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Endocrinology, doi:10.1210/en.2008-0651
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*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*4,4'-BISPHENOL A
*BISPHENOL A DISODIUM SALT
*DIETHYLSTILBESTROL
Endocrinology Vol. 149, No. 11 5848-5860
Copyright © 2008 by The Endocrine Society

Developmental Exposure to Bisphenol A Impairs the Uterine Response to Ovarian Steroids in the Adult

Jorgelina Varayoud, Jorge G. Ramos, Verónica L. Bosquiazzo, Mónica Muñoz-de-Toro and Enrique H. Luque

Laboratorio de Endocrinología y Tumores Hormonodependientes, School of Biochemistry and Biological Sciences, Universidad Nacional del Litoral, 3000 Santa Fe, Argentina

Address all correspondence and requests for reprints to: Enrique H. Luque, Ph.D., Laboratorio de Endocrinología y Tumores Hormonodependientes, School of Biochemistry and Biological Sciences, Casilla de Correo 242, 3000 Santa Fe, Argentina. E-mail: eluque{at}fbcb.unl.edu.ar.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Morphoregulator genes like members of the Hox gene family regulate uterine development and are associated with endocrine-related processes such as endometrial proliferation and differentiation in the adult uterus. Exposure to neonatal endocrine disruptors could affect signaling events governed by Hox genes, altering the developmental trajectory of the uterus with lasting consequences. We investigated whether neonatal exposure to bisphenol A (BPA) alters Hoxa10 and Hoxa11 mRNA uterine expression shortly after treatment as well as in the adult. Moreover, we studied whether xenoestrogen exposure may affect the adult uterine response to hormonal stimuli. Newborn females received vehicle, 0.05 mg/kg·d BPA, 20 mg/kgbulletd BPA, or diethylstilbestrol (0.2 µg/kgbulletd) on postnatal d 1, 3, 5, and 7). At postnatal d 8, real time RT-PCR assays showed a decrease in Hoxa10 and Hoxa11 expression in all xenoestrogen-treated groups. To evaluate the long-term effects, we used adult ovariectomized rats with hormonal replacement. The subepithelial stroma in BPA- and diethylstilbestrol-treated animals showed an impaired proliferative response to steroid treatment associated with a silencing of Hoxa10 but not associated with changes in the methylation pattern of the Hoxa10 promoter. BPA animals showed that the Hoxa10 reduction was accompanied by an increased stromal expression of the silencing mediator for retinoic acid and thyroid hormone receptor. The spatial coexpression of steroid receptors Hoxa10 and silencing mediator for retinoic acid and thyroid hormone receptor was established using immunofluorescence. Our data indicate that postnatal BPA exposure affects the steroid hormone-responsiveness of uterine stroma in adulthood. Whether this impaired hormonal response is associated with effects on uterine receptivity and decidualization is currently under investigation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PERINATAL UTERINE development is a critical period in development because exposure to endocrine disruptors can have long-term negative consequences for adult uterine function and reproductive health (1). Abnormal gene imprinting and impairment of steroid receptor-mediated responses are proposed as mechanisms underlying reproductive defects (2, 3).

From the 1940s to the 1970s, the xenoestrogen diethylstilbestrol (DES) was extensively prescribed to pregnant women to prevent miscarriage. Women exposed to DES in utero (DES daughters) exhibit genital tract abnormalities later in life (4). Perinatal exposure of laboratory rodents to DES generates a spectrum of reproductive tract lesions similar to those observed in humans (5). Using this model, many genes have been identified as critical components of the mechanism that causes DES-induced abnormalities. The discovery of an altered methylation pattern of estrogen-regulated gene promoters provides support for an epigenetic pathway by which DES permanently alters gene expression (6).

Hox genes are a family of transcription factors that are essential both for development of the reproductive tract during the embryonic period (organogenic differentiation) and adult function (functional differentiation) (7, 8). Two members of the Hox multigene family homeobox A, Hoxa10 and Hoxa11, are essential for female fertility (9). Gene expression profiling experiments reveal that Hoxa10 is an important regulator of implantation-associated events, such as uterine stromal cell proliferation and local immunosuppression. Hoxa10 is highly expressed in the proliferating and differentiated uterine stroma during the periimplantation period and progesterone (P) is the primary inducer of Hoxa10 in this tissue (10). Further investigations have revealed that the Hoxa11 gene is essential in female reproductive tract differentiation because Hoxa11 knockout animals have shown abnormal stromal and glandular cell differentiation during pregnancy (11).

Bisphenol A (BPA) is an endocrine disruptor chemical used in the manufacture of polycarbonate plastics and epoxy resins, and thus, it is present in a myriad of products: the interior coating of tins, milk containers, baby formula bottles, dental materials, etc. Recent data have shown widespread exposure to BPA among the U.S. population. In 2517 participants 6 yr of age or older in the 2003–2004 National Health and Nutrition Examination Survey, more than 92% of urine samples had detectable concentrations of BPA (12).

The route of BPA administration and the evaluated doses are important issues to determine BPA health risks in animal models (13). In fetuses and neonates, the low expression of the enzyme that conjugates BPA (uridine diphosphate-glucuronosyltransferase) implies that oral and nonoral administration of BPA during neonatal life give the same internal active dose (14). The Society of Plastic Industry and the U.S. Environmental Protection Agency have recommended using the current accepted lowest observed adverse effect level dose (50 mg/kg·d) of BPA to calculate a maximum acceptable or reference dose to be 0.05 mg/kg·d (15). In the present study, we examined the effects of two different doses of BPA, one of which was identical with the reference dose (0.05 mg/kg·d) and the other of which was 400-fold higher (20 mg/kg·d), although 2.5-fold lower than the declared lowest observed adverse effect level. Because low doses of classical estrogens are recommended to compare the effects of weak xenoestrogenic compounds (16), a low dose of the synthetic estrogen DES (0.2 µg/kg·d) was used as an endocrine disruptor control.

Different in vivo studies in laboratory rodents and wild life have established that BPA can induce alterations in reproductive functions (17, 18, 19, 20). We evaluated the reproductive effects of perinatal BPA exposure showing abnormal development of endocrine-related tissues in laboratory rodents (21, 22, 23, 24, 25, 26) and sex-reversal effects or altered ovarian development in the wild crocodile Caiman latirostris (27, 28).

