Identification of estrogen-regulated genes by microarray analysis of the uterus of immature rats exposed to endocrine disrupting chemicals
© Hong et al; licensee BioMed Central Ltd. 2006
Received: 17 June 2006
Accepted: 29 September 2006
Published: 29 September 2006
Environmental estrogenic compounds which bind to the estrogen receptor (ER) can block or alter endogenous functions of estrogen in reproductive and developmental stages. A microarray technology is a very valuable method for the prediction of hormone-responsive activities in various gene expressions. Thus, we investigated the altered gene expression by estrogen and endocrine disruptors (EDs) using microarray technology in the uterus of immature rats. In this study, the expression levels of only 555 genes (7.42%) among the 7636 genes spotted on microarray chips were enhanced by more than two-fold following treatment with estradiol (E2), suggesting that direct 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 complement related 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 these genes by real-time PCR were increased at follicular phase when E2 level was elevated during estrous cycle of adult female rats. In conclusion, these results indicate distinct altered expression of responsive genes following exposure to E2 and estrogenic compounds, and implicate distinct effects of endogenous E2 and environmental endocrine disrupting chemicals in the uterus of immature rats.
Environmental chemicals that disrupt endocrine function are suspected for their adverse effects on the reproductive system in wild animals and humans and are being increasingly assumed for their possible participation in inducing estrogenic effects. Furthermore, they are proposed to possess hormone-like properties, i.e., mimicking natural hormones, inhibiting the action of hormones, and inducing abnormal gene expressions. Environmental estrogenic compounds that bind to the estrogen receptors (ERs) can block or alter endogenous estrogen functions in reproductive and developmental stages via an ER-mediated response . Examples of suspected environmental estrogenic chemicals (endocrine disruptors; EDs) include polychlorinated hydroxybiphenyls, DDT and its derivatives, certain insecticides and herbicides (kepone and methoxychlor), plastic components (bisphenol A) and some components of detergents and their biodegradation products (alkylphenols etc.). Although the activity of most of these environmental estrogens is low when compared to endogenous or synthetic estrogens (17β-estradiol; E2 or ethinylestradiol), dietary or environmental exposure scenarios that led to the detection of significant quantities of these substances in human urine and tissue sample have been described .
The profound effects of E2 on cell growth, differentiation, and general homeostasis of reproductive and other systems are mediated mainly by the temporal and cell type-specific expression of different genes, whose products are the molecules controlling these molecular events [3, 4]. In rats, the concentration of E2 is consistently low throughout neonatal development and starts to increase after day 28 of age . The uterus and ovaries are two of the most sensitive tissues to E2, and both tissues express two forms of ER, ERα and ERβ. In particular, ERα is predominantly expressed in uteri while ERβ is expressed in ovaries [6, 7]. Diethylstilbestrol (DES) is a synthetic estrogen which can induce various reproductive alterations in humans [8, 9] and mice [10–12]. Numerous reproductive changes in wildlife populations can be caused by EDs which resemble DES . Alkylphenols (APs), such as octyl-phenol (OP) and nonylphenol (NP), were reported to bind directly to the ER in trout, stimulate citelogenin gene expression in trout hepatocytes, be mitogenic in MCF-7 cells and stimulate transcription in mammalian cells via ER . In vitro studies revealed that OP and NP are the most potent estrogenic alkylphenols, and the potency of OP has been shown to be approximately 10-3~10-7 relative to 17β-estradiol [12–15]. Furthermore, the binding affinity of BPA indicated that it is approximately 10000-fold less potent than E2 and 20000-fold less potent than DES for both ERα and ERβ receptors . In vivo estrogenic activities (400–1000 mg/kg/day) in immature or ovariectomized rats and mice have been recognized. Thus, the concentrations of EDs such as OP, NP, and BPA, in the present study are expected to have similar effects as those of steroid hormones. In addition, APs are weakly estrogenic in traditional uterotropic assays as evidenced by the increase in uterus weight . In vitro assays, E2 has been demonstrated to induce maximal proliferation of MCF-7 cells at 1 nM concentration, and OP and NP have been found to be considerably potent compounds as estrogenic chemicals at 1 and 10 μM, respectively. Treatments with OP and NP inhibited the binding affinity of E2 to ER in MCF-7 cells by a competitive ER binding assay . Bisphenol A (BPA) is a particularly important environmental estrogen. Not only in it widespread in the environment, but it is commonly ingested by humans, being released by polycarbonate plastics, the lining of food cans, and dental sealants . BPA only acts as an agonist of estrogen via ERβ whereas it has dual actions as an agonist and antagonist in some types of cells via ERα Thus, the activity of BPA may depend on the ER subtype and the tissue involved . ERβ has a higher relative binding affinity to genistein (Gen) in in vitro assays compare with ERα . Genistein is readily absorbed [21, 22] and act as a pharmacological estrogen both in vitro and in vivo via ERs [23–25]. Although an actual impact of environmental estrogens on reproductive health is not well defined thoroughly, these chemicals have the potential to disrupt the reproductive system and confirm its estrogen-like activity in vitro . Therefore, the changes in the expression of estrogen target genes are considered to be a useful index for evaluating the estrogenic activity of synthetic compounds. However, it is difficult to predict the full range of effects of estrogenic compounds from changes in the expression levels of only well-known estrogen target genes.
