Gene Expression Changes Induced in the Testis by Transplacental Exposure to High and Low Doses of 17-Ethynyl Estradiol, Genistein, or Bisphe

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     Miami Valley Innovation Center, The Procter and Gamble Company, Cincinnati, Ohio 45253

    ABSTRACT

    The purpose of this study was to determine (1) the transcriptional program elicited by exposure to three estrogen receptor (ER) agonists: 17 -ethynyl estradiol (EE), genistein (Ges), and bisphenol A (BPA) during fetal development of the rat testis and epididymis; and (2) whether very low dosages of estrogens (evaluated over five orders of magnitude of dosage) produce unexpected changes in gene expression (i.e., a non-monotonic dose-response curve). In three independently conducted experiments, Sprague-Dawley rats were dosed (sc) with 0.001–10 μg EE/kg/day, 0.001–100 mg Ges/kg/day, or 0.002–400 mg BPA/kg/day. While morphological changes in the developing reproductive system were not observed, the gene expression profile of target tissues were modified in a dose-responsive manner. Independent dose-response analyses of the three studies identified 59 genes that are significantly modified by EE, 23 genes by Ges, and 15 genes by BPA (out of 8740), by at least 1.5 fold (up- or down-regulated). Even more genes were observed to be significantly changed when only the high dose is compared with all lower doses: 141, 46, and 67 genes, respectively. Global analyses aimed at detecting genes consistently modified by all of the chemicals identified 50 genes whose expression changed in the same direction across the three chemicals. The dose-response curve for gene expression changes was monotonic for each chemical, with both the number of genes significantly changed and the magnitude of change, for each gene, decreasing with decreasing dose. Using the available annotation of the gene expression changes induced by ER-agonist, our data suggest that a variety of cellular pathways are affected by estrogen exposure. These results indicate that gene expression data are diagnostic of mode of action and, if they are evaluated in the context of traditional toxicological end-points, can be used to elucidate dose-response characteristics.

    Key Words: testis; epididymis; gene expression profiling; microarrays; 17--ethynyl estradiol; genistein; bisphenol A.

    INTRODUCTION

    The long-established view of estrogens (estradiol) as the "female hormones" and androgens (testosterone) as the "male hormones" has been modified in recent years. It is now recognized that estrogens have an important physiological role in the reproductive and other systems of the male as well as in the female (Akingbemi et al., 2003; Couse et al., 2001; Couse and Korach, 1999; Hess et al., 1997a, 2001; Jefferson et al., 2000; Luconi et al., 2002; O'Donnell et al., 2001; Robertson et al., 1999). Estrogens regulate the function of multiple cell types, not just in tissues from the reproductive system, but also in other tissues including bone, liver, brain, the cardiovascular and the immune system (Hall et al., 2001; Nilsson et al., 2001). In mammals, the predominant biological effects of estrogens are mediated through the activation of two distinct intracellular receptors: estrogen receptor (ER) alpha and ER-beta (Klinge, 2001; Nilsson et al., 2001). There is considerable variation in the expression levels of the two ER isoforms in the different target tissues of both females and males (Couse et al., 1997; Kuiper et al., 1997; O'Donnell et al., 2001). In the male reproductive system various cell types express ER. Leydig cells, which produce the primary male steroid hormone testosterone, express the two estrogen receptor subtypes, ER and ER, and have the capacity to convert testosterone to the natural estrogen 17 -estradiol. Thus, Leydig cells are subject to estrogen action (Akingbemi et al., 2003). ER is expressed in most of the cells of the epithelia of the efferent ducts and initial segment of the epididymis, while Sertoli cells, late spermatocytes, and early round spermatids express ER (Mowa and Iwanaga, 2001; Nie et al., 2002; Saunders et al., 1998, 2000; Shughrue et al., 1998; Turner et al., 2001; Van Pelt et al., 1999; Zhou et al., 2002). In fact, ER is required by the supporting somatic cells of the male reproductive tract to support the production of sperm (spermatogenesis) that are capable of fertilization (Mahato et al., 2001). By immunocytochemistry, it has been determined that the ER is expressed in some cells of the epithelium of efferent ductules (Fisher et al., 1997; Goyal et al., 1997; Hess et al., 1997a; Kwon et al., 1997), the stromal cells of the vas deferens, and the epithelial cells in the caput epididymis (Hess et al., 1997a). In mature males from different species the concentration of circulating 17 -estradiol, the natural endogenous estrogen, is relatively low, but it is highly concentrated in the testis and epididymis (Free and Jafee, 1979; Ganjam and Amann, 1976), where it promotes the reabsorption of luminal fluid, particularly in the head of the epididymis (Hess et al., 1997b). Also, in the male, 17 -estradiol is synthesized by at least three different cell types: Sertoli, Leydig, and germ cells (Hess et al., 2001; O'Donnell et al., 2001). The essential physiological role of estrogen in the reproductive system of the male has been further demonstrated by determining that male fertility is impaired in mice lacking ER, or aromatase, the enzyme responsible for estrogen biosynthesis (Eddy et al., 1996; Fisher et al., 1998; Robertson et al., 1999). Additionally, over-expression of aromatase in male mice increases estrogen production, causing infertility and an increased incidence of Leydig cell tumors in adulthood (Fowler et al., 2000).