Little is known regarding the effects of neonatal BPA exposure on developmental programming of the rat uterus. We specifically examined whether early postnatal exposure to BPA alters Hoxa10 and Hoxa11 mRNA uterine expression shortly after treatment. Next, we examined whether neonatal BPA exposure could adversely affect the uterine response to hormonal stimuli in adult rats. To eliminate variability in hormonal cycling between animals, we used ovariectomized (OVX) rats with exogenous ovarian steroid hormone treatment. If an adult OVX rat is primed with P treatment for 2 d before the administration of a single dose of 17β-estradiol (E), a significant proliferative response is detected in stromal cells but not the epithelium. This model provides a valuable paradigm to mimic the hormonal milieu of the rodent preimplantation period and to specifically evaluate the proliferative response of the subepithelial stromal compartment (29). During this period, P and E, acting primarily through their nuclear receptors, activate the transcription and translation of genes involved in uterine receptivity and decidualization, such as Hoxa10 and Hoxa11 (30).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All procedures were performed in accordance with the principles and procedures outlined in the Guide for the Care and Use of Laboratory Animals issued by the U.S. National Academy of Sciences. Rats of an inbred Wistar-derived strain bred at the Department of Human Physiology (Universidad Nacional del Litoral, Santa Fe, Argentina) were maintained under a controlled environment (22 ± 2 C; lights on from 0600 to 2000 h) and had free access to pellet laboratory chow (Nutrición Animal, Santa Fe, Argentina) and tap water. The concentration of phytoestrogens in the diet was not evaluated; however, because food intake was equivalent for control and experimental rats (our unpublished observations), we assumed that all animals were exposed to the same levels of food-borne phytoestrogens. To minimize additional exposures to endocrine-disrupting chemicals, rats were housed in stainless steel cages with wood bedding and tap water was supplied ad libitum in glass bottles with rubber stoppers surrounded by a steel ring.

Experimental design
Figure 1Go summarizes the experiment conducted. Pregnant rats were housed singly. Upon delivery pups were sexed according to anogenital distance and cross-fostering, distributing the female pups of each litter among different mothers (adjusting the number of pups to five females and five males whenever possible). These actions allowed us to minimize the use of siblings to avoid potential litter effects. Female pups from each foster mother were assigned to one of the following neonatal treatment (28–33 pups per group): 1) controls given corn oil vehicle alone, 2) DES (Sigma, St. Louis, MO) at 0.2 µg/kg·d, 3) BPA (99% purity; Aldrich, Milwaukee, WI) at 0.05 mg/kg·d (BPA.05), or 4) 20 mg/kg·d (BPA20). Treatments were given on postnatal days (PND) 1, 3, 5, and 7 (day of birth = 0) by sc injections in the nape of the neck. Male pups were used in another experiment. No signs of acute or chronic toxicity were observed, and no significant differences in weight gain between xenoestrogen-exposed and control pups were recorded during the experiment. For prepubertal studies, rats were killed on PND8, 24 h after the final injection (n = 20–25 pups/group), and the uterus was removed quickly, snap frozen, and stored at –80 C. Another set of female pups (eight rats/group) were weaned at 21 d of age and housed four per cage until 80 d of age (see Fig. 1Go). Female rats exposed to xenoestrogens did not exhibit advanced puberty, measured as early vaginal opening, compared with controls (data not shown).


Figure 1
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  		FIG. 1. Schematic representation of experimental protocol. IHQ-IF, Immunohistochemistry-immunofluorescence; X, time the animals were killed.

 
Ovariectomy and hormonal treatment
To evaluate the long-term effects of neonatal xenoestrogen exposure, we used a model of adult OVX rats with hormonal replacement treatment. In this model, P pretreatment followed by a single dose of E increases the number of proliferating uterine stromal cells. Previously this hormonal protocol has been exploited in an attempt to clarify the mechanism by which P exerts this effect (29). First, we performed a pilot experiment using adult rats that were OVX. Ten days after OVX, they were treated (sc) as follows: two daily doses of P (10 mg/kg; Sigma), followed by single doses of both P and E (4 µg/kg; Sigma) on the third day (pilot OVX-P+E group; n = 5). Another set of rats received daily doses of vehicle (pilot OVX group; n = 5). Sixteen hours after the last treatment dose, each rat was injected (ip) with the thymidine analog bromodeoxyuridine (BrdU; 60 mg/kg; Sigma). The set of rats neonatally exposed to corn oil, DES, and BPA (BPA.05 and BPA20), that had been housed until 80 d of age were OVX and treated with the above-described steroid replacement treatment (n = 8 rats/group). Uterine tissues were harvested 4 h after the BrdU injection. One uterine horn from each rat was placed immediately in liquid nitrogen and stored at –80 C for RNA or DNA extraction. The other uterine horn was weighed and then fixed by immersion in 10% buffered formalin for 6 h at 4 C, embedded in paraffin, and used for immunohistochemical and immunofluorescence staining.

