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From the * MRC Human Reproductive Sciences Unit,
Centre for Reproductive Biology, Edinburgh, United Kingdom; the
Laboratory of Medical Investigations,
Department of Radiology, School of Medicine, HUSC-University of Granada,
Granada, Spain; and the Institute of
Experimental Morphology & Anthropology, Bulgarian Academy of Science,
Sofia, Bulgaria.
| Correspondence to: Dr R. M. Sharpe, MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, Chancellor's Building, The University of Edinburgh, 49 Little France Crescent, Old Dalkeith Rd, Edinburgh EH16 4SB, United Kingdom (e-mail: r.sharpe{at}hrsu.mrc.ac.uk). |
| Received for publication November 12, 2002; accepted for publication February 26, 2003. |
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Abstract |
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) immunoexpression in the vas deferens and
seminal vesicles was evaluated. The coadministration of DES with TE prevented
the induction of all but one of the abnormalities induced by DES treatment on
its own, coincident with the restoration of normal/supranormal TE levels and
normal immunoexpression of the AR and ER- in the tissues studied. The
exception was DES-induced lumenal distension of the efferent ducts, which was
only partially prevented by the coadministration of DES with TE. These
evaluations were made on day 18, but the described abnormalities were already
somewhat evident by day 8 in DES-treated animals. It was therefore tested
whether a delay of TE replacement until days 8–12 was still able to
reverse the abnormalities already induced by DES treatment alone. A delayed
treatment with TE reversed the adverse changes in epithelial cell height and
in ER- and AR immunoexpression in the same tissues by day 18; however,
rete testis overgrowth was only partially prevented, and efferent duct
distension was not prevented at all. These results provide further evidence
that DES-induced disorders of reproductive tract development in the male
result from a disturbance of the androgen-estrogen balance rather than from
estrogen action alone.
Key words: Rete testis, efferent ducts, vas deferens, seminal vesicles, androgens, estrogens
One obvious interpretation of these findings is that estrogen-induced adverse effects in the developing male reproductive tract are due to their ability to suppress androgen action. However, this interpretation is not supported by the evidence showing that treatments that alter androgen production (administration of a gonadotropin-releasing hormone antagonist [GnRHa]) or androgen action (administration of the androgen antagonist ``flutamide'') neonatally do not exert comparable effects to estrogens on the reproductive tract apart from those that simply reflect developmental retardation (McKinnell et al, 2001; Williams et al, 2001b). The latter studies hypothesized that the estrogen induction of reproductive tract abnormalities in the neonatal male rat results from a disruption of the androgen-estrogen balance rather than from the absolute level of exposure to androgens or estrogens. If this is the case, high doses of estrogens could exert their effects by altering the androgen-estrogen balance (ie, by lowering androgen action and elevating estrogen action simultaneously) (McKinnell et al, 2001; Williams et al, 2001a,b).
Several pieces of evidence point to the importance of the androgen-estrogen balance in disorders such as gynecomastia (Braunstein, 1999; Mathur and Braunstein, 1997; Giwercman et al, 2000) and male infertility (Luboshitzky et al, 2002) in humans and ``clover'' disease in sheep (Bennett et al, 1946; Chamley et al, 1977). We have produced 2 pieces of experimental evidence to support the hypothesized importance of the androgen-estrogen balance in mediating DES-induced abnormalities in the developing male rat. First, we have shown that reproductive tract developmental abnormalities can be induced in neonatal rats by lowering androgen production (GnRHa) or action (flutamide) in combination with the administration of a dose of DES (0.1 µg) that is by itself incapable of inducing reproductive tract abnormalities (Rivas et al, 2002). Second, we have shown that the coadministration of testosterone (TE; 200 µg) with 10 µg of DES (DES + TE) appears to prevent at least some of the DES-induced morphological changes to the reproductive tract and to restore normal AR immunoexpression in the testis and epididymis (McKinnell et al, 2001). To expand on the latter observations, we have evaluated a wider range of reproductive tract abnormalities and have quantified several key endpoints (rete testis and efferent duct lumenal area and epithelial cell height of the efferent ducts and vas deferens) on day 18 in animals that have been treated neonatally with DES plus or minus TE. In addition, the same parameters have been evaluated in animals in which TE administration was delayed with respect to DES treatment, to establish whether a delayed TE treatment could reverse any of the abnormal changes in the reproductive tract induced by prior treatment with 10 µg of DES.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Rats from the various treatment groups were killed on day 18, a time at which DES-induced reproductive tract abnormalities are at their most prominent (Fisher et al, 1999; Atanassova et al, 2001; Williams et al, 2001a). Animals were anesthetized with flurothane, blood was collected by cardiac puncture, and the right testis was then dissected out, weighed, and fixed for ca 5 hours in Bouin fixative. The left testis was removed with the epididymis and proximal vas deferens still attached and was similarly fixed.