Reproductive organs are highly susceptible to hormonal exposure during organ development and sex differentiation. In our previous studies, we showed that NP, OP and BPA have estrogenic activity, resulting in uterotrophic effects in the uterus of rats treated with these compounds [26, 27]. In addition, we demonstrated that the CaBP-9k gene is not expressed in the uterus of immature rats, which do not obtain estrogen from ovaries, and that it is regulated through the binding of the ER/estrogen complex to the estrogen response element (ERE) in rats [26, 28]. CaBP-9k mRNA in the uterus is known to fluctuate during estrous cycle of rats depending on serum estrogen level. CaBP-9k mRNA at diestrus was not detectable, but increased at proestrus and reached the highest level at estrus and then decreased as metestrus . The aim of this study was to identify estrogen-responsive genes by E2 and endocrine disrupting chemicals in the uterus of rats and to determine whether estrogen responsive genes are differentially regulated following exposure to these estrogenic compounds by microarray analysis and real-time PCR. Finally, we evaluated the correlations between E2-induced and EDs-induced gene profiles, and confirmed the biomarker among altered gene expression for screening potential EDs.
Materials and methods
Animals and treatments
Immature Sprague-Dawley rats (2-weeks of age) with dams were obtained from Orient Co, Ltd. (Gyeonggi-do, Korea). All animals were housed in polycarbonate cages, and used after acclimation to an environmentally controlled room (temperature: 23 ± 2°C, relative humidity: 50 ± 10%, frequent ventilation and a 12 h day/night cycle). To determine the effect of EDs, each group of five animals (14-days old) was injected subcutaneously (sc, 0.1 ml per rat) 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/kg BW; Sigma-Aldrich Corp), and genistein (40 mg/kg BW; Sigma-Aldrich Corp) by a single dose daily for 3 days and euthanized 24 h after final injection. All chemicals were dissolved in corn oil (Sigma-Aldrich Corp) as a vehicle. The rats were injected with E2 (n = 3) as a positive control or 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 the injection.
It is believed that exposure to DES or endocrine disruptors might be detrimental to development and differentiation, although the effects may not be apparent until adulthood. In the present study, treatment with estrogenic compounds induced a significant increase in the mRNA expression of specific genes in the neonate rat uterus. To confirm altered gene expression profile by E2 or EDs in the uterus of immature rats, adult female rats were also employed to examine the elevated endogenous E2 in the induction of these genes at proestrus and estrus during estrous cycle. Estrous cycle was determined by the observation of three types of the cells derived from the vaginal smears: leukocytes, cornified cells and nucleated epithelial cells. Four stages of the estrous cycle (proestrus, estrus, metestrus and diestrus) were determined as following criteria: proestrus was characterized by many epithelial cells and few leukocytes; estrus by many cornified cells and no leukocytes; metestrus by some cornified cells and many leukocytes; and diestrus by few epithelial cells and many leukocytes. All animals were smeared daily, and the rats that had three regular cycles were selected. Forty female rats were randomly assigned to four groups according to the respective phase of estrous cycle (proestrus, estrus, metestrus and diestrus, n = 10 each) and euthanized immediately. The uteri were washed in cold sterile 0.9% NaCl solution (0.9% normal saline) and used for RNA extraction. All experimental procedures and animal use were approved by the Ethics Committee of the Chungbuk National University.
RNA isolation and cDNA microarray analysis
Total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA, USA) according to manufacturer's suggested procedure, and purified using RNeasy total RNA isolation kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. DNA was digested using an RNase-free DNase set (Qiagen) during RNA purification. Total RNA was quantified by spectrophotometer and its integrity was assessed by running on a 0.8% agarose gel. To make cDNAs from mRNAs for microarray analysis, the same quantity of each RNA sample from the treated groups (n = 5) or control groups (n = 3) was pooled. A cDNA microarray consists of 7636 cDNA spot including Incyte clones, housekeeping genes and Arabidopsis DNA as controls. The experiments were performed on the rat cDNA microarray prepared as previously described . The PCR reactions were prepared according to the standard protocol and reaction mixtures were subjected to be amplified at 35 cycles. The primer pair used for amplification was overlap primer-1 (5'-AAT TAA CCC TCA CTA AAG GG-3') and overlap primer-2 (5'-GTA ATA CGA CTC ACT ATA GGG C-3'). The size and amount of the PCR products were verified on 1% agarose gel. The PCR products were purified by ethanol precipitation, then resuspended 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 Technologies, CA) using 16-Stealth Micro spotting pins. The printed slides were processed according to CMT-GAPS α slide protocol. Briefly, the spots were re-hydrated with 1 × SSC for 1 min and then DNA linked using a UV crosslinker (Stratagene, CA). The slides were soaked in the succinic anhydride/sodium borate solution for 15 min with gentle agitation and then transferred to a 95°C water bath for 2 min. The slides were quickly transferred to 95% ethanol for 1 min and then dried using a centrifuge at 3000 rpm for 20 sec.
