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CIIT Centers for Health Research,3 Research Triangle Park, North Carolina 27709
Sanofi-Aventis Pharmaceuticals,4 Bridgewater, New Jersey 08807
National Institute for Statistical Sciences,5 Research Triangle Park, North Carolina 27709
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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adrenal, adrenal gland, cholesterol, Cyp11a1 (P450SCC), Cyp17a1, fetal development, male reproduction, male reproductive tract, phthalate esters, reproductive development, scavenger receptor, Steroid acute regulatory protein (Star), steroidogenesis, testis, testosterone, toxicology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Similar to the fetal testis, the fetal adrenal gland is steroidogenically active [12]. Steroid production by the fetal adrenal requires transport of cholesterol into the cell by SCARB1 [13], movement of cholesterol across the mitochondrial membrane by steroidogenic acute regulator protein (STAR), and removal of a six-carbon moiety from cholesterol by CYP11A1 [14]. Expression of each of these genes is repressed in the fetal testis following DBP exposure [8, 10].
The aim of the current studies was to determine the precise timing of the onset of DBP effects in the fetal testis and to use gene expression profiling to characterize the time course for gene expression changes in the fetal testis following DBP exposure. Additionally, we sought to determine whether expression of the Scarb1, Star, and Cyp11a1 genes, and consequently steroidogenesis, is similarly inhibited in the fetal adrenal gland after in utero exposure to DBP. We found that DBP treatment resulted in a rapid and transient induction of immediate early genes in the fetal testis. We also found that steroidogenesis was reduced in the fetal adrenal, although the reduction was statistically insignificant, and to a lesser extent than in the testis. Unlike the testis, expression of genes required for steroidogenesis was not affected in the adrenal gland.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This study was approved by the Institutional Animal Care and Use Committee of the CIIT Centers for Health Research and followed federal guidelines for the care and use of laboratory animals [15]. Pregnant timed-mated Sprague-Dawley outbred CD rats were purchased from Charles River Laboratories, Inc. (Raleigh, NC) on Gestation Day 0 (gd; gd 0 = the day sperm is detected in the vaginal smear). Animals were housed in the animal facility at CIIT, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, in a humidity- and temperature-controlled, high efficiency particulate air-filtered, mass air-displacement room. The room is maintained on a 12L:12D cycle at approximately 18 to 25°C with a relative humidity of approximately 30%–70%. Rodent diet NIH-07 (Zeigler Brothers, Gardners, PA) and reverse-osmosis water were provided ad libitum. Animals were acclimatized for the time period before dosing. Allocation of animals to control or DBP treatment groups (four dams per group) was performed by body weight randomization. Dams were dosed by oral gavage with 1 ml/kg corn oil (Sigma Chemical, Co., St. Louis, MO) or 500 mg/kg DBP (Aldrich Chemical Co., Milwaukee, WI). DBP was prepared at a concentration of 500 mg/ml corn oil. This dose level causes rapid decreases in gene and protein expression and testosterone production in the fetal testis [11]. In the first study, dosing with corn oil or DBP was performed at the following times before the animals were killed: 30 min; and 1, 2, 3, 6, 12, 18, and 24 h. In the second study, dosing with corn oil or DBP was performed daily from gd 12 to gd 19. Animals were killed on gd 19 by CO2 anesthesia and exsanguination by abdominal aorta transection. Fetuses were removed by Cesarean delivery and killed by decapitation. The sex of fetuses was determined by internal inspection of the gonads. All testes (from the first study) and adrenals (from the second study) were snap-frozen in liquid nitrogen and stored at –70°C.
