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Localization of estrogen and androgen receptors in male reproductive tissues of mice and rats.

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THE ANATOMICAL RECORD PART A 279A:768 –778 (2004)
Localization of Estrogen and
Androgen Receptors in Male
Reproductive Tissues of
Mice and Rats
SHUJI YAMASHITA*
Electron Microscope Laboratory, School of Medicine, Keio University, Tokyo, Japan
ABSTRACT
Using immunohistochemical methods, we studied the cell-type- and species-specific
expressions of estrogen receptor (ER) isoforms (ER␣ and ER␤) and androgen receptors (ARs)
in the male reproductive tract and accessory sex glands of mature mice and rats. ER␣ and
ER␤ showed cell-type- and species-specific distributions, respectively. In contrast, AR was
localized in the epithelial and stroma cells of all tissues examined in this study, in both
species. In mice, the epithelial cells of the ductuli efferentes showed a strong ER␣-immunoreaction, and those of the caput epididymis, coagulating glands, and prostate also exhibited
a positive reaction. Stroma cells, except in the ductuli efferentes, showed a positive ER␣immunostaining. In rats, ER␣ was detected in very few cell types: the epithelial cells of the
ductuli efferentes showed a strong reaction, and the stroma cells of the ampullary and
urethral glands exhibited a weak reaction. ER␤ was localized in the epithelial cells of the
prostate in mice, while the reaction was faint or negative in both the epithelial and stroma
cells of other tissues. In rats, the ER␤-immunoreaction was strongest in the epithelial cells of
the ventral prostate. The epithelial cells of the corpus and cauda epididymis, ductus deferens,
and urethral glands, and the stroma cells of the urethral glands were also positively ER␤immunostained. Almost the same AR distribution pattern was observed in both species. In
particular, strong AR-immunostaining was present in the epithelial cells of the caput and
corpus epididymis, seminal vesicle, and ventral prostate. These results indicate that species
and tissues differences should be taken into careful consideration in assessing the physiological and pharmacological effects of sex steroids (particularly estrogens) on the reproductive
tissues of male rodents. © 2004 Wiley-Liss, Inc.
Key words: estrogen receptor ␣; estrogen receptor ␤; androgen receptor; male
reproductive tissue
Androgens are essential for the normal development
and functional maintenance of male reproductive organs.
The actions of sex hormones are mediated through their
nuclear receptors. Hormone-occupied receptors bind directly to their corresponding hormone responsive elements of target genes, and subsequently recruit transcriptional cofactors to modulate the transcription (Evans,
1988; Beato and Sanchez-Pacheco, 1996; Moras and
Gronemeyer, 1998). Androgen receptors (ARs) have been
demonstrated in most cell types of male reproductive tissues (Schleicher et al., 1985; Sar et al., 1990; Zhou et al.,
2002). Two isoforms of estrogen receptors (ERs)—ER␣ and
ER␤— have also been localized in several male reproductive tissues (Iguchi et al., 1991; Kuiper et al., 1997; Pelletier, 2000; Atanassova et al., 2001; Zhou et al., 2002).
Although the roles of estrogens in male reproductive organs are still poorly understood, estrogens are widely be©
2004 WILEY-LISS, INC.
lieved to participate in normal and abnormal processes of
male physiology through ERs (Iguchi, 1992; Carreau et
al., 1999; Hess et al., 2001). Recent studies using ER␣knockout mice have demonstrated the presence of ER␣ in
Grant sponsor: Ministry of Education, Science and Culture,
Japan; Grant number: 11670030; Grant sponsor: Gijuku Academic Development Fund.
*Correspondence to: Shuji Yamashita, Electron Microscope
Laboratory, School of Medicine, Keio University, 35-Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Fax: 81-3-3353-3290.
E-mail: shuji@sc.itc.keio.ac.jp
Received 8 June 2003; Accepted 23 November 2003
DOI 10.1002/ar.a.20061
Published online 7 July 2004 in Wiley InterScience
(www.interscience.wiley.com).
