Miami Valley Laboratories, The Procter and Gamble Company, Cincinnati, Ohio 45253
Received November 11, 2002; accepted January 15, 2003
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ABSTRACT |
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Key Words: immature rat uterotrophic assay; gene expression profiling; microarrays; 17-ethynyl estradiol.
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INTRODUCTION |
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In the rat, the concentration of 17ß-estradiol, the natural hormone, is consistently low throughout prepubertal development and starts to increase after day 28 of age (Noda et al., 2002) with a drastic increase one day before the first ovulation (Noda et al., 2002
; Toorop et al., 1984
). However, exposure to estrogens during the prepubertal period can induce a uterotrophic response, a reversible modification of the morphology and physiology of the uterus. This uterotrophic response in immature rodents is being used as one of the standard assays to test estrogenicity of different compounds in vivo (Ashby and Tinwell, 1998
; Diel et al., 2000
; Kang et al., 2000
; Kanno et al., 2001
; Newbold et al., 2001
; Padilla-Banks et al., 2001
).
We have determined that transplacental exposure to estrogens of various potencies and dosages results in changes of the expression patterns of multiple genes in the rat fetal reproductive system (Naciff et al., 2002). From this data, we have identified a set of genes whose expression is consistently altered in the fetal uterus/ovaries by exposure to chemicals with estrogenic activity ("molecular fingerprint"). Although at this life stage, the female reproductive system is responsive to estrogen regulation, its response to this hormone is limited, since it is still in development. In order to understand better the susceptibility of reproductive tissues to estrogens and to clearly identify gene transcripts that could be used as biomarkers for estrogen exposure, it is necessary to identify the response of these tissues to exposure to this class of compounds when they are fully responsive to estrogen, such as during prepuberty. The present study covers this life stage. Although the comparison of the gene expression profiles elicited by estrogen exposure in these two developmental stages is beyond the scope of the present work, the use of 17
-ethynyl estradiol (EE) as a reference compound in the uterotrophic assay in immature rats and the identification of the gene expression changes induced by exposure to equivalent doses of EE used in the fetal study, will allow the identification of developmental stage-dependent as well as developmental stage-independent estrogen-responsive genes.
One of our goals is the identification of gene transcripts with potentially important roles in estrogen action that could be used as biomarkers for estrogen exposure. Thus, in the present study, we have used a version of the uterotrophic assay in the immature rat; one of the Tier I screening assays recommended for detecting the estrogenic properties of endocrine disrupting chemicals (OECD, 2001; U.S. Environmental Protection Agency, 1998
); to determine the uterotrophic potency of the different doses of EE studied, and the respective gene expression profile induced in the uterus and ovaries of prepubertal rats. In mammals, the predominant biological effects of estrogens are mediated through two distinct intracellular receptors: estrogen receptor ER-
and ER-ß (Klinge, 2001
; Nilsson et al., 2001
). There is considerable variation in the expression levels of the two ER isoforms in different tissues (Couse et al., 1997
; Kuiper et al., 1997
). The uterus and ovaries are two of the most sensitive tissues to estrogenic regulation, and both tissues express ER
and ERß, ER
being the predominant isoform in the uterus while ERß prevails in the ovaries (Couse et al., 1997
; Kuiper et al., 1997
). The different ratios of ER
/ERß expression in the uterus and ovaries make those tissues, in aggregate, ideal for identifying gene expression changes induced by activation of either of the ER isoforms. Therefore we have determined the changes in patterns of gene expression in the uterus and ovaries, pooled, of immature rats exposed to 17
-ethynyl estradiol.
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MATERIALS AND METHODS |
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Animals and treatments.
