213.10 transgenic mice expressed Pem protein only in Sertoli cells (in the nucleus and sometimes detectable in the cytoplasm), and not in germ cells, interstitial cells, or peritubular cells (Fig. 2
, CE). No germ cell staining was observed even when counterstain was omitted to permit higher sensitivity; instead, Pem was observed only in Sertoli cell nuclei and cytoplasm (Fig. 2F
). Thus, the Pem-213 transgene provided Sertoli cell-specific expression within the testis.

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Figure 2. Sertoli Cell- and Stage-Specific Expression from Pem Transgenes in Vivo
AF, Immunohistochemical analysis of wild type (WT) and Pem-213.10 adult testes sections using an antimouse Pem antisera. Panels AD show Pem-positive and -negative tubules at the indicated stages [periodic acid Schiff (PAS) and hemotoxylin counterstain; magnification: A, x400; BD, x200]. Panel E shows a high magnification view of a Pem-positive tubule (PAS and hemotoxylin counterstain; magnification, x400). Panel F shows a tubule with no counterstain (x200). The arrows point to selected Sertoli cell nuclei that stain for Pem. The arrowheads points to prominent regions of Sertoli cell cytoplasm that stains for Pem. G and H, Immunohistochemical analysis of Pem expression in Pem-214.10 adult testis sections (PAS and hemotoxylin counterstain). Panel G shows Pem-positive tubules at the indicated stages; panel H shows Pem high (left) and Pem low (right) tubules. The blunt arrows point to Sertoli cell nuclei that only stain modestly for Pem. IL, Immunohistochemical analysis of nontransgenic control and Pem-212.9 adult testes section using an antimouse GFP antisera (counterstained with PAS and hemotoxylin). Panel I shows GFP-negative tubules at the indicated stages; panels JL show GFP-positive and -negative tubules at the indicated stages (panels IK, magnification, x200; panel L, x400). The arrows and arrowheads point to Sertoli cell nuclei and cytoplasm, respectively, that stain for GFP. The double arrows point to nonspecific staining in interstitial cells. MO, Immunofluorescence analysis performed on a Pem-212.9 testicular cell suspension. GFP expression was detected by direct fluorescence and vimentin expression was detected with an antivimentin antibody. Panel M shows a phase-contrast view of all cells, panel N shows GFP-positive cells (green), and panel O shows vimentin-positive Sertoli cells (red).
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We also found that the Pem-213 transgene contained sufficient regulatory sequences to reproduce the developmentally regulated expression of Pem in the testis. RPA revealed that, like the endogenous Pem gene (17), the Pem-213 transgene was first expressed between d 6 and d 9 post partum (Fig. 1F
). In adult testes, the Pem transgene exhibited a stage-specific expression pattern virtually identical with that of the endogenous Pem gene (Fig. 2
, C and D). Immunohistochemical analysis of 100 tubules from adult Pem-213.10 transgenic mice showed Pem protein in Sertoli cell nuclei from stages IVVIII (5 of 6, 5 of 5, 11 of 11, 18 of 18, and 32 of 32 tubules were positive, respectively). All other stages of tubules had weak or no detectable Pem protein. Similarly, nontransgenic control littermate testes expressed Pem protein in stage IV or through VIII tubules (4 of 4, 8 of 8, 15 of 15, 14 of 14, and 20 of 20, respectively) but not in tubules from any other stage (Fig. 2
, A and B). The same stage-specific pattern of expression was also observed in Pem-213.6 mice. We conclude that the Pem-213 transgene contains sufficient sequences to provide the normal developmentally regulated expression pattern of Pem in both postnatal and adult testes.
