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From the * Department of Veterinary Biosciences,
University of Illinois, Urbana, Illinois;
Department of Urology, College of Medicine,
University of Illinois, Chicago, Illinois; MRC
Human Reproductive Sciences Unit, Edinburgh EH3 9ET, United Kingdom;
Departments of Cell and Structural Biology, and
Molecular and Integrative Physiology, University of Illinois, Urbana,
Illinois.
| Correspondence to: Dr Rex A. Hess, Veterinary Biosciences, University of Illinois, 2001 S. Lincoln, Urbana, IL 61802-6199 (e-mail: r-hess{at}uiuc.edu). |
| Received for publication March 6, 2002; accepted for publication June 6, 2002. |
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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and ERß were visualized by immunohistochemistry in adult male mice
reproductive tracts, including testes, efferent ductules; initial segment,
caput, corpus, and cauda epididymides; and vas deferens. Antibody specificity
was demonstrated by Western blot and antibody competition. In testis, AR was
expressed in Leydig cells, Sertoli cells, and most peritubular cells, but not
in germ cells; Sertoli cells showed more intense staining in stages VI-VII;
ER was present in Leydig and some peritubular cells; ERß was in
Leydig, some peritubular, all Sertoli and germ cells except in spermatids and
meiotic spermatocytes. In efferent ductules, AR was strongly expressed in
ciliated and nonciliated epithelial cells and in stromal cells; ER was
strongly expressed in ciliated and nonciliated epithelial cells; stromal cells
were negative; and ERß was strongly expressed in ciliated and nonciliated
epithelial cells and also in stromal cells. In epididymis, AR was strongly
expressed in all epithelial cells (not in intraepithelial lymphocytes);
ER was strongly expressed in apical, narrow, and some basal cells of
the initial segment, and in caput, principal cells of the caput, clear cells
of the distal caput through cauda; stromal cells were negative in the initial
segment, but more stromal cells were stained from caput to cauda; ERß was
strongly expressed in most of epithelial cells of the epididymis, but stromal
cells were inconsistently stained. In vas deferens, AR was weakly expressed or
absent in principal cells but moderately stained in basal cells, smooth muscle
cells of stroma were stained intensely, ER was absent in epithelial
cells but present in a subepithelial smooth muscle layer, and ERß was
strongly expressed in all epithelial cells and most stromal cells. This study
demonstrates that the reproductive tracts of male mice differ considerably
from those of rats in expression of ARs and ERs and that caution is needed
when extrapolating nuclear steroid receptor data across mammalian species.
Key words: Efferent ductule, epididymis, steroid hormone receptors, testis, vas deferens
and ß, are present in males
(Fisher et al, 1997;
Hess et al, 1997b; Couse and Korach, 1999;
Nie et al, 2002), but ERß
appears more abundant and in a greater number of cell types in the male
reproductive system (Saunders et al,
1998,
2001). The mouse is one of the most extensively used animals in biomedical research and a clear understanding of cellular localizations of AR and ER is necessary for appropriate interpretation of experiments focused on male reproduction. However, few studies have documented these steroid receptors by immunohistochemistry in this species (Iguchi et al, 1991; Zhou et al, 1996; Rosenfeld et al, 1998) and cell specificity is lacking in these reports.
A recent histological examination of the male reproductive tract in the
ER knockout mouse (ERKO) revealed several abnormalities in the
epididymis (Hess et al, 2000).
These abnormalities were cell-specific and corresponded to cells previously
shown to bind 3H-estradiol in mice
(Schleicher et al, 1984).
Thus, based on these observations, the presence of ER in the mouse
epididymal epithelium would be predicted. However, reports of steroid receptor
localizations have not been consistent across species nor among species. Some
studies have shown strong expression of ER in both testis and
epididymis, whereas other species have reduced expression in the testis and
sometimes no expression in the epididymis (Hess et al,
2001a,
2002). Our laboratory showed
epithelial expression in certain regions of the rat epididymis
(Hess et al, 1997b), whereas
another laboratory, using different antibodies, found ER expressed only
in the efferent ductules, with no expression in the epididymis
(Fisher et al, 1997). A
similar inconsistency has been noted in reports of ERß localization in
the testis of several species (van Pelt et
al, 1999; Pelletier et al,
2000; Makinen et al,
2001; Saunders et al,
2001).
Because of the significance of nuclear steroid receptors to our
understanding of reproductive biology, we thought it important to examine in
detail the comparative expression of androgen and estrogen receptors in the
reproductive tracts of important mammalian species. In the present study, the
pattern of immunohistochemical expression was examined in mice using
antibodies to AR, ER, and ERß. Antibody specificity was
demonstrated by antibody competition and Western blot analysis.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Tissue Processing
Animals were anesthetized and perfused with 10% neutral buffered formalin
(NBF) for 20 minutes. After tissues were postfixed in NBF overnight at
4°C, they were transferred to 70% ethanol and embedded in paraffin.
Sections were cut at 5 µm thickness and then dried at 37°C overnight.
Tissues evaluated were the testis, efferent ductules, epididymis, and vas
deferens.