Radioimmunoassay
Fetal glandular testosterone and corticosterone levels were determined as previously described [8]. Briefly, the testis or adrenal gland was homogenized in 100 µl of PBS-gel buffer followed by extraction with a mixture of ethyl acetate and chloroform (4:1). Extraction was performed three times using a total of 1 ml of ethyl acetate:chloroform. Extracts were dried under nitrogen and resuspended in 100 µl of radioimmunoassay (RIA) zero standard. Steroid concentrations were determined using the ImmuChem Coated Tube testosterone and corticosterone 125I RIA Kits (MP Biomedicals, Inc., Irvine, CA) according to the manufacturer's instructions. Samples were counted in a Cobra D5005 gamma counter (Packard Instrument Co., Downers Grove, IL).
RNA Isolation and cDNA Synthesis
Total RNA was isolated from frozen tissues using STAT-60 reagent (Tel-Test, Inc., Friendswood, TX) according to the manufacturer's instructions. Each total RNA sample was checked for integrity and DNA contamination by measurement of optical density on a NanoDrop ND 1000 (NanoDrop Technologies, Wilmington, DE) and size-fractionation of 18S and 28S rRNA on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). All reagents for reverse transcription (RT) were purchased from Applied Biosystems (Foster City, CA) unless otherwise noted. After isolation, total RNA was incubated for 1 h at 37°C in a reaction mixture containing RNase inhibitor, dithiothreitol, 5x transcription buffer, and RQ1 RNase-free DNase (Promega Corp., Madison, WI). DNase was inactivated by incubating for 5 min at 75°C. One microgram of total RNA was reverse-transcribed for 65 min at 42°C in a 20-µl reaction containing 5 mM MgCl2, 1x GeneAmp PCR buffer II (50 mM KCl, 10 mM Tris-HCl pH 8.3), 1 mM each dNTP, random hexamers, 20 U RNase inhibitor, and 50 U MuLV (Maloney murine leukemia virus) reverse transcriptase. The RT reaction was terminated by heating to 95°C for 5 min; 0.1 µl cDNA was used for subsequent polymerase chain reactions (PCRs).
Real-Time Quantitative RT-PCR
Real-time quantitative RT-PCR was performed on an ABI Prism 7900 HT Sequence Detection System (Applied Biosystems). Complementary DNA prepared as described above was amplified in a 25-µl reaction mix containing 1x SYBR Green PCR Master Mix (Applied Biosystems) and 64 nM each primer. Following a 10-min taq polymerase activation step at 95°C, reactions were subjected to 50 cycles of 15 sec denaturation at 94°C, and 1 min annealing/extension at 60°C. Primers were purchased from MWG Biotech (High Point, NC). Following PCR, reaction products were melted for 3 min at 95°C, and then the temperature was lowered to 50°C in 0.5°C increments, 10 sec per increment. Optical data were collected over the duration of the temperature drop, with a dramatic increase in fluorescence occurring when the strands reannealed. This was performed to ensure that only one PCR product was amplified per reaction. Relative expression of the RT-PCR products was determined using the method described by Pfaffl [16]. Each sample was run in triplicate and the mean Ct used for determination of relative expression. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used for normalization. Primer pairs appear in Table 1.
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Immunoblotting
For immunoblot analysis, total protein was extracted from paired testes or adrenals by homogenizing in 50 µl of lysis buffer (0.1 M Tris-HCl pH 8.0, 0.05 M EDTA, 0.1 M NaCl, 1% w/v SDS, and 1% w/v sarcosyl) supplemented with Complete Protease Inhibitor Cocktail (Roche Molecular Biochemicals, Mannheim, Germany). Total protein was quantitated using the bicinchoninic acid protein assay reagent (Sigma). Thirty micrograms of total protein was run on SDS-PAGE and transferred to polyvinylidene difluoride membranes. The following antibodies were used to probe the membrane: rabbit anti-mouse SCARB1 (Novus Biologicals, Inc., Littleton, CO), rabbit anti-rat cytochrome CYP11A1 (US Biological, Swampscott, MA), rabbit anti-rat STAR (Affinity Bioreagents, Inc., Golden, CO), and rabbit anti-porcine CYP17A1 (a kind gift from Dr. D.B. Hales, University of Illinois at Chicago) [17]. Immunoreactivity was detected using horseradish peroxidase-conjugated secondary antibodies to rabbit immunoglobulin G (Amersham Biosciences, Piscataway, NJ) and Enhanced Chemiluminescence Plus Western Blotting Detection Reagents (Amersham) and visualized with a MultiImage Light Cabinet (Alpha Innotech Corp., San Leandro, CA). Quantitation of expressed protein levels was performed using FluorChem 8000 software (Alpha Innotech).
