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Identification of estrogen-regulated genes by microarray analysis of the uterus of immature rats exposed… Hong, Eui-Ju; Park, Se-Hyung; Choi, Kyung-Chul; Leung, Peter C; Jeung, Eui-Bae Sep 29, 2006

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ralReproductive Biology and ssBioMed CentEndocrinologyOpen AcceResearchIdentification of estrogen-regulated genes by microarray analysis of the uterus of immature rats exposed to endocrine disrupting chemicalsEui-Ju Hong1, Se-Hyung Park2, Kyung-Chul Choi2, Peter CK Leung2 and Eui-Bae Jeung*1Address: 1Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, 361-763, Republic of Korea and 2Department of Obstetrics and Gynecology, Child and Family Research Institute, University of British Columbia, Vancouver, BC, V6H 3V5, CanadaEmail: Eui-Ju Hong - euijuhong@hotmail.com; Se-Hyung Park - sehyungpark@hotmail.com; Kyung-Chul Choi - kchoi@cw.bc.ca; Peter CK Leung - peleung@interchange.ubc.ca; Eui-Bae Jeung* - ebjeung@chungbuk.ac.kr* Corresponding author    AbstractEnvironmental estrogenic compounds which bind to the estrogen receptor (ER) can block or alterendogenous functions of estrogen in reproductive and developmental stages. A microarraytechnology is a very valuable method for the prediction of hormone-responsive activities in variousgene expressions. Thus, we investigated the altered gene expression by estrogen and endocrinedisruptors (EDs) using microarray technology in the uterus of immature rats. In this study, theexpression levels of only 555 genes (7.42%) among the 7636 genes spotted on microarray chipswere enhanced by more than two-fold following treatment with estradiol (E2), suggesting thatdirect or rapid response to E2 is widespread at the mRNA levels in these genes. In addition,elevated expression levels of the genes (over 2-fold) were observed by diethylstilbestrol (DES;9.01%), octyl-phenol (OP; 8.81%), nonyl-phenol (NP; 9.51%), bisphenol-A (BPA; 8.26%) or genistein(9.97%) in the uterus of immature rats. The expression levels of representative genes, i.e., calbindin-D9k (CaBP-9k; vitamin D-dependent calcium-binding protein), oxytocin, adipocyte complementrelated protein (MW 30 kDa), lactate dehydrogenase A and calcium binding protein A6 (S100a6;calcyclin), were confirmed in these tissues by real-time PCR. In addition, the mRNA levels of thesegenes by real-time PCR were increased at follicular phase when E2 level was elevated duringestrous cycle of adult female rats. In conclusion, these results indicate distinct altered expressionof responsive genes following exposure to E2 and estrogenic compounds, and implicate distincteffects of endogenous E2 and environmental endocrine disrupting chemicals in the uterus ofimmature rats.BackgroundEnvironmental chemicals that disrupt endocrine functioningly assumed for their possible participation in inducingestrogenic effects. Furthermore, they are proposed to pos-Published: 29 September 2006Reproductive Biology and Endocrinology 2006, 4:49 doi:10.1186/1477-7827-4-49Received: 17 June 2006Accepted: 29 September 2006This article is available from: http://www.rbej.com/content/4/1/49© 2006 Hong et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 12(page number not for citation purposes)are suspected for their adverse effects on the reproductivesystem in wild animals and humans and are being increas-sess hormone-like properties, i.e., mimicking natural hor-mones, inhibiting the action of hormones, and inducingReproductive Biology and Endocrinology 2006, 4:49 http://www.rbej.com/content/4/1/49abnormal gene expressions. Environmental estrogeniccompounds that bind to the estrogen receptors (ERs) canblock or alter endogenous estrogen functions in reproduc-tive and developmental stages via an ER-mediatedresponse [1]. Examples of suspected environmental estro-genic chemicals (endocrine disruptors; EDs) include poly-chlorinated hydroxybiphenyls, DDT and its derivatives,certain insecticides and herbicides (kepone and methoxy-chlor), plastic components (bisphenol A) and some com-ponents of detergents and their biodegradation products(alkylphenols etc.). Although the activity of most of theseenvironmental estrogens is low when compared to endog-enous or synthetic estrogens (17β-estradiol; E2 or ethi-nylestradiol), dietary or environmental exposurescenarios that led to the detection of significant quantitiesof these substances in human urine and tissue samplehave been described [2].The profound effects of E2 on cell growth, differentiation,and general homeostasis of reproductive and other sys-tems are mediated mainly by the temporal and cell type-specific expression of different genes, whose products arethe molecules controlling these molecular events [3,4]. Inrats, the concentration of E2 is consistently low through-out neonatal development and starts to increase after day28 of age [5]. The uterus and ovaries are two of the mostsensitive tissues to E2, and both tissues express two formsof ER, ERα and ERβ. In particular, ERα is predominantlyexpressed in uteri while ERβ is expressed in ovaries [6,7].Diethylstilbestrol (DES) is a synthetic estrogen which caninduce various reproductive alterations in humans [8,9]and mice [10-12]. Numerous reproductive changes inwildlife populations can be caused by EDs which resem-ble DES [2]. Alkylphenols (APs), such as octyl-phenol(OP) and nonylphenol (NP), were reported to binddirectly to the ER in trout, stimulate citelogenin geneexpression in trout hepatocytes, be mitogenic in MCF-7cells and stimulate transcription in mammalian cells viaER [12]. In vitro studies revealed