After fixation, tissue was transferred into 70% ethanol before being processed for 17.5 hours in an automated Leica TP1050 processor (Wetzler, Germany) and embedded in paraffin wax. 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 and/or image analysis as described below. All of the studies of the rete testis and reproductive tract described below used tissue sections of the left testis, which was fixed with the epididymis attached, so that minimal artifactual distortion was caused to the excurrent duct system.
Antibodies Used for Immunohistochemistry
Immunolocalization of the AR used a rabbit polyclonal antibody (Santa Cruz
Biotechnology Inc, Santa Cruz, Calif) raised against an epitope at the N
terminus of the human AR and was used at a dilution of 1:200. The estrogen
receptor alpha (ER-) was immunolocalized using a mouse monoclonal
antibody raised against a full-length human ER- recombinant protein
(Novocastra, Newcastle upon Tyne, United Kingdom) and was used at a dilution
of 1:20. The specificity of the AR and ER- antibodies has been detailed
in our previous studies (McKinnell et al,
2001; Saunders et al,
2001; Williams et al, 2001).
Immunohistochemistry
Unless otherwise stated, all incubations were performed at room
temperature. Sections were deparaffinized in Histoclear (National Diagnostics,
Hull, United Kingdom), rehydrated in graded ethanols, and washed in water. At
this stage, sections were subjected to a temperature-induced antigen-retrieval
step (Norton et al, 1994) in
0.01 M of citrate buffer, pH 6.0 (for the AR and ER-). After pressure
cooking for 5 minutes at full pressure, sections were left to stand,
undisturbed, for 20 minutes and were then cooled under running tap water
before being washed twice (5 minutes each) in Tris-buffered saline (TBS; 0.05
M of 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 (both from BDH Laboratory Supplies, Poole, Dorset, United Kingdom)
for 30 minutes, which was followed by two 5-minute washes in TBS. To block
nonspecific binding sites, sections were incubated for 30 minutes with the
appropriate normal serum diluted 1:5 in TBS containing 5% bovine serum albumin
(BSA; Sigma). For AR, normal swine serum (NSW) was used, and for ER-,
normal rabbit serum (NRS) was used (both from Scottish Antibody Production
Unit, Carluke, Scotland, United Kingdom). Primary antibodies were added to the
sections at the appropriate dilution in either NSW/TBS/BSA (for the AR) or
NRS/TBS/BSA (for ER-) and incubated overnight at 4°C in a
humidified chamber. After two 5-minute washes in TBS, sections were incubated
with a secondary antibody, namely a 1:500 dilution in the appropriate blocking
serum of biotinylated swine anti-rabbit immunoglobulin G (IgG; DAKO, High
Wycombe, United Kingdom) in the case of the AR or biotinylated rabbit
anti-mouse IgG (DAKO) for the ER. After 2 further 5-minute washes in TBS, all
sections were incubated for 30 minutes with avidin-biotin complex conjugated
to horseradish peroxidase (DAKO) diluted in 0.05 M of Tris-HCl, pH 7.4,
according to the manufacturer's instructions. Sections were washed twice (5
minutes each) in TBS, and immunostaining was developed using 0.05%
3,3'-diaminobenzidine (Sigma) in 0.05 M of Tris-HCl, pH 7.4, containing
0.01% (vol/vol) H2O2 until staining in positive control
tissues was optimal, at which time the reaction was stopped by immersing all
sections in distilled water. All sections were then lightly counterstained
with hematoxylin, dehydrated in graded ethanols, cleared in xylene, and
coverslipped using Pertex mounting medium (CellPath plc, Hemel Hempstead,
United Kingdom).
