Histochem Cell Biol (2004) 121:399–405

DOI 10.1007/s00418-004-0644-6

ORIGINAL PAPER

Naoko Kawano · Takehiko Koji ·

Yoshitaka Hishikawa · Kunihiko Murase ·
Ikuo Murata · Shigeru Kohno

Identification and localization of estrogen receptor

a- and b-positive cells in adult male and female mouse intestine
at various estrogen levels
Accepted: 16 March 2004 / Published online: 8 May 2004
Springer-Verlag 2004

Abstract Although estrogen is implicated in the regula- intestine in two ways; one is through interstitial macro-
tion of mammalian intestinal function, the presence and the phages and the other is through intestinal neurons.
distribution of estrogen receptor (ER)-positive cells in the
intestine are still controversial. The present study was Keywords Estrogen receptors · Intestine · Mouse ·
designed to localize ERa- and ERb-expressing cells in Histochemistry · Estrogen
female and male mouse intestines immunohistochemi-
cally under various estrogen conditions, especially in fe-
male mice, ovariectomized as well at various phases of Introduction
the estrous cycle. Western blot analysis detected both
ERa (66-kDa band) and ERb (56-kDa band). Immuno- Significant actions for female sex steroids have been
histochemical staining of paraffin-embedded sections af- noticed in the gastrointestinal tract in various experi-
ter antigen-retrieval treatment with autoclaving revealed mental animal models and human clinical settings. In
staining for ERa in submucosal interstitial cells, and rodents, it was reported that estrogen plays a protective
double staining identified these cells as a subtype of in- role against chemically induced colon carcinogenesis and
testinal macrophages. The number of these cells varied experimental colitis (Smirnoff et al. 1999; Verdu et al.
according to the estrous cycle phase. Administration of 2002). In women, the symptoms of irritable bowel dis-
17b-estradiol to ovariectomized mice resulted in a sig- eases fluctuate across the menstrual cycle (Heitkemper
nificant increase in the number of ERa-positive macro- and Jarrett 1992). Despite this evidence, the distribution
phages. On the other hand, the nuclei of nerve cells in of estrogen receptor (ER), which is thought to mediate
Auerbach and Meissner plexuses were positive for both estrogen action in mammalian intestines, remains con-
ERa and ERb, but the number of positive nerve cells was troversial.
not affected by estrogen. Our results indicate that estrogen In 1996, Kuiper et al. reported a new subtype of ER,
and estrogenic compounds may exert their actions on the which was discovered from the rat prostate cDNA library
and designated as ERb (Kuiper et al. 1996). Since it was
implied that different actions of estrogen among tissues
T. Koji ()) · Y. Hishikawa can be explained by the differential expression of the two
Department of Histology and Cell Biology,
Nagasaki University School of Medicine, classical ERs, currently known as ERa and ERb (Paech et
1-12-4 Sakamoto, 852-8523 Nagasaki, Japan al. 1997), a great deal of effort has been made to analyze
e-mail: [email protected] the expression of both ERs in various tissues including
Tel.: +81-95-8497027 pituitary glands (Nishihara et al. 2000), female repro-
Fax: +81-95-8497028 ductive organs (Pelletier and El-Alfy 2000), and male
N. Kawano · K. Murase · S. Kohno reproductive organs (Tsurusaki et al. 2003). In the in-
Second Department of Internal Medicine, testines, most of the studies in this field were conducted
Nagasaki University School of Medicine, using human colon cancer tissues (Singh et al. 1998;
Nagasaki, Japan Foley et al. 2000; Campbell-Thompson et al. 2001; Witte
et al. 2001; Oduwole et al. 2002; Qui et al. 2002) or cell
I. Murata lines (Fiorelli et al. 1999; Arai et al. 2000; Nakayama et
Department of Pharmacotherapeutics,
Graduate School of Biochemical Sciences, al. 2000; Qui et al. 2002), and confirmed the presence of
Nagasaki University, ERb, while there is still a controversy about the expres-
Nagasaki, Japan sion of ERa in the intestine. More importantly, however,
400 400

