help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2007-0028
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/7/3185    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Welsh, M.
Right arrow Articles by Sharpe, R. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Welsh, M.
Right arrow Articles by Sharpe, R. M.
Endocrinology Vol. 148, No. 7 3185-3195
Copyright © 2007 by The Endocrine Society

The Critical Time Window for Androgen-Dependent Development of the Wolffian Duct in the Rat

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

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

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Androgens are thought to separately regulate stabilization and differentiation of the Wolffian duct (WD), but the time windows for these effects are unclear. To address this, fetal rats were exposed to flutamide within either an early window (EW) [embryonic day 15.5 (E15.5) to E17.5], when the WD degenerates in the female, or a later window (LW) (E19.5–E21.5), when the WD morphologically differentiates in the male, or during the full window of WD development (FW) (E15.5–21.5). WDs were examined for abnormalities during fetal (E21.5) or postnatal life, and anogenital distance and prostate presence/absence were recorded. Exposure to FW- or EW-flutamide, but not to LW-flutamide, induced comparable abnormalities in the fetal WD at E21.5, namely reduced WD coiling, reduced cell proliferation, reduced epithelial cell height, altered epithelial vimentin expression, and reduced expression of smooth muscle actin in the WD inner stroma. Exposure to EW- or FW-flutamide, but not to LW-flutamide, resulted in incomplete/absent WDs in more than 50% of males by adulthood, although such abnormalities were infrequent in fetal life. These findings suggest that androgen action during the EW is sufficient to promote WD morphological differentiation several days later. Because the androgen receptor is expressed in the WD stroma but not in the epithelium during this EW, WD differentiation is likely to be dependent on androgen-mediated signaling from the stroma to the epithelium. In conclusion, the critical window for androgen action in regulating WD development in the rat is between E15.5 and E17.5. This window is also important for prostate formation and anogenital distance masculinization.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DURING MAMMALIAN development, before activation of SRY within the somatic cells of the genital ridge, the urogenital tract is initially identical in both sexes, comprising the Müllerian duct (MD) and the Wolffian duct (WD) (1, 2). In males, after differentiation of the different somatic cell types in the fetal testis, the Sertoli cells secrete anti-Müllerian hormone to induce MD degeneration, whereas the Leydig cells secrete testosterone, which acts via the androgen receptor (AR) to stabilize and rescue the WD (2, 3). Conversely, in females, the ovaries do not synthesize either androgens or anti-Müllerian hormone so the WD degenerates and the MD persists (1, 2, 3). In males, once the WD is stabilized, it then differentiates 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). This process is thought to be controlled by androgens because XY males with inactivating mutations in the AR have a female phenotype with intraabdominal testes, no prostate, and a lack of WD-derived tissues (6, 7, 8, 9). In the rat, epididymal differentiation becomes evident between embryonic day 19.5 (E19.5) to E21.5 and is characterized by transformation of the cranial segment of the simple straight WD into an intensely coiled structure (10). It is presumed that the patterning of this transformation is under the control of testosterone (11, 12), the secretion of which begins at approximately E15.5 in the fetal rat and peaks at E19.5 (13).

Previous researchers have shown that interfering with androgen action during fetal life, using either AR mutant animals or exposure to antiandrogenic compounds, results in abnormal reproductive tracts in adult males (for review, see Refs. 14, 15, 16, 17, 18, 19, 20, 21). Most studies have exposed rats to antiandrogenic compounds between E13 and E21, encompassing the onset of testosterone production by the fetal testis and the window of male reproductive development, and have then examined the effects of this in adulthood. In such males, the epididymis and/or vas deferens is often incomplete or absent altogether. It has been presumed that this was attributable to a failure of the male WD to stabilize early in fetal development, as occurs naturally in females. However, we demonstrated recently that exposure of pregnant rats to 100 mg/kg flutamide during the period E15.5–E21.5 did not induce regression of the male WD but did result in subsequent impairment of WD morphological differentiation (10). These studies and those reported previously in the literature suggest that fetal WD development may be a biphasic process, with stabilization and differentiation possibly under differential control. It has been presumed that androgen action is vital for both early stabilization of the WD and the later segmentation and differentiation of the WD into its adult derivatives, but the precise window of androgen action in WD development remains unknown.

One previous study in the mouse showed that exposure to hydroxyflutamide from E11 to E15 resulted in smaller epididymides and infertility, whereas exposure to hydroxyflutamide from E19 to E20 did not result in any obvious reproductive abnormalities or infertility in adulthood (22). A similar study using rats examined the effects of maternal exposure to a single dose of flutamide (50 mg/kg) on E16, E17, E18, or E19 and showed that this treatment resulted in a similar range of reproductive abnormalities in adulthood as did exposure to flutamide throughout the period of reproductive development (E12–E21) but at a lower incidence (23). Exposure of pregnant rats to another antiandrogen, vinclozolin (400 mg/kg), between E14 and E19 resulted in malformations of the external genitalia, permanent nipples, reduced anogenital distance (AGD), and reduced seminal vesicle, ventral prostate, and epididymal weights. When vinclozolin treatment was restricted to 2-d periods (e.g. E14–E15 or E18–E19), similar abnormalities were observed, but the incidence and severity was reduced and no obvious abnormalities were reported in adult WD-derived structures (24).

