Gene Expression Profile Induced by 17{alpha}-Ethynyl Estradiol in the Prepubertal Female Reproductive System of the Rat

Jorge M. Naciff1, Gary J. Overmann, Suzanne M. Torontali, Gregory J. Carr, Jay P. Tiesman, Brian D. Richardson and George P. Daston

Miami Valley Laboratories, The Procter and Gamble Company, Cincinnati, Ohio 45253

Received November 11, 2002; accepted January 15, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The profound effects of 17ß-estradiol on cell growth, differentiation, and general homeostasis of the reproductive and other systems, are mediated mostly by regulation of temporal and cell type-specific expression of different genes. In order to understand better the molecular events associated with the activation of the estrogen receptor (ER), we have used microarray technology to determine the transcriptional program and dose-response characteristics of exposure to a potent synthetic estrogen, 17{alpha}-ethynyl estradiol (EE), during prepubertal development. Changes in patterns of gene expression were determined in the immature uterus and ovaries of Sprague-Dawley rats on postnatal day (PND) 24, 24 h after exposure to EE, at 0.001, 0.01, 0.1, 1 and 10 µg EE/kg/day (sc), for four days (dosing from PND 20 to 23). The transcript profiles were compared between treatment groups and controls using oligonucleotide arrays to determine the expression level of approximately 7000 annotated rat genes and over 1740 expressed sequence tags (ESTs). Quantification of the number of genes whose expression was modified by the treatment, for each of the various doses of EE tested, showed clear evidence of a dose-dependent treatment effect that follows a monotonic response, concordant with the dose-response pattern of uterine wet-weight gain and luminal epithelial cell height. The number of genes whose expression is affected by EE exposure increases according to dose. At the highest dose tested of EE, we determined that the expression level of over 300 genes was modified significantly (p <= 0.0001). A dose-dependent analysis of the transcript profile revealed a set of 88 genes whose expression is significantly and reproducibly modified (increased or decreased) by EE exposure (p <= 0.0001). The results of this study demonstrate that, exposure to a potent estrogenic chemical during prepubertal maturation changes the gene expression profile of estrogen-sensitive tissues. Furthermore, the products of the EE-regulated genes identified in these tissues have a physiological role in different intracellular pathways, information that will be valuable to determine the mechanism of action of estrogens. Moreover, those genes could be used as biomarkers to identify chemicals with estrogenic activity.

Key Words: immature rat uterotrophic assay; gene expression profiling; microarrays; 17{alpha}-ethynyl estradiol.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The growth and development of the mammalian reproductive system is highly regulated by the steroid hormone 17ß-estradiol. At maturity, when the uterus and ovaries undergo the highly orchestrated cycles of proliferation and differentiation associated with estrous cycle, the activity of this steroid hormone is indispensable. Estrogens and other extracellular signals coordinate the estrous cycle through the tight regulation of key signaling molecules. The profound effects of 17ß-estradiol on cell growth, differentiation, and general homeostasis of the reproductive and other systems are mediated mainly by the regulation of temporal and cell type-specific expression of different genes, whose products are the molecules controlling those molecular events (Diel, 2002Go; Findlay et al., 2001Go; Hall et al., 2001Go; Nilsson et al., 2001Go).

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., 2002Go) with a drastic increase one day before the first ovulation (Noda et al., 2002Go; Toorop et al., 1984Go). 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, 1998Go; Diel et al., 2000Go; Kang et al., 2000Go; Kanno et al., 2001Go; Newbold et al., 2001Go; Padilla-Banks et al., 2001Go).

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., 2002Go). 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{alpha}-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, 2001Go; U.S. Environmental Protection Agency, 1998Go); 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-{alpha} and ER-ß (Klinge, 2001Go; Nilsson et al., 2001Go). There is considerable variation in the expression levels of the two ER isoforms in different tissues (Couse et al., 1997Go; Kuiper et al., 1997Go). The uterus and ovaries are two of the most sensitive tissues to estrogenic regulation, and both tissues express ER{alpha} and ERß, ER{alpha} being the predominant isoform in the uterus while ERß prevails in the ovaries (Couse et al., 1997Go; Kuiper et al., 1997Go). The different ratios of ER{alpha}/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{alpha}-ethynyl estradiol.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
17{alpha}-Ethynyl estradiol and peanut oil were obtained from Sigma Chemical Company (St. Louis, MO).

