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Endocrinology, doi:10.1210/en.2007-0930
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Endocrinology Vol. 148, No. 12 5851-5864
Copyright © 2007 by The Endocrine Society

CCAAT/Enhancer Binding Protein ß, But Not Steroidogenic Factor-1, Modulates the Phthalate-Induced Dysregulation of Rat Fetal Testicular Steroidogenesis

Adam J. Kuhl, Susan M. Ross and Kevin W. Gaido

The Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina 27709-2137

Address all correspondence and requests for reprints to: Adam J. Kuhl, The Hamner Institutes for Health Sciences, 6 Davis Drive, P.O. Box 12137, Research Triangle Park, North Carolina 27709-2137. E-mail: akuhl{at}thehamner.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prolonged in utero exposure of fetal male rats to dibutyl phthalate (DBP) can result in a feminized phenotype characterized by malformed epididymides, hypospadias, cryptorchidism, and retained thoracic nipples, among others. These symptoms likely result, in part, from decreased expression of steroidogenic enzymes and, therefore, reduced testosterone biosynthesis. However, the molecular mechanisms involved in these changes in gene expression profiles are unknown. To understand these mechanisms in rats, in vivo DNase footprinting was adapted to provide a semiquantitative map of changes in DNA-protein interactions in the promoter region of steroidogenic genes, including steroidogenic acute regulatory, scavenger receptor B-1, cytochrome P450 side chain cleavage, and cytochrome P450 17A1, that are down-regulated after an in utero DBP exposure. Regions with altered DNase protection were coordinated with a specific DNA binding protein event by EMSA, and binding activity confirmed with chromatin immunoprecipitation. Results demonstrated altered DNase protection at regions mapping to CCAAT/enhancer binding protein ß (c/ebp ß) and steroidogenic factor-1 (SF-1). Chromatin immunoprecipitation confirmed declines in DNA-protein interactions of c/ebp ß in DBP treated animals, whereas SF-1 was reduced in both diethyl phthalate (nontoxic) and DBP (toxic) treatments. These results suggest that inhibition of c/ebp ß, and not SF-1, is critical in DBP induced inhibition of steroidogenic genes. In addition, these observations suggest a pathway redundancy in the regulation of steroidogenesis in fetal testis. In conclusion, this study presents a snapshot of changes in the structure of transcriptional machinery and proposes a mechanism of action resulting from DBP exposure.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TRENDS IN DECLINING male reproductive health, including an increased incidence of cryptorchidism, hypospadias, reduced semen quality, and prostate and testicular cancer, have been recently reported (1). It has been suggested that environmental chemicals that disrupt the male endocrine system have contributed to the observed declines (2). Phthalate esters are widely used chemicals found in plastics, cosmetics, and other common consumer products. Studies have demonstrated that in rodents, maternal exposure to dibutyl phthalate (DBP) can induce cryptorchidism, hypospadias, and reduced fertility in the developing male fetus without any observable affect on the mother (3, 4, 5). Additional human studies have indicated a correlation between phthalate exposure and reproductive development of male fetuses (6).

The effects of DBP are similar to those observed with exposure to the antiandrogens linuron and flutamide. However, DBP and its primary metabolite do not interact with the androgen receptor (AR), unlike these other antiandrogens (7). These effects are instead a result of a reduction in testosterone synthesis at a developmental stage, when a testosterone surge is critical (8, 9). The normal development of the male reproductive tract is an androgen-driven process that requires the Leydig cells to produce steadily increasing testosterone levels from gestational d (GD) 17–20 (10). The signaling pathway that induces fetal testosterone production in rats is unknown. It is likely not to be under the influence of LH but, instead, under other paracrine and endocrine factors (11). This testosterone synthesis is critical for the stabilization and differentiation of the wolffian ducts into epididymides, vasa differentia, and seminal vesicles (12), and its inhibition adversely affects these developmental processes, resulting in malformations of the male reproductive tract (13).

Decreases in testosterone levels after phthalate exposure is due in part to the reduction in expression of genes in the testosterone biosynthesis pathway, including those coding for steroidogenic acute regulatory (StAR) protein and scavenger receptor B-1 (SR-BI), which are involved in cholesterol transport, as well as the steroid converting enzymes cytochrome P450 side chain cleavage (CYP11A1) and cytochrome P450 17A1 (CYP17A1). Using a testis explant system, DBP exposure leads to diminished function at each point in the testosterone biosynthesis pathway except 17ß- hydroxysteroid dehydrogenase (14). Furthermore, multiple studies have demonstrated a decreased expression of several genes involved in testosterone production after fetal DBP exposure (8, 9) at multiple time points (15) and doses (16). These results provide strong evidence for transcriptional inhibition of the steroidogenic pathway as one of the mechanisms for phthalate-induced testicular dysgenesis. However, the molecular mechanisms behind this coordinate down-regulation of these genes are unknown and are explored in this study.

Recent advances in toxicogenomics have contributed greatly to the study of global gene changes in response to xenobiotic exposure. Foremost in these advances has been the use of microarray technology, which measures the transcriptional activity of thousands of genes to determine potential gene pathways and networks involved in the toxic response. Although this technology can identify the etiology of a disease, microarrays do not provide direct information regarding the molecular mechanisms that manifest the changes in gene expression. The aim of the current study was to analyze protein-DNA interactions in the promoter regions of multiple down-regulated steroidogenic genes in an attempt to determine more directly the molecular mechanisms of DBP toxicity on gene expression. Transcription factors demonstrating changes in binding that are shared among genes in the testosterone biosynthesis pathway may suggest a common molecular mechanism of action for phthalate induced testicular dysgenesis.