ER␣, ER␤ AND AR IN MALE MICE AND RATS
the ductuli efferentes to be essential for luminal fluid
absorption from the testis (Hess et al., 1997; Lee et al.,
2000). In addition, it is well known that exogenous estrogen administration during the perinatal period elicits
morphological and functional changes in both the female
and male reproductive organs of immature and mature
rodents (McLachlan et al., 1975; Newbold et al., 1985;
Iguchi, 1992).
ER␣ and ER␤ appear to have unique and sometimes opposite roles (Paech et al., 1997; Hall and McDonnell, 1999;
Liu et al., 2002). ER␤ reportedly acts as a negative regulator
in epithelial proliferation in the uterus and prostate (Weihua
et al., 2000, 2001). Both isoforms of ERs and AR can also
modulate transcription indirectly through contact with various transcription factors, such as activator protein-1 (AP-1)
(Paech et al., 1997; Sato et al., 1997). Kushner et al. (2000)
demonstrated that ER␣ and ER␤ have differential interactions with AP-1, depending on the ligands used.
In reproductive tissues, sex steroids induce cell-typespecific responses. Several synthetic sex hormones and
antihormones also act in a tissue- and species-specific
manner (Campen et al., 1985; Rutqvist et al., 1995). These
differential responses to the ligands may be due to the
concentrations of ER isoforms and AR, transcriptional
cofactors, and other transcription factors (including AP-1)
in tissues and cells. In addition, Cunha and colleagues
(Bigsby and Cunha, 1986; Cooke et al., 1997; Kurita et al.,
2001) demonstrated that the presence of ERs and AR in
stroma cells is important for responses to sex steroids in
the epithelial cells of female and male reproductive tissues. Mice and rats are the animals most extensively used
to study the effects of sex steroids and the mechanisms of
hormone actions, and hormone target tissues from both
species are usually assumed to provide similar responses
to hormonal stimulation. However, systematic and comparative examinations concerning the distribution of ER
isoforms and AR have not been carried out in the male
reproductive tissues of mice and rats. Therefore, in the
present study, were studied cell-type- and species-specific
expressions of AR, ER␣, and ER␤ in the reproductive
tracts and accessory sex glands of mature male mice and
rats, using immunohistochemical techniques.
MATERIALS AND METHODS
Reagents
Anti-ER␣ rabbit antibody (MC-20, sc-542), anti-AR rabbit antibody (C-19, sc-815), and their immunizing peptides
(sc-542P and sc-815P) were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Anti-ER␤ rabbit antibody (PA1-310B) and the neutralizing peptide (PEP-007)
were obtained from Affinity Bioreagents (Golden, CO).
Peroxidase-linked anti-rabbit IgG F(ab)⬘2 fragment (from
donkey), HRP-F(ab)⬘2, was purchased from Amersham Japan (Tokyo, Japan). The blocking reagent was from Boehringer Mannheim (Mannheim, Germany), and the block
ace was from Dainippon Parmaceutical (Osaka, Japan). A
protease inhibitor cocktail (complete) and a protein quantification kit were obtained from Roche Diagnostics (Tokyo, Japan) and Dojindo Molecular Technologies (Gaithersburg, MD), respectively. Molecular-weight markers
(precision plus protein standards) were obtained from BioRad Laboratories (Hercules, CA), and the ELC Western
blotting detection kit was from Amersham Japan.
769
Animals and Tissue Preparation
Male CD-1 mice (8 weeks old), male Wistar rats (8 weeks
old), and female CD-1 mice (3 weeks old) were obtained from
Clea Japan (Tokyo, Japan). The uteri and ovaries from
3-week-old mice were used as a positive control and a standard for the immunohistochemistry and Western blot analysis of ER␣ and ER␤. In addition, we used 8-week-old male
mice that were injected with 17␤-estradiol (E2) or vehicle,
and killed 1 hr after the injection, to determine a suitable
fixing procedure for ERs, since we previously observed that
hormone-unoccupied ER␣ is easily extracted from frozen
sections during fixation (Yamashita and Korach, 1988). Tissues were mounted in OTC compound. They were then frozen in dry ice-cooled acetone for the immunohistochemistry,
or on a dry ice block for Western blotting, and were stored at
– 80°C. Six male mice, six male rats, and four female mice
were used for the immunohistochemistry. Tissues for the
Western blotting were collected from six male mice, six female mice, and four male rats.
