The authors have declared that no competing interests exist.
Conceived and designed the experiments: FVS CAR. Performed the experiments: JAT CAR AS KC HW. Analyzed the data: JAT KC TS CAR. Contributed reagents/materials/analysis tools: FVS TS TI . Wrote the paper: JAT FVS TS.
Developmental exposure of mouse fetuses to estrogens results in dose-dependent permanent effects on prostate morphology and function. Fetal prostatic mesenchyme cells express estrogen receptor alpha (ERα) and androgen receptors and convert stimuli from circulating estrogens and androgens into paracrine signaling to regulate epithelial cell proliferation and differentiation. To obtain mechanistic insight into the role of different doses of estradiol (E2) in regulating mesenchymal cells, we examined E2-induced transcriptomal changes in primary cultures of fetal mouse prostate mesenchymal cells. Urogenital sinus mesenchyme cells were obtained from male mouse fetuses at gestation day 17 and exposed to 10 pM, 100 pM or 100 nM E2 in the presence of a physiological concentration of dihydrotestosterone (0.69 nM) for four days. Gene ontology studies suggested that low doses of E2 (10 pM and 100 pM) induce genes involved in morphological tissue development and sterol biosynthesis but suppress genes involved in growth factor signaling. Genes involved in cell adhesion were enriched among both up-regulated and down-regulated genes. Genes showing inverted-U-shape dose responses (enhanced by E2 at 10 pM E2 but suppressed at 100 pM) were enriched in the glycolytic pathway. At the highest dose (100 nM), E2 induced genes enriched for cell adhesion, steroid hormone signaling and metabolism, cytokines and their receptors, cell-to-cell communication, Wnt signaling, and TGF- β signaling. These results suggest that prostate mesenchymal cells may regulate epithelial cells through direct cell contacts when estrogen level is low whereas secreted growth factors and cytokines might play significant roles when estrogen level is high.
The mouse prostate begins to differentiate from the urogenital sinus (UGS) at gestation day 17, soon after the onset of testosterone secretion by the fetal testes
We and others have shown that prenatal exposure of male mouse fetuses to estradiol-17β (E2), estrogenic drugs such as diethylstilbestrol (DES) and ethinylestradiol, or industrial estrogenic chemicals such as bisphenol A (BPA), induce an increase in the number of developing prostatic glands and an increase in prostate gland size during fetal life due to basal epithelial cell hyperplasia
All animal procedures were approved by the University of Missouri Animal Care and Use Committee (protocol number: 6489) and conformed to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The program is fully accredited by the Association for Assessment & Accreditation of Laboratory Animal Care, International (AAALAC).
CD-1 mice were purchased from Charles River Laboratories (Wilmington, MA) and maintained as an outbred stock at the University of Missouri. Animals were housed on corncob bedding in standard (11.5×7.5×5″) polypropylene cages. Water was purified by reverse osmosis and carbon filtration and provided in glass bottles
Timed-pregnant females were killed on gestation day (GD) 17 (mating = GD 0) by CO2 asphyxiation, and fetuses were removed from the uterine horns. The bladder and UGS were removed from male fetuses as previously described
First passage cells were used in these experiments and were seeded at 3.2×105 cells/well in 35 mm dishes. Cells were seeded in complete RPMI medium with endogenous hormones removed by substituting 5% (v/v) charcoal-stripped FBS and 5% (v/v) charcoal-stripped horse serum (Sigma, St. Louis, MO) for the 10% whole FBS, and further supplementing with ITS supplement (Cambrex, Walkersville, MD), for final concentrations of 10 µg insulin/ml, 10 µg transferrin/ml, and 10 ng selenium/ml. This medium was further supplemented with 690 pM DHT (200 pg/ml). Cells were treated with DHT rather than testosterone for two reasons. First, we wanted to control E2 exposure, since the developing prostate expresses aromatase
Total RNA was isolated from the Trizol lysate and purified with the RNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturers' instructions, and RNA quality was checked on an Agilent Bioanalyzer (Agilent, Palo Alto, CA). The transcriptomal profiles were determined using Affymetrix mouse ST 1.0 or 430A microarrays. Scanned image data were converted into numerical tables using Affymetrix GeneChip Operating Software and Gene Expression Console. Data analysis and mining, including gene ontology enrichment analysis, were performed using GeneSifter server (Giospiza Inc., Seattle, WA) and Partek Genomics Suite (Partek Inc., St. Louis, MO). Microarray data were deposited in NCBI Gene Expression Omnibus (accession numbers GSE16854 and GSE36630).