Many of the genes down-regulated after phthalate exposure are known to be regulated by the nuclear receptor steroidogenic factor 1 (SF-1), and, therefore, SF-1 is postulated as playing a role in phthalate toxicity (17). However, there are conflicting reports as to the importance of SF-1 in a phthalate response. Although some studies demonstrate a decrease in SF-1 expression after phthalate exposure (18), others have observed no change in protein or transcript levels (17, 19). Moreover, other factors have also been identified as possibly playing a role in phthalate toxicity, including the peroxisome proliferator activated proteins (reviewed in Ref. 20), steroid and xenobiotic nuclear receptor (21), pregnane X receptor, and constitutive androstane receptor (22), adding to the confusion of how phthalates influence transcriptional regulation. Therefore, in the study that follows, a detailed analysis of changes in DNA-protein interactions in the promoter of StAR, SR-BI, CYP11A1, and CYP17A1 was performed using multiple techniques to understand the role of SF-1 and other factors in the molecular mechanism of phthalate toxicity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
This study was done in accordance with and approved by the Institutional Animal Care and Use Committee of The Hamner Institutes for Health Research, and adhered to all federal regulations of care and use of laboratory animals. A total of 40 Sprague Dawley outbred rats was time mated at Charles River Laboratories, Inc. (Raleigh, NC) and shipped to The Hamner on GD 12. Dams were assigned to a treatment group by body weight randomization using Provantis (Instem LSS, Stone, UK), with 10 animals in each dose group. Animals were housed in The Hamner animal facility, accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, in a humidity and temperature-controlled, high-efficiency particulate air-filtered, mass air-displacement room. The room was maintained on a 12-h light/dark cycle at approximately 22 ± 4 C, with a relative humidity of approximately 30–70%. Animals were identified by ear tags and cage cards, and housed individually in polycarbonate cages with {alpha}-dri cellulose bedding (Shepherd Specialty Papers, Kalamazoo, MI). Rodent diet National Institutes of Health 07 (Zeigler Brothers, Gardener, PA) and reverse-osmosis water were provided ad libitum.

Dams were treated by oral gavage (1 ml/kg) on GD 18 with corn oil vehicle (Sigma Chemical Co., St. Louis, MO), DBP (Aldrich Chemical Co., Milwaukee, WI) in corn oil at 100 or 500 mg/kg, or 500 mg/kg diethyl phthalate (DEP) as a negative control. Purity and concentration of all doses were verified using a Hewlett Packard 5890 gas chromatograph (Hewlett Packard, Palo Alto, CA). Dose levels were chosen based on previous studies showing that 100 and 500 mg/kg DBP produced significant changes in gene expression in the male offspring without maternal toxicity or fetal death (9). DEP has induced no developmental abnormalities in the male fetus after a 750-mg/kg daily dosage from GD 14 to postnatal d 3 (23).

Twenty-four hours after treatment, all dams were euthanized on GD 19 by carbon dioxide asphyxiation. Fetuses were removed by cesarean section, and body weights were recorded. All fetuses were euthanized by decapitation and sexed by internal examination of the reproductive organs. The right and left testes were removed from male fetuses and separated using a dissecting microscope with transillumination. Testes were snap frozen in liquid nitrogen in separate vials and stored at –80 C.

Testosterone RIA
Testosterone was measured using a method modified from vom Saal (24), as performed previously in this laboratory (9, 14, 15, 16). Briefly, one pair of testes from eight replicate pups for each treatment was homogenized in 100 µl PBS with gelatin. A sample was removed for protein determination using the bicinchoninic acid protein assay (Sigma Chemical). Testosterone was then extracted using ethyl acetate-chloroform (4:1) for a total of 1 ml. The extracted layers were separated by centrifugation at 1500 x gfor 10 min at 4 C. The organic layer was removed from the inorganic layer. The organic layer was then dried under nitrogen gas. The samples were reconstituted in 100 µl of the zero standard provided in the I125 RIA coated tube kit from MP Biomedicals (Irvine, CA). Detection and measurement were conducted using the Testosterone I125 RIA kit (MP Biomedicals) according to manufacturer’s instructions.

Real-time quantitative RT-PCR
Total RNA was isolated from the testes of three pups per treatment group using RNA STAT-60 reagent (Tel-Test, Friendswood, TX). Subsequent RT reactions, quality control for RT reactions, and quantitative PCR were performed as described previously (9, 14, 15, 16). Real-time reactions were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. Preoptimized primers and probes were provided by Applied Biosystems, Inc. (Foster City, CA) (GAPDH, Rn99999916_s1; StAR, Rn00580695_m1; CYP11A1, Rn00568733_m1; SR-BI, Rn00580588_m1; and CYP17A1, Rn00562601_m1), and all reactions were performed on an ABI PRISM 7900HT Sequence Detection System using SYBR Green PCR and TaqMan Universal PCR Master Mix reagent kits according to the manufacturer’s instructions for quantification of gene expression.

In vivo DNase I footprinting
A protocol for nuclei isolation and DNase I footprinting described previously (25) was adapted from Schulte (26) and Bossard (27) et al., and performed in triplicate using testis from pups in separate litters. Briefly, one pair of testis was homogenized in a dounce homogenizer with the "B" pestle in 500 µl cold nuclei buffer (NB) [60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 15 mM Tris-HCl (pH 8.2), protease inhibitors, and 0.5 mM dithiothreitol (added fresh each time)]. The homogenate was pelleted and resuspended in 100 µl cold NB. DNA was digested with 20 U DNase I (Ambion, Austin, TX) for 2 min at 37 C. The reaction was stopped, and proteins were digested by adding 100 µl NB, 2 µl 0.5 M EDTA, 20 µg proteinase K, and 10 µl 20% sodium dodecyl sulfate, and incubating at 37 C for 3 h. RNA was removed by RNase A, and DNA was recovered with a phenol chloroform extraction and ethanol precipitation. DNA was quantified using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE).