    Exposure of mice and rats to relatively high concentrations of chemicals with estrogenic activity during fetal or post-natal development induces developmental abnormalities in the male reproductive system, and can cause a predisposition to abnormal function and disease after birth or during adulthood (Cupp and Skinner, 2001; Fielden et al., 2002; Majdic et al., 1996; McLachlan et al., 1975; O'Donnell et al., 2001; Rivas et al., 2002, 2003; Sharpe, 2003). We hypothesize that the largely latent developmental effects of estrogens are preceded by immediate changes in gene expression in the embryo and fetus. Since gene expression is integral to the signal transduction process for the estrogens, an approach to address the potential impact on the physiology of the male reproductive system to estrogenic exposure is to evaluate the gene expression changes elicited in the target organs (testis and epididymis) exposed to estrogen agonists. Therefore, one aim of the present study was to determine the gene expression changes induced in the testis and epididymis of the rat by transplacental exposure to three estrogens of different potency (17 -ethynyl estradiol, genistein, and bisphenol A) during organogenesis of the reproductive system. Microarray technology was used to facilitate the identification of gene transcripts with potentially important roles in estrogen action in the male reproductive system, many of which may have not been detected using traditional approaches.

    The second aim of this study was to evaluate whether changes in gene expression could be used to determine the shape of the estrogen dose-response curve at dose levels orders of magnitude below the no-observed effect level for frank toxicity. There has been some controversy over whether dosages of estrogens below the no-observed-effect level (NOEL) for morphological effects may produce changes in developing reproductive organs that are qualitatively different in nature than the effects produced above the NOEL. There have been conflicting reports about such low-dose effects; a panel of experts convened by the U.S. National Toxicology Program concluded that the weight-of-evidence suggested that estrogens do not produce these "low dose" effects, but could not definitively discount the studies that showed such effects (Melnick et al., 2002). The panel indicated that the parameters being measured—organ weights and morphology—are inherently variable, and that this variability is influenced by many different experimental factors, some of which are outside the control of the investigator. Therefore, while doing replicate experiments adds to the weight of evidence, these are insufficient to resolve the controversy.

    We have already demonstrated that gene expression is a more sensitive and less variable measure of estrogenic effects than morphological markers (Naciff et al., 2003); therefore, we hypothesized that it would be useful in defining the shape of the dose-response curve at low dosages. Because the effects of estrogens are brought about via changes in gene expression, evaluating gene expression is mechanistically relevant to higher-order effects on development. To carry out this aim of the study, we evaluated the effects on gene expression in male reproductive tissues of three estrogens given at dosages spanning five orders of magnitude. The gene expression changes induced by transplacental exposure to ER agonist, here identified, can be used to develop a less animal intensive, accurate, rapid, and cost effective method for assessing the potential estrogenicity of chemicals during development.

    MATERIALS AND METHODS

    Chemicals.

    Bisphenol A (BPA, 99% purity) was purchased from Aldrich Chemical Company (Milwaukee, WI). Peanut oil, 17--ethynyl estradiol, genistein (4',5,7-trihydroxyisoflavone), and dimethyl sulphoxide (DMSO) Hybri-Max were obtained from Sigma Chemical Company (St. Louis, MO).

    Animals and treatments.

    Five-month-old male and female Sprague Dawley rats weighing approximately 300 g (females) or 350–400 g (males) were used (Charles River VAF/Plus, Raleigh, NC). This rat strain was chosen because it is the most commonly used strain in reproductive and developmental toxicity studies, and offers historical comparisons of estrogen-dependent phenotypic changes that potentially can be correlated with some of the gene expression changes described in the present study and with other studies already published. (See for example Ashby et al., 2003; Barlow et al., 2004; Liu et al., 2005; Naciff et al., 2002, 2003; Shultz et al., 2001; Thompson et al., 2004, among others.) The rats were acclimated to the local vivarium conditions (24°C; 12-h light/12-h dark cycle) for two weeks. All rats were singly housed in 20 x 32 x 20-cm cages (stainless steel) during the experimental phase of the protocol. Animals were allowed free access to water and were fed a casein-based diet (soy and alfalfa-free diet, Purina 5K96; Purina Mills). 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.

    Breeding was carried out by co-housing one male and one female overnight. Successful mating was confirmed the following morning by the presence of sperm in vaginal smears. Sperm positive animals were considered to be at gestation day (GD) 0. The dams were randomized into six groups per chemical, and housed in individual cages. Each treatment group had a minimum of eight pregnant females.

    Starting on GD 11, the dams were dosed, by sc injection, for the dose-response study with 0.001, 0.01, 0.1, 1, or 10 μg/kg/day EE in peanut oil; or, 0.001, 0.01, 0.1, 10, and 100 mg genistein/kg/day in DMSO; or 0.002, 0.02, 0.5, 50, or 400 mg/kg/day BPA in DMSO. Animals

    The animals were sacrificed by CO2 asphyxiation 2 h after the last dosing, on GD 20. The fetuses were harvested and the fetal testes and epididymides, were removed and placed into RNAlater (50–100 mg/ml of solution; Ambion, Austin, TX), at room temperature. It has to be stressed that the epididymis, at this developmental stage, is really the efferent ducts and the differentiating Wolffian duct (pre-epididymis and pre-vas deferens) since in the rat the epididymis begins to differentiate by GD 20–21 and it is completed post-natally by day 44 (Sun and Flickinger, 1979). For simplicity, we have used the term epididymis throughout this article.

    In order to compare the expression profile induced by EE in the male versus the one induced in the female-derived tissues (uterus and ovaries, Naciff et al., 2002) we evaluated the gene expression changes induced by 0.5, 1, or 10 μg/kg/day EE in peanut oil on the uterus/ovaries and testis/epididymis from the same littermates of each dose group. In this case, the testes/epididymides were collected from littermates obtained from the same pregnant females used in our previous study (Naciff et al., 2002). Since the treatment was identical, potential gene expression changes induced by EE exposure in the female and male can be better compared.

