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(But Not Estrogen Receptor ß) during Postnatal Development of the Epididymis and Vas Deferens of the Rat and Disruption of This Pattern by Neonatal Treatment with Diethylstilbestrol1
Medical Research Council Human Reproductive Sciences Unit, Center for Reproductive Biology (N.A., C.M., K.W., K.J.T., J.S.F., P.T.K.S., M.R.M., R.M.S.), Edinburgh, Scotland EH3 9ET; and Institute of Experimental Morphology and Anthropology, Bulgarian Academy of Science (N.A.), 1113 Sofia, Bulgaria
Address all correspondence and requests for reprints to: Dr. R. M. Sharpe, Medical Research Council Human Reproductive Sciences Unit, Center for Reproductive Biology, 37 Chalmers Street, Edinburgh, Scotland EH3 9ET. E-mail: r.sharpe{at}hrsu.mrc.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(ER) and ERß in the efferent ducts,
epididymis, and vas deferens during postnatal development; 2) establish
whether ER expression changed after neonatal treatment with
diethylstilbestrol (DES); and 3) determine whether ER changes coincided
with abnormal epididymal/vas development. Rats were administered 10
µg DES or vehicle on days 2, 4, 6, 8, 10, and 12 and were sampled on
days 10, 18, 25, 35, and 90+. At all ages, ER was immunoexpressed
intensely in the efferent ducts. On day 10, immunoexpression of ER
was absent from the epididymis and vas, but was detectable on day 18 in
epithelial cells in the caput, corpus, and proximal cauda. Epithelial
expression of ER was absent from the distal cauda and in the
proximal and distal vas was confined to a band of periductal stromal
cells. Thus, on day 18, the site of ER expression delineated the
epididymis-vas boundary. On days 25–35, epithelial expression of ER
was absent, but stromal expression persisted in the vas and distal
cauda. In adults, immunoexpression of ER in the epididymis and vas
was absent. In contrast, ERß was immunoexpressed in epithelial cells
and some stromal cells in the efferent ducts, epididymis, and vas at
all ages. In the vas, stromal expression of ER and ERß was in
different layers.
DES treatment caused 1) underdevelopment of the epididymal duct and
reduced epithelial height in epididymis and vas; 2) coiling of the
extraepididymal vas; 3) thickening of the periductal actin-free stromal
layer in the distal cauda and vas; and 4) reduced cell proliferation on
day 18 in the epididymis and vas, based on incorporation of
bromodeoxyuridine, especially in the epithelium. These changes
coincided with abnormalities in cell- and region-specific
immunoexpression of ER, but not ERß. Thus, in DES-treated rats on
day 18, epithelial expression of ER occurred in all regions of the
epididymis and vas instead of being confined to the caput, corpus, and
proximal cauda as in controls. Similarly, stromal ER expression in
the vas of DES-treated rats was not confined to a periductal layer as
in controls, but occurred diffusely in the muscle layer. It is
suggested that 1) estrogens play a role in peripubertal development of
the epididymis and vas; 2) the cellular site of expression of ER
either plays a role in or reflects demarcation of the epididymal/vas
boundary; and 3) blurring of this boundary in DES-treated rats
coincides with altered ER immunoexpression.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(ER)-knockout mouse (ERKO) exhibits abnormalities of cellular
development in different regions of the epididymis, suggesting possible
abnormalities of epididymal function (9). Such changes
might go some way toward explaining the poor fertilizing ability of
spermatozoa that are recovered from young ERKO males (10).
Consistent with these findings, ERs, whether ER or ERß, are
expressed widely in the developing duct system of the male
(11). After birth, the rat epididymis goes through a phase
of relatively slow growth followed by rapid growth during and after
puberty, associated with the onset of differentiation of the adult
generation of Leydig cells and increasing testosterone levels
(12, 13, 14). A similar pattern of growth is found in the
human vas deferens (15). Although this fits with the
established importance of androgens in development of the
epididymis/vas, in such situations conversion of testosterone to
estradiol within target tissues might occur, so that the biological
effects attributed to androgens might also involve the effects of
locally produced estrogens (11).
In our own studies of the role of estrogens in development of the male
reproductive system, we noticed that neonatal estrogen treatment can
induce widespread abnormalities, including changes in the epididymis
and vas deferens, and that these changes are associated with abnormal
expression of progesterone receptor in stromal (but not epithelial)
cells that also coexpressed ERß (16). In addition, we
noticed that some of the distinctive morphological differences that
demarcated the cauda epididymal duct from the vas deferens appeared
less distinct in neonatally estrogen-treated animals. In our attempts
to interpret and explain these findings we became aware that detailed,
systematic analysis and description of ER expression throughout the
epididymis and vas deferens at different ages in the rat before and
after estrogen treatment was either lacking or conflicting. For
example, Hess et al. (17) reported that ER
was expressed in the adult epididymis and vas deferens, whereas our own
studies (18) concluded that epididymal expression of ER
is lost during sexual maturation in both the rat and the marmoset.