There is thus no consensus as to when androgens act to 1) stabilize the WD and 2) induce its differentiation (i.e. epididymal coiling) and establish the patterning of the segmentation of the WD into its adult derivatives. It is also unclear as to when a lack of fetal androgen action may result in loss of part or all of the WD and precisely when this loss first manifests because the studies to date have only examined adults. To address this deficit in understanding, we used a model system in which fetal rats were exposed to flutamide specifically within either an early window (EW), when the WD degenerates in the female (E15.5–E17.5), or in a later window (LW), when the WD morphologically differentiates in the male (E19.5–E21.5); the impact on WD development in fetal (E21.5) and postnatal [postnatal day 17 (PND17), PND42, and PND70] life was then studied. We previously published evidence highlighting WD development as a biphasic event with WD stabilization and differentiation dependent on either different levels of androgen action or on different mechanisms (10). The present study sought to establish the critical window of androgen action in both these processes and has found, surprisingly, that androgen action within the same early time window (E15.5–E17.5) is sufficient to induce both rescue and later morphological differentiation of the WD.


   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 established, and the presence of a vaginal plug was taken as evidence of mating; this was defined as E0.5.

Treatment and collection of tissues and measurement of AGD
A total of 36 pregnant dams were used for this study, with dams randomly allocated to treatment groups. Dams were dosed daily by oral gavage between 0830 and 1000 h with flutamide (Sigma, Poole, UK) at 100 mg/kg in 1 ml/kg corn oil/2.5% DMSO (Sigma); these doses were selected based on previously reported results (10, 17). Dosing was undertaken during critical windows that either 1) encompassed the period of WD stabilization, defined by the timing of WD regression in females (our unpublished data) (E15.5–E17.5, EW-flutamide, n = 7 litters) or 2) encompassed the period of WD morphological differentiation, defined by the appearance of coiling in the future epididymal segment of the WD (E19.5–E21.5, LW-flutamide, n = 8 litters) (Fig. 1Go). Flutamide exposure between E17.5 and E19.5 was not examined in this study because we aimed to examine the role for androgens during WD stabilization or during WD morphological differentiation rather than to investigate the role for androgens during the entire period of reproductive development.


Figure 1
View larger version (36K):
[in this window]
[in a new window]

  		FIG. 1. Summary of duration of flutamide exposure in relation to key events in reproductive development and testosterone (T) levels in the fetal male testis (4 13 38 39 ).

 
To compare the effects of short window flutamide exposure with the previously published effects of exposure to flutamide from the onset of androgen production by the testis until birth [E15.5–E21.5, full window (FW)-flutamide, n = 6 litters] (10), WDs from FW-flutamide exposed males were included in all analyses. To reduce animal usage, these WDs were taken from both archived datasets (10) as well as from litters generated specifically for this study; there were no obvious differences between the archived (n = 8 litters) and newly generated datasets (n = 23 litters). Control dams (n = 10 litters) were gavaged daily with the vehicle alone at 1 ml/kg corn oil/2.5% DMSO. Dams were checked daily for signs of toxicity, and dam weights were recorded daily throughout the dosing regimen. Male offspring were subsequently evaluated in late fetal life (E21.5) or at PND17, PND42, or PND70.

For recovery of fetal animals, dams were killed by inhalation of carbon dioxide and subsequent cervical dislocation. Pups were recovered, decapitated, and placed in ice-cold PBS (Sigma). Postnatal animals (PND17, PND42, and PND70) were killed by inhalation of carbon dioxide and subsequent cervical dislocation. Before recovery of reproductive tracts, AGD was measured in fetal (E21.5) and postnatal animals using digital calipers (Faithfull Tools, Kent, UK), because it is widely believed that AGD reflects the degree of masculinization of the animal (for review, see Ref. 25). Female littermates were also examined for AGD.

Our previous studies have shown that WD morphological differentiation is well established by E21.5, as evidenced by coiling in the future epididymal segment of the WD (10). WDs were collected from male pups by microdissection, examined with a Leica (Nussloch, Germany) MZ6 dissecting microscope and photographed using a Leica ICA camera to enable gross morphological evaluation and measurement of WD luminal length (see below). This study focused on the development of the future epididymis and vas deferens (i.e. the upper portions of the WD) rather than the seminal vesicles, which are believed to depend on both testosterone and dihydrotestosterone. For each pup, the future epididymis and vas deferens segments of one isolated WD was snap frozen in liquid nitrogen for subsequent RNA and protein analysis by Taqman and Western blot, respectively, whereas the other was fixed in Bouin’s fixative for 1 h before being transferred into 70% ethanol and processed for 17.5 h in an automated Leica TP1050 processor for later use in histological analysis. Fixed WDs were embedded horizontally in paraffin wax, sectioned (5 µm), floated onto slides coated with 2% 3-aminopropyltriethoxy-silane (Sigma), and dried overnight at 50 C before histological analysis (see below). Representative WDs from at least three animals from at least three litters from the aforementioned treatment groups were subsequently used for the studies detailed below.

To recover postnatal reproductive tracts, the abdomen of the supine male rat was opened, and testes, epididymides, and vas deferens were pulled out of the scrotal sac by the fat pad and removed from the animal.

Gross morphology and histological analysis
Reproductive tracts from control and flutamide-exposed males at E21.5 or PND17, PND42, and PND70 were analyzed microscopically, at the time of dissection, for any gross morphological abnormalities. Histological analysis was performed on WD sections stained with hematoxylin and eosin, using standard protocols. Note was taken of any histological abnormalities, including swollen lumens and epithelial malformation.

WD luminal length measurement
Differentiation of WDs at E21.5 was quantified by measuring the luminal length of WD from control and treated animals on photographs taken of WD at the time of dissection, as detailed previously (10). A line was digitally drawn through the lumen of the WD image taken at time of dissection using the NIH Image J program. To ensure reproducibility, luminal length was measured for WDs from 15–30 individuals from at least three different litters per treatment group.