    Histology.

    For the histological examination of the reproductive tissues, four fetuses obtained from different litters within the same dose-treatment group, were fixed in 10 % neutral buffered formalin immediately after removal from the dams, dehydrated, and embedded in paraffin. Serial 4–5 μm cross sections were made through the testis and stained with hematoxylin and eosin. To evaluate the serial sections for abnormalities we focused on gross anatomy of the testis, the efferent ducts and the differentiating Wolffian duct (pre-epididymis and pre-vas deferens), the proliferative state of the testicular cords, and on the determination of primitive spermatogonial cells in the region of the future lumen of seminiferous tubules, as well as in the interstitial cell content. However, it is not possible to distinguish morphologically with absolute certainty between mesenchyme cells and Leydig cells at this stage under a light microscope.

    Expression profiling.

    Our goal was to determine the gene expression profile induced by agonists of the estrogen receptors, in estrogen-regulated tissues of the reproductive tract of the male, with the ultimate purpose of providing information that will be useful in establishing a gene-based screening assay. We chose to evaluate the testis and epididymis as a pool because they are two clear targets of estrogenic regulation. While we realize that this may result in loss of information about gene expression in one or the other organ, we believe that from a screening perspective we have taken the best approach because these organs contain considerable variation in the expression levels of the two ER isoforms and consequently have the potential to represent gene expression changes induced by activation of either isoform. Therefore, the testis and epididymis of at least five littermates were pooled, to yield a representative litter sample (biological replica) for analysis, and total RNA was extracted 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 were converted into double-stranded cDNA by using SuperScript Choice system (Invitrogen, Carlsbad, CA) 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 using an RNeasy kit (QIAGEN) and 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 3 Array (Affymetrix, Inc.) to assess the overall quality of each sample. After determining the target cRNA quality, samples of pooled testis-epididymis from five individual dams (five independent biological replicates) from each treatment group (with high quality cRNA) were selected and hybridized to Affymetrix Rat Genome U34A high-density oligonucleotide microarrays for 16 h. 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) (Supplementary material: Table 11). Six independent samples from the EE study (including the corresponding controls and all the dose groups, with the exception of the 0.001 μg/kg/day group, where n = 7) were also hybridized to Affymetrix Rat Genome 230A high-density oligonucleotide microarrays, which contains 15,923 probe sets corresponding mostly to well annotated rat genes, although it has some ESTs (Supplementary material: Table 12). The microarrays were washed and stained on the Affymetrix Fluidics Station 400, using instructions and reagents provided by Affymetrix as described (Naciff et al., 2003).

    Real-Time RT-PCR.

    In order to confirm the relative changes in gene expression induced by estrogenic exposure identified on the oligonucleotide microarrays, we used a real-time (kinetic) quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) approach using selected transcripts, and using the same batch of RNA from the individual samples (biological replicas) evaluated by microarray, as described (Naciff et al., 2002). 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 tetracetic 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. The standard error (SE) has been calculated for each group of values from different studies (see for example Naciff et al., 2002, 2003) and ranged from 3 to 23%, the same SE values were obtained in the present study.

    Data analysis.

    Potential inter-individual variability was addressed by pooling the target tissues (testis and epididymis) from individual fetuses within each litter to yield a representative litter sample for analysis. We analyzed five individual litters, representing five independent samples, per dose-group. For gene expression analysis, scanned output files of all 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., 1996; www.affymetrix.com/index.affx). Arrays were scaled to an average intensity of 1500 units and analyzed independently. For the entire analysis, each probe set was analyzed as an individual entity, based upon its Affymetrix ID number, regardless of the multiplicity of probe sets representing any given gene product, and were considered as representing an individual gene until the completion of the analysis. The Microarray Analysis Suite (Affymetrix) was used to generate the data for statistical 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, Version 5.0. Probe sets for which an absent call was determined in all the samples, across the dose groups and their respective controls, for each chemical tested, were eliminated from further analysis. For the remaining probe sets, a series of statistical tests were conducted for each transcript separately. For each probe set and dose group, for each chemical, we conducted pairwise comparisons to vehicle controls using two-sample t-tests, comparing each treatment group to its control, and analysis of variance (ANOVAs) for general treatment effects 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 ANOVA, and a nonparametric test for dose-response trend, the Jonkheere-Terpstra test. Probe sets for which any of the tests had p 0.001 was taken as evidence that the expression of the genes represented by those probe sets was modified by the chemical-dose being tested. This procedure was done for each treatment vs. control, and for the full group of study results.

    In order to identify genes whose expression is regulated in a similar manner by exposure to chemicals with estrogenic activity during fetal development, data of each individual probe set from the three chemicals were pooled and analyzed. Here, we used linear models with terms for both study and treatment effects, on 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, stratification amounts to pooling within-study evidence of treatment effects. Genes regulated differentially among chemicals were identified by testing an interaction term in 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 0.001 for that particular gene transcript. After selecting all the significant probe sets, for any given dose and chemical being evaluated, we eliminated probe sets representing the same gene product, leaving only one probe set (with the most significant p value) per gene listed for each dose and chemical group. Fold-change summary values for genes were calculated as a signed ratio of mean signal values. Because fold-change values can become artificially large or undefined, when reference group (control) signal values approach zero, all the values <100 were made equal to 100 before calculating the mean signal values that were used in the fold-change calculation. For all statistical analyses we use the measured signal values for each probe set, even if they were smaller than 100 units.