only little information is available about the localiza- protocol (# 1009) was approved by the Biochemical Research
tion and characterization of ERa- (Singh et al. 1998) and Center, Center for Frontier Life Sciences, Nagasaki University.
Mice were housed two or three per cage in an air-conditioned
ERb- (Campbell-Thompson et al. 2001; Witte et al. 2001; and light-controlled room at the animal facility of Nagasaki Uni-
Oduwole et al. 2002) positive cells throughout the normal versity. Animals had free access to food pellets and water. Ten
intestine. intact male mice and seven to ten female mice at each estrous cycle
In the present study, we first investigated the distri- were used. The estrous cycle phase was estimated in each mouse by
bution of ERa- and ERb-positive cells in the intestines of examination of vaginal cell smears. Seven pregnant mice were
killed on pregnancy day 15. Bilateral ovariectomy was performed
normal male and female mice by immunohistochemistry in 11 female mice at 8 weeks of age, and five of them were treated
with antigen-retrieval protocols. Second, we examined with 17b-estradiol dissolved in corn oil and injected subcuta-
quantitatively the effects of various estrogen levels on the neously at a dose of 0.8 mg/mouse on days 1, 5, 9, and 13. On
number of ERa- and ERb-positive cells. The results re- day 14, all animals were killed and organs were removed as de-
scribed in Tissue collection and preparation.
vealed ERa expression in some stromal cells, which were Next, to investigate the influence of DES on mouse intestine,
later identified as macrophages, as well as in nerve cells five of ten male mice were treated with DES dissolved in 5%
in Auerbach and Meissner plexuses. The number of ERa- ethanol/corn oil, and the others were treated with 5% ethanol/corn
positive macrophages varied according to estrogen levels, oil as a control group. Injections were performed subcutaneously at
which increased gradually from diestrus to reach a peak at the dose of 20 mg/body weight on days 0, 5, 10, and 15. On day 20,
all animals were killed and organs were removed as described in
proestrus (Yoshinaga et al. 1969). In contrast, ERb was Tissue collection and preparation.
found only in nerve cells and its expression was not af-
fected by the estrogen status.
Tissue collection and preparation

