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Published as doi: 10.1096/fj.06-6635com.
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(The FASEB Journal. 2007;21:239-246.)
© 2007 FASEB

Xenoestrogen exposure imprints expression of genes (Hoxa10) required for normal uterine development

Caroline C. Smith* and Hugh S. Taylor{dagger},{ddagger},1

* Department of Epidemiology and Public Health,

{dagger} Department of Obstetrics, Gynecology, and Reproductive Sciences, and

{ddagger} Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut, USA

1Correspondence: Yale University School of Medicine, Division of Reproductive Endocrinology, 333 Cedar St., P.O. Box 208063, New Haven, CT 06520-8063, USA. E-mail: hugh.taylor{at}yale.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The developing reproductive tract is sensitive to endocrine perturbation. Bisphenol A (BPA), a xenoestrogen, is a common component of food storage plastics and dental composites. We tested the ability of BPA to alter expression of HOXA10, a gene necessary for uterine development. A dose-response increase in HOXA10 mRNA expression was demonstrated in Ishikawa cells treated with 0.1 nM to 25 µM BPA. To determine whether in utero BPA exposure resulted in a lasting alteration of uterine HOXA10 expression, mice were treated with 0.5–5.0 mg/kg BPA on gestational days 9–16. A dose-responsive increase was seen in stromal cell HOXA10 expression in 2- and 6-week-old mice exposed in utero. To discern the mechanism of BPA action, the HOXA10 estrogen response element (ERE) and autoregulatory element (ARE) were tested for BPA responsiveness. BPA drove luciferase expression from HOXA10-ERE and ARE reporter constructs. HOXA10 ERE mediated induction was blocked by ER antagonist ICI, while HOXA10 ARE induction was blocked by either ICI or HOXA10 antisense. BPA affects HOXA10 expression through the HOXA10 ERE and indirectly through the ARE. BPA initially alters HOXA10 expression through the ERE, however, the response is imprinted and uncoupled from estrogen stimulation in the adult. Several xenoestrogens alter HOX gene expression, indicating that HOX genes are a common target of endocrine disruption. In utero exposure to a xenoestrogen produces reproductive tract alterations by imprinting essential developmental regulatory genes.—Smith, C. C., Taylor, H. S. Xenoestrogen exposure imprints expression of genes (Hoxa10) required for normal uterine development.


Key Words: bisphenol A • BPA • endocrine disruption • HOX


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HUMANS ARE WIDELY EXPOSED to bisphenol A (BPA), a common component of plastics used to contain food products and in dental composite resins. BPA is a purported endocrine disruptor, first recognized as such when it leached from polycarbonate laboratory flasks and confounded an estrogenicity assay (1 , 2) . Reproductive tract development is altered in some rodent models by treatment with BPA (3 4 5 6 7) . In humans, exposure to BPA has also been associated with pregnancy loss (8) . The potential hazards that may stem from exposure to this xenoestrogen and its mechanism of action have not been fully characterized.

In utero exposure to relatively high levels of BPA results in multiple developmental defects in rodents. BPA treatment in utero reduces the number of days between vaginal opening and first estrus as well as the rate of postnatal growth in males and females (9 , 10) . In utero exposure to BPA also results in altered patterns of estrus cyclicity (11) . After in utero exposure, the vagina demonstrates long-term deleterious effects reminiscent of those induced by DES (14 , 15) . Additionally, BPA treatment in utero affects the mouse mammary gland, where it alters the rate of ductal migration to the stroma and significantly increases the number of ducts, terminal ducts, terminal end buds, and alveolar end buds (12 , 13) . Early onset of puberty, changes in hormone production, and histological changes in the vagina and mammary glands indicate that in utero exposure to BPA has multiple functionally significant developmental consequences in the female reproductive tract.

In utero exposure appears to have an effect on the uterus that persists in the adult. Low-dose gestational exposure results in decreased vol, increased proliferation, as well as increased estrogen receptor-alpha and progesterone receptor expression in the adult murine uterine endometrium (16) . Higher doses have produced similar morphological changes in the rat endometrium (17) .

