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Ontogenetic changes in the expression of estrogen receptor β in mouse duodenal epithelium
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ORIGINAL ARTICLE
Ontogenetic changes in the expression of estrogen receptor  in mouse duodenal epithelium Narantsog Choijookhuu , Shin-ichiro Hino , Phyu Synn Oo , Baatarsuren Batmunkh , Noor Ali Mohmand , Myat Tin Htwe Kyaw , Yoshitaka Hishikawa ∗ Division of Histochemistry and Cell Biology, Department of Anatomy, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan
Summary Estrogen is considered to be involved in duodenal function; however, the details of its receptor expression are largely unknown. The purpose of this study was to determine the expression and localization of estrogen receptors (ERs) in mouse duodenum. Male and female C57BL/6J mouse intestinal tissues were used to investigate the expression of ER␣ and ER by RT-PCR, western blotting, immunohistochemistry, and Southwestern histochemistry. ER, but not ER␣, was expressed in proximal duodenal epithelium, but not in jejunum and ileum. The expression of ER mRNA and protein were confirmed by RT-PCR and western blotting, respectively. At postnatal day 20, the transit period of suckling to weaning, the distribution of ER-positive cells was changed in the crypt-villus axis, and cytoplasm/nuclear staining changed to only nuclear staining. Moreover, Southwestern histochemistry was used to detect estrogen response element (ERE)-binding proteins, and their expression pattern was highly similar to that of ER. These results suggested that ER is the predominant ER type in mouse small intestine, and the highly similar co-localization of ERE-binding proteins reveals that ER is functionally active in mouse duodenum. The ER expression changes during postnatal development indicate that ER may be involved in the differentiation of duodenal epithelium. © 2015 Elsevier Masson SAS. All rights reserved.
Introduction ∗ Corresponding author. Division of Histochemistry and Cell Biology, Department of Anatomy, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan. Tel.: +81 985 85 1783; fax: +81 985 85 9851. E-mail address: [email protected] (Y. Hishikawa).
Estrogen plays a critical role in the maintenance of homeostasis and in the structure and function of various reproductive [1,2] and non-reproductive organs [3,4]. In general, the biological actions of estrogen are mediated through binding
http://dx.doi.org/10.1016/j.clinre.2015.01.004 2210-7401/© 2015 Elsevier Masson SAS. All rights reserved.
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to estrogen receptor ␣ (ER␣) and ER, which then bind to estrogen response elements (ERE), which are harbored in the promoter regions of various estrogen-dependent genes [5,6]. ERs are products of different genes and exhibit tissue and cell-type specific expression [7,8]. In contrast, the ER plays a dominant role in non-reproductive tissues such as the cardiovascular system [9] and large intestine [10], where it is expressed primarily in epithelial cells. In gastrointestinal tract, the presence of ER is mainly reported in large intestine such as human colon cancer [11,12] and colon cancer cell lines [13]. However in small intestine, only a report is the expression and localization of ER␣ and ER in enteric neurons of small intestine [3]. The presence of ERs in small intestinal epithelium has not been clarified, and this epithelium is critical for intestinal function. Although many reports have demonstrated the existence of functional ERs in the duodenum, the exact localization of ERs has not been confirmed. Clinical studies and animal models in rats and mice have indicated that estrogen has a protective role against duodenal ulcers [14—16]. The male to female sex ratio in the prevalence of duodenal ulcers is 4:1 in Asia [17], 1.7:1 in the United States [18], and 2.2:1 in Europe [19]. The global incidence of duodenal ulcers has gradually decreased in recent decades, but the prevalence is persistently higher in males [20]. Interestingly, a reduced frequency of duodenal ulcers has been found in pregnant women or women who taking oral contraceptives [21]. Moreover, basal and acid-stimulated duodenal bicarbonate secretions in premenopausal women are significantly higher than in postmenopausal women, suggesting that sex steroid hormone receptors are expressed in the duodenum and that they mediate biological activity [22]. In addition, decreased bicarbonate secretion after treatment with ICI 182,780 (an estrogen receptor antagonist) suggests that the duodenum expresses functional ERs [14]. In clinical settings, estrogen is well known to have diverse protective effects against osteoporosis [23], and cardiovascular and neurodegenerative diseases such as atherosclerosis [24] and Alzheimer’s [25]. Despite epidemiological studies suggesting that estrogen has a protective role in the duodenum, the distribution of ERs, which are thought to mediate estrogen action in the duodenum, remains to be clarified. In the present study, we first investigated the expression and localization of ER␣ and ER in mouse duodenum by immunohistochemistry. After confirming ER as the predominant ER type in the duodenum, we examined ER mRNA and protein expression using RT-PCR and western blotting, respectively. Finally, we examined ERE binding protein expression using Southwestern histochemistry.
Materials and methods
fluoride membrane (PVDF) was purchased from Bio-Rad (Hercules, CA, USA). 3,3 -Diaminobenzidine-4 HCl (DAB) was purchased from Dojindo Chemicals (Kumamoto, Japan). All other reagents used in this study were from Wako Pure Chemicals (Osaka, Japan) and were of high analytical grade.
Antibodies Mouse monoclonal antibody against ER␣ (1D5; 1.6 g/mL, dilution 1:100) and rabbit polyclonal antibody against ER (PA1—310B; 10 g/mL, dilution 1:100) were purchased from DAKO (Glostrup, Denmark) and Pierce Biotechnology (Rockford, IL, USA), respectively. Normal mouse and rabbit IgG, horseradish peroxidase (HRP)-goat anti-mouse IgG (dilution 1:100) and HRP-goat anti-rabbit IgG (dilution 1:200) were purchased from Dako. Mouse monoclonal antibody against -actin (AC-15; dilution 1:5,000) and normal goat IgG were purchased from Sigma (St. Louis, Mo, USA).
Animals and tissue preparation C57BL/6J male and female mice were examined at the various developmental period such as embryonic day 18, postnatal day 0; 1; 5; 10; 15; 20; 25; 28 and adult mice. In each sampling day points, three mice for both sexes were sampled and total 60 mice were used in this study. Mice were kept under constant 12 h dark/lighting condition and fed normal chow with drinking water ad libitum. The experimental protocol was approved by the Animal Ethics Review Committee of University of Miyazaki (#2012-502-3). After sacrifice, the intestinal tissues were removed, rinsed in icecold phosphate-buffered saline (PBS), opened through the mesenteric border and then divided into two pieces by longitudinal cutting. The first piece of the intestine was stripped off the mucosal layer by blunt dissection using the edge of a glass slide on ice-cold plate. Then tissues were snap frozen and kept at −80 ◦ C until used for RT-PCR and western blotting. The second piece of the intestine was fixed in 4% PFA in PBS (pH 7.4) at room temperature (RT) for 24 h and then embedded in paraffin.