Ligation-mediated (LM)-PCR
LM-PCR was performed as described by Mueller et al. (28) with the following modifications. First, strand synthesis was completed using 4 µg (~5 µl) genomic DNA, 0.3 pmol gene specific primer GSP1 (Table 1Go) (IDT, Inc., Coralville, IA), and 0.5 U Vent DNA polymerase (New England Biolabs, Ipswich, MA). PCR conditions consisted of one cycle of 5 min 95 C, 30 min 60 C, and 10 min 76 C. Blunt-end ligation of the linker adapter (annealed LM-PCR1 and LM-PCR2) was performed with overnight incubation at 17 C with T4 DNA ligase (New England Biolabs). DNA was ethanol precipitated, and segments were PCR amplified with 10 pmol LM-PCR2 and GSP2 (Table 1Go) primers, and 1 U Vent DNA polymerase for 29 cycles of 1 min 95 C denaturation, 2 min 66 C annealing, and 3 min 76 C extension. DNA was fluorescently labeled through a labeling PCR of three cycles of 1 min 95 C denaturation, 2 min 70 C annealing, and 3 min 76 C extension using 2.3 pmol of a FAM-labeled GSP3 (Table 1Go). After a phenol-chloroform extraction and ethanol precipitation, the pellet was resuspended in 1 µl water, 2.5 µl formamide, 0.5 µl loading dye, and 0.5 µl 400HD ROX size standard (Applied Biosystems). The samples were denatured by 3 min 95 C incubation, and 2.5 µl was loaded onto a 5% Lone Ranger Single sequencing gel (Cambrex, East Rutherford, NJ), and run on an ABI 377 automated sequencer (Applied Biosystems). For greater coverage upstream from the transcription start site, multiple primer sets were used to amplify proximal and distal regions of each promoter. Primer amplification efficiency determines upstream distance resolution.


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  		TABLE 1. Oligonucleotide sequences

 
Footprinting analysis
Applied Biosystems’ GeneScan Analysis system was used to detect size and intensity of peaks that corresponded to DNA fragments created by DNase I footprinting. These data were exported to SigmaPlot (Port Richmond, CA) where fragment sizes were corrected to account for linker adaptor and express sizes as distance upstream from the transcription start site. In addition, zero values were included for missing fragment sizes to complete the profile of the proximal promoter region of the gene. Peaks were aligned manually, and replicates were averaged to get a single pattern. Data were smoothed using loess smoothing with a 0.05 sampling proportion and plotted as a line graph. Peaks represent regions of DNase sensitivity, and valleys indicate likely protection from DNase digestion by protein binding. Changes in valley intensity indicate likely regions of altered transcription factor binding in response to phthalate treatment. Due to the nature of this assay, a detailed statistical analysis of DNase protection is not possible. Regions displaying potential changes in DNase protection were identified by visual inspection. Sequences that contained large differences in protection with relatively low noise and variability were further analyzed by EMSAs and chromatin immunoprecipitation (ChIP) to confirm transcription factor identity and binding activity. Potential transcription factors were identified by sequence analysis using the Transcription Element Search System, visual inspection for consensus binding sequences, and literature analysis.

ChIP
ChIP was performed to confirm changes in transcription factor binding. ChIP assays were performed using the ChIP-IT kit from Active Motif (Carlsbad, CA) following the manufacturer’s instructions with minor modifications. Briefly, two pairs of testes were pooled and fixed in 0.5 ml 1% formaldehyde solution. After the reaction was stopped with a glycine solution, testes were homogenized in a dounce homogenizer using a "B" pestle. DNA was sheared by sonication for five pulses of 10 sec each, followed by a 50-sec incubation on ice after each pulse. Sonication was performed by a VerTis VerSonic sonicator (VerTis Co., Gardiner, NY) at 10% power with a 3-mm tip. After sonication, samples were incubated with 1 µg antibody overnight at 4 C. Antibodies for SF-1 (catalog no. sc-28740x), CCAAT/enhancer binding protein ß (c/ebp ß) (sc-150x), and GATA4 (sc-1237x) were provided by Santa Cruz Biotechnology (Santa Cruz, CA), whereas anti-sp1 (catalog no. 07-645) was provided by Upstate (Charlottesville, VA). Samples were precipitated by binding to Protein G beads and washed. DNA fragments were eluted from the beads, treated to remove protein and RNA, and purified by DNA purification mini columns. Real-time quantitative PCR was performed in an ABI PRISM 7900HT Sequence Detection System using SYBR Green PCR Master Mix with primers designed for the proximal promoter region of StAR, SR-B1, CYP17A1, and CYP11A1 (Table 1Go). Each reaction was cycled 40 times at 94 C for 20 sec, 59 C for 30 sec, and 72 C for 1 min. All precipitations were preformed in triplicate as were all PCRs. Binding activity was calculated as percentage of preimmunoprecipitated input DNA. This is represented by 2–{Delta}Ct x 100, where {Delta}Ct represents the Ct of input DNA subtracted from the Ct of the immunoprecipitated sample. Manufacturer-provided controls were used for both positive and negative precipitation and PCR validation.

EMSAs
EMSAs were performed to confirm DNA-protein interactions at regions identified by in vivo footprinting and to confirm protein identity using the Pierce LightShift Chemiluminescent EMSA kit (Pierce, Rockford, IL). Briefly, 10 µg fetal testis nuclear extract was incubated in the provided reaction buffer supplemented with 20 mM EDTA and 1 µg BSA, with 20 fmol biotin end-labeled target DNA corresponding to regions of altered DNA binding. After a 20-min room temperature incubation, samples were separated on a 5% Tris-borate EDTA SDS-PAGE minigel (Bio-Rad Laboratories, Hercules, CA) and electrophoretically transferred to a nylon membrane (Bio-Rad Laboratories). Membrane was visualized using the Pierce chemiluminescent nucleic acid detection module. When antibodies were added for the supershift (or loss) of a specific protein-DNA interaction, the nuclear extracts were preincubated with 4 µg antiserum [SF-1, Santa Cruz Biotechnology (sc-28740x), c/ebp ß (sc-150x), and GATA4 (sc-1237x)] for 20 min at room temperature before the addition of the labeled DNA probe. Oligonucleotides used for each gene and binding region are listed in Table 1Go.

