Animal Treatments

Animals were treated according to the National Institutes of Health Care and Use of Laboratory Animals and all experiments were approved by the local ethical committee.

SCARKO and control mice were bred by crossing male mice homozygously expressing AMH-Cre (C57BL/6SJL) with female mice (Ar^fl/+) carrying a mutated AR with a floxed second exon (98% CD1). Genotyping was performed as described previously [2].

The effect of testosterone propionate (TP) and of the antiandrogens bicalutamide and flutamide was studied in 7-day-old male C57BL/6J@Rj mice or Wistar rats (Janvier). Bicalutamide was kindly provided by AstraZeneca and flutamide and TP were purchased from Fluka and Sigma respectively. Stock solutions of these compounds were prepared in ethanol and further diluted in arachis oil. Mice received 50 μg animal⁻¹ day⁻¹ (50 μl arachis oil containing 10% ethanol) and rats 100 μg animal⁻¹ day⁻¹ (100 μl) of each of the mentioned compounds for 5 days sc. Treatment groups consisted of five animals, except for the control group in the mouse experiment, which consisted of three animals. At Day 12, mice and rats were killed and individual testes were snap frozen in liquid nitrogen and weighed before RNA preparation. Urogenital systems were fixed in Bouin fluid and seminal vesicles were dissected and weighed after fixation.

Primary SC Cultures and Organotypic Testis Cultures

Primary cultures of SC were prepared from 19-day-old rat and mouse testes as described previously [13]. After 4 days of culture, cells were washed four times with PBS (Invitrogen) and treated as indicated. Organotypic cultures of prepubertal mouse testes were set up as described previously [14] and incubated for 48 h with Dulbecco modified Eagle medium (DMEM)/F12 medium (Invitrogen) supplemented with vehicle (ethanol), R1881 (Perkin Elmer), recombinant human follicle-stimulating hormone (rhFSH; Puregon, Organon), bicalutamide, ketoconazole (MP Biomedicals) or combinations of these agents as indicated.

Quantitative PCR

RNA was extracted with the RNeasy mini kit (Qiagen) according to the manufacturer's instructions, including an on-column DNaseI-treatment (Qiagen). For testicular tissue samples, 5 ng of luciferase mRNA (Promega) was added to the whole testis sample at the start of the RNA extraction procedure to control for the efficiency of RNA extraction, RNA degradation, and the reverse transcription step and to allow specific mRNA levels to be expressed per testis [15]. One microgram of total RNA was reverse transcribed with the Superscript II RNase H reverse transcription kit (Invitrogen) including RNaseOUT (Invitrogen), according to the manufacturer's instructions and using 150 ng random primers (Invitrogen) per reaction.

The 7500 Fast Real-time PCR system (Applied Biosystems) was used for sample cDNA quantification. Tubb isotypes, luciferase, Rn18s and Rpl19 were assayed using SybrGreen as a fluorescent dye. The relevant primers were described previously [4, 15] or are presented in Table 1. The quantitative RT-PCR (qPCR) two-step protocol was 2 min at 50°C followed by 2 min at 95°C. Subsequently, 40 cycles of 3 sec at 95°C and 30 sec at 60°C were performed. Each 10-μl real-time PCR reaction contained 1× Platinum SybrGreen qPCR Supermix-UDG (Invitrogen), 1× ROX dye (Invitrogen), 150 nM of each primer, and 2 μl of a 1:10 dilution of the cDNA reaction (1:1000 dilution for Rn18s). Dissociation curves confirmed uniqueness of each amplicon.

TABLE 1.

List of primers.

To create standards for the qPCR, gene-specific cDNAs were generated by RT-PCR. The fragments were then cloned into pGEM-T Easy (Promega), sequenced to confirm their identity, and quantified by spectrophotometry. All samples and standard curves were run in triplicate. Data were analyzed with 7500 Fast System SDS Software, version 1.4 (Applied Biosystems). Rn18s rRNA or Rpl19 mRNA was used as an internal control and expression levels were calculated as copy number per 10⁸ copies Rn18s rRNA or per 10³ copies Rpl19 mRNA. For testicular tissue samples, expression levels were calculated as copy number per 10⁸ copies luciferase mRNA.