RNA extraction and RT-PCR Total RNA was extracted from tissues using a QIAGEN RNA kit (QIAGEN, Hilden, Germany). RT-PCR assays were performed according to Hino et al. [26]. The oligonucleotides used for RT-PCR were as follows: ER␣: 5’-CTGCCAAGGAGACTCGCTACTGTGC-3’ 5’-GCTTGGCCAAAGGTTGGCAGCTCTCATG-3’; ER: 5’-GGAATCTCTTCCCAGCAGCA-3’ 5’-GGGAGCCCTCTTTGCTCTTACTGTC-3’; -actin: 5’-TCCTCCCTGGAGAAGAGCTAC-3’ 5’-TCCTGCTTGCTGATCCACAT-3’.
Chemicals and biochemicals Western blot analysis Paraformaldehyde (PFA) was purchased from Merck (Darmstadt, Germany). Trizma base, bovine serum albumin (BSA), 2-mercaptoethanol, 3-aminopropyl-triethoxysilane and Brij L23 were purchased from Sigma Chemical Co. (St Louis, MO, USA). Prestained protein marker was purchased from New England Biolab (Ipswich, MA, USA) and polyvinylidene
For western blot analysis of ER␣ and ER protein expression in duodenum, the stripped intestinal mucosa was lysed in hot sodium dodecyl sulfate that had been adjusted to contain 0.9% sodium dodecyl sulfate, 15 mM EDTA, 8 mM unlabeled methionine and a protease inhibitor cocktail, incubated in a
Please cite this article in press as: Choijookhuu N, et al. Ontogenetic changes in the expression of estrogen receptor  in mouse duodenal epithelium. Clin Res Hepatol Gastroenterol (2015), http://dx.doi.org/10.1016/j.clinre.2015.01.004
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ER expression in mouse duodenum boiling water bath for 10 min, cooled, diluted to 0.3% sodium dodecyl sulfate, and adjusted to contain 33 mM tris/acetate, pH 8.5, and 1.7% Triton X-100 [27]. The lysates were incubated on ice for 1 h and centrifuged at 12,000 g at 4 ◦ C for 30 min. Equal amounts of protein were subjected into 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred onto PVDF membranes. Then membranes were immunoblotted with ER␣ (0.8 g/mL, 1:200), ER (5 g/mL, 1:200) and -actin (0.4 g/mL, 1:5,000) antibodies. The membranes were washed with TBS/Tween-20, and then incubated with alkaline phosphatase-conjugated secondary antibody. The bands were visualized with 1 × 5bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) dye, till the bands of alkaline phosphatase were visible.
Immunohistochemistry Paraffin-embedded tissues were cut into 5 m thick sections and placed onto silane-coated slide glasses. The sections were deparaffinized with toluene, and rehydrated through graded ethanol series, and then autoclaved at 120 ◦ C for 15 min in 10 mM citrate buffer (pH 6.0) [28] and [29]. After inhibition of endogenous peroxidase activity with 0.3% H2 O2 in methanol for 15 min, the sections were pre-incubated with 500 g/mL normal goat IgG in 1% BSA/PBS for one hour to block non-specific binding of antibodies. Unless otherwise specified, all reactions were conducted at RT. Then, the sections were reacted with the primary antibodies for overnight. After washing with 0.075% Brij L23 in PBS, they were reacted with HRP-goat anti-mouse IgG or HRP-goat anti-rabbit IgG for 1 h. After washing in 0.075% Brij L23 in PBS, the HRP sites were visualized with DAB, Ni, Co, and H2 O2 according to the method of Adams [30]. As a negative control, normal mouse or rabbit IgG was used at the same concentration instead of the primary antibodies in every experiment.
Southwestern histochemistry Southwestern histochemistry was performed as described previously [31] and [32]. Double-stranded oligo-DNAs containing a complete palindromic estrogen responsive element (vERE: 5’-GATCCAGGTCACAGTGACCTGGATC-3’) of the chicken vitellogenin gene, and a mutated estrogen responsive element (mERE: 5’-GATCCAGATCACAGTGATCTGGATC3’) with a two base mutation with the digoxigenin labeling at the 3’-end. In briefly, deparaffinized sections were treated with autoclave at 120 ◦ C for 15 min in 10 mM citrate buffer (pH 6.0). The sections were washed in PBS and immersed in TMSE buffer containing 50 mM Tris/HCl buffer (pH 7.4), 5% nonfat dry milk, 400 mM NaCl and 1 mM EDTA for one hour [33]. Then, the sections were reacted with digoxigenin labeled oligo-DNA probe dissolved in TMSE buffer for overnight. For the detection of hybridized oligo-DNA probes, the sections were immunohistochemically stained with HRP-conjugated sheep anti-digoxigenin antibody, and the HRP sites were visualized according to the method of Adams [30].
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Figure 1 ER mRNA is expressed in mouse duodenum, but not in jejunum and ileum. RT-PCR analysis of ER␣ mRNA and ER mRNA in total RNA extracts of small and large intestinal mucosa from postnatal developing and adult mice. Small intestinal tissues were obtained from duodenum, jejunum, and ileum, respectively. Adult mouse testis was used as a positive control for both ER␣ and ER mRNA. -actin mRNA was used as a loading control.
Transfection of ER siRNA by electroporation Custom synthesized ER siRNA (Invitrogen) annealed duplexes were used for gene knock-down. Electroporatic transfection was performed as described previously [34]. Briefly, control or ER siRNA were injected into the center of testis and three pulses (poring pulse: 40 V, 30 ms of length with 50 ms interval, transfer pulse: 25 V, 80 ms of length with 50 ms interval) were delivered into testis using Nepa21 electroporator (Nepa Gene, Chiba, Japan). Testis was excised 24 h after the electroporation.
Image analysis The quantitative analysis for western blotting was performed by ImageJ 1.48 (NIH software). The staining of ERE sites were analyzed by a digital image analyzer (Winroof version 7.0, Mitani Corp, Tokyo, Japan). vERE and mERE slides were analyzed in same condition and positive cells were evaluated based on the staining density over the level of staining with the mERE probe.