Western blot analysis
For protein analysis of SF-1 and GATA4, whole testes from three individual fetuses per treatment group were solubilized in PBS, and protein concentration was determined using a bicinchoninic acid protein assay kit following the manufacturer’s protocol. Nuclear extract was prepared as described previously to analyze c/ebp ß-protein levels. An appropriate volume of protein (15, 15, and 20 µg for GATA4, c/ebp ß, and SF-1, respectively) was diluted in lamellae buffer and heated at 95 C for 5 min. Samples were then added to each lane on a 4–15% Tris-HCl SDS-PAGE minigel (Bio-Rad Laboratories). Proteins were electrophoretically transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories). Membranes were incubated for 1 h at room temperature in blocking Tris-buffered saline with 0.1% Tween 20 (TBST) buffer containing 5% nonfat milk. Immunoblot analyses of membranes were performed by incubating with the following primary antibodies at 4 C overnight at a 1:5000 concentration: SF-1, Santa Cruz Biotechnology (catalog no. sc-28740x); c/ebp ß (sc-150x); and GATA4 (sc-1237x). Membranes were washed in TBST for 45 min, followed by incubation with the appropriate secondary antibody conjugated with horseradish peroxidase (Amersham Biosciences, Piscataway, NJ). Positive bands were detected by chemiluminescence with the ECL Plus Western Blot Detection kit (Amersham Biosciences) after a final 1-h wash in TBST. Densitometry was performed using FluorChem version 2.0 analysis software (Alpha Innotech Corp., San Leandro, CA). All blots were normalized to GAPDH [Abcam, Cambridge, MA (catalog no. ab8245)] and expressed as percent control. Each replicate sample was analyzed on triplicate blots to account for blot-to-blot variability.

Statistics
Testosterone, relative gene expression, and relative DNA binding activity were compared among treatments by a one-way ANOVA. If a significant difference among groups was observed, the Tukey-Kramer pairwise multiple comparisons test was used to determine where significance occurred. Statistical significance was accepted at P < 0.05. All statistical analysis and graphing were completed using JMP 6.0 (SAS Institute Inc., Cary, NC). Protein levels from immunoblots were analyzed using ANOVA with subsampling, followed by Dunnett’s multiple comparisons determining statistical significance of difference from control treatments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DBP effects on testosterone concentration and mRNA transcript levels
Pregnant rats were treated with a single dose at GD 18 of 500 mg/kg DBP, 100 mg/kg DBP, 500 mg/kg DEP (a nontoxic phthalate), or corn oil (vehicle control), and killed 24 h later. Analysis showed a decrease in testosterone levels in the DBP-treated testes (Fig. 1Go). When adjusted for total protein concentration, dosing with 500 mg/kg DBP resulted in a significant (P < 0.05) 85% reduction in testosterone concentrations. DEP and 100 mg/kg DBP exposures resulted in no change from vehicle control. In addition, real-time quantitative RT-PCR demonstrated a significant decrease in mRNA of StAR, SR-B1, CYP17A1, and CYP11A1 in both 100 and 500 mg/kg DBP treatment groups (Fig. 2Go). DEP treated animals showed no difference in mRNA for any of the genes. These results are similar to previous studies that demonstrate a reduction in testicular testosterone and mRNA levels of testosterone biosynthesis genes after DBP exposure (14, 15, 16).


Figure 1
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  		FIG. 1. Whole testis testosterone concentration (mean ± SEM). Pregnant dams were treated with one dose of 500 and 100 mg/kg DBP, 500 mg/kg DEP, and vehicle control at GD 18, 24 h before pups were removed. Testes from the pups were snap frozen until use. Testosterone concentration was measured using a testosterone RIA and corrected for total protein concentration. Results were analyzed with ANOVA, followed by the Tukey-Kramer test. Letters represent differences between groups at a significance of P < 0.05.

 

Figure 2
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  		FIG. 2. Relative gene expression for StAR, SR-BI, CYP11A1, and CYP17A1 (mean ± SEM). Gene expression was quantified using real-time RT-PCR with total RNA isolated from whole testis. After RT reaction, transcript levels were measured using TaqMan chemistry, and preoptimized primers and probes from Applied Biosystems. Results were analyzed with ANOVA, followed by the Tukey-Kramer test. Letters represent differences between groups at a significance of P < 0.05.

 
Comparative DNase I footprinting demonstrates common changes in protein binding
A comparative DNase I footprinting assay that was previously adapted in this laboratory (25) was used to elucidate the mechanism behind the observed reduction in steroidogenesis by examining the promoter region of StAR, CYP17A1, CYP11A1, and SR-BI proteins for changes in protein-DNA interactions. Using the footprinting protocol described previously, a detailed map of binding activity was generated for the proximal promoter region of each gene (Fig. 3GoGo). In these maps, regions sensitive to DNase activity are represented by peaks and DNase protection (likely from protein binding) represented by valleys. The maps were then aligned with the DNA sequence of each proximal promoter to determine the location and sequence of protected regions and their putative DNA-binding proteins. Although the nature of this assay does not allow for a detailed statistical analysis, it does provide an excellent "starting off" point for changes in promoter activity when very little is known of the transcriptional regulatory mechanisms of a gene.


Figure 3
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  		FIG. 3. In vivo footprinting binding map of the proximal promoter region. A, StAR. B, CYP17A1. C, CYP11A1. D, SR-BI. Fetal rats were exposed in utero to either 500 mg/kg DBP, 100 mg/kg DBP, 500 mg/kg DEP, or corn oil. Fetal testes were removed, and promoter binding activity in the proximal promoter region was mapped using in vivo DNase footprinting. Maps were aligned to DNA sequence, and transcription factor binding sites were determined by consensus binding sequence and factors demonstrated to bind in the literature. Regions of differential binding activity are circled. Bp, Base pairs.

 

Figure 3A
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  		FIG. 3A. Continued.

 
For the StAR promoter (Fig. 3AGoGo), the protein binding locations corroborate previous reports of DNA binding factors locations (29, 30, 31, 32). These factors include SF-1 (–35, –100, and –140), c/ebp ß (–85 and –110), GATA4 (–60), and activating protein 1 (AP1) (–189). In addition, other potential binding sites are observed at –140, –210, and –250. Putative transcription factors for these sequences include sp1, c/ebp, and GATA. In addition, this assay compared the binding maps from controls with treated samples to identify four regions of differential protein binding activity. These regions include –37/–50, –57/–65, and –85/–95, corresponding to the binding sites of SF-1, GATA4, and c/ebp ß, respectively, all critical in the regulation of StAR transcription in Leydig cells (33).