that OP and NP are themost potent estrogenic alkylphenols, and the potency ofOP has been shown to be approximately 10-3~10-7 relativeto 17β-estradiol [12-15]. Furthermore, the binding affin-ity of BPA indicated that it is approximately 10000-foldless potent than E2 and 20000-fold less potent than DESfor both ERα and ERβ receptors [16]. In vivo estrogenicactivities (400–1000 mg/kg/day) in immature or ovariec-tomized rats and mice have been recognized. Thus, theconcentrations of EDs such as OP, NP, and BPA, in thepresent study are expected to have similar effects as thoseof steroid hormones. In addition, APs are weakly estro-genic in traditional uterotropic assays as evidenced by theincrease in uterus weight [17]. In vitro assays, E2 has beendemonstrated to induce maximal proliferation of MCF-7chemicals at 1 and 10 μM, respectively. Treatments withOP and NP inhibited the binding affinity of E2 to ER inMCF-7 cells by a competitive ER binding assay [18].Bisphenol A (BPA) is a particularly important environ-mental estrogen. Not only in it widespread in the environ-ment, but it is commonly ingested by humans, beingreleased by polycarbonate plastics, the lining of food cans,and dental sealants [19]. BPA only acts as an agonist ofestrogen via ERβ whereas it has dual actions as an agonistand antagonist in some types of cells via ERα Thus, theactivity of BPA may depend on the ER subtype and the tis-sue involved [20]. ERβ has a higher relative binding affin-ity to genistein (Gen) in in vitro assays compare with ERα[6]. Genistein is readily absorbed [21,22] and act as apharmacological estrogen both in vitro and in vivo via ERs[23-25]. Although an actual impact of environmentalestrogens on reproductive health is not well defined thor-oughly, these chemicals have the potential to disrupt thereproductive system and confirm its estrogen-like activityin vitro [6]. Therefore, the changes in the expression ofestrogen target genes are considered to be a useful indexfor evaluating the estrogenic activity of synthetic com-pounds. However, it is difficult to predict the full range ofeffects of estrogenic compounds from changes in theexpression levels of only well-known estrogen targetgenes.Reproductive organs are highly susceptible to hormonalexposure during organ development and sex differentia-tion. In our previous studies, we showed that NP, OP andBPA have estrogenic activity, resulting in uterotrophiceffects in the uterus of rats treated with these compounds[26,27]. In addition, we demonstrated that the CaBP-9kgene is not expressed in the uterus of immature rats,which do not obtain estrogen from ovaries, and that it isregulated through the binding of the ER/estrogen complexto the estrogen response element (ERE) in rats [26,28].CaBP-9k mRNA in the uterus is known to fluctuate duringestrous cycle of rats depending on serum estrogen level.CaBP-9k mRNA at diestrus was not detectable, butincreased at proestrus and reached the highest level atestrus and then decreased as metestrus [29]. The aim ofthis study was to identify estrogen-responsive genes by E2and endocrine disrupting chemicals in the uterus of ratsand to determine whether estrogen responsive genes aredifferentially regulated following exposure to these estro-genic compounds by microarray analysis and real-timePCR. Finally, we evaluated the correlations between E2-induced and EDs-induced gene profiles, and confirmedthe biomarker among altered gene expression for screen-ing potential EDs.Page 2 of 12(page number not for citation purposes)cells at 1 nM concentration, and OP and NP have beenfound to be considerably potent compounds as estrogenicReproductive Biology and Endocrinology 2006, 4:49 http://www.rbej.com/content/4/1/49Materials and methodsAnimals and treatmentsImmature Sprague-Dawley rats (2-weeks of age) withdams were obtained from Orient Co, Ltd. (Gyeonggi-do,Korea). All animals were housed in polycarbonate cages,and used after acclimation to an environmentally control-led room (temperature: 23 ± 2°C, relative humidity: 50 ±10%, frequent ventilation and a 12 h day/night cycle). Todetermine the effect of EDs, each group of five animals(14-days old) was injected subcutaneously (sc, 0.1 ml perrat) with E2 (40 μg/kg BW; Sigma-Aldrich Corp, St. Louis,MO, USA), DES (500 μg/kg BW; Sigma-Aldrich Corp), OP(600 mg/kg BW; Fluka Chemie, Buchs, Switzerland), NP(600 mg/kg BW; Sigma-Aldrich Corp), BPA (600 mg/kgBW; Sigma-Aldrich Corp), and genistein (40 mg/kg BW;Sigma-Aldrich Corp) by a single dose daily for 3 days andeuthanized 24 h after final injection. All chemicals weredissolved in corn oil (Sigma-Aldrich Corp) as a vehicle.The rats were injected with E2 (n = 3) as a positive controlor corn oil (n = 3) as a negative control daily for 3 days.The uteri were washed in cold sterile 0.9% NaCl solution(0.9% normal saline) and used for microarray and RT-PCR analyses. All rats were euthanized at 24 h after theinjection.It is believed that exposure to DES or endocrine disruptorsmight be detrimental to development and differentiation,although the effects may not be apparent until adulthood.In the present study, treatment with estrogenic com-pounds induced a significant increase in the mRNAexpression of specific genes in the neonate rat uterus. Toconfirm altered gene expression profile by E2 or EDs inthe uterus of immature rats, adult female rats were alsoemployed to examine the elevated endogenous E2 in theinduction of these genes at proestrus and estrus duringestrous cycle. Estrous cycle was determined by the obser-vation of three types of the cells derived from the vaginalsmears: leukocytes, cornified cells