After killing, the abdomen was opened, and the duodenum, jeju-
Materials and methods num, ileum, colon, rectum, ovary, and uterus were removed. Each
intestine sample was obtained from the proximal section. Ingesta of
Chemicals and biochemicals intestine were removed by rinsing in phosphate-buffered saline
(PBS). The intestines, ovaries, and uteri were cut into several small
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- pieces and divided into two groups. In the first group, the pieces
PAGE) reagents were purchased from Daiichi Pure Chemicals were quickly frozen and later used for western blotting. In the second
(Tokyo, Japan). Immobilon, polyvinylidene difluoride membra- group, tissue samples were fixed in 4% PFA in PBS at 4 C overnight
ne, was purchased from Millipore (Bedford, MA). Bovine serum and embedded in paraffin using a standard procedure.
albumin (BSA) minimum 98%, Brij 35, 2-mercaptoethanol, 3-
aminopropyltriethoxysilane, and 17b-estradiol were purchased
from Sigma (St. Louis, MO). Lima bean trypsin inhibitor was Preparations of tissue extract and western blotting
purchased from Worthington Biochemical (Freehold, NJ). Para-
formaldehyde (PFA) was purchased from Merck (Darmstadt, Intestines, ovaries, and uteri were homogenized in a lysis buffer
Germany), and 3,30 -diaminobenzidine-4HCl (DAB) and ethylene- containing 50 mM TRIS-HCl buffer (pH 7.5), 150 mM NaCl, 5 mM
diaminetetraacetic acid disodium salt dihydrate (EDTA) were EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 20 mM sodium
purchased from Dojin Chemicals (Kumamoto, Japan). Diethyl- molybdate, and 50 mg/ml lima bean trypsin as described previously
stilbestrol (DES) was purchased from ICN Biochemical (Aurora, (Mitchner et al. 1998). After centrifugation at 15,000 rpm for
OH). All other reagents used in this study were from Wako Pure 30 min at 4 C, the supernatants were collected and stored at 80 C.
Chemicals (Osaka, Japan) and were of analytical grade. Protein concentration was determined using a kit from BioRad
Laboratories according to the instructions provided by the manu-
facturer, using BSA as a standard. Twenty micrograms of the lysate
Antibodies used in the present study was mixed with the loading solution (200 mM TRIS-HCl, pH 8.0,
0.5 M sucrose, 5 mM EDTA, 0.01% bromophenol blue, 10%
A mouse monoclonal antibody against ERa (ER88; 1:160), which 2-mercaptoethanol, and 2.5% SDS), boiled for 5 min, and separated
recognizes the estrogen binding domain part as an epitope, was by SDS-PAGE with 10–20% gradient gel according to the method
purchased from BioGenex (San Ramon, CA). Rabbit polyclonal of Laemmli (1970), and electrophoretically transferred onto poly-
antibody against ERb (PA1–310B; 1:100) was purchased from vinylidene difluoride membranes. The membranes were blocked
ABR (Pennington, NJ). A rat monoclonal antibody against mouse with 10% nonfat milk in TRIS-buffered saline buffer (TBS; 20 mM
F4/80 (1:200) was purchased from Serotec (Raleigh, NC). A goat TRIS buffer, pH 7.6, and 150 mM NaCl) overnight at 4 C and
polyclonal antibody against mouse CD3- (1:200) was purchased incubated for 3 h with mouse anti-ERa or rabbit anti-ERb IgG,
from Santa Cruz Biotechnology (Santa Cruz, CA). A rat mono- diluted 1:100 and 1:80 with TBS/0.05% Triton X-100 buffer, re-
spectively. The membranes were subsequently washed three times
clonal antibody to mouse CD45R (1:200) was purchased from for 10 min each time with TBS/0.05% Triton X-100 buffer. As a
Caltag Laboratories (Burlingame, CA). Horseradish peroxidase second antibody, HRP-conjugated goat anti-mouse or anti-rabbit
(HRP)-conjugated goat anti-mouse IgG F(ab0 )2 (1:100) was pur- IgG F(ab0 )2 was reacted at 1:200 with 10% nonfat milk in TBS
chased from Chemicon International (Temecula, CA). HRP-con- buffer for 1 h and was again washed six times for 15 min each time
jugated goat anti-rabbit IgG F(ab0 )2 and HRP-conjugated goat anti- with TBS/0.05% Triton X-100 buffer. The bands were visualized
rat IgG F(ab0 )2 were purchased from MBL (Nagoya, Japan). Nor- with H2O2 and DAB in the presence of nickel and cobalt ions,
mal goat IgG, normal mouse IgG, and normal rat IgG were pur- according to the method of Adams (1981).
chased from Sigma.

Immunohistochemistry for ERa and ERb

Animals
Paraffin sections (5 mm) of the intestines and ovaries were cut
Young adult male, female, and pregnant ICR mice (7–9 weeks old) onto silane-coated glass slides, dewaxed with toluene, and rehy-
weighing 30–40 g were used in the present study. The experimental drated with serial ethanol solutions. The sections were autoclaved
401 401

at 120 C for 15 min in 10 mM citrate buffer (pH 6.0) (Ehara et al.

1995). After inactivation of endogenous peroxidase activity with
0.3% H2O2 in methanol for 30 min, the sections were preincubated
with 500 mg/ml normal goat IgG and 1% BSA in PBS for 1 h to block
nonspecific reaction with the first antibody. Then, the sec- tions were
reacted with anti-ERa (1:160) and anti-ERb (1:100) IgG
overnight. After washing with 0.075% Brij in PBS, HRP-labeled
goat anti-mouse and anti-rabbit IgG F(ab0 )2 were reacted for 1 h,
respectively, and the sites of HRP were visualized with DAB, Ni,
Co, and H2O2. As a negative control, some sections were reacted
with normal mouse IgG or normal rabbit IgG instead of the specific
antibody.