Promoter regions of other genes provided similar results. CYP17A1 demonstrated altered protein protection at –55/–80, –100/–120, and –180/–215 (Fig. 3BGoGo). The –55/–80 site corresponds to a SF-1 site in humans, rodents, and cow (34, 35, 36), and the –180/–215 site has been shown to bind AP2, sp1, and NR1C in thecal cell (37, 38) and contains a putative binding site for c/ebp ß. However, the –100/–120 site does not contain any significant transcription factor consensus binding motifs. CYP11A1 demonstrated regions of strong DNase protection at –30/–50, –75/–125, and –155/–185 (Fig. 3CGoGo), with the –30 and –160 sites displaying DBP-induced changes in protein binding strength. Others have demonstrated that the –30/–50 site binds SF-1 (39), and the –75/–125 likely binds a sp1/sp3 protein complex (40, 41). There is no previous published data of protein binding on the –160/195 site, but there is a strong putative match for c/ebp ß.

Finally, SR-BI contains regions of DNase protection at –45/–60, –70/–80, –100/–120, –150/–180, and –205/–230 (Fig. 3DGoGo). All of these regions besides the –100/–120 appear to have changes in binding strength after phthalate treatment. Sequence data suggest that sites at –45/–60 and –100/–120 bind sp1, whereas other putative regulatory sites include c/ebp ß (–70/–80 and –150/–180) and AP2, SRY, or c/ebp {alpha} (–205/–230).

Identification of DNA-binding proteins by gel shift assays
Regions that demonstrated possible changes in protein binding were then analyzed by electrophoretic gel shift assays. Biotin-labeled oligonucleotides corresponding to regions identified from footprinting as having differential protein binding were incubated with fetal testis nuclear extract.

To identify StAR transcription factors, oligonucleotides corresponding to –34/–58, –56/–77, and –73/–96 were incubated with nuclear extract from fetal rat testes and antibodies for SF-1, GATA4, c/ebp ß, or sp1. As shown in Fig. 4AGo, complexes were formed with all three oligonucleotides (lanes 1, 5, and 9). A supershift was induced after incubation with an SF-1 antibody (lane 4) that is not observed after incubation with {alpha}-c/ebp ß or {alpha}-GATA4 in the –34/–58 incubations (lanes 2 and 3). Similar incubations identified GATA4 as the binding protein for –56/–77, and c/ebp ß for –73/–96.


Figure 4
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  		FIG. 4. Conformation of DNA-binding proteins with EMSA. Nuclear extract from nontreated fetal rat testis was incubated with the double-stranded biotin-labeled DNA oligos corresponding to the suspected regions of DNase protection from StAR (A), CYP17A1 (B), SR-BI (C), and CYP11A1 (D). Band shifts (arrows) confirm protein DNA interactions at that site. Additional incubation with designated antibodies leads to a supershift (asterisk) or attenuation of the band and identifies the protein binding each oligo. The antibodies used in each reaction are listed above each lane.

 
Three regions of interest were identified from footprinting assays corresponding to –40/–70, –170/–220, and –235/–270 of the proximal CYP17A1 promoter. Figure 4BGo identifies the binding proteins using oligos designed for these regions. Each oligo formed a DNA-protein complex (lanes 1, 4, and 7). Incubation with specific antibodies identified the proteins in these complexes as SF-1, sp1, and c/ebp ß for –40/–70, –170/–220, and –235/–270, respectively (lanes 3, 6, and 8).

Figure 4CGo represents EMSA performed using oligos designed for SR-BI regions of interest (–40/–60, –140/–185, and –205/–230). Supershift after incubation of the protein-DNA complexes with antibodies for specific transcription factors identified the proteins bounds to –40/–60 and –140/–185 oligos as sp1 (lanes 3 and 7). However, band attenuation in the –205/–230 oligo-protein complex in both sp1 and c/ebp ß identified a possible dual binding region (lanes 9 and 11). Sequence data confirm the presence of a putative binding site for both sp1 and c/ebp ß.

Finally, footprinting detected three regions of interest for CYP11A1 at –30/–50, –108/–133, and –155/–185. As shown in Fig. 4DGo, oligos were incubated with testis nuclear extract (lanes 1, 4, and 6), and resulting protein-DNA complexes were further incubated with specific antibodies. Supershift or band attenuation identified SF-1, sp1, and c/ebp ß as the binding protein for –30/–50, –108/–133, and –155/–185, respectively (lanes 3, 5, and 7).

Confirming protein binding activity with ChIP
To confirm quantitative changes in protein-DNA interactions, transcription factor binding activity was assayed using ChIP assays (Fig. 5Go). Results indicated that both DBP and DEP exposure results in a 50–70% decrease in SF-1 binding (Fig. 5AGo) for StAR, CYP11A1, and CYP17A1. SR-BI resulted in no change in SF-1 binding. For DEP, results were significant for StAR and CYP17A1, and borderline significant CYP11A1. DBP induced a decrease in SF-1 binding activity in both 100 and 500 mg/kg treatment groups, but in only CYP17A1 was this decrease significant in the 500 mg/kg. Unlike SF-1, c/ebp ß demonstrated no difference in protein binding after DEP treatment but did demonstrate a decrease in binding activity in StAR, CYP11A1, and CYP17A1 in the 500-mg/kg exposures (Fig. 5BGo). The 100-mg/kg treatment group demonstrated a similar decrease in c/ebp ß binding, but it is not statistically significant. c/ebp ß binding in the SR-BI promoter resulted in a similar trend, but due to high variability in the corn oil treatments, it was not significant. Analysis of GATA4 binding resulted in no observable difference between treatments (Fig. 5CGo).