Single Receptor Staining
Tissues were stained for receptors as described previously
(Nie et al, 2002). To unmask
the receptor protein, sections were microwaved in a 0.01 M citrate buffer
solution pH 6.0 for 20 minutes. Tissues were respectively incubated with
AR-specific antibody PG21 at 1:500 diluted with phosphate-buffered saline
(PBS), ER-specific antibody NCL-ER-6F11 (Novocastra, Newcastle upon
Tyne, United Kingdom) at 1:1000 dilution, and ERß-specific antibody S-40
at 1:500 dilution for 12 hours at 4°C. Previous studies have described the
generation of the S-40 ERß and AR antibodies
(Prins et al, 1991;
Saunders et al, 2000). Three
other ERß antibodies were tested in this study to determine which to use
for optimum results. The antibodies included a rabbit anti-rat ERß
polyclonal antibody PA1-310 (Affinity BioReagents, Golden, Colo), and 2 mouse
anti-human monoclonal antibodies F-12 and E-12
(Choi et al, 2001). Antibody
bindings were visualized by using the avidin-biotin complex (ABC Kit, Vector
Laboratories, Burlingame, Calif), and the diaminobenzidine (DAB) chromogen.
Hematoxylin (Sigma Chemical Company, St Louis, Mo) was applied as a counter
stain. Sections incubated without the primary antibody but with PBS were used
as negative controls for color development on the same slide. Images were
captured with a Spot II digital camera (Diagnostic Instruments, Sterling
Heights, Mich) and compiled using Adobe Photoshop software (Adobe Systems, San
Jose, Calif).
Double Receptor Staining
Colocalization of ER and ERß in the efferent ductules and the
head of the epididymis was examined by double staining. After antigen
retrieval and blocking with 10% normal rabbit serum, sections were incubated
sequentially in the following solutions, with a PBS rinse in between:
ER (NCL-ER-6F11) mouse antibody (1:50), fluorescein isothiocyanate
(FITC)-conjugated antimouse immunoglobulin (Ig) G (1:100; Sigma), ERß,
S-40, sheep antibody (1:250), and Texas red—conjugated anti-sheep IgG
(1:100; Vector Laboratories). Sections incubated without the primary antibody
were used as the negative control. Tissues were examined with a fluorescence
microscope with suitable filters for FITC and Texas red, and images were
captured with the Spot II digital camera. In Adobe Photoshop, the individual
images for ER (FITC-green) and ERß (Texas red) were combined using
the overlay tool. Cells that contained both receptors stained various shades
of yellow-green to bright yellow.
Antibody Competition
AR21 (AR peptide), AR462 (AR unrelated peptide), recombinant human
ER protein (Panvera, Madison, Wis), and peptide P4 (the antigenic
peptide for S40) were used to perform antibody competition, respectively.
Briefly, 10-fold to 15-fold molar excess of protein or peptide were incubated
together with related antibodies overnight at 4°C, then were used as
primary antibody as described before for immunohistochemistry. Efferent
ductules and corpus epididymides were used for the competition tests of 3
antibodies.
Western Blot Analysis
Testis and epididymis were extracted for detection of AR protein. Mouse
epididymis and uterus (a positive control) were extracted for detection of
ER protein. Mouse epididymis was extracted for examination of ERß
protein. Human recombinant ERß was used as the protein standard for
ERß (Panvera). The method was modified based on a previous publication
(Choi et al, 2001). Protein
samples were separated on 10% sodium dodecyl sulfate—polyacrylamide gel
electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose membrane.
Nonspecific binding was blocked with 5% dried milk in Tris-buffered saline
(TBS; 50 mM Tris-Cl, pH 7.5/150 mM NaCl) containing 0.05% Tween-20 for 1.5
hours and then the membranes were incubated overnight at room temperature with
1:500 diluted AR antibody PG21, 1:50 diluted ER antibody NCL-ER-6F11
(Novocastra, England), and 1:2000 ERß antibody S-40 in TBS containing
1%-2% dried milk and 0.2% Tween-20. After washing in TBS containing 0.05%
Tween-20, the membranes were incubated with horseradish peroxidase-conjugated
secondary antibodies for 1 hour in the same buffer used for primary antibody
incubation. Peroxidase-conjugated secondary antibodies included goat
anti-rabbit IgG diluted at 1:2000 (Zymed, San Francisco, Calif), goat
anti-mouse IgG diluted at 1:4000 (Pierce, Rockford, Ill), or rabbit anti-sheep
IgG diluted 1:2000 (Sigma). Antibody bindings were visualized by using
diaminobenzidine (DAB) chromogen as substrate.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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in efferent ductules was defined as a baseline
strong staining. Evaluation of epididymal cell types was divided into
epithelial and stromal categories. Stromal cells contained smooth muscle or
peritubular smooth muscle cells, connective tissue cells (including
fibroblasts), and vascular endothelium. The only cell type in stroma that was
specifically identified was the peritubular smooth muscle cell or the myoid
cell (in testis). The peritubular myoid cells were confined to cells
underlying the seminiferous tubule. Peritubular smooth muscle cells were
identified as the cells lying immediately beneath the excurrent ductal
epithelium. The ERß antibodies gave identical nuclear staining patterns
in the excurrent ducts (from efferent ductules to vas deferens); however,
stain intensity was somewhat better with S40, and therefore it was used for
the illustrations. The results of immunostaining are summarized in
Table 1.
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Testis
AR immunoreactive staining was strong in most Leydig cells and in
approximately 95% of peritubular myoid cells
(Figure 1, a and m), regardless
of proximity to different stages of spermatogenesis
(Table 2). Sertoli cells showed
stage-specific staining, with the most intense staining of nuclei found in
stages VI—VII and the least amount of staining in stages I—III and
VIII—XII (Table 2). All
germ cells were AR-negative.
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ER immunostaining was intense in many Leydig cell nuclei, as well as
some peritubular myoid cells (Figure 1, b
and o). Sertoli cells and germ cells in all stages of
spermatogenesis were ER-negative. ERß was expressed in Leydig
cells and most peritubular myoid cells. Within the seminiferous tubule,
ERß staining was found in Sertoli cells and spermatogonia at all stages
of spermatocytes (Figure 1, c and
q). Spermatocytes were also ERß-positive, except for
spermatocytes in meiotic division.
