Journal of Reproduction and Development, Vol. 49, No. 4, 2003

—Research Note—

Effects of Oral Exposure of Bisphenol A on mRNA Expression of Nuclear Receptors in Murine Placentae Assessed by DNA Microarray

Satoshi IMANISHI1), Noboru MANABE1), Hanako NISHIZAWA1), Maki MORITA1), Miki SUGIMOTO1), Mineko IWAHORI1) and Hajime MIYAMOTO1)

1)Unit of Anatomy and Cell Biology, Department of Animal Sciences, Kyoto University, Kyoto 606–8502, Japan

Abstract. Bisphenol A (BPA), a candidate endocrine disruptor (ED), is considered to bind to estrogen receptors and to regulate expressions of estrogen responsive . It has also shown evidence of affecting the reproductive, immunological and nervous systems of mammalian embryos. However, the effects of BPA on placentae, a central organ of feto-maternal interlocution, are still unclear. To reveal the mechanisms of BPA effects on placentae in mammals, we compared the mRNA expression of 20 nuclear receptors between placentae of vehicle controls and those of orally BPA exposed pregnant mice by a DNA microarray technique. In murine placentae, mRNAs of 11 nuclear receptors were not detected. However, greater than 1.5 fold changes in mRNA expression of nine nuclear receptors between vehicle control and BPA treated mice were noted. Moreover, remarkable changes in mRNA expression of six non-nuclear were induced by BPA exposure. There were various differences in the effects of BPA on the expression of these mRNAs between the placentae with male embryos and those with female embryos. Such embryo-sex dependent differences are interesting and important pointers to understanding of the endocrine disrupting effect of BPA. The present data indicate that BPA affects the expression of mRNAs in placentae and may disrupt the physiological functions of placentae. Key words: Endocrine disruptor, Bisphenol A, DNA microarray, Murine placenta, Nuclear receptor (J. Reprod. Dev. 49: 329–336, 2003)