Immunohistochemistry
Immunohistochemistry was performed on Bouin’s-fixed WDs recovered from control and treated fetuses at E21.5 using previously published standard avidin-peroxidase protocols (10). The antibodies used for immunohistochemistry, their dilutions, and sources are listed in Table 1Go. Cellular sites of expression of AR, smooth muscle actin (SMA), and pan-cytokeratin were determined, and slides were photographed using a Provis AX70 (Olympus Optical, London, UK) microscope fitted with a Canon DS6031 camera (Canon Europe, Amsterdam, The Netherlands). 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 normal goat serum (NGS) alone, to ensure that any staining observed was specific; none of the antibodies used showed other than minor nonspecific staining.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Antibodies: source, dilution, retrieval, and species

 
Double-fluorescence immunohistochemistry for cytokeratin and vimentin
Sections from Bouin’s-fixed WDs were deparaffinized and rehydrated, and antigen retrieval was performed as detailed previously (10). Nonspecific binding sites were blocked by incubating sections in NGS (Autogen Bioclear UK Ltd., Wiltshire, UK) diluted 1:4 in PBS containing 5% BSA (Sigma) for 1 h and then incubated overnight at 4 C with anti-pan-cytokeratin antibody (Sigma) diluted 1:200 in NGS/PBS/BSA. This signal was detected by incubating sections for 1 h with goat antimouse secondary antibody directly conjugated with Alexa Fluor 546 (Invitrogen, Carlsbad, CA) diluted 1:200 in PBS to produce red fluorescence. Sections were washed well in PBS before blocking in NGS/PBS/BSA for 1 h. Because the anti-vimentin antibody (DakoCytomation, Ely, UK) and the pan-cytokeratin antibody were both raised in mice, the anti-vimentin antibody was directly labeled with Alexa Fluor 488 using an Alexa Fluor 488 labeling monoclonal antibody labeling kit (Invitrogen). This antibody was diluted 1:15 in NGS/PBS/BSA and incubated on sections for 2 h at room temperature. Because no secondary antibody detection was required, 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 (Welwyn Garden City, UK) LSM 510 Meta Axiovert 100M confocal microscope.

Frequency of cell mitoses in WDs
One randomly selected section from WDs recovered from control and flutamide-exposed fetuses at E21.5 was immunostained for phospho-histone H3 (Upstate Biotechnology, Dundee, UK) using a Bond-X automated immunostaining machine (Vision Biosystems, Newcastle, UK) as published previously (10). Phospho-histone H3-positive cells were counted in the epithelial compartment and the inner and outer stromal layers of the WD using an Olympus Optical (Tokyo, Japan) BH-2 microscope fitted with a Prior automatic stage (Prior Scientific Instruments, Cambridge, UK). Image-Pro Plus version 4.5.1 with Stereologer-Pro 5 plug-in software (Media Cybernetics, Wokingham, Berkshire, UK) was used as detailed previously (10). Positive cells were counted only in the future epididymal portion of the WD, and the number of positive cells was then related to the amount of epithelium seen in section and the total length of the WD lumen (10). Sections from 4–14 fetuses from three to five separate litters were analyzed from both control and flutamide-exposed animals.

Epithelial cell height measurement
WDs (E21.5) from six control fetuses and four to six fetuses from each of the flutamide treatment groups were sectioned and immunostained for pan-cytokeratin as detailed above to clearly label all epithelial cells. The software and stereological equipment noted above were used. Using a x63 objective, epithelial cell height was measured in every fifth epithelial cell per section. This was performed separately for the caput, corpus, and caudal regions of the future epididymal portion of the WD (10). 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.

Western blot analysis
Protein was harvested from frozen WDs using radioimmunoprecipitation assay lysis buffer as published previously (10). Protein concentration was determined using a Bio-Rad (Hemel Hempstead, UK) bicinchoninic acid protein assay kit according to the instructions of the manufacturer, and 20 µg of each protein extract was loaded onto a 7.5% polyacrylamide gel. Gels were subjected to electrophoresis at 100 V under reducing conditions. The proteins were then electrotransferred overnight at 20 V onto nitrocellulose membranes (Immobilon-P; Millipore, Bedford, MA). Membranes were washed in PBS before incubating in Odyssey blocking buffer (LI-COR, Lincoln, NE) diluted 1:1 in PBS for 1 h at room temperature to block nonspecific binding sites. Membranes were incubated overnight at 4 C with anti-SMA antibody (Sigma) diluted 1:10,000 and anti-ß-tubulin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:300 in Odyssey blocking buffer/PBS. The LI-COR buffer allows detection of more than one antibody at a time; therefore, both antibodies could be added at once as long as their host species differed, so they could be discriminated by secondary antibodies of different specificities. The anti-ß-tubulin antibody was used as a standardization loading control. Residual primary antibody was washed off with PBS with 0.1% Tween 20 (PBST) (Sigma) for 5 min (three times). Membranes were then incubated for 1 h at room temperature with the secondary antibodies diluted 1:5000 in Odyssey blocking buffer/PBST. Goat antirabbit secondary antibody conjugated with IRDye 800 (Rockland, Gilbertsville, PA) was used to detect ß-tubulin, whereas goat antimouse secondary antibody conjugated with Alexa Fluor 680 (Invitrogen) was added to detect SMA. Membranes were again washed in PBST for 5 min (three times) and then PBS (one time) to remove any residual Tween 20, before detecting the signal. The membrane was scanned using the LI-COR buffer, according to the instructions of the manufacturers.