Identification of cell type of ERa-positive cells

To identify ERa-positive cells, we performed double staining for

ERa and various cell surface markers in sections of the intestine.
After deparaffinization, the slides were immersed in PBS and au-
toclaved in 10 mM citrate buffer (pH 6.0) for 15 min at 120 C for
antigen retrieval. Then they were reacted with anti-F4/80 antibody, Fig. 1 Western blot analysis of estrogen receptor (ER)a and ERb
anti-CD45R antibody, or anti-CD3- antibody, and HRP-goat anti- expression. Twenty micrograms of extracts from normal ICR fe-
rat IgG (1:100) successively, according to the protocol described male mouse small and large intestines, as well as uterus and ovary
above. HRP sites were visualized with DAB and H2O2. The slides as a positive control, were subjected to SDS-PAGE. Western
were immersed three times with 0.1 M glycine-HCl buffer (pH 2.2) blotting was performed using a monoclonal antibody ER88 for ERa
for 30 min. After washing with Milli-Q water once and with PBS (left panel) and a polyclonal antibody for ERb (right panel). Ar-
three times, the sections were reacted with anti-ERa antibody and rows in the left and right panels indicate a 66-kDa band and a 55-
HRP-goat anti-mouse IgG (1:100) successively, similar to that kDa band, respectively
described above. The sections were visualized with 4-Cl naphthol
and H2O2 solution.
embedded sections of mice ovaries that had been auto-
claved for antigen retrieval. As expected (Wang et al.
Statistical analysis 2000) the ERa signal was detected in the nuclei of
granulosa cells in degenerative follicles as well as stromal
ERa-positive cells were counted per square millimeter. Data are cells, whereas ERb staining was found in the nuclei of
expressed as mean € SD. Differences between groups were ex-
amined for statistical significance using Student’s t-test. P<0.05 granulosa cells of healthy developing follicles (Fig. 2).
denoted the presence of a statistically significant difference. In the next step, we examined the expression of ERa
and ERb in the intestines of male and female mice using
the same protocol. As shown in Fig. 3, immunostaining
Results for ERa was observed in some stromal cells of lamina
propria mucosae, and in the nuclei of nerve cells of
Analysis of ERa and ERb expression in extracts of nor- Auerbach and Meissner plexuses. The stromal cell signal
mal mouse intestine by western blotting for ERa was localized predominantly in the cytoplasm
but not the nuclei. Interestingly, the number of stromal
First, to confirm the specificity of the antibodies used for ERa-positive cells in female mice was higher than in
the following immunohistochemistry of ERa and ERb, male mice. In contrast, the signal for ERb was detected in
western blot analysis of mouse intestinal tissue extracts the cytoplasm of nerve cells in Auerbach and Meissner
was performed. In the experiment, we used extracts of plexuses. There was no difference in the intensity of im-
normal mouse uterus and ovary as a positive control for munostaining for ERb as well as in the number of ERb-
ERa and ERb, respectively. As shown in Fig. 1, anti-ERa positive cells between male and female mice. As negative
antibody (ER88) detected a single band corresponding to control, when some sections were reacted with normal
66-kDa band in the uterine extract, and anti-ERb reacted IgG at the same dilution instead of the specific antibody,
specifically with a 55-kDa band in the ovarian extract. In no staining was observed (data not shown).
the next step, when the extract of intestine was analyzed
by western blotting in the same way, both the 66- and 55-
kDa bands for ERa and ERb, respectively, were also Effects of estrous cycles on the number
detected. of ERa-positive cells in the large intestine of female mice

To analyze the relationship between ERa-positive cells

Immunohistochemical localization of ERa and ERb and estrogen level, the number of ERa-positive cells in
in intestines of normal male and female mice the lamina propria mucosae was counted and compared
based on the stage of the estrous cycle (Fig. 4). The
To assess the specificity of anti-ERa and anti-ERb anti- density (number per square millimeter) of ERa-positive
bodies under our standard protocol of immunohisto- cells in the large intestine, but not the small intestine,
chemistry, we tried to localize ERa and ERb in paraffin- varied according to the stage of the estrous cycle; the
402 402

