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Age-, Cell- and Region-Specific Immunoexpression of Estrogen Receptor (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

	Abstract

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This study in rats sought to 1) characterize immunoexpression of estrogen receptor (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.

	Introduction

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STABILIZATION OF THE Wolffian duct and its subsequent development and differentiation into the epididymis, vas deferens, and seminal vesicles are widely accepted as being driven by testicular androgens (1, 2). However, there are several pieces of evidence that also point toward a role for estrogens in at least some aspects of development of the duct system, particularly in its peripubertal/postnatal differentiation. For example, neonatal exposure of rodents to exogenous estrogens can result in varying degrees of atrophy/underdevelopment of the epididymis accompanied by changes in the relative amounts of epithelial and stromal tissue (3, 4, 5, 6). Epididymal abnormalities, including epididymal cysts, have also been found in adult human males who were exposed to diethylstilbestrol (DES) in utero (7, 8). More recently, it has been shown that the estrogen receptor (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.

	Materials and Methods

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Animals, treatments, sample collection, and processing
Wistar rats bred in our own animal house were maintained under standard conditions on a standard diet (rat and mouse breeding diet no. 3, SDS, Dundee, Scotland, UK) that contains 15.5% soy-meal. All-male litters of 8–12 pups were generated by cross-fostering pups on day 1 (day of birth). Beginning on postnatal day 2, rats were subjected to one of the following two treatments: 1) sc injection of DES (Sigma, Poole, UK) at a dose of 10 µg in 20 µl corn oil on days 2, 4, 6, 8, 10, and 12; or 2) sc injection of 20 µl corn oil alone (control). Rats from these two treatment groups were subsequently sampled on days 10, 18, 25, 35, and 90–105 (adults). At 1 h before death, rats were injected sc with 100 mg/kg bromodeoxyuridine (BrdU; Sigma) to label cells in the S phase of the cell cycle. Animals were anesthetized with flurothane, and the left testis was removed with the epididymis and proximal vas deferens still attached and fixed for 5.5 h in Bouin’s fixative. From some animals on day 18, the corpus and cauda epididymis from the right side were not fixed, but were used instead for protein extraction as described below. The corpus and cauda only were used to exclude possible contamination with efferent duct tissue.

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.

View this table: [in this window] [in a new window]   Table 1. Summary of age-and region-dependent changes in immunoexpression of ER1 in epithelial (EP) and stromal (STR) cells in the epididymis and vas deferens of control rats and animals treated neonatally with diethylstilbestrol (DES)

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.

	Results

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Delineation of the boundary region between the cauda epididymis and proximal vas deferens
To orientate the reader, the main region of focus in the present studies is shown in Fig. 1. 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.

View larger version (109K): [in this window] [in a new window]   Figure 1. Sagittal section through the cauda epididymis and proximal vas deferens of a day 18 control rat to highlight the regions on which the present studies are focused. Regions I–IV are referred to in later figures that describe region-specific changes in the immunoexpression of ER. Note that region II incorporates a gradual transition from the distal cauda epididymal duct to the proximal vas deferens. The box highlights an area in which the duct of the proximal cauda and the proximal vas deferens run side by side, and such a comparison is used in several of the later figures. The section has been immunostained for -actin to show smooth muscle (in black).

View larger version (125K): [in this window] [in a new window]   Figure 2. Effect of neonatal treatment of rats with DES on gross morphology of the cauda epididymis and vas deferens (A–H) and cell proliferation as indicated by incorporation of BrdU (brown staining; J–Q) on day 18. Sections in the two lefthand columns have been double immunostained for -actin (brown-red color) and for cytokeratins (blue color) to highlight smooth muscle and epithelial tissues, respectively. Note the changes in DES-treated rats: 1) underdevelopment of the epididymal duct (E vs. A); 2) coiling of the initial extraepididymal region of the vas deferens (F vs. B); 3) reduced epithelial height in the proximal vas deferens (C vs. G, and D vs. H); 4) enlargement of the periductal stromal layer (arrows), which is immunonegative for -smooth muscle actin and cytokeratin in the cauda epididymis and vas deferens (B–H); 5) reduced cell proliferation, as judged by BrdU incorporation at 18 days in the caput (J vs. N), corpus (K vs. O), and proximal cauda (L vs. P) as well as in the proximal vas deferens (M vs. Q). The inset in J shows a section in which BrdU antiserum was omitted. Scale bars, 500 µm (A, B, E, and F), 200 µm (C and G), or 100 µm (D, H, and J–Q).