and nucleated epithe-lial cells. Four stages of the estrous cycle (proestrus, estrus,metestrus and diestrus) were determined as following cri-teria: proestrus was characterized by many epithelial cellsand few leukocytes; estrus by many cornified cells and noleukocytes; metestrus by some cornified cells and manyleukocytes; and diestrus by few epithelial cells and manyleukocytes. All animals were smeared daily, and the ratsthat had three regular cycles were selected. Forty femalerats were randomly assigned to four groups according tothe respective phase of estrous cycle (proestrus, estrus,metestrus and diestrus, n = 10 each) and euthanizedimmediately. The uteri were washed in cold sterile 0.9%NaCl solution (0.9% normal saline) and used for RNAextraction. All experimental procedures and animal usewere approved by the Ethics Committee of the ChungbukRNA isolation and cDNA microarray analysisTotal RNA was extracted with Trizol (Invitrogen, Carlsbad,CA, USA) according to manufacturer's suggested proce-dure, and purified using RNeasy total RNA isolation kit(Qiagen, Valencia, CA, USA) according to the manufac-turer's instructions. DNA was digested using an RNase-free DNase set (Qiagen) during RNA purification. TotalRNA was quantified by spectrophotometer and its integ-rity was assessed by running on a 0.8% agarose gel. Tomake cDNAs from mRNAs for microarray analysis, thesame quantity of each RNA sample from the treatedgroups (n = 5) or control groups (n = 3) was pooled. AcDNA microarray consists of 7636 cDNA spot includingIncyte clones, housekeeping genes and Arabidopsis DNAas controls. The experiments were performed on the ratcDNA microarray prepared as previously described [30].The PCR reactions were prepared according to the stand-ard protocol and reaction mixtures were subjected to beamplified at 35 cycles. The primer pair used for amplifica-tion was overlap primer-1 (5'-AAT TAA CCC TCA CTAAAG GG-3') and overlap primer-2 (5'-GTA ATA CGA CTCACT ATA GGG C-3'). The size and amount of the PCRproducts were verified on 1% agarose gel. The PCR prod-ucts were purified by ethanol precipitation, then resus-pended in 15 μl of hybridization solution (GenoCheck,Korea), and spotted onto CMT-GAPS α silane slide glass(Corning, NY) with a pixsys 5500 arrayer (Cartesian Tech-nologies, CA) using 16-Stealth Micro spotting pins. Theprinted slides were processed according to CMT-GAPS αslide protocol. Briefly, the spots were re-hydrated with 1 ×SSC for 1 min and then DNA linked using a UVcrosslinker (Stratagene, CA). The slides were soaked in thesuccinic anhydride/sodium borate solution for 15 minwith gentle agitation and then transferred to a 95°C waterbath for 2 min. The slides were quickly transferred to 95%ethanol for 1 min and then dried using a centrifuge at3000 rpm for 20 sec.Hybridization with fluorescent DNA probeTotal RNA was extracted from the treated and untreatedtissues from immature or adult rats at the indicated timepoints using the TRI-REAGENT (MRC, OH) according tothe manufacturer's instructions. Fluorescent labeledcDNA probes were prepared from 50 §P of total RNA byoligo (dT)18-primed polymerization using SuperScript αreverse transcriptase (Invitrogen, NY) in a total reactionvolume of 30 μl. The reverse transcription mixtureincluded 400 U Superscript RNase H-reverse transcriptase(Invitrogen), 0.5 mM dATP, dTTP and dGTP, 0.2 mMdCTP and 0.1 mM Cy3 or Cy5 labeled dCTP (NEN LifeScience Product Inc.). After reverse transcription, the sam-ple RNA was degraded by adding 5 μl of stop solution (0.5M NaOH/50 m EDTA) and incubating at 65°C for 10Page 3 of 12(page number not for citation purposes)National University. min. The labeled cDNA mixture was then concentratedusing the ethanol precipitation method. After determin-Reproductive Biology and Endocrinology 2006, 4:49 http://www.rbej.com/content/4/1/49ing the target cDNA quality, cDNA samples derived fromthe pooled uteri of five individual neonate rats from eachtreated group were selected and hybridized. The concen-trated Cy3 and Cy5 labeled cDNAs were resuspended in10 μl of hybridization solution (GenoCheck). After twolabeled cDNAs were mixed, the mixture was denaturized95°C for 2 min and then incubated in 45°C water cham-ber for 20 min. The cDNA mixture was then placed atthree spotted slide positions and covered by a cover slip toassess the overall quality of each sample. The slides werehybridized for 12 h at 62°C hybridization chamber. Thehybridized slides were washed in 2 × SSC, 0.1% SDS for 2min, 1 × SSC for 3 min, and then 0.2 × SSC for 2 min atroom temperature. The slides were centrifuged at 3000rpm for 20 sec to be dried.Scanning and image analysisHybridized slides were scanned with the Axon Instru-ments GenePix 4000B scanner and the scanned imageswere analyzed with the software program GenePix Pro 5.1(Axon, CA) and GeneSpring 7.1 (Sillicongenetics, CA). Inorder to allow algorithm to eliminate all bad spots, nodata points were eliminated by visual inspection from theinitial GenePix image. For signal normalization, house-keeping genes (β-actin) and positive control genes (A.thaliana genes) were spotted onto each slide. The signalsof these spots were used for normalization. To filter outthe unreliable data, spots with signal-to-noise (signal –background – background SD) below 100 were notincluded in the data. Data were normalized by global,lowess, print-tip and scaled normalization for data relia-bility. Data were sorted of above 2-fold altered genesusing GeneSpring 7.1 (Sillicongenetics) and a hierarchicalclustering analysis was performed using Pearson correla-tion. The statistical significance of