Efferent Ductules
Ciliated and nonciliated epithelial cells exhibited intense staining for AR
(Figure 1, d and n), ER
(Figure 1, e and p), and
ERß (Figure 1, f and r).
Among stromal cells, AR was strongly positive in peritubular smooth muscle and
most other cell types. ER was negative in most stromal cells but weakly
positive in a few. Most stromal cells were strongly positive for ERß.
Initial Segment of the Epididymis
In this region, AR and ERß shared a similar strong staining pattern.
AR (Figure 1, g and s) and
ERß (Figure 1, i and w) were strongly positive in nuclei of all epithelial cells, including apical,
narrow, basal, and principal cells. There appeared to be no difference in
staining intensity between principal and basal cells. Intraepithelial
lymphocytes (halo cells) were negative (observed, but not illustrated). Some
apical and narrow cells were also intensively positive for ER, but
other nuclei of these cell types stained weakly positive. Principal cells were
weakly positive or negative for ER. Most basal cells were
ER-negative or weakly positive, but an occasional basal cell showed an
intense staining for ER (Figure 1, h
and u). In the stroma, many cells were weakly to moderately
positive for AR and ERß but were essentially negative for ER.
Caput Epididymis
AR was expressed in all epithelial cells
(Figure 1, j and t). Most
principal cells were moderately to strongly positive for ER
(Figure 1, k and v) and
ERß (Figure 1, l and x)
except for some basal cells, and the strongest ER immunostaining was
distributed in apical cells (Figure
1k, unlabeled arrows). There was a distinct increase in staining
intensity for ER in the caput epididymis compared with the initial
segment.
In the stromal area, AR was positive in peritubular cells and some other
stromal cells. Most stromal cells were weakly positive to negative for
ER, and ERß was generally absent in the stroma.
Corpus Epididymis
AR (Figure 2, a and j) and
ERß (Figure 2, c and n)
were expressed abundantly in all epithelial cells lining the corpus epididymal
duct. In contrast, ER was present only in clear cells of the epithelium
and some peritubular cells of the stroma
(Figure 2, b and l). In the
stroma, AR and ERß were positive in some cells but negative in
others.
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Cauda Epididymis
Epithelial cells stained intensely for AR
(Figure 2, d and k) and
ERß (Figure 2, f and o)
but ER staining (Figure 2, e and
m) was identical to the corpus epididymis, where only clear cells
and some peritubular smooth muscle cells were strongly positive. Many stromal
cells were positive for all three receptors.
Vas Deferens
In the epithelium, AR was expressed weakly in epithelial cells but with
moderate strength in basal cells (Figure 2,
g and p). ER was absent within the epithelium
(Figure 2, h and r), whereas
ERß was abundant in all epithelial cell types
(Figure 2, i and t). In the
stroma, AR was expressed in smooth muscle and connective tissue cells, but
ER was found only in the outer layer of smooth muscle cells, whereas
ERß was abundant throughout the stroma.
Double Staining
It was obvious from microscopic observation that both ER and
ERß were colocalized in the same cells in various regions of the male
tract. Therefore, we selected to examine the efferent ductules and initial
segment of epididymis using colocalization of fluorescent signals to determine
whether the same cells express the 2 receptors. Both ER (green) and
ERß (red) were expressed in epithelial nuclei of the efferent ductules
(Figure 3, a and b).
Colocalization of the 2 receptors in the same cell was detected by an orange
to yellowish color, with variations in color intensities indicating
differences in the proportion of the 2 receptors in an individual cell nucleus
(Figure 3c). In the overlay
view of the proximal efferent ductule epithelium
(Figure 3c), some cells
remained green, indicating expression of only ER; some stained
intensely yellow, indicating equivalent expression of ER and ERß;
and an occasional cell stained only red, indicating an expression of only
ERß.
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A transition zone from efferent ductules to the initial segment of the
epididymis can be viewed in Figure 3,
d-f. The one tubule that stained intensely for both ER and
ERß belongs to the common efferent duct that opens into the initial
segment epididymis. This duct shows less variation in overlay staining
(Figure 3f) than does the
proximal ductules (Figure 3c),
indicating nearly equal expression of the 2 receptors in all epithelial cells.
In the same photographs, a region of the initial segment is also noted. The
only fluorescence detected for ER in the initial segment was the apical
and narrow cells (Figure 3d).
ERß was expressed in all epithelial cell types
(Figure 3e). The overlay shows
considerable variation in staining of apical and narrow cells
(Fig. 3f; note the unlabeled
arrows), similar to what was observed with single receptor staining
(Figure 1, h and i).
Antibody Competition
AR21 peptide competed away the nuclear staining given by the AR antibody
PG21 reaction to efferent ductule (Figure
2q) and epididymal tissues (not shown), whereas the unrelated
peptide AR462 was not able to ward off the specific nuclear staining (not
shown). Recombinant human ER protein competed away all nuclear staining
given by monoclonal antibody 6F11 reaction
(Figure 2s), and peptide P40
also competed away all staining given by the ERß antibody S40
(Figure 2u).