To ensure the reproducibility of findings, tissue sections from a minimum
of 3–6 animals in each treatment group were evaluated, and this was
performed on at least 2 separate occasions, with similar results obtained.
Further confirmation was obtained by undertaking immunohistochemistry with
tissue sections from control and treated animals on the same slide next to
each other. The specificity of immunostaining was checked for each antibody
using previously established procedures. This involved demonstrating that an
incubation of the primary antibody with either 10x wt/wt of the peptide
immunogen (AR: Santa Cruz peptide sc-816P) or with the respective recombinant
protein (ER-: McKinnell et al,
2001; Saunders et al,
2001; Williams et al, 2001) overnight at 4°C was able to block
immunostaining.
Immunostained sections were examined and photographed using a Provis microscope (Olympus Optical, London, United Kingdom) fitted with a Kodak DCS330 camera (Eastman Kodak, Rochester, NY). Captured images were stored on a G4 computer (Apple MacIntosh) and compiled using Photoshop 5.0 before being printed using an Epson Stylus 750 color printer (Seiko Epson Corp, Nagano, Japan).
Measurement of Rete Testis Lumenal Area (Rete Testis
Distension/Overgrowth)
The rete testis lumenal area was quantified as a measure of rete testis
distension/overgrowth (Rivas et al,
2002). Sections immunostained for ER- as described above
were used together with an Olympus BH2 microscope fitted with a 4x plan
achromat objective and a 3.3x phototube. The image was captured using an
xC77CE video camera (Sony, Tokyo, Japan) linked to a personal computer with a
frame grabber and Image Pro Image Analysis software (Media Cybernetics, Silver
Spring, Md). To ensure consistency regarding the cross section of the rete
testis that was measured, sections were chosen in which the region of the rete
testis draining into the efferent ducts could be viewed in the plane of
sections. Using the count/size tool, the area was measured by outlining the
edges of the rete lumens. The total rete area in the plane of sections was
determined in at least 3–6 animals in each treatment group.
Measurement of Efferent Duct Lumenal Area (Efferent Duct
Distension)
The efferent duct lumenal area was measured as an index of efferent duct
distension (Fisher et al, 1999;
McKinnell et al, 2001;
Rivas et al, 2002). Sections
immunostained for ER- as described above were used, and the
quantification method was similar to that described for the rete testis
lumenal area, except that a 20x plan achromat objective was used. Only
round, symmetrical efferent ductule cross sections were selected for
measurement to avoid errors due to the plane of sectioning of individual
ductules. Using the count/size tool, the edges of the lumen of individual
ductules were outlined, and the area was measured. For each animal, 10 cross
sections were measured, and a mean value per animal was then calculated.
Measurement of Epithelial Cell Height
To determine whether neonatal treatment altered the height of epithelial
cells in the efferent ducts and vas deferens, cross sections from 3–15
rats from control and treated cohorts were evaluated using image analysis.
Sections immunostained for ER- as described above were used. The height
of the epithelial cells within the efferent ducts and vas deferens was
measured using a 40x plan achromat and 20x objective for the
efferent ducts and vas deferens, respectively. Only round or oval cross
sections were selected for measurement. Using the length tool, the height of
the epithelium was measured by drawing a line at right angles to the base of
the cell adjacent from the basement membrane to the lumenal surface of the
cell. After measuring the length, the angle of the line was measured to ensure
that it was at 90 degrees. For each animal, at least 24 cells were measured,
with sampling from a number of different duct profiles, and the mean value was
then calculated for each animal.
Measurement of Plasma TE Levels
Plasma levels of TE were measured using an enzyme-linked immunosorbent
assay adapted (Rivas et al,
2002) from an earlier radioimmunoassay method
(Corker and Davidson, 1981).
The limit of detection was 12 pg/mL, and the intra-assay coefficient of
variation was less than 10%. All samples were assayed together in 1 run.