Fifteen-day-old female Sprague Dawley rats were obtained (Charles River VAF/Plus) in groups of 10 pups per surrogate mother. This rat strain was chosen because it is a commonly used strain in reproductive and developmental toxicity studies. The rats were acclimated to the local vivarium conditions (24°C; 12-h light/dark cycle) for five days. Starting on postnatal day (PND) 20 and during the experimental phase of the protocol, all rats were singly housed in 20 x 32 x 20-cm plastic cages. The experimental phase was run in two stages: during the first one we tested the effects of EE at the relatively high dosages of 0.1, 1, and 10 µg EE/kg/day (HDUA set, Table 2), in animals fed a standard laboratory rodent diet (Purina 5001, Purina Mills). The Purina 5001 diet contains phytoestrogens, mostly genistein and daidzein derived from soy and alfalfa (Thigpen et al., 1999
), at levels that may have an impact on the gene expression profile, however, those levels are not uterotrophic (evaluated by the traditional end points: uterine weight gain and increase in luminal epithelial cell high) (see Results). Nevertheless, we chose to use this diet to avoid a potential negative shifting of the baseline data, thereby diminishing the value of historical comparisons of estrogen-dependent gene expression data already published. While this raised the possibility of a modest background estrogenic effect, we believe it negligible since it was diluted at the highly active dose levels of EE used in this set (HDUA). During the second experimental phase, we tested the effects of EE at the lower doses of 0.001, 0.01, and 0.1 µg EE/kg/day (LDUA set, Table 2
), and compared with vehicle-treated controls. The LDUA animals were given a casein-based diet, essentially phytoestrogen-free (Purina 5K96) from PND 16 onward, to override any possible effects of the standard rodent diet (Purina 5001) used by the animal supplier. The casein-based diet consistently contains less than 1 ppm aglycone equivalent of genistein, daidzein, and glycitein (Purina Mills). All the animals were allowed free access to water and specific pelleted commercial diet (Purina 5001 or casein-based 5K96; Purina Mills, St. Louis, MO). The experimental protocol was carried out according to Procter and Gambles animal care-approved protocols, and animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
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Histology.
Reproductive tissues from two animals in each dose group were fixed in 10% neutral buffered formalin immediately after weighing, then dehydrated, and embedded in paraffin. Serial 45 µm cross sections were made through the ovaries, oviducts and uterine horns, and stained with hematoxylin and eosin. To evaluate the morphological changes induced by EE exposure in the uterus, we focused on the proliferative state of the endometrial stroma and luminal epithelium along the uterine horns, while in the ovaries we evaluated the developing follicles, focusing in granulosa cells, primary, secondary, and mature follicles, and the potential presence of corpora lutea. Tissue sections from the mid-region of each uterine horn were evaluated for epithelial cell height in five different areas of the epithelial lining of the lumen along the uterus, using a light microscope (Nikon Optiphot-2, Nikon) interfaced with a Fujix HC-2000 high-resolution digital camera (Fuji Photo Film Co., Ltd., Tokyo, Japan).
Expression profiling.
Total RNA was extracted from uterus and ovaries from individual animals (combining only the tissues from the same animal) using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH). Total RNA was further purified by RNeasy kit (QIAGEN, Valencia CA). Ten µg of total RNA from each pool of tissue sample (individual animals) was converted into double-stranded cDNA, using the SuperScript Choice system (GIBCO BRL, Rockville, MD) with an oligo-dT primer containing a T7 RNA polymerase promoter. The double-stranded cDNA was purified by phenol/chloroform extraction, and then used for in vitro transcription, using ENZO BioArray RNA transcript labeling kit (Affymetrix, Inc. Santa Clara, CA). Biotin-labeled cRNA was purified by RNeasy kit (QIAGEN), and a total of 20 µg of cRNA were fragmented randomly to 200 bp at 94°C for 35 min (200 mM Tris-acetate, pH 8.2, 500 mM KOAc, 150 mM MgOAc). Labeled cRNA samples were hybridized to the Affymetrix GeneChip Test 2 Array (Affymetrix, Inc. Santa Clara, CA) to assess the overall quality of each sample. After determining the target cRNA quality, samples of uteri-ovaries from five or six individual females (replicates) from each treatment group for the HDUA and LDUA sets, respectively (with high quality cRNA) were selected and hybridized to Affymetrix Rat Genome U34A high-density oligonucleotide microarrays for 16 h. The microarrays were washed and stained by streptavidin-phycoerythrin (SAPE) to detect bound cRNA. The signal intensity was amplified by second staining with biotin-labeled anti-streptavidin antibody and followed by streptavidin-phycoerythrin staining. Fluorescent images were read using the Hewlett-Packard G2500A Gene Array Scanner.