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 2Go), 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., 1999Go), 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 2Go), 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 Gamble’s 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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TABLE 2 Effect of 17{alpha}-Ethynyl Estradiol on Uterine and Ovarian Wet-Weight Gain
 
Starting on PND 20, the animals were dosed, by subcutaneous injection, with 0.001, 0.01, 0.1, 1, or 10 µg/kg/day 17{alpha}-ethynyl estradiol in peanut oil. Animals received 5-ml/kg body weight of dose solution once a day for four days. A four-day dosing regime was selected to optimize detection of any effect of EE exposure at the low-dose range, both at the histological level as well as at the gene expression level. The dose was administered between 8 and 9 A.M. each day. Controls received 5 ml/kg of peanut oil once a day for four days. Doses were administered on a µg/kg body weight basis and adjusted daily for weight changes. Body weight (nearest 1.0 g) and the volume of the dose administered (nearest 0.1 ml) were recorded daily. The exact time of the last dose was recorded, to establish a 24-h waiting period before tissue collection. The animals were sacrificed by CO2 asphyxiation 24 h after the last dosing, on PND 24. The body of the uterus was cut just above its junction with the cervix, and, leaving the ovaries attached to it, was carefully dissected free of adhering fat and mesentery and weighed as a whole. The ovaries were dissected free, and the uterine and ovarian wet weight was recorded; both the uterus and ovaries were then placed into RNAlater (50–100 mg/ml of solution; Ambion, Austin, TX), at room temperature.

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 4–5 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., 2002Go). 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., 2001Go). 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 1Go 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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TABLE 1 Primers Used to Validate the Array-based Gene Expression Changes Induced by 17ß-Ethynyl Estradiol by QRT-PC
 
Data analysis.
Potential inter-individual variability was addressed by using independent samples of each dose group (n = 6 for the LDUA set, and n = 5 for the HDUA set) for analysis. For the uterine/ovarian weight determination, we evaluated ten female tissues (uterus/ovaries) per dose-group (Table 2Go). For gene expression analysis, scanned output files of affymetrix microarrays were visually inspected for hybridization artifacts and then analyzed using Affymetrix Microarray Suite (version 5.0) and Data Mining Tool (version 3.0) software, as described (Lockhart et al., 1996Go; http://www.affymetrix.com/index.affx). Arrays were scaled to an average intensity of 1500 units and analyzed independently. The affymetrix rat genome U34A microarrays used in this study have 8740 probe sets corresponding to ~7000 annotated rat genes and 1740 expressed sequence tags (ESTs). Each gene or EST is represented by 16–20 pairs of 25-mer oligonucleotides that span the coding region. Each probe pair consists of a perfect match sequence that is complementary to the cRNA target and a sequence that is mismatched by a single base change at the middle of the nucleotide, a region critical for target hybridization. The mismatched oligonucleotide serves as a control for nonspecific hybridization. The Microarray Analysis Suite software (affymetrix) was used to generate the data for comparative analysis. Distinct algorithms made an absolute call, present/marginal/absent, for each transcript, and calculated the average difference between perfect match and mismatch probe pairs, signal value. The mathematical definitions for each algorithm are described in the Affymetrix Microarray Suite User’s Guide, Ver. 5.0. Transcripts, for which an absent call was determined in all the samples across the dose groups for a given compound and their respective controls, were eliminated from further analysis. For the remaining transcripts, a series of statistical tests were conducted for each transcript separately.

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 2Go), 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.).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 in Table 2Go) in animals fed with a standard laboratory rodent diet (Purina 5001; Purina Mills). During the second experimental phase, we tested the effects of EE at the relatively low doses of 0.001, 0.01 and 0.1 µg EE/kg/day (LDUA set in Table 2Go), in animals fed with a soy- and alfalfa-free diet (casein-based diet, 5K96; Purina Mills). While we realized that the difference in phytoestrogen content of the two diets may have an impact on the gene expression profile identified, we chose to use the typical rodent diet to feed the animals used for the evaluation of the EE exposure on gene expression at high-dose ranges, to avoid a potential negative shifting of the baseline data, which would diminish the value of historical comparisons of estrogen-dependent gene expression data already published. For the characterization of the gene expression changes induced by low doses of EE (LDUA set in Table 2Go), females were switched to a casein-based diet (Purina 5K96) five days before starting the dosing protocol (PND 16) and during the entire experimental phase (PND 20 to 24).

