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Endocrinology Vol. 147, No. 10 4820-4830
Copyright © 2006 by The Endocrine Society

Androgen-Dependent Mechanisms of Wolffian Duct Development and Their Perturbation by Flutamide

Michelle Welsh, Philippa T. K. Saunders, Nicholas I. Marchetti and Richard M. Sharpe

Medical Research Council Human Reproductive Sciences Unit, Centre for Reproductive Biology, The Queen’s Medical Research Institute, Edinburgh EH16 4TJ, Scotland, United Kingdom

Address all correspondence and requests for reprints to: Michelle Welsh, MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, The Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, Scotland, United Kingdom. Email: m.welsh{at}hrsu.mrc.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Androgens play a vital role in Wolffian duct (WD) development, but the mechanisms that underlie this are unknown. The present study used in utero exposure of pregnant rats to the androgen receptor antagonist flutamide (50 or 100 mg/kg) to explore possible mechanisms. Pregnant rats were treated from embryonic d 15.5 (E15.5), and WDs were isolated from fetuses from E17.5–E21.5 and from adults. WD morphology was evaluated, and total length of the duct lumen was determined in fetal samples. Fetal WDs were immunostained for androgen receptor and stromal (inner and outer) and/or epithelial-cell-specific markers and analyzed for cell proliferation and apoptosis. In adulthood, most flutamide-exposed males lacked proximal WD-derived tissues, whereas at E18.5–E19.5, a time when the WD has completely regressed in females, a complete normal WD was present in all flutamide-exposed animals. This suggests that flutamide, at doses of 50 or 100 mg/kg, interferes with WD differentiation, not stabilization. Consistent with this, WD elongation/coiling increased in controls by 204% between E19.5 and E21.5 but increased less significantly (103%) in flutamide-exposed animals. This was associated with reduced cell proliferation, but there was no increase in apoptosis or change in expression of androgen receptor mRNA or protein. Flutamide treatment impaired differentiation of inner stromal cells, shown by decreased expression of smooth muscle actin, before effects were noted in the epithelium, consistent with androgens driving WD development via stromal-epithelial interactions. In conclusion, WD differentiation is far more susceptible to blockade of androgen action than is its initial stabilization, and these effects may be mediated by disruption of stromal-epithelial interactions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DURING MAMMALIAN DEVELOPMENT, the urogenital tract is identical in both sexes and is made up of two duct systems, the Müllerian duct and the Wolffian duct (WD) (1, 2). In males, Sertoli cells secrete anti-Müllerian hormone, which causes the Müllerian duct to degenerate, whereas Leydig cells secrete testosterone, which stabilizes and rescues the WD (2, 3). In females, the lack of androgens prevents stabilization of the WD, resulting in its degeneration (3). Once stabilized, the male WD begins to differentiate to form its adult derivatives; the cranial portion of the WD convolutes to form the adult epididymis, the central portion remains a relatively simple straight duct and forms the vas deferens, whereas the seminal vesicles bud off the distal segment (4, 5).

It is widely accepted that WD development is under the control of testosterone (2, 6), which is synthesized by the Leydig cells of the testis (7) and is delivered directly from the testis down the lumen of the WD (8). Testosterone secretion begins at around E15.5 in the fetal rat with a peak at E19.5 (9). In some cells, testosterone can be metabolized by 5{alpha}-reductase to form the more potent androgen dihydrotestosterone (DHT) or aromatized to form estrogens (2). However, it is unlikely that either of these hormones are involved in WD development because aromatase could not be detected in the fetal WD (unpublished findings) and estrogen receptor knockout mice and aromatase knockout mice have epididymides present in adulthood (10, 11). Furthermore, DHT has not been detected in the WD until after epididymal differentiation is complete (12, 13), and 5{alpha}-reductase-deficient patients show normal WD differentiation (14), whereas rats exposed to finasteride, a 5{alpha}-reductase inhibitor, show normal WD development (15).

Androgen action is mediated via the androgen receptor (AR), which is a member of the superfamily of ligand-activated steroid hormone receptors (16). The AR binds both testosterone and its metabolite DHT with high specificity and affinity; however, DHT dissociates less easily from the AR and is thus more effective at stabilizing the receptor in its active conformation (2, 17). Patients with mutations in the AR affecting its activity and/or expression exhibit a range of phenotypic abnormalities (18). In the case of complete androgen insensitivity, genetic XY males are born with a female phenotype, intraabdominal testes, no prostate, and a lack of WD-derived tissues (19, 20, 21).

Simple columnar epithelial cells line the lumen of the WD with stromal cells surrounding this epithelium. The AR is first expressed in the rat WD stroma at E16.5 and at low levels in the epithelial cells by E17.5 (22, 23). Studies in both male and female reproductive tracts show that mesenchymal cells can determine the morphological fate of the overlying epithelium, possibly via local production of growth factors (24, 25, 26, 27). It has been proposed that stromal cells are the primary target for androgen action and that testosterone may induce proliferation and differentiation of epithelial cells through stromal-epithelial interactions (24, 28).

Administration of the AR antagonist flutamide during pregnancy has been shown to dose-dependently impair masculinization. For example, treatment with 18 mg/kg flutamide resulted in complete feminization of external genitalia but normal WD differentiation (15). However, at doses above 100 mg/kg flutamide, the vas deferens was absent unilaterally or bilaterally, and only small remnants of the epididymis were present in adults (15). It is generally presumed that the latter effect results from interference with WD stabilization but this has not been shown directly during fetal life.