Real-Time RT-PCR.
In order to corroborate the relative changes in gene expression induced by estrogenic exposure of the fetal uterus and ovaries of the rat in selected genes identified by the oligonucleotide microarrays, we used a real-time (kinetic) quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) approach, as described (Naciff et al., 2002). QRT-PCR evaluates product accumulation during the log-linear phase of the reaction, and it is currently the most accurate and reproducible approach for transcript quantification (Rajeevan et al., 2001
). This approach also allowed us to evaluate the "basal level" of expression of individual genes in samples derived from animals exposed to the two different diets used in our study. QRT-PCR was used to compare the transcript level of selected genes in samples derived from animals exposed to Purina 5001, with the levels of the same transcript found in equivalent samples derived from animals exposed to casein-based diet (controls and 0.1 EE µg/kg/day groups in both the HDUA and LDUA experimental blocks). To confirm the amplification specificity from each primer pair, the amplified PCR products were size-fractioned by electrophoresis in a 4% agarose gel in Tris borate ethylene diamine tetraacetic acid (TBE) buffer and photographed after staining with ethidium bromide. Table 1
shows the nucleotide sequences for the primers used to test the indicated gene products. Preliminary experiments were done with each primer pair to determine the overall quality and specificity of the primer design. After RT-PCR, only the expected products, at the correct molecular weight, were observed.
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For each transcript and dose group, we conducted pair-wise comparisons to vehicle controls using two-sample t-tests, comparing each treatment group to its control, and using analysis of variance for general treatment effects (ANOVAs) on the signal value (that serves as a relative indicator of the level of expression of a transcript) and the log of the signal value. General treatment effects were evaluated by analysis of variance, and a nonparametric test for dose-response trend (the Jonkheere-Terpstra test). Genes for which any of the tests had p 0.001 was taken as evidence that the expression of those genes was modified by the EE dose being tested. This procedure was done for each treatment vs. control, and for the full group of study results for each set, LDUA and HDUA (vehicle vs. low-, mid-, and high-dose of each set). Data were also analyzed for dose response. For the combined analysis of the two sets (LDUA and HDUA), stratified nonparametric tests were conducted that were focused on detecting genes showing a dose response in both EE dose ranges (LDUA and HDUA), or where there was a consistent treatment effect versus vehicle for the EE dose in common to both studies (0.1 µg EE/kg/day). Here, we used linear models with terms for both study and treatment effects, on average differences (signal values) and their log transformation, as well as stratified forms of the Wilcoxon-Mann-Whitney nonparametric statistic and a stratified form of the Jonkheere-Terpstra nonparametric statistic for dose response. In the linear model analysis, study-to-study differences are adjusted for by the presence of a term for study effects in the model and in the nonparametric statistics, and stratification amounts for pooling within-study evidence of treatment effects. Genes regulated differentially by low or high doses of EE were identified by the addition of an interaction term to the linear model analyses.
In all of these pooled analyses, the expression of a gene was considered affected when any of the relevant tests had p 0.0001 for that particular gene. Fold-change summary values for genes were calculated as a signed ratio of mean signal values (for each EE-dose group compared with the appropriate control). Because fold-change values can become artificially large or undefined when mean signal values approach zero, all the values <100 were made equal to 100 before calculating the mean signal values that are used in the fold-change calculation. Note that all statistical analyses used the measured signal values, even if they were smaller than 100 units.
In order to compare the gene expression profiles induced by the different EE doses tested, and to address possible issues of diet-induced differences (due to different phytoestrogen-content), the average value of the signal values, a relative indicator of the level of expression of a transcript, was compared between the two groups of independent controls and the 0.1 µg EE/kg/day which was used in both experimental blocks (LDUA and HDUA, respectively, Table 2), for all the 8740 transcripts represented on the array. Our analysis indicated that approximately 2% of those transcripts showed a significant change on their level of expression, which can be correlated to the diet used to feed the animals (J.M.N., manuscript in preparation). No significant changes were found at the transcript level, for selected estrogen-regulated genes, by QRT-PCR. Importantly, none of the genes that were identified as being responsive to EE exposure in a dose-dependent manner was affected by the diet.