Statistical analysis All data were expressed as mean ± SEM. Statistical significance was assessed by Student’s t-test. P < 0.05 was considered statistically significant. All analyses were performed with The Statistical Package for Social Sciences (version 17.0; Chicago, IL, USA).
Results ER mRNA and protein were expressed in duodenal mucosa The expressions of ER␣ and ER mRNA were analyzed by RTPCR throughout the intestinal tract in postnatal day (PND) 10 mice and in 8-week-old mice. ER␣ mRNA was not expressed along the intestinal tract. Interestingly, in small intestine,
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Figure 3 ER decreases after ER knockdown in mouse testis. Western blot analysis of ER in mouse testis tissue after electroporatic transfection of ER siRNA. Arrow indicates decreased ER protein expression in ER-knocked down testis, whereas asterisk indicates the same level of non-specific stainings. The loading of equal amounts of total protein was confirmed by -actin.
which was consistent with ER␣ mRNA expression. ER was expressed in mouse ovary and uterus, and a weak band was detected in duodenal mucosa of both PND 10 and 8week-old mice. There has no significant difference in ER expression in PND 10 and 8-week-old mice (Fig. 2C). High background staining was found in ER-antibody reacted membrane. Therefore, to determine the specificity of ER antibody, the ER gene was knocked-down by ER siRNA in mouse testis (Fig. 3). In ER knocked-down testis, the ER expression was significantly decreased comparing to control testis, while there had similar background staining was observed in same height (asterisk in Fig. 2 and Fig. 3).
Figure 2 ER, but not ER␣, is expressed in mouse duodenum. A. Western blot analysis of ER␣ and ER in mouse duodenum, ovary, and uterus. Duodenal epithelium was obtained from PND 10 and 8-week-old mouse intestine by scraping as described in the section materials and methods. Arrows indicate ER␣ (66 kDa), ER (55 kDa), and -actin (42 kDa). B,C. Densitometry analysis of western blotting. ER protein expression was normalized by -actin.
ER mRNA was expressed in scraped duodenal tissues of both PND 10 and 8-week-old mice, but not expressed in jejunum and ileum (Fig. 1). Moreover, ER mRNA was expressed in the large intestines of both PND 10 and 8-week-old mice. Testis samples were used as a positive control for both ER␣ and ER mRNA. We next measured ER␣ and ER protein expression in mouse duodenum using western blot analysis. Mouse ovary and uterus were used as a positive control. ER␣ expression was found in mouse ovary and uterus, but not in duodenal mucosa of either PND 10 and 8-week-old mice (Fig. 2),
ER is a major estrogen receptor in mouse duodenal epithelium H&E staining revealed the gastroduodenal junction and the duodenal epithelium (Fig. 4A). Next, we examined the expression of ER␣ and ER in PND 10 mouse small intestine. As shown in Fig. 4B, ER␣ was not detected in male and female mice small intestine from each sampling day. Interestingly, ER was localized in duodenal epithelial cells (Fig. 4C), but not in jejunum and ileum (data not shown). ER-positive cells were stained in both nuclei and cytoplasm. A magnified microphoto is shown in Fig. 4D. The control sections treated with normal mouse or rabbit IgG were negative (data not shown). To assess the specificity of ER␣ and ER antibodies under our standard immunohistochemistry protocol, we stained for ER␣ and ER in paraffin-embedded sections of mouse uterus and ovaries. As expected [1], the ER␣ signal was detected in the nuclei of glandular (arrow) and luminal (arrowhead) epithelium as well as stromal cells (Fig. 4E), whereas ER staining was found in the nuclei of granulosa cells of ovarian follicles (Fig. 4F).
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Figure 4 ER is the predominant estrogen receptor in mouse duodenum. Immunohistochemical localization of ER␣ and ER in mouse duodenum. Paraffin-embedded sections of PND 10 mouse duodenum were used for H&E staining and immunohistochemistry. A. H&E staining revealed the gastroduodenal junction and proximal duodenum of PND 10 mice. Arrow indicates the first part of the duodenal epithelium at the gastroduodenal junction. Immunohistochemistry for ER␣ and ER in duodenum are shown in B and C, respectively. Black arrow indicates ER-positive epithelial cells at the gastroduodenal junction. A magnified microphoto is shown in D. ER-positive epithelial cells were stained in the nuclei and cytoplasm (n = 3). E. In mouse uterus, ER␣ was expressed in the nuclei of epithelial cells as well as somatic cells. Red arrow and arrowheads indicate the glandular and luminal epithelium of uterus, respectively. F. ER was expressed in granulosa cells of mouse ovarian follicles. Magnification, × 100 (A—C, E, F); scale bar, 500 m (A—C, E, F).
ER protein expression was evaluated by western blotting in age matched male and female mice duodenum (Fig. 5A). The quantitative analysis revealed ER protein expression has no significant difference between male and female mice duodenum (Fig. 5B).
ER localization changed in duodenal epithelium during postnatal development Next, we examined ER expression during ontogenesis, including the embryonic period, during postnatal development and in adult mouse duodenum. Interestingly, ER was expressed at embryonic day 18 in mouse duodenal
epithelium. Positive cells were localized in the lower part of the villi and were strongly stained in the nuclei and cytoplasm (Fig. 6A). Moreover, ER expression was found at PND 1 and PND 5 which indicates that ER is consistently expressed before duodenal crypt formation (Fig. 6B, C). After crypt formation, PND 10 and PND 15 mice results revealed that the ER-positive cells expanded toward the transit-amplifying cells (Fig. 6D, E). Surprisingly, at PND 20, which is the transit time of suckling to weaning, there were two major changes in the ER distribution pattern: ERpositive cells expanded throughout the duodenal villi, and ER was expressed only in the nuclei, but not in the cytoplasm (Fig. 6F). During the prepubertal and adult stages of the mice, ER expression was localized along the villi
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Figure 5 Comparision of ER expression in male and female mouse duodenum. A. Western blot analysis of ER in ovary, uterus and male, female mouse duodenum. Duodenal epithelium was obtained from 8-week old mouse intestine by tissue scraping. Arrows indicate ER (55 kDa) and -actin (42 kDa). B. Densitometry analysis of western blotting. ER protein expression was normalized by -actin.