Immunohistochemistry
Frozen sections (7 ␮m thick) were fixed with 4% paraformaldehyde dissolved in 0.15 M phosphate buffer (pH
7.4) for 20 min at room temperature (Yamashita, 2002).
The fixed sections were washed with phosphate-buffered
saline (PBS), 10 mM phosphate buffer, and pH 7.4 containing 0.85% NaCl, and were then treated with 0.2%
glycine in PBS for 30 min. After the sections were treated
with the blocking solution (1% bovine serum albumin and
1% blocking reagent dissolved in PBS) for 60 min, they
were incubated with each antibody overnight at 4°C. AntiER␣ antibody was diluted 500-fold, and other antibodies
were diluted 200-fold. All primary antibodies and HRPF(ab)⬘2 were diluted with the blocking solution. Primary
antibodies that were preabsorbed with their immunizing
peptides were employed for the control immunostaining.
The diluted antibodies (1 ml) were incubated with 5 ␮l of
each peptide solution overnight at 4°C. The sections were
then incubated with HRP-F(ab)⬘2 diluted 50-fold for 60
min at room temperature. Immunoreaction was visualized
with an imidazol-DAB solution.
Western Blot Analysis
The frozen tissues were homogenized with a glassTeflon homogenizer in ice-cold 50 mM Tris-HCl buffer (pH
7.4) containing 0.4 M NaCl, 0.1 mM EDTA, 0.1 mM EGTA,
and the protease inhibitor cocktail, and centrifuged at
14,000 ⫻ g for 10 min at 4°C. The protein concentration of
the supernatant was measured with the protein quantification kit, and the supernatant was aliquoted and stored
at – 80°C. Each sample was subjected to sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE),
with a 10% gel, and the precision plus protein standards
were used as molecular-weight markers. The separated
polypeptides were then transblotted onto a PVDF membrane. The membrane was treated with the block ace for 2
hr at room temperature, and then with the first antibodies
overnight at 4°C with a gentle shaking (anti-ER␣ antibody, 1:2000; anti-ER␤ antibody, 1:500; anti-AR antibody,
1:400). The membrane was subsequently incubated with
HRP-F(ab⬘)2, 5,000-fold dilution, for 1 hr at room temperature. All of the antibodies were diluted with the blocking
solution employed for the immunohistochemistry. The enzyme activity of HRP was detected with the use of an ELC
770
YAMASHITA
Western blotting detection kit and a Lumivision Imager
(HSII; Aisin Seiki, Aichi, Japan).
stronger ER␤-immunoreaction than mice in tissues in
which ER␤ was detected.
RESULTS
Immunohistochemical Procedures and
Specificities of Antibodies
Reproductive tract. The epithelium of the ductuli
efferentes exhibited the strongest ER␣-immunoreaction in
the male reproductive tissues of both species (Fig. 3a and
b); however, no clear ER␤-immunostaining was present in
the epithelium (Fig. 4a).
In the mouse epididymal duct, ER␣-immunoreaction
was strongest in the epithelial cells of the caput (Fig. 3a).
However, in the initial segment, ER␣-immunostaining of
the principal cells was faint, although apical cells and
narrow cells exhibited moderate staining (Fig. 3a). ER␣immunoreaction in epithelial cells gradually diminished
from the corpus to the cauda, and only clear cells showed
a positive reaction in the distal portions (Fig. 3c). Stroma
cells showed a positive ER␣-immunoreactivity throughout
the epididymal duct, with the cauda exhibiting the strongest reaction. In the rat epididymis, ER␣-immunoreaction
was negative in both epithelial and stromal cells (Fig. 3b
and e). The epididymal epithelium of mice showed a faint
ER␤-immunoreaction, whereas those of the corpus and
cauda epididymis were weakly immunostained for ER␤ in
rats (Fig. 4a and b).
In the ductus deferens, ER␣-immunoreaction was positive in the stroma cells, but not in the epithelium, of both
species (Fig. 3d and f), while a faint to weak ER␤-immunoreaction was seen in both cell types (Fig. 4c and d).