Similarly, adult exposure to BPA results in reproductive alterations. Functional effects are seen in exposed female mice; BPA exposure results in a reduction in pregnancy rate and litter size, as well as an increase in embryo resorption (18 19 20 21) . In humans, exposure to BPA has been associated with recurrent miscarriage (8) . Paradoxically, lower BPA serum levels are associated with complex endometrial hyperplasia in women (22) . Despite the human and animal data associating BPA with anomalous reproductive tract development, cancer, and pregnancy loss, the mechanism of action is not understood.

Previously, we and others have demonstrated that two endocrine disruptors alter Hox gene expression in the female reproductive tract (25 26) . HOX genes are highly conserved developmental regulators that govern the segmental differentiation of the anterior-posterior body axis (27) . HOX genes direct the development of the fallopian tube, uterus, cervix, and upper vagina, all of which derive from the undifferentiated paramesonephric duct (28) . Hoxa9, Hoxa10, Hoxa11, and Hoxa13 are expressed in the developing oviducts, uterus, uterine cervix, upper vagina, respectively (28) . Hoxa10 (mouse)/HOXA10 (human) in particular is the target of endocrine disruption by diethylstilbestrol (DES) both in mice and in human reproductive tract cell lines (24 25 26) . Unlike estradiol, DES induces alteration of Hoxa10 expression in vitro and causes posterior shifts in Hox gene expression after in utero exposure (24) . The molecular mechanism by which DES exerts this effect involves the HOXA10 estrogen response element (ERE) (25 , 29) . Similarly, neonatal exposure to another endocrine disruptor, methoxychlor, permanently reduces uterine HOXA10 expression (23) .

We hypothesized that the purported endocrine disruptor BPA would impact the expression of HOXA10, a gene required for normal development of the uterus. Because of the necessary role of HOXA10 in uterine development and adult function, aberrant expression of this gene may underlie BPA-mediated endocrine disruption. Humans are widely exposed to estrogenic endocrine disrupting chemicals; disturbances in the normal endocrine regulation of HOX genes may be a common mechanism of estrogenic endocrine disruption. Further, we identify a potential mechanism of persistently altered gene expression after xenoestrogen exposure.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
Human Ishikawa cells, a well-differentiated endometrial adenocarcinoma cell line, were obtained from R. Hochberg (30 , 31) . HOX gene expression in this cell line has been previously well characterized (32 33 34) . Estrogen receptor status was verified using ELISA according to the manufacturer’s instructions. Cells were maintained in Dulbecco’s Modified Eagles Medium (DMEM, Sigma, St. Louis, MO, USA) supplemented with 10% FBS, sodium pyruvate, sodium bicarbonate, and antibiotic-antimycotic (Life Technologies, Inc. Invitrogen, Carlsbad, CA, USA). Cells were grown in 37°C in a 5% CO2 atmosphere to 95% confluence in 10 cm culture dishes, and were trypsinized and plated at ~2.5 x 105 cells per well of a 24-well plate for treatment. Cells were transferred to phenol red free medium supplemented with 10% charcoal-stripped calf serum and L-glutamine for 24 h and then treated in steroid-free medium with concentrations of bisphenol A (Sigma), which ranged from 0.1 nM to 25 µM, 1 µM ER antagonist ICI 182780 (Sigma) or vehicle control (DMSO) for an additional 24 h. RNA was isolated using the Qiagen RNeasy Kit (Valencia, CA, USA).

Semiquantitative and quantitative polymerase chain reaction (PCR)
Semiquantitative reverse-transcriptase-polymerase chain reaction (RT-PCR) was performed as we have previously described (35) . Reverse transcriptase was used to generate cDNA from the isolated RNA. The total reaction vol was 20 µl, consisting of RNase-free water, 0.25 µg RNA, oligo dT, 5x buffer, dNTPs, Rnase inhibitor, and RT-avian myeloblastosis virus. Generation of cDNA was performed in a Eppendorf Mastercycler Gradient thermal cycler (Hamburg, Germany) at 42°C for 1 h and 95°C for 5 min. The PCR reaction vol was 52 µl, consisting of 10x PCR buffer, 25 mm Mg2+, HOXA10 and G3PDH forward and reverse primers, RNase-free water, and Taq polymerase. The thermal profile for the PCR reaction was 95°C for 5 min, 95°C for 45 s, 72°C for 45 s, and a final extension at 72°C for 7 min. PCR products were run in a 1.8% agarose gel, and bands were visualized with ethidium bromide (Bio-Rad, Hercules, CA, USA).