Hybridization with fluorescent DNA probe
Total RNA was extracted from the treated and untreated tissues from immature or adult rats at the indicated time points using the TRI-REAGENT (MRC, OH) according to the manufacturer's instructions. Fluorescent labeled cDNA probes were prepared from 50 §P of total RNA by oligo (dT)18-primed polymerization using SuperScript α reverse transcriptase (Invitrogen, NY) in a total reaction volume of 30 μl. The reverse transcription mixture included 400 U Superscript RNase H-reverse transcriptase (Invitrogen), 0.5 mM dATP, dTTP and dGTP, 0.2 mM dCTP and 0.1 mM Cy3 or Cy5 labeled dCTP (NEN Life Science Product Inc.). After reverse transcription, the sample RNA was degraded by adding 5 μl of stop solution (0.5 M NaOH/50 m EDTA) and incubating at 65°C for 10 min. The labeled cDNA mixture was then concentrated using the ethanol precipitation method. After determining the target cDNA quality, cDNA samples derived from the pooled uteri of five individual neonate rats from each treated group were selected and hybridized. The concentrated Cy3 and Cy5 labeled cDNAs were resuspended in 10 μl of hybridization solution (GenoCheck). After two labeled cDNAs were mixed, the mixture was denaturized 95°C for 2 min and then incubated in 45°C water chamber for 20 min. The cDNA mixture was then placed at three spotted slide positions and covered by a cover slip to assess the overall quality of each sample. The slides were hybridized for 12 h at 62°C hybridization chamber. The hybridized slides were washed in 2 × SSC, 0.1% SDS for 2 min, 1 × SSC for 3 min, and then 0.2 × SSC for 2 min at room temperature. The slides were centrifuged at 3000 rpm for 20 sec to be dried.
Scanning and image analysis
Hybridized slides were scanned with the Axon Instruments GenePix 4000B scanner and the scanned images were analyzed with the software program GenePix Pro 5.1 (Axon, CA) and GeneSpring 7.1 (Sillicongenetics, CA). In order to allow algorithm to eliminate all bad spots, no data points were eliminated by visual inspection from the initial GenePix image. For signal normalization, housekeeping genes (β-actin) and positive control genes (A. thaliana genes) were spotted onto each slide. The signals of these spots were used for normalization. To filter out the unreliable data, spots with signal-to-noise (signal – background – background SD) below 100 were not included in the data. Data were normalized by global, lowess, print-tip and scaled normalization for data reliability. Data were sorted of above 2-fold altered genes using GeneSpring 7.1 (Sillicongenetics) and a hierarchical clustering analysis was performed using Pearson correlation. The statistical significance of differential expression was assessed by computing a q-value for each gene. To determine the q-value, we used a permutation procedure, and for each permutation a two-sample t-statistic was computed for each gene. The result was considered significant when the logarithmic gene expression ratio of three independent hybridizations was more than twofold the difference in the expression level. The accuracy of microarray analysis in this study was confirmed by real-time PCR.
Confirmation of microarray analysis with real-time PCR
Oligonucleotide sequences with predicted sizes of respective PCR product
vitamin D-dependent calcium binding protein
Adipocyte complement related protein of 30 kDa
Lactate dehydrogenase A
S100 calcium binding protein A6 (calcyclin)
Data are presented as the mean ± SD. The data were analyzed by non-parametric procedure of the Kruskal Wallis test, followed by Dunnett's test for two-pair comparisons. The each value of Dunnett's test was converted to rank for statistical analysis. All statistical analyses were performed with SAS. P < 0.05 was considered statistically significant.
Global analysis of estrogen-regulated genes compared with endocrine disruptors
Altered genes induced by E2, DES and OP in the uterus of immature rats
Seizure related 6 homolog (mouse)
Epithelial membrane protein 1
Discs, large homolog 3 (Drosophila)
Decay accelarating factor 1
Gamma-aminobutyric acid (GABA) A receptor, pi
X transporter protein 3
Integrin, alpha E, epithelial-associated
Rous sarcoma oncogenes
Secretory leukocyte peptidase inhibitor
Inter alpha-trypsin inhibitor, heavy chain 4
Regulated endocrine-specific protein 18
Lactate dehydrogenase A
Immunoglobulin superfamily, member 6
Myristoylated alanine rich protein kinase C substrate
TRAF family member-associated Nf-kappa B activator
Altered genes induced by NP, BPA, and Gen in the uterus of immature rats
Solute carrier family 22 (organic anion transporter), member 6
Rabphilin 3A homolog (mouse)
Aldehyde dehydrogenase family 5, subfamily A1
Solute carrier family 21, member 1
Inhibitor of kappaB kinase beta
Aminolevulinic acid synthase 1
Lipopolysaccharide binding protein
Serine/threonine kinase 17b (apoptosis-inducing)
Kelch repeat and BTB (POZ) domain containing 10
ATP synthase, H+ transporting, O subunit
Transcribed locus, strongly similar to XP_130951.1 dolichyl-phosphate mannosyltransferase polypeptide 3 [Mus musculus]
Eph and elk-related kinase
Transcribed locus, moderately similar to XP_525535.1 similar to Yippee-like protein 1 (DiGeorge syndrome-related protein FKSG3)
Growth hormone releasing hormone
Mast cell protease 1
Lymphocyte cytosolic protein 2
Heat shock 70 kD protein 1A
RAB14, member RAS oncogene family
Sodium-dependent neutral amino acid transporter ASCT2
Non-metastatic cells 7, protein expressed in
Chemokine (C-X-C motif) ligand 10
ATPase, H+ transporting, lysosomal (vacuolar proton pump), subunit 1
In this study, we extended a large-scale of microarray analysis to six other estrogenic compounds, and identified crucial estrogen-responsive genes, which were either common in all or specific to each or some of the compounds. The results with all six compounds are shown in Fig. 2C using a hierarchical clustering algorithm of the mean values of triplicate slides and a pseudo-color visualization matrix. The gene profile in red color shows a higher expression than the control, while the gene profile in green color shows a lower expression (Fig. 2C). These genes might be universal estrogen – responsive genes among various tissues. These compounds exhibited a reasonable hierarchical clustering; DES or OP resembled to E2. However, NP-induced gene profile is similar to that of BPA or genistein.