We examined various fixing protocols and antigen-retrieval methods to obtain a suitable immunohistochemical
procedure. As the concentration of phosphate buffer-dissolving formaldehyde was increased from 0.05 M to 0.2 M,
the extraction of the ERs was suppressed, and hormonetreated and -untreated tissues began to show a similar
ER-immunoreaction intensity accompanied by a reduction
in immunostaining. Therefore, in this study, we fixed frozen sections with 4% formaldehyde in 0.15 M phosphate
buffer at room temperature to reduce the extraction of
hormone-unoccupied ERs, and to obtain a relatively high
immunoreaction sensitivity. Antigen retrieval procedures
by heating were ineffective for the immunostaining of
ER␣, ER␤, and AR. When the frozen sections fixed with
formaldehyde were soaked in boiling solutions or microwaved in solutions at pH 3.5, 6.0, or 9.0, the immunoreactions of these receptors were completely lost or reduced.
ER␣, ER␤, and AR proteins were exclusively localized in
the nuclei in the standard tissues (Fig. 1a, c, and e) and all
tissues examined in this study. Uterine gland epithelia
from 3-week-old mice were used as a standard for ER␣immunostaining and for scoring the intensity of staining
(Fig. 1a). Granulosa cells of 3-week-old mouse ovary
served as a standard for ER␤-immunostaining intensity
(Fig. 1c), and epithelial cells of seminal vesicles in 8-weekold rats were used as a standard for AR-immunostaining
(Fig. 1e). In every specimen, immunostaining by antibodies preabsorbed with their respective immunizing peptides almost completely disappeared (Fig. 1b, d, and f).
The specificities of the antibodies were further confirmed
by Western blot analyses. Anti-ER␣ antibody recognized the
main 67-kDa band (which corresponds to the molecular
weight of ER␣ protein) and minor 56- and 50-kDa bands
(which may be degradation products of ER␣ (Korach et al.,
1988)) in extracts from a 3-week-old mouse uterus (Fig. 2A,
lane 1). These bands were recognized in the mouse ductuli
efferentes (lane 2), epididymis (lane 3), and ventral prostate
(lane 4). In the rat tissues, 67- and 50-kDa bands and a few
very faint bands were detected in the ductuli efferentes (lane
5), but not in the epididymis (lane 6) or ventral prostate (lane
7). ER␣-antibody pretreated with the antigenic peptide
showed a faint reaction with 67-kDa polypeptide only in the
mouse uterus. On the Western blots, anti-ER␤ antibody
recognized a single band in the ovary with an apparent
molecular weight of 54 kDa (which corresponds to that of
ER␤ protein (Kuiper et al., 1996)), whereas preabsorbed
antibody with the immunizing peptide provided a negative
reaction (Fig. 2B, lane 1). Polypeptides, 140 and 105, 54 and
35 kDa, from the mouse caput epididymis reacted with anti-AR antibody (Fig. 2C, lane 1). The molecular weight of AR
is reported to be about 110 kDa (Weihua et al., 2002). Male
reproductive tissues showed a differential molecular weight
of positive bands, although 54 kDa was a major band (data
not shown). Antibody treated with the immunizing peptide
showed a negative immunoreaction (lane 2).
Localization of ERs
ER␣ was localized in many reproductive tissues in mice,
but in few cell types in rats. In contrast, rats showed a
Accessory glands. In mice, epithelial cells contained
relatively high amounts of ER␣ proteins in the coagulating glands (Fig. 3i) and a low level of ER␣ protein in
the ventral and dorsal prostate (Fig. 3g). A faint to weak
ER␣-immunoreaction was seen in the stroma cells of
seminal vesicles, coagulating glands, prostate glands,
and urethral glands (Fig. 3g and i). Stromal cells of the
ampullary gland exhibited a moderate reaction. In rats,
ER␣-immunoreaction was barely recognizable in the
epithelial cells of the accessory glands (Fig. 3h and j).
Stroma cells in the proximal portion of the ampullary
and urethral glands showed a positive ER␣-immunoreaction. ER␤-immunoreaction was present in the epithelial
cells of the prostate in both species, with the ventral prostate
showing a stronger reaction than the dorsal prostate (Fig.