Other researchers have reported the presence of ER in the epididymis of
the adult ram (19) and human (20, 21). In the
rabbit, epididymal expression of ER was reported to decline, but not to
disappear, during maturation (22, 23). However, some of
these studies used tissue estrogen binding assays and had been
undertaken before the existence of ERß had been established, with the
likelihood that ERß as well as or instead of ER had been detected
by the binding assays. Additionally, measurements that involved
extraction of epididymal tissue might not have completely excluded the
efferent ducts, which are now established to have a high level of
expression of ER (17, 18). Studies conducted at
different developmental stages/ages or species-specific differences in
ER expression in the epididymis could also account for some of the
reported differences in findings. Finally, reported differences in
ER immunolocalization might reflect differential cross-reaction of
different ER antibodies with ER and ERß in the various
studies.
The present studies had two aims: first to characterize in detail the
postnatal changes with age in the cellular sites of immunoexpression of
ER and ERß throughout the epididymis and vas deferens of the
rat using antibodies that we have shown to be specific for ER and
ERß (16, 24), and second, to establish what effect
neonatal treatment with DES had on this pattern of expression, using a
dose that we knew to cause epididymal abnormalities during puberty. Our
results demonstrate that ER, but not ERß, shows a region-, cell-,
and age-specific pattern of immunoexpression during epididymal
development in the rat and that this pattern is disrupted in a very
discrete way by neonatal DES treatment.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After fixation, the epididymis with efferent ducts and rete region of the testis attached were separated from the main bulk of the testis by cutting with a razor blade. This tissue was transferred into 70% ethanol before being processed for 17.5 h in an automated Shandon processor and embedded in paraffin wax. Before embedding, the epididymis was oriented such that full-length sections of the efferent ducts, epididymis, and vas could be obtained at sectioning. Sections of 5 µm thickness were cut and floated onto slides coated with 2% 3-aminopropyltriethoxy-silane (Sigma) and dried at 50 C overnight before being used for immunohistochemistry as described below.
For protein extraction, small pieces of unfixed corpus and caudal epididymal tissue were snap-frozen in liquid nitrogen and stored at -70 C. The tissue was subsequently ground in a pestle and mortar under liquid nitrogen, and then resuspended in ice-cold buffer comprising 10 mM HEPES, pH 7.8 (Sigma); 0.1 mM EDTA; 0.1 mM EGTA; 1 mM dithiothreitol (all from Sigma); and a protease inhibitor cocktail (Complete, Roche, Lewes, UK). The protein concentration was measured by absorbance at 280 nm, and the protein extracts were stored at -70 C. At least two separate experiments were performed for each of the treatments specified; comparable results were obtained in each experiment.
Antibodies
Immunolocalization of ER was determined using a mouse
monoclonal antibody raised to full-length recombinant human ER
(NCL-ER-6F11, Novocastra, Newcastle upon Tyne, UK). To confirm findings
with this antibody, limited studies using another monoclonal antibody
raised against a peptide from the N-terminal (A/B) region of the human
(clone 1D5, DAKO Corp., High Wycombe, UK) was also used.
Both antibodies were used at a dilution of 1:20. ERß was
immunolocalized using polyclonal antipeptide IgGs raised in sheep
against a specific peptide in the D region of human ERß, as
previously described in detail (24); it was used at a
dilution of 1:1000. The specificities of both ER antibodies have been
detailed in previous studies (16, 24) and are reaffirmed
below.
Immunolocalization of -smooth muscle actin used a mouse monoclonal
antibody (Sigma) raised against the
NH2 terminal synthetic decapeptide of -smooth
muscle actin; it was used at a dilution of 1:5000. A mouse antidesmin
monoclonal antibody (clone DE-R-11; DAKO Corp.) was used
at a dilution of 1:50. A mouse monoclonal anti-pan cytokeratin antibody
mixture that recognized human cytokeratins 1, 4, 5, 6, 8, 10, 13,18,
and 19 was employed for localization of cytokeratins as markers of
epithelial tissue; it was used at a dilution of 1:50. A mouse
monoclonal antibody against rat proliferating cell nuclear antigen
(PCNA; DAKO Corp.) was used at a dilution of 1:1000. For
BrdU labeling, a mouse monoclonal antibody against BrdU
(Roche) was used at a dilution of 1:30.
Immunohistochemistry
Unless otherwise stated, all incubations were performed at room
temperature. Sections were deparaffinized in Histoclear (National
Diagnostics, Hull, UK), rehydrated in graded ethanols, and washed in
water. At this stage, sections used for immunostaining of ER, ERß,
desmin, PCNA, and BrdU were subjected to a temperature-induced antigen
retrieval step using a domestic pressure cooker (25)
heated on a halogen hotplate. The pressure cooker contained either 0.01
M citrate buffer, pH 6.0 (for ER, desmin, BrdU), or 0.05
M glycine buffer, pH 3.5, and 0.01% EDTA (for ERß,
PCNA). After pressure cooking for 5 min at full pressure, sections were
left to stand, undisturbed, for 20 min, then were cooled under running
tapwater before being washed for 5 min in Tris-buffered saline (TBS;
0.05 M Tris-HCl, pH 7.4, and 0.85% NaCl). Endogenous
peroxidase activity was blocked by immersing all sections in 3%
(vol/vol) H2O2 in methanol
for 30 min, followed by two 5-min washes in TBS. To block nonspecific
binding sites, sections were incubated for 30 min with normal rabbit
serum (NRS; Scottish Antibody Production Unit, Carluke, Scotland)
diluted 1:5 in TBS containing 5% BSA (Sigma). The primary
antibodies were added to the sections at appropriate dilutions in
blocking serum and incubated overnight at 4 C or for 2 h at room
temperature (for BrdU) in a humidified chamber. After two 5-min washes
in TBS, all sections were incubated with a secondary antibody for 30
min, namely, a 1:500 dilution in blocking serum of either biotinylated
rabbit antisheep IgG (Vector Laboratories, Inc.,
Peterborough, UK) in the case of ERß or biotinylated rabbit antimouse
IgG (DAKO Corp.) for all other antibodies. After two
additional 5-min washes in TBS, sections were incubated for 30 min with
avidin-biotin conjugated to peroxidase (DAKO Corp.)