differential expressionwas assessed by computing a q-value for each gene. Todetermine the q-value, we used a permutation procedure,and for each permutation a two-sample t-statistic wascomputed for each gene. The result was considered signif-icant when the logarithmic gene expression ratio of threeindependent hybridizations was more than twofold thedifference in the expression level. The accuracy of micro-array analysis in this study was confirmed by real-timePCR.Confirmation of microarray analysis with real-time PCRThe standard curve was generated for a standard RNApreparation by serial dilution (1, 1/10, 1/100, 1/1000, 0).A real-time PCR reaction was carried out in a 25 μl finalvolume containing 12.5 μl of 2× premix (TaKaRa BioInc.), 0.3 μl of each of forward and reverse primers, 1 μl ofcDNA, and distilled water up to 10.9 μl. The oligonucle-otide sequences of primers were employed to detect vari-ous genes as shown Table 1. Polymerase chain reaction ofamplification using the Smart Cycle System (TaKaRa BioInc.) began with an initial denaturation at 95°C for 30sec. Each of the 35 amplification cycles consisted of dena-turation at 95°C for 5 sec, annealing at 55°C for 15 sec,and extension at 72°C for 15 sec. Relative expression lev-els of each sample were calculated based on the cyclethreshold (Ct) and monitored for an amplification curve.The PCR amplification curves were evaluated by fluores-cence of the double-stranded DNA-specific dye, SYBRGreen, versus the amount of standardized PCR product.All gene expressions were normalized to Cytochrome oxi-dase subunits I mRNA (IA, Housekeeping gene) as con-trols.Data analysisData are presented as the mean ± SD. The data were ana-lyzed by non-parametric procedure of the Kruskal Wallistest, followed by Dunnett's test for two-pair comparisons.The each value of Dunnett's test was converted to rank forstatistical analysis. All statistical analyses were performedwith SAS. P < 0.05 was considered statistically significant.ResultsGlobal analysis of estrogen-regulated genes compared with endocrine disruptorsFollowing individual treatment of immature rats withEDs, total RNAs from the uteri were isolated and proc-essed by microarray analysis as described. Based on thenumber of genes expressed in control versus treated sam-ples, the overall pattern of altered gene expression wassimilar between the control (vehicle-treated) and estro-genic compound-treated (E2, DES, OP, NP, BPA or genis-tein) rats. Although the number of genes whoseexpression is altered by any chemical tested is modest,there is substantial change in the gene expression alteredor modified by exposure to each compound. The meanTable 1: Oligonucleotide sequences with predicted sizes of respective PCR productAccession No. Gene name Forward sequence Reverse sequence SizeNM_012521 vitamin D-dependent calcium binding protein 5'-aagagcatttttcaaaaata-3' 5'-gtctcagaatttgctttatt-3' 314NM_012996 Oxcytocin 5'-gaccttcatcatcgtactgg-3' 5'-gagttgctcttcttgctgac-3' 275NM_144744 Adipocyte complement related protein of 30 kDa 5'-gtgttcttggtcctaagggt-3' 5'-tgtacaccgtgatgtggtag-3' 287NM_017025 Lactate dehydrogenase A 5'-aggtgacactgactcctgac-3' 5'-gtgggattgtcacactaacc-3' 285Page 4 of 12(page number not for citation purposes)AF140232 S100 calcium binding protein A6 (calcyclin) 5'-cttctcgtggctatcttcc-3' 5'-actggacttgactgggatag-3' 289Reproductive Biology and Endocrinology 2006, 4:49 http://www.rbej.com/content/4/1/49values of the intensities of each spot in this study were cal-culated, and are plotted in Fig. 1. The data shown in Fig. 1are typical of the three self vs. self comparisons, whichshowed that a more than 2-fold change would be neces-sary to overcome the intrinsic variability introduced bythe hybridization procedure. For this reason, the data wasfurther filtered to select for genes that showed an absolutedifferential of at least two-fold or greater. Of the 7479genes examined, the change in the mRNA expressions of85 genes was listed in the Additional file 1. In addition,treatment with E2 resulted in the induction of 555 genesby more than two-fold. About 7.42% of all genes were up-regulated by E2 (Fig. 2A), causing no significant differencein the expression of the remaining 92.58% of the genes.As shown in Fig. 2A, the expression level significantlyincreased in the uterus when treated with DES (9.01%) orOP (8.81%). Furthermore, Table 2 shows the common 21genes induced by E2, DES, and OP in the uterus of imma-ture rats. In addition, a single treatment of NP (9.51%),BPA (8.26%) or genistein (9.97%) induced a significantincrease in gene expressions in this tissue (Fig. 2B), andthere are 30 common genes by these EDs (Table 3). Theseresults indicate that significant gene profile is altered inthe uteri by E2 and EDs.In this study, we extended a large-scale of microarray anal-ysis to six other estrogenic compounds, and identified cru-cial estrogen-responsive genes, which were eithercommon in all or specific to each or some of the com-pounds. The results with all six compounds are shown inFig. 