    The data obtained from the samples evaluated in the Affymetrix Rat Genome 230A high-density oligonucleotide microarrays were also analyzed as described above, but only to identify EE-effect and to better delineate the characteristics of the dose-response curve. Direct comparison between the U34A and the 230A microarrays is not feasible.

    Online supplemental materials.

    Affymetrix image files for the sixty chip hybridizations (U34A microarrays) of the testis-epididymis, the thirty chip hybridizations on the new 230A microarrays (EE study) and the absolute analysis results of the entire study are available at ArrayExpress-EBI (Accession no. E-TABM-12).

    RESULTS

    Maternal and Fetal Toxicity

    There were no detectable effects on maternal body weights or numbers of live fetuses per litter at any of the doses tested of the three ER agonists. However, adverse effects were seen with the highest dose tested of EE (10 μg/kg/day), namely vaginal bleeding and early parturition in one of eight dams exposed to this dose. In the fetuses exposed to this high dose of EE or the highest dose of BPA (400 mg/kg/day), prominent nipples/areolas were observed in both females and males. Genistein did not have any of these effects in the dams or their fetuses, at any dose tested. Histological examination of fetal testes and epididymides revealed no changes in micromorphology or gross abnormalities of these organs induced by any of the chemicals, at any of the doses tested. The histology of representative tissue sections of fetuses exposed to EE is shown in Figure 1.

    Gene Expression Changes Induced by 17 -Ethynyl Estradiol, Genistein, and Bisphenol A

    RNA samples from the fetal testes and epididymides were analyzed using Affymetrix Rat Genome U34A high-density oligonucleotide microarrays. The pooled tissues from five litters were used as independent experimental samples (biological replicas) for each treatment group.

    The gene expression profile of testis and epididymis was modified by exposure to these three ER-agonists in a consistent way. Transplacental exposure to these three estrogenic compounds, results in both gene induction (up-regulation) and gene repression (down-regulation). Based on the number of genes expressed in control versus treated samples, as well as on the level of expression of individual genes, the overall gene expression pattern was similar between control (vehicle-treated, peanut oil vs. DMSO treated) and treated (vehicle-treated vs. EE, Ges, or BPA) fetal tissues. Of the 8740 transcripts analyzed in this study, the highest dosage of EE, Ges, or BPA produced a significant change in expression level of 83, 46, and 67 genes, respectively. Tables 2, 3, and 4 show a partial list of genes whose expression is significantly and reproducibly (p 0.001, t-test and ANOVAs) modified by EE, Ges, and BPA, respectively. The relative increase or decrease on the expression of those genes to each dose of the indicated ER-agonists is also shown. Only the genes for which expression is modified by at least 1.5-fold (up- or down-regulated) by each chemical are shown (Tables 2–4), however there are some genes whose expression is only changed by 20–50% in a very robust manner (p 0.0001), and were identified in the analysis of the entire set of samples.

    Dose-dependent analysis of the transcript profile elicited by each chemical, just by itself, revealed that the expression of 59 genes is significantly modified by EE (230A microarray), while only 23 genes and 15 genes are dose responsive to Ges or BPA (U34A microarray), respectively. However, for the majority of the genes whose expression is modified by each chemical, the dose-responsiveness is mostly seen towards the higher dose end of the curve (Tables 2–4). The dose-response for the genes showing the most robust response to EE-, Ges-, or BPA-exposure (U34A microarray) is shown in Figure 2.

    Since the dose-responsiveness is mostly seen towards the higher dose end of the curve, for each ER-agonist, for the global comparison of the effect of EE, Ges, and BPA exposure we only considered for the analysis the data from the three highest doses, including the appropriate control. This dose-dependent analysis indicated that the expression of 50 unique genes change in the same direction, although at a different magnitude, after exposure to any of the three ER-agonists. Table 5 shows the complete list of the 50 genes, showing a statistically significant (p 0.0001) change in their expression by estrogenic exposure, along with their accession number and fold change (average calculated by comparing treatment versus control, with an n = 5 in each case). It has to be stressed that there are very few genes showing an effect at the lower doses (Tables 2–4), and only the data at the three highest doses of each chemical was analyzed to identify a common set of genes affected by transplacental exposure to EE, Ges, and BPA (Table 5). The fold change induced in the expression level of these genes is, in some cases, only 20% different than the control; however, this response is statistically significant (p 0.0001). When comparing the transcript profile elicited by EE vs. BPA, there are 37 genes whose expression is modified by at least 1.5-fold by both chemicals, in the same direction, while the same comparison between EE and Ges, or BPA and Ges identified only 14 and 18 genes, respectively, with the same parameters (magnitude and direction of the change; Table 5).

    Dose-Response Characteristics of Gene Expression Changes Induced by Exposure to Chemicals with Estrogenic Activity

    Most of the changes in gene expression were restricted to the higher dosages of each ER-agonist. Both the number of genes significantly changed and the magnitude of the transcriptional change on each affected gene decreased with decreasing dose (Figs. 2 and 3). From this set of data it is evident that most of the genes whose expression is modified by exposure to chemicals with estrogenic activity do so when the organism is exposed to relatively high concentrations. There was not a unique set of genes expressed in response to low doses of ER-agonists (Tables 2–4 ).