Sertoli Cell-Specific Expression in Testes Requires Only 0.3-kb Pem Pp 5'-Flanking Sequence
To further localize the Pem Pp regulatory elements that drive Sertoli cell-specific and developmentally regulated expression, we generated a construct identical with Pem-213 except that it contained only 0.3-kb Pem Pp 5'-flanking sequence (Pem-214; Fig. 3A
). Analysis of mice from two independent transgenic lines containing the Pem-214 transgene indicated that this shorter transgene was specifically expressed in testes (Fig. 3B
) and epididymis (data not shown). These Pem-214.8 and 214.10 transgenic lines expressed the transgene at levels 17- and 13.7-fold higher, respectively, than that of endogenous Pem transcripts in the testis (Fig. 3C
). Western blot analysis showed much higher protein expression in Pem-214.10 mice testes compared with normal control mice testes (Fig. 1E
). Immunohistochemical analysis demonstrated that both Pem-214 transgenic lines expressed Pem only in Sertoli cells within the testis (Fig. 2
, G and H), just as was the case for the Pem-213 transgenic mice.

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Figure 3. A Transgene Harboring 0.3-kb Pem Pp 5'-Flanking Sequence Is Expressed at High Levels in Testes in Vivo
A, Schematic diagram of the Pem-214 transgene. BD, RPA of total cellular RNA (10 µg) from Pem-214.8 and Pem-214.10 mice analyzed in the same manner as described in Fig. 1 . The data in panels B and D are from Pem-214.10 mice. E, Schematic diagram showing the two regulatory regions we have defined upstream of the Pem Pp transcription start site. Consensus GATA and AR transcription factor-binding sites are indicated. F, Southern-blot RT-PCR analysis of Pem Pp and Pem Pd transcripts in normal mouse embryos at the stages indicated. The positive control for Pem Pp and Pem Pd transcripts was adult mice testis and placenta, respectively. The size of the Pem Pp, Pem Pd, and ß-actin transcripts was 420, 180, and 220 nt, respectively, as expected based on the primers pairs used. The internal control, ß-actin, was amplified from the same cDNA under limiting cycling conditions to provide linearity (20 cycles). G, Southern blot RT-PCR analysis of Pem Pp transcripts from the Pem-214 transgene in Pem-214 mouse embryos (e18.5). Adult Pem-214.10 epididymis was included as a positive control. The cDNA generated as described in panel F was used for PCR either undiluted (neat) or diluted in buffer by the amount indicated. The size of the transgene band is approximately 230 nt, as expected based on the primer pairs used. The ß-actin band was stained with ethidium bromide.
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However, in contrast to the Pem-213 transgene, we found that the Pem-214 transgene exhibited abnormal developmental regulation. Pem-214 transcripts were expressed prematurely after birth (at least as early as d 6 post partum; Fig. 3D
), and Pem protein was expressed in Sertoli cell nuclei (and to a lesser extent in their cytoplasm) during all seminiferous epithelial cycle stages (Fig. 2
, G and H) in both Pem-214 transgenic lines. However, tubules varied in the amount of Pem that they expressed. Intriguingly, all stage VIVIII tubules had high Pem expression (13 of 13, 16 of 16, and 29 of 29 tubules examined, respectively) whereas all tubules from other stages had low Pem expression (Fig. 2H
). Manipulations that decreased the Pem signal (anti-Pem diluted to 1:2000 or shorter incubations with diaminobenzidine) did not alter this high-low expression pattern in Pem-214 mice; i.e. it did not give the high-none expression pattern that we observed in Pem-213 mice. Taken together, the data from Pem-213 and -214 transgenic mice imply that there are at least two regulatory regions that control Pem Pp transcription: region I houses regulatory elements that confer Sertoli cell-specific expression highest during stages VIVIII of the seminiferous epithelial cycle (Fig. 3E
). Region II has a negative regulatory element that inhibits Pem expression in inappropriate stages of the seminiferous epithelial cycle (Fig. 3E
). These two regions collaborate to generate the highly stage-specific expression pattern characteristic of Pem.
We also examined whether the Pem-214 transgene was expressed during embryogenesis. Figure 3F
shows that the Pem Pp is not normally expressed (from the endogenous Pem gene) during mouse embryogenesis, whereas the distal promoter of Pem (Pem Pd) is highly active in mouse embryos. Consistent with this, we found that the Pem-214 transgene was not detectably expressed at any of the various times of embryogenesis that we tested between 7.5 d and 18.5 d post coitum (Fig. 3G
and data not shown).