RT and real-time quantitative PCR analysis (QPCR)
An optimized RT-PCR protocol was used to analyze the relative expression levels of Hoxa10 and Hoxa11 mRNA in PND8 rats and in adult animals assigned to the model of OVX + steroid treatment (OVX-P+E groups). Relative quantification allows for the evaluation of changes in the relative expression of Hox genes normalized to an invariant control gene (ribosomal protein L19). Two procedures were used for RNA extraction depending on tissue volume. Five pools of PND8 rat uterus (n = 4–5 uteri/pool) or eight individual uterine horn samples from adult animals were homogenized in TRIzol (Invitrogen, Carlsbad, CA), and RNA was prepared according to the manufacturer’s protocol. The concentration of total RNA was assessed by A260, and RNA was stored at –80 C until needed. Equal quantities (4 µg) of total RNA were reverse transcribed into cDNA with avian myeloma virus reverse transcriptase (12.5 U; Promega, Madison, WI) using 200 pmol of random primers (Promega). Twenty units of ribonuclease inhibitor (RNAout; Invitrogen Argentina, Buenos Aires, Argentina) and 100 nmol of a deoxynucleotide triphosphate (dNTP) mixture were added to each reaction tube in a final volume of 30 µl of 1x avian myeloma virus-reverse transcriptase buffer. Reverse transcription was performed at 42 C for 90 min. Reactions were terminated by heating at 97 C for 5 min and cooling on ice, followed by dilution of the reverse-transcribed cDNA with ribonuclease-free water to a final volume of 60 µl. Samples were analyzed in triplicate, and a sample without reverse transcriptase was included to detect contamination by genomic DNA. Primer pairs used for amplification of Hoxa10, Hoxa11, and the ribosomal protein L19 (housekeeping gene) cDNAs are shown in Table 1Go (accession no. XM_347220, XM_575479, NM_031103, respectively). cDNA levels were detected using QPCR with the DNA Engine Opticon system (Bio-Rad Laboratories, Inc., Waltham, MA) and SYBR Green I dye (Cambrex Corp., East Rutherford, NJ).


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  		TABLE 1. Primers used for real-time quantitative PCR

 
For cDNA amplification, 5 µl of cDNA were combined with a mixture containing 2.5 U Taq-DNA polymerase (Invitrogen), 2 mM MgCl2 (Invitrogen), 0.2 mM of each of the four dNTPs (Promega), and 10 pmol of each primer (Invitrogen) in a final volume of 25 µl of 1x SYBR Green I PCR Taq buffer. After initial denaturation at 97 C for 1 min, the reaction mixture was subjected to successive cycles of denaturation at 97 C for 45 sec, annealing at 57 C (Hoxa10 and Hoxa11) and 55 C (L19) for 1 min, and extension at 72 C for 1 min. Product purity was confirmed by dissociation curves and random samples were subjected to agarose gel electrophoresis. No DNA template controls were included in any of the assays, yielding no consistent amplification. All PCR products were cloned using a TA cloning kit (Invitrogen Argentina), and specificity was confirmed by DNA sequencing (data not shown). Calculation of the relative expression levels of each target was conducted based on the cycle threshold (CT) method (31). The CT for each sample was calculated using the Opticon monitor analysis software (MJ Research, Waltham, MA) with an automatic fluorescence threshold setting. The efficiency of PCRs was assessed for each target by amplification of serial dilutions (more than 5 orders of magnitude) of cDNA fragments of the transcripts under analysis. Depending on specific PCR conditions, efficiencies ranged from 90 to 120%. Accordingly, the fold expression over control values was calculated for each target by the equation 2–{Delta}{Delta}CT, where {Delta}CT is determined by subtracting the corresponding L19 CT value (internal control) from the specific CT of each target and experimental condition, and {Delta}{Delta}CT is obtained by subtracting the {Delta}CT of each experimental group from that of the control group (taken as reference value 100). No significant differences in CT values were observed for L19 between the different experimental groups.

Antibodies
To evaluate steroid receptor coregulator expression, we used affinity-purified rabbit polyclonal antibodies generated and tested in our laboratory using previously described protocols (32). The antigens were expressed in Escherichia coli JM109 (Stratagene Corp., La Jolla, CA) as glutathione-S-transferase fusion proteins using a pGEX4T-3 vector (Stratagene). The steroid receptor coactivator (SRC)-3 antigen included the region corresponding to amino acids 581–650 of the rat sequence (accession no. XP_215947) and the silencing mediator for retinoic acid and thyroid hormone receptor (SMRT) antigen was composed of amino acids 1946–1995 (accession no. XP_341073). The specificity of the antiserum was tested via Western blot as previously described (32). For immunohistochemical analysis, antibodies were purified using antigen linked affinity chromatography (Hi-Trap NHS activated HP column; GE Healthcare, Buenos Aires, Argentina) and were used at a 1:50 dilution for anti-SMRT and a 1:150 dilution for anti-SRC-3. For specificity validation tests, the antigenic peptides for both coregulatory factors were used to preadsorb SRC-3 and SMRT antibodies by incubating 1 µg antibody with 10–20 µg peptide for 24 h at 4 C. The antibody-antigen complexes were applied in immunohistochemical assays to positive control tissues.

The affinity-purified antibodies for estrogen receptor (ER)-{alpha} (clone 6F-11, 1:200 dilution) and BrdU (clone 85-2C8, 1:100 dilution) were purchased from Novocastra (Newcastle upon Tyne, UK). The anti-Hoxa10 antibody (sc-17159, 1:50 dilution) was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The antibody for progesterone receptor (PR) (A0098, 1:500 dilution) was purchased from Dako Corp. (Carpinteria, CA). Antirabbit and antimouse secondary antibodies (biotin conjugate, B8895/B8774, 1:200 dilution) were purchased from Sigma; antigoat secondary antibody (biotin conjugate, sc-2042, 1:200 dilution) was purchased from Santa Cruz Biotechnology. For dual-immunofluorescence staining, the secondary antibodies were AlexaFluor 488 goat antirabbit (green) (A-11034, 1:100 dilution; Invitrogen), Cy2-conjugated goat antimouse (155-225-003, green, 1:100 dilution), and tetramethylrhodamine isothiocyanate-conjugated antirabbit (016-020-084, red, 1:200 dilution; Jackson ImmunoResearch, West Grove, PA).

Immunohistochemistry
Uterine sections (5 µm in thickness) were deparaffinized and dehydrated in graded ethanols. BrdU incorporation to detect cells in the S phase of the cell cycle was evaluated as previously described (33). In brief, an acid hydrolysis for DNA denaturation and microwave pretreatment for antigen retrieval were performed as described previously according to routine immunohistochemical procedures (34). Endogenous peroxidase activity and nonspecific binding sites were blocked. Primary antibodies were incubated overnight at 4 C. After incubation with biotin-conjugated secondary antibodies for 1 h, the reactions were developed using a streptavidin-biotin peroxidase method and diaminobenzidine (Sigma) as a chromogen substrate. Samples were counterstained with Harris or Mayer hematoxylin (Biopur, Rosario, Argentina) and mounted with permanent mounting medium (PMyR, Buenos Aires, Argentina). Each immunohistochemical run included negative controls. Negative controls replacing the primary antibody with nonimmune goat serum (Sigma) were performed for each immunohistochemical staining. In addition, for BrdU immunodetection we performed the technique in samples from animals that did not receive BrdU.