isphenol A: 2,2-bis (BPA: 4-hydroxyphenyl [8] and resistance to bacteria infection [9]. In the propane), a plasticizer synthetic resin and a embryos of aromatase- knockout mice, in candidate endocrine disruptor (ED), has which no estradiol-17β is produced and whose physiological effects on reproductive, phenotype is bone-less, BPA exposure restored the immunological and nervous systems in mammals bone-less phenotype as well as estradiol-17β [1–8]. For instance, in rodents, prenatal or neonatal production [10], indicating BPA may bind with exposure to BPA stimulated mammary glands [1, 2] estrogen receptors (ERs). When compared with and uteri [3], caused abnormality of prostate estradiol-17β, however, the affinities of BPA to ERs development [4, 5] and decreased the number and are very low (approximately 1/10,000), and so it is fertilization quality of sperm [6, 7]. Also, BPA considered that additional mechanisms are suppressed the mitogenic activity of lymphocytes involved in the BPA activity seen in the knockout mice. Accepted for publication: May 29, 2003 Small lipophilic molecules such as steroid Correspondence: N. Manabe (e-mail:[email protected]) hormones, thyroid hormones, the active forms of 330 IMANISHI et al. vitamin A (retinoids) and vitamin D play important on a 12-h light, 12-h dark cycle. All animals were roles in growth, differentiation, reproduction, and given free access to tap water and CM rodent feed morphogenesis in mammals [11–13]. Most cellular (Oriental Yeast, Tokyo, Japan) ad libitum. They actions of these molecules are mediated through received humane care as outlined in the “Guide for binding to nuclear receptors that act as ligand- the Care and Use of Laboratory Animals” (Kyoto inducible transcription factors. Histologically, once University Animal Care Committee according to a nuclear receptor for a steroid hormone has been NIH No. 86–23; revised 1999). After mating, cloned, other nuclear hormone receptors have been females detected with a vaginal plug were rapidly cloned and their target sequences on DNA considered as 0.5 day-post coitum (dpc) of identified [11–13]. Our knowledge of regulation of pregnancy. From 6.5 to 17.5 dpc, pregnant females by nuclear receptors has grown were daily administerd BPA (2 µg/kg body spectacularly during the last few years, mainly due weight/day, 12 times; ICN Biomedicals, Aurora, to the realization that not only the interaction of the OH, USA) dissolved in purifed olive oil (Wako receptors with DNA are important for Pure Chemicals, Osaka, Japan) or purified olive oil transcriptional responses, but also that many (5 ml/kg/day) as vehicle control. At 18.5 dpc (24 h regulators (activators and repressors) are crucial for after the final administration), pregnant mice were transducing the hormonal signal to the sacrificed under anesthesia by diethylether in the transcriptional machinery. This knowledge has afternoon (12:00 to 14:00). The litter size was revealed the major toxicological mechanisms of recorded, and then each placenta with male or EDs including BPA which are as follows: (1) an ED female embryos was removed, weighed, rapidly molecule directly binds with nuclear receptor frozen in liquid N2, and stored at –80 C until DNA (s) and disrupts the hormonal signal microarray analysis. transduction; (2) an ED molecule directly interacts with the regulator(s) and disturbs the nuclear DNA microarray analysis receptor dependent-transcriptional responses; (3) For DNA microarray analysis, frozen placenta an ED molecule affects the production samples were homogenized, and total RNA was (transcription and/or translation) of nuclear isolated using an Atlas glass total RNA isolation kit receptor protein(s) (mechanism involved in non- (Clontech, Palo Alto, CA, USA) [14–16]. A part of DNA binding-dependent regulation by cross-talk each extracted total RNA sample was with other signal transduction pathways); and (4) electrophoresed in 1.2% (w/v) agarose gel (Sigma an ED molecule affects the production of Aldrich Chemicals, St. Louis, MO, USA) containing regulator(s) of the nuclear receptor. Moreover, that 1.23 M formaldehyde (Wako) and 1 mg/ml orphan receptors also play key roles in embryonic ethidium bromide (Wako). After electrophoresis, development, homeostasis and disease has been the gels were recorded with a digital recorder proven by targeted deletion in mice and by their (LAS-1000; Fuji Film, Tokyo, Japan), and the RNA association with different diseases including levels of the two brightest bands (28S and 18S atherosclerosis, cancer, diabetes and lipid disorders ribosoml RNA bands at approximately 4.5 and 1.9 [13]. In the present study, we focused on the effects kb, respectively) were quantified using of BPA on mRNA expression of nuclear receptors ImageGauge (Fuji Film) on a Macintosh computer. by examining the effects of BPA on mRNA The ratio of fluorescent intensities of the 28S and expression of nuclear receptors in