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 then quantified, with the area of exposure equating to the amount of labeled protein present in the sample. Protein expression level was corrected for loading using ß-tubulin. To ensure reproducibility of results, the Western blot was performed at least twice, and protein was isolated from at least three WDs from two to 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’s post hoc test, using GraphPad Prism version 4 (GraphPad Software, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of flutamide exposure on AGD and the reproductive tract
Maternal exposure to flutamide during the entire window of reproductive development (E15.5–E21.5, FW-flutamide) prevented normal masculinization of fetal external genitalia, with AGD significantly reduced (P < 0.001) at E21.5 and PND17 to a length comparable with that of control females. Exposure to flutamide (100 mg/kg) early in WD development (E15.5–E17.5, EW-flutamide) reduced AGD by the same amount as did FW-flutamide exposure, whereas maternal exposure to flutamide late in WD development (E19.5–E21.5, LW-flutamide) did not result in any significant change in male AGD at E21.5 or PND17 when compared with age-matched control males (Fig. 2Go).


Figure 2
View larger version (27K):
[in this window]
[in a new window]

  		FIG. 2. AGD in males and females from control and flutamide-exposed litters at E21.5 and PND17. Note that AGD was significantly smaller in control females than in males; AGD in males exposed to flutamide between E15.5 and E21.5 (FW; white bars) or between E15.5 and E17.5 (EW; striped bars) was reduced compared with control males and was comparable with female AGD. Exposure to LW-flutamide (E19.5–E21.5; checkered bars) did not affect male AGD. ***, P < 0.001 compared with control male AGD. Values are means ± SEM for 6–23 animals.

 
Maternal exposure to FW-flutamide (E15.5–E21.5) or EW-flutamide (E15.5–E17.5) prevented normal prostate and penis formation in males and resulted in the presence of a vaginal pouch, whereas exposure to LW-flutamide (E19.5–E21.5) had no such effects (data not shown).

Effects on fetal and postnatal WD development of impaired androgen action during critical windows of development
Fetal.
Exposure to FW-flutamide reduced WD development and coiling compared with age-matched controls, as published previously (10). Exposure to EW-flutamide inhibited coiling (WD differentiation) at E21.5 to a similar extent as did FW-flutamide exposure, whereas exposure to LW-flutamide had no obvious effect on WD coiling at E21.5 compared with age-matched controls (Fig. 3Go). This difference was confirmed quantitatively (Fig. 3Go), highlighting that there was no significant difference in luminal length between WDs from litters exposed to FW-flutamide and those exposed to EW-flutamide. Exposure to LW-flutamide did not significantly reduce WD luminal length compared with age-matched controls.


Figure 3
View larger version (68K):
[in this window]
[in a new window]

  		FIG. 3. Representative WDs at E21.5 from control and flutamide-exposed fetuses. Note that coiling is evident in the future epididymal segment of the control WD (A) and that exposure to flutamide between E15.5 and E21.5 (FW; B) reduced this coiling. Exposure to flutamide between E15.5 and E17.5 (EW; C) reduced WD coiling to a similar extent as did exposure to FW-flutamide (B). In contrast, exposure to LW-flutamide (E19.5–E21.5; D) did not obviously reduce WD coiling compared with age-matched controls. * indicates efferent ducts. Scale bar, 1 mm. E shows quantification of coiling (luminal length) of E21.5 WDs from control and flutamide-exposed litters. Note the significant reduction in WD luminal length in animals exposed in utero to FW-flutamide (white bar) or EW-flutamide (striped bar) when compared with controls (black bar). Exposure to LW-flutamide (checkered bar) did not reduce WD luminal length. Values are mean ± SEM for 15–30 animals per group. ***, P < 0.001 compared with the respective control value.

 
As well as the reduction in coiling, exposure to FW-flutamide resulted in some WDs appearing incomplete at E21.5 (Table 2Go), with thinning of the epithelium and missing corpus/cauda segments, as reported previously (10). This phenotype of missing segments was not noted at E21.5 in fetuses exposed to either EW- or LW-flutamide (Table 2Go).


View this table:
[in this window]
[in a new window]

  		TABLE 2. Percentage of rats with an intact WD (E21.5) or epididymis and vas deferens (i.e. WD-derived tissues, PND17, PND42, adult) after exposure to flutamide (100 mg/kg) during specific time windows in fetal life

 
Postnatal.
Consistent with our findings in the fetus and with previous reports (23, 26, 27), FW- or EW-flutamide exposure resulted in abnormal morphology of the adult male reproductive tract with underdeveloped or incomplete epididymides, lack of a vas deferens, and underdeveloped or vestigial seminal vesicles. At PND17 (early puberty), all males exposed to either EW- or LW-flutamide treatment had complete epididymides and vasa deferentia, but, by late puberty (PND42), 40% of males exposed to EW-flutamide had incomplete reproductive tracts with missing or incomplete epididymides and/or vas deferentia (Table 2Go). By adulthood (PND70), 50% of males exposed to EW-flutamide had incomplete epididymides and/or vasa deferentia (Table 2Go); an additional 25% of such males had underdeveloped or abnormal epididymides/vasa deferentia (data not shown). This phenotype was similar to that observed in males exposed to FW-flutamide, in which 83% of males examined in adulthood had incomplete WD-derived tissues (Table 2Go). In contrast, males exposed to LW-flutamide all had complete epididymides and vas deferens at PND42 and in adulthood (Table 2Go).