Quantification of cell proliferation and protein expression by image analysis
Tissue sections were evaluated using an BH2 microscope (illumination: 12-V halogen lamp, 100 W, equipped with a stabilized light source; Olympus, Tokyo, Japan) with the Dplan x40 objective (numerical aperture = 0.65; Olympus). The proliferation index was obtained using a point counting procedure as previously described (35). To obtain the data, a glass disk with a square grid was inserted into a focusing eyepiece of the microscope and the volume fractions (Vv) of the BrdU(+) subepithelial stromal cells were calculated by applying the method described by Weibel (36).

To measure the integrated OD (IOD) of PR, ER{alpha}, SMRT, and SRC-3 immunostaining in the subepithelial stromal cells, image analysis was performed using the Image Pro-Plus 4.1.0.1 system (Media Cybernetics, Silver Spring, MD), as previously described (23, 24, 25, 35). In brief, the images were recorded with a Spot Insight version 3.5 color video camera, attached to an Olympus BH2 microscope, using a Dplan x40 objective (at least 10 fields were recorded in each section and two sections per animal were evaluated). The microscope was set up properly for Koehler illumination. Correction of unequal illumination (shading correction) and the calibration of the measurement system were done with a reference slide. The images of immunostained slides were converted to gray scale, and the subepithelial stromal compartment was delimited (a 300 µm wide area adjacent to the epithelium, from the basement membrane toward the outer layers). Using the Auto-Pro macro language (Media Cybernetics) an automated standard sequence operation was created to measure the IOD as a linear combination between the average gray intensity and the relative area occupied by positive cells. Because IOD is a dimensionless parameter, the results were expressed as arbitrary units.

Dual-immunofluorescent staining
Uterine sections were deparaffinized, rehydrated, and submitted to microwave antigen retrieval. To minimize nonspecific background, sections were blocked for 1 h with normal donkey serum (Hoxa10) or goat serum (PR, ER{alpha}, SMRT; Sigma). The incubation with primary antibodies was performed overnight at 4 C. The secondary antibodies and rhodamine (tetramethylrhodamine isothiocyanate) (red)-conjugated streptavidin (1:150 dilution Jackson ImmunoResearch Laboratories) were incubated for 1 h, and then sections were washed for a total of 45 min with three baths of PBS. Finally, all sections were washed in PBS and mounted with Vectashield fluorescent mounting medium (Vector Laboratories, Inc., Burlingame, CA) with 4',6-diamidino-2-phenylindole dihydrochloride (Fluka; Sigma) and stored in the dark at 4 C. Controls included uterine sections incubated using primary antibody buffer solution (3% BSA, 0.1% Tween 20 in PBS) in place of the primary antibody to control for nonspecfic staining. All immunostained sections were examined using an Olympus BX-51 microscope equipped for epifluorescence detection and with the appropriate filters (Olympus). Images were recorded using a high-resolution USB 2.0 digital color camera (QImaging Go-3; QImaging, Surrey, British Columbia, Canada).

DNA extraction and bisulfite modification
DNA was isolated from the uterine horns of rats neonatally exposed to xenoestrogen and submitted to the model of OVX + hormone steroid treatment (OVX-P+E groups) by using the Wizard genomic DNA purification kit (Promega). DNA (2 µg) in a volume of 50 µl was denatured with NaOH for 20 min at 37 C. Thirty microliters of 10 mM hydroquinone (Sigma) and 520 µl of 3 M sodium bisulfite (Sigma) (pH 5), freshly prepared, were added and mixed, and samples were incubated in mineral oil at 55 C for 16 h (37). Modified DNA was purified using Wizard DNA purification resin according to the manufacturer (Promega) and eluted into 50 µl of heated water (60–70 C). Modification was completed by NaOH treatment for 15 min at 37 C, followed by ethanol precipitation. DNA was resuspended in water and used immediately or stored at –20 C.

Real-time quantitative methylation-specific PCR (QMSP)
Using the MethPrimer software (http://www.urogene.org/methprimer) (38), we identified a CpG-rich fragment in the 5'upstream region of exon 1 of the rat Hoxa10 gene. This CpG island was recognized in the region corresponding to –279 and –58 bp, considering +1 the translation start site (accession no. NW_047692). After sodium bisulfite conversion, genomic DNA was analyzed using the Engine Opticon system (Bio-Rad) and SYBR Green I dye (Cambrex) as described previously with some modifications (39). Briefly, two sets of primers designed specifically for bisulfite-converted DNA, were used: 1) one primer set (M) that anneals to the methylated sequence of the Hoxa10 promoter region: (sense: 5'-GTTTTTGGGTTATAGGTGTTAGGC-3', antisense: 5'-ACTCCCAATTTAATTTCGTAAACG-3' and 2) a reference set for the L19 ribosomal protein gene that lacks any CpG dinucleotides (sense 5'-TGTGGTTGGATTTTAATGAAATTAA-3', antisense 5'-AACAATACCCTTCCTCTTCCCTATA-3'). This set allows for equal amplification, regardless of the methylation state of the bisulfite-converted DNA and provides an index of the level of modified DNA in each sample (sample normalization). The PCR mixture contained 4 mM MgCl2 (Invitrogen), 0.2 mM of each of the four dNTPs (Promega), 20 pmol of each primer (Invitrogen), and bisulfite-modified DNA (1 µl) in a final volume of 25 µl of 1x SYBR Green PCR buffer. Reactions were hot started at 95 C for 5 min before the addition of 2.5 U Taq-DNA polymerase (Invitrogen). Amplification was carried out for 35 cycles (15 sec at 95 C, 15 sec at 57 C, 15 sec at 72 C). Specifically for methylation-specific RT-PCR, the fluorescence measurements were performed at 75 C because of the low-melting temperature of the PCR products. The reactions were run in triplicate for each individual sample.