To confirm the relative changes in gene expression induced by estradiol, we used a real-time quantitative reverse transcriptase-polymerase chain reaction (qPCR) approach for selected transcripts
The relative concentrations of specific mRNAs in each sample were normalized to total RNA per well, as described
Exposure of the primary culture mouse prostate mesenchymal cells significantly affected expression of 628 genes (ANOVA, p<0.01, unadjusted). Benjamini-Hochberg correction of multiple testing eliminated these effects, reflecting the relatively low statistical power of the present analysis due to the limited numbers of samples in each group.
These 628 genes were subjected to hierarchical clustering, which classified them into seven groups, based on induction or suppression of gene activity and on relative sensitivity to E2 (
Analysis of GO enrichment of E2 inducible genes (
Set 1: E2-inducible high sensitivity. Set 2: E2- inducible moderate sensitivity. Set 3: E2-inducible low sensitivity. Select genes of interest are highlighted. The figures show raw p values as well as, where indicated, Benjamini-Hochberg corrected p values.
Set 5: E2-suppressible high sensitivity. Set 6: E2-suppressible low sensitivity. The figures show raw p values as well as, where indicated, Benjamini-Hochberg corrected p values.
For the 34 genes showing an inverted U-shape dose response (
A) Set 7: Inverted U-curve. Both raw p values and Benjamini-Hochberg corrected p values are given. B) Genes identified as part of the glucose metabolic pathway in panel A depicted as relative values to illustrate the high association between dose and gene expression.
Highlighted genes (outlined in red) were influenced by lower dose estradiol treatment (10 pM and 100 pM) in an inverted U manner, suggesting enhancement of glycolysis by 10 pM E2 but suppression by 100 pM E2.
After filtering the data in GeneSifter, using a 1.5-fold expression ratio criterion between control and estradiol treatment and a statistical cutoff at P≤0.05, and discarding genes with expression levels less than 10 fluorescence units in both treated and control samples, it was determined that 181 genes were activated by 100 nM E2 exposure and 86 genes were repressed.
The results of Gene Ontology functional enrichment analysis, within the categories of Biological Process and Molecular Function, are shown in
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Category | Gene Ontology Term | up-regulated | down- regulated | up-regulated | down- regulated |
Biological process | metabolic process | 121 | 69 | −2.08* | −1.43 |
biological regulation | 100 | 40 | 2.70** | −0.99 | |
growth | 17 | 3 | 5.11** | −0.03 | |
reproduction | 11 | 9 | 0.94. | 2.02* | |
reproductive process | 5 | 6 | 0.42. | 2.42* | |
rhythmic process | 6 | 1 | 3.92** | 0.23 | |
Molecular function | catalytic activity | 135 | 76 | −2.3* | 0.82 |
transporter activity | 53 | 35 | 2.76** | 1.42 |
The expression patterns of several genes demonstrated to be up- or down-regulated by estradiol treatment using microarray analysis were validated in independent samples using quantitative PCR. The genes selected were: Ar, Bmp4, Capn6, Cyp7b1, Esr1, Sfrp4, and Thbs2. Ar and Esr1 were chosen because we have shown by qPCR
Gene expression in cells treated with 100 nM 17β-estradiol (grey bars) is compared to that in untreated control cells (black bars). * Control vs. treated cells statistically different, p<0.05. The qPCR data were previously published elsewhere
The effects of fetal E2 exposure on prostate development do not follow a monotonic dose-response
Non-monotonic dose responses were seen in our initial examination of the effects of estradiol and BPA on Ar and Esr1 expression in fetal mouse UGS mesenchyme