4e– g and i). In the urethral glands, ER␤-immunoreaction
was seen in the epithelial and stromal cells of both mice and
rats (Fig. 4h and j).
Localization of AR
The epithelial cells in rats exhibited nearly the same or
a stronger intensity of AR-immunostaining in male reproductive tissues compared to those in mice, except in the
coagulating and ampullary glands.
Reproductive tract. AR-immunoreaction was seen in
the epithelial cells and stroma cells throughout the male
reproductive tract in mice and rats.
The ductuli efferentes showed faint to weak AR-immunoreaction in both species. The caput and corpus
epididymis showed a strong AR-immunoreaction in the
epithelial cells (Fig. 5a and b). In the initial segment of
the caput epididymis, the apical cells showed a slightly
weaker AR-immunoreactivity than the principal and
clear cells (Fig. 5a and b). The reaction slightly decreased in the distal portions of the reproductive tract
ER␣, ER␤ AND AR IN MALE MICE AND RATS
771
Fig. 1. Specificities of antibodies and localization of ER isoforms and
AR in the tissues used as standards. a: The uterus of a 3-week-old CD-1
mouse was used as a standard tissue for ER␣-immunostaining. Arrows
indicate glandular epithelium showing intense staining (⫹⫹⫹⫹). b: The
antibody pretreated with the immunizing peptide was used as the control
immunostaining. ER␤ localized in the ovaries of a 3-week-old mouse
was employed for a standard of ER␤-immunoreaction in the male reproductive tissues. The sections were treated with anti-ER␤ antibody (c), or
the antibody preabsorbed with the immunizing peptide (d). Strong ER␤immunostaining (⫹⫹⫹) corresponds to the reaction in the granulosa
cells. e: Seminal vesicles of 8-week-old Wistar rats were used as a
standard tissue for AR-immunoreaction; immunostaining in the epithelium was scored as strong (⫹⫹⫹). f: Anti-AR antibody preabsorbed with
the antigenic peptide was employed for the control immunostaining.
Bar ⫽ 50 ␮m.
(i.e., the cauda epididymis and ductus deferens; Fig. 5c
and d). The AR-immunoreaction in the stroma cells was
nearly homogeneous throughout the reproductive tracts
of both species.
The epithelia of the ventral prostate showed a stronger
reaction than those of the dorsal prostate, and the lobespecific expression of AR was significant in mice (Fig. 5e– g
and i). The epithelial cells of the coagulating, ampullary,
and urethral glands also exhibited a positive AR-immunoreaction (Fig. 5h and j). AR-immunostaining in the stroma
cells was almost constant in the accessory glands of both
species.
Accessory glands. A strong AR-immunoreaction was
present in the epithelial cells of the seminal vesicles and
ventral prostate in both species (Figs. 1e, and 5e and f).
772
YAMASHITA
Fig. 2. Western blot analysis of receptor proteins. A: ER␣ expression
in the reproductive tissues of 8-week-old male mice and rats, and the
uterus of a 3-week-old mice used as a standard. Gels were loaded with
100 ␮g protein per lane: mouse uterus (lane 1), ductuli efferentes (lane 2),
caput epididymis removing (or eliminating) the initial segment (lane 3),
ventral prostate (lane 4), rat ductuli efferentes (lane 5), caput epididymis
removing (or eliminating) the initial segment (lane 6), and ventral prostate
(lane 7). Blots were stained with ER␣ antibody (bottom blot) and the
antibody preabsorbed with the immunizing peptide (upper blot), respectively. B: ER␤ in the mouse ovary. Proteins (200 ␮g) were subjected to
SDS-PAGE. ER␤ was detected with anti-ER␤ antibody (lane 1) and
antibody preincubated with the immunizing peptide (lane 2). C: AR in the
mouse caput epididymis removing (or eliminating) the initial segment.
Proteins (125 ␮g) were separated by SDS-PAGE. AR was detected with
anti-AR antibody (lane 1) and the antibody absorbed with the antigenic
peptide (lane 2). Arrows show the positions of the standard proteins for
molecular weight. Arrowheads indicate bands detected with the respective antibodies in the mouse uterus (A), ovary (B), and epididymis (C).