(Fig. 6G, H). All of these findings were observed in three mice from both sexes at every sampling point of the experiments.
ERE binding proteins were co-localized with ER We investigated the DNA binding sites for EREs using Southwestern histochemistry. The localization of ERE binding proteins were detected in E 18 (Fig. 7A), PND 10 (Fig. 7D), and 4-week-old mouse duodenal epithelium (Fig. 7G). Positive staining was processed by a DAB-image analyzer and is shown in Fig. 7B, E, and H. No staining was detected when adjacent sections were reacted with a mutant ERE probe (Fig. 7C, F, I), which indicates high specificity of the probes. Moreover, duodenal adjacent sections were reacted without probe and revealed no staining (data not shown). Interestingly, the staining pattern and localization of ERE binding protein by Southwestern histochemistry was highly similar to that of ER protein expression (Fig. 7A, D, G).
Discussion The major finding of this study was the localization of ER in mouse duodenal epithelium. During ontogenesis, ER was consistently expressed in duodenal epithelium; however, the ER expression pattern changed around PND 20, which is the time when mice shift from breast milk to solid food. Moreover, ERE binding proteins were co-localized with ER expression in mouse duodenum. In the present study, we first detected the expression and localization of ER in duodenal epithelium. Indeed, both ER␣ and ER were examined throughout the intestinal tract, and only ER, but not ER␣, was detected in mouse intestine. The level of circulating estrogen may have an important role in the stimulation of estrogen signaling via ER in the duodenal epithelium. In our previous study, the upregulation of sodium hydrogen exchanger-3 (NHE3) expression via ER was found in mouse proximal colon, in parallel with the increase of circulating estrogen levels during pregnancy [34].
Figure 6 ER expression changes in mouse duodenum during ontogenetic development. Paraffin-embedded mouse duodenal sections were used for immunohistochemistry. ER was revealed in E 18 (A), PND 1 (B), PND 5 (C), PND 10 (D), PND 15 (E), PND 20 (F), 4-week-old (G), and 8-week-old (H) mouse duodenum (n = 3). Negative control is shown in the inset. Magnification, × 400 (A—F) and ×200 (G—H); scale bar, (A—F) 50 m and (G—H) 100 m.
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Figure 7 ERE binding protein is localized in mouse duodenum. A. Duodenal serial sections were used for detection of estrogen responsive elements (ERE) by Southwestern histochemistry in E18 (A—C), PND 10 (D—F), and 4-week-old (G—I) mice duodenum. Digoxigenin labeled vitellogenin ERE (vERE) oligo-DNA probe (A, D, G); vERE slides were processed by DAB image analyzer and the red color represents positive cells (B, E, H). Digoxigenin labeled mutated ERE (mERE) oligo-DNA probe (C, F, I). Magnification, × 400 (A-F) and × 200 (G—H); scale bar, (A—F) 50 m and (G—H) 100 m.
Moreover, circulating estrogen-dependent activation has been reported for both reproductive and non-reproductive organs [1,35]. In the present study, ER expression was found at each sampling point irrespective of sex. These findings demonstrate that ER is persistently expressed in both male and female mouse duodenal epithelium. However, the functional activation may depend upon the circulating level of estrogen ligand. Reports on sex-specific duodenal ulcer incidence supports our hypothesis that the ligand-dependent activation of estrogen signaling in female duodenum [17]. In our findings, ER was localized in proximal duodenal epithelium, the place that has the most active acid neutralization. This result suggests that estrogen may be important for duodenal mucosal protection against acidinduced injury. The duodenal epithelium plays a major role in the neutralization of acidic content to maintain normal homeostasis of luminal pH. In contrast, patients with duodenal ulcers have significantly decreased proximal duodenal mucosal bicarbonate secretion, which implies that the protective role of estrogen signaling may be related to stimulation of bicarbonate secretion [22]. Although the estrogen signaling mechanism in the duodenum is still not clear, the reduced frequency of duodenal ulcers in pregnant women or women taking oral contraceptives directly indicates a protective function of estrogen in the duodenum [21]. In addition, Tuo et al. reported that genistein, an isoflavone phytoestrogen, stimulates duodenal bicarbonate secretion through the PI3K pathway in mice, which confirms the presence of functional ER in mouse duodenum [36]. Kawano et al. reported that ER␣ and ER are localized in
the enteric neurons of Auerbach’s and Meissner’s plexuses; this was the limit of information about ER distribution in the small intestine, until now [3]. In the present study, we found the expression and localization of ER in the duodenum, which may be involved in the protective function of estrogen signaling in duodenal epithelium. Surprisingly, major changes were observed in the ER distribution pattern at PND 20, which is the transition time of suckling to weaning. In fact, intestinal epithelial changes in the suckling to weaning period are considered to result from major changes in diet, gut microflora [37], and levels of circulating hormones such as glucocorticoid [38]. Our results indicate that ER may be involved in epithelial cell differentiation and intestinal maturation. The ER-ligand complex binds to specific consensus sequences known as EREs, located in various target gene promoters, and stimulates gene transcription [31]. We found that ERE binding proteins were localized in duodenal crypts by Southwestern histochemistry. Interestingly, the highly similar expression pattern of ERE binding proteins paralleled that of ER during ontogenesis, indicating that ER may exert transcriptional activity in proximal duodenal epithelium. Only in the intestinal crypts of 4-week and older mice was a mismatch between ER and ERE binding protein localization observed. These findings indicate that not only ER, but also other receptors may mediate estrogen signaling in mouse duodenum. Although we examined the expression of estrogen related receptor alpha (ERR␣) and gamma (ERR␥), orphan receptors that could activate an ERE-dependent pathway, we did not find expression patterns that paralleled with ERE binding
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proteins. Therefore, more detailed analysis is necessary to investigate the mechanism of estrogen signaling in duodenal epithelium. In summary, our findings suggest that estrogen has a protective role in the duodenum that may be mediated through ER in mice. Although we did not provided the direct mechanism of estrogen protection, our findings concerning ER may helpful for the design of new treatment strategies for duodenal ulcers. Further functional investigations are necessary to unravel the protective mechanism of estrogen signaling via ER in the duodenum.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgments This study was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 21590216 and No. 24590255 to Y. Hishikawa and No. 23790340 to S.-I. Hino).