0.6-kb Pem Pp 5'-Flanking Sequence Is Sufficient to Drive Green Fluorescent Protein (GFP) Expression in Sertoli Cells in Vivo
Because it is possible that Pem exon or intron sequences contribute to the expression pattern of Pem Pp transcripts, we examined the expression pattern of 0.6 kb of Pem Pp 5'-flanking sequence driving a reporter GFP gene (construct Pem-212; Fig. 4A
). Of three independent transgenic lines that we developed containing Pem-212, two expressed the transgene in testis, as judged by RT-PCR (data not shown). We focused our analysis on line Pem-212.9, as it expressed the highest levels of transgene mRNA. These mice expressed the transgene in the testis and the epididymis but not in any other tissue tested (Fig. 4B
and data not shown). Immunohistochemical analyses with anti-GFP antisera demonstrated that Sertoli cells during stages IVIX expressed GFP, but not any germ cell type (Fig. 2
, JL). Note that the anti-GFP staining of some interstitial cells is nonspecific, as nontransgenic mice also exhibited this staining pattern (Fig. 2I
). Analysis of 100 tubules from adult Pem-212.9 transgenic mice showed that GFP protein was mostly common in Sertoli cell nuclei from tubules in stages IVVIII (3 of 4, 8 of 9, 15 of 15, 9 of 11, and 19 of 21 positive tubules, respectively). In contrast, tubules from stages I, II, III, IX, X, XI, and XII exhibited a frequency of GFP expression that ranged from none to modest (0 of 4, 1 of 3, 2 of 5, 5 of 10, 1 of 9, 1 of 5, and 0 of 3 positive tubules, respectively). We found that GFP was expressed in the majority of Sertoli nuclei in tubules from stages IVVIII, whereas its expression was restricted to only a few Sertoli cell nuclei in tubules from other stages. We conclude that 0.6 kb of the Pem Pp 5'-flanking sequence provides the most consistent expression in stages in which Pem is normally expressed but that it does not completely prevent expression in other stages.

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Figure 4. Male Reproductive Tract-Specific Expression in Vivo from a Transgene Harboring 0.6 kb of the Pem Pp 5'-Flanking Sequence Driving GFP Expression
A, Schematic diagram showing the Pem-212 transgene and location of probe C. B, RPA of total cellular RNA (10 µg) from adult tissues from Pem-212.9 mice using probe C and analyzed as in Fig. 1 . The protected band was approximately 190 nt.
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To further characterize the cell populations expressing GFP protein, immunocytochemical analysis was performed on testicular cells in suspension using an antivimentin antibody as a marker for Sertoli cells (Fig. 2
, MO). We found that all GFP-positive cells (green) were also vimentin positive (red) (100 of 100 GFP-positive cells counted), suggesting that Pem expression from the Pem-212 transgene was restricted to Sertoli cells. In contrast, not all vimentin-positive cells were GFP positive (70 of 113 vimentin-positive cells were GFP positive), consistent with our immunohistochemical analysis showing that the Pem-212 transgene is expressed in a stage-specific manner.
Androgen-Dependent Expression in Vitro and in Vivo
We previously reported that Pem expression from the Pem Pp is androgen dependent in testes in vivo (13, 17, 19, 20). To examine whether 0.3-kb Pem Pp 5'-flanking sequence is sufficient to permit androgen-dependent expression, we performed testosterone inhibition and add-back experiments on the Pem-214 transgenic mice. In one group of these mice we implanted peristaltic pumps containing Nal-Glu and pellets containing flutamide to block testosterone production and action, respectively. In another group of mice, we implanted the pumps and also gave them sufficient testosterone to overcome the effects of the Nal-Glu and flutamide. A third group of mice were sham treated. RPA demonstrated that combination treatment with flutamide and Nal-Glu dramatically decreased expression from both the endogenous Pem gene (data not shown) and the Pem-214 transgene (Fig. 5A
). Expression from the endogenous Pem gene and the Pem-214 transgene was restored in the mice supplemented with testosterone. The efficacy of these treatments was determined by testing intratesticular testosterone levels. Nal-Glu and flutamide treatment decreased testosterone levels by approximately 10-fold (8.2 ng/g of testis compared with 87.9 ng/g of testis in sham-treated mice). Testosterone supplementation increased testosterone levels by more than 4-fold (38.5 ng/g of testis) but less than sham-treated animals, indicating that suboptimal testosterone levels are sufficient to maintain Pem Pp expression. We conclude that region I contains sufficient regulatory elements to permit androgen-dependent regulation.