Although it is obvious that androgens play a role in WD development, the molecular and cellular events that underlie its stabilization and subsequent differentiation have received little attention to date. To address this, we have used a model system in which WD development can be altered, thus enabling the investigation of WD stabilization and differentiation with the aim of elucidating the cellular mechanisms responsible for androgen-dependent WD development. This is the first report to have taken this approach, and it was used to address the following specific questions. 1) Does flutamide-induced blockade of androgen action affect stabilization of the male WD, as occurs naturally in females, and/or its differentiation? 2) Do androgens control WD development by altering cell proliferation and/or apoptosis, and does this preferentially target one cell compartment? 3) Does altered expression of AR play a role in flutamide-induced inhibition of WD development? 4) Are the effects of flutamide treatment first detectable in the stromal compartment, consistent with the view that epithelial development is mediated by androgen effects on the stroma?


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Wistar rats were bred and maintained in our own animal house under standard conditions according to United Kingdom Home Office guidelines. Animals had access ad libitum to water and a soy-free breeding diet (SDS, Dundee, UK). Time matings were set up, and the presence of a vaginal plug was taken as evidence of mating; this was defined as embryonic d 0.5 (E0.5).

In vivo treatment
Pregnant dams (n = 70) were randomly allocated to treatment groups and dosed daily by oral gavage between 0830 and 1000 h with flutamide (Sigma, Poole, UK) at 50 mg/kg (n = 30 dams) and 100 mg/kg (n = 18) in 1 ml/kg corn oil/2.5% dimethylsulfoxide (Sigma) or vehicle alone (n = 22). Dosing was undertaken from E15.5, the time at which the fetal testis begins androgen synthesis, until the day before cull. Flutamide doses were selected based on results previously reported (15). Dams were weighed daily and checked for signs of toxicity.

Dams were killed by inhalation of carbon dioxide and subsequent cervical dislocation. Fetuses were recovered at E17.5–E21.5, decapitated, and placed in ice-cold PBS (Sigma). WDs were collected from male fetuses by microdissection, examined with a Leica MZ6 dissecting microscope (Leica Microsystems UK Ltd., Milton Keynes, UK) and photographed using a Leica ICA camera. For each fetus, one WD was snap frozen in liquid nitrogen, whereas the other was fixed in Bouin’s for 1 h, transferred into 70% ethanol, and processed for 17.5 h in an automated Leica TP1050 processor. Fixed WDs were embedded horizontally in paraffin wax and processed and sectioned onto coated slides using standard procedures. Representative WDs from the aforementioned litters were subsequently used for the studies detailed below; at least three animals from at least three litters were studied per age/treatment group. Only samples that were analyzable were studied; for example, WDs from flutamide-exposed animals with incomplete lumens (7% of animals at E21.5) were not included in the analysis of luminal length. Thus, results presented for luminal length are likely to underestimate the overall effect of flutamide exposure.

Gross morphology and histological analysis
Reproductive tracts from control and flutamide-exposed males and control females were analyzed microscopically, at the time of dissection, for any gross morphological abnormalities. Considerable variation was seen in the severity of malformation of the WDs after flutamide exposure; therefore, careful note was taken of any macroscopic abnormalities. Reproductive tracts from control female fetuses were also recovered, and the degree of regression of the WD was noted. Gross histological analysis was performed on WD sections stained with hematoxylin and eosin, using standard protocols.

WD luminal length measurement
Differentiation of WDs was quantified by measuring the luminal length of the epididymal segment of WDs from control and treated animals; a line was digitally drawn through the lumen of the WD image taken at the time of dissection using the Image J program (National Institutes of Health, Bethesda, MD). This line was drawn from the head of the epididymis, where the efferent ducts end, to the tail of the epididymal section just before the start of the vas deferens. To ensure reproducibility and to correct for individual variation, luminal length was measured for WDs from 15–37 animals from at least three different litters per treatment group.

Immunohistochemistry
Immunohistochemistry was performed on WDs recovered from control and treated fetuses at E17.5–E21.5 using standard avidin peroxidase protocols (see Ref. (29). For AR, pan-cytokeratin, and cleaved caspase 3, antigen retrieval was performed using 0.01 M citrate buffer (pH 6.0). Sections were pressure cooked for 5 min at full pressure, left to stand for 20 min, and then cooled under running water. Endogenous peroxidase activity was blocked by washing sections in 3% H2O2 in methanol for 30 min at room temperature. All washes comprised two 5-min washes at room temperature in Tris-buffered saline (TBS) (0.05 M Tris-HCl, pH 7.4, and 0.85% NaCl). Nonspecific binding sites were blocked by incubating sections in normal goat serum (NGS) (Autogen Bioclear UK Ltd., Wiltshire, UK) diluted 1:4 in TBS containing 5% BSA (Sigma). Sections were incubated overnight at 4 C with primary antibodies diluted in NGS/TBS/BSA: AR at 1:50 (Santa Cruz Biotechnology, Santa Cruz, CA), smooth muscle actin (SMA, Sigma) at 1:10,000, pan-cytokeratin (Sigma) at 1:200, and cleaved capsase 3 (Cell Signaling Technology, Beverly, MA) at 1:200. Control sections were incubated with blocking peptide when available or blocking serum alone to confirm antibody specificity. Sections were incubated with the appropriate secondary antibody, either biotinylated goat antirabbit or biotinylated goat antimouse, diluted 1:500 in NGS/TBS/BSA for 30 min at room temperature before incubation for 30 min with avidin-biotin conjugated with peroxidase diluted in 0.05 M Tris-HCl (pH 7.4) according to the manufacturer’s instructions (ABC-HRP; Dako, Ely, UK). Antibody localization was determined using 3,3'-diaminobenzidine (liquid DAB+; Dako) until staining was optimally detected in control sections; the reaction was stopped by immersing the sections in distilled water. Sections were counterstained in Harris’s hematoxylin and mounted using Pertex (Cell Path, Hemel Hempstead, UK). Cellular sites of expression of AR, SMA, pan-cytokeratin, and cleaved caspase 3 were determined and slides photographed using a Provis AX70 (Olympus Optical, London, UK) microscope fitted with a Canon DS6031 camera (Canon Europe, Amsterdam, The Netherlands).