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 2Go). 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 2Go). 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 2Go), on wet uterine weight gain or uterine epithelial cell height (Figs. 1–3GoGoGo). 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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FIG. 1. Representative uterine transversal sections from equivalent regions of vehicle-treated control immature rats (PND 24) (A) or animals EE-treated with 0.001 (B), 0.01 (C), 0.1 (D), 1 (E), or 10 (F) µg/kg/day. The immature female rats on PND 20 were treated with a single daily injection (sc) with the indicated doses of EE, for four days; 24 h after the last dose, the uterus and ovaries were removed, weighed, and processed for histological examination, as described in Materials and Methods. The dose-dependent uterotrophic response to EE exposure is evident at the highest-dose range (A–D vs. E–F), but not at the low-dose range (A vs. B–D). Scale bar, 0.08 mm, for all the photomicrographs. Uterine lumen (L), gland (g), estroma (ES), and smooth muscle layer (M) are indicated.

 


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FIG. 2. Representative luminal epithelial cell height of the uterus from immature rats exposed to vehicle control (A, E) or various doses of EE (B–D, F–H). Tissue sections from the mid-region of each uterine horn, at equivalent areas, and with clear representation of the epithelium lining the lumen along the uterus, were used to determine the response of the luminal epithelial cells to EE exposure. The unstimulated uterus of vehicle or low-dose EE-treated (0.001 to 0.1 µg EE/Kg/day) immature rats shows two to three layers of cuboidal cells lining the uterine lumen (A, E vs. B-D, F), while the exposure to higher doses of EE (1–10 µg EE/Kg/day) increases dramatically the epithelial cell height and turns the epithelia into a pseudo-stratified columnar layer of 6–8 cylindrical-shaped, vacuolated cells (G, H). Feeding the animals with a normal rodent diet (E–H; Purina 5001), or a casein-based diet (A–D; Purina 5K96), does not have an impact on the histological changes induced by the various doses of EE. Scale bar, 0.01 mm, for all the photomicrographs. The uterine (L) and glandular lumens (G) are indicated.

 


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FIG. 3. Luminal epithelial cell height of the uterus from immature rats exposed to vehicle control (low or high dose, LD and HD, respectively, fed with casein-based diet or Purina 5001) or various doses of EE (in animals fed with a casein-based diet, 0.001–0.1 µg EE/kg/day, or a normal rodent diet, 0.1–10 µg EE/kg/day). Values were obtained from tissue sections from the mid-region of each uterine horn, at equivalent areas, and with clear representation of the epithelium lining the lumen along the uterus (as shown in Fig. 2Go). Cell height was determined by obtaining five measurements from five areas from two animals for each treatment group. These values were used to determine the mean cell height ± standard deviation (SD) for each treatment group. Treatment with 1 and 10 µg EE/Kg/day induced a significant increase (*) on luminal epithelial cell height compared with vehicle-treated animals (p <= 0.001, or p <= 0.05). The difference in phytoestrogen content of the two diets used does not have an impact on this parameter (compare control LD vs. control HD).