diluted in 0.05 M Tris-HCl, pH 7.4, according to the
manufacturer’s instructions. Sections were washed twice (5 min each
time) in TBS, and immunostaining was developed using
3,3'-diaminobenzidine (liquid DAB; DAKO Corp.) according
to the manufacturer’s instructions until staining in controls was
optimal, at which time the reaction was stopped by immersing all
sections in distilled water. All sections were then lightly
counterstained with Harris’s hematoxylin, dehydrated in graded
ethanols, cleared in xylene, and coverslipped using Pertex mounting
medium (CellPath plc, Hemel Hempstead, UK).
Double immunostaining method for -smooth muscle actin and
cytokeratins
To delineate the structural changes induced by DES treatment and
to facilitate the precise localization of ER expression, both smooth
muscle and epithelial tissues were labeled immunohistochemically on the
same sections. After development of -smooth muscle actin
immunostaining with DAB as described above, some sections were again
incubated with blocking serum (NRS/TBS/BSA) for 30 min. They were then
incubated with mouse monoclonal anti-pan cytokeratin antibody overnight
at 4 C in a humidified chamber. After two 5-min washes in TBS, sections
were incubated in rabbit antimouse IgG (DAKO Corp.) at a
1:60 dilution in blocking serum for 30 min. After twice rinsing in TBS,
mouse alkaline phosphatase antialkaline phosphatase (DAKO Corp.) was applied to the slides at a 1:100 dilution in blocking
serum for 30 min. After two additional 5-min washes in TBS, the slides
were given a final 5-min wash in 100 mM Tris buffer, pH
9.5, containing 100 mM NaCl and 50 mM MgCl
before the addition of 337.5 µg/ml 4-nitro blue tetrazolium chloride
(Roche, Mannheim, Germany), 175 µg/ml 5-bromo-4
chloro-3-indolylphosphate (Roche) and 0.001%
levamisole (Sigma) in 10 ml Tris-MgCl buffer to develop
color at the sites of antibody localization. The slides were incubated
until the color developed to the required intensity in control
sections, when the reaction was stopped by immersion in distilled
water. Slides were very lightly counterstained in Harris’s hematoxylin
before being dehydrated rapidly in absolute ethanol and cleared in
xylene. Sections were coverslipped using Pertex mounting medium.
Double immunostaining method for ER and -smooth muscle
actin
To facilitate the cellular localization of ER, both ER and
smooth muscle tissue were labeled immunohistochemically on the same
sections. After development of ER immunostaining with DAB as
described above, some sections were again incubated with blocking serum
(NRS/TBS/BSA) for 30 min. Sections were then incubated with mouse
monoclonal anti -smooth muscle actin antibody at a 1:1000 dilution
overnight at 4 C in a humidified chamber. After two 5-min washes in
TBS, sections were incubated in rabbit antimouse IgG at a 1:60 dilution
in blocking serum for 30 min. After twice rinsing in TBS, mouse
alkaline phosphatase antialkaline phosphatase was applied to the slides
at a 1:100 dilution in blocking serum for 30 min. After two additional
5-min washes in TBS the immunostaining was developed using 1 mg/ml Fast
Blue (Sigma) dissolved in 100 mM Tris, pH 8.2,
containing 0.02% naphthol AS-MX phosphate (Sigma) and 2%
(vol/vol) dimethylformamide (Sigma). The sections were
incubated until the color developed to the required intensity in
control sections, and the reaction was then stopped by immersion in
distilled water. Sections were coverslipped using aqueous mounting
medium (Permafluor, Immunotech, Marseilles, France). For
observations under high power magnification (x1000), some sections
were dehydrated by blotting onto filter paper and rapidly cleared in
xylene followed by coverslipping using Pertex mounting medium.