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ORIGINAL ARTICLE
Ontogenetic changes in the expression of estrogen receptor  in mouse duodenal epithelium Narantsog Choijookhuu , Shin-ichiro Hino , Phyu Synn Oo , Baatarsuren Batmunkh , Noor Ali Mohmand , Myat Tin Htwe Kyaw , Yoshitaka Hishikawa ∗ Division of Histochemistry and Cell Biology, Department of Anatomy, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan
Summary Estrogen is considered to be involved in duodenal function; however, the details of its receptor expression are largely unknown. The purpose of this study was to determine the expression and localization of estrogen receptors (ERs) in mouse duodenum. Male and female C57BL/6J mouse intestinal tissues were used to investigate the expression of ER␣ and ER by RT-PCR, western blotting, immunohistochemistry, and Southwestern histochemistry. ER, but not ER␣, was expressed in proximal duodenal epithelium, but not in jejunum and ileum. The expression of ER mRNA and protein were confirmed by RT-PCR and western blotting, respectively. At postnatal day 20, the transit period of suckling to weaning, the distribution of ER-positive cells was changed in the crypt-villus axis, and cytoplasm/nuclear staining changed to only nuclear staining. Moreover, Southwestern histochemistry was used to detect estrogen response element (ERE)-binding proteins, and their expression pattern was highly similar to that of ER. These results suggested that ER is the predominant ER type in mouse small intestine, and the highly similar co-localization of ERE-binding proteins reveals that ER is functionally active in mouse duodenum. The ER expression changes during postnatal development indicate that ER may be involved in the differentiation of duodenal epithelium. © 2015 Elsevier Masson SAS. All rights reserved.
Introduction ∗ Corresponding author. Division of Histochemistry and Cell Biology, Department of Anatomy, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan. Tel.: +81 985 85 1783; fax: +81 985 85 9851. E-mail address: [email protected] (Y. Hishikawa).
Estrogen plays a critical role in the maintenance of homeostasis and in the structure and function of various reproductive [1,2] and non-reproductive organs [3,4]. In general, the biological actions of estrogen are mediated through binding
http://dx.doi.org/10.1016/j.clinre.2015.01.004 2210-7401/© 2015 Elsevier Masson SAS. All rights reserved.
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to estrogen receptor ␣ (ER␣) and ER, which then bind to estrogen response elements (ERE), which are harbored in the promoter regions of various estrogen-dependent genes [5,6]. ERs are products of different genes and exhibit tissue and cell-type specific expression [7,8]. In contrast, the ER plays a dominant role in non-reproductive tissues such as the cardiovascular system [9] and large intestine [10], where it is expressed primarily in epithelial cells. In gastrointestinal tract, the presence of ER is mainly reported in large intestine such as human colon cancer [11,12] and colon cancer cell lines [13]. However in small intestine, only a report is the expression and localization of ER␣ and ER in enteric neurons of small intestine [3]. The presence of ERs in small intestinal epithelium has not been clarified, and this epithelium is critical for intestinal function. Although many reports have demonstrated the existence of functional ERs in the duodenum, the exact localization of ERs has not been confirmed. Clinical studies and animal models in rats and mice have indicated that estrogen has a protective role against duodenal ulcers [14—16]. The male to female sex ratio in the prevalence of duodenal ulcers is 4:1 in Asia [17], 1.7:1 in the United States [18], and 2.2:1 in Europe [19]. The global incidence of duodenal ulcers has gradually decreased in recent decades, but the prevalence is persistently higher in males [20]. Interestingly, a reduced frequency of duodenal ulcers has been found in pregnant women or women who taking oral contraceptives [21]. Moreover, basal and acid-stimulated duodenal bicarbonate secretions in premenopausal women are significantly higher than in postmenopausal women, suggesting that sex steroid hormone receptors are expressed in the duodenum and that they mediate biological activity [22]. In addition, decreased bicarbonate secretion after treatment with ICI 182,780 (an estrogen receptor antagonist) suggests that the duodenum expresses functional ERs [14]. In clinical settings, estrogen is well known to have diverse protective effects against osteoporosis [23], and cardiovascular and neurodegenerative diseases such as atherosclerosis [24] and Alzheimer’s [25]. Despite epidemiological studies suggesting that estrogen has a protective role in the duodenum, the distribution of ERs, which are thought to mediate estrogen action in the duodenum, remains to be clarified. In the present study, we first investigated the expression and localization of ER␣ and ER in mouse duodenum by immunohistochemistry. After confirming ER as the predominant ER type in the duodenum, we examined ER mRNA and protein expression using RT-PCR and western blotting, respectively. Finally, we examined ERE binding protein expression using Southwestern histochemistry.
Materials and methods
fluoride membrane (PVDF) was purchased from Bio-Rad (Hercules, CA, USA). 3,3 -Diaminobenzidine-4 HCl (DAB) was purchased from Dojindo Chemicals (Kumamoto, Japan). All other reagents used in this study were from Wako Pure Chemicals (Osaka, Japan) and were of high analytical grade.
Antibodies Mouse monoclonal antibody against ER␣ (1D5; 1.6 g/mL, dilution 1:100) and rabbit polyclonal antibody against ER (PA1—310B; 10 g/mL, dilution 1:100) were purchased from DAKO (Glostrup, Denmark) and Pierce Biotechnology (Rockford, IL, USA), respectively. Normal mouse and rabbit IgG, horseradish peroxidase (HRP)-goat anti-mouse IgG (dilution 1:100) and HRP-goat anti-rabbit IgG (dilution 1:200) were purchased from Dako. Mouse monoclonal antibody against -actin (AC-15; dilution 1:5,000) and normal goat IgG were purchased from Sigma (St. Louis, Mo, USA).
Animals and tissue preparation C57BL/6J male and female mice were examined at the various developmental period such as embryonic day 18, postnatal day 0; 1; 5; 10; 15; 20; 25; 28 and adult mice. In each sampling day points, three mice for both sexes were sampled and total 60 mice were used in this study. Mice were kept under constant 12 h dark/lighting condition and fed normal chow with drinking water ad libitum. The experimental protocol was approved by the Animal Ethics Review Committee of University of Miyazaki (#2012-502-3). After sacrifice, the intestinal tissues were removed, rinsed in icecold phosphate-buffered saline (PBS), opened through the mesenteric border and then divided into two pieces by longitudinal cutting. The first piece of the intestine was stripped off the mucosal layer by blunt dissection using the edge of a glass slide on ice-cold plate. Then tissues were snap frozen and kept at −80 ◦ C until used for RT-PCR and western blotting. The second piece of the intestine was fixed in 4% PFA in PBS (pH 7.4) at room temperature (RT) for 24 h and then embedded in paraffin.