Microarray Hybridization
Testes from individual fetuses were homogenized in RNA Stat-60 reagent (Tel-Test) and RNA was isolated using the RNAeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer's protocol. RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies). Complementary DNA was synthesized from 2.5 µg of total RNA and purified using the RiboAmp OA 1 Round RNA Amplification kit (Arcturus, Mountain View, CA) according to the manufacturer's protocol. Equal amounts of purified cDNA per sample were used as the template for subsequent in vitro transcription reactions for cRNA amplification and biotin labeling using the BioArray High Yield RNA Transcript Labeling Kit (Enzo Life Sciences, Inc., Farmingdale, NY). Complementary RNA was purified and fragmented according to the protocol provided with the GeneChip Sample Cleanup Module (Affymetrix, Santa Clara, CA). All GeneChip arrays were hybridized, washed, stained, and scanned using the Complete GeneChip Instrument System according to the Affymetrix Technical Manual.
Statistical Analyses
The Student t-test or one-way analysis of variance (ANOVA) with Tukey post hoc analysis were performed using JMP statistical software (SAS Institute, Inc., Cary, NC) version 5.0.1. A P value < 0.05 was considered to be statistically significant. Array analysis was as previously described [18]. Probe level data were first summarized by robust singular value decomposition. After normalization, the first robust principal component of the perfect match was extracted and the mean of the fitted value was used as the signal intensity. A log-2 transformation was performed to stabilize the variance. Simultaneous t-tests (multiple comparisons) were performed between control and DBP treatment for each time point on a gene-by-gene basis. The error variance was estimated from ANOVA. The Bonferroni procedure was applied to control the family-wise error rate. An adjusted P value < 0.05 was considered to be statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To investigate the molecular alterations resulting in the initial decrease in steroidogenesis and the eventual repression of Star, Scarb1, Cyp11a1, and Cyp17a1, we performed gene expression profiling on fetal testes at discrete time points after DBP exposure. There were 106 genes in the DBP-treated groups that were significantly different than time-matched controls (Table 2). The gene expression profile revealed clear time-related patterns (Fig. 2). Six genes were significantly elevated within 1 h of DBP exposure. An additional 43 genes were up-regulated 3 h after DBP exposure and five genes were down-regulated at this time. The rapid induction of these genes was a transient effect; none of the genes up-regulated 1 h after treatment were still significantly different than control 6 h after DBP treatment and only nine genes showed significant changes from control between the 3- and 6-h time points. Prior to 6 h after DBP exposure, the majority of the changes in expression had reflected increased transcription. At 6 h, 19 genes were significantly decreased in expression and 17 were increased in expression.