Fluorescence immunohistochemistry
To delineate stromal and epithelial compartments, fluorescence immunohistochemistry was used to colocalize three proteins in WDs recovered from control and treated fetuses at E17.5–E21.5. Antigen retrieval was performed as detailed above. All washes were two 5-min washes at room temperature in PBS (Sigma). At each stage, control sections were incubated with blocking serum without antibody to confirm antibody specificity. Nonspecific binding sites were blocked by incubating sections in NGS diluted 1:4 in PBS containing 5% BSA (Sigma). Sections were incubated overnight at 4 C with anti-pan-cytokeratin antibody (Sigma) diluted 1:200 in NGS/PBS/BSA and was detected by incubating sections for 1 h with goat antimouse secondary antibody directly conjugated with Alexa Fluor 488 (Molecular Probes, Poort Gebouw, The Netherlands) diluted 1:200 in PBS to produce green fluorescence. Sections were incubated for 30 min with mouse IgG (Sigma) diluted 1:2000 in NGS/PBS/BSA to block any remaining mouse IgG sites and prevent subsequent nonspecific binding. Nonspecific binding sites were blocked again by incubating sections with NGS/PBS/BSA for 30 min before incubating overnight at 4 C with anti-AR (Santa Cruz Biotechnology) diluted 1:50 in NGS/PBS/BSA. AR immunostaining was detected using biotinylated goat antirabbit IgG secondary antibody (Dako) diluted 1:500 in NGS/PBS/BSA for 30 min followed by incubation for 1 h with streptavidin-conjugated Alexa 546 (Molecular Probes), producing red fluorescence. Nonspecific binding sites were blocked again with NGS/PBS/BSA for 30 min before incubating overnight at 4 C with anti-SMA antibody (Sigma) diluted 1:500 in NGS/PBS/BSA. Sections were incubated for 1 h with goat antimouse labeled with Cy5 (Amersham Biosciences, Little Chalfont, UK) diluted 1:60 in PBS, producing blue fluorescence. Sections were counterstained for 10 min with a nuclear-specific blue fluorescent label, 4',6-diamidino-2-phenylindole (Sigma), diluted 1:1000 in PBS and then mounted in Mowiol mounting medium (Calbiochem, Lutterworth, UK). Fluorescent images were captured using a Zeiss LSM 510 Meta Axiovert 100M confocal microscope (Carl Zeiss Ltd., Welwyn Garden City, UK).

To ensure reproducibility of results and allow accurate comparison of immunostaining between treatment groups, sections of WDs from control and treated animals were processed in parallel on at least three occasions; sections of WDs from at least three animals in each treatment group were run on each occasion. Appropriate negative controls were included, whereby the primary antibody was replaced by blocking peptide or NGS alone, to ensure staining observed was specific; none of the antibodies used showed other than minor nonspecific staining.

Apoptosis analysis
Cleaved caspase 3 immunostaining was performed on WDs from control and flutamide-exposed fetuses using standard methods, as detailed above. Very few cleaved caspase-3-positive cells were detected; therefore, a detailed stereological analysis was not appropriate and all positive cells were manually counted in each WD using an Axiolab microscope (Carl Zeiss Ltd.).

Frequency of cell mitoses in WDs
To determine whether cell proliferation in WD compartments was affected by flutamide treatment, various cell cycle markers and analytical methods were investigated. For technical reasons, it was considered that determination of the proliferation index was impractical for the densely packed stromal cell compartment. Instead, a method was devised to allow enumeration of the total number of mitotic cells in each compartment, as outlined below.

WD sections from control and flutamide-exposed fetuses at E19.5–E21.5 were immunostained for phospho-histone H3 (Upstate Biotechnology, Dundee, UK) using a Bond-X automated immunostaining machine (Vision Biosystems, Newcastle, UK) and a polymer high-contrast program. Briefly, after high-pressure antigen retrieval, slides were peroxidase blocked for 5 min and incubated for 2 h with the primary antibody diluted 1:1000 in the diluent supplied and then with the post-primary reagent for 15 min. Control sections were incubated with diluent alone to confirm antibody specificity. Sections were then incubated with the polymer reagent for 15 min to increase sensitivity of detection before DAB detection for 10 min, counterstained in hematoxylin for 5 min, dehydrated, and mounted as detailed above.

Phospho-histone H3-positive cells were counted in the epithelial compartment and the inner and outer stromal layers of the WD using the x20 objective on an Olympus BH-2 microscope fitted with a Prior automatic stage (Prior Scientific Instruments Ltd., Cambridge, UK). Image-Pro Plus version 4.5.1 with Stereologer-Pro 5 plug-in software (Media Cybernetics UK, Wokingham, UK) was used for analysis. Positive cells were counted only in the future epididymal portion of the WD, not in the efferent ducts or vas deferens. Because differences were noted in the degree of coiling along the length of the future epididymal portion of the WD, phospho-histone H3-positive cells were initially counted in each region of the future epididymis individually, defined as in the adult epididymis as caput, corpus, and cauda (5) (Fig. 1DGo). However, no consistent difference was seen in phospho-histone H3 staining between the different regions of the epididymal portion of the WD; therefore, proliferation was subsequently analyzed in the epididymal portion of the WD as a whole.