Table 1 summarizes the localizations of the ER isoforms
and ARs in the male reproductive tissues of mature mice
and rats.
DISCUSSION
Western blot and immunohistochemical studies demonstrated that the antibodies used in this study are specific
for each receptor protein, since the antibodies preabsorbed
with their respective immunizing peptides exhibited no
immunostaining in the tissue sections or blots (although a
faint reaction with ER␣ antibody suggestive of an incomplete absorption was seen in the mouse uterus). On the
Western blot, anti-ER␣ antibody reacted with 67-kDa
ER␣ protein in the mouse ductuli efferentes, epididymis,
ventral prostate, and rat ductuli efferentes, but not in the
rat epididymis or ventral prostate. These results are in
good agreement with those obtained by immunohistochemistry. The predominant 54-kDa band recognized with
anti-AR antibody in the mouse epididymis may be a proteolytic fragment of a 105-kDa receptor. Several polypeptides that are assumed to be degradation products of intact receptor proteins were recognized with antibodies in
the blots, when the male reproductive tissues were analyzed with antibodies to AR and ER␤. The molecular
weights and amounts of the polypeptides varied among
tissues, which may be due to differences in activities and
the kinds of proteases contained in the tissues.
AR proteins were localized in both the epithelial and
stromal cells of all of the male reproductive tissues examined in the present study, which is essentially in accordance with results obtained previously by immunohistochemistry and steroid autoradiography (Schleicher et al.,
1985; Sar et al., 1990; Prins and Birch, 1993; Pelletier et
al., 2000; Zhu et al., 2000; Zhou et al., 2002). On the other
hand, ER␣ and ER␤ were expressed in cell-type- and
species-specific manners. Previous studies (Lubahn et al.,
1993; Eddy et al., 1996; Krege et al., 1998) observed no
significant morphological changes in the reproductive tissues of young male ER␣ and ER␤ knockout mice. In addition, Fisher et al. (1998) reported that knockout mice for
aromatase had no severe lesions in male reproductive
organs, and were capable of breeding. These results indicate that androgens and ARs are essential for the development and normal functioning of male reproductive tissues, and that ERs apparently act as modulators of
androgen action.
This study demonstrates that ER␣ is highly expressed
in the epithelium of the ductuli efferentes in mice and rats
(Fig. 3a and b). ER␣ and ER␤ are undetectable in the rete
testis of both species (S. Yamashita, unpublished observation). ER␣ has also been localized primarily in the epithelium of the ductuli efferentes in several species, including
mice and rats (West and Brenner, 1990; Iguchi et al.,
1991; Fisher et al., 1997; Goyal et al., 1997; Hess et al.,
1997; Zhu et al., 2000; Zhou et al., 2002). These findings
support previous results obtained using ER␣ knockout
mice, which indicated that ER␣ is essential for the fluidreabsorbing function of the ductli efferentes (Hess et al.,
1997; Lee et al., 2000); however, the mechanism of fluidreabsorption through the ER␣ system is still unclear.
Zhou et al. (2002) demonstrated strong and nearly homogeneous ER␤-immunostaining throughout the epithelial cells of the male reproductive tract in mice. Atanassova et al. (2001), employing the same antibody used
by Zhou et al. (2002), demonstrated similar ER␤ localization in Wistar rats. However, the present study demonstrates an ER␤ distribution pattern for both species that is
quite different from those previous results, although the
cell-type-specific distribution patterns of ER␣ and AR are
in good agreement with those obtained by Zhou et al.
(2002) in mice. The antibody used by Zhou et al. (2002)
may have a high titer for ER␤ proteins, and thus may be
able to detect a low concentration of ER␤. However, ER␤
mRNA levels analyzed by Northern blot were shown to be
much lower in the epididymis and ductus deferens than in
the ovaries of mice and rats (Couse et al., 1997; Kuiper et
al., 1997; Jefferson et al., 2000), and steroid autoradiography demonstrated nuclear labeling of [3H]E2 in the ep-
ER␣, ER␤ AND AR IN MALE MICE AND RATS
Fig. 3. Localization of ER␣ in male reproductive tissues. Tissues from
8-week-old rodents were used for immunohistochemistry of ER␣. ER␣
was localized in the mouse ductuli efferentes and caput epididymis (a),
cauda epididymis (c), ductus deferens (d), ventral prostate (g), and
coagulating gland (i), and in the rat ductuli efferentes and caput epidid-
773
ymis (b), caput epididymis (e), ducts deferens (f), ventral prostate (h), and
coagulating gland (j). Arrows (c) indicate clear cells. DE, ductuli efferentes; Ei, initial segment of caput epididymis; Ec, caput epididymis.