Statistics
A comparison of the different parameters for the various treatment groups
was made using analysis of variance after logarithmic transformation of the
data to obtain a normal distribution. When significant differences between
groups were indicated, subgroup comparisons also used analysis of variance but
used the variance from the experiment as a whole (for that parameter) as the
measure of error.
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Results |
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Efferent Duct Lumenal Area and Epithelial Cell Height
The efferent ducts showed lumenal dilation after neonatal treatment with 10
µg of DES, as reported previously
(Fisher et al, 1999; McKinnell et al, 2001;
Rivas et al, 2002). Coincident
with the increase in lumenal area, a significant decrease in epithelial cell
height occurred after DES treatment (Figures
1 and
3). TE treatment alone caused a
much smaller, but significant, increase in lumenal area and a small but
significant increase in epithelial cell height
(Figure 3). Efferent duct
lumenal area and epithelial cell height differed in their response to combined
DES and TE treatment. Epithelial cell height was restored to control levels in
DES plus TE–treated rats (Figures
1 and
3). However, in animals treated
with DES plus TE, a ninefold increase in lumenal area of the efferent ducts
was still observed. Nevertheless, this increase was significantly smaller than
that (13-fold) caused by DES administered alone (P < .001)
(Figure 3).
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Vas Deferens Epithelial Cell Height
Epithelial cell height in the proximal and distal vas deferens was reduced
by 63% and 80%, respectively, in rats treated with 10 µg of DES alone when
compared with controls (Figure
4). Treatment with TE alone caused a small but insignificant
increase in epithelial cell height in the vas deferens. Treatment with DES
plus TE was able to completely prevent the underdevelopment of the epithelium
in both the proximal and distal vas deferens when compared with animals
treated with DES alone (P < .001)
(Figure 4).
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Immunoexpression of ER-
Efferent Ducts—
As expected (Atanassova et al,
2001), the nuclei of epithelial cells in the efferent ducts showed
a constant and very intense immunoexpression of ER-. None of the
treatments induced any detectable change in ER- immunoexpression in the
efferent ducts (data not shown).
Vas Deferens—
As we reported previously (Atanassova et
al, 2001), immunoexpression of ER- in the day 18 control
animals was localized to a periductal stromal layer in the proximal and distal
vas deferens (Figure 5). In
contrast, neonatal treatment with 10 µg of DES induced epithelial
immunoexpression of ER- in the vas deferens, and stromal
immunoexpression was markedly reduced in intensity when compared with that in
controls and did not localize to the immediate periductal region
(Figure 5). Treatment with DES
plus TE prevented DES-induced immunoexpression of ER- in epithelial
cells of the vas deferens (Figure
5).
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Seminal Vesicles—
As reported previously (Williams et al,
2001a) in the day 18 control animals, ER- was weakly
immunoexpressed in a small number of stromal cells in the seminal vesicles.
Treatment with DES induced a marked increased in ER- immunoexpression
in stromal cells but not in epithelial cells
(Figure 5). Treatment with DES
plus TE suppressed the DES-induced increase in immunoexpression of ER-
in stromal tissues of the seminal vesicles
(Figure 5).
Immunoexpression of the AR
As reported previously (McKinnell et
al, 2001; Rivas et al,
2002), treatment with 10 µg of DES alone induced a marked
reduction in AR immunoexpression in all regions of the reproductive tract
(data not shown). Treatment with DES plus TE completely prevented this change
and restored the normal intensity of AR immunoexpression in all tissues
studied (data not shown).