Western Blot Analysis
A single dominant band of approximately 110 kd was detected for AR in both
mouse testis and epididymal extracts
(Figure 4a), which is the
reported molecular size for AR (Prins et
al, 1991). A comigration band of ER with mouse uterus was
shown in mouse epididymal extract. The molecular size of this band was
approximately 66 kd (Figure
4b). Mouse uterine extract was used as a positive control for
ER. Two bands were observed on mouse epididymal extract, which also
comigrated with the purified human recombinant ERß
(Figure 4c). The molecular
weight of the purified protein was 54 kd. One band comigrated with this
positive marker, the other band is approximately 59 kd, as reported for the
long form of ERß (Kuiper et al,
1998).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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in a region and cell-specific manner in the mouse demonstrates that
species differences exist for ER expression in the adult male.
Testis
Although there is a substantial decrease in AR in the male mouse
reproductive tract tissues from birth to adulthood
(Gallon et al, 1989),
androgens remain the primary steroid hormones for maintenance of male
reproductive function in the adult (Ezer
and Robaire, 2002), and the common presence of AR in the male
would be expected. There is nearly universal agreement across species that in
the adult testis, AR is present in Sertoli cells, peritubular cells, and
Leydig cells (Sar et al, 1990;
Bremner et al, 1994;
Vornberger et al, 1994; Van Roijen et al, 1995; Goyal
et al,
1997a,b;
Suarez-Quian et al, 1999; Pelletier et al, 2000;
Zhu et al, 2000). Expression
of AR in Sertoli cells supports previous reports that androgens regulate
Sertoli cell function and are essential for spermatogenesis
(Sharpe, 1994). The present
study found a stage-dependent expression of AR in Sertoli cells, similar to
other studies in rats and humans (Bremner
et al, 1994; Vornberger et al,
1994; Suarez-Quian et al,
1999).
AR was not found in germ cells of mouse testis. However, the presence of AR in testicular germ cells has been controversial, with most studies in other species having indicated a lack of AR, but some studies showing AR-positive spermatogonia (Kimura et al, 1993; Zhou et al, 1996) or stage-specific elongate spermatids (Vornberger et al, 1994). In support of our findings in the mouse, a recent study using spermatogonial stem cell transplant technology demonstrated that germ cells of the mouse testis do not require a functional AR to complete spermatogenesis (Johnston et al, 2001). Therefore, if Sertoli cells in certain stages also express no AR or low concentrations, it is likely that testosterone provides indirect stimulation of spermatogenesis in those stages through the AR-positive peritubular cells. AR expression in Sertoli cells appears to have more androgen dependency than in Leydig and peritubular cells (Zhu et al, 2000).
In the testes of adult mice, we detected ER in Leydig cells and
peritubular myoid cells, whereas Sertoli and germ cells of the seminiferous
epithelium were negative. This is consistent with a recent report that the
presence of ER in germ cells of the testis is not required for their
development (Mahato et al,
2000). It is surprising that in testes of adult humans, macaques,
marmosets, and goats, no ER immunoexpression was detected in any cell
type (Goyal et al, 1997a;
Makinen et al, 2001;
Saunders et al, 2001). However, in another study of marmosets, ER was reported in interstitial
(Leydig) cells (Fisher et al,
1997). In cats and dogs (Nie
et al, 2002) and rats (Sar and
Welsch, 2000), ER has also been detected in the
interstitial (Leydig) cells but not in peritubular cells.
In the present study, ERß was expressed more extensively than
ER from the testis to the vas deferens, both in cell types and in
number of positive cells. Its expression in the testis is controversial, with
considerable variation across species and even within species. We found in
mouse testis, using the S40 antibody, that ERß was expressed in Sertoli
cells, spermatogonia, and spermatocytes (except for cells in meiotic
division). Spermatids and spermatozoa were negative. These results are similar
to previous studies in several species
(Enmark et al, 1997; Rosenfeld et al, 1998;
Saunders et al, 1998,
2001; Pelletier et al,
1999,
2000;
van Pelt et al, 1999;
Makinen et al, 2001;
Nie et al, 2002), but
contrasts with other reports. In one study of rats, ERß was found only in
Sertoli cells (Pelletier et al,
2000) and in a study of humans, Makinen et al
(2001) found that ERß was
expressed only in germ cells. Rosenfeld
(1998) found that in mice,
ERß was also expressed in elongated spermatids.
Efferent Ductules
There are few reports of AR localization in efferent ductules, but in all
species studied to date, AR appears to be underexpressed in efferent ductule
epithelium, compared to the epididymis, except in mice, as reported here.
Ungefroren et al (1997)
reported no AR messenger RNA or immunohistochemical signal in humans. In
rhesus monkeys, AR was expressed but was less abundant in efferent ductule
than in caput and corpus epididymis
(Roselli et al, 1991). Goats
also showed lower expression, with no appearance in ciliated cells and
variable staining in nonciliated cells
(Goyal et al, 1997a). The
function of efferent ductules in fluid reabsorption appears to be similar in
all species (Hess, 2002);
however, this difference in AR expression in mice compared to other species
raises serious questions regarding interspecies comparisons in toxicological
studies. Further study of AR expression in this region of the male is needed
in other species, and in particular in rats, which are used extensively in
toxicology. One study in rats to day 18 postpartum showed AR expression that
was abated with perinatal treatment with diethylstilbestrol
(McKinnell et al, 2001).
Collectively, these data are in agreement with our previous studies showing
the importance of ER abundance in these tubules and the major role of
ER, rather than AR, in regulation of efferent ductule function
(Hess et al, 2002).