Figure 5
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  		FIG. 5. Quantitative assessment of DNA-protein interactions by ChIP (mean ± SEM). Sheared DNA-protein complexes were incubated with antibodies raised against SF-1 (A), c/ebp ß (B), GATA4 (C), and sp1 (D). Precipitated DNA was amplified by real-time PCR using SYBR Green chemistry. Binding activity was calculated as percentage of preimmunoprecipitated input DNA as represented by 2–{Delta}Ct x 100, where {Delta}Ct represents the Ct of input DNA subtracted from the Ct of the immunoprecipitated sample. Results were analyzed with ANOVA, followed by the Tukey-Kramer test. Letters represent differences between groups at a significance of P < 0.05.

 
On the other hand, sp1 demonstrated a different pattern of expression from SF-1 and c/ebp ß (Fig. 5DGo). Here, although statistically insignificant, there is a trend toward increasing protein binding in phthalate-treated samples. This binding activity appears to be the strongest in the DEP treated samples but also seems greater in the DBP treated samples. These results were consistent for all four genes examined.

These results suggest that although both c/ebp ß and SF-1 were responsive to phthalate exposure, c/ebp ß was responsive to DBP only and, therefore, likely involved in the DBP response mechanism.

Protein expression of SF-1, GATA4, and c/ebp ß
To determine whether changes in protein-DNA binding were the result of decreased protein levels, SF-1, c/ebp ß, and GATA4 levels in fetal testis after DBP exposure were quantified by Western blot analysis (Fig. 6Go). Similar to protein binding results, GATA4 demonstrated no changes in protein levels after toxic and nontoxic phthalate exposure. In addition, there is no detectable change in the protein levels of SF-1 after phthalate exposure, which is consistent with previous results that show no change in SF-1 mRNA transcripts after DBP exposure (9). Although not quite significant (P = 0.066), relative protein levels of c/ebp ß, however, were diminished in the 500-mg/kg exposures.


Figure 6
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  		FIG. 6. Analysis of transcription factor protein levels. Western blot analysis of SF-1 (A), c/ebp ß (B), and GATA4 (C) was performed as described in Materials and Methods. Whole cell lysate (SF-1 and GATA4) or nuclear extract (c/ebp ß) was loaded on a 4–12% SDS-PAGE gel and separated. Proteins were transferred to a polyvinylidene difluoride membrane and visualized with the appropriate antibody. GAPDH was used as a loading control. Control extracts (lane 1) were nuclear extracts from MA-10 (SF-1), F9 (GATA4), and HeLa (c/ebp ß). Relative protein levels of corn oil (lane 2), 500 mg/kg DEP (lane 3), 100 mg/kg DBP (lane 4), and 500 mg/kg DBP (lane 5) were quantified and plotted in a bar graph (D). Bars represent mean with SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The in utero development of the male reproductive tract is dependent on testosterone biosynthesis, and is required for the development of the wolffian duct, epididymides, and other male sex characteristics. Exposure to antiandrogenic chemicals that either block testosterone from binding to the AR or induce changes in testosterone synthesis results in the abnormal development of the male reproductive tract and testicular dysgenesis (3, 4, 5, 42, 43). Toxic phthalate esters do not bind to the AR (7) but, instead, exert their effects by targeting the transcriptional regulation of genes involved in cholesterol transport and testosterone biosynthesis pathways (8, 9, 16, 19). Multiple phthalates (DBP, mono-2-ethylhexyl phthalate, benzyl butyl phthalate, and dipentyl phthalate) induce male reproductive toxicity through this mechanism, whereas other nontoxic phthalates, including DEP, dimethyl phthalate, and dioctyl tere-phthalate, do not induce changes in testosterone levels, expression of steroidogenic genes, or developmental malformations (19, 23). However, the molecular mechanism by which DBP and other reproductively toxic phthalates cause a coordinate reduction in steroidogenic gene expression is unknown and examined here. In this study an examination of changes in DNA-protein interactions in promoters of multiple genes in the testosterone biosynthesis pathway after exposure to a toxic (DBP) and nontoxic (DEP) phthalate indicate that c/ebp ß and not SF-1 inhibition is the likely cause of transcriptional inhibition.

Testosterone biosynthesis begins with the transport of cholesterol first into the cell, mediated by SR-BI (44), and then to the inner mitochondrial membrane by StAR (45). Analysis of the transcriptional regulation of human SR-BI, and both human and rodent StAR has revealed that SF-1 can bind to proximal promoter sites and is required for transcription (29, 46, 47, 48). However, in the current study, we were unable to identify any SF-1 binding regions in the proximal 5' untranslated region of rat SR-BI. This confirms other work that identifies no SF-1 binding sites in the rat proximal promoter, but possible sites have been observed at an upstream site (+28) in the first exon and at a distal site (–516) (49). There are conflicting reports as to the importance of these sites in transcriptional regulation (49, 50), and these sites were not included in our examination of the rat SR-BI promoter. Cotransfection experiments of SF-1 did not affect the promoter activity of SR-BI constructs, indicating that SF-1 is not essential for SR-BI expression (50). SF-1 is also not the sole factor responsible for StAR transcriptional activation. Mutations in the SF-1 binding sites resulted in decreased StAR expression in in vitro reporter gene assays but did not abolish activity (33, 51). In addition, ChIP analysis in the current study demonstrates a loss of SF-1 binding in StAR after both DEP and DBP exposure. As mentioned earlier, DEP does not induce a reduction in fetal testosterone levels nor does it down-regulate SR-BI or StAR gene transcription. Together, the lack of an SF-1 binding site in the rat SR-BI promoter and no transcriptional inhibition of StAR after the loss of SF-1 binding suggest that although SF-1 may contribute, it is not essential in transcriptional inhibition of cholesterol transport genes by DBP.

Conversely, researchers have demonstrated that sp1, c/ebp, and GATA4 play a role in SR-BI and/or StAR transcription in rat (49, 50, 51, 52). In this study we identify a novel c/ebp ß binding site at –215 in SR-BI and confirm a binding site at –72 in StAR. c/ebp ß is a positive regulator of steroidogenesis, and mutation of the –72 site in StAR results marked reduction in StAR transcription, indicating that c/ebp ß binding is required for high levels of expression of StAR (53). Therefore, c/ebp ß may contribute to the surge in testosterone production observed between GD 16 and 19 in the rat developing male fetus. In addition, we see here that in contrast to SF-1 ChIP results, loss of c/ebp ß binding was only observed after DBP exposure. These observations suggest that c/ebp ß may be an important factor in SR-BI transcriptional regulation and a point of interference in DBP induced inhibition.