AR expression
Androgens act through the AR; therefore, it was important to establish whether flutamide exposure affected AR expression. In control animals, AR expression was initially restricted to the WD stroma at E15.5 (Fig. 4Go) and E16.5 (data not shown), but, by E17.5, a few epithelial cells were weakly immunopositive for AR (Fig. 4Go). AR was expressed in many epithelial cells by E19.5 and in the majority of epithelial cells by E21.5 (Fig. 4Go). Exposure to either EW- or LW-flutamide did not alter AR expression in either the stroma or the epithelium at E21.5 or PND17 (data not shown). Additional analysis of AR expression using Taqman quantitative RT-PCR confirmed that there was no significant difference in AR mRNA expression in flutamide-exposed WDs at E21.5 compared with controls (data not shown). This is in agreement with results reported previously, showing that FW-flutamide exposure did not alter AR expression in the fetal WD (10) or postnatally (our unpublished data).


Figure 4
View larger version (155K):
[in this window]
[in a new window]

  		FIG. 4. Immunoexpression of AR in the developing WD from control animals at E15.5–E21.5. AR (immunostained brown) was confined to the WD stroma at E15.5 (A and B). AR was first detected in a few epithelial cells at E17.5 (C and D) and was strongly expressed in the epithelium by E19.5 (E and F). AR was intensely expressed in both the stroma and the epithelium by E21.5 (G and H). Scale bars, 50 µm.

 
Frequency of cell mitoses in WDs
Cell proliferation was detected in all compartments of WDs from both control and flutamide-exposed animals at E21.5. Immunostaining for phospho-histone H3 detected fewer proliferating cells in WDs from pups exposed to FW- or EW-flutamide compared with age-matched controls, but there was no apparent change in WDs exposed to LW-flutamide (data not shown). This was confirmed quantitatively (Fig. 5Go). Cell proliferation data were expressed as the overall number of proliferating cells in the complete epididymal portion of the WD rather than per 100 µm epithelium, so as to take into account any treatment-induced changes in WD length.


Figure 5
View larger version (21K):
[in this window]
[in a new window]

  		FIG. 5. Total number of mitotic epithelial and stromal cells in the entire epididymal segment of the WD at E21.5 from control and flutamide-exposed fetuses. Note the significant reduction in the number of mitotic cells in WDs from animals exposed to either FW-flutamide (white bars) or EW-flutamide (striped bars) compared with controls (black bars); this was apparent in all cellular compartments. Note also that exposure to LW-flutamide (checkered bars) did not affect WD cell proliferation at E21.5 compared with age-matched controls; these values were significantly higher than those from EW-flutamide WDs. *, P < 0.05 compared with respective EW-flutamide value; ***, P < 0.001 compared with respective control value. Values are means ± SEM for 3–16 animals per group.

 
Reduction in epithelial cell height after flutamide exposure
Exposure to FW-flutamide resulted in epithelial abnormalities in some areas of the WD in some animals at E21.5, for example, shorter epithelium, missing cells, and widened lumens, as reported previously (10). These abnormalities were also evident in WDs from animals exposed to EW-flutamide, but histological examination revealed no obvious abnormalities in the epithelium of WDs from fetuses exposed to LW-flutamide compared with age-matched controls (Fig. 6Go). These differences in WD epithelial cell height at E21.5 were confirmed quantitatively (Fig. 6Go).


Figure 6
View larger version (103K):
[in this window]
[in a new window]

  		FIG. 6. Representative gross abnormalities seen in the epithelium of WDs (immunostained for cytokeratin) from control (A) and flutamide-exposed (B–D) fetuses at E21.5. Note the apparent reduction in epithelial cell height (arrow) in WDs from males exposed to FW-flutamide (B) or EW-flutamide (C) compared with age-matched controls (A). This was not obvious in WDs from males exposed to LW-flutamide (D). Scale bar, 100 µm. E, Quantification of epithelial cell height in WDs at E21.5 from males exposed to FW-flutamide (white bar) or EW-flutamide (striped bar) or LW-flutamide (checkered bar) compared with age-matched controls (black bar). Values are means ± SEM for 3–19 WDs per age/treatment, each from different litters. *, P < 0.05 and ***, P < 0.001 compared with E21.5 controls.

 
Vimentin expression
Vimentin protein expression was examined in control and flutamide-exposed WDs because vimentin is an early mesenchymal intermediate filament, the expression of which is lost as cells undergo differentiation; aberrant expression of vimentin can result in abnormal cell behavior (28, 29). Vimentin was immunolocalized to the WD stroma of both control and flutamide-exposed fetuses at all ages examined (Fig. 7Go). Vimentin was also detected at the basolateral edges of epithelial cells in the WD at E17.5; this "spiking" was seen along the full length of the WD at E15.5 (data not shown) and E17.5 (Fig. 7AGo). In contrast, at E19.5 and E21.5, vimentin expression was rarely seen in the epithelium of the caput region in control WDs (Fig. 7Go, B and C), although it was still evident in the epithelium of the caudal region at both E19.5 and E21.5 (data not shown). Exposure to flutamide did not affect expression of vimentin in the stroma but affected its distribution in the epithelium (Fig. 7Go). For example, vimentin spiking was apparent in the epithelium at the caput region as well as in the caudal region (Fig. 7Go) in WDs from E21.5 fetuses exposed to FW- or EW-flutamide; this spiking was rarely seen at the caput region of WDs from fetuses exposed to LW-flutamide (Fig. 7Go) but was still noted in the epithelium of the caudal region, as in age-matched controls (data not shown).