2C using a hierarchical clustering algorithm of themean values of triplicate slides and a pseudo-color visual-ization matrix. The gene profile in red color shows ahigher expression than the control, while the gene profileA scattergram of gene expression analyzed by DNA microarrayFigure 1A scattergram of gene expression analyzed by DNA microarray. For each gene, the relative mRNA level in the control is given on the x axis and the expression level for the same transcript in the experimental sample (estrogenic compound exposed) is plotted on the y axis. Each graph displays two lines indicating 2-fold up-regulation or down-regulation in the expression level of Page 5 of 12(page number not for citation purposes)each individual probe set comparing treated vs. control sample. The linear line (P < 0.05) was fitted to the microarray data.Reproductive Biology and Endocrinology 2006, 4:49 http://www.rbej.com/content/4/1/49in green color shows a lower expression (Fig. 2C). Thesegenes might be universal estrogen – responsive genesamong various tissues. These compounds exhibited a rea-sonable hierarchical clustering; DES or OP resembled toE2. However, NP-induced gene profile is similar to that ofBPA or genistein.Confirmation of estrogen-regulated genes by real-time PCRuterus are listed in Fig. 3. The expression patterns of sev-eral genes up-regulated by estrogen were confirmed to ver-ify the results of the microarray analysis using real-timePCR analysis. The rats were injected with endocrine dis-ruptors to determine their effect on the induction of calbi-ndin D9k (CaBP-9k) mRNA in the immature uterus. Theexpression level of CaBP-9k mRNA significantly increasedwhen treated with OP (12.2-fold vs. vehicle), NP (14.2-fold) and BPA (1.6-fold) for 3 days (Fig. 3). In addition,Venn diagram showing the number of genes induced by E2 and EDsFigure 2Venn diagram showing the number of genes induced by E2 and EDs. The altered gene profiles by E2, OP and DES (overlapped 21 genes; A) or NP, BPA and Gen (overlapped 30 genes B), respectively, were summarized. A hierarchical clustering analysis (C) was performed following treatments with E2 and other EDs in the uterus of immature rats. Two-dimensional hierarchical clustering was applied to the expression data from 7.5 k genes, which showed significant changes in the balanced differential expression. Increased expression levels are shown in red and decreased expression levels are shown in green.Page 6 of 12(page number not for citation purposes)The primer sets used in the RT-PCR are listed in Table 1,and the altered genes which showed a high increase in theCaBP-9k mRNA was markedly induced by a single dose ofE2 (9.1-fold) or DES (21.4-fold) as seen in Fig. 3. TheReproductive Biology and Endocrinology 2006, 4:49 http://www.rbej.com/content/4/1/49induction of uterine oxytocin by estrogenic compoundswas further assessed by real-time RT-PCR. An increase inoxytocin was observed in the uterus of rats as expectedwhen treated with a single of OP (13.6-fold), NP (17.4-fold) and BPA (1.5-fold) for 3 days Furthermore, treat-ment with a single dose of E2 (9.9-fold) or DES (30.5-fold) resulted in an increase of oxytocin in this tissue.Increased expression level of adipocyte complementrelated protein (30 KDa; Acrp30) was observed whentreated with OP (14.1-fold), NP (9.4-fold) and BPA (1.7-fold). In addition, a single dose of E2 (4.1-fold) or DES(19.1-fold) induced a marked increase in Acrp30 (Fig. 3).The treatment with E2 (2.3-fold), DES (3.1-fold), OP (2.6-fold), and NP (2.5-fold) altered the expression level of lac-tate dehydrogenase A (Ldha). As shown in Fig. 3, theexpression level of calcium binding protein A6 (S100a6)in this tissue significantly increased when treated with OP(3.1-fold) and NP (4.2-fold). In addition, a single dose ofE2 (2.8-fold) and DES (5.8-fold) treatment induced a sig-nificant increase in the expression of calcium binding pro-tein A6 mRNA in the uterus of rats. These results indicatethat estrogenic compounds in parallel with E2 in theuterus regulate the expression levels of various genes.Expression of estrogen-regulated genes during estrous cycleTo investigate the regulation of various genes in the uterusof rats during estrous cycle, the expression levels of CaBP-uterus in a follicular phase (proestrus and estrus) com-pared to a luteal phase (metestrus and diestrus) as shownin Fig. 4. The level of CaBP-9k mRNA at follicular phaseincreased up to 70.5-fold compared to that at luteal phase(Fig. 4). In parallel with CaBP-9k mRNA, the expressionlevels of oxytocin (13.4-fold, vs. luteal phase), Acrp30(3.1-fold, vs. luteal phase), Ldha (3.3-fold, vs. lutealphase), and S100a6 (2.7-fold, vs. luteal phase) mRNAs atfollicular phase were up-regulated in this tissue, indicat-ing that they are dominantly expressed during follicularphase and weakly detected at luteal phase in the uterus ofmature rats.DiscussionThe microarray technique is a very valuable method forthe prediction of hormone-responsive activity in variousgene expressions. Estrogen plays an important role in var-ious molecular events, but the molecular mechanismsthat are regulated by estrogen in the uterus remain largelyunknown. Therefore, the identification of estrogen-induced gene expression is essential to understandinghow estrogenic compounds regulate uterine physiologyand pathology at the cellular level. In the present study,microarray analysis showed that only 7.42% (555 genes)of all genes spotted on the microarray slide were up-regu-lated by more than two-fold following estrogen treatment,suggesting that direct or rapid responses to estrogen iswidespread at the mRNA level. Furthermore, treatmentsTable 2: Altered genes induced by E2, DES and OP in the uterus of immature ratsAccession No. Gene name Gene symbol E2 DES OPAF154245 3.20 4.47 2.66AF494463 Seizure related 6 homolog (mouse) Sez6 3.32 2.47 3.44NM_012843 Epithelial membrane protein 1 Emp1 2.24 2.24 3.21AW915015 Discs, large homolog 3 (Drosophila) Dlgh3 2.19 3.29 2.75AF039584 Decay accelarating factor 1 Daf1 6.35 6.46 8.47NM_031029 Gamma-aminobutyric acid (GABA) A receptor, pi Gabrp 2.59 2.90 4.10NM_133296 X transporter protein 3 Xtrp3 2.25 2.11 4.12AF020045 Integrin, alpha