Figure 1
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  		FIG. 1. Age-dependent increase in coiling/elongation of the WD between E18.5 and E21.5. Representative WDs recovered from control fetuses (A and D) and fetuses exposed to 50 mg/kg flutamide (B and E) or 100 mg/kg flutamide (C and F) at E18.5 (top) and E21.5 (bottom) are illustrated. Note the reduced coiling in WDs from flutamide-exposed animals at E21.5 (E and F) when compared with the age-matched control (D). Note the demarcation of the epididymal regions of a control WD at E21.5 (D) into the caput (Cap), corpus (Cor), and cauda (Cau), as in the adult epididymis. Scale bar, 1 mm. E, Prospective epididymis; ED, efferent ducts; V, prospective vas deferens. The graph (G) illustrates quantification of coiling (luminal length) of the WDs. Note the progressive increase in luminal length of WDs from controls between E18.5 and E21.5 (black bars). In WDs from animals exposed in utero to either 50 mg/kg (white bars) or 100 mg/kg (striped bars) flutamide, there was a significant reduction in luminal length at E20.5 and E21.5 when compared with controls, whereas at E18.5 and E19.5, no difference was evident. Values are mean ± SEM for 15–36 animals per group. ***, P < 0.001 in comparison with respective value for preceding day; a, P < 0.001 in comparison with respective control value.

 
Variation was noted in the length of the WD visible in each section as well as the amount of epithelium visible per section; this was a result of both treatment effects and the plane of sectioning. To correct for this variation, the overall length of the WD and the length of epithelium visible in each section were measured. The number of phospho-histone H3-positive cells in the epithelial compartment was then divided by the total length of epithelium visible in the section to calculate proliferation per micron of epithelium. Because flutamide exposure reduces coiling and WD luminal length, it was important to take this into account when analyzing cell proliferation. The number of positive epithelial cells per micron of epithelium was therefore multiplied by the luminal length for that specific WD (calculated at the time of dissection, method detailed above) to calculate total epithelial proliferation in the whole epididymal portion of the WD, not just cell proliferation in the section being analyzed. Similarly, the number of phospho-histone H3-positive cells in the stromal compartment was divided by the length of WD epithelium visible in the section to calculate stromal proliferation per micron of stroma and hence correct for variation in the length of the WD. This was then multiplied by the luminal length of the WD to calculate total stromal proliferation in the whole epididymal portion of the WD. Sections from eight to 14 fetuses from three to five separate litters were analyzed from both control and flutamide-exposed animals at each age. To confirm that this analysis was likely to reflect the cell proliferation/mitotic index, the total number of phospho-histone H3-positive and negative epithelial cells were counted (500–700 cells) in three control and three F100 E21.5 WDs, and an epithelial mitotic index was derived (percent phospho-histone H3-positive cells/total number of cells x 100%).

Epithelial cell height measurement
WD sections from six control fetuses and six fetuses from each of the flutamide treatment groups at E21.5 were randomly selected and immunostained for pan-cytokeratin as detailed above to clearly label all epithelial cells. Using a x63 objective, epithelial cell height was measured in every fifth epithelial cell per section, using the software and stereological equipment noted above. This was performed separately for the caput, corpus, and cauda regions of the future epididymal portion of the WD. Only epithelial cells in which the nucleus could be clearly identified were measured, thus excluding from analysis any epithelial cells from the flutamide treatment groups that were severely flattened or disintegrating. In a subset of these animals, epithelial cell width was also measured using the same method.

Quantitative RT-PCR
RNA was isolated from frozen WDs using the RNeasy Mini extraction kit (QIAGEN, Crawley, UK) according to the manufacturer’s instructions. RNA was DNase treated during extraction using RNase-free DNase on the column digestion kit (QIAGEN), and random hexamer primed cDNA was prepared using the Applied Biosystems TaqMan RT kit (Applied Biosystems, Foster City, CA). Real-time PCR was performed with the ABI Prism 7900 Sequence Detection System (Applied Biosystems). Expression of AR mRNA was determined using the Assay-On-Demand Gene Expression system for rat AR (Rn00560747_m1; Applied Biosystems). The expression level of AR mRNA was corrected using an internal control, 18S rRNA, and related to rat uterus expression levels. Results shown are the mean of a minimum of three WDs per treatment group performed in triplicate on at least two occasions.

Western blot analysis
Protein was harvested from frozen WDs using RIPA [1% Triton X-100, 15 mM HEPES-NaOH (pH 7.5), 0.15 mm NaCl, 1% sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, 10 mM EDTA, and 0.5% protease inhibitor cocktail (Sigma)] lysis buffer. WDs were homogenized in 75 µl RIPA buffer and then incubated on ice for 1 h. Samples were centrifuged at 2500 rpm for 10 min and the supernatant collected. The protein concentration was determined using a Bio-Rad BCA protein assay kit according to the manufacturer’s instructions (Bio-Rad Laboratories, Hemel Hempstead, UK). Proteins were denatured by boiling with SDS loading buffer for 5 min before loading 20 µg of each protein extract onto a 7.5% polyacrylamide gel. Gels were subjected to electrophoresis at 100 V under reducing conditions for 2 h. The proteins were electrotransferred overnight at 20 V onto nitrocellulose membranes (Immobilon-P; Millipore, Bedford, MA). Membranes were washed in TBS containing 0.1% Tween 20 (TBST) (Sigma) before incubating in TBST with 5% milk for 1 h at room temperature to block nonspecific binding sites. Membranes were washed twice in TBST for 5 min and then incubated overnight at 4 C with anti-AR antibody (Santa Cruz Biotechnology) diluted 1:200 in TBST, anti-SMA antibody (Sigma) diluted 1:10,000, or an anti-ß-tubulin antibody (Sigma) as a standardization control at 1:300 in TBST. After washing with TBST, membranes were incubated for 1 h at room temperature with antirabbit horseradish peroxidase or antimouse horseradish peroxidase (SAPU, Carluke, UK), respectively, at 1:5000 in TBST/5% milk. The signal was detected using ECL plus Western blot detection reagents according to the manufacturer’s instructions (Amersham Biosciences). Signals were visualized using high-performance chemiluminescence imaging film (Amersham Biosciences) and developed using a Xograph compact x4 imaging system (Xograph, Tetbury, UK). Antibody specificity was confirmed by the detection of only one band at the expected size when visualizing each antibody. The intensity of the bands was quantified using the Typhoon 9400 variable mode imager (Amersham Biosciences). The expression level of protein was corrected for loading using ß-tubulin and related to E19.5 control protein levels. To ensure reproducibility of results, the Western blot was performed three to six times, and protein was isolated from at least three WDs from three different litters each time.