Figure 7
View larger version (105K):
[in this window]
[in a new window]

  		FIG. 7. Coexpression of vimentin (green) and cytokeratin (red) protein in the caput region of WDs from control and flutamide-exposed animals. Note that vimentin was expressed in the stroma at E17.5 (A), E19.5 (B), and E21.5 (C–F) and did not vary with flutamide treatment (D, FW-flutamide; E, EW-flutamide; F, LW-flutamide). Vimentin was also detected at the basolateral edges of the WD epithelium (arrow) at E17.5 (A) but was absent by E19.5 (B) and E21.5 (C) in control animals. However, vimentin spiking in the epithelium was still apparent in the caput region of the WD at E21.5 in fetuses exposed to FW-flutamide (D) or EW-flutamide (E) but was not evident in WDs exposed to LW-flutamide (F).

 
Evidence for flutamide effects on inner stromal cells
Immunostaining for SMA, which is expressed in the inner stromal compartment and believed to be androgen dependent (10, 30), revealed a reduction in the SMA-positive layer in WDs from E21.5 fetuses that had been exposed to FW- or EW-flutamide compared with age-matched controls (Fig. 8Go). No obvious change was noted in this SMA-positive layer in WDs from males exposed to LW-flutamide (Fig. 8DGo). The SMA-positive stromal compartment varied considerably in breadth between sections of WDs, so the reduction in SMA protein expression in WDs recovered from fetuses exposed to FW- or EW-flutamide was confirmed quantitatively by Western blot analysis (Fig. 8EGo). This confirmed that SMA protein levels in WDs from EW- and FW- treatments, but not those from LW-flutamide fetuses, were significantly different from that in age-matched controls (Fig. 8EGo).


Figure 8
View larger version (89K):
[in this window]
[in a new window]

  		FIG. 8. Comparative expression of SMA at E21.5 in WDs from controls (A) and animals exposed to FW-flutamide (B), EW-flutamide (C), or LW-flutamide (D). Note the apparent reduction in SMA immunoexpression (brown) in WDs from fetuses exposed to FW- or EW-flutamide but not in WDs from fetuses exposed to LW-flutamide. E, Quantitative data from Western blot analysis comparing expression of SMA protein at E21.5 in WDs from control (C) and flutamide-exposed animals. Note the reduced levels of SMA protein in WDs from fetuses exposed to FW-flutamide (white bar) or EW-flutamide (striped bar), although no obvious difference can be seen in SMA protein expression in WDs from fetuses exposed to LW-flutamide (checkered bar) compared with age-matched controls (black bar). n = 3–5 WDs per age/treatment, from at least three different litters. Loading was corrected using ß-tubulin protein expression. *, P < 0.05; **, P < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is widely accepted that androgens play a critical role in WD development. A recent study published by our group confirmed the role for androgens in male fetal reproductive development and highlighted WD development as a biphasic process, with fetal exposure to flutamide (100 mg/kg) inhibiting development, elongation, and differentiation of the WD but not its earlier stabilization (10). Abnormalities in WD-derived structures persisted and worsened by adulthood, confirming that the patterning of the reproductive tract is established in the fetus and that interfering with androgen action during this critical period permanently impairs reproductive tissues. It has been assumed that androgen-driven gene activity is essential during the period of both WD stabilization and morphological differentiation, but the precise time window within which androgens act in males to mediate WD stabilization and/or differentiation remains unclear. In the present study, we used an in utero model system in which fetal rats were exposed to flutamide on either E15.5–E17.5 (EW), to interfere with androgen action during the "stabilization window" when the WD is degenerating in the female, or E19.5–E21.5 (LW), during the "differentiation window" when WD morphologically differentiates into the epididymis in the male. The impact of these treatments on WD morphology and functional differentiation in fetal (E21.5) and postnatal (PND17, PND42, and PND70) life was also investigated.

The main findings of these studies are, first, that androgen action during the early time window (E15.5–E17.5), which is several days before any sign of WD morphological differentiation, is sufficient to promote later WD segmentation and development (coiling/functional differentiation). Second, exposure to EW-flutamide was able to induce similar gross and histological abnormalities in the fetal and adult WD as those observed after exposure to FW-flutamide. Because the AR is expressed in the WD stroma but not in the epithelium during this "early" window, this suggests that AR-dependent androgen action must signal via the stroma to pattern later WD development and differentiation. Finally, exposure to flutamide during the "late" window (E19.5–E21.5), the period in which WD morphological differentiation actually occurs, did not result in any obvious gross or histological WD abnormalities at this or later times, thus suggesting that the pattern of WD segmentation is already established by E19.5 and that its morphological differentiation is no longer dependent on high levels of androgen action. The critical window for high levels of androgen action in establishing the pattern of WD development in rats is therefore between E15.5 and E17.5.