RNA extraction and RT-PCR Total RNA was extracted from tissues using a QIAGEN RNA kit (QIAGEN, Hilden, Germany). RT-PCR assays were performed according to Hino et al. [26]. The oligonucleotides used for RT-PCR were as follows: ER␣: 5’-CTGCCAAGGAGACTCGCTACTGTGC-3’ 5’-GCTTGGCCAAAGGTTGGCAGCTCTCATG-3’; ER: 5’-GGAATCTCTTCCCAGCAGCA-3’ 5’-GGGAGCCCTCTTTGCTCTTACTGTC-3’; -actin: 5’-TCCTCCCTGGAGAAGAGCTAC-3’ 5’-TCCTGCTTGCTGATCCACAT-3’.
Chemicals and biochemicals Western blot analysis Paraformaldehyde (PFA) was purchased from Merck (Darmstadt, Germany). Trizma base, bovine serum albumin (BSA), 2-mercaptoethanol, 3-aminopropyl-triethoxysilane and Brij L23 were purchased from Sigma Chemical Co. (St Louis, MO, USA). Prestained protein marker was purchased from New England Biolab (Ipswich, MA, USA) and polyvinylidene
For western blot analysis of ER␣ and ER protein expression in duodenum, the stripped intestinal mucosa was lysed in hot sodium dodecyl sulfate that had been adjusted to contain 0.9% sodium dodecyl sulfate, 15 mM EDTA, 8 mM unlabeled methionine and a protease inhibitor cocktail, incubated in a
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ER expression in mouse duodenum boiling water bath for 10 min, cooled, diluted to 0.3% sodium dodecyl sulfate, and adjusted to contain 33 mM tris/acetate, pH 8.5, and 1.7% Triton X-100 [27]. The lysates were incubated on ice for 1 h and centrifuged at 12,000 g at 4 ◦ C for 30 min. Equal amounts of protein were subjected into 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred onto PVDF membranes. Then membranes were immunoblotted with ER␣ (0.8 g/mL, 1:200), ER (5 g/mL, 1:200) and -actin (0.4 g/mL, 1:5,000) antibodies. The membranes were washed with TBS/Tween-20, and then incubated with alkaline phosphatase-conjugated secondary antibody. The bands were visualized with 1 × 5bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) dye, till the bands of alkaline phosphatase were visible.
Immunohistochemistry Paraffin-embedded tissues were cut into 5 m thick sections and placed onto silane-coated slide glasses. The sections were deparaffinized with toluene, and rehydrated through graded ethanol series, and then autoclaved at 120 ◦ C for 15 min in 10 mM citrate buffer (pH 6.0) [28] and [29]. After inhibition of endogenous peroxidase activity with 0.3% H2 O2 in methanol for 15 min, the sections were pre-incubated with 500 g/mL normal goat IgG in 1% BSA/PBS for one hour to block non-specific binding of antibodies. Unless otherwise specified, all reactions were conducted at RT. Then, the sections were reacted with the primary antibodies for overnight. After washing with 0.075% Brij L23 in PBS, they were reacted with HRP-goat anti-mouse IgG or HRP-goat anti-rabbit IgG for 1 h. After washing in 0.075% Brij L23 in PBS, the HRP sites were visualized with DAB, Ni, Co, and H2 O2 according to the method of Adams [30]. As a negative control, normal mouse or rabbit IgG was used at the same concentration instead of the primary antibodies in every experiment.
Southwestern histochemistry Southwestern histochemistry was performed as described previously [31] and [32]. Double-stranded oligo-DNAs containing a complete palindromic estrogen responsive element (vERE: 5’-GATCCAGGTCACAGTGACCTGGATC-3’) of the chicken vitellogenin gene, and a mutated estrogen responsive element (mERE: 5’-GATCCAGATCACAGTGATCTGGATC3’) with a two base mutation with the digoxigenin labeling at the 3’-end. In briefly, deparaffinized sections were treated with autoclave at 120 ◦ C for 15 min in 10 mM citrate buffer (pH 6.0). The sections were washed in PBS and immersed in TMSE buffer containing 50 mM Tris/HCl buffer (pH 7.4), 5% nonfat dry milk, 400 mM NaCl and 1 mM EDTA for one hour [33]. Then, the sections were reacted with digoxigenin labeled oligo-DNA probe dissolved in TMSE buffer for overnight. For the detection of hybridized oligo-DNA probes, the sections were immunohistochemically stained with HRP-conjugated sheep anti-digoxigenin antibody, and the HRP sites were visualized according to the method of Adams [30].
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Figure 1 ER mRNA is expressed in mouse duodenum, but not in jejunum and ileum. RT-PCR analysis of ER␣ mRNA and ER mRNA in total RNA extracts of small and large intestinal mucosa from postnatal developing and adult mice. Small intestinal tissues were obtained from duodenum, jejunum, and ileum, respectively. Adult mouse testis was used as a positive control for both ER␣ and ER mRNA. -actin mRNA was used as a loading control.
Transfection of ER siRNA by electroporation Custom synthesized ER siRNA (Invitrogen) annealed duplexes were used for gene knock-down. Electroporatic transfection was performed as described previously [34]. Briefly, control or ER siRNA were injected into the center of testis and three pulses (poring pulse: 40 V, 30 ms of length with 50 ms interval, transfer pulse: 25 V, 80 ms of length with 50 ms interval) were delivered into testis using Nepa21 electroporator (Nepa Gene, Chiba, Japan). Testis was excised 24 h after the electroporation.
Image analysis The quantitative analysis for western blotting was performed by ImageJ 1.48 (NIH software). The staining of ERE sites were analyzed by a digital image analyzer (Winroof version 7.0, Mitani Corp, Tokyo, Japan). vERE and mERE slides were analyzed in same condition and positive cells were evaluated based on the staining density over the level of staining with the mERE probe.
Statistical analysis All data were expressed as mean ± SEM. Statistical significance was assessed by Student’s t-test. P < 0.05 was considered statistically significant. All analyses were performed with The Statistical Package for Social Sciences (version 17.0; Chicago, IL, USA).