Western Blotting

Testes were homogenized with a Dounce homogenizer in 1× sample buffer (Invitrogen) containing β-mercaptoethanol. For cells, dishes were washed once in PBS, cells were scraped in 1× sample buffer, and the homogenate was passed 10 times through an 18G needle. Protein concentration was determined using a bicinchoninic acid procedure (Pierce Biochemical Company). Twenty micrograms of protein was separated on a NuPAGE 3%–8% Tris-Acetate gel (Invitrogen) and blotted onto a Hybond ECL nitrocellulose membrane (GE Healthcare). Membranes were blocked in 2% casein in TBS-T (25 mM Tris-HCL, 0.9% NaCl, 0.1% Tween, pH 7.4) and were incubated overnight with antibodies against TUBB3 (SDL.3D10, 1:5000, Sigma), TUBB5 (SAP.4G5, 1:20 000, Sigma), TUBB2C (7B9, 1:1000, Sigma), TUBB2A and TUBB2B (ONS.1A6, 1:400, Sigma), general β-tubulin (tub 2.1; 1:2000, Sigma), or β-actin (1:10 000; Cell Signaling Technology) in 5% nonfat dry milk in TBS-T. Immunoreactive signals were detected by incubation with Mouse TrueBlot ULTRA horseradish peroxidase-conjugated anti-mouse IgG (eBioscience Ltd.) followed by chemiluminescent detection using the Western Lightning Chemiluminescence Reagent Plus Kit (Perkin Elmer). The monoclonal antibodies used are specifically directed against a synthetic peptide representing the isotype-defining carboxyterminal amino acid sequence of β-tubulins [16–18]. Given that the ONS.1A6 antibody is raised against C-terminal peptide that is common to TUBB2C and TUBB4, it theoretically recognizes both proteins [16]. However, it was shown previously that TUBB4 is not expressed in the testis [19].

Immunohistochemistry

Mice (control and SCARKO at Days 10 and 140) and adult rats were killed and testes were fixed in Bouin solution for 6 h at room temperature. Tissues were processed into paraffin using standard techniques and sectioned at 5-μm thickness. Beta-tubulin proteins were detected by immunohistochemistry using standard methods described in detail previously [20]. Briefly, antigen retrieval in 0.01 M citrate buffer, pH 6.0, was performed, followed by endogenous peroxidase activity blocking by incubation of slides in 3% (vol/vol) H₂O₂ in methanol. Slides were incubated overnight at 4°C with primary antibody against TUBB3 (T2200, 1:20 000 dilution for mouse; SDL.3D10, 1:10 000 dilution for rat; Sigma), TUBB5 (SAP.4G5, 1:100 000, Sigma), TUBB2C (7B9, 1:100, Sigma), TUBB2A and TUBB2B (ONS.1A6, 1:300, Sigma), and general β-tubulin (tub 2.1; 1:500, Sigma). Slides were then incubated with biotinylated goat-anti-rabbit or rabbit-anti-mouse secondary antibody (DAKO) followed by incubation with streptavidin-conjugated horseradish peroxidase (DAKO) and visualization of immunostaining using diaminobenzidine (Liquid DAB+; DAKO). To ensure reproducibility of results and accurate comparison of immunostaining between treatment groups, sections from different genotypes were run in parallel on at least three occasions. Representative sections were photographed using a Leica DMR microscope (Leica) fitted with a Sony DXC 9100P 3CCD color video camera (Sony) and captured with Leica LIDA software version 1.60 (Leica Microsystems Imaging Solutions Ltd.). Images were compiled using Coreldraw 9 (Corel Corporation).