The specificity of the reactions for methylated DNA was confirmed separately using genomic rat DNA extracted from the hippocampus (in this sample, the Hoxa promoter is completely unmethylated) and SssI (New England Biolabs, Beverly, MA)-treated genomic rat DNA (in this sample, the Hoxa promoter is 100% methylated). No amplification products were obtained when the unmethylated DNA was amplified with the M primer set. Standard curves were generated by amplifying (with the M set of primers) serial dilutions of the in vitro methylated DNA with increasing quantities of unmethylated DNA (the final concentration of the total DNA, methylated + unmethylated, was exactly the same in each reaction tube). The calibration curves were used to quantify the relative degree of methylation of each experimental DNA sample expressed as the ratio of the level of methylated DNA to the level of modified DNA from the L19 quantification.

Statistics
All data were calculated as the mean ± SEM. We have performed a Kruskal-Wallis analysis to assess the overall significance (testing the hypothesis that the response was not homogeneous across treatments), and the Dunn post hoc test was used to compare each experimental group with the control group. P < 0.05 was accepted as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BPA and DES altered Hoxa10 and Hoxa11 mRNA levels in prepubertal rats
The acute effects of neonatal BPA or DES exposure on Hoxa10 and Hoxa11 expression were assessed via analysis of mRNA levels in uterine tissue collected 24 h after the last xenoestrogen injection (PND8). As shown in Fig. 2Go, xenoestrogen exposure induced changes in the uterine levels of mRNAs encoding Hoxa10 and Hoxa11, compared with control rats. A significant reduction of Hoxa10 and Hoxa11 mRNA expression on PND8 (P < 0.001), was observed in animals exposed to BPA or DES.


Figure 2
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  		FIG. 2. Levels of mRNAs encoding for Hoxa10 (A) and Hoxa11 (B) in the uterus of prepubertal (8 d old) female rats exposed during neonatal life to corn oil (control), DES, BPA.05 (0.05 mg/kg·d), or BPA20 (20 mg/kg·d). Relative mRNA levels were measured via real-time QPCR and fold expression from control values were calculated for each target by the equation 2–{Delta}{Delta}CT. Control values were assigned to a reference level of 100 and values are given as mean ± SEM. A total of five pools of PND8 rat uterus (4–5 uteri/pool) was assayed in triplicate. Ribosomal protein L19 was used as internal control. *, P < 0.001 vs. control.

 
Long-lasting effects of BPA or DES on uterine response to ovarian steroid hormones
Next, we studied whether neonatal BPA or DES exposure affects the long-term uterine response to hormonal steroid stimuli in adult rats. We used an OVX model (denoted OVX-P+E), in which the main endogenous P and E source was absent. Results from the pilot study performed to characterize the model of OVX in rats revealed that animals treated with P+E (pilot OVX-P+E group) exhibited: 1) a dramatic increase in BrdU incorporation specifically in subepithelial stromal cells (Fig. 3AGo), 2) up-regulation of PR and ER{alpha} expression in the same cellular compartment (Fig. 3Go, B and C), 3) induction of Hoxa10 and Hoxa11 mRNA (Fig. 3Go, D and E) compared with OVX adult rats injected with vehicle (pilot OVX group), 4) a clear induction of Hoxa10 protein localized in subepithelial stromal cells (Fig. 3Go, F vs. G). These results allowed us to determine the subepithelial stromal response to steroid hormones in the model of OVX adult rats.


Figure 3
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  		FIG. 3. Uterine events mediated by ovarian steroids evaluated in an OVX adult rat model in response to hormone replacement. Stromal proliferative activity (A) was expressed as volume fractions (Vv x 100). PR (B), and ER{alpha} (C) stromal immunostaining were expressed as IOD that consists of a linear combination between the intensity and the relative area occupied by positive cells. Because the IOD is a dimensionless parameter, the results were expressed as arbitrary units. Hoxa10 (D) and Hoxa11 (E) mRNA levels are expressed as 2-{Delta}{Delta}CT. Asterisks denote P < 0.001. Photomicrographs show Hoxa10 protein induction with the P+E regimen in the uterine subepithelial stroma. Pilot OVX (F) and pilot OVX-P+E (G) are shown. Original magnification, x600.

 
Stromal cell proliferation.
To test whether stromal cell proliferation was disrupted after neonatal exposure to xenoestrogen, a set of animals was subjected to the OVX-P+E protocol, injected with BrdU, and killed 4 h later. A noteworthy observation was that after P+E treatment, the BrdU incorporation in the subepithelial stromal cells (expressed as Vv x 100) in neonatal controls (oil exposed rats) was 28.04 ± 2.36, whereas BrdU incorporation in the BPA.05, BPA20, and DES-exposed animals was 12.56 ± 1.04, 11.14 ± 2.59, and 17.87 ± 4.77, respectively (Figs. 4AGo and 5Go, A–D). The proportion of subepithelial stromal cells that incorporated BrdU in animals treated with DES and both doses of BPA was significantly lower than control animals (P < 0.001). The decrease in stromal cell proliferation was not associated with changes either in gross morphological features or uterine weight.


Figure 4
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  		FIG. 4. Long-term effects of neonatal exposure to BPA or DES on uterine stroma ovarian steroid-mediated events evaluated in a model of adult OVX rats in response to hormone replacement. Rate of proliferation (A), PR (B), and ER{alpha} (C) expression in the subepithelial stromal compartment is shown. Control group, OVX-P+E. Asterisk, P < 0.05 (n = 8/group).

 

Figure 5
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  		FIG. 5. Effect of neonatal exposure to DES or BPA on proliferative activity and steroid receptor expression in the uterus of OVX rats after P+E replacement. Representative uterine tissue section samples immunostained for BrdU, PR, and ER{alpha}. BrdU incorporation (A vs. B–D) was clearly affected in DES-, BPA20-, and BPA.05-exposed rats. PR (E vs. F) shows a down-regulation only in DES animals. In addition, DES and BPA.05 but not BPA20 rats failed to up-regulate ER{alpha} in response to P+E (I vs. J and L). GE, Glandular epithelium; LE, luminal epithelium; ST, subepithelial stroma. Original magnification, x400.