Bar ⫽ 50 ␮m.
774
YAMASHITA
Fig. 4. Immunohistochemistry of ER␤ in male reproductive tissues.
ER␤ proteins were localized in the mouse ductuli efferentes and caput
epididymis (a), ductus deferens (c), ventral prostate (e), dorsal prostate
(g), and urethral gland (h). In rats, ER␤ was immunostained in the corpus
epididymis (b), ducts deferens (d), ventral prostate (f), dorsal prostate (i),
and urethral gland (j). DE, ductuli efferentes; Ei, initial segment of caput
epididymis; Ec, caput epididymis. Bar ⫽ 50 ␮m.
ER␣, ER␤ AND AR IN MALE MICE AND RATS
Fig. 5. Expression of AR proteins in male reproductive tissues. ARimmunostaining is shown in the mouse ductuli efferentes and caput
epididymis (a), ducts deferens (c), ventral prostate (e), dorsal prostate
(g), and coagulating gland (h), and in the rat caput epididymis (b), ductus
775
deferens (d), ventral prostate (f), dorsal prostate (i), and coagulating
gland (j). DE, ductuli efferentes; Ei, initial segment of caput epididymis;
Ec, caput epididymis. Bar ⫽ 50 ␮m.
776
YAMASHITA
TABLE 1. ER␣, ER␤ and AR expression in the reproductive tissues of mice and rats*
ER␣
Tissues
Ductuli efferentes
Epithelium
Stroma
Epididymis
Caput
epididymis
Epithelium
Stroma
Corpus
epididymids
Epithelium
Stroma
Cauda
epididymis
Epithelium
Stroma
Ductus deferens
Epithelium
Stroma
Seminal vesicles
Epithelium
Stroma
Ampullary glands
Epithelium
Stroma
Coagulating
glands
Epithelium
Stroma
Prostate
Ventral
prostate
Epithelium
Stroma
Dorsal prostate
Epithelium
Stroma
Urethral glands
Epithelium
Stroma
ER␤
AR
Mouse
Rat
Mouse
Rat
Mouse
Rat
⫹⫹⫹⫹
–
⫹⫹⫹
–
–
–
–
–
⫾⬃⫹
⫾
⫹
⫾
⫾⬃⫹⫹a
⫾⬃⫹
–
–
⫾
–
⫾
–
⫹⫹
⫾
⫹⫹⬃⫹⫹⫹
⫾⬃⫹
⫾⬃⫹
⫾⬃⫹
–
–
⫾
–
⫾⬃⫹
–
⫹⫹
⫹
⫹⫹⫹
⫹
⫾b
⫹⬃⫹⫹
–
–
⫾
⫾
⫾⬃⫹
–
⫹
⫾⬃⫹
⫹⫹
⫹
–
⫹⫹
–
⫹
⫾
⫾
⫹
⫾
⫹
⫹
⫹⫹
⫹⬃⫹⫹
⫾
⫾
–
–
–
–
⫾
–
⫹⫹⫹
⫹
⫹⫹⫹
⫹
–
⫹⫹
–
⫾⬃⫹⫹c
–
–
–
–
⫹⫹
⫹
⫹⬃⫹⫹
⫹
⫹⬃⫹⫹
⫹
–
–
⫾
–
⫾
–
⫹
⫹
⫾⬃⫹
⫹
⫹
⫾
–
–
⫹
⫾
⫹⫹
–
⫹⫹⬃⫹⫹⫹
⫹
⫹⫹⬃⫹⫹⫹
⫹
⫹
⫾
–
–
⫾
⫾
⫾⬃⫹
–
⫹
⫹
⫹⫹
⫹
–
⫹
–
⫹
⫾
⫾
⫹
⫹
⫹⫹
⫹
⫹⫹
⫹⫹
*The intensity of ER␣-immunostaining was scored using uterine sections from 3-week-old mice (Fig. 1a): ⫹⫹⫹⫹ (intense),
corresponds to the staining in the glandular epithelium; ⫹⫹⫹ (strong), ⫹⫹ (moderate); ⫹ (weak); ⫾ (faint), – (negative). The
ovaries of 3-week-old mice served as a standard for ER␤-immunostaining intensity (Fig. 1c): ⫹⫹⫹ (strong); corresponds to the
ER␤-immunostaining in the granulosa cells of growing follicles. AR-immunostaining was also described as ranging from strong
(⫹⫹⫹) to negative (–): ⫹⫹⫹; corresponds to staining in the epithelial cells of seminal vesicles in rats (Fig. 1e).