E, epithelial-associated Itgae 6.54 3.01 2.15NM_031698 Ribophorin II Rpn2 3.45 2.09 3.16NM_031977 Rous sarcoma oncogenes Src 2.28 2.29 2.13NM_021702 Ataxin 3 Atxn3 2.61 5.68 2.79AF151982 Secretory leukocyte peptidase inhibitor Slpi 2.11 2.89 2.26NM_019188 Beta-microseminoprotein Msmb 2.29 3.14 3.42NM_019369 Inter alpha-trypsin inhibitor, heavy chain 4 Itih4 2.43 2.47 3.12U06752 Mucin 4 Muc4 3.99 5.84 4.10NM_019278 Regulated endocrine-specific protein 18 Resp18 2.37 4.15 3.78NM_017025 Lactate dehydrogenase A Ldha 2.17 2.06 2.10NM_134377 Calsyntenin 2 Cstn2 2.60 2.46 2.42AJ223184 Immunoglobulin superfamily, member 6 Igsf6 7.61 2.23 2.38AI556074 Myristoylated alanine rich protein kinase C substrate Marcks 2.08 2.80 2.43NM_145788 TRAF family member-associated Nf-kappa B activator Tank 3.00 3.05 2.82Page 7 of 12(page number not for citation purposes)9k, oxytocin, Acrp30, Ldha, and S100a6 mRNA were ana-lyzed by real-time PCR. They were highly expressed in thewith DES (9.01%), OP (8.81%), NP (9.51%) BPA(8.26%) or genistein (9.97%) showed an induction of dis-Reproductive Biology and Endocrinology 2006, 4:49 http://www.rbej.com/content/4/1/49tinct genes by more than two-fold in the uterus. Recently,gene profile patterns by microarray technology were deter-mined in the developing uterus and ovaries of Sprague-Dawley rats at different stages of the developmentexposed to graded dosages (sc) of 17alpha-ethynyl estra-diol (EE), Gen, or BPA [31,32]. This analysis of the tran-script profile in a dose-dependent manner revealed that acommon set of genes are altered, but some of the genes aredifferentially changed by these estrogenic chemicals [31].Interestingly, 592 genes of immature rat showed strongand consistent changes in expression after EE exposure[33], indicating that previously identified estrogen-sensi-tive genes are sensitive to EE exposure and that they aretargets of the estrogenic action of EDs in both the uterusand the ovary. In actual, the level of EDs in nature is lowcompared with using dosage in the present study. Forexample, several studies have been performed to assessthe presence of EDs in milk. EDs were determined in milkand infant formula at concentrations from 0.4 to 81 μg/kgin NP [34], or 0.1 to 13.2 μg/kg in BPA [35], respectively.using cDNA microarray to detect estrogenicity in vivo fol-lowing treatment with EDs.Uterine CaBP-9k may be involved in controlling myome-trial activity which is affected by the intracellular calciumlevel; however, the exact role of CaBP-9k in the uterus isstill under investigation by us and other research groups[36]. Recently, it has been demonstrated in our previousstudies that both CaBP-9k mRNA and protein could be anovel biomarker for estrogenic compounds in the uterusof immature rats [26,27]. Based on the previous results,the present study was performed to further investigateEDs-induced specific gene expression in the uterus follow-ing treatment with DES, E2 and estrogenic compoundssuch as OP, NP, BPA and genistein. Thus, the expressionlevels of CaBP-9k mRNA were analyzed to confirm theestrogenic effect of these compounds in this tissue. E2 is amajor factor controlling CaBP-9k gene expression in therat uterus. The CaBP-9k gene is not expressed in the uterusof mature ovariectomized and immature rats which doTable 3: Altered genes induced by NP, BPA, and Gen in the uterus of immature ratsAccession No. Gene name Gene symbol NP BPA GenNM_017224 Solute carrier family 22 (organic anion transporter), member 6 Slc22a6 2.95 2.09 2.12NM_012500 N-acylaminoacyl-peptide hydrolase Apeh 3.93 2.53 2.08U12571 Rabphilin 3A homolog (mouse) Rph3a 2.48 2.04 4.11L34821 Aldehyde dehydrogenase family 5, subfamily A1 Aldh5a1 5.27 2.33 2.77AF053317 Solute carrier family 21, member 1 Slc21a1 2.05 2.21 3.82AF115282 Inhibitor of kappaB kinase beta Ikbkb 2.19 2.58 3.64NM_024484 Aminolevulinic acid synthase 1 Alas1 15.07 3.65 5.16NM_017208 Lipopolysaccharide binding protein Lbp 2.68 12.17 4.15NM_133392 Serine/threonine kinase 17b (apoptosis-inducing) Stk17b 3.12 3.91 2.18X55969 Apolipoprotein B Apob 2.31 2.41 2.35NM_019333 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 Pfkfb4 2.48 2.81 2.51NM_031698 Ribophorin II Rpn2 2.81 3.15 10.86AJ293948 Kelch repeat and BTB (POZ) domain containing 10 Kbtbd10 2.50 2.43 2.71D13127 ATP synthase, H+ transporting, O subunit Atp5o 2.55 3.02 2.40NM_021689 Epiregulin Ereg 2.31 3.07 2.32NM_022709 SMR2 Smr2 8.56 24.93 68.75BF549833 Transcribed locus, strongly similar to XP_130951.1 dolichyl-phosphate mannosyltransferase polypeptide 3 [Mus musculus]3.36 2.20 4.24X59290 Eph and elk-related kinase LOC60589 2.16 2.75 4.81BF521799 Transcribed locus, moderately similar to XP_525535.1 similar to Yippee-like protein 1 (DiGeorge syndrome-related protein FKSG3)5.14 2.72 3.61NM_031577 Growth hormone releasing hormone Ghrh 3.72 5.51 2.39NM_017145 Mast cell protease 1 Mcpt1 2.57 2.19 2.98U57062 Granzyme C Gzmc 6.65 2.50 2.08NM_130421 Lymphocyte cytosolic protein 2 Lcp2 2.72 2.50 2.14NM_031971 Heat shock 70 kD protein 1A Hspa1a 3.62 2.25 2.29M83680 RAB14, member RAS oncogene family Rab14 3.03 4.36 3.06AJ132846 Sodium-dependent neutral amino acid transporter ASCT2 Slc1a5 2.77 2.19 2.73NM_138532 Non-metastatic cells 7, protein expressed in Nme7 3.63 2.38 2.96AF168795 CDK107 Slfn3 3.03 3.04 6.29U22520 Chemokine (C-X-C motif) ligand 10 Cxcl10 2.05 2.19 3.87NM_031785 ATPase, H+ transporting, lysosomal (vacuolar proton pump), subunit 1 Atp6ap1 3.81 3.52 3.93Page 8 of 12(page number not for citation purposes)Although we are aware that we used the high dose of EDs,this study focused to describe estrogen specific