 
PR and ER{alpha} expression.
Both isoforms of PR (PRA and PRB) and ER{alpha} were identified by immunohistochemistry and quantified by image analysis to determine the IOD of both receptors in the uterine subepithelial stroma. A high expression of PR and ER{alpha} in stromal cells was observed in controls (neonatally exposed to oil and OVX-P+E). In rats neonatally exposed to DES, the long-term response to ovarian steroids was impaired, showing a lower expression of both steroid receptors in the subepithelial stromal cells relative to control rats (P < 0.05, Fig. 4Go, B and C). There were no statistically significant differences in PR expression between BPA-exposed animals (BPA20 and BPA.05) and controls (Fig. 4BGo). However, BPA.05 but not BPA20 rats failed to up-regulate ER{alpha} in response to P+E (Fig. 4CGo). Photomicrographs illustrating these results are shown in Fig. 5Go, E–L.

Homeotic genes.
To evaluate whether the impaired proliferative response to P+E could be associated with changes in Hoxa10 and/or Hoxa11 expression, we evaluated the uterine expression of both homeotic genes by QPCR in OVX hormone-treated adult rats neonatally exposed to xenoestrogens. Notably, the ovarian steroid induction of Hoxa10 mRNA expression was significantly attenuated in neonatally BPA.05-, BPA20-, and DES-exposed rats compared with controls exposed to corn oil (P < 0.001, see Fig. 6AGo). Regarding Hoxa11 mRNA, rats neonatally exposed to BPA20 or DES showed no differences compared with controls, whereas in the low dose of BPA.05, a significant down-regulation was observed (P < 0.05, Fig. 6BGo).


Figure 6
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  		FIG. 6. Long-term effects of neonatal exposure to BPA or DES on levels of mRNA encoding for Hoxa10 (A) and Hoxa11 (B) in the uterus of adult OVX rats in response to steroid hormone replacement. Values were normalized with L19 and expressed as 2-{Delta}{Delta}CT (n = 8 rats/group). *, P < 0.05.

 
The methylation status of the Hoxa10 promoter was not affected after neonatal exposure to xenoestrogens
Cytosine methylation of CG dinucleotides is a stable marker on DNA associated with decreased gene expression and has been proposed as a possible mechanism underlying the xenoestrogen effects on gene expression. We examined the methylation status of CpG sites across the Hoxa10 promoter using real-time QMSP assays on DNA extracted from uterine tissue of animals neonatally exposed to xenoestrogen and included in the OVX model. Partial methylation of the Hoxa10 promoter region was observed in all DNA samples, without detecting differences between experimental groups (Fig. 7Go).


Figure 7
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  		FIG. 7. Methylation status of the Hoxa10 promoter. A, The CpG island indicated with lollipop signs and consists of a region corresponding to –279 and –58 bp, considering +1 the translation start site (accession no. NW_047692). B, Quantitative evaluation of Hoxa10 methylation by QMSP. The results are expressed as relative levels of Hoxa10 methylation. Control values were assigned to a reference level of 1 and values are given as mean ± SEM (n = 8 rats/group).

 
Expression of steroid receptor coregulators
To obtain more information regarding the P and E pathways, we analyzed the expression of two nuclear receptor coregulators. SRC-3 and SMRT were chosen as representative members of coactivator and corepressor families respectively, and their expression was evaluated by immunohistochemistry and quantitative image analysis. Characterization of antiserum against SMRT and SRC-3 is shown in Fig. 8Go. In Western blot assays of rat uterus, specific bands of 160 and 150 kDa were detected using anti-SRC-3 and anti-SMRT, respectively (Fig. 8AGo). The spatial pattern of expression of both proteins in the uterine tissue was defined by immunohistochemistry. In control conditions, SRC-3 was primarily found in the nuclei of subepithelial stromal cells (Fig. 8BGo), whereas SMRT showed a weak nuclear and cytoplasmic (perinuclear) expression (Fig. 8CGo). Even though the distribution pattern of SMRT in the uterine subepithelial stroma was not affected by neonatal xenoestrogen exposure, an increased intensity of expression was found in samples from rats exposed to BPA.05 and BPA20 and submitted to the OVX-P+E protocol (P < 0.05, Figs. 8EGo and 9BGo). No differences were observed in the SRC-3 expression across experimental groups (Fig. 8Go, B vs. D, and Fig. 9AGo). Specific staining was absent when primary antibodies were preincubated with peptides used as immunogens (Fig. 8Go, F and G). Similarly, immunostaining was absent when antibodies were replaced by normal rabbit serum (data not shown).


Figure 8
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  		FIG. 8. Western blot and immunohistochemistry of SRC-3 and SMRT antisera in the rat uterus. Antibodies performed successfully in Western blot, detecting specifically the SRC-3 (160 kDa) and the SMRT (150 kDa) native proteins (A). Expression of SRC-3 (left panel) and SMRT (right panel) in uterine samples of OVX-P+E controls (B and C) and neonatally BPA20-exposed rat (D and E). Nuclear SRC-3 and mainly cytoplasmic (perinuclear) SMRT staining were detected in uterine tissue. Primary antibodies were preabsorbed with peptides used as immunogens for SRC-3 (F) and SMRT (G) (both photomicrographs were obtained from sections without hematoxylin counterstaining). Original magnification x1000.

 

Figure 9
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  		FIG. 9. Immunohistochemical quantification of SRC-3 (A) and SMRT (B) in the uterine stromal compartment of rats submitted to the OVX-P+E protocol. SCR-3-positive staining was not different between control and xenoestrogen-treated animals. A significant increase in SMRT expression was observed in the uterine subepithelial stroma of BPA.05 and BPA20 neonatally exposed rats. *, P < 0.05 vs. control (n = 8/group).