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Figure 5. Androgen-Regulated Expression in Vitro and in Vivo from Transgenes Harboring 0.3 kb of the Pem Pp 5'-Flanking Sequence
A, RPA of total cellular RNA (10 µg) from adult testes from Pem-214.10 mice using probe A. The mice were treated as described in Materials and Methods (N, Nal-Glu; F, flutamide; T, testosterone). The protected band was approximately 180 nt. B, RPA of total cellular RNA (10 µg) from the cell lines shown using probe D (Fig. 1 ). The protected band was approximately 140 nt. C, RPA of total cellular RNA (10 µg) from cells cotransfected with Pem-214, a ß-globin expression plasmid as an internal control, and either AR expression plasmid (+) or empty vector (-) and incubated with testosterone (+) or media alone (-). The bands protected by probe B were approximately 180 nt and approximately 130 nt for endogenous and transgene transcripts, respectively. D, RPA of total cellular RNA (10 µg) from MSC1 cells cotransfected with Pem-215, ß-globin expression plasmid, and either AR expression plasmid (+) or empty vector (-) and incubated with testosterone (+) or media alone (-). The band protected by probe C was approximately 190 nt.
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Although these findings indicated that Pem Pp expression requires androgen in vivo, they did not distinguish between direct androgen action on Sertoli cells and indirect androgen action via another AR-positive cell type (e.g. peritubular cells). To assess whether androgen dependence of Pem Pp was the direct result of androgen on Sertoli cells, we elected to use a transient transfection in vitro approach. For this analysis, we used two immortalized Sertoli cell lines: 15P-1 and MSC1. The MSC1 cell line expresses endogenous Pem Pp transcripts whereas the 15P-1 cell line does not (Fig. 5B
). The differential ability of these cell lines to express the Pem Pp was not the result of differences in endogenous androgen receptor (AR) levels, as both cell lines expressed similar (albeit low) levels of AR mRNA (data not shown). To determine whether region I (Fig. 3E
) is sufficient to drive Pem Pp expression in these immortalized Sertoli cell lines, we performed transient transfection experiments with the Pem-214 construct in the presence or absence of testosterone. Transfected Pem-214 transcribed Pem transcripts in an androgen-dependent manner in MSC1 cells (Fig. 5C
). In contrast, 15P-1 cells failed to transcribe detectable levels of Pem Pp transcripts from transfected Pem-214, either in the absence or presence of testosterone. To determine whether region I is sufficient to provide androgen-dependent expression, we used the Pem-215 construct that has GFP downstream of the promoter, rather than the Pem coding region. We found that addition of testosterone led to an increase in Pem-215 expression, which was further up-regulated in the presence of AR (Fig. 5D
). Interestingly, we found that transfected AR alone increased the levels of both endogenous and transfected (Pem-214 and Pem-215) Pem Pp transcripts (Fig. 5
, C and D). This induction in the absence of exogenous testosterone suggests that AR acts in a ligand-independent manner, that a ligand other than testosterone can stimulate AR activity, or that MSC1 Sertoli cells secrete their own androgen, permitting autocrine regulation.
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DISCUSSION
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We have identified promoter sequences from the Pem homeobox gene that provide high levels of expression in Sertoli cells of the testis. Sertoli cell-specific expression within the testis was obtained with only 0.3-kb 5'-flanking sequence (region I; Fig. 3E
). To our knowledge, this is the first report of promoter sequences that confer Sertoli cell-specific expression in postnatal and adult testes. In contrast, the Müllerian inhibiting substance promoter, which has been widely used to study fetal Sertoli gene expression and genes involved in sexual determination, is dramatically down-regulated in rat testes after birth (22, 23) and is undetectable in adult mouse testes (24). Although a transgene driven by the Müllerian inhibiting substance promoter was reported to be expressed in adult mouse testes (25), it was not clear whether this expression was restricted to Sertoli cells.