Evaluation of ER immunostaining and its semiquantitation
To ensure the reproducibility of findings for immunostaining,
tissue sections from a minimum of four to six animals in each
age/treatment group were evaluated, and this was performed for at least
two separate experiments. Sections from control and DES-treated rats or
from controls of different ages were run in parallel, and where
differences in immunoexpression pattern or intensity were apparent,
confirmation was obtained by undertaking immunohistochemistry with
tissue sections from control and treated animals on the same slide. A
subjective scoring method was used to score the intensity of
immunostaining for ER or ERß throughout the length of the
epididymis and proximal vas deferens. As an internal reference in each
section, immunoexpression of the relevant ER in the nuclei of
epithelial cells in the efferent ducts was used, as no detectable
change in immunoexpression of either ER (scored ++++) or ERß
(scored +++) was observed in this tissue with change in age or
treatment (see Table 1
). The
immunoexpression of ERß did not change greatly in intensity (scored
++ to +++) in different cell types or regions of the epididymis and vas
in relation to age or treatment, so these data were not tabulated. In
contrast, the immunoexpression of ER exhibited major changes in
intensity according to age, region, and treatment, ranging from
undetectable (-) to ++++ (i.e. equivalent to the level of
immunoexpression in the efferent ducts). The average scores for
intensity of ER immunoexpression reported in Table 1 are based on
systematic analysis of a total of at least six animals from two
separate experiments.
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Western analysis
Western blotting was used to confirm the specificity of the
antibodies used for immunolocalization of ER and ERß. Protein
extracts of corpus and cauda epididymal tissue from 18-day-old control
rats were separated using SDS-PAGE. Acrylamide gradient gels (4–20%)
were purchased from Novex (Frankfurt, Germany) and used
according to the manufacturer’s instructions. Gels were loaded with
100 µg protein extract, 0.25 µg recombinant human ER protein
(P2187; 66 kDa), and 0.25 µg recombinant human ERß protein (P2718;
59 kDa; both from Pan Vera, Madison, WI). Prestained molecular weight
markers (Bio-Rad Laboratories, Inc., Hemel Hempstead, UK)
were run in parallel. Gels were run at 100 mA for approximately 1
h before blotting onto a polyvinylidene difluoride membrane
(Immobilon-P, Millipore Corp., Watford, UK) for 90 min at
30 V. Membranes were blocked for 2–3 h in TBS containing 0.05%
Tween-20 (Sigma) and 5% skimmed milk powder (Marvel,
Premier Brands Ltd., Moreton, UK). The ER antibody (NCL-ER-6F11) was
added at a dilution of 1:200 in TBS/Tween containing 5% normal rabbit
serum, and the ERß antibody was used at 1:2000 in TBS/Tween
containing 5% normal donkey serum. Blots were incubated overnight at
room temperature. After repeated washing with TBS/Tween, blots were
incubated for 2 h with a peroxidase-conjugated secondary antibody,
namely a 1:4000 dilution in TBS/Tween of rabbit antimouse IgG
(DAKO Corp.) in the case of ER or donkey antisheep IgG
(DAKO Corp.) for ERß. After repeated extensive
washes in TBS/Tween, specific signals were detected using the ECL
detection system (Amersham Pharmacia Biotech) and
Hyperfilm (Kodak) following the manufacturers’
instructions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. This
illustrates a sagittal section through the caudal region of the
epididymis of a control day 18 rat immunostained for -smooth muscle
actin. The latter staining clearly differentiates the proximal caudal
epididymal duct from the proximal vas deferens based on the increased
thickness of the muscle layer (-actin-stained) surrounding the vas.
There is also a conspicuous difference in cross-sectional size of the
duct in the two regions. As described below, there is a clear
difference in the site of immunoexpression of ER in these two
regions in 18-day-old rats. The region intermediate between the
proximal cauda and proximal vas, i.e. the distal cauda,
shows a gradual transition in terms of thickness of the actin-positive
layer and the size of the duct in cross-section. The box in
Fig. 1 indicates the area in which comparison of proximal cauda and
proximal vas can be compared side by side in the same section, and this
comparison is used in several places in the results described below. In
the figure legends, the region of sampling is indicated and corresponds
to regions I–IV shown in Fig. 1.
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, A and E; also
illustrated in other figures below). As a result of these changes, the
cauda epididymal region of DES-treated rats took on a characteristic
spherical or bulb shape, in contrast to the more elongated shape in
control animals (Fig. 2, A and E).
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) was coiled, but as it
emerged from the cauda (region IV in Fig. 1 and more distally), it
became a more or less straight duct (Fig. 2B). In contrast, the
extraepididymal portion of the vas (region IV) in 70% of DES-treated
rats remained coiled (Fig. 2F), and this extended for 1–2 cm along the
vas (not shown). This abnormal coiling was still evident in 35-day-old
and adult DES-treated animals (not shown).
Another change induced by DES was underdevelopment (reduction in cell
height) of the epithelium of the cauda epididymis (Fig. 2, C and G) and
vas deferens (Fig. 2, D and H). This change was also evident in the
caput and corpus regions of the epididymis (not shown), but was less
pronounced than in the cauda and vas.
In the cauda (Fig. 2, C and G) and vas (Fig. 2, D and H) there was a
notable thickening of the immediate periductal stromal layer adjacent
to the basal membrane in which there was no immunostaining for
-smooth muscle actin. This actin-free periductal layer was first
detected in control animals on day 18, but was only evident in the
distal cauda (region II) and more distally and was most pronounced in
the vas. The layer was still evident on day 25, but became far less
conspicuous or was absent on day 35 and in adulthood (not shown). It is
tentatively concluded that this actin-free layer is comprised of
fibroblast-like cells, because it did not stain for -smooth muscle
actin using an antibody that recognizes myofibroblasts and smooth
muscle cells, but not fibroblasts. In addition, the layer was not
stained by a pan-cytokeratin antibody (Fig. 2, F–H) that recognizes
all epithelial cell types, but does not react with fibroblasts,
myofibroblasts, or smooth muscle cells, or by an antibody for desmin
(not shown).