 
Colocalization of ER{alpha}, PR, Hoxa10, and the corepressor SMRT
Finally, to determine whether the studied proteins were colocalized in the same cell of the subepithelial uterine stroma, we performed dual immunofluorescent staining. The comparative expression pattern of PR/ER{alpha}, PR/SMRT, and Hoxa10/SMRT between uterine tissues of controls and BPA20-exposed animals is shown in Fig. 10Go. Our dual-immunofluorescence studies for PR/ER{alpha} revealed that there is an intense nuclear colocalization of both receptors in the subepithelial stroma in controls and BPA20-exposed animals (Fig. 10AGo). Moreover, the high levels of SMRT expression in the subepithelial cells in BPA20-exposed animals were colocalized with PR protein (Fig. 10BGo). In addition, control animals showed high Hoxa10 protein expression, mainly in the nucleus of subepithelial cells, which colocalized with a low expression of SMRT (Fig. 10CGo). In contrast, the uteri of BPA20 rats showed an inverted pattern of expression: a higher expression of SMRT associated with a low expression of Hoxa10 in the same subepithelial cells (Fig. 10CGo).


Figure 10
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  		FIG. 10. Dual-immunofluorescence staining for PR/ER{alpha} (A), PR/SMRT (B), and Hoxa10/SMRT (C) in the rat uterus. Substantial coexpression of PR and ER{alpha} in the nuclei of subepithelial stromal cells in both control and BPA20 animals, evidenced as yellow nuclei in merged images is shown in A. In B the higher expression of SMRT in BPA20 animals is coincident with the expression of PR in the same cells. In addition, the lower expression of Hoxa10 in BPA20 animals is associated with a higher expression of SMRT in the same compartment (C). Original magnification, x600; merge, x1000.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The developmental programming hypothesis suggests that aberrant stimuli encountered during critical periods of development can permanently reprogram normal physiological responses and thus, give rise to reproductive consequences later in life (40). In the present study, we show that exposure to BPA or DES during neonatal uterine development reprograms the normal ovarian steroid hormone responsiveness of target cells leading to dysregulation of normal ovarian steroid control of uterine stromal cell proliferation. We demonstrate that environmentally relevant xenoestrogen exposure during the early postnatal period alters the expression of homeobox genes in rat uteri. In particular, the Hoxa10 alteration persists until adulthood as a hormone-mediated activation of this gene failed in the adult female. In these animals, not only is the transcription of Hoxa10 affected but some downstream events associated with its functions in the uterus were also fully disrupted. We have shown a close correlation between Hoxa10 disruption and stromal proliferation failure during adulthood. In parallel, an abnormal overexpression of the corepressor SMRT was found in the same stromal cells in which Hoxa10 induction failed, suggesting that neonatal xenoestrogen exposure alters the transcriptional machinery of steroid-dependent genes.

Because the true impact of endocrine disruptors on human health is difficult to assess, it is important to test their effect under controlled exposure conditions in animal models (19). The model of DES-induced reproductive tract malformations and cancers serves as a novel paradigm to study the pathological consequences that arise in adults exposed early in life to hormonally active substances (41). There is no clear sign that any gene has undergone a permanent mutation in response to DES exposure. Instead, a transient disruption of normal gene expression occurs during a critical period that impacts all subsequent and normal development. It has been shown that DES potently repressed a number of developmental control genes, including several Hox and Wnt genes, during critical periods of reproductive tract patterning. However, these effects were not present in ER{alpha} knockout mice, clearly suggesting that the xenoestrogen disruption is observed only in the presence of an intact ER pathway (3).

We investigated the effects of neonatal xenoestrogen exposure in the rat uterus early in development (PND8) and in adulthood (OVX-P+E model). This model allowed us to investigate uterine events that are under ovarian steroid control. Specifically, we investigated whether a brief neonatal exposure to BPA disrupts transcriptional control of the development-related genes Hoxa10 and Hoxa11. Hoxa10 and its neighbor in the Hoxa gene cluster, Hoxa11, are abdominal B type homeobox genes, which normally regulate differentiation of the Müllerian duct (42). Down-regulation of both Hox genes has been detected using mice as a model of postnatal exposure to DES (3). Methoxychlor, a pesticide that has adverse effects on the reproductive capability of mice, decreased Hoxa10 mRNA levels in Ishikawa cells in vitro and diminished uterine Hoxa10 expression in mice treated in vivo (43). At this moment, little is known about BPA effects on Hox gene expression. A recent report showed a dose-response increase in Hoxa10 levels in the uterus of 2- and 6-wk-old mice exposed in utero to BPA (44). In the present study, we demonstrate that BPA or DES exposure during early postnatal days decreased Hoxa10 and Hoxa11 expression in the prepubertal rat uterus. Taken together, the above-mentioned results show that Hox genes are a common target of endocrine disruption (43) and suggest that exposure during different developmental periods could lead to a different characteristic effect.

Previous results showed that mutant mice lacking normal Hoxa10 expression show defective P-dependent uterine stromal cell proliferation (10). In the present study, we used a model system in which P pretreatment followed by a physiological dose of E increased the number of synchronously proliferating uterine stromal cells. We found that neonatally DES- or BPA-exposed rats showed an impaired response to normal ovarian steroid-mediated induction of uterine stromal cell proliferation. In addition, the DES and BPA groups showed that the steroid-mediated activation of Hoxa10 failed in the adult females, indicating that the Hoxa10 alteration persists until adulthood. It is notable that increased cell number and endometrial tissue volume may provide the necessary infrastructure for optimal embryo implantation (45). In support of this, in humans, increasing endometrial thickness correlates with higher implantation rates (46). Recently a decreased volume of the endometrial lamina propria in adult CD-1 mice exposed in utero to low doses of BPA was observed (47). Alterations in the proliferative status of uterine stromal cells in response to steroid hormones described here could result in asynchrony between the endometrium and the embryo, likely leading to decreased fertility. These alterations have been demonstrated in mice in which the P-pathway was affected with clear consequences on fertility (48). Furthermore, several studies have supplied evidence about the possible relationship between this impaired proliferative response and the development of endocrine-related diseases like endometriosis and endometrial tumors (49).