 
The uterotrophic response of the immature uterus to EE at the histological level is shown in Figures 1 and 2GoGo. The weight increase induced by high doses of EE is the result of hypertrophy and hyperplasia, as well as accumulation of fluid in the lumen. Uterine histology was very consistent with the dose response in uterine wet-weight gain (Table 2Go vs. Fig. 2Go). Hypertrophy of luminal epithelial, stromal, and myometrial cells, thickening of stromal layer, and some stromal inflammatory reaction were the morphological changes induced by high doses of EE (Figs. 1 and 2GoGo). Those changes were undetectable at doses of 0.001 and 0.01 µg EE/kg/day (Figs. 2B and 2CGoGo), mildly detected in animals exposed to 0.1 µg EE/kg/day (Figs. 1C, 2D, and 2FGoGo), and clearly and consistently observed in animals exposed to 1 or 10 µg EE/kg/day (Figs. 1E –1FGo and 2G–2HGo). The response of the uterine epithelial cell height to the various doses of EE tested is shown in Figure 2Go at the histological level, and its quantification is shown in Figure 3Go. In control animals, the uterine epithelium consists of two to three layers of cuboidal cells (Figs. 2A and 2EGo). After exposure to EE, these cells change from cuboidal- to cylindrical-shaped vacuolated cells (Figs. 2G–2HGo). The thickness of the luminal epithelium increases to six to eight layers of cells, and consequently the luminal epithelial cell height increases in a statistically significant way (p <= 0.001) only in animals exposed to 1 and 10 µg EE/kg/day, compared with vehicle-treated controls (Fig. 3Go). The different diets have no effect on uterine histology (Fig. 2Go).

Exposure to the different dosages of EE did not induce significant changes in the ovarian weight when compared to vehicle-treated animals (Table 2Go). 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 2Go). At the histological level there was not a difference between controls and EE-treated animals (Fig. 4Go). 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. 4Go). Theca cells, oocytes, and interstitial cells were also identical in vehicle-treated controls and EE-treated animals (Fig. 4Go).



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FIG. 4. Representative ovarian transverse sections from equivalent regions of vehicle-treated control immature rats (PND 24) (A, E) or animals treated with 0.001 (B), 0.01 (C), 0.1 (D, F), 1 (G), or 10 (H) µg/kg/day EE. PND 20 rats were treated with a single daily injection (sc) for four days. The uterus and ovaries were removed 24 h after the last dose, weighed, and processed for histological examination as described in Materials and Methods. The ovaries are maturing normally, regardless of the EE exposure, at the doses tested, as is evidenced by the multiple primordial and growing large healthy maturing pre-antral and antral follicles (*) containing the characteristic granulose cell layers and a single oocyte, present in control or EE-treated animals fed a regular rodent diet (Purina 5001, E–H) or a casein-based diet (A-D). No corpora lutea were observed, even in animals exposed to the highest EE concentration (10 µg EE/kg/day, H). Scale bar, 0.08 mm, for all the photomicrographs.

 
Gene expression changes induced by EE exposure.
In order to reduce the probability of identifying false positive gene expression changes and to have better leverage on our statistical analysis, six (LDUA) and five (HDUA) independent tissue samples (pool of uterus and ovaries from individual animals) were used to evaluate the effect of EE exposure in each treatment group.

We identified clear evidence of a dose-dependent treatment effect on gene expression (Fig. 5Go). 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. 5Go). There was a good correspondence between the EE doses that were uterotrophic, and the effects on gene expression (compare Table 2Go and Figs. 2 and 3GoGo vs. Fig. 5Go). 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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FIG. 5. Number of statistically significant gene-expression changes induced in the uterus/ovaries of immature rats by 17{alpha}-ethynyl estradiol exposure, in a dose-dependent way. The number of genes whose expression was statistically significant modified by EE exposure, as compared with the corresponding vehicle-treated control, was determined using the t-test, at two levels of significance (p <= 0.001, and p <= 0.0001). To calculate the p value, the signal value (t-test) or the log of the signal value (LogTest), that represents the relative expression level for each of the 8743 genes represented on the array, were used as described in Materials and Methods. At p <= 0.001 one gene per every 1000 should be expected as a false positive, while at p <= 0.0001 only one gene per 10,000 should be expected as a false positive. For the low-dose range set (control and 0.001–0.1 µg EE/kg/day) n = 6, while for the high-dose range set (control and 0.1–10 µg EE/kg/day), n = 5. Feeding the animals a normal rodent diet (**Purina 5001), or a casein-based diet (*Purina 5K96), does not have an impact on the number of genes whose expression is significantly affected by exposure to the different doses of EE.

 
Although a number of gene-expression changes can be correlated with the two rodent diets used in this study, the presence of a higher phytoestrogen content in one of them (Purina 5001) did not induce a large number of gene-expression changes that can be correlated with estrogenic activity. Comparing the number of genes affected in tissues from animals exposed to the same dose of EE (0.1 µg EE/kg/day) but fed different diets, and their respective control groups (Fig. 5Go) did not result in statistically different number of genes being affected.