Fig. 2A–D Immunohistochem-

ical localization of ERa (A, B)
and ERb (C, D) in paraffin-
embedded sections of the
mouse ovary. ERb was detected
in granulosa cell nuclei of
healthy follicles, while ERa
was found in the nuclei of
granulosa cells of degenerating
follicles and of interstitial cells.
Arrows indicate positive cells.
Magnification 200 (A, C),
400 (B, D)

highest number of such cells was observed at diestrus and In these experiments, immunostaining for ERa and
proestrus. No such tendency was observed in the small ERb in nerve cells did not change in response to exoge-
intestine. nous 17b-estradiol or DES. In addition, there was essen-
tially no change in the staining pattern of these ERs in
various parts of the intestine.
Effects of exogenous estrogen on the number
of ERa-positive cells in ovariectomized mice
Identification of ERa-positive cells
To confirm the effect of estrogen on the density of ERa- in mouse intestinal stroma
positive cells, we prepared ovariectomized mice and
compared the density of ERa-positive cells in the stroma Finally, to identify ERa-positive stromal cells, we per-
of intestines in mice injected with or without exogenous formed immunohistochemical analyses of various sur-
17b-estradiol. As shown in Fig. 5, administration of 17b- face markers such as F4/80 (a marker for macrophages;
estradiol significantly increased the density of ERa-pos- Fig. 6), CD3 (for pan-T cells), and CD45R (for B cells) in
itive cells compared with the control group injected with the paraffin sections of normal mouse intestine. Com-
the vehicle, corn oil, alone. In addition, ovariectomy re- parison of the expression of these surface antigens and
sulted in a decrease in the density of ERa-positive stromal ERa expression showed that a population of macrophages
cells (data not shown). in the intestinal stroma, which were positive to anti-F4/80,
was also immunopositive for ERa.

Effects of exogenous DES on the number

of ERa-positive cells Discussion
We then investigated the effect of DES, a powerful es- In the present study, we examined the expression of ERa
trogenic endocrine disruptor (Wiggins et al. 1980), on the and ERb in the mouse intestine and also addressed the
density of ERa-positive cells. As shown in Fig. 5, the effects of estrogen on the distribution of ERa- and/or
density of ERa-positive cells in mice injected with DES ERb-positive cells. Firstly, we confirmed the presence of
was higher than that of the control group. This significant both ERa and ERb in the intestines by western blotting
increase was seen in the large intestine but not in the and then applied immunohistochemistry to localize these
small intestine. receptors. Our results showed positive staining for ERa
in certain stromal cells and nerve cells in Auerbach and
403 403

Fig. 3A–E Immunohistochemi-

cal localization of ERa (A–C)
and ERb (D, E) in paraffin-
embedded sections of mouse
small (A, C, D) and large in-
testines (B, E). Arrows indicate
positive cells. A, B Staining for
ERa was observed in the nuclei
and cytoplasm of stroma cells
of lamina propria mucosae. C–
E ERa and ERb were detected
in the nucleus and cytoplasm of
nerve cells, respectively. Mag-
nification 400

Fig. 4A, B Quantitative analy-

sis of the density of ERa-posi-
tive stroma cells at different
estrogen levels in the small (A)
and large (B) intestines of nor-
mal mice. Data are mean € SD.
* P<0.05

Meissner plexuses, while ERb was exclusively expressed The presence of ERa in mammalian intestines has
in the latter cells. Moreover, the number of ERa-positive been a controversial issue in the last few years (Mary et
cells, which were later confirmed to be a subgroup of al. 1993). Although a specific estrogen binding activity
intestinal macrophages, was influenced by estrogen. was detected in intestinal extracts, immunohistochemical
404 404

Fig. 5A, B Quantitative analy-

sis of the density of ERa-posi-
tive stroma cells in the presence
of exogenous estrogen. A small
intestine, B large instestine.
Data are mean € SD. * P<0.05