Plasmids

All genomic Tubb3 fragments tested were derived from the BAC clone RP23-34O22 (obtained from CHORI,  http://bacpac.chori.org/home.htm). Fragments were isolated either by restriction digestion or by PCR amplification (for primers see Table 1). Luciferase reporter plasmids were constructed using the pGEM-4Z as a basic vector. A minimal promoter containing the human E1b TATA box [21] was inserted in this vector in front of the luciferase reporter gene from the pGL4 vector (Promega) [22]. All PCR fragments were cloned into the basic reporter vector using the NheI restriction site. For DNA fragments obtained by restriction digestion, fragments and linearized vector were blunted before ligation. Mutations were introduced using the QuickChange II site-directed mutagenesis kit (Stratagene) with appropriate primers (Table 1).

Transfection Experiments

COS-7 (American Type Culture Collection) and SK11 cells (obtained from Dr. P. Saunders, Edinburgh, U.K.) were plated in a 96-well plate at 10⁵ and 5.10⁴ cells/well respectively in DMEM glutamax medium (Invitrogen) supplemented with 10% fetal calf serum (Hyclone) and antibiotics. COS-7 cells were maintained at 37°C, and SK11 cells were grown at 34°C [23]. After 24 h, cells were transfected using GeneJuice transfection reagent (Novagen) in 96-well plates with 100 ng luciferase reporter vector, 10 ng of the hAR expression plasmid [24], and 5 ng of CMV-β-galactosidase plasmid (Stratagene) per well as a control for transfection efficiency. For each reporter construct, triplicate wells were treated for 24 h with 10 nM R1881 or vehicle, starting 24 h after transfection. After correction for transfection efficiency, induction factors were calculated as the ratios of the average value of the luciferase value of the hormone-stimulated samples vs. vehicle-treated samples. An empty luciferase reporter vector and a 4xTAT-GRE construct [25] were included as negative and positive control respectively. As a supplementary negative control, a mirror experiment was set up in which no hAR expression plasmid was cotransfected. None of the investigated constructs was able to stimulate expression of the luciferase reporter after addition of R1881 in the absence of AR.

Electrophoretic Mobility Shift Analysis

Band shift assays were performed essentially as described previously [26] using cellular extracts from COS-7 cells transiently transfected with the hAR expression plasmid [27]. Partially overlapping oligonucleotides containing the ARE motifs or mutations thereof (Table 1) were radiolabeled with [α-³²P]dCTP via a Klenow fill-in reaction. In-house anti-AR rabbit polyclonal antibody was added to the binding mixture to prove specific interaction between the AR and the probe. Competition experiments were performed with a 50- or 200-fold excess of unlabeled double-stranded oligonucleotide (wild-type ARE as the specific competitor; mutated ARE as the nonspecific competitor; Table 1).

Chromatin Immunoprecipitation

The detailed procedure for isolation of chromatin from adult mouse testes and the subsequent chromatin immunoprecipitation (ChIP) reaction is described in the Supplemental Materials and Methods (all Supplemental Data are available online at  www.biolreprod.org). In short, immunocomplexes were pulled down after overnight incubation of the chromatin with anti-AR antibody (PG-21; Millipore) or rabbit IgG (negative control; Cell Signaling Technology). After washes, reverse cross-linking, and phenol-chloroform extraction of DNA, binding of AR to the Tubb3 ARE region was determined by qPCR analysis. The presence of the Tubb3 ARE region (primers were chosen to span ARE1; see Table 1) and a control region (Tubb3 Ctrl; Table 1) in the samples was quantified with respect to the input. Recovered DNA in all samples was expressed as a percentage of the input, and the magnitude of recruitment for both genomic loci was calculated as the ratio of percentage of recovered DNA for the specific antibody sample over the percentage of recovered DNA for the nonspecific antibody sample.

Regulatory Sequences Analysis Tools

The Matrix-scan tool of the Regulatory Sequences Analysis Tools software package ( http://rsat.ulb.ac.be/rsat/) [28] was used to screen for putative AREs using default settings and different thresholds for the P value as described earlier [22].