ER expression in efferent ductule epithelium has been consistent
across all species studied, including rats, mice, roosters, dogs, cats, goats,
monkeys, and humans (West and Brenner,
1990; Ergun et al,
1997; Fisher et al,
1997; Goyal et al,
1997a; Hess et al,
1997b; Kwon et al,
1997; Rosenfeld et al,
1998; Saunders et al,
2001; Nie et al,
2002). However, in some species, the ciliated cells were
ER-negative (West and Brenner,
1990; Ergun et al,
1997; Goyal et al,
1997a; Saunders et al,
2001), but in mice, all epithelial cells were ER-positive
in these ductules. ER showed the highest intensity of reproductive
tract staining in the efferent ductules, consistent with our previous studies
showing that ER is responsible for regulating fluid reabsorption in
these ducts (Hess et al,
1997a) through the control of epithelial ion transporters
(Lee et al, 2001;
Zhou et al, 2001). Efferent
ductules are responsible for reabsorbing more than 90% of the fluid entering
from the rete testis (Clulow et al,
1998). This action of estrogen in the efferent ductules is now
recognized as being essential for male fertility
(Eddy et al, 1996;
Hess et al, 2001b; Oliveira et
al, 2001,
2002;
Zhou et al, 2001).
The expression of ERß throughout the excurrent ducts is ubiquitous
even though it seems more predominant in epithelia than stroma. The expression
profile of ERß in mouse tract is more similar to AR than it is to
ER. Due to its unclear physiological role in the male, further study is
need to clarify ERß action in the testis and its excurrent ducts
(Krege et al, 1998; Couse et al, 1999;
Dupont et al, 2000).
Epididymis
AR has been demonstrated by various techniques to be present in the
epididymis of numerous species (Younes and
Pierrepoint, 1981; Schleicher
et al, 1984; Toney and Danzo,
1988; Gallon et al,
1989; Tekpetey et al,
1989; Sar et al,
1990; Cooke et al,
1991; Roselli et al,
1991; Goyal et al,
1997b; Ungefroren et al,
1997; You and Sar,
1998; Pelletier,
2000), and dependence of the epididymis on androgens for structure
and function is well known (Ezer and
Robaire, 2002). In mice, immunostaining for AR demonstrates a
decrease in intensity from the efferent ductule epithelium to the vas
deferens, with the caput, corpus, and cauda regions showing equivalent
staining, but the vas exhibiting very low expression of AR. Some species
variation is seen in the ram, in which there also appears to be regional
differences in expression (Carreau et al,
1984; Tekpetey et al,
1989). In the present study, AR was expressed equally in principal
and basal cells in mice, in contrast to that of rats
(Zhu et al, 2000).
Although AR appears to be dominant in the epididymal epithelium, the
ERKO male demonstrated epididymal abnormalities in the absence of a
functional ER (Hess et al,
2000). It is interesting that the same cell types that were
abnormal in ERKO clearly show ER-positive immunoreactivity in
the present study, in particular in narrow cells of the initial segment,
apical cells of the caput, and in clear cells. Principal cells of the caput
were also ER-positive in mice, but they did not show gross
abnormalities in ERKO mice. These data are consistent with an earlier
autoradiography study in mice (Schleicher
et al, 1984), which showed much higher binding of
3H-estradiol in the initial segment and caput epididymis than in
the corpus through vas deferens, with greater binding in apical/narrow cells
and in clear cells. What is not understood is that ERß is abundant
throughout the epididymal epithelium of mice, and yet the autoradiographic
data show little evidence of equivalent binding of estradiol throughout the
epididymal epithelium. Because ER is absent in principal cells of the
corpus, cauda, and vas, 3H-estradiol binding shown in these cells
(Schleicher et al, 1984) must
represent the presence of ERß. In adult rats, inconsistent results for
epididymal ER have been reported, apparently due to the different
antibodies applied (Fisher et al,
1997; Hess et al,
1997b; Sar and Welsch,
2000; Atanassova et al,
2001). These data are consistent with messenger RNA hybridization
studies in situ (Mowa and Iwanaga,
2001). Sar and Welsch
(2000) reported all stroma to
be positive but only some epithelial cells in the rat were positive. In
contrast, the entire epididymis, both epithelium and stroma, were found to be
negative in several species, including rats
(Fisher et al, 1997; Atanassova et al, 2001), dogs
(Nie et al, 2002), goats
(Goyal et al, 1998) and
marmoset monkeys (Fisher et al,
1997). Other studies have shown variable amounts of staining by
the epididymal epithelium with the same antibodies
(Saunders et al, 2001;
Nie et al, 2002), which
suggests there are major species difference for ER expression in the
adult epididymis.
ERß was expressed throughout the male mouse reproductive tract epithelium, similar to what has been reported in other species (Sar and Welsch, 2000; Atanassova et al, 2001; Saunders et al, 2001). There did not appear to be cell-specific expression of ERß; however, the epithelium showed more intense staining than did the stroma in all regions, except for the cauda and vas deferens.
Vas Deferens
Although there is considerable evidence for androgen action in the vas
deferens (Schindelmeiser et al,
1988; Dassouli et al,
1995; Darne et al,
1997), few reports have described AR presence. The vas deferens
epithelium in humans shows only weak staining for AR
(Ungefroren et al, 1997),
which is similar to our findings in mice and corresponds to data showing a
decrease in AR from neonatal to adult ages in rats
(Gallon et al, 1989). However,
goat, rat (Goyal et al,
1997b), and human epithelium
(Sar et al, 1990) expressed AR
with intense immunostaining. In the mouse, there was abundant expression of AR
in the stroma, which is consistent with autoradiographic studies
(Weaker and Sheridan,
1983).
As observed in most species, (Goyal et
al, 1997b; Hess et al,
1997b; Jefferson et al,
2000; Nie et al,
2002), the vas deferens epithelium was ER-negative. Only
the cat vas deferens shows ER-positive staining
(Nie et al, 2002). ERß is
abundant in both the epithelium and stroma of the vas deferens, similar to all
species examined (Jefferson et al,
2000; Nie et al,
2002).