Quantitative real-time RT-PCR (qRT-PCR) was performed using the Lightcyle SYBR Green RT-PCR kit (Roche, Indianapolis, IN, USA) as described previously (23) . Total RNA (1 µg) was reverse-transcribed in 20 µl of reaction mixture containing l0 mM each of dATP, dCTP, dGTP, and dTTP; 20 pmol oligo(dT); 40 U/µl ribonuclease inhibitor, 10 U/µl avian myeloblastosis virus (AMV)-reverse transcriptase, and 10x AMV-RT buffer for 30 min at 61°C. qPCR was used to amplify and quantify Hoxa10 and ß-actin expression as described previously. The Hoxa10 intron spanning primers were selected using the primer selection program Primer3 developed by the Whitehead Institute for Biomedical Research and had the following sequences:

Sense: 5'-GCCCTTCCGAGAGCAGCAAAG-3',

Antisense: 5'-AGGTGGACGCTGCGGCTAATCTCTA-3'

All primers were synthesized by the Yale University School of Medicine, Department of Pathology. The primers were designed to yield a 211 bp reaction product from mRNA, and a 1389 bp DNA reaction product from DNA, which allowed control for possible DNA contamination.

The Roche Light Cycler monitored the increasing fluorescence of PCR products during amplification. A quantitative standard curve was then created. Quantitation of samples was determined with the Roche Light Cycler and adjusted to the quantitative expression of ß-actin from these same samples. Melting curve analysis was conducted to determine the specificity of the amplified products and to ensure the absence of primer-dimer formation. All products obtained yielded the predicted melting temperature. Three independent experiments were conducted at each concentration.

Animal care and immunohistochemistry
Adult reproductive age CD1 mice were obtained from Charles River and were housed in the Yale University Animal Care facilities. All mice were administered food and water ad libitum. In all breeding studies, 1–3 females were housed with a proven fertile male. The day of vaginal plug detection was designated Day 1 of pregnancy, when females were removed from the male and housed in groups of 2–5.

Timed pregnant female cluster of differentiation (CD)-1 mice were injected intraperitoneally (i.p.) with 0.5, 1.0, 5.0, 50, or 200 mg/kg BPA from gestational days 9–16. At least three pregnant females received each dose of BPA. Female offspring exposed in utero were euthanized 2 or 6 weeks after birth, and reproductive tracts were resected, fixed in 10% formalin, and paraffin-embedded. Ten mice from at least three separate litters were examined for each exposure and each time point. Slides were deparaffinized and rehydrated in a series of xylene and ethanol washes. The sections were permeabilized in a 95% ethanol wash and steamed in 0.1 M sodium citrate buffer at 90°C for 20 min, followed by washing for 3 min in PBS. The sections were circumscribed with a hydrophobic pen and washed in 0.1% PBS Tween 20 (PBST), and the endogenous peroxidase was inhibited with a 3% peroxide wash. The sections were blocked with 1.5% horse serum in PBST for 1 h at room temperature. The primary antibodies goat polyclonal HOXA10 or goat IgG isotype control (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA) were applied overnight at 4°C. After washing, the biotinylated secondary horse anti-goat antibody (Ab) (Vector, Burlingame, CA, USA) was applied for 1 h at 4°C. Slides were washed in PBST and then incubated with avidin-biotin complex (ABC) Elite (Vector) for 30 min at room temperature. The slides were washed with PBST and incubated in 3,3'-diaminobenzidine (Vector) for 5 min. Hematoxylin was used to counterstain the cell nuclei. The slides were then dehydrated through a reverse of the ethanol and xylene washes. The coverslip was mounted with permount.

Hoxa10 immunostaining was quantified by H score (36) . The H score was determined on each endometrial sample in a blinded fashion by two observers. The HSCORE was calculated using the following equation: HSCORE = {Sigma} Pi (i+1), where i is the intensity of staining (1=weak, 2=moderate, and 3=strong) and Pi is the percentage of stained epithelial cells for each intensity (0–100%). Interobserver and intraobserver variations have been reported previously using this approach (37) . All animal experimentation was conducted in accord with accepted standards of humane animal care under an approved institutional animal care and use protocol.