Results ER mRNA and protein were expressed in duodenal mucosa The expressions of ER␣ and ER mRNA were analyzed by RTPCR throughout the intestinal tract in postnatal day (PND) 10 mice and in 8-week-old mice. ER␣ mRNA was not expressed along the intestinal tract. Interestingly, in small intestine,
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Figure 3 ER decreases after ER knockdown in mouse testis. Western blot analysis of ER in mouse testis tissue after electroporatic transfection of ER siRNA. Arrow indicates decreased ER protein expression in ER-knocked down testis, whereas asterisk indicates the same level of non-specific stainings. The loading of equal amounts of total protein was confirmed by -actin.
which was consistent with ER␣ mRNA expression. ER was expressed in mouse ovary and uterus, and a weak band was detected in duodenal mucosa of both PND 10 and 8week-old mice. There has no significant difference in ER expression in PND 10 and 8-week-old mice (Fig. 2C). High background staining was found in ER-antibody reacted membrane. Therefore, to determine the specificity of ER antibody, the ER gene was knocked-down by ER siRNA in mouse testis (Fig. 3). In ER knocked-down testis, the ER expression was significantly decreased comparing to control testis, while there had similar background staining was observed in same height (asterisk in Fig. 2 and Fig. 3).
Figure 2 ER, but not ER␣, is expressed in mouse duodenum. A. Western blot analysis of ER␣ and ER in mouse duodenum, ovary, and uterus. Duodenal epithelium was obtained from PND 10 and 8-week-old mouse intestine by scraping as described in the section materials and methods. Arrows indicate ER␣ (66 kDa), ER (55 kDa), and -actin (42 kDa). B,C. Densitometry analysis of western blotting. ER protein expression was normalized by -actin.
ER mRNA was expressed in scraped duodenal tissues of both PND 10 and 8-week-old mice, but not expressed in jejunum and ileum (Fig. 1). Moreover, ER mRNA was expressed in the large intestines of both PND 10 and 8-week-old mice. Testis samples were used as a positive control for both ER␣ and ER mRNA. We next measured ER␣ and ER protein expression in mouse duodenum using western blot analysis. Mouse ovary and uterus were used as a positive control. ER␣ expression was found in mouse ovary and uterus, but not in duodenal mucosa of either PND 10 and 8-week-old mice (Fig. 2),
ER is a major estrogen receptor in mouse duodenal epithelium H&E staining revealed the gastroduodenal junction and the duodenal epithelium (Fig. 4A). Next, we examined the expression of ER␣ and ER in PND 10 mouse small intestine. As shown in Fig. 4B, ER␣ was not detected in male and female mice small intestine from each sampling day. Interestingly, ER was localized in duodenal epithelial cells (Fig. 4C), but not in jejunum and ileum (data not shown). ER-positive cells were stained in both nuclei and cytoplasm. A magnified microphoto is shown in Fig. 4D. The control sections treated with normal mouse or rabbit IgG were negative (data not shown). To assess the specificity of ER␣ and ER antibodies under our standard immunohistochemistry protocol, we stained for ER␣ and ER in paraffin-embedded sections of mouse uterus and ovaries. As expected [1], the ER␣ signal was detected in the nuclei of glandular (arrow) and luminal (arrowhead) epithelium as well as stromal cells (Fig. 4E), whereas ER staining was found in the nuclei of granulosa cells of ovarian follicles (Fig. 4F).
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Figure 4 ER is the predominant estrogen receptor in mouse duodenum. Immunohistochemical localization of ER␣ and ER in mouse duodenum. Paraffin-embedded sections of PND 10 mouse duodenum were used for H&E staining and immunohistochemistry. A. H&E staining revealed the gastroduodenal junction and proximal duodenum of PND 10 mice. Arrow indicates the first part of the duodenal epithelium at the gastroduodenal junction. Immunohistochemistry for ER␣ and ER in duodenum are shown in B and C, respectively. Black arrow indicates ER-positive epithelial cells at the gastroduodenal junction. A magnified microphoto is shown in D. ER-positive epithelial cells were stained in the nuclei and cytoplasm (n = 3). E. In mouse uterus, ER␣ was expressed in the nuclei of epithelial cells as well as somatic cells. Red arrow and arrowheads indicate the glandular and luminal epithelium of uterus, respectively. F. ER was expressed in granulosa cells of mouse ovarian follicles. Magnification, × 100 (A—C, E, F); scale bar, 500 m (A—C, E, F).
ER protein expression was evaluated by western blotting in age matched male and female mice duodenum (Fig. 5A). The quantitative analysis revealed ER protein expression has no significant difference between male and female mice duodenum (Fig. 5B).
ER localization changed in duodenal epithelium during postnatal development Next, we examined ER expression during ontogenesis, including the embryonic period, during postnatal development and in adult mouse duodenum. Interestingly, ER was expressed at embryonic day 18 in mouse duodenal
epithelium. Positive cells were localized in the lower part of the villi and were strongly stained in the nuclei and cytoplasm (Fig. 6A). Moreover, ER expression was found at PND 1 and PND 5 which indicates that ER is consistently expressed before duodenal crypt formation (Fig. 6B, C). After crypt formation, PND 10 and PND 15 mice results revealed that the ER-positive cells expanded toward the transit-amplifying cells (Fig. 6D, E). Surprisingly, at PND 20, which is the transit time of suckling to weaning, there were two major changes in the ER distribution pattern: ERpositive cells expanded throughout the duodenal villi, and ER was expressed only in the nuclei, but not in the cytoplasm (Fig. 6F). During the prepubertal and adult stages of the mice, ER expression was localized along the villi
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Figure 5 Comparision of ER expression in male and female mouse duodenum. A. Western blot analysis of ER in ovary, uterus and male, female mouse duodenum. Duodenal epithelium was obtained from 8-week old mouse intestine by tissue scraping. Arrows indicate ER (55 kDa) and -actin (42 kDa). B. Densitometry analysis of western blotting. ER protein expression was normalized by -actin.
(Fig. 6G, H). All of these findings were observed in three mice from both sexes at every sampling point of the experiments.