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Real-time RT-PCR analysis was used to confirm distinct patterns of rapid and transient transcriptional alterations following exposure to DBP. None of the genes surveyed differed from control at 30 min after DBP exposure. The immediate early gene Fos (cellular oncogene fos; also known as C-fos) and the putative mRNA destabilizing gene Zfp36 (zinc finger protein 36) demonstrate peak expression 1 h after DBP exposure (Fig. 3) and other immediate early genes demonstrated peak expression 2 h post-DBP administration (Fig. 4). There were two distinct patterns of expression change in genes demonstrating peak expression 3 h posttreatment with DBP (Fig. 5). Cebpd (CCAAT/enhancer binding protein C/EBP delta), Cxcl1 (chemokine [C-X-C motif] ligand 1), and Nr4a3 (nuclear receptor subfamily 4, group A, member 3) rapidly increased between 2 and 3 h, while all other 3-h peak genes showed a more gradual increase in expression. Tsp1 (thrombospondin 1) was unique in that it showed a high relative expression (approximately 25-fold) at 3 h, with expression returning to baseline levels at 6 h. All of the genes that had peaked by 3 h returned to basal expression levels by 12 h after DBP exposure. Of the three genes showing peak expression 6 h after exposure to DBP, two (Nfil3 [nuclear factor, interleukin 3 regulated] and Stc1 [stanniocalcin]) were still elevated 24 h after exposure. Genes demonstrating peak expression elevation 12 h after DBP exposure did not demonstrate the same magnitude of expression elevation observed in the genes altered at the earlier time points. The steroidogenesis-associated genes Nr0b1 (nuclear receptor subfamily 0, group B, member 1) and Nr4a1 (nuclear subfamily 4, group A, member 1; also known as Nur77) were elevated at this time point (Fig. 6).
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A small number of genes exhibited decreased expression shortly after DBP exposure (Fig. 7). The expression alteration was also transient in this group of down-regulated genes; two of the genes (Tcf1 [transcription factor 1] and Sgk [serum/glucocorticoid regulated kinase]) had returned to baseline expression levels by 3 h post-DBP exposure with the other two genes, Sostdc1 (uterine sensitization-associated gene 1 protein) and Hes6_predicted (hairy and enhancer of split 6 predicted) returning to controls levels within 6 h.
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Following exposure to DBP over gd 12 to gd 19, there were decreases in adrenal corticosterone concentrations of 48% and 42%, respectively, in the male and female fetuses (Fig. 8A). These results were not, however, statistically significant. In contrast to the situation in the fetal testis, adrenal mRNA expression of Star, Cyp11a1, and Scarb1 was unaffected by DBP (Fig. 8B). Protein expression showed some gender-specific discordance with mRNA expression, because STAR and SCARB1 proteins were diminished exclusively in male fetuses exposed to DBP (Fig. 8C). This effect was statistically significant only for STAR protein. The magnitude of change in steroid concentration and protein expression was considerably less in the fetal adrenal than had been observed previously in the fetal testis (approximately 87% decrease for both testosterone and protein).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A rapid alteration in the availability of cholesterol, the common precursor to both gonadal and adrenal steroids, could account for the decrease in steroidogenesis observed in both tissues. Phthalate esters can act as peroxisome proliferators [19], a diverse group of chemicals that have been shown to regulate cholesterol homeostasis [20, 21]. The effects of peroxisome proliferators are mediated through activation of the peroxisome proliferator activated receptor (PPAR) [21]. DBP is capable of activating both PPAR 
and 
isoforms. However, changes in expression of genes targeted by PPAR and are not observed until at least 3 h after DBP treatment [22], suggesting that the early repression of steroidogenesis observed in this study is not mediated via PPAR activation. The peroxisome proliferator troglitazone inhibits cholesterol production in CHO cells through a mechanism independent of PPAR activation [23]. DBP may similarly be limiting cholesterol availability to the steroidogenic pathway.
Given that previous studies have shown predominantly repressed transcription of testicular genes following DBP exposure [8, 10, 11, 18], an unexpected finding was that most early (within 3 h) gene expression changes observed in DBP-exposed fetal testis reflected increased transcription. Many of these induced genes belong to a class of genes known as immediate-early genes that are rapidly induced upon exposure to growth and differentiation factors as well as a variety of external stimuli [24].