Statistical analysis
Values have been expressed as means ± SEM. Data were analyzed using one-way ANOVA followed by the Bonferroni post hoc test, using GraphPad Prism version 4 (Graph Pad Software Inc., San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Normal development of the WD
Figure 1Go shows images of WDs isolated at E18.5 and E21.5. At E18.5 (Fig. 1AGo), the epididymal portion of the WD is a simple, straight uncoiled duct. Coiling first becomes evident at the cranial and caudal ends of the future epididymal portion of the WD at E20.5, whereas the corpus remains relatively uncoiled (not shown). By E21.5, the full length of the epididymal portion of the WD appears highly convoluted, including the corpus (Fig. 1DGo). This age-dependent increase in coiling and development was confirmed quantitatively by the demonstration of an increase in luminal length (Fig. 1GGo). No significant increase (P > 0.05) in WD luminal length was seen between E18.5 and E19.5, but a highly significant increase was noted on consecutive days thereafter.

Impact of flutamide treatment on gross WD morphology
Consistent with previous studies (30) that have blocked androgen action in utero, considerable variation between animals was noted in the degree of WD coiling after exposure to flutamide; this was more apparent at later fetal ages (E20.5–E21.5) when WDs are more differentiated in fetuses from control mothers. To take account of this variability, WDs were analyzed from 19–37 animals from at least three litters per age/treatment group.

As in control WDs, an age-dependent increase was noted in the degree of coiling and hence differentiation of WDs from flutamide-exposed fetuses, but the degree of coiling was reduced compared with age-matched controls (Fig. 1Go). As in controls, no coiling was seen in WDs from flutamide-exposed animals at E18.5 (Fig. 1Go, B and C). At E20.5 and E21.5, but not at E18.5 or E19.5, flutamide-exposed animals showed a highly significant reduction in WD coiling and luminal length compared with controls (Fig. 1GGo). At E21.5, coiling was more dramatically reduced in fetuses exposed to 100 mg/kg flutamide than those exposed to 50 mg/kg (Fig. 1Go, F compared with E, respectively). As well as this reduction in coiling, some WDs from flutamide-exposed fetuses appeared incomplete at E21.5 with thinning of the epithelium and missing corpus segments; this was not seen in control WDs. Missing corpus segments were first noted at E21.5 whereby 5 and 11% of WDs recovered from animals exposed to low- and high-dose flutamide, respectively, had incomplete lumens (Table 1Go). These fetuses were from the same litter within each treatment group. This was not seen in WDs at E18.5–E20.5 from flutamide-exposed animals. This is in contrast to the natural regression of WDs in females, in which by E18.5 the WD has fully regressed and is no longer visible (Fig. 2BGo).


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  		TABLE 1. Number of incomplete WD in control and flutamide-exposed animals

 

Figure 2
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  		FIG. 2. Natural regression of the WD in female rats between E17.5 (A) and E18.5 (B) in comparison with the effect of flutamide (100 mg/kg) treatment on WDs in the male (C). Note the presence of both the Müllerian duct (arrow) and WD (arrowhead) in the female at E17.5 (A), whereas the WD has regressed by E18.5 (B). In comparison, in the flutamide-exposed male at E18.5 (C), the WD is still present and is comparable to its age-matched control (see Fig. 1AGo). Scale bar, 1 mm.

 
Impact of fetal exposure to flutamide on male reproductive tract morphology
Exposure to either dose of flutamide prevented normal masculinization of the fetal external genitalia, with anogenital distance reduced in E21.5 flutamide-exposed males to a length similar to that of control females (data not shown). Flutamide-exposed E21.5 males had no prostate and a vaginal pouch was present, but testis descent was largely unaffected (data not shown). Consistent with previous reports (31, 32, 33), these abnormalities were still evident postnatally. Considerable variation was noted in the degree of abnormality of WD-derived adult tissues, including underdeveloped or incomplete epididymides, lack of vas deferens, and underdeveloped or vestigial seminal vesicles (data not shown). As in the fetus, the corpus segment of the epididymis was often absent with 83% of epididymides studied being incomplete (Table 1Go). In five animals, there was near-complete absence of all epididymal segments and the vas deferens.

Histology
The WD is composed of two cellular compartments, simple columnar epithelial cells lining the lumen of the duct and stromal cells surrounding this epithelium, and this did not vary along the length of the WD or with age. The mesenchymal cells can be easily separated into two distinct populations, those directly surrounding the epithelial cells, which are more densely packed and termed here as inner stroma, and those that make up the rest of the body of the WD, referred to here as outer stoma. This can easily be seen by hematoxylin and eosin staining (Fig. 3AGo), but this distinction was confirmed by staining for SMA, which is expressed only in the inner and not the outer stromal cells of the WD (Fig. 3BGo). SMA protein expression switched on in these cells between E17.5 and E18.5 and increased dramatically between E18.5 and E19.5 (data not shown).


Figure 3
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  		FIG. 3. Demarcation of the E21.5 WD stroma into the inner and outer stromal compartments (IS and OS, respectively). Note that the IS can be identified as the more dense compartment immediately proximal to the epithelium (arrow; A). This regionalization was confirmed by immunostaining for SMA, which is expressed only in the inner stroma (brown; B). Scale bar, 100 µm.

 
Examination of WDs revealed no obvious histological differences between WDs from control and flutamide-exposed fetuses at E18.5 or E19.5 (data not shown). However, at E21.5 after flutamide exposure (high and low dose), the WD epithelium often looked abnormal compared with that of controls, appearing more flattened and sometimes fibroblast-like. The extent of this flattening varied between WDs but was not seen in controls or flutamide-exposed WDs at any earlier age.