a
Immunoreaction was moderate in the principal cells of caput epididymis, while reaction was faint in the principal cells and
weak to moderate in apical and narrow cells in the initial segment.
b
Clear cells exhibited moderate staining but principal cells showed negative reaction.
c
Stromal cells were moderately immunostained in the proximal portion and faintly stained in the distal portion.
ithelial cells of the corpus and cauda epididymis and the
ductus deferens to be very low or absent (Schleicher et al.,
1984). The ER␤-immunostaining pattern in the present
study supports the results obtained by Northern blot analyses and steroid autoradiography in mice and rats.
The rodent prostate has been used as a model system
to study the mechanism of hormone actions and hormone-dependent carcinogenesis in the male reproductive tissues (Prins and Birch, 1993; Paris et al., 1994;
Yeh et al., 1998). In rodents, lobe-specific responses to
androgens and estrogens have been reported (Prins and
Birch, 1993; Banerjee et al., 2001; Risbridger et al.,
2001a). The coagulating glands (anterior prostate) ap-
pear to be most sensitive to estrogens (as in the case of
squamous metaplasia induced by the administration of
exogenous estrogen into mature mice (Risbridger et al.,
2001a)). In the young CD-1 mice, the epithelial cells of
the coagulating gland exhibited the strongest ER␣-immunostaining (Fig. 3i). Iguchi et al. (1991) reported a
similar ER␣ distribution in mature C57BL mice. These
results suggest that responses to exogenous estrogens
depend on the concentration of ER␣, and support the
conclusion that ER␣ is the predominant ER isoform in
ER␣ knockout mice for mediating estrogen actions, including estrogen-induced squamous metaplasia in the
mouse prostate (Risbridger et al., 2001b).
ER␣, ER␤ AND AR IN MALE MICE AND RATS
It has been reported that both testosterone (T) and
estrogen are required for the development of benign prostatic hyperplasia and prostate cancer in both rodents and
humans (Wang and Wong, 1998; Cunha et al., 2003;
Steiner and Raghow, 2003). Cunha et al. (2003) demonstrated that the presence of AR and ER␣ is essential in
hormonal carcinogenesis elicited by treatment with T plus
E2 in rodents, based on experiments employing ER␣ and
ER␤ knockout mice. The contribution of ER isoforms has
not yet been elucidated in the human prostate (Linja et
al., 2003; Steiner and Raghow, 2003). However, several
reports have suggested that a lower expression of ER␤,
which may act as a negative regulator for the proliferation
of prostate epithelia, is related to benign prostate hyperplasia and cancer in humans (Horvath et al., 2001; Leav et
al., 2001; Fixemer et al., 2003; Tsurusaki et al., 2003).
In conclusion, AR proteins were localized in both the
epithelial and stromal cells of all of the male reproductive
tissues from 8-week-old mice and rats examined in this
study. However, ER␣ and ER␤ showed cell-type- and species-specific distributions. Recently, Harris et al. (2002)
reported that the ligand-binding profiles of ER␣ and ER␤
are species-dependent. Furthermore, distributions of coactivators and corepressors of nuclear receptors may differ
among species and strains of rodents. Therefore, differences in species, strains, ages, and tissues should be taken
into consideration in assessing the physiological and pharmacological effects of sex steroids (particularly estrogens)
on the male reproductive tissues of rodents.
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