Statistical Analysis

Gene expression between different groups and treatments were compared by ANOVA followed by Fisher least significant difference (LSD) test using NCSS 2000 software (NCSS, Statistical Analysis and Data Analysis Software) unless indicated otherwise.

Tubb3, But Not Other Beta-Tubulins, Is Downregulated in SCARKO Testes

We previously identified Tubb3 as an AR-regulated gene in the testis based on microarray analysis [4]. Tubb3 transcript levels were assessed by qPCR in testes derived from 10-day-old control and SCARKO mice, together with the transcripts encoding all other known β-tubulin isotypes. As shown in Figure 1A, the data confirm that ablation of the AR significantly decreases the expression levels of Tubb3 mRNA (by 60%), whereas expression of other β-tubulin isotypes is not significantly affected. The data also demonstrate that Tubb3 is one of the most prominent β-tubulin isotypes in the testis at this age. The reduction in Tubb3 mRNA level is paralleled by a comparable reduction (down to 30%) in TUBB3 protein level (Fig. 1B). Quantitative RT-PCR measurements on Days 35 and 140 were performed to verify whether the reduction in Tubb3 expression persists throughout puberty and adulthood (Fig. 1C). As the cellular composition of SCARKO and control testes differs significantly after d10, an external standard (luciferase mRNA) was added to the samples to allow normalization of Tubb3 mRNA levels per testis. Figure 1C shows that Tubb3 mRNA levels per testis remain significantly lower in d35 and d140 SCARKO testes than in control testes.

FIG. 1.

Testicular expression of β-tubulin isotypes in SCARKO and control mice. A) The expression of different β-tubulin isotypes was assessed by qPCR. Expression levels were measured in control and SCARKO testes of 10-day-old animals. Data were normalized to Rpl19 mRNA. Values represent the mean ± SEM of nine measurements. B) TUBB3 expression in 10-day-old control and SCARKO testes as determined by Western blotting (n = 6 per genotype). A representative Western blot is shown on the left. Beta-actin (ACTB) was used as a loading control. C) Tubb3 mRNA expression in pubertal and adult (Day 35 and Day 140) testes of control and SCARKO mice as measured by qPCR. Data were normalized to a standard of exogenously added luciferase mRNA. Values represent the mean ± SEM of seven measurements. ND, not detectable; *P < 0.001 in comparison with the respective control group (AMHCre).

Antiandrogens Suppress Tubb3 Gene Expression in Wild-Type Prepubertal Mice and Rats

To confirm androgen regulation of Tubb3 expression in an independent experimental paradigm, 7-day-old wild-type mice were treated for 5 days either with the antiandrogens flutamide or bicalutamide or with TP. Rhox5 expression levels were measured as a positive control. Antiandrogen administration markedly decreased and TP administration increased seminal vesicle weight confirming the efficacy of treatment (Supplemental Table S1). Bicalutamide decreased Rhox5 and Tubb3 mRNA levels to 4.7% and 31.1% of those observed in vehicle-treated controls, respectively (Fig. 2, left panel). Flutamide caused a similar decrease in transcript levels (to 9.6% and 40.3% respectively).

FIG. 2.

Antiandrogen and androgen treatment reduce Rhox5 and Tubb3 expression in prepubertal mice and rats. Quantitative RT-PCR analysis of testes from 12-day-old mice and rats treated for 5 days with bicalutamide (Bic), flutamide (Flut), testosterone proprionate (TP), or vehicle as described in Materials and Methods. Gene expression was normalized to luciferase mRNA added to the sample as an external standard. Results are expressed as a percentage of the expression observed in the controls (arbitrarily assigned a value of 100; dashed line). Values shown are means ± SEM. *P < 0.05 in comparison with the respective control group.

Unexpectedly, TP also significantly reduced the expression of Tubb3 (to 38.1%) and of Rhox5 (to 54.3%). A plausible explanation for the reduction in expression in these AR-dependent genes, as well as for the 22% reduction in the relative testis weight after TP administration, is that already at this early age, androgen administration reduces intratesticular androgen levels through a negative feedback effect on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion (as demonstrated below for the rat).