Online supplemental materials.
Affymetrix image files for the forty-four-chip hybridizations, the analysis of the two control groups, and the absolute analysis results of each dose group are available upon request (J.M.N.).
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RESULTS |
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EE effects on uterine/ovarian and uterine wet weight and uterine luminal epithelial cell height.
Treatment with the various doses of EE was well tolerated by all the animals. No evidence of overt toxicity, and no clinical signs of toxicity were observed. A nonstatistically significant decrease in body weights was observed in animals exposed to the higher doses of EE (Table 2). Premature vaginal opening was not detected in any of the animals exposed to even the highest dose of EE, nor was there a change in the number of uterine glands. There was a dose-dependent increase in wet, absolute, and relative uterine weight (Table 2
). Significant increases in wet uterine weight were determined only at doses of 1 and 10 µg EE/kg/day (p
0.01 or p
0.05), producing 3.8- and 4.3-fold increases in wet uterine weight, respectively, compared with vehicle-treated controls. The uterus of animals exposed to 1 or 10 µg EE/kg/day showed clear accumulation of fluid in the lumen.
The phytoestrogen content in the two rodent diets did not compromise our ability to detect the effect of the different doses of EE tested, even at the lower-dose range (LDUA, Table 2), on wet uterine weight gain or uterine epithelial cell height (Figs. 13
). There were no significant changes in any of the biological end points evaluated, comparing the two diet control groups or the two groups of animals exposed to 0.1 µg EE/kg/day, but fed with different diet.
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Exposure to the different dosages of EE did not induce significant changes in the ovarian weight when compared to vehicle-treated animals (Table 2). However, a non-statistically significant decrease in ovarian weight was observed in animals exposed to the highest dose of EE tested (10 µg/kg/day) (Table 2
). At the histological level there was not a difference between controls and EE-treated animals (Fig. 4
). Granulosa cells, primary, secondary and mature follicles, including pre-antral and antral follicles, were indistinguishable from either control or EE-treated females. Even at the relatively high EE-doses tested (1 and 10 µg EE/kg/day) we did not identify corpora lutea in those ovaries (Fig. 4
). Theca cells, oocytes, and interstitial cells were also identical in vehicle-treated controls and EE-treated animals (Fig. 4
).
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We identified clear evidence of a dose-dependent treatment effect on gene expression (Fig. 5). Exposure to low dosages of EE (0.001 or 0.01 µg EE/kg/day) did not induce statistically significant gene expression changes, even at a less stringent significance value (p
0.001, two-sample t-tests), while dosages of 0.1, 1, or 10 µg EE/kg/day affected the expression pattern of a number of genes in the uterus and ovaries of immature rats (Fig. 5
). There was a good correspondence between the EE doses that were uterotrophic, and the effects on gene expression (compare Table 2
and Figs. 2 and 3
vs. Fig. 5
). However, there is a discrepancy at 0.1 µg EE/kg/day, where no statistically significant uterotrophic effect was detected, but there are a number of genes whose expression was modified.
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Global analysis of the gene expression changes induced by EE exposure (LDUA and HDUA sets) indicated that the expression of over 342 genes was changed, 203 being upregulated and 139 downregulated by the highest dose. Trend analysis of the data from all the doses of EE tested indicated that the expression of 88 genes was statistically and significantly changed by EE in a dose-dependent manner (p 0.0001). A list of those genes, along with their accession numbers, gene symbols, and the average fold changes induced by the different doses of EE (average of 6 or 5 independent samples, calculated by comparing treatment versus control) is shown in Table 3
.
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Exposure of immature female rats to EE at the low-dose range (0.001, 0.01 or 0.1 µg EE/kg/day) induced statistically significant changes in the expression of 39 genes from the uterus/ovaries (ANOVA, trend or t-test, p 0 .0001). A list of these genes, along with their accession number, gene symbol, and the average fold change induced by the different doses of EE (average of 6 independent samples, calculated by comparing treatment versus control) is shown in Table 4
. The expression of 5 genes from the group of 39 shows a significant dose-response (trend analysis, p
0.001). Those genes are: initiation factor-2 kinase (eIF-2a), creatine kinase B, nerve growth factor-induced factor A, osteopontin (also called sialoprotein), and the EST AA924772. Evaluating the fold change induced by the different doses, with a few exceptions (indicated 5 genes), the expression of the genes listed in Table 4
changed when the animals were exposed to 0.1 µg EE/kg/day, but not when the exposure dose was lower (0.001 or 0.01 µg EE/kg/day).