A second factor possibly involved in StAR regulation is GATA4 (48, 53). In our footprinting studies, a GATA4 binding region in StAR shows reduced protein-DNA interactions, yet ChIP assays show no changes in GATA4 binding activity. This apparent discrepancy can be accounted for by observations that SF-1 and c/ebp ß cooperate to regulate murine StAR expression (29). The putative GATA4 site that shows reduced binding activity (–55 to –65) is located between the binding sites for SF-1 and c/ebp ß. Protein-protein binding between these transcription factors might protect this region of DNA from DNase activity. DBP disrupts this protein-protein cooperation and, therefore, exposes this region of DNA for DNase digestion. In addition, no other gene contained GATA4 binding sites or changes in GATA4 binding activity. Therefore, it can be concluded that GATA4 is not involved in fetal testosterone biosynthesis.

sp1 recognition sites have also been important in regulation of these genes (48, 50, 53, 54). In our study no significant changes in DNase protection were observed at any sp1 site. However, there is a possible increase of sp1 binding in phthalate-exposed testis, especially DEP, as observed in ChIP assays. Interestingly, researchers have also demonstrated an sp1 binding site in the murine StAR promoter that overlaps with an SF-1 site (–146/–132). This site has demonstrated dual binding capacity for both the SF-1 and sp1 factors (53), and they may compete for binding at this site. Furthermore, the SF-1 binding site at –40 is also a putative binding site for sp1. The possible presence of dually active sites could account for our observation that DEP exposure results in a decrease in SF-1 binding activity with no subsequent decrease in StAR transcription and testosterone levels. DEP treatments also resulted in the strongest increase in sp1 binding, which is also a demonstrated enhancer of StAR transcription (54). This increase in sp1 binding may compensate for the decrease in SF-1 binding to prevent a DEP induced reduction in StAR regulation. However, no other dually active sequences were observed in other steroidogenic enzymes.

After transport into the cell, the testosterone biosynthesis enzymes, including cytochrome P450scc and P450c17 (coded by CYP11A1 and CYP17A1, respectively), convert the cholesterol into testosterone. In several species, proximal SF-1 binding sites have been identified in both genes that are critical to the basal and cAMP mediated transcription in steroidogenic tissues (55, 56, 57, 58, 59, 60). Results from the present study confirm SF-1 binding in both CYP11A1 and CYP17A1, whereas DNase I footprinting demonstrates a possible change in binding strength after phthalate exposure. However, like StAR we also demonstrate that inhibition of SF-1 binding by DEP exposure does not alter transcription of either gene, further emphasizing the role of other factors. This implies that other factors are needed for transcriptional regulation (61). These factors may include sp1, sp3, Ku, AP2, GATA4, GATA6, chicken ovalbumin upstream promoter transcription factor, and SET, which have all been shown as potential transcriptional regulators of CYP11A1 and CYP17A1 in various tissues and species (56, 62, 63, 64, 65, 66). Our studies demonstrated no changes in footprint strength at sp1 binding sites for either gene, yet both may show an increase in sp1 binding activity in DEP exposed animals. This is similar to observations in the cholesterol transport genes and may suggest a common mechanism.

In addition, this study identified novel c/ebp ß binding regions in both CYP11A1 and CYP17A1 proximal promoters (–165/–205 and –240/–270, respectively). Like with the cholesterol transport genes, ChIP assays confirm DBP-dependent changes in binding activity of this transcription factor, suggesting a role for c/ebp ß in transcription of testosterone biosynthesis enzymes. However, further tests to examine whether they are functional binding sites are necessary.

By examining multiple genes in the steroidogenic pathway, we have observed that SF-1 is not a critical regulator steroidogenic gene expression in the fetal Leydig cell. This is in contrast to other studies that demonstrated the down-regulation of multiple SF-1 regulated genes, including those examined here, after fetal phthalate exposure in rats and, therefore, suggested that phthalate toxicity is SF-1 mediated (17, 18). However, evidence for the SF-1 mediated regulation of many of these genes is from in vitro transfection studies and may not reflect the in vivo regulation in fetal testis. Other evidence for the importance of SF-1 in fetal development is that knockout mice are lethal shortly after birth, with all offspring developing no adrenals or gonads. There is a persistence of müllerian ducts and female external genitalia, regardless of genetic sex (67, 68). Although these knockouts demonstrate the importance of SF-1 in the differentiation and development of the gonads, the lack of steroidogenic tissue development prevents the examination of SF-1’s role in fetal steroidogenesis (69). Multiple studies have also reported no change in SF-1 mRNA or protein levels in the interstitial tissue of DBP testis (9, 17). Finally, reporter constructs in a mouse Leydig cell line (MA-10) demonstrated that mono-butyl phthalate, the primary toxic metabolite of DBP, has no effect on SF-1 mediated transcription of StAR, SR-BI, and CYP17A1 (14). This indicates that myelin basic protein does not interfere with SF-1 mediated transcription in these cells. Taken with the results of the current study, we can conclude that DBP-induced inhibition of steroidogenesis is not mediated by SF-1 regulation of steroidogenic genes, and SF-1 does not play an important role in the regulation of fetal testosterone biosynthesis in rats.