Confirmation of estrogen-regulated genes by real-time PCR
Expression of estrogen-regulated genes during estrous cycle
The microarray technique is a very valuable method for the prediction of hormone-responsive activity in various gene expressions. Estrogen plays an important role in various molecular events, but the molecular mechanisms that are regulated by estrogen in the uterus remain largely unknown. Therefore, the identification of estrogen-induced gene expression is essential to understanding how estrogenic compounds regulate uterine physiology and 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-regulated by more than two-fold following estrogen treatment, suggesting that direct or rapid responses to estrogen is widespread at the mRNA level. Furthermore, treatments with DES (9.01%), OP (8.81%), NP (9.51%) BPA (8.26%) or genistein (9.97%) showed an induction of distinct genes by more than two-fold in the uterus. Recently, gene profile patterns by microarray technology were determined in the developing uterus and ovaries of Sprague-Dawley rats at different stages of the development exposed to graded dosages (sc) of 17alpha-ethynyl estradiol (EE), Gen, or BPA [31, 32]. This analysis of the transcript profile in a dose-dependent manner revealed that a common set of genes are altered, but some of the genes are differentially changed by these estrogenic chemicals . Interestingly, 592 genes of immature rat showed strong and consistent changes in expression after EE exposure , indicating that previously identified estrogen-sensitive genes are sensitive to EE exposure and that they are targets of the estrogenic action of EDs in both the uterus and the ovary. In actual, the level of EDs in nature is low compared with using dosage in the present study. For example, several studies have been performed to assess the presence of EDs in milk. EDs were determined in milk and infant formula at concentrations from 0.4 to 81 μg/kg in NP , or 0.1 to 13.2 μg/kg in BPA , respectively. Although we are aware that we used the high dose of EDs, this study focused to describe estrogen specific genes using cDNA microarray to detect estrogenicity in vivo following treatment with EDs.
Uterine CaBP-9k may be involved in controlling myometrial activity which is affected by the intracellular calcium level; however, the exact role of CaBP-9k in the uterus is still under investigation by us and other research groups . Recently, it has been demonstrated in our previous studies that both CaBP-9k mRNA and protein could be a novel biomarker for estrogenic compounds in the uterus of immature rats [26, 27]. Based on the previous results, the present study was performed to further investigate EDs-induced specific gene expression in the uterus following treatment with DES, E2 and estrogenic compounds such as OP, NP, BPA and genistein. Thus, the expression levels of CaBP-9k mRNA were analyzed to confirm the estrogenic effect of these compounds in this tissue. E2 is a major factor controlling CaBP-9k gene expression in the rat uterus. The CaBP-9k gene is not expressed in the uterus of mature ovariectomized and immature rats which do not have circulating E2 from ovaries . Using microarray, the expression level of uterine CaBP-9k mRNA was significantly increased when treated with E2 (4.2-fold), DES (7.32-fold), OP (3.09-fold), and NP (2.57-fold) in the uterus of immature rats [see Additional file 1]. In pregnant rats, a relative potency of estrogenic compounds indicated OP = NP > BPA . Despite different potency in estrogenicity, E2 and the estrogenic compounds tested in this study induced a significant increase of CaBP-9k mRNA in the uterus of immature rats. The expression profile potentially provides a wealth of data about the differences in gene expression between experimental samples; however, these differences do not always reflect realistic mRNA levels. The induction of uterine CaBP-9k by estrogenic compounds was further assessed by real-time RT-PCR. Although the gene expression levels by microarray analysis were not identical to those obtained PCR analysis, the expression patterns of these genes obtained by these two types of analysis were largely similar. In agreement with a previous study , there was no change in the expression pattern of CaBP-9k mRNA during the estrous cycle. Estrogen stimulated the number of uterine oxytocin binding sites, and oxytocin receptor mRNA expression in ovariectomized virgin rats [39, 40]. In rats, during the terminal stages of pregnancy, the myometrium is extremely sensitive to oxytocin and this increase in uterine responsiveness occurs in parallel with increases in the number of uterine oxytocin binding sites . This leads to increased uterine responsiveness to oxytocin and the onset of the uterine contractions that facilitate parturition. The change in uterine responsiveness to oxytocin involves an increase in the quantity of oxytocin receptor protein per cell and the number of smooth muscle cells that express oxytocin receptors . Based on the treatments with EDs, increased expression levels of oxytocin mRNA were observed when rats were treated with OP (14.76-fold) and NP (9.54-fold), and a single dose treatment with DES (5.70-fold) and E2 (5.83-fold) for 3 days. However, treatments with BPA and genistein for 3 days failed to detect the expression level of oxytocin mRNA [see Additional file 1]. These observations raise the possibility that the increase in the oxytocin mRNA level shown in Figure 3, occurs, at least in part, as a result of the injected estrogenic compounds and that the PCR data and microarray data are in general agreement. Furthermore, the present study indicated at least that oxytocin mRNA which can be amplified with the primer set increase about 2-fold on proestrus compared with the value on metestrus. The mRNA levels of oxytocin increased 2-fold between metestrus and proestrus by Northern blot analysis during estrous cycle, while oxytocin binding increased more than 10-fold within this same interval in the uterus of rats .