    In order to identify as many genes as possible that can be associated with estrogen exposure, and to better delineate the characteristics of the dose-response curve, the samples from the EE study (including the corresponding controls) were hybridized to Affymetrix Rat Genome 230A high-density oligonucleotide microarrays, which contain 15,923 probe sets corresponding mostly to well annotated rat genes. Using this approach, we determined that the expression of 142 genes was modified by transplacental exposure to 10 μg EE/kg/day, in a robust and significant manner (p 0.001, t-test). Trend analysis of these gene expression changes indicated that the expression of 59 shows some dose-responsiveness, as was found with EE, Ges, and BPA on the U34A microarrays, with the response being restricted to the higher end of the dose-response curve (Fig. 3, Table 2). Further, evaluation of the expression of 16,000 genes indicates that the testis/epididymis show no response to transplacental exposure to low doses of a potent ER-agonist.

    Gene Expression Changes Induced by EE in Both Female and Male-Derived Tissues

    In a previously reported study we analyzed female tissues (ovaries and uterus) from the same litters as the EE-exposed males (Naciff et al., 2002). When thinking in sex-specific response to estrogen exposure, one question that arises is: which genes are uniquely responsive to ER-agonist in the reproductive tissues of the male And consequently, which ones are in common with the female reproductive tissues To explore these questions, we compared the gene expression changes induced by EE-exposure in the females with those induced in the testis and epididymis (U34A microarrays). This analysis indicated that the expression of 48 genes was consistently and significantly regulated in the same direction (p 0.0001), although at a different magnitude, by EE (Table 6) in both female- and male-derived tissues.

    Prenatal exposure to EE resulted in the alteration of the expression of 19 transcripts exclusively in the reproductive system of the male during fetal development, suggesting that those transcripts represent male-specific estrogenic regulatory targets. Table 7 lists these 19 genes showing a statistically significant (p 0.001, t-test) change in their expression by estrogenic exposure, along with their accession number and fold change (average calculated by comparing treatment versus control, with an n = 5 in each case). With the exception of three genes (IgE binding protein, phosphatidyletahnolamine binding protein, and the EST197830 -AA894027), these genes are not detectable in the female fetus-derived tissues by microarray analysis (Table 7, denoted as absent -A).

    There is also a set of genes for which the expression is significantly modified only in the fetal uterus/ovaries (female-specific), additional evidence of the gender specificity of estrogen. Listed in Table 8 there are 28 of those gene transcripts and many more have been described (Naciff et al., 2002). The expression of most of these genes can be detected in the male-derived tissues but they do not show estrogen regulation. For example, exposure to EE induces the expression of the uterus-ovary specific putative transmembrane protein and the down-regulation of apolipoprotein C1, in a dose dependent manner, only in the uterus/ovaries while in the testis/epididymis the expression of those genes is undetectable (Table 8, denoted as absent -A). The estrogen-sensitivity of these, and other genes, may be correlated to the physiological function of their products and sexual differentiation of the tissues.

    The reliability of the gene expression changes identified here was independently corroborated by two approaches. First by identification of the same gene expression changes using two different microarrays (U34A and 230A; Table 2 vs. Table 5), from independent in life studies, and by real-time quantitative (kinetic) reverse transcriptase-polymerase chain reaction (QRT-PCR) analysis of selected genes in identical samples used in the microarray experiments. As shown in Table 9, the expression of IL4R, PrgR, 11-HSD, Cyp17, StAR, Dax-1, and the EST AA957003 [GenBank] mRNAs based on QRT-PCR analysis, followed essentially the same expression profile in the testis/epididymis induced by the different doses of EE as determined by microarray analysis. No significant changes in the expression of two control genes (VaACTIN–Table 9, and mitochondrial cytochrome oxidase subunits I, II, III genes, ATPase subunit 6 gene, data not shown) were identified by microarray or QRT-PCR analysis. The same verification approach has been performed with a selected group of genes on the samples obtained from the female-derived tissues and has been already published (Naciff et al., 2002).

    DISCUSSION

    The data reported in this study shows that transplacental exposure to chemicals with estrogenic activity during critical stages of development induced changes in the transcriptional program of the testis/epididymis of the rat. This change is only observed in a selected group of genes, and it is induced by relatively high doses of the ER-agonists, without causing overt anatomical malformations in the target tissues (at GD 20). Further, our results show that a non-physiological ER activation may result in modification of gene expression during a critical developmental period. Comparing the response of the female versus the male-derived tissues, the expression of a subset of genes is exclusively modified in the reproductive system of the male fetus. Based upon the robustness of the response, the transcripts identified could be valuable markers of estrogen exposure in the male. However, this possibility needs to be further explored using many more chemicals with known estrogenic activity, as well as with known chemicals lacking this activity. In addition, the transcripts we have identified can be used to generate testable hypothesis to improve the understanding of the molecular pathways associated with physiological and pathophysiological responses of the reproductive system of the male which may result after exposure to chemicals with estrogenic properties.

    Although we did not observe overt morphological changes in the testis/epididymis, we did not determine specific cell numbers, of any cell type, in our study. Thus, we cannot rule out that the estrogen exposures caused differences in absolute number of specific cell types. Some studies have shown that estrogen exposure disturbs the differentiation and/or proliferation of Leydig (Abney, 1999; O'Donnell et al., 2001) and Sertoli cells (Atanassova et al., 1999), directly or via perturbation of the hypothalamo-pituitary axis (Pinilla et al., 1992). Changes in cell numbers could have a confounding effect on the interpretation of the gene expression data, as the possibility exists that the observed changes in gene expression are the result of cell populations being present at different relative proportions, rather than the result of a direct effect of estrogens on gene expression. We believe that this interpretation is unlikely because (1) there are no obvious histological changes (i.e., if there are changes in cell populations, they are subtle); (2) there is an overlap in gene expression between males and females, suggesting that the genes expressed in common are directly controlled by estrogens; and (3) many of the genes for which expression was changed are known to be estrogen-responsive. The expression of 3-beta hydroxysteroid dehydrogenase or hydroxy-delta-5-steroid dehydrogenase (Hsd3b1), a gene that has been proposed as a marker of fetal and adult Leydig cells (Ariyaratne et al., 2000; Murray et al., 2000), was not modified by transplacental exposure to EE, Ges, or BPA. This may indicate that the number of Leydig cells is not modified by exposure to these chemicals. Even if the gene expression changes determined in this study are induced by extra-gonadal influences, they still are elicited by transplacental exposure to chemicals with estrogenic activity and can be used to better understand the effects of estrogen receptor activation on the target tissues.