AR expression
AR was immunolocalized to cell nuclei throughout the WD at all ages examined (E17.5–E21.5). Stromal cells directly surrounding the epithelium showed the most intense immunostaining, whereas the epithelial cells showed weaker staining (Fig. 4Go). WDs from flutamide-exposed animals displayed a similar pattern of AR expression to controls at all ages studied with AR expressed in the stroma by E16.5 and epithelial expression switching on in some cells by E17.5 (data not shown). Additional analysis using Western blotting and TaqMan quantitative real-time PCR confirmed that there was no significant difference in either AR protein or mRNA expression in control compared with flutamide-exposed WDs at E17.5–E21.5 (Fig. 4Go, F and G). Variation was seen in the levels of AR mRNA and protein within each age/treatment; however, this was not significant and presumably represents variation between individual WDs.


Figure 4
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  		FIG. 4. AR expression in the different compartments of the WD caput at E21.5. Note that the inner stroma is identified by SMA immunostaining (blue) and the epithelium by cytokeratin staining (green). No obvious difference was seen in AR expression between WDs from control animals (A) and animals exposed to 50 mg/kg (B) or 100 mg/kg (C) flutamide. Image D shows AR only staining from Fig. 5AGo, highlighting that AR protein was expressed (red) more intensely in the stroma than in the epithelium (arrows) of the control WD at E21.5 (D), and flutamide treatment had no obvious effect on this pattern (B and C). Scale bar, 50 µm. E, Representative Western blot for AR protein expression in WDs at E17.5–E21.5 (n = 3 WDs per age/treatment) with its loading control below. F, Quantitative analysis (mean ± SEM) of AR protein from Western blots confirming no significant change in AR expression in WDs at E17.5–E21.5 from control (black bars) and flutamide-exposed (white bars, 50 mg/kg; striped bars, 100 mg/kg) rats (n = 6 Western blots, each using a pool of three WDs per age/treatment from at least three litters). Protein expression was corrected for loading and expressed relative to levels in E19.5 control WD. G, No significant difference is shown in AR mRNA expression between control and flutamide-exposed WD at E18.5–E21.5, as determined by TaqMan quantitative real-time PCR (means ± SEM; n = 3–5 WDs per age/treament, each from different litters).

 
Role for apoptosis in flutamide-induced impairment of WD development
Apoptotic cells were rarely detected (typically one cell per section) in WDs from either control or flutamide-exposed fetuses at any age studied (data not shown).

Frequency of cell mitoses in WDs
Cell mitosis was evident in all compartments of WDs from control and flutamide-exposed animals at all ages (Fig. 5Go, A and B). Immunostaining for phospho-histone H3 suggested fewer mitotic cells were present in WDs from flutamide-exposed fetuses compared with controls, and this was confirmed by quantitation that showed a significant reduction in numbers of mitotic epithelial and stromal cells per WD compared with controls (Fig. 5Go). This reduction was seen at all ages studied; however, results are presented here only for E21.5. Cell proliferation data are expressed as the overall number of mitotic cells in the complete epididymal portion of the WD rather than per 100 µm epithelium so as to take into account the treatment-induced reduction in WD length. If this correction was not applied, a similar treatment-induced reduction in cell mitosis was evident but was of smaller magnitude.


Figure 5
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  		FIG. 5. Frequency of mitotic cells (immunopositive for phospho-histone H3; black staining) in representative WDs from a control (A) and an animal exposed in utero to 50 mg/kg flutamide (B) at E21.5. Note the numerous mitotic cells in the inner stroma (arrows), outer stroma (arrowhead), and epithelium. Panel C shows the total number of mitotic epithelial and stromal cells in the entire epididymal segment of the WD at E21.5 in controls (black bars) and animals exposed to 50 mg/kg (white bars) or 100 mg/kg (striped bars) flutamide. ***, P < 0.001 in comparison with respective control value. Values are means ± SEM for 12–16 animals.

 
Analysis of the epithelial mitotic index yielded comparable results, with epithelial mitosis reduced (P < 0.05) in flutamide-exposed WDs (0.2 ± 0.03%, mean ± SEM; n = 3), compared with control WDs (0.3 ± 0.02%, mean ± SEM; n = 3), indicating that the analytical method used reflects the mitotic index.

Reduction in epithelial cell height after flutamide exposure
Upon visual inspection, areas of the epithelial compartment in some animals at E21.5 appeared abnormal after flutamide exposure, with shorter epithelium, missing cells, and widened lumens (Fig. 6BGo); this was not seen in controls (Fig. 6AGo). This difference was confirmed quantitatively at E21.5 as flutamide exposure reduced WD epithelial cell height (Fig. 6CGo). This reduction was more dramatic in the corpus and caudal portions of the WD and was most pronounced in animals exposed to 100 mg/kg flutamide. No significant difference was noted in the width of WD epithelial cells in flutamide-exposed animals compared with controls (data not shown).


Figure 6
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  		FIG. 6. Epithelial cell height at E21.5 in WDs from controls (A and black bars in C) and animals exposed to 50 mg/kg (B and white bars in C) or 100 mg/kg (striped bars) flutamide. Note that the epithelium (immunostained for cytokeratin) is flatter in WDs in the flutamide-exposed animal. Images in A and B are both from the caput region of a WD, the segment of the WD where epithelial height is least reduced by flutamide exposure. Scale bar, 25 µm. Panel C shows quantification of epithelial cell height in WDs from controls (black bars) and from animals exposed to either 50 mg/kg (white bars) or 100 mg/kg (striped bars) flutamide. Note that epithelial cell height is reduced more in the corpus and caudal portions of the future epididymis after flutamide treatment. *, P < 0.05; **, P < 0.01 in comparison with respective control value. Values are means ± SEM for five to seven animals per treatment from at least three different litters.