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 analysis 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 Acrp30 mRNA in the neonatal uteri following injection with EDs. Although the rates of Acrp induction were not identical from microarray analysis, treatment with E2, DES, OP and NP resulted in significant increases in Acrp30 mRNA. Furthermore, our results showed that Acrp30 fluctuated during the estrous cycle, suggesting that steroid hormones play a role in the regulation of Acrp30. Acrp30 is expressed exclusively in the adipocytes, its hormone is exclusively secreted by differentiated adipocytes , and its protein was secreted and detected in plasma . The levels of the adipocyte hormones, leptin and adiponectin, appear to be correlated with the cell proliferation index and sex steroid receptor abundance . Furthermore, OVX in young cycling mice induced plasma Acrp30, and E2 implants reversed the effect . However, our results indicate that E2, DES, OP and NP up-regulated its level, suggesting that it does not imply any reverse effect in this tissue.
The lactate dehydrogenase-A (Ldha) is hormonally regulated in rodents and in highly expressed in the rat mammary gland during pregnancy and lactation, and Ldha mRNA also increases during mammary gland tumorigenesis . E2 induces lactate dehydrogenase activity in MCF-7 human breast cancer cells, and is elevated in estrogen receptor positive or progesterone receptor positive tumors . The synthesis of Ldha isoenzyme was found to increase significantly in the uterus of immature mice, and expression from the mouse lactate dehydrogenase-A promoter fused to the cat gene in Chinese hamster ovary cells was also induced by E2 and DES . Using microarray, the expression level of uterine Ldha mRNA was significantly increased when treated with E2 (2.17-fold), DES (2.06-fold), OP (1.77-fold), and NP (2.72-fold) in the uterus of immature rats [see Additional file 1]. In addition, we further investigated the expression of Ldha mRNA related to estrogen during estrus cycle. A significant increase in Ldha mRNA was detected at proestrus and estrus compared with metestrus and diestrus. The expression pattern of Ldha mRNA during the estrous cycle was in parallel with CaBP-9k mRNA, whereas others appeared to peak at estrus compared to their level at proestrus.
Calcyclin (S100A6), a small acidic protein that weighs about 10 kDa, belongs to the S100 calcium-binding protein family . These family members share a common S100 calcium-binding motif and are implicated in several regulatory functions that include protein phosphorylation, some enzyme activities, the dynamics of cytoskeletal components, transcription factors, and Ca2+ homeostasis, and also cell proliferation and differentiation . The effect of E2 on the expression level of calcyclin mRNA in uteri was further examined to elucidate a relationship between expression levels of calcyclin and estrogenic compounds. E2 resulted in an induction of uterine calcyclin mRNA in immature rats. In addition, treatment with estrogenic compounds resulted in a significant increase in the expression of calcyclin mRNA, whose level paralleled those of oxytocin and Acrp30, as shown in the Additional file 1 and Fig. 3. After adjusting the data, it is clear that major alterations in gene profiles were induced by estrogenic compounds, such as E2, DES, OP, and NP, and that these differences could have a significant effect on uterine function in reproductive tissues during the estrous cycle.
Taken together, we demonstrated that an alteration in various mRNAs of gene profiles is one of the most significant factors at the transcriptional level in the reproductive tissue following E2, DES or estrogenic compounds. In addition, the expression patterns of CaBP-9k, oxytocin, Acrp, Ldha, and calcyclin mRNAs were altered in the uterus of immature rats during the estrous cycle. In conclusion, these results indicate distinct altered expression of responsive genes following exposure to estrogen and estrogenic compounds, and implicate differential effects of estrogen and environmental endocrine disrupting chemicals in the uterus of immature rats.
This 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.