murine placentae 18S rRNA bands in each sample was between 1.5:1 by a DNA microarray technique. to 2.5:1. Protein contaminants were removed using a Quick clean resin (Clontech). Each microarray experiment (i.e. hybridization) compared the Materials and Methods relative level of gene expression for a placenta with male or female embryos in the BPA group (Cy5- Animals and BPA treatment labeled cDNA) to that of a placenta with male or Female and male ICR mice were obtained from female embryos in the vehicle control group (Cy3- Clea Japan (Tokyo, Japan), housed in labeled cDNA). To synthesize Cy3- and Cy5- polycarbonate cages and maintained in a humidity labeled cDNA, each total RNA sample was reverse- (30–40%) and temperature (24 C)-controlled room transcribed with dNTP oligo primers and labeled EFFECT OF BPA ON MOUSE PLACENTA 331 by the incorporation of Cy3-dUTP or Cy5-dUTP Results (Amersham Biosciences, Piscataway, NJ, USA) into first strand cDNA using an Atlas glass fluorescent Effect of BPA on the expression of nuclear receptor labeling kit (Clontech). The Fluor-labeled probes mRNAs in murine placentae were purified with the Qiagen PCR purification kit Twenty mRNAs of nuclear receptors including (Qiagen, Valencia, CA, USA). The Cy3-labeled orphan receptors were arrayed on the Atlas glass cDNA was mixed with Cy-5-labeled cDNA, mouse 3.8 I DNA-microarray slide (3,800 genes vacuum dried, and resuspended in hybridization were applied) used in this study (Table 1). In buffer, 40% (v/v) formamide, 4 × standard saline murine placentae, mRNAs of thyroid hormone citrate, 1% (w/v) sodium 0.5% (w/v) dodecyl receptor β, peroxisome proliferator activated sulfate, polyadenylate and 0.5% (w/v) mouse COT- receptor (PPAR) α, PPAR γ, constitutive androstane 1 DNA as competitor (Clontech). The labeled receptor (also called as MB67), , cDNA mixture was heated at 95 C for 2 min before chicken ovalbumin upstream promoter hybridization to the microarray in a humidified (COUP-TF) β, testis receptor β, microarray-hybridization chamber (Clontech). The estrogen related receptor γ, aryl hydrocarbon microarray (Atlas glass mouse 3.8 I DNA- receptor (AhR), small heterodimer partner, or microarray; Clontech) was hybridized for 18 h at dosage-sensitive sex reversal (DAX-1) were not 42 C. Each slide was washed, dried by detected. No expression of neuron-derived orphan centrifugation, and scanned promptly using a receptor (NOR)-1 or retinoic acid related orphan microarray scanner (Clontech) at 532 and 635 nm receptor γ (RORγ; also called as retinoid Z receptor for Cy3 and Cy5, respectively. Images were γ) mRNA was detected in placentae with male analyzed using Atlas navigator software (Clontech) embryos. In placentae with female embryos, on an IBM compatible computer as suggested in the however, BPA treatment increased NOR-1 mRNA manufacturer’s instructions. All the above expression (35% in comparison with the samples of procedures were performed according to the vehicle controls), but BPA treatment decreased manufacturer’s protocols. RORγ mRNA expression (–21%). No expression of estradiol receptor (ER)-β or (LXR) α Statistical analysis mRNA was detected in placentae with female To assess the degree of treatment-induced embryos. In placentae with male embryos, BPA variation in gene expression measurements, treatment increased both of them (64 and 65%, Pearson correlation coefficients r were calculated to respectively). BPA treatment increased the compare the similarity between sample expression of (PR) mRNA in measurements within and across hybridizations placentae with male embryos (27%). In contrast, in using StatView-4.5 program (Abacus Concepts, placentae with female embryos, BPA decreased PR Berkely, CA) on a Macintosh computer. mRNA expression (–25%). In placentae with male Correlation coefficients were normalized with a embryos, BPA exposure decreased COUP-TF α, Fisher’s z transformation to calculate 95% nuclear factor (GCNF), steroidogenic confidence intervals surrounding r. Differentially factor-1 (SF-1; also called as Fushi Tarazu factor-1) expressed genes were identified by computing and photoreceptor-specific nuclear receptor (PNR) paired t statistics and P values. When significant (–41, –41, –41 and –7%, respectively), but those difference (P<0.05) was identified between the BPA expression levels in placentae with female embryos treated group and the vehicle control group, the were increased by BPA administration (15, 8, 34 change in expression level of mRNA in the BPA and 83%, respectively). group was represented as percent of increase or decrease vs vehicle control group. Effect of BPA on the expression of non-nuclear receptor protein mRNAs Notable changes in mRNA expression levels of six non-nuclear receptor proteins in murine placentae were induced by BPA exposure (Table 2). Briefly, in placentae with female embryos, BPA treatment increased the expression of fast skeletal 332 IMANISHI et al.