 
Evidence for earliest effects of flutamide treatment on inner stromal cells
Because the data presented above indicated an impact of flutamide treatment on both stromal and epithelial tissues of the WD, we sought evidence of stromal-specific effects using SMA, a stromal cell marker. Immunostaining suggested a reduction in this SMA-positive layer in flutamide-exposed WDs compared with age-matched controls, although this varied considerably between WDs. This decrease was confirmed quantitatively by Western blot analysis, which revealed that the biggest reduction was at E19.5 (Fig. 7Go), a time point at which no epithelial damage or change in luminal length was detectable in flutamide-exposed animals (Fig. 1Go).


Figure 7
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  		FIG. 7. Comparative expression of SMA at E19.5 in WDs from controls (A) and animals exposed in utero to 50 mg/kg (B) or 100 mg/kg (C) flutamide. Note the reduction in SMA expression in WDs after flutamide exposure. D, Representative Western blot for SMA protein in WDs from control (C) and flutamide-exposed animals (F, 50 mg/kg; HF, 100 mg/kg) at E17.5–E21.5 (n = 3–5 WDs per age/treatment from at least three different litters). Testes (T; E21.5) were used as a positive control for SMA expression. E, Quantitative data expressed relative to SMA expression in control WDs at E19.5 (corrected for loading using ß-tubulin). Note the increase in SMA protein expression between E18.5 and E19.5 in controls and the decrease in SMA expression after flutamide exposure (white bars, 50 mg/kg; striped bars, 100 mg/kg) compared with controls (black bars). *, P < 0.05; ***, P < 0.001 in comparison with respective control value. Values are means ± SEM for two to three Western blots, each using a pool of three WDs per age/treatment from at least three litters.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is widely accepted that androgens play a vital role in WD development, but little is known about the molecular and cellular events that underlie this (2, 34, 35). The present study set out to use an in utero model system in which epididymal development can be altered to investigate the cellular mechanisms responsible for androgen-dependent WD development and allow better understanding of the mechanisms behind epididymal abnormalities in adults after reduced fetal androgen action. The main findings of these studies are, first, that in utero exposure to 50 or 100 mg/kg flutamide inhibits development, elongation, and differentiation of the WD but not its stabilization, contrary to previous interpretations. Second, these flutamide effects are mediated by effects on cell mitosis, but not apoptosis, and do not involve altered AR expression. Last, flutamide treatment appears to impair/delay differentiation of inner stromal cells at a stage in development before effects on the epithelium, consistent with the view that androgens drive WD development via stromal-epithelial interactions.

In males, the WD differentiates during neonatal life to form the epididymis, vas deferens, and seminal vesicles (4). In female rats, the WD regresses naturally between E16.5 and E18.5; this is believed to be because of a lack of androgens, whereas in males at this age, androgen action is believed to stabilize the WD, allowing it to subsequently differentiate into its adult derivatives (2, 3). In our rat colony, differentiation is first evident in the future epididymis by E20.5, suggesting that the developmental window for WD differentiation is between E19.5 and birth, coinciding with the peak in testosterone seen in male rats at E19.5 (9). This is in agreement with timings seen in previous studies (36).

It has been suggested that the pattern of AR expression reflects the androgen responsiveness of the tissue (23). We have mapped the AR to the epithelia from E17.5 and stroma of the WD from E16.5; however, epithelial AR expression was less intense than in stromal cells, particularly before E20.5. This is in agreement with other studies using antibodies demonstrating that ARs are expressed in the nuclei of both the stroma and epithelial cells of the rat WD by E17.5 (23). However, studies in mice using [3H]dihydrotestosterone steroid autoradiography reported stromal expression from E13, but epithelial expression was not detected until E19, much later than in rats (37). This difference in timing of expression may be because of species differences or different methodologies. The more intense immunoexpression of AR in the stroma compared with the epithelium would be consistent with the view that androgen effects on early WD development occur mainly through stromal AR and that androgens stimulate the stroma to signal to the epithelium to indirectly control epithelial fate (24). Flutamide exposure did not alter AR expression; therefore, WDs from flutamide-exposed animals retained the potential for AR-mediated androgen action. This conclusion is in contrast to findings by others who were unable to detect AR protein by immunohistochemistry in WDs from E21.5 fetuses exposed to 100 mg/kg flutamide (23). This difference is unexplained, although it could be because of rat strain difference or to the use of different anti-AR antibodies.

Previous studies have reported that flutamide exposure in utero results in abnormal epididymides in adults, but in these studies, it was not possible to identify whether the WD formed and stabilized but segments were later lost during differentiation or whether it failed to form and/or stabilize (31, 32, 33). In the present studies, WDs from flutamide-exposed males were present at E18.5 and E19.5 and appeared normal in all animals, with no obvious morphological differences or reduction in luminal length compared with their age-matched controls. This suggests that flutamide-exposed WDs initially form normally and are morphologically stabilized; however, it is unknown whether these WDs are functional at the biochemical level, and this needs additional investigation. In the regressing female WD, apoptosis is apparent, suggesting that the WD is dying rather than not differentiating (unpublished findings) (38, 39). In contrast, minimal cell apoptosis was detected in WDs from flutamide-exposed males at any age studied. Together these findings suggest that the doses of flutamide administered do not completely block the AR and so may not reduce androgen action sufficiently to impair WD stabilization and so cause it to regress, as occurs in the female (38). This difference could reflect the higher levels of testosterone reported to be available at E18.5 when the WD is undergoing stabilization than at E20.5 when the WD is differentiating (40), thus making it more difficult for flutamide to block androgen action. Alternatively, WD stabilization may require relatively low levels of androgens, less than that required for differentiation, or WD stabilization and differentiation may be regulated by different mechanisms. This is the first report that we are aware of highlighting WD development as a two-phase process, each of which may be differentially controlled. Insight from patients with complete androgen insensitivity syndrome and testicular feminization mice might further elucidate this as AR signaling is blocked in each case. Reports have shown that in both instances, adults lack any WD-derivatives (41, 42), however, no definitive evidence is published regarding the status of the fetal WD.