Effect of Delayed TE Treatment on DES-Induced Reproductive Tract
Abnormalities
Another goal of the present study was to evaluate whether the delayed
administration of TE was able to reverse the DES-induced abnormal changes. By
day 8, when the TE was first administered, treatment with DES since day 2 had
already induced abnormalities in the reproductive tract such as rete testis
distension (Figure 1). Delayed
administration of TE was still effective in preventing the adverse changes in
epithelial cell height induced by treatment with DES alone in the efferent
ducts (Figure 3) and vas
deferens (Figure 4). Even
though TE levels in this treatment group were not significantly different from
those of controls (Figure 2),
rete testis distension/overgrowth was only partially prevented
(Figure 2). Additionally,
immunoexpression of ER- and AR was restored to normal in the same
tissues (data not shown). In contrast, the lumenal distension of the efferent
ducts induced by DES treatment was not prevented at all by delayed TE
treatment (Figure 3).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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in the vas deferens and seminal vesicles were
completely prevented by the coadministration of TE with DES and coincided with
the restoration of normal/supranormal TE levels and normal AR immunoexpression
in the same tissues (see also McKinnell et
al, 2001). In contrast, efferent duct lumenal distension was only
partially prevented by the coadministration of DES with TE. It therefore
appears that for potent estrogens to induce major reproductive tract
abnormalities, there must not only be an increase in estrogen action, there
must also be a coincident suppression of androgen action (reduced AR
expression and TE levels). These results strongly support our working
hypothesis that it is the disturbance of the balance between androgen and
estrogen action that determines abnormalities of reproductive tract
development, rather than the absolute levels of either hormone alone
(McKinnell et al, 2001; Rivas et al, 2002). Traditionally, hormones are thought to exert their effects in a strictly dose-dependent manner, such that increasing levels of hormone lead to an increasing magnitude of response until a maximal effect is achieved. We have recently (McKinnell et al, 2001; Rivas et al, 2002) used 2 opposing approaches that suggest that, as far as abnormalities of the developing male reproductive system are concerned, the androgen-estrogen balance may be more important than the absolute level of either hormone. First, we have shown (Rivas et al, 2002) that the administration of a low dose of DES (0.1 µg), which is largely ineffective on its own in inducing reproductive tract abnormalities, can induce a spectrum of reproductive tract abnormalities similar to a high dose of DES (10 µg) on its own if TE production (GnRHa administration) or action (flutamide administration) is suppressed at the same time. In contrast, the simple suppression of androgen production or action on its own is incapable of inducing the same abnormalities (McKinnell et al, 2001; Rivas et al, 2002). Second, we have demonstrated (McKinnell et al, 2001) that at least some of the adverse effects on the male reproductive tract induced by 10 µg of DES administered neonatally can be prevented by coadministration with TE, though this study did not quantify any of the relevant endpoints. Because we have now shown that a more rigorous approach is to quantify the different DES-induced abnormalities (rete testis, efferent ducts lumen area, and epithelial cell height of efferent ducts and vas deferens) (Rivas et al, 2002), we considered it important to apply similar quantification to tissues from animals treated with DES plus or minus TE.
Our results show that, with one exception (efferent duct lumenal area), all
endpoints are completely normalized by the coadministration of DES with TE.
Detailed quantification of the efferent duct lumenal area clearly showed that
the coadministration of DES with TE only partially ameliorated (about
ninefold) DES-induced lumenal distension (
13-fold), an observation not
made previously. This particular finding fits with the fact that the efferent
ducts are recognized to be a major site of fluid resorption and estrogen
action (high levels of immunoexpression of ER-) within the male
reproductive tract (Hess et al,
2001). Studies using ER- knockout mice
(Lee et al, 2000; Zhou et al, 2000;
Hess et al, 2001) have shown
that ER- is essential for fluid reabsorption and that, in the absence
of expression, the male is infertile. These findings, together with our
results, suggest that efferent duct fluid resorption is more sensitive to
estrogens than are other regions of the male reproductive tract and that
estrogen action per se, rather than the androgen-estrogen balance, may be the
deciding factor in determining the normality (or otherwise) of
development.