Transfection and luciferase assay
The HOXA10 ERE and ARE were tested for BPA responsiveness in pGL3-promotor using a luciferase assay as described previously (25 , 38 , 58) . In brief, Ishikawa cells were grown to 50–60% confluence and transfected with the appropriate plasmid (pGL3/HOXA10-ERE, pGL3/HOXA10-ARE, or empty pGL3) using Lipofectamine 2000 in serum-free Optimem (Invitrogen) medium. After 5 h, the cells were washed and cultured in steroid-free medium for an additional 24 h. Cells were then treated with BPA at 1 nM, 1 µM, and 25 µM 17 beta-estradiol (100 nM); 1 µM ER antagonist ICI 182780; or HOXA10 antisense as described previously (59 , 60) . The cells were washed with cold PBS and lysed with Reporter lysis Buffer (Promega, Madison, WI, USA), and the lysates were collected. Luciferase activity was measured in cell lysates using the Luciferase Assay Kit (Promega), and a luminometer. A Renilla luciferase reporter construct was cotransfected to correct for variations in transfection efficiency. Reporter activity was normalized to Renilla expression. Normalized expression was compared using the t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Treatment with BPA alters HOXA10 in vitro
Semiquantitative RT-PCR was performed to screen the effect of endocrine disruptor treatment on HOXA10 gene expression of human Ishikawa cells. Ishikawa cells were treated with concentrations of BPA from 0.1 to 25 µM or with DMSO. RNA was subsequently isolated, reverse-transcribed, and amplified using a previously verified semiquantitative RT-PCR protocol (35) . As demonstrated in Fig. 1 , DMSO (control diluent) exerted no effect on HOXA10 expression. An increase in HOXA10 gene expression was seen with increasing concentrations of BPA treatment.


Figure 1
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  		Figure 1. Semiquantitative RT-PCR amplification of HOXA10 mRNA after BPA exposure. RNA was isolated from BPA-treated Ishikawa cells. HOXA10 and G3PDH were amplified using semiquantitative RT-PCR. Results are shown after exposure of cells to concentrations of BPA from 100 nM to 25 µM. Images are representative of three independent experiments. MW: MW markers, BPA: Bisphenol A, DMSO: dimethyl sulfoxide.

To confirm and quantify the positive semiquantitative results, real-time (quantitative) RT-PCR was performed with the RNA isolated from the treated cells. As shown in Fig. 2 , the expression of HOXA10 was elevated in a dose-responsive fashion in BPA-treated cells. HOXA10 expression was initially elevated after treatment with 1 uM BPA. An ~15-fold increase in HOXA10 expression was seen as BPA concentration was increased from 0.1 nM to 25 µM. Concomitant treatment with ICI blocked the response to BPA.


Figure 2
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  		Figure 2. A) Real-time RT-PCR confirms increased expression of HOXA10 after BPA treatment. Reverse transcription and PCR were performed in a single reaction using mRNA isolated from BPA- or ICI-treated cell culture. HOXA10 and ß-actin were coamplified and HOXA10 expression normalized to actin presented. N > 3 for each treatment. DMSO: dimethyl sulfoxide, BPA: bisphenol A. The treatment groups were compared with the vehicle-treated control using the unpaired two-tailed Student’s t test. *P < 0.05 compared with DMSO-treated control. **P < 0.001. B) ICI prevents the increase in HOXA10 expression in the presence of BPA. BPA indicates treatment with 10 µM Bisphenol A. *P < 0.01.