In controls, WDs showed a significant age-dependent increase in coiling and differentiation, as determined by quantification of luminal length; this is believed to be an androgen-dependent process. Unsurprisingly, flutamide interrupted this differentiation, thus confirming the vital role for androgens in differentiation of the stabilized WD. The most obvious explanation for this reduction in WD development could be an increase in cell apoptosis and/or a decrease in cell mitosis. Because apoptosis was minimal in WDs from flutamide-exposed animals, it was considered likely that altered cell proliferation was responsible. This is the first study to report the impact of androgen blockade on cell mitosis in the differentiating WD, showing androgen regulates both stromal and epithelial cell proliferation but that the consequences of reduced AR signaling can be noted earlier in the stromal compartment.

At E19.5, WDs from all flutamide-exposed animals were morphologically intact, suggesting that AR-mediated signaling was incompletely blocked fetally, even at 100 mg/kg flutamide, thus allowing WD rescue. Conversely, in adults that had been similarly exposed to flutamide in utero, the majority of the WD-derived tissues were largely absent. This is in agreement with previous studies showing that exposure to antiandrogenic compounds in utero results in a high frequency of epididymal malformations in adult rats (15, 31, 32, 33, 43). The contrast between WD abnormalities at E19.5 and in adulthood in flutamide-exposed animals demonstrates that the major effect of flutamide is on epididymal differentiation rather than stabilization, contrary to what has been presumed to occur by earlier researchers (15, 33). The increased prevalence of epididymal abnormalities as development proceeds may be a result of failure to establish normal patterning of the WD fetally. Interfering with androgen action within a critical window of development can therefore impair WD patterning and hence differentiation into its adult derivatives, resulting in irrecoverable malformation of the reproductive tract and likely impairment of fertility.

By E21.5, WDs from flutamide-exposed animals show abnormalities in both gross morphology and histology, including shorter, flatter epithelia, occasional missing corpus segments of the future epididymis, reduced coiling, and incomplete lumens. This is not seen until E21.5; thus the epithelium initially forms normally in flutamide-exposed animals but degenerates during differentiation, possibly because of interrupted androgen-driven signaling between the stroma and epithelium. In multiple organ systems, it is believed that steroid hormones control the fate of epithelial cells via interactions with the underlying stroma, and hormone withdrawal can result in epithelial-mesenchymal transformation (24, 26, 44, 45, 46). Because the epithelium in incomplete regions often appeared flattened with some cells even looking more fibroblast-like, it may be that a lack of androgen signaling resulted in dedifferentiation of some epithelial cells into mesenchymal-like cells, thus contributing to the impaired development of the WD. These missing segments were noted only in the corpus segment, the region of the WD that coils last and the segment of the adult epididymis that is least coiled. The androgen signal may be weaker in the corpus than in the caput or cauda, and thus flutamide treatment could have more impact on its development. The caput is closest to the testis and is therefore likely to be exposed to high levels of locally delivered androgens, but it is not obvious why the cauda should be less affected by flutamide treatment than the corpus. It is possible that the cauda may obtain testosterone from the blood as well as directly from the testis. However, there is unlikely to be a role for the more potent androgen DHT, because Wilson and Lasnitzki (12) and Siiteri and Wilson (13) both reported that DHT was not detectable in the WD until after epididymal differentiation was complete.

Blocking androgen action by flutamide exposure impaired stromal differentiation as indicated by reduced expression of SMA, an androgen-responsive stromal differentiation marker (47). This reduction in smooth muscle differentiation may explain the reduction in WD coiling seen after flutamide exposure. The flutamide-induced reduction in SMA expression was noted as early as E19.5, before any obvious signs of impaired epithelial development were apparent. This is consistent with previous studies in various organ systems that have shown that the stromal cells, via paracrine interactions, play a critical role in controlling epithelial proliferation, differentiation, and development (25, 26, 48, 49, 50). The mechanisms underlying these paracrine interactions are poorly understood, but growth factors and/or the extracellular matrix are likely to be involved.

There is substantial literature on the effects of reduced androgen action on the male reproductive tract using knockout mice and AR antagonists; however, most have studied the effects in adult males, and few have looked at how this impacts on development in fetal life. Because interfering with androgen action during fetal life, using 50 or 100 mg/kg flutamide, did not prevent stabilization of the WD but impaired its subsequent convolution and differentiation into its adult derivatives, the current study has highlighted WD development as a two-phase process with WD stabilization apparently requiring lower androgen action than WD differentiation. Why WD stabilization is not affected by administration of high doses of flutamide, approximately 5-fold higher than levels that induce complete agenesis of the prostate and feminization of the external genitalia, is something of a mystery and merits additional study. Future studies will also focus on using the present model system to investigate possible signaling pathways involved in WD patterning and differentiation. This should allow a clearer insight into the mechanisms behind interrupted epididymal development and function.


   Acknowledgments
 
We thank Sheila MacPherson and Mike Millar for their expert technical assistance in confocal microscopy and Mark Fisken for expert animal husbandry.


   Footnotes
 
This study was funded by the UK Medical Research Council.

Disclosure summary: M.W., P.T.K.S., N.I.M., and R.M.S. have nothing to declare.

First Published Online June 29, 2006

Abbreviations: AR, Androgen receptor; DHT, dihydrotestosterone; E0.5, embryonic d 0.5; NGS, normal goat serum; SMA, smooth muscle actin; TBS, Tris-buffered saline; TBST, TBS containing 0.1% Tween 20; WD, Wolffian duct.

Received February 6, 2006.

Accepted for publication June 19, 2006.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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