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The reliability of the microarray approach we have used in this study was independently corroborated by real-time quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) analysis of selected genes in independent samples, used for transcript profiling by microarray analysis, from each treatment group. The relative expression levels of complement component 3 (CC3), intestinal calcium-binding protein (InCaBP), progesterone receptor (PrgR), 11 ß-hydroxylsteroid dehydrogenase type 2 (11ß-HSD), osteopontin, and the EST AA924772 (AA924772) that has high homology to growth inhibitory factor, a metallothionein homolog, were evaluated by QRT-PCR. As shown in Table 6, the relative expression level of CC3, InCaBP, PrgR, 11ß-HSD, osteopontin, and the EST-AA924772 mRNAs, based on QRT-PCR analysis, followed essentially the same expression profile induced by the different doses of EE, as determined by microarray analysis. No significant changes on the expression levels of two control genes, vascular
-actin (VaACTIN) and cyclophilin B, were identified by QRT-PCR or microarray analysis.
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DISCUSSION |
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The identification and characterization of estrogen-regulated genes in reproductive tissues is an essential step toward understanding the mechanism of action of natural and synthetic chemicals with estrogenic activity, both in normal reproductive system development and in diseases. In this study, we have identified a set of genes whose expression is regulated by 17-ethynyl estradiol, a pure estrogen receptor agonist, in the immature uterus and ovaries of the prepubertal rat. The number of genes affected by EE exposure shows a clear monotonic dose response to this chemical, and their products are implicated in multiple cellular pathways.
While some of the genes whose expression is modified by EE exposure in the uterus and ovaries of the immature rat have been previously identified as estrogen-regulated genes by traditional methodologies, in the present study we have identified many more genes that are regulated, directly or indirectly, by estrogen exposure. These genes might constitute a transcript profile that is characteristic of potent estrogen-receptor agonists. The expression of those genes could be directly regulated by either of the two estrogen receptor isoforms (ER or ERß) through the classical ER receptor pathway requiring the binding to an estrogen-response element (reviewed by: Klinge, 2001
; Nilsson et al., 2001
), or through the alterative pathway requiring the participation of activating protein 1, AP-1 (Kushner et al., 2000
; Paech et al., 1997
); or indirectly via one of the multiple gene products induced by estrogen, through the regulation of regulatory factors such as insulin-like growth factor 1, TGFß, etc., or even by actions of estrogen modifying the activity of other regulators of transcription, such as NF-
B mediated gene expression changes (Bodine et al., 1999
; Evans et al., 2002
). Our results do not permit us to distinguish among these possibilities for every gene identified; however, when applicable, sequence analysis of the promoter region of each of those genes might allow the identification of specific regulatory elements within such promoters.
The dose-response analysis, at the gene-expression level, indicates that dosages of EE equal or higher than 0.1 µg/kg/day are required to elicit a detectable response (Fig. 5; Tables 3 and 4
). Of the genes whose expression is regulated by low doses of EE, with a significant response to 0.1 µg/kg/day (trend analysis, p
0.001), with the possible exception of the creatine kinase B and the EST AA924772 (which has high homology to growth inhibitory factor, a metallothionein homolog), the product of the other three genes (initiation factor-2 kinase
(eIF-2a), nerve growth factor-induced factor A and osteopontin also called sialoprotein) are regulators of cell growth, differentiation, or apoptosis, key cellular functions to account for uterotrophicity. A selected number of genes whose expression shows a dose response to EE exposure, and whose products affect cell growth, differentiation, and/or apoptosis, among other functions, are listed in Table 7
. Selecting individual genes, from the ones listed (Tables 35
and Table 7
), to perform kinetic analysis of specific genes expression changes elicited by estrogens of various potencies, and correlating them with physiological parameters, would result in mechanistically enriched information to better understand the mechanism of action of chemicals with estrogenic activity.