Table 1. Effect of bisphenol A on expression of nuclear receptor mRNAs in murine placentae with male or female embryos

Nuclear receptors Placentae with Placentae with male embryos female embryos Thyroid β ND ND Peroxisome proliferator activated receptor α ND ND Peroxisome proliferator activated receptor γ ND ND Constitutive androstane receptor ND ND Farnesoid X receptor ND ND Chicken ovalbumin upstream promoter ND ND transcription factor β Testis receptor β ND ND Estrogen related receptor γ ND ND Aryl hydrocarbon receptor ND ND Small heterodimer partner ND ND Dosage-sensitive sex reversal ND ND Neuron-derived orphan receptor 1 ND 35 Retinoic acid related orphan receptor γ ND –21 Estradiol receptor β 64 ND Liver X receptor α 65 ND Progesterone receptor 27 –25 Chicken ovalbumin upstream promoter –41 15 transcription factor α –41 8 –41 34 Photoreceptor-specific nculear receptor –7 83 Each value represents mean value of percentage of increase vs each vehicle control.

Table 2. Effect of bisphenol A on expression of non-nuclear receptor mRNAs in murine placentae with male or female embryos Placentae with Placentae with Genes male embryos female embryos Fast skeletal troponin C –20 340 Probasin –41 220 RNA-specific adenosine deaminase –11 220 ADAM25/testase 2 –74 32 α-fetoprotein –60 –24 Kinesin light chain 1 –70 –10 Each value represents mean value of percentage of increase vs each vehicle control. troponin C, probasin, RNA-specific adenosine Discussion deaminase and ADAM25/testase 2 (340, 220, 220 and 32%, respectively). However, the expression Nuclear receptors including ERs bind to levels of these mRNAs were decreased by BPA lipophilic ligands (ex. steroid hormones, retinoic treatment in placentae with male embryos (–20, acid metabolites, thyroid hormone etc.) and act as –41, –11 and –74%, respectively). mRNA transcriptional factors [11–13]. The nuclear expression levels of α fetoprotein (–60 and –24% in receptors play crucial roles in embryogenesis, cell placentae with male and those with female growth, cell differentiation, cell death and embryos, respectively) and kinesin light chain 1 numerous physiological events. To date, many (–70 and –10% in those with male and female members of nuclear receptor families have been embryos, respectively) were decreased by BPA found [11–13], but the endogenous ligands of most treatment. of these orphan receptors have not been identified EFFECT OF BPA ON MOUSE PLACENTA 333 yet. BPA may bind with not only ERs but also detected. BPA exposure decreased the COUP-TFα unknown nuclear receptors, with the result, that mRNA expression level in placentae with male the physiological activities of BPA are induced. embryos, but the COUP-TFα mRNA expression Thus, revealing the effects of BPA on the expression level in placentae with female embryos was of nuclear receptor mRNAs is important for increased by BPA administration. Since COUP understanding the mode of action of BPA. family receptors act as a modulator for nuclear BPA exposure increased mRNA expression of receptor signaling, BPA effects on the expression of ERβ, a class III nuclear receptor for estradiol-17β COUP-TFα mRNA may cause critical toxicity in [13], in placentae with male embryos, but no placentae. As with COUP-TFα, BPA decreased the expression was detected in placentae with female expression level of GCNF, a class VI orphan nuclear embryos. In murine placentae, ERβ mRNA receptor [13], SF-1, which acts as a transcriptional expression was demonstrated in decidual cells [17]. factor of cholesterol side-chain cleavage Moreover, BPA increased mRNA expression of PR, cytochrome P450 (steroidogenic acute regulatory a class III nuclear receptor [13], in placentae with protein) and of other steroidogenic factors [13, 23, male embryos and decreased it in placentae with 24], and PNR, which is a class II orphan nuclear female embryos. These findings reveal that BPA receptor expressed in photoreceptors of retinae effects on mRNA expression of ERβ and PR are [25], in placentae with male embryos, and increased different between placentae with male and female them in placentae with female embryos. The embryos. ERβ induced by BPA may cause higher regulation of mRNA expression of these nuclear reactivity/sensitivity to endogenous estradiol-17β, receptors may be sex dependent, at least in suggesting BPA indirectly shows an estrogenic placentae. GCNF plays critical roles in maturation effect in placentae with male embryos. of germ cells and normal Progesterone is a key hormone for maintaining [26–29]. A recent targeted mutation study has pregnancy in mammals. Since BPA changes the shown that GCNF is required for anteroposterior expression levels of PR mRNA in placentae, BPA axis formation and somatogenesis in mice [29]. In may affect the function of placentae in late murine placentae, GCNF is specifically expressed pregnancy and disrupt the embryonic by the labyrinthine layer [30], but the physiological development. As with ERβ, LXRα, which is a class role of GCNF in placentae remains to be studied. I nuclear receptor and whose endogenous ligand is No expression of SF-1 mRNA was reported in oxysterol [13], was also detected only in placentae human trophoblasts [31], rat choriocarcinoma with male embryos. In contrast, NOR-1 and RORγ, Rcho-1 cells and rat mid-pregnancy placentae [32], ligand unknown orphan receptors, were detected but we observed SF-1 mRNA expression in murine only in placentae with female embryos. LXRα placentae at 18.5 dpc. SF-1 may play key roles in regulates various physiological functions, mouse late-pregnancy placentae, and its mRNA cholesterol homeostasis, lipid metabolism, expression is disrupted by BPA exposure. In adult adipogenesis, development of the central nervous animals, PNR mRNA is definitely expressed in system and immunity [15, 18, 19], and is activated retinae, but strong expression of it was confirmed by endogenous cholesterol metabolites. Increase of in placentae with female embryos, and BPA LXRα mRNA expression induced by BPA exposure decreased this expression. Although the detailed may cause the disruption of steroid metabolism in function of PNR in placentae is unknown, PNR placentae, which plays very important roles for may play important roles in placentae with female normal embryonic development and for embryos, and BPA disrupts its function. It is maintenance of pregnancy. supposed that these orphan receptors also act as COUP-TFα, a class II orphan nuclear receptor modulators, activators and/or repressors, for [13], plays a critical role in the development of the nuclear receptor signaling [13] and play crucial nervous system [20, 21] and also acts as a regulator roles in placentae. Differences in the effect of BPA in sex differentiation [22] through interactions with on the expression of these orphan receptor mRNAs promoters of DAX-1, a class 0 orphan nuclear between the placentae with male embryos and receptor [13], and SF-1, a class V oxysterol receptor those with female embryos are interesting and [13]. In placentae with male and female embryos, important for understanding the endocrine high expression of COUP-TFα mRNA was disrupting effect of BPA. 334 IMANISHI et al.