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).

View larger version (57K): [in this window] [in a new window]   Figure 3. Specificity of the antisera used for immunolocalization of ER (left panel) and ERß (right panel), as determined by Western blotting. Note that the ER antibody detects recombinant ER, but not recombinant ERß (left panel), and that, correspondingly, the antibody to ERß detects recombinant ERß, but not recombinant ER (right panel). Both antisera detected a protein band corresponding in size to the respective full-length ER in protein extracts of the corpus and cauda epididymis from 18-day-old control rats (arrows). Figures in the middle indicate the positions of molecular mass markers (in kilodaltons).

Immunoexpression of ER

View larger version (126K): [in this window] [in a new window]   Figure 4. Region- and age-related changes in immunoexpression of ER in the epididymis and proximal vas deferens of rats during progression through puberty. Note at all ages the constant high immunoexpression of ER in nuclei of epithelial cells in the efferent ducts (A–D), whereas immunoexpression in the epididymis and proximal vas deferens varies with age and region. ER is not immunoexpressed in the caput (E), cauda (J), or proximal vas (J) on day 10, whereas on day 18, there is immunoexpression in epithelial cell nuclei in the caput (F), corpus (not shown), and proximal cauda (K), switching to stromal cell immunoexpression (arrows) in the proximal vas (K). By day 25 (G and L) and on day 35 (H and M) and at later ages (not shown), ER immunoexpression in epithelial cells of the epididymis is lost, but stromal cell immunoexpression (arrows) in the vas persists (L and M). The sections in the third row (J–M) correspond to the boxed region in Fig. 1. Clearer delineation of the region-specific pattern of ER immunoexpression (brown staining) on day 18 is shown in the bottom row in sections that have been double stained for -actin (blue) to show smooth muscle cells; arrows point to periductal stromal expression of ER. Insets in A–D and F show higher power views of the epithelium of individual duct cross-sections; the inset in E shows immunoexpression of ER after preabsorption of the primary antibody with recombinant ER. Scale bars, 100 µm, except in the insets in which they represent 20 µm.

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).

View this table: [in this window] [in a new window]   Table 2. Demarcation of tissue boundaries in the epididymis and vas deferens on d18, based on the cellular sites and intensity of immunoexpression of ER

View larger version (128K): [in this window] [in a new window]   Figure 5. Effect of neonatal treatment of rats with DES on immunoexpression of ER (A–H) and ERß (J–Q) on day 18 in the efferent ducts (top row), caput epididymis (second row), proximal cauda/proximal vas (third row), and the extraepididymal region of the vas deferens (bottom row). Sections shown in the third rowcorrespond to the boxed region in Fig. 1. Note that, unlike in controls (C and D), epithelial expression of ER in DES-treated rats extends into the proximal (G) and extra-epididymal (H) regions of the vas deferens; immunoexpression was unaffected in the efferent ducts (A vs. E), caput (B vs. F), and proximal cauda (C vs. G). Note also the reduction in height of the epithelium in DES-treated rats (red bars in D, H, and L–Q). Note that ERß immunoexpression occurs in epithelial and stromal cells in all regions and was unaffected by DES treatment (J–Q). Insets in B and K show negative controls in which the primary antibody was preabsorbed with the appropriate recombinant protein. Asterisks indicate lumenal distension of the efferent (E and N) and caput epididymal (F and O) duct in DES-treated animals. Scale bars, 50 µm.

Effect of neonatal administration of DES on immunoexpression of ER and ERß

Immunoexpression of ER.

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.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our findings demonstrate that ER 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.

View this table: [in this window] [in a new window]   Table 11. Summary of age- and region-dependent changes in immunoexpression of ER in epithelial (EP) and stromal (STR) cells in the epididymis and vas deferens of control rats and animals treated neonatally with diethylstilbestrol (DES)

View this table: [in this window] [in a new window]   Table 21. Demarcation of tissue boundaries in the epididymis and vas deferens on day 18, based on the cellular sites and intensity of immunoexpression of ER

	Acknowledgments

	Footnotes

 
¹ This work was supported in part by the European Center for the Ecotoxicology of Chemicals and by a Strategic Research Fund Award from AstraZeneca plc.

² Recipient of a Royal Society/NATO fellowship.

Received August 16, 2000.

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