Exposure to BPA in utero leads to persistently increased HOXA10 expression
To determine whether in utero BPA treatment would produce alteration in Hoxa10 expression that persists in the adult, we dosed timed-pregnant female mice with BPA and examined their female pups. The pregnant mice were injected with one of five concentrations of BPA on days 9–16 of gestation; at either 2 or 6 weeks after birth, female pups were euthanized and reproductive tracts were resected, fixed, and paraffin-embedded. As shown in Fig. 3 , immunohistochemistry was performed on sections of the reproductive tracts from female offspring. The control IgG-treated slides showed no nuclear staining. The uteri obtained from vehicle-treated controls revealed the expected basal expression of Hoxa10 protein. Attempts to dose pregnant females with BPA at 50 mg/kg resulted in one stillbirth followed by death and one death without parturition. Treatment with 200 mg/kg resulted in death of all pregnant females. After administration of 0.5 mg/kg to 1.0 mg/kg BPA, a dose-responsive increase in uterine stromal Hoxa10 expression was detected. A 5-, 7-, and 10-fold increase in Hoxa10 protein expression was seen in the 0.5, 1.0, and 5.0 mg/kg BPA-treated mice, respectively, compared with vehicle-treated controls as determined by H-score at the 2 week time point. At 6 weeks a 5-, 9-, and 12-fold increase was seen in the 0.5, 1.0, and 5.0 mg/kg BPA-treated mice. The results were statistically significant; P < 0.01 compared with controls in the 1.0 and 5.0 mg/kg treated groups at 2 weeks, and the 0.5, 1, and 5 mg/kg groups at 6 weeks using ANOVA on ranks. After in utero treatment, no gross anomalies of the reproductive tract were observed in any of the female offspring. No positional shifts were noted in the spatial expression of Hoxa10 in the uterus or other female reproductive tract tissues examined by IHC (oviduct, cervix, vagina: data not shown). Additionally, no anatomic defects were observed in nonreproductive tissues known to be developmentally regulated by Hoxa10, including the spine and limbs. Reproductive success has been previously reported and was not evaluated here.


Figure 3
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  		Figure 3. Immunohistochemical analysis demonstrates Hoxa10 immunoreactivity in reproductive tracts of 2- and 6-week-old female mice exposed in utero to BPA. Uteri were obtained from mice exposed in utero to either vehicle-treated control (inset: goat IgG isotype control), BPA 0.5 mg/kg, BPA 1.0 mg/kg, or BPA 5.0 mg/kg. Photomicrographs in (A) demonstrate Hoxa10 expression in uteri from 2-week-old mice, while those in (B) are from 6-week-old mice. Uterine stromal cells show increasing nuclear Hoxa10 protein expression with increased exposure to BPA. The increase persists at 6 weeks of age. Images are representative of 10 mice from at least three separate litters per treatment group. BPA: Bisphenol A.

Three mice from the 5 mg/kg treated group and three controls underwent ovariectomy (OVX) at 6 weeks. Two weeks after OVX, uterine endometrium was examined in the same fashion as described above. Those mice exposed to BPA in utero continued to display higher levels of uterine Hoxa10 expression than controls. Hoxa10 expression was 10-fold higher in the BPA-treated mice than controls. The increased Hoxa10 expression persisted despite absence of estrogen stimulation. Hoxa10 expression was imprinted by BPA.

BPA action is mediated through the HOXA10 ERE and ARE
HOXA10 is regulated by an ERE and an autoregulatory element (ARE) (24 , 58) . To determine whether the BPA-driven increase HOXA10 expression was mediated through the HOXA10 ERE, transient transfection and luciferase reporter gene assays were performed. Briefly, Ishikawa cells were transfected with the pGL3/HOXA10-ERE or control plasmid, treated with varying concentrations of BPA, estradiol (E2), or diluent control, and luciferase activity was measured in cell lysates. As shown in Fig. 4 , treatment with E2 resulted in a dose-responsive increase in luciferase activity. Activity was detected starting at 10–12 M E2 treatment with a Kd 50 of 10–10 M. Treatment with BPA resulted in an induction of luciferase activity compared with controls. BPA-induced reporter expression was first detected at 10–8 M with a Kd 50 of 10–6 M. Excess ICI (10–6 M) treatment inhibited luciferase induction in response to either E2 or BPA.


Figure 4
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  		Figure 4. Activity of BPA compared with estradiol (E2) on the HOXA10 ERE. pGL3/HOXA10-ERE was used to transfect Ishikawa cells, which were subsequently treated with BPA, E2, or vehicle-control medium. A Renilla luciferase reporter construct was cotransfected to control for transfection efficiency. Reporter activity was normalized to Renilla expression. Relative luciferase activity obtained after treatment with E2 is designated by small squares, while activity after BPA treatment is indicated by large circles. The mean ± SD is shown, n = 4 for each compound at each concentration.