- Korach KS, Chae K, Gibson M, Curtis S: Estrogen receptor stereochemistry: ligand binding and hormonal responsiveness. Steroids. 1991, 56 (5): 263-270. 10.1016/0039-128X(91)90045-W.View ArticlePubMedGoogle Scholar
- Crisp TM, Clegg ED, Cooper RL, Wood WP, Anderson DG, Baetcke KP, Hoffmann JL, Morrow MS, Rodier DJ, Schaeffer JE, Touart LW, Zeeman MG, Patel YM: Environmental endocrine disruption: an effects assessment and analysis. Environ Health Perspect. 1998, 106 Suppl 1: 11-56.View ArticlePubMedGoogle Scholar
- Hall JM, Couse JF, Korach KS: The multifaceted mechanisms of estradiol and estrogen receptor signaling. J Biol Chem. 2001, 276 (40): 36869-36872. 10.1074/jbc.R100029200.View ArticlePubMedGoogle Scholar
- Nilsson S, Makela S, Treuter E, Tujague M, Thomsen J, Andersson G, Enmark E, Pettersson K, Warner M, Gustafsson JA: Mechanisms of estrogen action. Physiol Rev. 2001, 81 (4): 1535-1565.PubMedGoogle Scholar
- Noda S, Sawaki M, Shiraishi K, Yamasaki K, Yamaguchi R: Age-related changes of genital systems in the female Crj:CD (SD) IGS rats during sexual maturation. J Vet Med Sci. 2002, 64 (4): 315-319. 10.1292/jvms.64.315.View ArticlePubMedGoogle Scholar
- Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson JA: Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta. Endocrinology. 1997, 138 (3): 863-870. 10.1210/en.138.3.863.PubMedGoogle Scholar
- Couse JF, Lindzey J, Grandien K, Gustafsson JA, Korach KS: Tissue distribution and quantitative analysis of estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta) messenger ribonucleic acid in the wild-type and ERalpha-knockout mouse. Endocrinology. 1997, 138 (11): 4613-4621. 10.1210/en.138.11.4613.PubMedGoogle Scholar
- Bibbo M, Gill WB, Azizi F, Blough R, Fang VS, Rosenfield RL, Schumacher GF, Sleeper K, Sonek MG, Wied GL: Follow-up study of male and female offspring of DES-exposed mothers. Obstet Gynecol. 1977, 49 (1): 1-8.PubMedGoogle Scholar
- Wilcox AJ, Baird DD, Weinberg CR, Hornsby PP, Herbst AL: Fertility in men exposed prenatally to diethylstilbestrol. N Engl J Med. 1995, 332 (21): 1411-1416. 10.1056/NEJM199505253322104.View ArticlePubMedGoogle Scholar
- McLachlan JA, Newbold RR, Bullock BC: Long-term effects on the female mouse genital tract associated with prenatal exposure to diethylstilbestrol. Cancer Res. 1980, 40 (11): 3988-3999.PubMedGoogle Scholar
- Newbold R: Cellular and molecular effects of developmental exposure to diethylstilbestrol: implications for other environmental estrogens. Environ Health Perspect. 1995, 103 Suppl 7: 83-87.View ArticlePubMedGoogle Scholar
- White R, Jobling S, Hoare SA, Sumpter JP, Parker MG: Environmentally persistent alkylphenolic compounds are estrogenic. Endocrinology. 1994, 135 (1): 175-182. 10.1210/en.135.1.175.PubMedGoogle Scholar
- Bolger R, Wiese TE, Ervin K, Nestich S, Checovich W: Rapid screening of environmental chemicals for estrogen receptor binding capacity. Environ Health Perspect. 1998, 106 (9): 551-557.PubMed CentralView ArticlePubMedGoogle Scholar
- Safe SH, Pallaroni L, Yoon K, Gaido K, Ross S, Saville B, McDonnellc D: Toxicology of environmental estrogens. Reprod Fertil Dev. 2001, 13 (4): 307-315. 10.1071/RD00108.View ArticlePubMedGoogle Scholar
- Soto AM, Sonnenschein C, Chung KL, Fernandez MF, Olea N, Serrano FO: The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic environmental pollutants. Environ Health Perspect. 1995, 103 Suppl 7: 113-122.View ArticlePubMedGoogle Scholar
- Maruyama S, Fujimoto N, Yin H, Ito A: Growth stimulation of a rat pituitary cell line MtT/E-2 by environmental estrogens in vitro and in vivo. Endocr J. 1999, 46 (4): 513-520.View ArticlePubMedGoogle Scholar
- Bicknell RJ, Herbison AE, Sumpter JP: Oestrogenic activity of an environmentally persistent alkylphenol in the reproductive tract but not the brain of rodents. J Steroid Biochem Mol Biol. 1995, 54 (1-2): 7-9. 10.1016/0960-0760(95)00118-J.View ArticlePubMedGoogle Scholar
- Kwack SJ, Kwon O, Kim HS, Kim SS, Kim SH, Sohn KH, Lee RD, Park CH, Jeung EB, An BS, Park KL: Comparative evaluation of alkylphenolic compounds on estrogenic activity in vitro and in vivo. J Toxicol Environ Health A. 2002, 65 (5-6): 419-431. 10.1080/15287390252808082.View ArticlePubMedGoogle Scholar