Receptors in Stromal Tissue
The interaction of epithelia and stroma has been studied in many tissues
(Cunha et al, 1985;
Cooke et al, 1986), and there
is clear evidence that stroma plays an influential role in determining the
fate of epithelial differentiation and function through paracrine regulation
(Cooke et al, 1998,
1997;
Prins et al, 2001). Our data
demonstrate a predominant presence of AR in stroma of the entire excurrent
ductal system, and that ER and ERß exhibit more variation in
expression. For example, there is little expression of ER in stroma of
efferent ductules, but ERß is expressed as strongly as that of AR. There
is a tendency for decreased expression of both ER and ERß in
stroma, going from the head of the epididymis through the vas. Even though
ERß appears to be present in more cell types, ER maintains much
stronger intensity of staining in a cell-specific manner than does ERß.
Therefore, these data suggest that further study is needed not only to
determine the interactions between receptors and their specific hormone
ligands, but also to determine the interactions between epithelial and stromal
cells in the epididymal region.
Conclusions
This study demonstrates that the male mouse reproductive tract is
substantially different from that reported in rats for the expression of ARs
and ERs. This finding further supports the need to be cautious when
extrapolating nuclear steroid receptor data across species in the study of
male reproductive tract biology. All epithelial and most stromal cells
contained AR, except for the germ cells and some vas deferens cells. ER
was abundant in efferent ductules, similar to all other species, but its
presence in specific cell types along the epididymis was novel, because it
differs from that seen in rats. ERß distribution was similar to that of
AR, except that ERß alone was prominent in germ cells and vas deferens
epithelium. Many cells expressed all three steroid receptors, including Leydig
cells, peritubular myoid cells surrounding the seminiferous tubules, all
epithelial cells from the efferent ductules, apical and narrow cells of the
initial segment, and clear cells of the epididymis. The coexistence of
multiple receptors in the same cells raises important questions regarding
steroid hormone interactions and receptor cross-talk in the control of male
reproductive tract function.
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Acknowledgments |
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References |
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 S Kerkhofs, S Denayer, A Haelens, and F Claessens Androgen receptor knockout and knock-in mouse models J. Mol. Endocrinol., January 1, 2009; 42(1): 11 - 17. [Abstract] [Full Text] [PDF]
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K. Toda, T. Okada, Y. Hayashi, and T. Saibara Preserved tissue structure of efferent ductules in aromatase-deficient mice J. Endocrinol., October 1, 2008; 199(1): 137 - 146. [Abstract] [Full Text] [PDF]
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R. Sirianni, A. Chimento, C. Ruggiero, A. De Luca, R. Lappano, S. Ando, M. Maggiolini, and V. Pezzi The Novel Estrogen Receptor, G Protein-Coupled Receptor 30, Mediates the Proliferative Effects Induced by 17{beta}-Estradiol on Mouse Spermatogonial GC-1 Cell Line Endocrinology, October 1, 2008; 149(10): 5043 - 5051. [Abstract] [Full Text] [PDF]
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F. Yasuhara, G. R. O. Gomes, E. R. Siu, C. I. Suenaga, E. Marostica, C. S. Porto, and M. F. M. Lazari Effects of the Antiestrogen Fulvestrant (ICI 182,780) on Gene Expression of the Rat Efferent Ductules Biol Reprod, September 1, 2008; 79(3): 432 - 441. [Abstract] [Full Text] [PDF]
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A. Wahlgren, K. Svechnikov, M.-L. Strand, K. Jahnukainen, M. Parvinen, J.-A. Gustafsson, and O. Soder Estrogen Receptor {beta} Selective Ligand 5{alpha}-Androstane-3{beta}, 17{beta}-Diol Stimulates Spermatogonial Deoxyribonucleic Acid Synthesis in Rat Seminiferous Epithelium in Vitro Endocrinology, June 1, 2008; 149(6): 2917 - 2922. [Abstract] [Full Text] [PDF]
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H. Nishida, S. Miyagawa, M. Vieux-Rochas, M. Morini, Y. Ogino, K. Suzuki, N. Nakagata, H.-S. Choi, G. Levi, and G. Yamada Positive Regulation of Steroidogenic Acute Regulatory Protein Gene Expression through the Interaction between Dlx and GATA-4 for Testicular Steroidogenesis Endocrinology, May 1, 2008; 149(5): 2090 - 2097. [Abstract] [Full Text] [PDF]
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T. F.G Lucas, E. R Siu, C. A Esteves, H. P Monteiro, C. A Oliveira, C. S Porto, and M. F. M Lazari 17Beta-Estradiol Induces the Translocation of the Estrogen Receptors ESR1 and ESR2 to the Cell Membrane, MAPK3/1 Phosphorylation and Proliferation of Cultured Immature Rat Sertoli Cells Biol Reprod, January 1, 2008; 78(1): 101 - 114. [Abstract] [Full Text] [PDF]
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 S. Aquila, E. Middea, S. Catalano, S. Marsico, M. Lanzino, I. Casaburi, I. Barone, R. Bruno, S. Zupo, and S. Ando Human sperm express a functional androgen receptor: effects on PI3K/AKT pathway Hum. Reprod., October 1, 2007; 22(10): 2594 - 2605. [Abstract] [Full Text] [PDF]