Transgenic mice studies on promoters that are normally expressed in adult Sertoli cells have so far not yielded 5'-flanking sequences that confer normal regulation restricted to the male reproductive tract. For example, the FSH receptor (FSHR) is expressed from a well studied promoter that is normally selectively expressed in Sertoli cells in male mice, but when expressed as a transgene containing approximately 5 kb of 5'-flanking sequences, it was also expressed in brain and sometimes in other tissues (26, 27). Furthermore, this FSHR transgene did not display normal developmental regulation, as it was not expressed on d 10 post partum when endogenous FSHR transcripts normally first appear. A FSHR transgene containing a smaller 5'-flanking region [198 nucleotides (nt)] was better restricted in its expression to the testis, but its temporal expression pattern was abnormal and it was expressed mostly, if not entirely, in germ cells (9). Another promoter studied in transgenic mice is the inhibin-
promoter, which is normally expressed in Sertoli cells, granulosa cells, and adrenal gland cells. An inhibin-
promoter transgene containing a 6-kb upstream sequence was found to display normal expression in these cell types but was also aberrantly expressed in Leydig cells (10, 28). Other promoters studied in transgenic mice are those from the human sex hormone-binding globulin (SHBG) and rat androgen-binding protein (ABP) genes, which encode related gene products. The SHBG promoter is normally most highly expressed in liver but is also expressed in testes, uterus, and placenta (29, 30, 31). A transgene composed of the entire SHBG gene and 11 kb of upstream sequences was faithfully expressed in Sertoli cells and exhibited selectivity by virtue of it not being expressed in brain or spleen (29). However, it may not be a useful tool for studies on Sertoli cells, as it was expressed at much higher levels in liver and kidney than it was in testes (29). A rat ABP transgene gene containing 1.5-kb 5'-flanking sequences was shown to express transcripts in the testes displaying an in situ hybridization staining pattern consistent with Sertoli cell expression (32). However, the selectivity of expression from this ABP transgene was unclear, as its expression was only examined in one transgenic line, and the Northern blot analysis of this line suffered from high background hybridization (32). We conclude that it remains uncertain as to whether any of the promoter sequences described in the literature provide high levels of expression in Sertoli cells without also being transcribed in cells outside of the male reproductive tract.
We found that region I from the Pem Pp conferred not only Sertoli cell-specific expression in the testis but also androgen-dependent expression (Fig. 5
). This androgen dependence may explain part of the Sertoli cell specificity of Pem Pp, as Sertoli cells express AR, and it is generally accepted that germ cells in the testis do not (33, 34). However, because AR is expressed by Pem-negative peritubular cells in the testis, as well as by cells in organs that do not express Pem, androgen receptivity cannot be the sole determinant of Pem expression. Other factors that may also contribute to the specific expression of Pem in Sertoli cells are GATA transcription factors, as regions I and II have several consensus GATA-binding sites (Fig. 3E
). GATA-1, -4, and -6 are known to be expressed in developing and mature Sertoli cells (35, 36), where they are known to regulate some genes (37, 38). GATA-1 is a particularly good candidate to regulate Pem, as it is expressed in Sertoli cells during the same stages of the seminiferous epithelial cycle as Pem and, like Pem, does not require the presence of germ cells for its expression in Sertoli cells (17, 36). However, because all members of the GATA transcription factor family have identical or nearly identical DNA-binding specificity and some members are expressed in cell types that do not express Pem (e.g. GATA-6 and GATA-4 are also expressed in Leydig cells), it is clear that GATA transcription factors cannot be entirely responsible for the Sertoli cell-specific expression of Pem Pp. Future studies will be required to determine whether GATA transcription factors collaborate with AR to provide the Sertoli cell-specific expression of Pem or whether other factors are also involved.