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The identification of the various cellular pathways affected by EE exposure offers the possibility to characterize the molecular mechanisms involved in the action of natural and synthetic estrogenic compounds, providing information on interrelationships among the responsive genes. For example, we have identified osteopontin (also known as sialoprotein, and secreted phosphoprotein 1) as one of the genes the expression of which is downregulated by EE exposure (Tables 36). Osteopontin is an arginine-glycine-aspartic acid (RGD)containing glycoprotein that interacts with integrins and CD44 as major receptors. Osteopontin has been shown to be multifunctional, with activities in cell migration, cell survival (it promotes apoptosis through the activation of the cell-death receptor CD44), inhibition of calcification, regulation of immune cell function, and control of tumor cell phenotype (Ashkar et al., 2000
; Morimoto et al., 2002
). It has also been implicated in tissue remodeling in vivo, by affecting cell migration, survival, and angiogenesis during extracellular matrix reorganization (Liaw et al., 1998
). The expression of osteopontin is primarily induced in response to progesterone, whereas the ß 3 integrin subunit, one of its receptors, is upregulated by epidermal growth factor (EGF) or heparin-binding EGF (Apparao et al., 2001
). Osteopontin has been identified as a novel substrate for two matrix metalloproteinases (MMPs), MMP-3 (stromelysin-1) and MMP-7 (matrilysin) (Agnihotri et al., 2001
). Concurrently, we have found that EE exposure at high doses (1 to 10 µg/kg/day) promotes the expression of matrilysin (MMP-7) in the immature reproductive system of the rat (Table 5
), and downregulates the expression of osteopontin (Tables 4 and 6
). Assuming that the increase in matrilysin mRNA induced by EE results in the increase of this metalloprotease, this in turn will decrease further the protein levels of osteopontin, resulting in a net promotion of the uterine growth observed after estrogen exposure. Furthermore, matrilysin and other matrix metalloproteinases are enzymes implicated in normal and pathologic tissue remodeling processes, and the uterine response to estrogen requires extensive tissue remodeling. Recently, Yu et al. (2002
), determined that matrilysin interacts directly with CD44, a widely expressed heparan sulfate proteoglycan implicated in cell adhesion and trafficking, tumor survival and progression, as well as in apoptosis, and which expression is upregulated by EE-exposure (Table 3
). These authors found that CD44 heparan sulfate proteoglycan (CD44HSPG) recruits proteolytically active matrilysin and heparin-binding epidermal growth factor precursor (pro-HB-EGF) to form a complex on the surface of the uterine smooth muscle, among other tissues. The HB-EGF precursor within this complex is processed by matrilysin, and the resulting mature HB-EGF binds and activates its receptor, ErbB4 (a member of the epidermal growth factor receptor family of tyrosine kinases), promoting cell survival (Yu et al., 2002
) among other activities. The upregulation of matrilysin and CD44, and down-regulation of osteopontin, at uterotrophic doses of EE is clearly a synchronized, and perhaps required, response to estrogenic stimulation.
Although we have not gathered the complete information to understand estrogen action in its totality, the transcript profile induced by EE, here identified clearly, shows important points about the estrogen mechanisms of action. The comprehensive analysis of the physiological implications for each of the genes whose expression is regulated by estrogens is beyond the scope of this work. However, our findings will be valuable to design experimental approaches to determine the mechanism of action of estrogens in the context of the biological response to this class of chemicals.
In all, our data indicate that exposure to a potent synthetic estrogen EE, at and above 0.1 µg EE/kg/day, can induce gene expression changes that may have an impact on the uterotrophic response. If the same results are obtained by exposure of female immature rats to other chemicals with estrogenic activity at dosages of equivalent potency, then the evaluation of gene expression changes induced in the reproductive system of the immature rat could be used to identify the estrogenic properties of novel compounds, such as we have proposed for fetal transcript profiles (Naciff et al., 2002). The transcript profile elicited in the immature uterus and ovaries of the prepubertal rat by exposure to 17
-ethynyl estradiol provides important information to determine the molecular mechanism of action of this class of chemicals.
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ACKNOWLEDGMENTS |
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NOTES |
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