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 J. S. Rocha, M. S. Bonkowski, L. R. Franca, and A. Bartke Mild Calorie Restriction Does Not Affect Testosterone Levels and Testicular Gene Expression in Mutant Mice Exp Biol Med, September 1, 2007; 232(8): 1050 - 1063. [Abstract] [Full Text] [PDF]
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M L Gould, P R Hurst, and H D Nicholson The effects of oestrogen receptors {alpha} and {beta} on testicular cell number and steroidogenesis in mice Reproduction, August 1, 2007; 134(2): 271 - 279. [Abstract] [Full Text] [PDF]
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J. Cheng, S. C. Watkins, and W. H. Walker Testosterone Activates Mitogen-Activated Protein Kinase via Src Kinase and the Epidermal Growth Factor Receptor in Sertoli Cells Endocrinology, May 1, 2007; 148(5): 2066 - 2074. [Abstract] [Full Text] [PDF]
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 S. M. Eacker, J. E. Shima, C. M. Connolly, M. Sharma, R. W. Holdcraft, M. D. Griswold, and R. E. Braun Transcriptional Profiling of Androgen Receptor (AR) Mutants Suggests Instructive and Permissive Roles of AR Signaling in Germ Cell Development Mol. Endocrinol., April 1, 2007; 21(4): 895 - 907. [Abstract] [Full Text] [PDF]
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 K. Schauwaers, K. De Gendt, P. T. K. Saunders, N. Atanassova, A. Haelens, L. Callewaert, U. Moehren, J. V. Swinnen, G. Verhoeven, G. Verrijdt, et al. Loss of androgen receptor binding to selective androgen response elements causes a reproductive phenotype in a knockin mouse model PNAS, March 20, 2007; 104(12): 4961 - 4966. [Abstract] [Full Text] [PDF]
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J. Merlet, C. Racine, E. Moreau, S. G. Moreno, and R. Habert Male fetal germ cells are targets for androgens that physiologically inhibit their proliferation PNAS, February 27, 2007; 104(9): 3615 - 3620. [Abstract] [Full Text] [PDF]
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C. V. Teixeira, D. Silandre, A. M. de Souza Santos, C. Delalande, F. J B Sampaio, S. Carreau, and C. da Fonte Ramos Effects of maternal undernutrition during lactation on aromatase, estrogen, and androgen receptors expression in rat testis at weaning J. Endocrinol., February 1, 2007; 192(2): 301 - 311. [Abstract] [Full Text] [PDF]
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T. Hoshii, T. Takeo, N. Nakagata, M. Takeya, K. Araki, and K.-i. Yamamura LGR4 Regulates the Postnatal Development and Integrity of Male Reproductive Tracts in Mice Biol Reprod, February 1, 2007; 76(2): 303 - 313. [Abstract] [Full Text] [PDF]
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C. Zhang, S. Yeh, Y.-T. Chen, C.-C. Wu, K.-H. Chuang, H.-Y. Lin, R.-S. Wang, Y.-J. Chang, C. Mendis-Handagama, L. Hu, et al. Oligozoospermia with normal fertility in male mice lacking the androgen receptor in testis peritubular myoid cells PNAS, November 21, 2006; 103(47): 17718 - 17723. [Abstract] [Full Text] [PDF]
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J. F. Couse, M. M. Yates, K. F. Rodriguez, J. A. Johnson, D. Poirier, and K. S. Korach The Intraovarian Actions of Estrogen Receptor-{alpha} Are Necessary to Repress the Formation of Morphological and Functional Leydig-Like Cells in the Female Gonad Endocrinology, August 1, 2006; 147(8): 3666 - 3678. [Abstract] [Full Text] [PDF]
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P. Sipila, D. A. Pujianto, R. Shariatmadari, J. Nikkila, M. Lehtoranta, I. T. Huhtaniemi, and M. Poutanen Differential Endocrine Regulation of Genes Enriched in Initial Segment and Distal Caput of the Mouse Epididymis as Revealed by Genome-Wide Expression Profiling Biol Reprod, August 1, 2006; 75(2): 240 - 251. [Abstract] [Full Text] [PDF]
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S. F. Sneddon, N. Walther, and P. T. K. Saunders Expression of Androgen and Estrogen Receptors in Sertoli Cells: Studies Using the Mouse SK11 Cell Line Endocrinology, December 1, 2005; 146(12): 5304 - 5312. [Abstract] [Full Text] [PDF]
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J. Meng, R. W. Holdcraft, J. E. Shima, M. D. Griswold, and R. E. Braun Androgens regulate the permeability of the blood-testis barrier PNAS, November 15, 2005; 102(46): 16696 - 16700. [Abstract] [Full Text] [PDF]
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H. Baines, M. O Nwagwu, E. C Furneaux, J. Stewart, J. B Kerr, T. M Mayhew, and F. J P Ebling Estrogenic induction of spermatogenesis in the hypogonadal (hpg) mouse: role of androgens Reproduction, November 1, 2005; 130(5): 643 - 654. [Abstract] [Full Text] [PDF]
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Y. Li, C. A. Putnam-Lawson, H. Knapp-Hoch, P. J. Friel, D. Mitchell, R. Hively, and M. D. Griswold Immunolocalization and Regulation of Cystatin 12 in Mouse Testis and Epididymis Biol Reprod, November 1, 2005; 73(5): 872 - 880. [Abstract] [Full Text] [PDF]