A further minor observation in DES-treated rats was that the smooth
muscle region surrounding the distal cauda and vas deferens appeared
more loosely organized than in controls (compare Fig. 2, C and D, with
Fig. 2, G and H, respectively).
Effect of neonatal DES treatment on cell proliferation in the
epididymis and proximal vas deferens on day 18
The relative changes in epithelial and stromal tissue in
DES-treated rats, as well as the appearance of the actin-free
periductal layer described above, suggested that changes in cell
proliferation may have occurred compared to that in controls. This was
assessed grossly by immunostaining for BrdU. Typical results for the
caput (Fig. 2, J and N), proximal cauda (Fig. 2, K and O), distal cauda
(Fig. 2, L and P), and proximal vas deferens (Fig. 2, M and Q) are
illustrated. These showed clearly that rates of cell proliferation in
all regions of the epididymis and proximal vas deferens were notably
lower in DES-treated rats, based on immunostaining for BrdU, and this
was confirmed further by immunostaining for PCNA (not shown). It was
also notable that in controls it was the epithelial cells that were
most actively proliferating, and there was a gradient of proliferation
from the caput (highest rate) to the distal cauda/proximal vas (lowest
rate) (Fig. 2, J–Q). These findings suggest that the relative changes
in proportion of epithelial and stromal tissue in DES-treated rats
(Fig. 2, A and E) are the result of underproliferation of epithelial
cells rather than the overproliferation of stromal cells.
Developmental immunoexpression of ERs in the epididymis and vas
deferens
As an initial approach to understanding how the DES-induced
changes described above might have arisen, we sought to systematically
investigate the cell-, region-, and age-specific pattern of
immunoexpression of ERs in the epididymis and vas deferens of control
rats. Firstly, the specificity of the ER antibodies was confirmed by
Western blotting. Thus, the ER antibody (NCL-ER-6F11) detected
recombinant ER protein, but not recombinant ERß, and also detected
a band corresponding to the size of ER in protein extracts of the
corpus/cauda epididymis from a day 18 control rat (Fig. 3). Similarly, the ERß antibody
recognized recombinant ERß protein, but not recombinant ER, and
also detected a band corresponding to the size of the ERß in
corpus/cauda epididymal extracts (Fig. 3).
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are summarized in Table 1 and shown in Fig. 4. The results
illustrated are based on studies using antibody NCL-ER-6F11, but
comparable results were obtained using antibody ID5 (see
Materials and Methods). The nuclei of epithelial cells in
the efferent ducts showed a constant and very intense immunoexpression
of ER at 10 days of age and throughout postnatal life to adulthood
(17, 18). Therefore, this tissue was used as 1) an
internal ER-positive control in each section (Fig. 4, A–D), and 2)
an internal reference (scored ++++) for semiquantitation of the
intensity of ER immunostaining throughout the epididymis and vas at
each age. Compared with the efferent ducts, the intensity of
immunoexpression of ER in the epididymis and vas was rarely better
than moderate (++++) to weak (+; Table 1).
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), the entire epididymis and vas (Fig. 4, E and J) were negative for
ER immunostaining.
Day 18 epididymis/vas. Weak to moderate immunostaining was
observed in the caput within the nuclei of many, but not all,
epithelial cells (Fig. 4F), and a similar pattern of immunoexpression
was noted in the corpus (not shown) and proximal cauda epithelium (Fig. 4, K and N). However, epithelial immunoexpression of ER was absent
from the distal cauda (Fig. 4O), coincident with thickening of the
periductal muscle layer in this region described earlier. In the
proximal vas and more distally, immunoexpression of ER reappeared,
but was localized not to epithelial cells, but to a layer of stromal
cells surrounding the duct and close to the basal membrane (Fig. 4, K
and P). By reference to staining for -smooth muscle actin, the
position of the ER-immunopositive cells probably corresponds to
cells within the narrow actin-free layer described above (Fig. 2, C and
D) and to some actin-positive cells at the edge of the muscle stroma
(Fig. 4, P and Q).
Day 25 epididymis/vas. Epithelial immunoexpression of ER
was no longer evident in the caput (Fig. 4G), and was confined to a few
isolated cells in the cauda (Fig. 4L). Stromal cell immunoexpression
was also absent from the caput at this age (Fig. 4G), and was confined
to a few isolated cells in the proximal cauda (Fig. 4L). However,
immunoexpression was evident in periductal stromal cells in the distal
cauda and was more intense in the vas (Fig. 4L).
Day 35 epididymis/vas. Epithelial cell immunoexpression of
ER was completely absent from all regions of the epididymis and the
vas (Fig. 4, H and M), and periductal stromal cell immunoexpression of
ER at this age was largely confined to the distal cauda (Fig. 4M),
with just a few immunopositive cells in the vas (Fig. 4M).
Adult epididymis/vas. No epithelial or stromal cell
immunoexpression of ER was detected in any region of the epididymis
or vas (not shown, but see Table 1).