It can be argued that, in the present study involving the coadministration
of DES with TE from days 2–12, the dose of TE administered resulted in
supranormal levels of TE in vivo, which accelerated maturational development
of the reproductive tract, and that this ``protected'' it against the adverse
effects of high levels of estrogen. To test this possibility, we administered
DES without TE up to an age (day 8) when adverse effects of DES treatment
became evident. Treatment with DES plus TE from days 8–12 was still able
to restore to normal most of the parameters assessed (TE levels, efferent duct
lumenal area, efferent duct and vas deferens epithelial cell height, and
immunoexpression of the ER- and AR in the same tissues). In contrast,
rete testis overgrowth was only partially prevented by the delayed TE
treatment, and efferent duct distension was not prevented at all. However, the
latter endpoint was also only partially returned to normal, even when TE was
coadministered with DES from day 2, reinforcing the view that this parameter
reflects estrogen action rather than the androgen-estrogen balance. The
inability of the delayed TE treatment to completely prevent/reverse
DES-induced overgrowth of the rete testis is perhaps not surprising, as this
is a gross structural change that persists for life in animals treated with 10
µg of DES alone (Fisher et al,
1999). The ability of the delayed TE treatment to partially
prevent DES-induced rete overgrowth at day 18 probably reflects inhibition of
overgrowth between days 8 and 18, as a comparison of rete size at these 2 ages
in animals treated with DES alone showed that overgrowth was more pronounced
at day 18 than at day 8 (see Figure
1). Nevertheless, the ability of the delayed TE treatment to
reverse most of the DES-induced abnormalities by day 18 leads us to conclude
that this does not result in any simple way by exerting a ``protective
effect'' on the reproductive tract.
We have now accumulated substantial evidence (Williams et al, 2000, 2001a,b; Atanassova et al, 2001; McKinnell et al, 2001; Rivas et al, 2002; present study) demonstrating that major developmental abnormalities of the male reproductive tract associated with exposure to potent estrogens such as DES occur only when there is coincident suppression of androgen action. The latter results from suppression of TE levels (Sharpe et al, 2003), from Leydig cell development (Sharpe et al, 2003), and, most importantly, from loss of AR expression (McKinnell et al, 2001; Rivas et al, 2002). The present findings, which show that TE administration with consequent restoration of normal androgen action can block most of the effects of treatment with 10 µg of DES when administered alone, are thus straightforward to interpret in this context. We can see no other unifying explanation for these various findings other than it is the androgen-estrogen balance, rather than the absolute levels of either hormone, that is the deciding factor in determining whether or not abnormal reproductive tract development occurs after DES treatment.
Although our various findings emphasize that DES treatment has major
effects on androgen production and action, we have also shown that treatment
with 10 µg of DES can alter estrogen action by affecting the distribution
of ER-. Thus, treatment with 10 µg of DES can switch on aberrant
expression of ER- in the epithelium of the proximal vas deferens
(Atanassova et al, 2001) and
increase stromal immunoexpression of ER- in the seminal vesicles
(Williams et al, 2001a). Other
researchers have shown that neonatal estrogen treatment can also up-regulate
ER- expression in the neonatal rodent testis
(Sato et al, 1994) and
prostate (Prins and Birch,
1997). These changes, which enable increased estrogen action, are
associated with coincident down-regulation of AR expression in all the
affected tissues (Prins and Birch,
1995; McKinnell et al,
2001; Williams et al,
2001a,b)
and may thus contribute to distortion of the androgen-estrogen balance. The
present findings show that the coadministration of TE with DES is able to
block the induction of aberrant ER- immunoexpression in both the vas
deferens and the seminal vesicles. This change will presumably aid in
preventing an abnormal balance in androgen and estrogen action, but it also
demonstrates that androgens (or the androgen-estrogen balance) are able to
influence the pattern of ER- expression. A notable exception is,
however, the efferent ducts, in which intense ER- expression is
maintained irrespective of treatment or the androgen-estrogen balance
(Atanassova et al, 2001; McKinnell et al, 2001; present
study).
In summary, our results strongly suggest that distortion of the androgen-estrogen balance is the most important factor in determining whether abnormal reproductive tract development occurs in the male after DES treatment. This is supported by our ability to block all but one of the abnormalities induced by high-dose DES treatment by coadministration with TE. Only when there is concomitant alteration of androgen production/action are estrogens able to induce major adverse effects in the male reproductive tract. Remarkably, provided that androgen action is maintained (by TE treatment), even the administration of an extremely high dose of DES (10 µg per injection, equivalent to >300 µg/kg/d) to neonatal male rats is without significant effects on the developing reproductive tract, with the exception of fluid balance in the efferent ducts, which is probably a mainly estrogen-regulated phenomenon.
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Footnotes |
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