genesnot have circulating E2 from ovaries [37]. Using microar-ray, the expression level of uterine CaBP-9k mRNA wasReproductive Biology and Endocrinology 2006, 4:49 http://www.rbej.com/content/4/1/49significantly increased when treated with E2 (4.2-fold),DES (7.32-fold), OP (3.09-fold), and NP (2.57-fold) inthe uterus of immature rats [see Additional file 1]. In preg-nant rats, a relative potency of estrogenic compoundsindicated OP = NP > BPA [38]. Despite different potencyin estrogenicity, E2 and the estrogenic compounds testedin this study induced a significant increase of CaBP-9kmRNA in the uterus of immature rats. The expression pro-file potentially provides a wealth of data about the differ-ences in gene expression between experimental samples;however, these differences do not always reflect realisticmRNA levels. The induction of uterine CaBP-9k by estro-genic compounds was further assessed by real-time RT-PCR. Although the gene expression levels by microarrayanalysis were not identical to those obtained PCR analy-sis, the expression patterns of these genes obtained bythese two types of analysis were largely similar. In agree-ment with a previous study [29], there was no change inthe expression pattern of CaBP-9k mRNA during theestrous cycle. Estrogen stimulated the number of uterineoxytocin binding sites, and oxytocin receptor mRNAexpression in ovariectomized virgin rats [39,40]. In rats,ine responsiveness occurs in parallel with increases in thenumber of uterine oxytocin binding sites [39]. This leadsto increased uterine responsiveness to oxytocin and theonset of the uterine contractions that facilitate parturition.The change in uterine responsiveness to oxytocin involvesan increase in the quantity of oxytocin receptor proteinper cell and the number of smooth muscle cells thatexpress oxytocin receptors [41]. Based on the treatmentswith EDs, increased expression levels of oxytocin mRNAwere observed when rats were treated with OP (14.76-fold) and NP (9.54-fold), and a single dose treatmentwith DES (5.70-fold) and E2 (5.83-fold) for 3 days. How-ever, treatments with BPA and genistein for 3 days failedto detect the expression level of oxytocin mRNA [see Addi-tional file 1]. These observations raise the possibility thatthe increase in the oxytocin mRNA level shown in Figure3, occurs, at least in part, as a result of the injected estro-genic compounds and that the PCR data and microarraydata are in general agreement. Furthermore, the presentstudy indicated at least that oxytocin mRNA which can beamplified with the primer set increase about 2-fold onproestrus compared with the value on metestrus. TheConfirmation of gene profiles by real-time PCR analysisFigure 3Confirmation of gene profiles by real-time PCR analysis. Relative values of expression of the altered genes quantified by real-time PCR are shown in graphs, indicating the comparison of fold change determined by real-time PCR analysis by E2, DES, OP, NP, BPA, and Gen in the uteri of immature rats. The representative genes are CaBP-9k, oxytocin, adipocyte complement related protein (MW 30 kDa), lactate dehydrogenase A and calcium binding protein A6 (calcyclin). Total RNAs from the uterus following treatment with estrogenic compounds were used to quantify altered gene expression normalized by cytochrome oxi-dase subunits I (1A) as a control.Page 9 of 12(page number not for citation purposes)during the terminal stages of pregnancy, the myometriumis extremely sensitive to oxytocin and this increase in uter-mRNA levels of oxytocin increased 2-fold between mete-strus and proestrus by Northern blot analysis duringReproductive Biology and Endocrinology 2006, 4:49 http://www.rbej.com/content/4/1/49estrous cycle, while oxytocin binding increased more than10-fold within this same interval in the uterus of rats [40].In the present study, the expression level of adiponectin(adipocyte complement-related protein of 30 kDa,Acrp30) in the uterus of rats increased in microarray anal-ysis following with E2 (5.86-fold), DES (6.26-fold), OP(5.97-fold), and NP (10.05-fold) treatments. Using real-time PCR analysis, we confirmed the expression of Acrp30mRNA in the neonatal uteri following injection with EDs.Although the rates of Acrp induction were not identicalfrom microarray analysis, treatment with E2, DES, OP andNP resulted in significant increases in Acrp30 mRNA. Fur-thermore, our results showed that Acrp30 fluctuated dur-ing the estrous cycle, suggesting that steroid hormonesplay a role in the regulation of Acrp30. Acrp30 isexpressed exclusively in the adipocytes, its hormone isexclusively secreted by differentiated adipocytes [42], andits protein was secreted and detected in plasma [43]. Thelevels of the adipocyte hormones, leptin and adiponectin,appear to be correlated with the cell proliferation indexand sex steroid receptor abundance [44]. Furthermore,OVX in young cycling mice induced plasma Acrp30, andE2 implants reversed the effect [45]. However, our resultsindicate that E2, DES, OP and NP up-regulated its level,suggesting that it does not imply any reverse effect in thisThe lactate dehydrogenase-A (Ldha) is hormonally regu-lated in rodents and in highly expressed in the rat mam-mary gland during pregnancy and lactation, and LdhamRNA also increases during mammary gland tumorigen-esis [46]. E2 induces lactate dehydrogenase activity inMCF-7 human breast cancer cells, and is elevated in estro-gen receptor positive or progesterone receptor positivetumors [47]. The synthesis of Ldha isoenzyme was foundto increase significantly in the uterus of immature mice,and expression from the mouse lactate