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 3Go.


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TABLE 3 Partial List of Genes Whose Expression Is Significantly Regulated by 17{alpha}-Ethynyl Estradiol in a Dose-Dependent Way
 
Dose-response characteristics of gene expression changes induced by EE exposure.
Analyzing the dose-response characteristics of EE exposure (Fig. 5Go and Table 3Go), including the data from all the dose groups, it was clear that most of the genes change their expression level by exposure to the higher dosages. To discriminate the uterine/ovarian dose-response further at the gene expression level, we analyzed the data collected from the two independent sets: low-dose and high-dose ranges, respectively (as indicated in Table 2Go), comparing each independent set with their respective control.

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 4Go. 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 4Go 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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TABLE 4 Genes Whose Expression is Regulated by Low Doses of 17{alpha}-Ethynyl Estradiol
 
Exposure of immature rats to EE at a high-dose range (0.1 to 10 µg/kg/day) induced statistically significant changes in the expression of 216 genes (ANOVA, p <= 0.0001) from the uterus/ovaries. Gene induction is the most prevalent effect of EE exposure (152 genes are upregulated), although downregulation is also promoted (64 genes are downregulated). Trend analysis indicated that the expression of 123 genes shows a significant dose-response effect (p <= 0.0001). A list of these genes, along with their accession numbers, gene symbols, and the average fold change induced by the different doses of EE (average of 5 independent samples, calculated by comparing treatment versus control) is shown in Table 5Go. In this analysis, both gene induction and repression are represented (65 and 58 genes, respectively). From this set of data, it is evident that most of the genes whose expression is modified by EE exposure do so when the organism is exposed to relatively high concentrations of this estrogenic compound (1 to 10 µg EE/kg/day). The expression of multiple genes such as complement component 3 and its precursor, polymeric immunoglobulin receptor, cathepsin S, small proline-rich protein, FSH receptor, follistatin, and hydroxysteroid dehydrogenase 17ß type 1 among others, seems to be fully affected by 1 µg EE/kg/day, up- or downregulated, although exposure to the highest dose of EE (10 µg/kg/day) alters the expression of a larger number of genes. The number of genes showing a statistically significant change in their expression increases from very few (3, by t-test at p <= 0.001) at the low dose (0.1 µg EE/kg/day) to a larger number at the mid- to high-dose (159 and 252 genes, respectively; by t-test analysis at p <= 0.001). This dose dependency on the number of genes affected by EE exposure correlates very well with the uterotrophic response, at the same dosages.


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TABLE 5 Genes Whose Expression Is Regulated by High Doses of 17{alpha}-Ethynyl Estradiol
 
Although most of the genes we have identified as being regulated by estrogen exposure (Tables 3–5GoGoGo) are novel genes not previously identified as being estrogen-responsive (including annotated genes and still uncharacterized ESTs), on this list are included genes known to be directly regulated by estrogens, such as complement component 3 (and its precursor, pre-pro-complement component 3), intestinal calcium-binding protein, 11 ß-hydroxylsteroid dehydrogenase type 2, creatine kinase B, and others, which corroborates published data (Darnel et al., 1999Go; Diel et al., 2000Go; Hyder et al., 1999Go; Newbold et al., 2001Go; Rivera-Gonzalez et al., 1998Go), and at the same time validates our approach using microarrays to identify estrogen-regulated genes. Furthermore, there was a high consistency on the gene expression changes from animal to animal, within the same dose-group (n = 6 or n = 5, for each set, LDUA and HDUA, respectively; individual signal values are available upon request, J.M.N.).

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 6Go, 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 {alpha}-actin (VaACTIN) and cyclophilin B, were identified by QRT-PCR or microarray analysis.