To explore whether androgen regulation of Tubb3 could also be confirmed in another species, a qPCR assay was developed for the rat homologues of Rhox5 and Tubb3 and 7-day-old rats were exposed to the above described treatment protocol with antiandrogens and TP. Efficacy of treatment was again confirmed by the observed changes in seminal vesicle weight (Supplemental Table S1). The effects of the studied treatments on rat Rhox5 and Tubb3 transcript levels were comparable to those observed in mice, although the antiandrogen-induced decrease in transcript levels was less pronounced, particularly for Rhox5 (Fig. 2, right panel). The bicalutamide- and flutamide-induced decrease in transcript levels occurred despite an approximately 2-fold and 4-fold increase in serum LH levels (Supplemental Fig. S1), which may have counteracted the effects of the antiandrogens by raising the intratesticular level of androgens. As in mice, TP treatment also significantly decreased both Rhox5 and Tubb3 expression levels. The decrease in relative testicular weight (by 26%) together with the decrease in LH and FSH serum levels associated with TP treatment (Supplemental Table S1 and Supplemental Fig. S1) confirm that the hypothalamo-pituitary-gonadal axis is functional at this age, and, accordingly, that TP treatment may have suppressed intratesticular androgen levels.

To avoid the confounding secondary changes (e.g., in the secretion of gonadotropins) inherent to in vivo systems, we also studied the effects of androgens and antiandrogens on Tubb3 expression in a recently developed organotypic culture system [14]. As shown in Figure 3A, bicalutamide treatment for 48 h significantly reduced Tubb3 expression in testicular explants from 8-day-old mice. In contrast, incubation with the synthetic androgen R1881 did not result in transcript levels exceeding those observed in the vehicle treated control (or in 8-day-old mice testes) indicating that Tubb3 is maximally stimulated by endogenous androgens, as demonstrated for other androgen-regulated genes [14]. However, a clear stimulatory effect of androgens on Tubb3 expression in this system was demonstrated upon treatment with R1881 after inhibition of endogenous androgen production by ketoconazole for 24 h (Fig. 3B). Interestingly, experiments with rhFSH revealed a dual mechanism of action for this hormone. First, rhFSH alone stimulated Tubb3 mRNA level (without increasing testosterone production in the explants [14]); the level rose to that in 10-day-old mice testes (Fig. 3A). This effect was largely counteracted by bicalutamide, suggesting that rhFSH mainly acts through AR-dependent signaling to regulate Tubb3 expression in SCs. Second, FSH increased Tubb3 expression in explants derived from SCARKO mice (data not shown), indicating that FSH can stimulate Tubb3 expression independently of AR. Further supporting this concept was the finding that explants cotreated with rhFSH and bicalutamide had significantly higher Tubb3 expression than those treated with bicalutamide alone (Fig. 3A). In conclusion, our data indicate that androgens and FSH cooperate to control Tubb3 transcription.

FIG. 3.

Hormonal control of Tubb3 expression in organotypic cultures. A) Testicular explants from 8-day-old mice were cultured for 48 h in control medium (Vehicle) or in medium supplemented with R1881 (10⁻⁸ M), bicalutamide (Bic; 10⁻⁵ M), rhFSH (20 ng/ml) or combinations of these agents, as indicated. Expression levels of Tubb3 were measured by qPCR (grey bars). As a control, expression levels were also measured in testis samples freshly derived from 8-day-old (day 8) and 10-day-old (day 10) mice processed in parallel (open bars). All measurements were normalized to the level of Rn18s and were expressed as a percentage of the values measured in testes from 10-day-old mice, arbitrarily set at 100 (dashed line). Values indicated are the mean ± SEM of five independent samples. B) Testicular explants from 8-day-old mice were pretreated for 24 h with control medium or with ketoconazole (Keto; 2.10⁻⁵ M) as indicated. Thereafter, incubations were either stopped (STOP) or continued after replacement of the medium by control medium or by medium supplemented with R1881 (10⁻⁸ M) and incubation was continued for another 24 h. Expression levels were determined using qPCR and are expressed relative to 10⁸ copies of Rn18s as an internal control. The values are the mean ± SEM of four or five independent samples. ANOVA followed by Fisher LSD test was used to compare expression levels in cultured explants after log transformation. Values derived from freshly prepared Day 8 and Day 10 testes were not included in the statistical analysis. Values that differ significantly between each other (P < 0.05) are indicated by different letters (a, b, c, and d).