Based on these findings, it was concluded that on day 18
(although not at other ages) the morphological boundaries of the
epididymal duct and the vas deferens (Fig. 1) were also distinguishable
by the switch in immunoexpression of ER from the epithelium in the
caput, corpus, and proximal cauda to stromal cells in the proximal vas
(Table 2). Conveniently, the distal cauda
epididymis (region II in Fig. 1) marked a transitional zone, in that
epithelial immunoexpression of ER was absent, and only a few
isolated stromal cells were ER immunopositive. Therefore, regions I,
II, and III in Fig. 1 could be demarcated based on the sites of
immunoexpression of ER on day 18 (Table 2).
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. Immunoexpression
was detected in the nuclei of epithelial cells and in some, but not all
periductal stromal cells, from the efferent ducts through the
epididymis and throughout the vas deferens (see Fig. 5, J–M). In the
vas deferens, it was notable that stromal immunoexpression of ERß
localized to the muscle stroma layer, in contrast to ER, which
localized mainly to the immediate periductal (non-muscle) layer
(compare Fig. 5, C and D, with L and M).
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and ERß. Neonatal treatment with DES failed
to induce any significant change in ER immunoexpression on day 10,
except for the appearance of isolated, weakly positive stromal cells
scattered in the intertubular space (not shown, but see Table 1).
Similarly, DES treatment had no effect on epithelial immunoexpression
of ER in the efferent ducts (Fig. 5, A and E), caput (Fig. 5, B and
F), corpus (not shown), or proximal cauda epididymis (Fig. 5, C and G)
on day 18. However, the switch in immunoexpression of ER from
epithelium to stroma that occurred at the cauda/vas boundary in control
animals at this age (Fig. 5C and Table 2), did not occur in DES-treated
animals (Fig. 5G). Instead, epithelial immunoexpression of ER
persisted in the distal cauda (not shown, see Table 1) and continued
out into the proximal (Fig. 5G) and distal (Fig. 5H) vas, and in the
latter region the intensity of expression approached that seen in the
efferent ducts. In addition, stromal immunoexpression of ER in the
vas of DES-treated rats at this age was markedly reduced compared with
that in controls and did not localize to the immediate periductal
region as in controls, but to the muscle layer in which ERß also
localized (compare Fig. 5, G and P, with Fig. 5, C and L, and compared
Fig. 5, H and Q, with Fig. 5, D and M).
The persistence of epithelial immunoexpression of ER in the vas of
DES-treated rats on day 18 was associated with a marked reduction in
height of the epithelium compared with controls (Fig. 5, D, H, and
L–Q). On day 25, DES treatment had little or no effect on epithelial
immunoexpression of ER in any region of the epididymis, whereas the
stromal immunoexpression seen in the distal cauda and vas of control
animals at this age was eliminated by treatment with DES (Table 1). On
day 35, DES treatment had no major effect on ER immunoexpression,
apart from the appearance of immunostaining in a few isolated stromal
cells within the caput and proximal cauda and a slight reduction in
immunoexpression in the periductal stromal cells of the distal cauda
(Table 1). By adulthood, epithelial and stromal expression of ER in
all regions of the epididymis and vas of DES-treated animals was mainly
absent, as in controls (Table 1), with the exception of one animal in
which there was persistence of strong epithelial immunoexpression of
ER in the vas (not shown).
Immunoexpression of ERß. In contrast to the effect of
neonatal DES administration on the immunoexpression of ER, the
intensity and pattern of immunoexpression of ERß were unchanged on
day 18 in the efferent ducts (Fig. 5, J and N), caput (Fig. 5, K and
O), cauda, and vas (Fig. 5, L–Q). Similarly, at all later ages studied
there was no major or consistent change in the immunoexpression of
ERß in any region of the epididymis or vas after treatment with DES,
and the pattern of expression remained comparable to that shown in Fig. 5.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is immunoexpressed in the
epididymis and vas deferens of the rat during puberty in a highly
cell-, region-, and age-specific manner. The most notable observations
were that 1) immunoexpression of ER in epithelial cells of the
epididymal duct was confined to the caput, corpus, and proximal cauda
within a relatively narrow time-frame of puberty, in contrast to the
uniformly high immunoexpression at all ages in the efferent ducts. 2)
Transition of the caudal epididymal duct into the proximal vas deferens
was associated with a switch from epithelial to periductal (stromal)
immunoexpression of ER that persisted along the length of the vas
deferens during puberty before disappearing by adulthood. 3) This
switch in cellular site of ER immunoexpression was most distinct on
day 18, but did not occur in rats treated neonatally with a high dose
of DES. In the latter, epithelial immunoexpression of ER in treated
animals was evident throughout both the proximal and distal cauda and
continued out into the vas deferens. 4) The latter change was
associated with a number of abnormal morphological changes induced by
DES treatment, such as reduced epithelial height of the duct and
convolution of the initial, extraepididymal region of the vas deferens.