ERE binding proteins were co-localized with ER We investigated the DNA binding sites for EREs using Southwestern histochemistry. The localization of ERE binding proteins were detected in E 18 (Fig. 7A), PND 10 (Fig. 7D), and 4-week-old mouse duodenal epithelium (Fig. 7G). Positive staining was processed by a DAB-image analyzer and is shown in Fig. 7B, E, and H. No staining was detected when adjacent sections were reacted with a mutant ERE probe (Fig. 7C, F, I), which indicates high specificity of the probes. Moreover, duodenal adjacent sections were reacted without probe and revealed no staining (data not shown). Interestingly, the staining pattern and localization of ERE binding protein by Southwestern histochemistry was highly similar to that of ER protein expression (Fig. 7A, D, G).
Discussion The major finding of this study was the localization of ER in mouse duodenal epithelium. During ontogenesis, ER was consistently expressed in duodenal epithelium; however, the ER expression pattern changed around PND 20, which is the time when mice shift from breast milk to solid food. Moreover, ERE binding proteins were co-localized with ER expression in mouse duodenum. In the present study, we first detected the expression and localization of ER in duodenal epithelium. Indeed, both ER␣ and ER were examined throughout the intestinal tract, and only ER, but not ER␣, was detected in mouse intestine. The level of circulating estrogen may have an important role in the stimulation of estrogen signaling via ER in the duodenal epithelium. In our previous study, the upregulation of sodium hydrogen exchanger-3 (NHE3) expression via ER was found in mouse proximal colon, in parallel with the increase of circulating estrogen levels during pregnancy [34].
Figure 6 ER expression changes in mouse duodenum during ontogenetic development. Paraffin-embedded mouse duodenal sections were used for immunohistochemistry. ER was revealed in E 18 (A), PND 1 (B), PND 5 (C), PND 10 (D), PND 15 (E), PND 20 (F), 4-week-old (G), and 8-week-old (H) mouse duodenum (n = 3). Negative control is shown in the inset. Magnification, × 400 (A—F) and ×200 (G—H); scale bar, (A—F) 50 m and (G—H) 100 m.
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Figure 7 ERE binding protein is localized in mouse duodenum. A. Duodenal serial sections were used for detection of estrogen responsive elements (ERE) by Southwestern histochemistry in E18 (A—C), PND 10 (D—F), and 4-week-old (G—I) mice duodenum. Digoxigenin labeled vitellogenin ERE (vERE) oligo-DNA probe (A, D, G); vERE slides were processed by DAB image analyzer and the red color represents positive cells (B, E, H). Digoxigenin labeled mutated ERE (mERE) oligo-DNA probe (C, F, I). Magnification, × 400 (A-F) and × 200 (G—H); scale bar, (A—F) 50 m and (G—H) 100 m.
Moreover, circulating estrogen-dependent activation has been reported for both reproductive and non-reproductive organs [1,35]. In the present study, ER expression was found at each sampling point irrespective of sex. These findings demonstrate that ER is persistently expressed in both male and female mouse duodenal epithelium. However, the functional activation may depend upon the circulating level of estrogen ligand. Reports on sex-specific duodenal ulcer incidence supports our hypothesis that the ligand-dependent activation of estrogen signaling in female duodenum [17]. In our findings, ER was localized in proximal duodenal epithelium, the place that has the most active acid neutralization. This result suggests that estrogen may be important for duodenal mucosal protection against acidinduced injury. The duodenal epithelium plays a major role in the neutralization of acidic content to maintain normal homeostasis of luminal pH. In contrast, patients with duodenal ulcers have significantly decreased proximal duodenal mucosal bicarbonate secretion, which implies that the protective role of estrogen signaling may be related to stimulation of bicarbonate secretion [22]. Although the estrogen signaling mechanism in the duodenum is still not clear, the reduced frequency of duodenal ulcers in pregnant women or women taking oral contraceptives directly indicates a protective function of estrogen in the duodenum [21]. In addition, Tuo et al. reported that genistein, an isoflavone phytoestrogen, stimulates duodenal bicarbonate secretion through the PI3K pathway in mice, which confirms the presence of functional ER in mouse duodenum [36]. Kawano et al. reported that ER␣ and ER are localized in
the enteric neurons of Auerbach’s and Meissner’s plexuses; this was the limit of information about ER distribution in the small intestine, until now [3]. In the present study, we found the expression and localization of ER in the duodenum, which may be involved in the protective function of estrogen signaling in duodenal epithelium. Surprisingly, major changes were observed in the ER distribution pattern at PND 20, which is the transition time of suckling to weaning. In fact, intestinal epithelial changes in the suckling to weaning period are considered to result from major changes in diet, gut microflora [37], and levels of circulating hormones such as glucocorticoid [38]. Our results indicate that ER may be involved in epithelial cell differentiation and intestinal maturation. The ER-ligand complex binds to specific consensus sequences known as EREs, located in various target gene promoters, and stimulates gene transcription [31]. We found that ERE binding proteins were localized in duodenal crypts by Southwestern histochemistry. Interestingly, the highly similar expression pattern of ERE binding proteins paralleled that of ER during ontogenesis, indicating that ER may exert transcriptional activity in proximal duodenal epithelium. Only in the intestinal crypts of 4-week and older mice was a mismatch between ER and ERE binding protein localization observed. These findings indicate that not only ER, but also other receptors may mediate estrogen signaling in mouse duodenum. Although we examined the expression of estrogen related receptor alpha (ERR␣) and gamma (ERR␥), orphan receptors that could activate an ERE-dependent pathway, we did not find expression patterns that paralleled with ERE binding
Please cite this article in press as: Choijookhuu N, et al. Ontogenetic changes in the expression of estrogen receptor  in mouse duodenal epithelium. Clin Res Hepatol Gastroenterol (2015), http://dx.doi.org/10.1016/j.clinre.2015.01.004
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proteins. Therefore, more detailed analysis is necessary to investigate the mechanism of estrogen signaling in duodenal epithelium. In summary, our findings suggest that estrogen has a protective role in the duodenum that may be mediated through ER in mice. Although we did not provided the direct mechanism of estrogen protection, our findings concerning ER may helpful for the design of new treatment strategies for duodenal ulcers. Further functional investigations are necessary to unravel the protective mechanism of estrogen signaling via ER in the duodenum.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgments This study was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 21590216 and No. 24590255 to Y. Hishikawa and No. 23790340 to S.-I. Hino).
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Please cite this article in press as: Choijookhuu N, et al. Ontogenetic changes in the expression of estrogen receptor  in mouse duodenal epithelium. Clin Res Hepatol Gastroenterol (2015), http://dx.doi.org/10.1016/j.clinre.2015.01.004