the production of nNOS can be regulated by estrogen

through both ERs. Furthermore, these findings suggest
that the produced NO might act as a “second messenger”
for the absorbed estrogenic compounds. On the other
hand, the cellular localization of ERa in the stromal cells
was mostly cytoplasmic. Since ERa is usually found in
nuclei irrespective of ligand-free or -bound states (Eche-
verria et al. 1994), the present results suggest a different
role for the cytoplasmic ER from what is expected. In this
context, it should be noted that plasma membrane-type
ERa was recently identified and implicated in the growth
regulation of human breast cancer cells (Marquez and
Pietras 2001).
In the present study, the number of ERa-positive mac-
rophages in the intestine fluctuated and was dependent on
estrogen level. In the normal estrous cycle, the density of
these cells in the large intestine was highest at the stage of
Fig. 6 Double staining for F4/80 and ERa in paraffin-embedded diestrus. Administration of estrogen to ovariectomized
sections of normal female mouse intestine. F4/80-positive cells are
stained brown, whereas ERa-positive cells are stained blue. Some mice and treatment of intact male mice with DES result-
of the F4/80-positive cells are ERa-positive cells. Arrow indicates ed in a marked increase in the density of ERa-positive
double-positive cell. Magnification 400 macrophages in the large intestine, but not in the small
intestine. These results indicate that the increased density
of ERa-positive macrophages in the large intestine rep-
and histochemical approaches failed to identify the re- resents a reaction to the high level of estrogen.
sponsible cells. However, a recent study reported the pres- Since immune cells like macrophages can migrate, un-
ence of ERa mRNA-expressing cells in the stroma (lam- derstanding the reason for the increased density is
ina propria mucosa) of normal human colon by in situ somewhat complicated. In fact, it is known that estrogen
hybridization (Singh et al. 1998). Our study confirmed the promotes the migration of macrophages and plasma cells
above findings in the mouse intestine and further identi- into mouse uterus and that the distribution of immune
fied the cells as a subtype of macrophages. On the other cells in mouse uterus and vagina is dependent on the
hand, since the discovery of ERb in 1996 (Kuiper et al. levels of estrogen and the estrous cycle (Yoshinaga et al.
1996), several reports indicated the expression of this 1969). It is true that estrogen can upregulate the expres-
receptor subtype in normal and malignant intestines, al- sion of ERa in some cases including primate uterus (Koji
though identification of positive cells in the normal in- and Brenner 1993), but this is not the case in our study
testine remained to be clarified. because the staining intensity of macrophages was con-
The roles of these ERs in the intestine remain largely stant even at the stage of the highest density. Furthermore,
unknown. It is possible that estrogenic compounds ab- in the present study, there was an apparent discrepancy in
sorbed from the intestinal epithelium exert their first ef- the peak time between the migration of immune cells and
fects through these intestinal receptors. Previous studies the concentration of estrogen in peripheral blood (Yoshi-
in rats reported the presence of immunohistochemical naga et al. 1969). In nonreproductive tissues of transgenic
staining of neuronal nitric oxide synthase (nNOS) in mouse that expresses a luciferase reporter gene under the
nerve cells of the Auerbach plexus in the colon and that control of activated estrogen receptors, maximal lucifer-
the intensity of the staining of nNOS as well as the ase activity occurred at diestrus (Ciana et al. 2003), while
number of the positive cells increased with estrogen the number of immune cells reached a peak at proestrus in
treatment (Shah et al. 2001). Considering the presence of the female reproductive organs (Mara Suburo et al. 1995;
both ERa and ERb in the nerve cells, it is not strange that
405 405

Kaeoket et al. 2001). This temporal difference could be Mara Suburo A, Chaud M, Franchi A, Polak JM, Gimeno MA (1995)
due to tissue-specific responses to estrogen. In this regard, Distribution of neuronal and non-neuronal NADPH di-
aphorases and nitric oxide synthases in rat uterine horns under
English et al. (1999) reported that 17b-hydroxysteroid different hormonal conditions. Biol Reprod 52:631–637
dehydrogenase is present in human colon mucosa, indi- Marquez DC, Pietras RJ (2001) Membrane-associated binding sites
cating that estrogen level in the colon tissues can be lo- for estrogen contribute to growth regulation of human breast
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Finally, in inflammatory bowel diseases (IBD), acti- Mary LT, Xiaomeng XU, Andrea MN, Cheryl SW (1993) The
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vation of monocyte migration from blood to the intestine cells. Endocrinology 132:426–430
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Nakayama Y, Sakamoto H, Satoh K, Yamamoto T (2000) Tam-
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treatment of IBD patients. Further studies on human telomerase expression through estrogen receptor beta mediated
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Yamashita S, Koji T (2000) Ontogenetic changes in the ex-
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