dehydrogenase-Apromoter fused to the cat gene in Chinese hamster ovarycells was also induced by E2 and DES [48]. Using micro-array, the expression level of uterine Ldha mRNA was sig-nificantly increased when treated with E2 (2.17-fold),DES (2.06-fold), OP (1.77-fold), and NP (2.72-fold) inthe uterus of immature rats [see Additional file 1]. In addi-tion, we further investigated the expression of LdhamRNA related to estrogen during estrus cycle. A significantincrease in Ldha mRNA was detected at proestrus andestrus compared with metestrus and diestrus. The expres-sion pattern of Ldha mRNA during the estrous cycle wasin parallel with CaBP-9k mRNA, whereas others appearedto peak at estrus compared to their level at proestrus.Calcyclin (S100A6), a small acidic protein that weighsabout 10 kDa, belongs to the S100 calcium-binding pro-tein family [49]. These family members share a commonS100 calcium-binding motif and are implicated in severalregulatory functions that include protein phosphoryla-tion, some enzyme activities, the dynamics of cytoskeletalcomponents, transcription factors, and Ca2+ homeostasis,and also cell proliferation and differentiation [50]. Theeffect of E2 on the expression level of calcyclin mRNA inuteri was further examined to elucidate a relationshipbetween expression levels of calcyclin and estrogenic com-pounds. E2 resulted in an induction of uterine calcyclinmRNA in immature rats. In addition, treatment withestrogenic compounds resulted in a significant increase inthe expression of calcyclin mRNA, whose level paralleledthose of oxytocin and Acrp30, as shown in the Additionalfile 1 and Fig. 3. After adjusting the data, it is clear thatmajor alterations in gene profiles were induced by estro-genic compounds, such as E2, DES, OP, and NP, and thatthese differences could have a significant effect on uterinefunction in reproductive tissues during the estrous cycle.Taken together, we demonstrated that an alteration in var-ious mRNAs of gene profiles is one of the most significantfactors at the transcriptional level in the reproductive tis-sue following E2, DES or estrogenic compounds. In addi-tion, the expression patterns of CaBP-9k, oxytocin, Acrp,Ldha, and calcyclin mRNAs were altered in the uterus ofimmature rats during the estrous cycle. In conclusion,Expression of CaBP-9k, oxytocin, Acrp30, Ldha and calcyclin mRNA  in the uterus of adult rats during estrous cycleFigure 4Expression of CaBP-9k, oxytocin, Acrp30, Ldha and calcyclin mRNAs in the uterus of adult rats during estrous cycle. To investigate the expression of these genes during estrous cycle, the expression levels of these genes in the uterus of adult female rats were analyzed by real-time PCR. Data were analyzed by non-parametric procedure of the Kruskal Wallis test, followed by Dunnett's test for two-pair comparisons. The values represent means ± SD. a, P < 0.05 vs. Metestrus and Diestrus.Page 10 of 12(page number not for citation purposes)tissue. these results indicate distinct altered expression of respon-sive genes following exposure to estrogen and estrogenicReproductive Biology and Endocrinology 2006, 4:49 http://www.rbej.com/content/4/1/49compounds, and implicate differential effects of estrogenand environmental endocrine disrupting chemicals in theuterus of immature rats.Additional materialAcknowledgementsThis work was supported by Korea Research Foundation Grant (KRF 2004-041- E00335), the Research Project on the Production of Bio-ograns (No. 200508010701) Ministry of Agriculture and Forestry and the Ministry of Education and Human Resources Development (MOE), the Ministry of Commerce, Industry and Energy (MOCIE) and the Ministry of Labor (MOLAB) through the fostering project of the lab of Excellency. In addition, the authors appreciate a graduate fellowship provided by the Brain Korea 21 Project in 2006. This work was also supported by Canadian Institutes of Health Research to PCKL. References1. Korach KS, Chae K, Gibson M, Curtis S: Estrogen receptor ster-eochemistry: ligand binding and hormonal responsiveness.Steroids 1991, 56(5):263-270.2. Crisp TM, Clegg ED, Cooper RL, Wood WP, Anderson DG, BaetckeKP, Hoffmann JL, Morrow MS, Rodier DJ, Schaeffer JE, Touart LW,Zeeman MG, Patel YM: Environmental endocrine disruption: aneffects assessment and analysis.  Environ Health Perspect 1998,106 Suppl 1:11-56.3. Hall JM, Couse JF, Korach KS: The multifaceted mechanisms ofestradiol and estrogen receptor signaling.  J Biol Chem 2001,276(40):36869-36872.4. Nilsson S, Makela S, Treuter E, Tujague M, Thomsen J, Andersson G,Enmark E, Pettersson K, Warner M, Gustafsson JA: Mechanisms ofestrogen action.  Physiol Rev 2001, 81(4):1535-1565.5. 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Microarray analysis in the uterus of imma-ture rats following treatments with E2, DES, OP, NP, BPA or GenClick here for file[http://www.biomedcentral.com/content/supplementary/1477-7827-4-49-S1.doc]Page 11 of 12(page number not for citation purposes)11. Newbold R: Cellular and molecular effects of developmentalexposure to diethylstilbestrol: implications for other envi-tive system of the rat.  Toxicol Sci 2003, 72(2):314-330.Publish with BioMed Central   and  every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime."Sir Paul Nurse, Cancer Research UKYour research papers will be:available free of charge to the entire biomedical communitypeer reviewed and published immediately upon acceptancecited in PubMed and archived on PubMed Central Reproductive Biology and Endocrinology 2006, 4:49 http://www.rbej.com/content/4/1/4933. 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