We also identified sequences that conferred the normal developmentally regulated expression of Pem in Sertoli cells (Fig. 2
). Our results indicate that region I drives high expression during the normal stages of Pem expression in adult testis (stages VIVIII), whereas region II appears to act as a negative regulator that inhibits expression during the stages when Pem is not normally expressed (Fig. 3E
). The combined action of regions I and II provides the highly restricted pattern of expression of Pem in adult Sertoli cells during the seminiferous epithelial cycle. Region II also directs postnatal developmental expression in Sertoli cells when the adjacent germ cells are undergoing the first wave of spermatogenesis, as we found that deletion of region II caused premature Pem expression (Fig. 3
). Interestingly, the induction of Pem expression in Sertoli cells postnatally and in the adult corresponds to the initiation of meiosis in the adjacent germ cells. This expression of Pem during meiosis may be functionally significant, as we have found that overexpression of Pem in Sertoli cells in vivo increases the frequency of preleptotene spermatocytes with DNA strand breaks (39). Our finding that the same region directs activation of Pem expression during both the first wave of spermatogenesis and its stage-specific expression in the adult is intriguing, but it remains to be determined whether the same or different transcription factors that bind to region II govern these two events. To our knowledge, neither cis- nor trans-acting factors that confer developmentally regulated expression for any gene in Sertoli cells in vivo have been previously defined.
Our finding that the regulatory region responsible for androgen-dependent expression (region I) is the same as that required for high levels of expression in stages VIVIII is consistent with the long-standing view that these stages are the androgen-dependent stages (40, 41, 42). That Pem is directly regulated by androgen is supported by our finding that testosterone in combination with AR induced Pem Pp expression in the MSC1 Sertoli cell line (Fig. 5
), thereby ruling out paracrine regulation involving more than one cell type. Further support for direct regulation comes from Barbulescu et al. (43) who recently identified two AREs at the -247 and -85 positions that drive androgen-dependent expression in transfected cells. In addition, we have identified two other putative AREs at positions -299 and -182 that have sequence identity at all or most of the critical positions in the half-site consensus sequence.
A surprising observation was that region I does not act alone to direct stage-specific regulation of Pem but that region II is also necessary. We found that deletion of region II permitted Pem expression in virtually all stages of the seminiferous epithelial cycle in vivo (Fig. 2
, G and H), suggesting that it acts as a negative regulator of expression during the stages in which Pem is not normally expressed. It will be intriguing in future studies to determine the mechanism by which region II participates with region I in generating very tight stage-specific regulation of Pem.
The promoter that we have defined here could be useful for several applications. One application would be to use this promoter to selectively knock out genes specifically in Sertoli and epididymal cells using the Cre/loxP system. A second would be to use it to selectively overexpress or ectopically express genes in Sertoli and epididymal cells to determine the functions of such genes. A third application would be to express one or more germ cell-inactivating proteins, which could form the basis of a gene therapy approach of male contraception. Finally, the Pem-212 mice described herein might be used as a source of stage-specific GFP-positive Sertoli cells that can be purified by flow cytometry for experiments designed to investigate the molecular and cellular characteristics of this Sertoli cell subset. In summary, the Pem Pp is a useful tool that may allow a better understanding of Sertoli cells at both the molecular and cellular levels.
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MATERIALS AND METHODS
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Plasmids
We generated four plasmids for this study: Pem-212, -213, -214, and -215. Pem-212 was made by replacing the cytomegalovirus (CMV) immediate early promoter in the GFP-containing expression vector enhanced GFP (EGFP)-hMT2A-N1 (G-312) with Pem Pp 5'-flanking sequences (-655 to -1 with respect to the Pem start ATG codon in exon 3). This construct lacks exons 1 and 2 and hence does not have the Pem Pd. The Pem Pp 5'-flanking sequences were amplified from the plasmid Pem-121 (39) by PCR with primers containing AseI and KpnI sites to allow cloning into the expression vector. EGFP-hMT2A-N1 is identical with EGFP-N1 (CLONTECH Laboratories, Inc. Palo Alto, CA) except that it has hMT2A intron 1 inserted in the EGFP coding region at nt 898 (kindly provided by Dr. Gilbert Cote, M.D. Anderson Cancer Center). Pem-213 was made by inserting a 4.5-kb SalI/NotI fragment (from Pem-121) that contains Pem sequences extending from intron 2 (-655 with respect to the start ATG in exon 3) to exon 6 along with bovine GH (bGH) polyadenylation sequences downstream into the plasmid pGEM-11Zf (Promega Corp. Inc., Madison, WI). Pem-214 was made by inserting a 4.2-kb EcoRV/NotI fragment (from Pem-121) that contains Pem sequences extending from intron 2 (-353 with respect to the start ATG in exon 3) to exon 6, along with bGH polyadenylation sequences downstream, into the plasmid Bluescript KS+ (Stratagene, Inc., San Diego, CA). Pem-215 was made by substituting the CMV promoter with Pem Pp 5'-flanking sequences (-353 to -1 with respect to the Pem start ATG) in the plasmid EGFP-hMT2A-N1. This was accomplished by digesting with AseI and XhoI to remove the CMV promoter, religating after polishing the 5'-overhangs with polymerase I, and then inserting the Pem Pp 5'-flanking sequences (amplified by PCR from Pem-121) at the EcoRI and KpnI sites.