Once activated through the ERE, we hypothesized that HOXA10 expression would activate the ARE and maintain the increased expression. To determine whether the BPA- driven increased HOXA10 expression was further mediated through the HOXA10 ARE, transient transfection and luciferase reporter gene assays were performed. Briefly, Ishikawa cells were transfected with the pGL3/HOXA10-ARE, control plasmid, with or without ICI or HOXA10 antisense, and treated with varying concentrations of BPA or diluent control. Luciferase activity was measured in cell lysates. As shown in Fig. 5 , treatment with BPA resulted in an induction of luciferase activity compared with controls. BPA-induced reporter expression was first detected at 10–8 M. ICI blocked the ability of ICI to induce reporter expression. HOXA10 antisense treatment similarly inhibited luciferase induction from the ARE in response to BPA, indicating an indirect response; BPA mediated ARE activation through increased HOXA10. ICI blocked HOXA10 induction by BPA, while HOXA10 AS blocks HOXA10 expression and therefore HOXA10 activation of the ARE. Our model of HOXA10 endocrine disruption is shown in Fig. 6 . HOXA10 expression is initially attained through the ERE and subsequently maintained by an autoregulatory loop involving the ARE.


Figure 5
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  		Figure 5. Activity of BPA on the HOXA10 autoregulatory element (ARE). pGL3/HOXA10-ARE was used to transfect Ishikawa cells, which were subsequently treated with BPA, ICI, HOXA10 antisense (AS), or vehicle control. A Renilla luciferase reporter construct was cotransfected to control for transfection efficiency. Reporter activity was normalized to Renilla expression. Relative luciferase activity is shown. n = 4 for each compound at each concentration. HOXA10 ARE activity is induced in a dose-responsive fashion by BPA. This effect is blocked by either ICI or HOXA10 AS, indicating that reporter activity is mediated through BPA induction of HOXA10 expression.


Figure 6
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  		Figure 6. Model of Xenoestrogen driven HOXA10 imprinting. A) Initially BPA or other xenoestrogens drive HOXA10 expression through the estrogen response element (ERE). B) BPA-induced HOXA10 expression subsequently drives further HOXA10 expression through the autoregulatory element (ARE). C) In the absence of continued BPA exposure, HOXA10 expression is maintained through the ARE.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Endocrine disruption continues to be a significant health concern. The paradigm was established based on the untoward effects seen in women exposed in utero to the nonsteroidal estrogen DES. DES, given to women to prevent miscarriages, instead caused disruption of reproductive tract development. Many of the daughters of women given DES during pregnancy were found to have reproductive tract anomalies, and some of these women developed vaginal adenocarcinoma (39) . DES-mediated endocrine disruption during gestation results in developmental anomalies that persist into adulthood. More recently, endocrine disruption by multiple agents has been documented in humans, laboratory animals, and diverse species of wildlife (40 , 41) . BPA is a major component of polycarbonate plastics and a demonstrated xenoestrogen. BPA-containing plastics are used extensively in packaging of food products and are also found in the material used for dental composite fillings and sealants, which has resulted in numerous opportunities for human exposure to a synthetic estrogen.

In vitro treatment with BPA resulted in elevated levels of HOXA10 gene expression. BPA concentrations used to treat uterine cell culture included the average levels detected in the serum of nonpregnant women (2.0 ng/ml, 8.8 nM), women in early pregnancy (1.5 ng/ml, 6.6 nM), women in late pregnancy (1.4 ng/ml, 6.1 nM), and fetal serum (2.2 ng/ml, 9.6 nM) (42) . Even higher concentrations are reported in human amniotic fluid (up to 8.3 ng/ml, 36 nM) (43 , 44) . Based on the relative bioavailability demonstrated in rats, the lowest BPA doses administered here to pregnant mice would have been expected to achieve average serum levels over 24 h that are comparable with those reported after human exposure (45 46 47 , 53) . Further, human serum BPA levels vary widely, indicating potential for an increase in response with greater-than-average human exposure.

Altered HOXA10 expression in women is seen in association with a number of common medical conditions. Diminished HOXA10 expression has been reported in polycystic ovary syndrome (PCOS), endometriosis, hydrosalpinx, and improper implantation (32 , 54 55 56 57) . Elevated HOXA10 expression is seen in ectopic pregnancy (55) . HOXA10 expression is tightly regulated, and optimal reproductive success likely depends on appropriate levels of the HOXA10 gene product. The alterations in HOXA10 by BPA reported here have the potential to influence reproductive potential.