    Many of the gene expression changes induced by transplacental exposure to EE identified in male tissues were also identified in the fetal uterus/ovaries in our previous study (Naciff et al., 2002) and are concordant with published data for chemicals able to interact with the ERs in the mouse or the rat (Barlow et al., 2004; Fertuck et al., 2003; Fielden et al., 2002; Liu et al., 2005; Moggs et al., 2004; Shultz et al., 2001; Thompson et al., 2004). For example: the induction of ICaBP also know as Calbindin-D(9k) (An et al., 2004; Lee et al., 2004), IL4R (Rivera-Gonzalez et al., 1998), progesterone receptor (Ashby and Odum, 2004; Williams et al., 2000), and the repression of glutathione S-transferase A5 (Gsta5) (Benbrahim-Tallaa et al., 2002), Cyp17, and StAR, (Govoroun et al., 2001; Majdic et al., 1996) have been determined in the reproductive system of the female and/or the male. Specifically in the male, transplacental exposure to DES and 4-octylphenol, at relatively high doses (600 μg/kg, and 500 mg/kg, respectively) reduce Cyp17 mRNA and protein levels in the rat fetal testis (Majdic et al., 1996) and high doses of di-n-butyl phthalate (500 mg/kg/day) as well as other developmentally toxic phthalates (benzyl butyl, dipentyl, or diethylexyl phthalate) induces the down-regulation of scavenger receptor class B-1, P450 side chain cleavage, Cyp17, StAR and seminal vesicle secretion 5 (Liu et al., 2005; Shultz et al., 2001). Treatment of rainbow trout with 17-estradiol significantly decreases Cyp17, 3HSD, and P45011beta mRNAs from the testis after 10 days of treatment (Govoroun et al., 2001). However, many of the gene expression changes induced by EE exposure we have identified have not been described in the developing tissues of the male, nor have they been identified as estrogen-regulated genes in any other tissue. In fact, multiple genes of the testis/epididymis whose expression is significantly regulated by transplacental exposure to EE, Ges, and BPA are ESTs (Table 2–5), some of which have homology to known genes (shown in Tables 2–5) although their identity and function have yet to be determined.

    Although the elucidation of the biological significance of each affected gene is beyond the scope of the present study, the gene expression changes we have identified in the developing male show that a variety of cellular pathways could be affected. For example, some of the genes whose expression changes by exposure to the three chemicals encode products implicated in steroidogenesis. The expression of these genes is specifically down-regulated by EE, Ges, and BPA, a response that could result in decreased androgen levels (testosterone), thereby affecting both gonadal differentiation and subsequent reproductive function. In agreement with our results, in the mouse testis, gestational and lactational exposure to diethylstilbestrol (DES; 0, 0.1, 1, and 10 μg/kg maternal body weight) results in early and latent alterations in the expression of genes involved in steroidogenesis, such as steroidogenic factor 1, Cyp17, Cyp11a, StAR, and scavenger receptor class B1 (Fielden et al., 2002). The observation that exposure to high doses of EE (10 μg/kg/day) or BPA (400 mg/kg/day) induced areolae retention in male fetuses suggests interference with androgen signaling (Mylchreest et al., 1999). The biosynthesis of androgens requires the activity of StAR and the steroidogenic enzymes cholesterol side-chain cleavage enzyme (P450scc or Cyp11A), Cyp17, 3-HSDII, 17-HSDIII, and 5 -reductase. Deficiencies have been described in each of these enzymes and have been associated with male pseudohermaphroditism (Martin et al., 2003; Miller, 2002; Sharpe, 2003; Yucel and Polat, 2003). Particularly in the rat, it has been determined that transplacental exposure to compounds that reduce testosterone synthesis and/or interfere with androgen signaling, alters epididymal development, frequently resulting in the absence of whole sections of this organ (McIntyre et al., 2002; Mylchreest et al., 2000). We have determined that transplacental exposure to EE, Ges, or BPA induced the down-regulation of StAR, Cyp17, Cyp11A, the scavenger receptor class B, and 7-dehydrocholesterol reductase. In all, these gene expression changes point towards a decrease in steroid synthesis. The scavenger receptor class B binds HDL and mediates the selective transfer of cholesteryl esters from HDL to cells, and may deliver HDL-cholesterol to the liver and to nonplacental steroidogenic tissues (Landschulz et al., 1996). In agreement with our data, in the liver of the rat, EE down-regulates the expression of scavenger receptor class B, probably by increasing the activity of the LDLR (Stangl et al., 2002). In various steroidogenic tissues (including gonads and adrenal cortex), StAR interacts with the outer mitochondrial membrane and facilitates the rate-limiting transfer of cholesterol to the inner mitochondrial membrane (Sandhoff et al., 1998; Sugawara et al., 1996) where Cyp11A1 converts this cholesterol into pregnenolone. Hormone-induced StAR expression is regulated through the cAMP-dependent pathway involving activation of protein kinase A (Tuckey et al., 2002). However the down-regulation on StAR expression induced by estrogen seems to be mediated indirectly by an AP-1 site in the StAR promoter (Manna et al., 2004) and not through an estrogen response element (ERE). However, little is known about the estrogen regulation of the other genes whose products are required for biosynthesis of androgens.