- Olea N, Pulgar R, Perez P, Olea-Serrano F, Rivas A, Novillo-Fertrell A, Pedraza V, Soto AM, Sonnenschein C: Estrogenicity of resin-based composites and sealants used in dentistry. Environ Health Perspect. 1996, 104 (3): 298-305.PubMed CentralView ArticlePubMedGoogle Scholar
- Kurosawa T, Hiroi H, Tsutsumi O, Ishikawa T, Osuga Y, Fujiwara T, Inoue S, Muramatsu M, Momoeda M, Taketani Y: The activity of bisphenol A depends on both the estrogen receptor subtype and the cell type. Endocr J. 2002, 49 (4): 465-471. 10.1507/endocrj.49.465.View ArticlePubMedGoogle Scholar
- Chang HC, Churchwell MI, Delclos KB, Newbold RR, Doerge DR: Mass spectrometric determination of Genistein tissue distribution in diet-exposed Sprague-Dawley rats. J Nutr. 2000, 130 (8): 1963-1970.PubMedGoogle Scholar
- Degen GH, Janning P, Diel P, Bolt HM: Estrogenic isoflavones in rodent diets. Toxicol Lett. 2002, 128 (1-3): 145-157. 10.1016/S0378-4274(02)00009-7.View ArticlePubMedGoogle Scholar
- Farmakalidis E, Hathcock JN, Murphy PA: Oestrogenic potency of genistin and daidzin in mice. Food Chem Toxicol. 1985, 23 (8): 741-745. 10.1016/0278-6915(85)90268-6.View ArticlePubMedGoogle Scholar
- Markiewicz L, Garey J, Adlercreutz H, Gurpide E: In vitro bioassays of non-steroidal phytoestrogens. J Steroid Biochem Mol Biol. 1993, 45 (5): 399-405. 10.1016/0960-0760(93)90009-L.View ArticlePubMedGoogle Scholar
- Song TT, Hendrich S, Murphy PA: Estrogenic activity of glycitein, a soy isoflavone. J Agric Food Chem. 1999, 47 (4): 1607-1610. 10.1021/jf981054j.View ArticlePubMedGoogle Scholar
- An BS, Kang SK, Shin JH, Jeung EB: Stimulation of calbindin-D(9k) mRNA expression in the rat uterus by octyl-phenol, nonylphenol and bisphenol. Mol Cell Endocrinol. 2002, 191 (2): 177-186. 10.1016/S0303-7207(02)00042-4.View ArticlePubMedGoogle Scholar
- An BS, Choi KC, Kang SK, Hwang WS, Jeung EB: Novel Calbindin-D(9k) protein as a useful biomarker for environmental estrogenic compounds in the uterus of immature rats. Reprod Toxicol. 2003, 17 (3): 311-319. 10.1016/S0890-6238(03)00003-0.View ArticlePubMedGoogle Scholar
- Jeung EB, Leung PC, Krisinger J: The human calbindin-D9k gene. Complete structure and implications on steroid hormone regulation. J Mol Biol. 1994, 235 (4): 1231-1238. 10.1006/jmbi.1994.1076.View ArticlePubMedGoogle Scholar
- Krisinger J, Dann JL, Currie WD, Jeung EB, Leung PC: Calbindin-D9k mRNA is tightly regulated during the estrous cycle in the rat uterus. Mol Cell Endocrinol. 1992, 86 (1-2): 119-123. 10.1016/0303-7207(92)90182-6.View ArticlePubMedGoogle Scholar
- Jung JW, Park JS, Hwang JW, Kang KS, Lee YS, Song BS, Lee GJ, Yeo CD, Kang JS, Lee WS, Jeon KS, Um CH, Kim YS, Oh MJ, Youn JP, Li P, Park JE, Hwang SY: Gene expression analysis of peroxisome proliferators- and phenytoin-induced hepatotoxicity using cDNA microarray. J Vet Med Sci. 2004, 66 (11): 1329-1333. 10.1292/jvms.66.1329.View ArticlePubMedGoogle Scholar
- Naciff JM, Jump ML, Torontali SM, Carr GJ, Tiesman JP, Overmann GJ, Daston GP: Gene expression profile induced by 17alpha-ethynyl estradiol, bisphenol A, and genistein in the developing female reproductive system of the rat. Toxicol Sci. 2002, 68 (1): 184-199. 10.1093/toxsci/68.1.184.View ArticlePubMedGoogle Scholar
- Naciff JM, Overmann GJ, Torontali SM, Carr GJ, Tiesman JP, Richardson BD, Daston GP: Gene expression profile induced by 17 alpha-ethynyl estradiol in the prepubertal female reproductive system of the rat. Toxicol Sci. 2003, 72 (2): 314-330. 10.1093/toxsci/kfg037.View ArticlePubMedGoogle Scholar
- Naciff JM, Torontali SM, Overmann GI, Carr GJ, Tiesman JP, Daston GP: Evaluation of the gene expression changes induced by 17-alpha-ethynyl estradiol in the immature uterus/ovaries of the rat using high density oligonucleotide arrays. Birth Defects Res B Dev Reprod Toxicol. 2005, 74 (2): 164-184. 10.1002/bdrb.20032.View ArticlePubMedGoogle Scholar
- Guenther K, Heinke V, Thiele B, Kleist E, Prast H, Raecker T: Endocrine disrupting nonylphenols are ubiquitous in food. Environ Sci Technol. 2002, 36 (8): 1676-1680. 10.1021/es010199v.View ArticlePubMedGoogle Scholar