Taking into account that P priming is an absolute requirement for uterine stromal cell proliferation in response to E and that this event requires the presence of PR and Hoxa10 (10), we wanted to know whether BPA or DES neonatal exposure could produce long-term effects by affecting the normal induction of PR expression in the adult uterine stroma. In OVX rats with steroid hormone replacement (OVX-P+E), we detected differential uterine stromal PR induction, depending on the xenoestrogen used. Whereas DES-exposed rats failed to induce PR in the stroma after P+E treatment, BPA.05- and BPA20-exposed rats showed an up-regulation of PR similar to control animals. These results indicate that each xenoestrogen would affect the PR signaling pathways by different mechanisms. It has been shown that PR and ERs display low corepressor binding in the absence of hormone but gain an increased ability to bind corepressors, depending on the presence of ubiquitous transcription cofactors, promoter context, and chromatin acetylation levels (50). Postnatal exposure to BPA affected uterine stromal SMRT expression in response to P+E treatment in adults, showing a clear up-regulation of this corepressor in the subepithelial compartment. Moreover, in BPA-exposed animals, the increased expression of SMRT without changes in steroid receptor expression may provide a possible explanation for the reduced responsiveness to hormone replacement due to the fact that SMRT is a limiting factor that inhibits the transcriptional activity of steroid hormone receptors (50). In our experiments, the high expression of SMRT in BPA-treated animals was accompanied by a low expression of the Hoxa10 gene in the same cells. In light of this, the following hypothesis can be proposed: the exposure to xenoestrogen chemicals during critical periods of perinatal life changes the uterine hormonal response during adulthood by disrupting the transcription machinery’s assembly of PR- and ER-dependent genes (51). Future studies will address this issue, studying whether neonatal exposure to BPA affects the transcription factor assembly in the Hoxa10 promoter region.

The term selective ER modulator is applied to chemicals, such as BPA, that have tissue-specific and species-specific effects (52). With regard to tissue-specific actions, it was shown that the effects of BPA are virtually identical with estradiol, ethinyl estradiol, and DES in the fetal mouse prostate (53) but markedly different from estradiol in the uterus (54). In a model of immature rats other authors showed that BPA, unlike estradiol, had no effect on uterine weight; but like estradiol, both the peroxidase activity and PR levels were elevated (55). In the present study, we confirmed that BPA acts as a selective ER modulator, differing from DES effects in some but not all the end points evaluated.

Our results showed a biphasic response to BPA in relation to ER{alpha} and Hoxa11 expression after steroid treatment in OVX adult animals. These results suggest that, at the low dose, BPA appears to produce the major long-lasting effects, whereas at the higher dose, the disruption in expression of these two genes could not be detected. These findings suggest an inverted U effect and are consistent with many reports in the BPA literature. A nonmonotonic dose-response curve, either U or inverted U shaped, challenges the default linear model, which has been in practice for decades (52, 56). Mechanisms underlying these complex endocrine behaviors have not been fully understood. In a recent report, we found that BPA exposure alters the promoter selection of the ER{alpha} gene, modifying the abundance of mRNAs isoforms and protein in the rat brain (26).

Previous studies have shown that endocrine disruptor exposure induces a hormonal imprint by means of a methylation pattern in specific gene promoter regions (2). Abnormal expression of lactoferrin and c-fos in the endometrium of adult CD-1 mice developmentally exposed to DES correlates with an altered pattern of CpG methylation in the promoter regions of these genes (6). However, the pattern of methylation of Hoxa10 and Hoxa11 genes did not show modifications in response to DES in the same animal model (57). In the present study, using a methylation-specific real-time PCR approach, no differences in the methylation pattern of the Hoxa10 promoter between xenoestrogen exposed and control rats were found. Based on this finding, we suggest that the differential methylation of CG-rich regions of the Hoxa10 promoter might not be the mechanism that induces the silencing of this gene. However, different technical approaches such as DNA-sequencing analysis after sodium bisulfite treatment would be necessary to confirm this result.

Postnatal uterine morphogenesis is governed by a variety of hormonal, cellular, and molecular mechanisms. Some regulatory factors have been defined, such as Hox genes and ovarian growth factor systems. The importance of the ovarian-derived activin-follistatin system was demonstrated in the control of adenogenesis in neonatal ovine uterus development (58). In rodents, from PND10 to PND14, uterine growth depends on the presence of the ovaries, suggesting the attractive hypothesis that neonatal exposure to BPA could disrupt the ovarian control of early uterine development. In addition, during pregnancy, uterine-synthesized activin-A has been suggested as a regulator of trophoblast growth and differentiation as well as embryo implantation (59, 60, 61). Taking these findings into account, the failed uterine hormone response observed in BPA-exposed animals could be related to not only Hoxa10 gene silencing but also a disruption in the paracrine action of local activin.

In summary, it is suggested that the disruption of Hoxa10 and Hoxa11 expression due to xenoestrogen exposure during early postnatal life could reprogram the normal responsiveness of uterine stromal cells to E and P during adulthood. We found that not only is the transcription of Hoxa10 affected, but also some downstream events associated with its functions in the uterus were fully disrupted. The close correlation between Hoxa10 disruption and stromal cell proliferation failure described here might affect embryo implantation.


   Acknowledgments
 
We are grateful to Mr. Juan Grant and Mr. Juan C. Villarreal for technical assistance and animal care. We thank Dr. Pablo Beldomenico for advice on statistics and Dr. Laura Kass for her helpful with immunofluorescence staining.


   Footnotes
 
This work was supported by grants from the Universidad Nacional del Litoral (CAI+D program) and the Argentine National Agency for the Promotion of Science and Technology. V.L.B. is a fellow and J.V., J.G.R., and E.H.L. are Career Investigators of the Argentine National Council for Science and Technology.

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 24, 2008

Abbreviations: BPA, Bisphenol A; BrdU, bromodeoxyuridine; CT, cycle threshold; DES, diethylstilbestrol; dNTP, deoxynucleotide triphosphate; E, 17β-estradiol; ER, estrogen receptor; IOD, integrated OD; PCR; OVX, ovariectomized; P, progesterone; PND, postnatal day; PR, progesterone receptor; QMSP, real-time quantitative methylation-specific PCR; QPCR, real-time quantitative PCR; SMRT, silencing mediator for retinoic acid and thyroid hormone receptor; SRC, steroid receptor coactivator; Vv, volume fractions.

Received May 2, 2008.

Accepted for publication July 16, 2008.


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 Introduction
 Materials and Methods
 Results
 Discussion
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