Cell Culture and Transient Transfection Assays
The MSC1 and 15P-1 Sertoli cell lines were maintained in DMEM supplemented with 10% fetal bovine serum and 50 mg/ml of both penicillin and streptomycin. All cell culture reagents were obtained from Life Technologies, Inc. (Gaithersburg, MD). Transient transfection of all cells was performed using LipofectAmine (Life Technologies, Inc.) according to the manufacturers recommendations. Cells were plated on 150-mm culture dishes and then cotransfected with 5.6 µg of Pem plasmid, 1.1 µg of either the AR expression plasmid mARpcDNAI/Neo (G-194) obtained from Dr. Vijay Kumar (Mayo Clinic, Rochester, MN) or pcDNAI/Neo (Invitrogen Corp., Carlsbad, CA), and 0.25 µg of human ß-globin (G-1F) as a transfection control. DNA was incubated with the cells in serum-free DMEM for 8 h, and then replaced with DMEM supplemented with 10% charcoal-stripped bovine serum (HyClone Laboratories, Inc. Logan, UT) in the presence or absence of 1 nM testosterone and harvested 48 h later.
Transgenic Mice
DNA fragments containing the transcription units of Pem-212, Pem-213, and Pem-214 were excised from the vector backbone using the appropriate restriction enzymes, gel purified, and then injected into the male pronuclei of C57/BL6 mouse embryos by the M.D. Anderson Cancer Center transgenic mouse core laboratory. Two Pem-213 (213.6, 213.10), two Pem-214 (214.8, 214.10), and three Pem-212 (212.9, 212.12, 212.25) founder lines containing transgene DNA were generated, as detected by PCR using tail DNA as template and primers that directed amplification between nt 487 and nt 986 (with respect to the start ATG of EGFP) for Pem-212 and nt 3840 and nt 4182 (with respect to the Pem ATG) for the other two transgenic constructs.
Hormone Treatment
The GnRH antagonist Nal-Glu ([AC-D2-Nal1,D4-Cl-Phe2,D3-Pal3,Arg5,D4-p-methoxybenzoyl-2-amino butyric acid (6),D-Ala (10)GnRH) was supplied by Dr. H. K. Kim (National Institutes of Child Health and Development, Bethesda, MD). It was suspended in distilled water at a concentration of 8 mg/ml and used to fill an osmotic pump with a delivery period of at least 2 wk (ALZET model 2002; Alza Corp., Palo Alto, CA). The pump was placed under the back skin where it delivered a dose of 2500 µg/kg/d. Subcutaneous 168-mg flutamide pellets with a delivery period of at least 4 wk (SA-152, Innovative Research of America, Sarasota, FL) were used as an antiandrogen. Testosterone replacement was accomplished by inserting subcutaneously 0.5-cm testosterone capsules (44, 45). Male mice, 68 wk of age, were treated for a duration of 2 wk. Animals were then killed and their testes were collected. Intratesticular testosterone levels were measured by RIA, as described previously (46, 47). All experiments involving mice were performed in accordance with NIH guidelines for care and use of animals and as recommended by the M.D. Anderson Cancer Center Animal Care Committee.