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 J. Igarashi-Migitaka, A. Takeshita, N. Koibuchi, S. Yamada, R. Ohtani-Kaneko, and K. Hirata Differential expression of p160 steroid receptor coactivators in the rat testis and epididymis Eur. J. Endocrinol., October 1, 2005; 153(4): 595 - 604. [Abstract] [Full Text] [PDF]
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C. Staub, M. Rauch, F. Ferriere, M. Trepos, I. Dorval-Coiffec, P. T. Saunders, G. Cobellis, G. Flouriot, C. Saligaut, and B. Jegou Expression of Estrogen Receptor ESR1 and Its 46-kDa Variant in the Gubernaculum Testis Biol Reprod, October 1, 2005; 73(4): 703 - 712. [Abstract] [Full Text] [PDF]
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 J. M. Naciff, K. A. Hess, G. J. Overmann, S. M. Torontali, G. J. Carr, J. P. Tiesman, L. M. Foertsch, B. D. Richardson, J. E. Martinez, and G. P. Daston Gene Expression Changes Induced in the Testis by Transplacental Exposure to High and Low Doses of 17{alpha}-Ethynyl Estradiol, Genistein, or Bisphenol A Toxicol. Sci., August 1, 2005; 86(2): 396 - 416. [Abstract] [Full Text] [PDF]
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S. Filippi, A. Morelli, L. Vignozzi, G. B. Vannelli, M. Marini, P. Ferruzzi, R. Mancina, C. Crescioli, N. Mondaini, G. Forti, et al. Oxytocin Mediates the Estrogen-Dependent Contractile Activity of Endothelin-1 in Human and Rabbit Epididymis Endocrinology, August 1, 2005; 146(8): 3506 - 3517. [Abstract] [Full Text] [PDF]
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X. Ye, S. J. Han, S. Y. Tsai, F. J. DeMayo, J. Xu, M.-J. Tsai, and B. W. O'Malley Roles of steroid receptor coactivator (SRC)-1 and transcriptional intermediary factor (TIF) 2 in androgen receptor activity in mice PNAS, July 5, 2005; 102(27): 9487 - 9492. [Abstract] [Full Text] [PDF]
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Q. Zhou, J. E. Shima, R. Nie, P. J. Friel, and M. D. Griswold Androgen-Regulated Transcripts in the Neonatal Mouse Testis as Determined Through Microarray Analysis Biol Reprod, April 1, 2005; 72(4): 1010 - 1019. [Abstract] [Full Text] [PDF]
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D. M. Selva, O. M. Tirado, N. Toran, C. A. Suarez-Quian, J. Reventos, and F. Munell Estrogen Receptor {beta} Expression and Apoptosis of Spermatocytes of Mice Overexpressing a Rat Androgen-Binding Protein Transgene Biol Reprod, November 1, 2004; 71(5): 1461 - 1468. [Abstract] [Full Text] [PDF]
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Z.M. Lei, S. Mishra, P. Ponnuru, X. Li, Z.W. Yang, and Ch.V. Rao Testicular Phenotype in Luteinizing Hormone Receptor Knockout Animals and the Effect of Testosterone Replacement Therapy Biol Reprod, November 1, 2004; 71(5): 1605 - 1613. [Abstract] [Full Text] [PDF]
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C. M. Hill, M. D. Anway, B. R. Zirkin, and T. R. Brown Intratesticular Androgen Levels, Androgen Receptor Localization, and Androgen Receptor Expression in Adult Rat Sertoli Cells Biol Reprod, October 1, 2004; 71(4): 1348 - 1358. [Abstract] [Full Text] [PDF]
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T. R. Chauvin and M. D. Griswold Androgen-Regulated Genes in the Murine Epididymis Biol Reprod, August 1, 2004; 71(2): 560 - 569. [Abstract] [Full Text] [PDF]
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C. A Oliveira, G. A B Mahecha, K. Carnes, G. S Prins, P. T K Saunders, L. R Franca, and R. A Hess Differential hormonal regulation of estrogen receptors ER{alpha} and ER{beta} and androgen receptor expression in rat efferent ductules Reproduction, July 1, 2004; 128(1): 73 - 86. [Abstract] [Full Text] [PDF]
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 R. W. Holdcraft and R. E. Braun Androgen receptor function is required in Sertoli cells for the terminal differentiation of haploid spermatids Development, January 15, 2004; 131(2): 459 - 467. [Abstract] [Full Text] [PDF]
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H. Johnston, P. J. Baker, M. Abel, H. M. Charlton, G. Jackson, L. Fleming, T. R. Kumar, and P. J. O'Shaughnessy Regulation of Sertoli Cell Number and Activity by Follicle-Stimulating Hormone and Androgen during Postnatal Development in the Mouse Endocrinology, January 1, 2004; 145(1): 318 - 329. [Abstract] [Full Text] [PDF]
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S.Y. Man, J. Clulow, and R.C. Jones Signal Transduction in the Ductuli Efferentes Testis of the Rat: Inhibition of Fluid Reabsorption by Cyclic Adenosine 3', 5'-Monophosphate Biol Reprod, November 1, 2003; 69(5): 1714 - 1718. [Abstract] [Full Text] [PDF]
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Y. Li, P. J. Friel, D. J. McLean, and M. D. Griswold Cystatin E1 and E2, New Members of Male Reproductive Tract Subgroup Within Cystatin Type 2 Family Biol Reprod, August 1, 2003; 69(2): 489 - 500. [Abstract] [Full Text] [PDF]
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 G. Castoria, M. Lombardi, M. V. Barone, A. Bilancio, M. Di Domenico, D. Bottero, F. Vitale, A. Migliaccio, and F. Auricchio Androgen-stimulated DNA synthesis and cytoskeletal changes in fibroblasts by a nontranscriptional receptor action J. Cell Biol., May 12, 2003; 161(3): 547 - 556. [Abstract] [Full Text] [PDF]
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