Because exposure to FW-flutamide did not prevent stabilization of the male WD but impaired WD differentiation (i.e. coiling) between E19.5 and E21.5 (10), we anticipated that reducing androgen action during WD morphological differentiation would allow WD "rescue" but would prevent subsequent WD differentiation, whereas exposure to EW-flutamide would not have any major impact on WD coiling or functional differentiation. Contrary to these expectations, reduced androgen action early in male reproductive tract development (E15.5–E17.5) resulted in a similar phenotype to that documented after exposure to FW-flutamide, with males having a vaginal pouch instead of a normal prostate, impaired WD development, and an AGD the same as that in normal females. This phenotype was not observed in male fetuses exposed to LW-flutamide. These results suggest that prostate formation, WD patterning, and masculinization of AGD is established between E15.5 and E17.5, several days before any sign of morphological differentiation of any of these tissues, and that high levels of androgen action later in fetal development are not essential for maintaining this male phenotype. These findings support and extend those of a recent study in which pregnant rats were exposed to a single dose of flutamide (50 mg/kg) between E16 and E19, and the impact on male phenotype of adult rats was examined. The results revealed that exposure to flutamide on E16 or E17 resulted in missing epididymides in adults, whereas exposure on E18 or E19 only resulted in smaller epididymides (23). These authors highlighted that the peak incidence of abnormal prostate development was noted in adults exposed to flutamide on E17 or E18, similar to our observations in males exposed to flutamide between E15.5 and E17.5 (23). Furthermore, they noted that exposure to a single dose of flutamide on any one day between E16 and E19 resulted in a similar range of reproductive abnormalities as did exposure to flutamide throughout the period of reproductive development (E12–E21), although the incidence was lower (23). We also noted a reduced incidence of reproductive abnormalities after exposure to EW-flutamide compared with that observed in animals exposed to FW-flutamide in animals examined at a range of different ages.

It was surprising that high levels of androgen action did not appear to be essential during the window of morphological differentiation of the WD (E19.5–E21.5). Instead, it appears that, once the pattern of WD segmentation has been established by earlier androgen exposure, the WD continues to develop normally, even if androgen action is reduced during the period of morphological differentiation. It is worth noting that, as reported previously (10), exposure to this level of flutamide may not completely block androgen action; therefore, we cannot completely rule out a role for low levels of androgens during this LW of WD development (E19.5–E21.5). Flutamide was used to interfere with androgen action because it has been shown to be a strong competitive AR antagonist and exposure during fetal development interrupts male reproductive development (17, 26, 27, 31, 32). Flutamide is believed to be cleared from the body relatively quickly and has a plasma half-life of 5–6 h in man (33), suggesting that fetuses are not exposed to flutamide for more than 24 h after the final gavage. Studies in the mouse have shown that exposure to flutamide on E19 and E20 did not result in any obvious reproductive abnormalities or infertility in adulthood, whereas exposure on E11–E15 or E15–E20 resulted in smaller epididymides and infertility or lack of a prostate, respectively (22). These studies again confirm the long-term deleterious effects of EW short-term androgen blockade. It remains unclear what role, if any, androgens play late in fetal male reproductive development, and additional investigation of more endpoints would be required in males from litters exposed to flutamide between E19.5 and E21.5.

In the present study and as reported previously (10), exposure to flutamide during the FW of male reproductive development (E15.5–E21.5) resulted in incomplete WDs in 11% of males at E21.5, 50% of males at PND17, and 83% of adult males. This is in agreement with previous studies showing that exposure to antiandrogenic compounds in utero results in a high frequency of epididymal malformations when studied in adulthood (17, 23, 26, 27, 34). Exposure to LW-flutamide did not result in incomplete WDs at any time point studied. In contrast, EW-flutamide did not show evidence of loss of WD during fetal life, but 40% of pubertal males (PND42) and 50% of adults had incomplete epididymides and/or vasa deferentia. This has provided compelling evidence that androgen action early in WD development (E15.5–E17.5) is essential to establish the pattern of WD development and that interfering with this has long-term consequences for the adult male reproductive tract. However, it remains to be shown why completion of puberty should result in delayed degeneration or loss of WD-derived tissues in animals exposed to EW- or FW-flutamide.

To confirm that the disturbances in WD development seen in animals exposed to flutamide during the early treatment window involved similar mechanisms to those seen in animals exposed to flutamide throughout reproductive development (10), various endpoints were examined (summarized in Table 3Go). As we reported previously (10), exposure to flutamide between E15.5 and E21.5 did not alter expression of the AR in the stroma or epithelium of the WD at any age during fetal life or postnatally (our unpublished data). Similarly, in the present study, exposure to flutamide during either defined window (early or late) did not interrupt AR expression in the fetus or at PND17, confirming that these WDs are still capable of responding to androgens; because AR is expressed in the postnatal (PND17, PND42, and PND70) WD derivatives from animals exposed to flutamide between E15.5 and E21.5 (our unpublished data), it is unlikely that exposure to flutamide during the EW or LW would alter AR expression. This is in contrast to findings by Bentvelsen et al. (31), who were unable to detect AR protein by immunohistochemistry in WDs from E21.5 fetuses exposed to 100 mg/kg flutamide. This difference may be attributable to differences in rat strain or to the use of different anti-AR antibodies and immunohistochemistry techniques. Exposure to FW-flutamide resulted in reduced epithelial cell height and abnormal epithelial development in the WD by E21.5 (10). The same epithelial disturbance was not found in WDs from males exposed to LW-flutamide but was seen in WDs from males exposed to EW-flutamide. These effects on the epithelium were not evident until E21.5, suggesting that, although the epithelium initially forms normally, flutamide exposure results in subsequent abnormalities during differentiation, possibly attributable to impaired androgen-driven signaling between the stroma and epithelium. As well as the impact of flutamide exposure on epithelial differentiation, we reported previously that exposure to flutamide between E15.5 and E21.5 impaired WD stromal cell differentiation, as evidenced by a reduction in SMA protein expression at E19.5–E21.5 (10). In the present studies, a similar reduction was noted in WDs from fetuses exposed to flutamide during the EW but not the LW of WD development, highlighting the role for androgens between E15.5 and E17.5 in regulating stromal cell differentiation as well as epithelial cell differentiation.