    Multiple genes responsive to estrogen exposure identified in the present study have not been previously identified and are not detectable in the female. In the list of genes up-regulated (Tables 2–5) and showing the most robust response are multiple ESTs, e.g., BF403853 [GenBank] , AI599419 [GenBank] , BI281680 [GenBank] , and BF417784 [GenBank] , which have no homology to characterized genes at the time of writing this article. Annotated genes include glutathione S-transferase Yc1, carboxypeptidase A1, phosphatidylethanolamine binding protein and seminal vesicle mRNA for SVS-protein F also known as SVS5 (Tables 2–5). These gene transcripts are undetectable in the fetal uterus/ovaries, even at the highest EE dose tested (10 μg/kg/day). Susceptibility to endocrine disruption during development could be different in females than in males, depending upon the specific-chemical exposure, and from a screening standpoint, the identification of a sex-specific estrogenic fingerprint may be important. The gender-specificity in the response to estrogen exposure can help to enhance our understanding of the mechanism of action of chemicals with estrogenic activity, by delineating the cellular pathways being affected preferentially in the male or in the female.

    As an example, one gene susceptible to estrogen regulation in a relative modest way is discussed: Dax1 gene, a nuclear receptor from the subfamily 0, group B, member 1 (Dax1/Nr0b1). The expression of DAX1 is down-regulated between 30 to 70% by microarray or QRT-PCR analysis, respectively (Table 9), when male fetuses are exposed to 10 μg EE/kg/day, and its mRNA is undetectable in female fetuses (Tables 8). The expression of this gene is also down-regulated in the fetal testis of rats transplacentally exposed to developmentally toxic phthalates (Liu et al., 2005). DAX-1 is a nuclear hormone receptor, which has been implicated in mammalian gonad development and sex determination (Liu et al., 2005; Meeks et al., 2003a). Thus, reduction of its expression during development may have an important physiological impact on testicular development and postnatal maturation. The expression of the gene in the gonad follows a dynamic pattern in time and place in the embryo and the adult. Mutations of the Dax1 nuclear receptor gene cause adrenal hypoplasia congenita, an X-linked disorder characterized by adrenal insufficiency and hypogonadotropic hypogonadism (reviewed in Meeks et al., 2003a). Targeted deletion of Dax1 in mice also reveals primary testicular dysgenesis, which is manifested by obstruction of the rete testis by Sertoli cells and hyperplastic Leydig cells, leading to seminiferous tubule dilation and degeneration of germ cells (Yu et al., 1998). Dax1 is expressed early in gonadal development. In Dax1(-/Y) male mice, the gonad develops normally until 12.5 dpc, however by 13.5 dpc the testis cords are disorganized and incompletely formed in Dax1-deficient mice (Meeks et al., 2003b). It has been suggested that Dax1 plays a crucial role in testis differentiation by regulating the development of peritubular myoid cells and the formation of intact testis cords. There are no reports in the literature of the estrogenic regulation of this gene, in this sense our findings are novel. Hoyle et al. (2002) have recently identified that Dax1 expression is dependent on steroidogenic factor 1 (SF-1) in the developing gonad. SF-1 (NR5A1) is an orphan nuclear receptor that is expressed in the adrenal gland, gonads, spleen, ventromedial hypothalamus and pituitary gonadotroph cells, and seems to be involved in the regulation the expression of different genes. However, Majdic et al. (1997) determined that transplacental exposure to DES or 4-octylphenol (OP), only at high concentrations (500 μg DES/kg or 600 mg OP/kg), decreased the expression of SF-1/Ad4BP in the fetal rat testis. In preliminary experiments, by RT-PCR analysis we have seen that transplacental exposure to EE also induced the down-regulation of SF-1. To better delineate this pathway, the effect of EE exposure on SF-1 expression has to be determined, since its expression is required for normal development of the gonads. Targeted disruption of SF-1 (FTZF1) in mice prevents gonadal and adrenal development and causes male-to-female sex reversal (revised by Ozisik et al., 2003). As we have discussed above, the SF-1 is a constitutive activator, and its activity is repressed by Dax-1 (NR0B1). Furthermore, Dax-1 directly inhibits the AR function. Recently, Holter et al. (2002) have provided evidence that indicates a direct interaction of the two receptors, Dax-1 and AR, that involve the N-terminal repeat domain of DAX-1 and the C-terminal ligand-binding and activation domain of AR. Clearly understanding the function of Dax-1 requires further investigation, since changes in its expression level has the potential to induce malformation of the male reproductive system. In the male rat, at 20 days of gestation, with the exception of retention of the areolae in the males, which points to an anti-androgenic effect, we could not determine any overt detrimental effects (malformations) as a result to EE, Ges, or BPA exposure during critical periods of the reproductive system development (GD 11 to GD 20; Fig. 1). However, the gene expression changes induced by EE, Ges, and BPA could account for the delayed phenotype (latency, postnatal development) and warrant further investigation. It is possible that the coincidental change in the expression of the genes identified in this study, and other still unidentified, is in fact a requirement for estrogen to elicit, directly or indirectly, a normal physiological response in the male and/or to induce pathological changes in the reproductive system of the male, dependent on the estrogen level.