Although the early gene expression changes present no definitive mechanism for repression of steroidogenesis, a compelling mode of regulation is suggested by the increased expression of Zfp36. Zfp36 is a member of the tristetraprolin family of CCCH zinc finger proteins [25] that regulate mRNA levels by binding class II AU-rich elements in the 3'-untranslated region of target transcripts and consequently promoting degradation of these transcripts [26]. Induction of Zfp36 has been observed in response to growth factor stimulation, and promoter analysis of Zfp36 has revealed a consensus binding site for the immediate-early gene Egr1 (early growth response 1) [27]. Egr1 is a zinc finger transcription factor identified independently by several laboratories searching for genes that are rapidly expressed in response to mitogenic stimuli [28]. Activation of the mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase (ERK) pathway leads to increased expression of Egr1 [29], which in turn, influences growth, differentiation, and development in myriad cell types through regulation of Egr1 target genes [28, 30]. Egr1 exhibits rapidly increased expression after DBP exposure. Exposure to DBP may initially result in stimulation of the MAPK/ERK pathway in the fetal testis, which is supported by the slightly increased expression of steroidogenic genes and testosterone production 30 min after DBP treatment. The resultant expression of Egr1 and Zfp36 would lead to destabilization of mRNAs required for testicular steroidogenesis. The Star mRNA contains the AU-rich element targeted by Zfp36 [31], while Scarb1 and Cyp11a1 contain near consensus AU-rich sequences. Further studies in our laboratory will focus on the early signaling events initiated by exposure to DBP and the potential destabilization of mRNAs by Zfp36.
A possible point of regulation for several genes altered by DBP in the fetal testis is the Edg3 (endothelial differentiation sphingolipid G-protein-coupled receptor 3) signaling system. Edg3 is the G protein-coupled receptor for sphingosine-1-phosphate (S-1-P) [32], a phospholipid that acts as both an intracellular and extracellular signaling molecule [33]. Ligand binding by Edg3 leads to activation of MAPKs and consequent induction of immediate-early genes, including Fos and Egr1 [34, 35]. S-1-P activation of Edg3 also leads to activation of Nf-kb (nuclear factor-kappa beta) [36], which can, in turn, lead to increased expression of Btg2 (B-cell translocation gene 2, antiproliferative) [37], Sostdc1 [38], and Ler3 (immediate early response 3) [39], each of which was rapidly induced following DBP exposure. Edg3 mRNA is itself rapidly induced in the testis following exposure to DBP. In a recent study, S-1-P was shown to inhibit steroidogenesis in a Leydig cell tumor line [40]. Given the demonstrated effects of phthalate esters on lipid homeostasis [41, 42], further investigation of the effects of DBP on the S-1-P/EDG3 signaling cascade in the fetal testis is warranted.
Notably, all of the genes showing peak expression in the first 3 h post-DBP exposure return to baseline expression levels within 12 h. The distinct patterns of gene expression and turnover underscore the importance of including multiple time points in the examination of gene expression profiles in response to toxicants.
In summary, these studies demonstrate that steroidogenic genes repressed in the fetal testis following DBP exposure are not similarly affected in the fetal adrenal gland. There is a statistically insignificant decrease in adrenal steroidogenesis following DBP exposure that is similar in magnitude to the diminution of testosterone production in the fetal testis 1 h after the administration of DBP to the pregnant dam. The decreased steroidogenesis observed in the testis at the 1-h time point precedes inhibition of Star, Scarb1, Cyp11a1, or Cyp17a1. Future studies in our laboratory will focus on determining the mechanism of testicular steroidogenesis repression by DBP.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Kevin W. Gaido, CIIT Centers for Health Research, P.O. Box 12137, Research Triangle Park, NC 27709. FAX: 919 558 1300; gaido{at}ciit.org
Received: 4 April 2005.
First decision: 28 April 2005.
Accepted: 29 June 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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P < 0.05 compared to control by one-way ANOVA with Tukey post hoc analysis. Real-time RT-PCR was performed to assess expression of DBP-sensitive genes. Results are expressed as mean normalized expression relative to controls killed 30 min after exposure to corn oil. Error bars show SEM; n = 4–5 fetuses per group, with each fetus taken from a separate litter. *P < 0.05 compared to 30-min control by one-way ANOVA with Tukey post hoc analysis. B) Total protein lysates were prepared and 30 µg of each sample was electorphoresed on 10% SDS-PAGE gels. Pictures are representative of four separate blots