TUBB3 Protein Is Specifically Expressed in SCs in an AR-Dependent Manner

TUBB3 immunohistochemical staining of control and SCARKO testes of 10-day-old mice revealed intensive staining around SC nuclei with radial extensions to the base and center of the tubules. No staining was observed in developing germ cells, interstitial cells, or peritubular myoid cells (Fig. 4A, second row). Concordant with these data, staining was more intense in control than in SCARKO SCs (Fig. 4A, top row).

FIG. 4.

Expression of TUBB3 protein in mouse and rat testes. A) Top row: immunostaining of TUBB3 (brown) in day 10 control (left) and SCARKO (right) mouse testes. Dashed rectangle: area selected for magnification in second row. Second row: high-power magnification of pictures in the top row for identification of cell types. Third row: immunostaining of TUBB3 in day 140 control (left) and SCARKO (right) mouse testes. Dashed rectangle: area selected for magnification in bottom row. Bottom row: high-power magnification of pictures in third row for identification of cell types. Insets: negative control. Arrows, SCs; arrowheads, germ cells. Bar = 10 μm. B) TUBB3 expression in 140-day-old control and SCARKO testes as determined by Western blot analysis. Densitometric quantification demonstrated decreased TUBB3 expression in SCARKO testes relative to control testes (n = 3 per genotype). C) Immunostaining of TUBB3 in adult rat testis. Arrows, SCs; arrowheads, germ cells. Bar = 10 μm.

In testes from 140-day-old control mice, TUBB3 staining was also clearly confined to SCs. Staining was in a dense, spoke-like pattern radiating from the base of the seminiferous epithelium to the lumen. Bundles of TUBB3 staining touched the periphery of the tubules and clearly surrounded SC nuclei; extensive branching of stained bundles was seen around elongating spermatid heads. No staining was observed in interstitial cells, peritubular myoid cells, developing germ cells, or released spermatozoa (Fig. 4A, bottom row). Day 140 SCARKO testes also had SC staining but, as expected, the signal was weaker than in control testes (Fig. 4A, third row). The pattern of staining was also more variable. Some tubules show spoke-like structures comparable to those observed in control testes, whereas others displayed a more irregular honeycomb pattern of staining. No obvious difference in TUBB3 expression levels was observed between the different stages of the seminiferous epithelial cycle. Western blotting analysis confirmed lower TUBB3 protein expression in SCARKO vs. control testes (Fig. 4B).

Staining of adult rat testis for TUBB3 revealed a pattern of preferential SC localization very similar to that observed in mice (Fig. 4C). In rats, however, some staining was also noticed in the interstitial compartment.

To explore whether other β-tubulin isotypes are also expressed in SCs, adult mouse testes were also stained using antisera directed against TUBB2A/2B, TUBB2C, and TUBB5. An antiserum recognizing all β-tubulins was included as a control. As shown in Supplemental Figure S2, selective SC staining was also noted for TUBB2A/2B and TUBB5, whereas germ cell staining was noted for TUBB2C. The expression of TUBB2A/2B, TUBB3, and TUBB5 in SCs was confirmed by Western blotting experiments on SCs isolated from 19-day-old mice. Purified rat SCs expressed the same profile of β-tubulin isotypes (Supplemental Fig. S2).