In murine placentae, mRNAs of AhR, which play expression of α-fetoprotein induced by BPA a crucial role in dioxin toxicity, were not detected. exposure may strengthen estrogen activity in AhR knock-out mice showed no toxic effect to embryos. Fast skeletal troponin C and kinesin light dioxin administration [33], indicating dioxin has no chain-1 are strongly expressed in placenta and BPA toxic effect on placentae. Moreover, as mentioned treatment strongly disrupted the expression of above, BPA showed varied effects on mRNA these mRNAs, indicating that BPA affects cellular expression in murine placentae, so BPA toxicity is skeletons and inner cellular transport. not dependent on AhR. The present results imply some novel Additionally, in the present study, we detected mechanisms of BPA toxicity in murine placentae, remarkable changes in mRNA expression of non- especially since there are embryo-sex dependent nuclear receptor proteins. BPA showed anti- differences. However, there is a long way to go to androgenic activity in an in vitro yeast based assay reach complete comprehension of BPA toxicity. [34], but showed androgenic activity, assessed as Recently, it has become known that the modulatory increased cell proliferation in a prostate domain of nuclear receptors including orphan adenocarcinoma cell line, LNCaP cell [35]. Kim et nuclear receptors is the target for phosphorylation al. [36], however, reported that BPA has no mediated by different signaling pathways, and androgenic or anti-androgenic activity. such modification can significantly affect Interestingly, we showed in the present study that transcriptional activity [40–42]. For example, BPA exposure decreased mRNA expression levels nuclear receptors such as the ERs are of probasin, an androgen-regulated protein [37] in phosphorylated at serine and/or threonine placentae with male embryos. In contrast, BPA residues by the mitogen-activated protein kinase largely increased the expression in placentae with (MAPK) in vitro [40], and in cultured cells treated female embryos, indicating that BPA has bifacial with growth factors that stimulate the Ras-MAPK androgenic effects on murine placentae. BPA cascade, and this phosphorylation enhances decreased the mRNA expression level of RNA- transcriptional activity. To elucidate BPA toxicity, specific adenosine deaminase, which catalizes the changes in mRNA expression levels of such adenine of mRNA into inosine [38] in placentae modulation factors are being examined in our with male embryos, but increased it in placentae laboratory. with female embryos, indicating that BPA disrupts the editing of mRNAs. Similarly, BPA exposure decreased the mRNA expression level of Acknowledgements ADAM25/testase 2, a critical factor of spermatogenesis and fertilization in male animals This work was supported by Grant-in-Aid for [39], whose function in placenta is still unknown in Creative Scientific Research (13GS0008) and for placentae with male embryos, but increased it in Scientific Research on Priority Areas (A) (13027241) placentae with female embryos. α-fetoprotein, a to N. M. from the Ministry of Education, Culture, major fetal serum protein, binds with estrogens and Sports, Science and Technology of Japan. regulates estrogen activities. A decrease in mRNA

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