- Biles JE, White KD, McNeal TP, Begley TH: Determination of the diglycidyl ether of bisphenol A and its derivatives in canned foods. J Agric Food Chem. 1999, 47 (5): 1965-1969. 10.1021/jf9810867.View ArticlePubMedGoogle Scholar
- Mathieu CL, Burnett SH, Mills SE, Overpeck JG, Bruns DE, Bruns ME: Gestational changes in calbindin-D9k in rat uterus, yolk sac, and placenta: implications for maternal-fetal calcium transport and uterine muscle function. Proc Natl Acad Sci U S A. 1989, 86 (9): 3433-3437. 10.1073/pnas.86.9.3433.PubMed CentralView ArticlePubMedGoogle Scholar
- L'Horset F, Perret C, Brehier A, Thomasset M: 17 beta-estradiol stimulates the calbindin-D9k (CaBP9k) gene expression at the transcriptional and posttranscriptional levels in the rat uterus. Endocrinology. 1990, 127 (6): 2891-2897.View ArticlePubMedGoogle Scholar
- Hong EJ, Choi KC, Jeung EB: Maternal-fetal transfer of endocrine disruptors in the induction of Calbindin-D9k mRNA and protein during pregnancy in rat model. Mol Cell Endocrinol. 2003, 212 (1-2): 63-72. 10.1016/j.mce.2003.08.011.View ArticlePubMedGoogle Scholar
- Fuchs AR, Periyasamy S, Alexandrova M, Soloff MS: Correlation between oxytocin receptor concentration and responsiveness to oxytocin in pregnant rat myometrium: effects of ovarian steroids. Endocrinology. 1983, 113 (2): 742-749.View ArticlePubMedGoogle Scholar
- Larcher A, Neculcea J, Breton C, Arslan A, Rozen F, Russo C, Zingg HH: Oxytocin receptor gene expression in the rat uterus during pregnancy and the estrous cycle and in response to gonadal steroid treatment. Endocrinology. 1995, 136 (12): 5350-5356. 10.1210/en.136.12.5350.PubMedGoogle Scholar
- Ivell R, Kimura T, Muller D, Augustin K, Abend N, Bathgate R, Telgmann R, Balvers M, Tillmann G, Fuchs AR: The structure and regulation of the oxytocin receptor. Exp Physiol. 2001, 86 (2): 289-296. 10.1113/eph8602185.View ArticlePubMedGoogle Scholar
- Scherer PE, Williams S, Fogliano M, Baldini G, Lodish HF: A novel serum protein similar to C1q, produced exclusively in adipocytes. J Biol Chem. 1995, 270 (45): 26746-26749. 10.1074/jbc.270.45.26746.View ArticlePubMedGoogle Scholar
- Berg AH, Combs TP, Scherer PE: ACRP30/adiponectin: an adipokine regulating glucose and lipid metabolism. Trends Endocrinol Metab. 2002, 13 (2): 84-89. 10.1016/S1043-2760(01)00524-0.View ArticlePubMedGoogle Scholar
- Shively CA, Register TC, Grant KA, Johnson JL, Cline JM: Effects of social status and moderate alcohol consumption on mammary gland and endometrium of surgically postmenopausal monkeys. Menopause. 2004, 11 (4): 389-399. 10.1097/01.GME.0000109312.11228.62.View ArticlePubMedGoogle Scholar
- Combs TP, Berg AH, Rajala MW, Klebanov S, Iyengar P, Jimenez-Chillaron JC, Patti ME, Klein SL, Weinstein RS, Scherer PE: Sexual differentiation, pregnancy, calorie restriction, and aging affect the adipocyte-specific secretory protein adiponectin. Diabetes. 2003, 52 (2): 268-276.View ArticlePubMedGoogle Scholar
- Richards AH, Hilf R: Effect of estrogen administration on glucose 6-phosphate dehydrogenase and lactate dehydrogenase isoenzymes in rodent mammary tumors and normal mammary glands. Cancer Res. 1972, 32 (3): 611-616.PubMedGoogle Scholar
- Nagai MA, Sonohara S, Brentani MM: Estrogen control of lactate dehydrogenase isoenzyme-5 in human breast cancer. Int J Cancer. 1988, 41 (1): 10-16.View ArticlePubMedGoogle Scholar
- Li SS, Hou EW: Estrogen-induced expression of mouse lactate dehydrogenase-A gene. Cell Biol Int Rep. 1989, 13 (7): 619-624. 10.1016/0309-1651(89)90112-4.View ArticlePubMedGoogle Scholar
- Heizmann CW, Cox JA: New perspectives on S100 proteins: a multi-functional Ca(2+)-, Zn(2+)- and Cu(2+)-binding protein family. Biometals. 1998, 11 (4): 383-397. 10.1023/A:1009212521172.View ArticlePubMedGoogle Scholar
- Donato R: Functional roles of S100 proteins, calcium-binding proteins of the EF-hand type. Biochim Biophys Acta. 1999, 1450 (3): 191-231. 10.1016/S0167-4889(99)00058-0.View ArticlePubMedGoogle Scholar
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