Tubb3 Intron 1 Has an ARE

To explore whether the Tubb3 gene may be directly regulated by androgens, we performed a functional screen for AREs in the region extending from −7028 to +101 bp relative to the Tubb3 transcription initiation site. To this end, fragments of the promoter, varying from 655 up to 7128 bp, were cloned into a luciferase reporter plasmid (Supplemental Fig. S3, A and B) and transfected into either COS-7 or SK11 cells together with an hAR expression plasmid. In COS-7 cells, the presence of the promoter fragments caused a limited (∼8-fold) increase in luciferase activity when compared to empty reporter vector (Supplemental Fig. S3B), but no further increase was observed after stimulation with the synthetic androgen R1881 (Supplemental Fig. S3C). In SK11 cells, an SC-derived cell line, the presence of the promoter fragments caused a marked (∼55-fold) increase in the basal expression level. The highest levels were obtained with the 655-bp proximal promoter fragment, suggesting that this fragment contains regulatory elements enhancing basal transcription, particularly in SCs. As in COS-7 cells, however, none of the constructs tested was able to mediate a further increase in luciferase expression after addition of R1881 (Supplemental Fig. S3, B and C).

Because AREs have also been described in the first intron of some androgen-regulated genes [29–31], we next screened a 5262-bp BamHI-XhoI-fragment (+647 to +5808) spanning nearly the entire Tubb3 intron 1 (Fig. 5A) for its ability to enhance luciferase expression in the presence of an activated AR. As shown in Figure 5B, transfection of the 5262-bp fragment in COS-7 cells resulted in a ∼24-fold induction of luciferase activity after stimulation with R1881. Examination of the activity of different subfragments within and upstream of the 5262-bp fragment revealed that the activity was situated in a 965-bp region at the 3′ end of the intron (+4958 to +5922; ∼22-fold stimulation). Similar results were obtained in transfected SK11 cells, although the magnitudes of induction for the 5262-bp and the 965-bp fragments were slightly lower (∼8.0-fold and ∼10-fold respectively).

FIG. 5.

Identification of a functional ARE in the first intron of Tubb3. A) Restriction map of the Tubb3 promoter and intron 1. Exons 1 and 2 are shown as rectangles. Arrow, start of transcription. B) Different fragments of Tubb3 intron 1 were cloned into a luciferase reporter vector and tested for androgen responsiveness. In short, COS-7 and SK11 cells were treated 24 h after transfection with 10⁻⁸ M R1881 or vehicle for 24 h, as described in Materials and Methods. All results are presented as mean ± SEM of induction factors measured in four (COS-7) or five (SK11) independent experiments. A positive control (4xTAT-GRE) and a negative control (empty reporter vector) were included in each experiment. All fragments are identified by their length, as indicated. C) Subfragments of a 965-bp fragment were cloned into a luciferase reporter vector and tested for androgen responsiveness as described in B. Results are presented from six (COS-7) or five (SK11) independent experiments. Putative AREs are shown as squares, mutated ARE 1 is shown as a cross. Luc, luciferase reporter gene.

The 965-bp fragment was screened for consensus AREs using the matrix-scan tool of the Regulatory Sequences Analysis Tools software package [28]. With the P value threshold set at ≤10⁻⁴, one consensus-like ARE motif was identified, located at position +5439 to +5453 (AGAAGGctgTGTTCT; designated ARE1; P = 9.2 × 10⁻⁵). When the P value threshold was set at ≤ 10⁻³, three additional ARE motifs were identified, all located in the neighborhood of ARE1 at position +5497 to +5511 (TAGTTTtggTGTTCC; ARE2; P = 6.5 × 10⁻⁴), +5366 to +5380 (TAGTCTgcaTGTCCT; ARE3; P = 8.1 × 10⁻⁴) and +5588 to +5602 (AGAACGcaaTGCTCC; ARE4; P = 1.0 × 10⁻³).