RNA Isolation and Analysis
Total tissue RNA was isolated by guanidinium thiocyanate lysis and centrifugation over a 5.7 M CsCl cushion as described previously (48). RPA was performed as described (20) using the following probes. Probe A contains 61 nt of Pem exon 6 and 250 nt of the bGH 3'-untranslated region (UTR) and was generated by digesting Pem-121 with NdeI. Probe B contains 92 nt of Pem 3'-UTR and was generated by digesting a plasmid containing mouse Pem cDNA (Pem-27) with MslI. probe C, which contains between nt 482 and nt 693 (with respect to the ATG) of the GFP coding region, was amplified by PCR from Pem-212. Probe D, which contains 433 nt of the Pem 5'-UTR, 81 nt of exon 3, and 198 nt of intron 3, was generated by digesting a plasmid containing mouse genomic Pem sequences (Pem-132) with EcoRV. The ß-actin probe, which contains 34 nt of human ß-actin exon 3, was prepared by linearizing plasmid G-98, which contains human ß-actin (GenBank accession no. X00351), with BanI. RT-PCR analysis was performed by first generating cDNA from RNA (0.5 µg) using reverse transcription, as per the manufacturers instructions (Omniscript Reverse Transcription kit 50, QIAGEN Inc.). The following primer pairs were used to specifically amplify transgene Pem Pp, endogenous Pem Pd, and endogenous Pem Pp transcripts: MDA-870 (exon 6)/-871 (bGH 3'UTR), MDA-1286 (exon 1)/-1288 (exon 3), and MDA-245 (intron 2)/-268 (exon 4), respectively. PCR was performed 1 min at 94 C, 1 min at 58 C, and for 1 min at 72 C for 35 cycles. To increase sensitivity the PCR products were run out on a 1%-agarose gel, blotted, and hybridized with transgene-, Pem Pd-, and Pem Pp-specific probes.
Immunohistochemistry and Western Blot Analysis
Histological and immunohistochemical analyses were performed as described (18, 49). Anti-Pem antisera (21), antivimentin monoclonal antibody (provided by Dr. Elizabeth Grimm, M.D. Anderson Cancer Center), and anti-GFP antisera (CLONTECH Laboratories, Inc.) were used at 1:500, 1:500, and 1:5000 dilutions, respectively. Biotinylated antirabbit IgG was incubated with streptavidin-horseradish peroxidase (Vectastatin ABC kit; Vector Laboratories, Inc., Burlingame, CA) and the substrate 3,3'-diaminobenzidine peroxidase. For Immunofluorescence analysis, an antimouse IgG linked with Alexa 488 fluorochrome (Molecular Probes Inc., Eugene, OR) was used as secondary antibody. Western blot analysis was performed on testicular homogenates (40 µg protein/lane; assayed by the BCA protein assay kit, Pierce Chemical Co.), subjected to SDS-PAGE (10% acrylamide), and then electroblotted to nitrocellulose membranes. The membranes were probed overnight at 4 C with anti-Pem antibody (18) at a dilution of 1:10,000. The membranes were given three 10-min washes with 1 x PBS/0.1% Tween-20 at room temperature and then incubated for 45 min at room temperature with a peroxidase-linked antirabbit IgG secondary antibody at a dilution of 1:5,000. Protein bands reactive with antibodies were visualized using the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Arlington Heights, IL). The images of the protein bands were optimized, captured, and analyzed by ImageMaster VDS gel documentation system (Kodak Inc., Rochester, NY).
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FOOTNOTES
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This work was supported by NIH Grant CA-78023.
Abbreviations: ABP, Androgen-binding protein; AR, androgen receptor; ARE, androgen-responsive element; bGH, bovine GH; EGFP, enhanced green fluorescent protein; FSHR, FSH receptor; GFP, green fluorescent protein; nt, nucleotide; Pem Pp, Pem proximal promoter; RPA, ribonuclease protection analysis; SHBG, sex hormone-binding globulin; UTR, untranslated region.
Received for publication July 3, 2002.
Accepted for publication November 7, 2002.
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