In contrast, this study observed that a loss of c/ebp ß binding in ChIP assay corresponded to decreased transcription in all four genes examined, which when combined with previous reports of down-regulation of c/ebp ß after exposure (19), suggests a role for this factor in DBP toxicity. c/ebp ß is a leucine zipper transcription factor that regulates multiple cellular functions, including cell proliferation, differentiation, and apoptosis (70, 71). As mentioned previously, it has been implicated in the regulation of several steroidogenic genes, and it has been detected in both immature and adult rat Leydig cell preparations (72). In addition, c/ebp ß mediates cellular responses environmental stressors, including ozone (73), hypoxia (74), and asbestos (75). Mice that are c/ebp ß null are mainly characterized by immune deficiencies (76), but females are sterile due to the inability of granulosa cells to transgress to the luteal stage (77). However, male c/ebp ß-null mice appear to develop normally and are fertile (77), suggesting that c/ebp ß is not critical for fetal testicular steroidogenesis in the mouse. This observation is interesting that in contrast to rats, recent observations in this laboratory have demonstrated that testosterone biosynthesis in the mouse is unaffected by DBP exposure (78).

In addition, we see that sp1 may play a role in fetal testosterone biosynthesis. sp1 is a ubiquitously expressed transcription factor shown to be a positive regulator of steroidogenesis at multiple steps (50, 56, 62, 79). sp1 regulates transcription by interacting with a variety of different transcription factors, including SF-1 (30, 56, 79), c/ebp ß (80), and GATA4 (65). Along with coregulator recruitment, phosphorylation (81) and alternate splicing (82) provide the widely expressed sp1 sensitive control over cell- and stage-specific gene transcription. In the current study, the presence of dually active SF-1/sp1 sites and a possible DEP induced increase in sp1 binding suggest a role for sp1 in phthalate response. sp1 binding and subsequent interaction with c/ebp ß may compensate for the loss of SF-1 and c/ebpß/SF-1 protein-protein interactions.

The mechanism that induces fetal testosterone production is not known, but recently there has been evidence that steroidogenesis in fetal testis is regulated independently from pituitary LH (11, 83). A variety of nongonadotropic paracrine and endocrine factors instead regulate testosterone production in fetal Leydig cells. These factors include vasoactive intestinal peptide (11), pituitary adenylate cyclase-stimulating polypeptide (84), and several natriuretic peptides (atrial natriuretic, brain natriuretic, and C-type natriuretic peptides) (85). Vasoactive intestinal peptide and pituitary adenylate cyclase-stimulating polypeptide are stimulants of the cAMP signaling pathway, which results in stimulation of testosterone biosynthesis (11, 84, 86) and increase transcription of steroidogenic enzymes, including CYP11A1 and CYP17A1 in ovaries (87). However, the natriuretic peptides stimulate fetal testosterone biosynthesis through the cyclic GMP (cGMP) second messenger pathway and have no effect on the cAMP pathway (85). The ability of multiple peptides to stimulate steroidogenesis through different signaling pathways suggests a redundancy in stimulation and reflects the critical nature of steroidogenesis in development.

SF-1 mediated activation of steroidogenic enzymes requires an activation of the cAMP signaling cascade and protein kinase A. This cAMP activation is unlikely to occur through increased SF-1 transcription but through SF-1 interaction with other transcription factors and cofactors, and direct phosphorylation of SF-1 (88). Conversely, modulation of c/ebp ß-activity is controlled by phosphorylation through several signal transduction pathways, including cAMP and cGMP (72, 89). Treatment of mouse Leydig cells with multiple natriuretic peptides stimulated the production of testosterone biosynthesis precursors through the cGMP pathway (90). Therefore, in the fetal rat testis, the increase in cGMP and subsequent protein kinase G activity from binding of natriuretic peptides may increase c/ebp ß binding activity and, thus, c/ebp ß-regulated transcription of steroidogenic genes. This presence of cAMP and cGMP signaling pathways mediated by SF-1 and c/ebp ß, respectively, suggests a possible mechanism of action for phthalate-induced inhibition of testosterone biosynthesis that will be explored in future experiments. Although DEP can inhibit the activity of cAMP/SF-1 mediated transcriptional regulation, this pathway is either not critical for fetal testosterone production, or the c/ebpß/cGMP pathway compensates to regulate transcription. In contrast, DBP inhibits either the c/ebpß/cGMP or both the SF-1/cAMP and c/ebpß/cGMP mediated pathway, resulting in reduced transcription.

In conclusion, the study presented here identifies c/ebp ß and, possibly, sp1 as critical factors involved in transcriptional modulation of fetal rat testosterone production after phthalate exposure. Inhibition of c/ebp ß binding corresponds to a loss of transcriptional activity of multiple steroidogenic enzymes, whereas loss of SF-1 binding resulted in no change, possibly from sp1 compensation. Future experiments will focus on how phthalates influence c/ebp ß-activation potential to determine the initial molecular point of action in DBP induced testicular dysgenesis. This information will not only provide the molecular mechanism of phthalate action but also shed some light on the basic biology of fetal testosterone production in testicular development.


   Acknowledgments
 
We thank Kim Lehmann and Janan Hensley for their assistance with animal necropsies and gene expression. Dennis House also provided critical statistical assistance. The authors also thank Drs. Kamin Johnson and Jacques Robidoux for their critical reading of this manuscript.


   Footnotes
 
This study was supported by National Institutes of Health Grant R01ES011803.

Disclosure Statement: The authors have nothing to declare.

First Published Online September 20, 2007

Abbreviations: AP, Activating protein; AR, androgen receptor; c/ebp ß, CCAAT/enhancer binding protein ß; cGMP, cyclic GMP; ChIP, chromatin immunoprecipitation; CYP17A1, cytochrome P450 17A1; CYP11A1, cytochrome P450 side chain cleavage; DBP, dibutyl phthalate; DEP, diethyl phthalate; GADH, glyceraldehyde-3-phosphate dehydrogenase; GD, gestational day; LM, ligation-mediated; NB, nuclei buffer; SF-1, steroidogenic factor-1; SR-BI, scavenger receptor B-1; StAR, steroidogenic acute regulatory; TBST, Tris-buffered saline with 0.1% Tween 20.

Received July 12, 2007.

Accepted for publication September 7, 2007.


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E. Lague and J. J. Tremblay
Antagonistic Effects of Testosterone and the Endocrine Disruptor Mono-(2-Ethylhexyl) Phthalate on INSL3 Transcription in Leydig Cells
Endocrinology, September 1, 2008; 149(9): 4688 - 4694.
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