These microarray experiments were performed as a hypothesis generation step for a study of effects of estrogens on prostate development and differentiation, and the sample size is small. Because of this, the data must be seen as preliminary, but the results do indicate activation of different patterns of gene expression and dominance of different pathways at low, physiologically relevant, compared to high, pharmacological, doses of E2. Results from the lowest (10 pM and 100 pM) doses of E2 treatments indicate E2-inducible genes within pathways related to cell adhesion, actin cytoskeleton reorganization, EGF-like calcium binding, sterol biosynthesis and lipoprotein metabolism, and E2-suppressible genes within pathways related to growth factor signaling, tube development and additional effects on cell adhesion. At the high (100 nM) concentration, E2 induced genes enriched for steroid hormone signaling and metabolism, cytokines and their receptors, cell-to-cell communication, and TGF-β signaling (
Pathway/Category | Direction | Ratio | Gene Identifier | Gene Name |
Cell Communication | Up | 2.50 | AV239646 | Gjb2 |
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Up | 1.96 | BE197934 | Krt1-14 |
Up | 2.62 | AV330726 | Gja1 | |
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Up | 4.48 | L06421 | Thbs2 | |
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Up | 1.83 | BI455189 | Col6a2 | |
Androgen and estrogen metabolism | Down | 7.25 | NM_023135 | Sult1e1 |
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Up | 2.42 | NM_007825 | Cyp7b1 |
Up | 8.12 | NM_01378 | Hsd17b9 | |
TGF-beta signaling pathway | Down | 1.93 | NM_010496 | Idb2 |
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Down | 1.83 | NM_008046 | Fst |
Down | 1.75 | NM_007554 | Bmp4 | |
Down | 3.27 | BM230984 | Tgfb14i | |
Up | 3.67 | BB353211 | Inhbb | |
Up | 4.48 | L06421 | Thbs2 | |
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Steroid hormone receptors | Up | 3.34 | NM_007956 | Esr1 |
Up | 5.29 | NM_008829 | Pgr | |
Wnt signaling | Down | 2.22 | NM_009519 | Wnt11 |
Up | 3.72 | NM_009526 | Wnt6 | |
Up | 1.89 | W29605 | Wnt7b | |
Up | 5.85 | NM_020265 | Dkk2 | |
Up | 2.18 | BB221995 | Sfrp4 | |
Cytokine-cytokine Receptor interaction | Up | 2.77 | NM_019583 | Il17rb |
Up | 2.21 | NM_011330 | Ccl11 | |
Up | 6.53 | NM_021443 | Ccl8 | |
Up | 3.67 | BB353211 | Inhbb | |
Up | 3.85 | AF000304 | Il4ra | |
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Hedgehog signaling | Down | 2.22 | NM_009519 | Wnt11 |
Down | 1.75 | NM_007554 | Bmp4 | |
Up | 3.72 | NM_009526 | Wnt6 | |
Up | 1.89 | W29605 | Wnt7b | |
Apoptosis | Up | 2.07 | BF137345 | Birc4 |
Down | 2.85 | NM_007603 | Capn6 | |
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Prostate cancer | Up | 3.40 | BC010786 | Creb3l3 |
Up | 2.21 | AJ252157 | Foxo1 | |
Up | 2.60 | NM_019739 | Foxo1 | |
Basal cell carcinoma | Down | 2.22 | NM_009519 | Wnt11 |
Down | 1.75 | NM_007554 | Bmp4 | |
Up | 3.72 | NM_009526 | Wnt6 | |
Up | 1.89 | W29605 | Wnt7b |
All genes listed are significantly altered at P<0.05. Where the z-score for the entire pathway was significant, the score is given below the pathway name. Where multiple probes for the same gene are represented in these lists (indicated by italics), agreement was good between the probes.
Importantly, an inverted U (non-monotonic) response was seen within the low-dose results, with enhancement of glycolysis observed at 10 pM E2 but significant suppression at 100 pM E2 (
Only 29 genes out of those screened were influenced by all doses of estradiol examined (
Log2 fold expression relative to control | ||||||
Low-dose cluster group | Gene ID | No E2 | 10 pM E2 | 100 pM E2 | 100 nM E2 | Monotonic trend? |
Inducible_moderate | Sprr1a | 0.00 | 0.98 | 2.23 | 5.57 | Y |
Inducible_moderate | Angpt2 | 0.00 | 1.20 | 2.25 | 3.77 | Y |
Inducible_moderate | Dkk2 | 0.00 | 0.67 | 2.21 | 2.55 | Y |
Inducible_moderate | Pgr | 0.00 | 0.98 | 2.22 | 2.40 | Y |
Inducible_low | Fabp7 | 0.00 | 0.75 | 2.18 | 2.26 | Y |
Inducible_low | Fbxo32 | 0.00 | −0.33 | 1.52 | 1.57 | Y |
Inducible_moderate | Esr1 | 0.00 | 1.00 | 2.19 | 1.74 | I |
Inducible_moderate | Rgs4 | 0.00 | 0.64 | 2.09 | 1.81 | I |
Inducible_moderate | Thbs2 | 0.00 | 1.49 | 2.20 | 1.78 | I |
Inducible_moderate | Btbd3 | 0.00 | 0.95 | 2.25 | 1.05 | N |
Inducible_moderate | Gja1 | 0.00 | 0.66 | 2.21 | 1.51* | N |