In contrast to these variations in the pattern of immunoexpression of
ER, no major cell-, region-, age-, or DES-related changes in
immunoexpression of ERß were observed. Our findings provide
additional support for the view that estrogens play a role in pubertal
development of the epididymis and vas deferens, mediated either via
ER or differential expression of ER and ERß. This may include a
role in morphological demarcation of the boundaries of the epididymal
duct and the vas deferens. Our present demonstration of underdevelopment of the epithelium of the epididymal duct together with relative overgrowth of stromal tissue after neonatal DES treatment is not a new finding, as similar changes have been described previously after early estrogen exposure (3, 4, 5, 6, 26). Similar changes have also been reported in the prostate (27, 28) and seminal vesicles (16) of rats treated neonatally with estrogens. One morphometric study of the epididymis (4) reported an increase in the proliferative activity of stromal cells and decreased proliferative activity of epithelial cells on day 15 after treatment on the day of birth with 500 µg estradiol benzoate. Our findings with BrdU labeling on day 18 confirm these earlier findings with respect to decreased proliferation of epithelial cells, but fail to confirm increased proliferation of stromal cells. It is possible that increased stromal cell proliferation could have occurred at some earlier time point in our studies, although our preliminary analysis of BrdU labeling on day 10, when the animals were still receiving DES treatment, also failed to find evidence of increased proliferation (unpublished data). Other changes, such as increased deposition of extracellular matrix (or even edema), might perhaps account for the apparent increase in stromal tissue in the present study rather than cellular proliferation. Similar changes and/or altered differentiation of the periductal layer of fibroblast-like cells into myoid cells, might underlie the notable increase in size of the periductal actin-free layer in the distal cauda and vas deferens of DES-treated rats. Alterations in periductal stromal cell differentiation have also been reported in the prostate after neonatal estrogen treatment (29).
The present finding that the majority of DES-treated rats exhibited
abnormal coiling of the extraepididymal portion of the proximal vas
deferens has, to our knowledge, not been reported previously. However,
similar changes have been reported in transgenic mice in which
inactivating mutations of the posterior (Abd) Hox genes
Hoxa-10 (30) or Hoxa-11
(31) have been introduced. The abnormal coiling of the vas
in the Hoxa-10 and Hoxa-11 mutant mice was
interpreted by these researchers as being a homeotic change,
i.e. the partial conversion of the vas to a more anterior
structure, namely the coiled epididymal duct. As there is also evidence
that DES treatment of the female can suppress expression of
Hoxa-10 in the reproductive tract (32), it is
not unreasonable to consider that a similar sequence of events in
DES-treated males in the present studies might underlie the apparent
blurring of the epididymal:vas boundary. Not only was the latter change
evident from the coiling of the proximal vas deferens, it was also
evident at the cellular level, based on the altered pattern of
immunoexpression of ER on day 18, which was expressed in epithelial,
rather than stromal, cells in the vas, in contrast to controls.
Induction of epithelial expression of ER messenger RNA and protein
in the vas deferens has also been reported in mice after neonatal
estrogen treatment (33). It is arguable whether the switch
in site of expression of ER from epithelium to stroma in the cauda
and vas is related causally to delineation of these regions during
neonatal/peripubertal development. The fact that immunoexpression of
ER in these regions was not apparent until some time between days 10
and 18 is perhaps more consistent with this pattern of immunoexpression
being a consequence rather than a cause of the tissue demarcation.
However, our results do not exclude the possibility that the pattern of
ER immunoexpression is established much earlier during development,
but the level of expression is too low to be detected using our methods
(see below).
Our findings show that the cellular site of immunoexpression of ER
provides a useful biochemical marker for distinguishing the distal
cauda epididymis and the proximal vas deferens, at least on day 18.
Furthermore, morphological abnormalities in this transition, such as
that induced by neonatal DES treatment, are manifest by a change in the
pattern of immunoexpression of ER. The switch from epithelial
immunoexpression of ER in the cauda epididymis to stromal cell
immunoexpression in the vas in controls on day 18 coincided with
morphological changes in the epithelium and stroma (thickness of the
smooth muscle layer). The site of immunoexpression of ER in the vas,
in cells adjacent to the basement membrane of the duct, means that
these cells are well located to influence the development of both the
neighboring stromal smooth muscle and the epithelium of the duct when
activated by estrogens. This is in keeping with other studies that
suggest that smooth muscle cells may be an important target for
estrogens in the male reproductive tract (28, 34).
Additionally, the timing of the transient immunoexpression of ER
described here coincides with the period when the epididymal epithelium
proliferates and differentiates into its constituent cell types
(12, 14) to establish the major functions (secretion and
endocytosis) that typify the adult epididymis (35). These
changes are associated with a rise in testosterone levels at puberty.
Assuming that aromatase is expressed locally in the epididymis (there
is no evidence for or against, but see Refs. 11 and
36), it seems likely that local conversion of androgens to
estrogens could play a role in cellular and functional differentiation
of the epididymis and vas during puberty. Recent findings of structural
and functional abnormalities in the epididymides of ERKO mice
(9) support this contention. It is emphasized, however,
that whatever the roles of estrogen may be in the development of the
epididymis/vas, it is unquestionable that androgens play the dominant
regulatory role (1, 2, 37, 38). This is reinforced by our
own observations, which show that most of the DES-induced changes to
the developing reproductive system of the male, including the changes
to the epididymis and vas described in the present studies, are
associated with loss of expression of the androgen receptor
(39).