In this study, in vivo administration of BPA to pregnant mice increased HOXA10 expression in the uterine stromal cells of female pups exposed in utero. Dysfunction of the reproductive system attributable to the abnormal expression of HOXA10 has been well-documented in rodents. HOXA10 is necessary for normal decidualization and pregnancy. Increased Hoxa10 expression results in altered endometrial pinopods and microvillie as well as increased litter size (49 , 50) . Hoxa10 (–/–) mice are infertile due to a defect in uterine decidualization leading to failure of embryo implantation (48 49 50 51) . BPA exposure has similarly been associated with uterine decidualization defects and embryo resorption, in mice (52) . The similar phenotype shared among mice exposed to BPA and those with altered expression of HOXA10 suggest that decidualization defects may be mediated by the altered HOXA10 expression after BPA exposure. Either decreased or persistently elevated Hoxa10 expression can impair endometrial function.

HOX genes have a necessary role in the development of the mouse and human reproductive tract and may be a common target of endocrine disruption. DES and methoxychlor abnormally regulate the expression of those HOX genes that direct differentiation of the uterus from the paramesonephric duct (24 , 25) . DES exposure in mice decreases Hoxa10 expression in its normal domain and shifts its expression posteriorly along the reproductive axis. Similarly, Methoxychlor reduces uterine Hoxa10 expression. Multiple xenoestrogens, including BPA, may potentially act as endocrine disruptors by altering expression of this gene. Interestingly, while DES or methoxychlor exposure decreases Hoxa10 expression, here BPA treatment increased it. The final mechanism of action in vivo differs between these estrogenic substances despite all three activating reporter expression by the Hoxa10 ERE. In vivo this effect is compound-dependent, leading to either activation or inhibition gene expression. Differential coactivator or corepressor recruitment likely underlies this differential regulatory function. The net activities of xenoestrogens are more complex than predicted based on in vitro reporter assays and likely depend on cellular and developmental context.

The effect of BPA on HOXA10 expression persisted long after in utero exposure and in the absence of estrogen stimulation. This suggests the existence of an imprinting mechanism that maintains expression at altered levels, even in the absence of ligand. Several tissues and the expression of multiple genes are permanently altered by transient in utero exposure to DES. DES and BPA appear to share the ability to imprint developmental genes in utero. Endocrine disruptors may change a critical set point in the regulation of gene expression. BPA likely alters HOXA10 expression by inappropriate activation of the ERE at a time when it is normally silent. This activation may result in epigenetic modifications of autoregulatory loops, which then become fixed at distinct set points and subsequently determine gene expression throughout life. This model is demonstrated in Fig. 6 ; HOXA10 is initially regulated by estrogens or xenoestrogens. This initial activation initiates autoregulation, however the mechanism by which the level of expression driven from this loop is ultimately set is not yet characterized; however, it does not appear to involve methylation of HOX genes (61) . Changes in transcription of a gene have been shown to alter the rate of its epigenetic modification (62) . Imprinting of HOXA10 by BPA is likely mediated indirectly through the premature activation of the autoregulatory element, which then inappropriately remains persistently active. Inappropriate activation in the wrong developmental context likely exposes the element to proteins that have the ability to alter the set point. Multiple developmental genes that are autoregulated or involve complex regulatory loops may be similarly imprinted. Endocrine disruptors alter a critical set point that is later uncoupled from ERE activation.

Exposure to BPA is nearly universal due to its widespread use in plastics. Functional consequences of BPA exposure have been documented in both animals and humans (63) . A change in HOXA10 gene expression provides one mechanistic explanation to explain the effects of environmental xenoestrogen exposures. We have previously demonstrated that both DES and methoxychlor also alter HOXA10 expression (23 24 25) . Imprinting of HOX gene expression by xenoestrogens may be a common mechanism contributing to endocrine disruption.


   ACKNOWLEDGMENTS
 
The authors wish to thank Jennifer Sarno, Xiaolan Fei, and Hongling Du for expert technical assistance. This work was supported by National Institutes of Health ES10610, The Society for Gynecologic Investigation, and the Jan A. J. Stolwijk Fellowship.

Received for publication June 8, 2006. Accepted for publication July 24, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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