Inducible_moderate | Npy1r | 0.00 | 1.14 | 2.16 | 1.49 | N |
Inducible_low | Perp | 0.00 | 0.21 | 2.06 | 0.71 | N |
Suppressible_low | Sult1e1 | 0.00 | 0.20 | −1.81 | −2.86 | Y |
Suppressible_low | Lcn2 | 0.00 | 0.49 | −1.63 | −2.44 | Y |
Suppressible_low | Egfl6 | 0.00 | −0.20 | −2.04 | −1.93 | Y |
Suppressible_low | Pdlim3 | 0.00 | −0.14 | −1.96 | −1.95 | Y |
Suppressible_low | Cdkn1c | 0.00 | −0.19 | −1.99 | −1.16 | Y |
Suppressible_low | Capn6 | 0.00 | −0.43 | −1.93 | −1.51 | I |
Suppressible_low | Igfbp2 | 0.00 | 0.15 | −1.87 | −0.96 | I |
Suppressible_low | Wnt11 | 0.00 | −0.55 | −2.11 | −1.15 | I |
Suppressible_low | Cyb561 | 0.00 | 0.57 | −1.52 | −0.93 | I |
Suppressible_low | Gda | 0.00 | −0.49 | −2.06 | −0.77 | I |
Suppressible_low | Dpep1 | 0.00 | 0.97 | −1.19 | −0.67 | I |
Suppressible_low | Zfp161 | 0.00 | 0.10 | −1.70 | 0.74 | N |
Suppressible_low | Sfrp4 | 0.00 | 0.16 | −1.77 | 1.12 | N |
Suppressible_low | Penk1 | 0.00 | −0.53 | −1.95 | 1.06 | N |
Suppressible_low | Enpp2 | 0.00 | 0.90 | −1.17 | 1.85 | N |
U-curve | Cd80 | 0.00 | −1.04 | 1.12 | 0.65 | I |
For each gene the log2 value of the fold change is given, and thus up-regulated and down-regulated genes are reflected in positive and negative numbers respectively. Genes are sorted first according to the cluster groups identified for the low-dose treatments (see Methods), and then by whether the trend at the high dose (100 nM) is consistent with the results seen at lower doses. Y = monotonic trend; gene expression at 100 nM E2 continues the trend at lower doses or has reached a plateau at that point. N = trend is clearly not monotonic; gene expression at 100 nM E2 is in the reverse direction of the trend at lower doses. I = suggestion of non-monotonic trend; gene expression at 100 nM shows slight reversal of trend at lower doses. *Value is average value for all probes for this gene (n = 5).
Also of interest in this 29-gene subset are the clear inverse U effects on Perp and Gja1 expression. Perp is typically upregulated during apoptosis
The Wnt signaling pathway was influenced at all E2 doses examined, but with an emphasis toward up-regulation of canonical Wnt/β-catenin stabilization signaling at the high dose, and non-canonical (PCP) signaling at lower doses. The high-dose effect may be mediated through the known association of β-catenin with AR and ER. Truica
At the high dose of E2 we observed changes in genes related to steroid hormone metabolism, and alterations in steroid hormone signaling that would lead in turn to disruption of the normal expression of other developmentally important genes. Of particular interest was the observed up-regulation of Cyp7b1, which catalyzes the metabolism of the DHT metabolites 3α-Adiol and 3β-Adiol, and is thought to control cellular levels of both androgens and estrogens
It is important to note that the intracellular concentration of E2 within the urogenital sinus during development is still unknown. The dose of E2 that reaches ER in male mouse UGS mesenchyme cells would depend not only on E2 uptake from the blood but also on local aromatization of testosterone to E2. Because of this issue, we administered E2 over a wide dose range, but also ensured that the opportunity for aromatization was controlled by the use of DHT rather than testosterone in the culture medium. Total testosterone circulates in the range of 5–8 nM in the male rat and mouse fetus during prostate differentiation
The up-regulation of Pgr by all doses of E2 administered here to UGS mesenchyme cells is in general agreement with Risbridger et al., who reported up-regulation of progesterone receptors (PR) in the adult mouse prostate after estrogen treatment
Neonatal estrogen treatment is known to affect the expression of several genes critical to prostate development. Notable examples are Hoxb13, Nkx3.1, Shh, Fgf10 and Bmp4
Developmental estrogen exposure has the potential to acutely stimulate abnormal growth and induction of hyperplasia in the developing prostate
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