    The results obtained from the present study demonstrate that transplacental exposure to a potent (EE), medium potency (Ges), or a weak (BPA) ER-agonist, at equipotent doses, changes the gene expression profile of estrogen-sensitive tissues of the male, but only at relatively high doses. There are some reports in the literature (Nagel et al., 1997; vom Saal et al., 1997) indicating that the effects of exposure to low doses of estrogens are qualitatively different than the effects produced after exposure to high doses. For example, vom Saal's laboratory has reported that prostate weights in mice were persistently increased by BPA or diethylstilbestrol at low doses but have opposite effects (DES) or no effect (BPA) at high doses, producing a non-monotonic dose-response curve resembling an inverted U-shape. Attempts to replicate these studies have failed to do so (Ashby et al., 1999; Cagen et al., 1999a,b), despite the fact that these later studies had greater statistical power.

    A panel of scientists convened by the U.S. National Toxicology Program to resolve the controversy was unable to definitively determine which sets of studies were valid, although it did conclude that the greater weight of evidence was on the side of the later studies that showed no effects at low doses (Melnick et al., 2002). The reason that the panel was unable to make definitive conclusions is that the endpoints being measured were variable in nature, and that small differences in any of a number of experimental variables, some of which are beyond the control of the investigator, could make enough of a difference to account for the disparate results. Given this conclusion, simply repeating the same studies with more animals will not resolve the controversy. Since gene expression is an integral part of the signal transduction pathway of estrogens, and can be measured with great sensitivity, we investigated the possibility that changes in gene expression would provide a more reliable basis for determining the nature of the dose-response curve at low and high levels of exposure during fetal development of the male. Although the response of the rat testis could entail some differences, compared with the response determined in the mouse prostate, the results presented in this study do not support the hypothesis that the shape of the dose-response curve, particularly at low doses, for the effects of chemicals with estrogenic activity on male reproductive system development, is different than at high doses. Our results indicate that the dose-response curve for gene expression changes is monotonic for EE, Ges, or BPA, with both the number of genes significantly changed and the magnitude of change, for each gene, decreasing with decreasing dose. Thus, for the three ER-agonist here tested it can be concluded that the exposure to low doses do not elicit a robust quantifiable response at the gene expression level. In support of our results, during the re-evaluation of the uterotrophic activity of BPA in the immature rat (over the dose range 2 μg/kg-800 mg/kg/day), Ashby and Odym (2004) determined that BPA induces an uterotrophic response only in the dose range of 200–800 mg/kg BPA. These authors determined that over the dose range of 2 μg/kg–20 mg/kg BPA there was no uterotrophic response and no increase in the expression of three estrogen responsive uterine genes: complement component 3, lipocalin 2, and progesterone receptor (determined using real-time RT-PCR). Further, the down-regulation of the expression of Cyp17a1, StAR, and Cyp11a has been also observed in the testis of the mouse, after gestational and lactational exposure to diethylstilbestrol (0, 0.1, 1, and 10 μg/kg maternal body weight; Fielden et al., 2002). Changes in the expression of progesterone receptor, Cyp17a1, StAR, and Cyp11a, among other genes, is only induced by relatively high doses of the three ER-agonist here tested, and these data does not support the hypothesis of a different genomic response after exposure to low doses of ER-agonists.

    Gene expression changes are elicited by transplacental exposure to EE, Ges, or BPA, at doses where there appears to be no discernable tissue changes. That would argue against the NOTEL being the same as the NOEL. The concept of NOTEL, or No Observed Transcriptional Effect Level, has been put forward to demonstrate the utility of gene expression profiling as a means to identify concentrations that do not elicit a change in gene expression (Lobenhofer et al., 2004). Our data, particularly in relationship to the low dose effect, will still support this concept. We identify gene expression changes induced by ER agonists only after exposure to relatively medium to high dosages, but not at low doses. The lack of significant gene expression changes determined in the fetal testis/epididymis after exposure to relatively low doses of any of the three ER-agonists here reported, supports the concept of NOTEL. This concept can be used to establish gene expression changes that can be used as biomarkers of exposure or effect.

    We conclude that prenatal exposure to chemicals with estrogenic activity alters the fetal gene expression pattern of the rat reproductive system both in the male (testis/epididymis) as well as in the female (uterus/ovaries), resulting in a characteristic and gender-specific molecular fingerprint. These results indicate that gene expression data are diagnostic of mode of action at doses below a threshold for morphologically detectable effects. The functional characterization of the genes whose expression is modified by ER-agonist will help to understand the physiological and pathophysiological changes induced by exposure to chemicals with estrogenic activity during development. These transcripts, or a subset, could be valuable markers of estrogen exposure in the male, or the female, since changes in their expression can be elicited by chemicals with various potencies as estrogen receptor agonist. The same genomic response is predicted after equivalent exposure to chemicals that inhibit estrogen metabolic enzymes that bring forth higher levels of endogenous estrogen (-estradiol), such as inhibitors of estrogen sulfotransferase (Kester et al., 2000; Qian et al., 2001). However, before we can pinpoint biomarkers of estrogen exposure during development of the male, the molecular fingerprint we have identified has to be validated.

    SUPPLEMENTARY DATA

    Supplementary tables containing all the Affymetrix Probe IDs, together with the listing of the GenBank accession number, gene title, and gene symbol associated with each probe set represented in the U34A (Table 11) and 230A (Table 12) microarrays are available online at www.toxsci.oupjournals.org.

    ACKNOWLEDGMENTS

    The authors wish to thank Drs. William Owens and Tyra Leazer for their helpful discussions. Conflict of interest: none declared.

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