In the present studies immunoexpression of ER in the epididymis was
confined to a relatively short window of peripubertal development, in
contrast to the uniformly high level of immunoexpression in the
adjacent efferent ducts at all ages to adulthood. These findings
confirm our earlier preliminary data (18) and are
consistent with other reports in the literature (23, 40).
We also obtained confirmation of the present findings using a second
antibody to ER. Although there is good agreement across all species
studied for efferent duct immunoexpression of ER from fetal life
through to adulthood (16, 17, 41, 42), our findings of
transient immunoexpression of ER in the epididymis and vas conflict
with several pieces of previous data for the rat (17) and
other species (19, 20, 21). Most of these studies used
estradiol-specific binding as their end point, so it is likely that the
studies were also detecting binding to ERß. Our present findings
demonstrate widespread immunoexpression of this receptor at relatively
constant levels throughout all stages of life in the epididymis and vas
of the rat. However, the disparity between our findings and those of
Hess et al. (17), who also used
immunohistochemistry to localize ER, remain unexplained. The latter
study did not provide evidence that the ER antibody used did not
cross-react with ERß, so this is one possible (although unlikely)
explanation. Alternatively, the method of tissue fixation and
processing for immunocytochemistry in that study was fundamentally
different from that used in our own studies. Thus, the researchers used
preliminary microwaving of whole tissue before freezing it, followed
again by microwaving of frozen tissue sections and then fixation,
whereas our study used fixation in Bouin’s fixative for 6 h,
followed by microwaving of tissue sections. It is likely that the
method of Hess et al. (17) will be more
efficient than our own approach in unraveling folded and complexed
proteins and thus revealing their antigenic epitopes. It is therefore
possible that their detection of ER immunoexpression in a wider
range of ages in the rat than in our studies is because of greater
sensitivity. Alternatively, at certain ages in the epididymis and vas,
ER might be complexed or conformed in such a way that it is not
recognizable by either of the antibodies used in the present study. We
recognize these as possible explanations for our findings. Regardless
of whether such factors affect the antigenicity of ER in the
epididymis and vas, the fact that our studies show constant
immunoexpression of ER in the efferent ducts in the same sections in
which region-specific or no immunostaining is found in the epididymis
and vas at specific ages argues that the changing pattern of
immunoexpression of ER must be indicative of fundamental changes to
ER (whether in expression or conformation) that are important in
development of the epididymis. This conclusion may apply to other
species as well, as our studies of the epididymis and vas of the
marmoset and human (18) (unpublished data) also show lack
of immunoexpression of ER in adulthood when using the same
antibodies and similar fixation/antigen retrieval methods as those used
in the present studies.
In contrast to ER, no consistent change in immunoexpression of ERß
with age, epididymal region, or treatment was detected. Indeed, ERß
was expressed in most, if not all, epithelial cells and many periductal
stromal cells throughout the efferent ducts, epididymis, and vas at all
ages studied. This widespread expression pattern confirms and expands
on earlier reports (11, 16, 17, 43, 44, 45). More importantly,
it points to a key role for ER, for if estrogens play a role in
development of the epididymis and vas, then it is the dynamic changes
in expression of ER that coincide with these changes rather than the
uniform unchanging expression of ERß. This interpretation does not
exclude the possibility that differential heterodimerization of ER
and ERß could also be an important factor in this respect. The
absence of reported abnormalities in the reproductive tract of
ERß-knockout (BERKO) mice (46) is consistent with our
interpretation, although as abnormalities in the epididymis of ERKO
mice have only just been reported (9), more detailed
scrutiny of BERKO mice will be required before final conclusions can be
reached.
In summary, the present findings add to the growing evidence indicating
a role for estrogens in the development and function of the male
reproductive system. The current working hypothesis invoked to account
for this change in thinking is that it is the androgen:estrogen balance
that is of central importance in male reproductive development, as
opposed to just androgens alone (11, 39). Disturbance of
this balance, in particular lowering the androgen side of the balance
at the same time as elevating the estrogen side of the balance, can
result in reproductive tract abnormalities such as those described in
the present studies. The fact that these abnormalities are associated
with changes in the expression of both the androgen receptor
(39) and the ER (present studies) reinforces the close
interrelationship between androgen and estrogen action in male
reproductive development. Under physiological conditions it is proposed
that estrogens also contribute to the development of the male
reproductive system, but this role is largely masked by the dominant
role of androgens. Studies in ERKO mice together with studies of the
kind reported here should enable these roles to be delineated.
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Acknowledgments |
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Footnotes |
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2 Recipient of a Royal Society/NATO fellowship.
Received August 16, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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knockout mice. J Androl 21:107–121[Abstract]
and ß)
expression in the excurrent ducts of the adult male rat reproductive
tract. J Androl 18:602–611
(ER) within the testis and excurrent ducts of the rat and marmoset
monkey from perinatal life to adulthood. J Endocrinol 153:485–495 and –ß and androgen
receptor in the ovaries of marmoset and human. Biol Reprod 63:1098–1105 (ER) and estrogen receptor-ß (ERß) messenger
ribonucleic acid in the wild-type and ER-knockout mouse.
Endocrinology 138:4613–4621
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