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TABLE 6 Selected Gene Expression Changes Validated by Quantitative Real-Time PCR
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The histological changes induced in the immature uterus of the rat by exposure to a model compound with estrogenic properties (EE) were characteristic of the response expected for a potent ER agonist (Kanno et al., 2001Go; Newbold et al., 2001Go; O’Connor et al., 1998Go; Odum et al., 1997Go). The dose response in the biological end points evaluated, namely absolute and relative uterine wet weight gain, accumulation of fluid in the uterine lumen, and increase in uterine epithelial cell height, were also concordant with published data (Ashby and Tinwell, 1998Go; Diel et al., 2000Go; Kang et al., 2000Go; Kanno et al., 2001Go; O’Connor et al., 1996Go, 1998Go; Newbold et al., 2001Go; Padilla-Banks et al., 2001Go).

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{alpha}-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{alpha} or ERß) through the classical ER receptor pathway requiring the binding to an estrogen-response element (reviewed by: Klinge, 2001Go; Nilsson et al., 2001Go), or through the alterative pathway requiring the participation of activating protein 1, AP-1 (Kushner et al., 2000Go; Paech et al., 1997Go); 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-{kappa}B mediated gene expression changes (Bodine et al., 1999Go; Evans et al., 2002Go). 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. 5Go; Tables 3 and 4GoGo). 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 {alpha} (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 7Go. Selecting individual genes, from the ones listed (Tables 3–5GoGoGo and Table 7Go), 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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TABLE 7 Selected Genes Whose Expression Is Modified by Ee Exposure, Up- (+) or Downregulated (–), with Functional Implications in Cell Growth, Differentiation and Apoptosis
 
Our findings show clear evidence of the diverse cellular pathways affected by estrogen exposure. As indicated above, among the genes regulated by the estrogenic doses of EE are genes whose products are involved in cell growth (the {alpha} subunit of the eukaryotic initiation factor 2, GFR bound protein 14, TGF-ß masking protein; prolactin receptor, insulin-like growth factor binding protein 2 and 3, guanylate cyclase, follicle stimulating hormone receptor, etc.), differentiation (CRP-ductin/ebnerin/hensin, f-spondin, nerve growth factor-induced factor A, progesterone receptor, small proline-rich protein, SmLIM, phosphatidylethanolamine binding protein, prolactin receptor, etc.), stress response (glutathione S-transferase, non-neuronal enolase, mismatch-repair protein, heat shock protein 60, etc.), apoptosis (amyloid precursor protein-binding protein, IGF-binding protein 2, ryudocan, osteopontin or sialoprotein, etc.), and inflammation (complement component 3, eotaxin, cathepsin S, fibronectin, CD36, CD37, and polymeric Ig receptor). EE also regulates genes encoding structural proteins, such as small proline-rich protein, tensin, actin (the isoform gamma-enteric smooth muscle), ß-tropomyosin or fibroblast tropomyosin 1, collagen {alpha} 1 type V, keratin 8 and claudin 9; as well as different enzymes, such as 11-ß-hydroxylsteroid dehydrogenase type 2, cretine kinase-B, 3-hydroxy-3-methylglutaryl-CoA synthase, adrenodoxin reductase, 3 ß-hydroxysteroid dehydrogenase isomerase type II, and 17{alpha}-hydroxylase cytochrome P450 (Cyp17), among others.

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 3–6GoGoGoGo). 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., 2000Go; Morimoto et al., 2002Go). It has also been implicated in tissue remodeling in vivo, by affecting cell migration, survival, and angiogenesis during extracellular matrix reorganization (Liaw et al., 1998Go). 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., 2001Go). Osteopontin has been identified as a novel substrate for two matrix metalloproteinases (MMPs), MMP-3 (stromelysin-1) and MMP-7 (matrilysin) (Agnihotri et al., 2001Go). 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 5Go), and downregulates the expression of osteopontin (Tables 4 and 6GoGo). 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. (2002Go), 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 3Go). 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., 2002Go) 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., 2002Go). The transcript profile elicited in the immature uterus and ovaries of the prepubertal rat by exposure to 17{alpha}-ethynyl estradiol provides important information to determine the molecular mechanism of action of this class of chemicals.


    ACKNOWLEDGMENTS
 
We thank Marilyn J. Aardema and Frank Gerberick for their helpful discussions.


    NOTES
 
1 To whom correspondence should be addressed at The Procter and Gamble Co., Miami Valley Laboratories, P.O. Box 538707 #805, Cincinnati, OH 45253-8707. Fax: (513) 627-0323. E-mail: naciff.jm{at}pg.com. Back


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