To elucidate which, if any, of the predicted AREs mediated androgen responsiveness of the 965-bp fragment and to explore the possibility of other hidden nonconsensus AREs, we subcloned different overlapping parts of the 965-bp fragment in the reporter vector. In COS-7 cells, a prominent androgen response was observed with the 545-bp, 198-bp-a, and 101-bp fragments (92-, 184- and 38-fold stimulation, respectively). All these fragments contained the ARE1 sequence (Fig. 5C). Much lower degrees of stimulation (1.7- to 3.5-fold) were observed with fragments containing none, one, or more than one of the other predicted AREs (222 bp, 200 bp, 196 bp, 198 bp-b, and 156 bp). As with COS-7 cells, SK11 cells exhibited androgen induction with the 545-bp, the 198-bp-a, and the 101-bp fragments (11-, 9- and 3-fold induction, respectively). No consistent activity was observed with the other fragments investigated.

Taken together, these data suggest that ARE1 is the most active response element in the studied region. Interestingly, its activity seems to be enhanced by ARE3, as a reporter plasmid including both AREs (198 bp-a) showed a 4.9-fold (in COS-7 cells) and 2.3-fold (in SK11-cells) higher induction factor than a construct with ARE1 alone (101 bp). To definitively determine whether ARE1 promotes androgen responsiveness, we mutated the “TGTTCT” half-site of ARE1 to “TTTTCT” in the 545-bp fragment (545 bp-mut). This reduced androgen induction in COS-7 cells and SK11 cells by a factor of 27- and 12-fold respectively (Fig. 5).

Next, we investigated the ability of the identified AREs to interact with the AR in vitro using electrophoretic mobility shift analysis (EMSA). As shown in Figure 6A, addition of a cell extract containing AR clearly delayed the migration of a labeled probe containing ARE1 and ARE4. A supershift was observed in the presence of an AR antiserum for both AREs, confirming that the delay in migration was due to interaction with the AR. No binding was observed for ARE2 and ARE3 (Supplemental Fig. S4A). As evidence for specificity, competition experiments were performed in which a 50- and 200-fold excess of unlabeled WT and mutated ARE probes was added to the reaction (Supplemental Fig. S4B). Mutated ARE probes were designed to contain a 1-bp mutation that disrupts one half site of the ARE consensus sequence (see Table 1). For both AREs, a 50-fold excess of unlabeled WT probe was able to compete for binding of the AR to the labeled WT probe, an effect that was even more outspoken when a 200-fold excess unlabeled probe was applied. In contrast, competition with an unlabeled mutated probe (either 50-fold or 200-fold excess) did not affect binding of the AR to the labeled WT probe, demonstrating that the AR specifically binds to the ARE DNA sequence.

FIG. 6.

AR-Tubb3 ARE Binding. A) EMSA using full-size AR constructs were performed with wild-type ARE1 and ARE4 radiolabeled probes, as described in Materials and Methods. Anti-AR antibody was added where indicated to demonstrate specificity of AR binding. Arrow, DNA-receptor complex; arrowhead, supershifted complex. B) ChIP analysis of adult mouse testes chromatin. Fold enrichment is expressed as the ratio of recovered DNA in the specific antibody sample over the nonspecific antibody sample (1-fold enrichment = no enrichment) as explained in Materials and Methods. Values are given as mean ± SEM for five independent experiments for the Tubb3 ARE region (ARE1) and a negative control region located 4.2 kb upstream of the Tubb3 ARE1 (Ctrl).

Finally, we performed ChIP assays on chromatin from adult testicular tissue with an antibody against AR to demonstrate in vivo binding of the AR to the Tubb3 ARE region. Importantly, as the complete Tubb3 ARE cluster spans only 237 bp, the resolution of the present ChIP experiment (with an average chromatin fragment size of 300 to 400 bp) does not permit identification of the specific ARE(s) bound, but rather allows determination of binding to the Tubb3 ARE region as a whole. As demonstrated in Figure 6B, the Tubb3 ARE region (ARE1) is significantly enriched 2.2-fold when ChIP is performed with a specific anti-AR antibody compared to a nonspecific antibody. In contrast, a control genomic region (Ctrl) situated 4.2 kb downstream of the Tubb3 ARE1 is not enriched (factor 0.9). In conclusion, the data suggest that the AR binds specifically to the Tubb3 ARE region in vivo.