View this table:
[in this window]
[in a new window]

  		TABLE 3. Summary of endpoints affected by exposure to flutamide during specific time windows in fetal life compared with controls at E21.5

 
Vimentin, an early mesenchymal intermediate filament, was expressed in the WD stroma at all ages studied (E15.5–E21.5), and its expression in the stroma was not obviously affected by flutamide exposure. This stromal vimentin is likely to be providing mechanical support to the developing WD and may also mediate signaling through interactions with and organization of the cytoskeleton (35). Vimentin was also expressed on the basolateral boundary of the epithelial cells in some regions of the developing WD. It is thought that vimentin expression often precedes expression of other intermediate filaments during differentiation, for example desmin. Vimentin is initially expressed in the epithelium along the full length of the WD at E15.5 but becomes progressively more restricted such that, by E21.5, it is confined to the caudal region in control WDs. This may mean that vimentin expression in the epithelium is replaced by another intermediate filament in a craniocaudal direction as development proceeds. At E21.5, epithelial spiking of vimentin was detected along the full length of the epididymal segment of WDs from fetuses exposed to FW- or EW-flutamide rather than just in the cauda. This pattern is in contrast to WDs from fetuses exposed to LW-flutamide, which were similar to controls. The alteration in epithelial vimentin expression could be attributable to failure of the epithelial cells to differentiate and thus to switch off epithelial vimentin expression and may mean that these epithelial cells retain some mesenchymal-like properties. This might explain the reduced epithelial cell height and loss of attachment to the basement membrane seen in flutamide-exposed WDs. Vimentin is required for cell motility, and increased vimentin expression is seen in prostate cancer cells as they become more invasive (28). It could therefore be hypothesized that these vimentin-positive WD epithelial cells may be gaining motility but there is no definitive evidence for this, and additional investigation of earlier and later time points might provide some insight. Studies in the equine endometrium have shown that, in cases of endometriosis, cytokeratin and vimentin become coexpressed in the epithelium, and it has been suggested that this could be attributable to increased proliferation and a loss of cell to cell contact (36). Other researchers have also shown that altered hormone action could induce epithelial vimentin expression (37).

It is concluded that the pattern of WD coiling and its subsequent ability to develop fully during postnatal life is established by androgen action early in fetal reproductive development (E15.5–E17.5) and that, by E19.5, androgen-dependent WD patterning is already established and is no longer dependent on high levels of androgen action. Reduced androgen action during this early time window inhibited coiling at E21.5 to the same extent as did exposure from E15.5 to E21.5 and resulted in a similar high incidence of epididymal loss/abnormalities in late puberty and adulthood. Exposure later in fetal life (E19.5–E21.5), the period in which WD morphological differentiation actually occurs, did not impact WD coiling at E21.5 or subsequent epididymal development postnatally. The critical window for high levels of androgen action in establishing the pattern of WD development is therefore between E15.5 and E17.5 in the rat.


   Acknowledgments
 
We thank Sheila MacPherson and Mike Millar for their expert technical assistance in confocal microscopy and fluorescent labeling of the vimentin antibody and Mark Fisken for expert animal husbandry. We also thank Dr. Lee Smith for his constructive comments and advice in the preparation of this manuscript.


   Footnotes
 
This study was funded by the Medical Research Council.

M.W., P.T.K.S., and R.M.S. have nothing to declare.

First Published Online April 12, 2007

Abbreviations: AGD, Anogenital distance; AR, androgen receptor; E, embryonic day; EW, early window; FW, full window; LW, late window; MD, Müllerian duct; NGS, normal goat serum; PBST, PBS with 0.1% Tween 20; PND, postnatal day; SMA, smooth muscle actin; WD, Wolffian duct.

Received January 10, 2007.

Accepted for publication April 2, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Drews U 2000 Local mechanisms in sex specific morphogenesis. Cytogenet Cell Genet 91:72–80[CrossRef][Medline]
  2. George FW, Wilson J 1994 The physiology of reproduction. 2nd ed. New York: Raven Press
  3. Huhtaniemi I 1994 Fetal testis—a very special endocrine organ. Eur J Endocrinol 130:25–31[Abstract]
  4. Wilson JD, George FW, Griffin JE 1981 The hormonal control of sexual development. Science 211:1278–1284[Free Full Text]
  5. Robaire B, Hermo L 1988 Efferent ducts, epididymis, and vas deferens: structure, functions, and their regulation. In: Physiology of reproduction. 2nd ed. New York: Raven Press
  6. Quigley CA 2002 Editorial: the postnatal gonadotropin and sex steroid surge-insights from the androgen insensitivity syndrome. J Clin Endocrinol Metab 87:24–28[Abstract/Free Full Text]
  7. Tsuji M, Shima H, Cunha GR 1991 In vitro androgen-induced growth and morphogenesis of the Wolffian duct within urogenital ridge. Endocrinology 128:1805–1811[Abstract]
  8. Brinkmann AO 2001 Molecular basis of androgen insensitivity. Mol Cell Endocrinol 179:105–109[CrossRef][Medline]
  9. McPhaul MJ 2002 Androgen receptor mutations and androgen insensitivity. Mol Cell Endocrinol 198:61–67[CrossRef][Medline]
  10. Welsh M, Saunders PT, Marchetti NI, Sharpe RM 2006 Androgen-dependent mechanisms of Wolffian duct development and their perturbation by flutamide. Endocrinology 147:4820–4830[Abstract/Free Full Text]
  11. Veyssiere G, Berger M, Jean-Faucher C, de Turckheim M, Jean C 1982 Testosterone and dihydrotestosterone in sexual ducts and genital tubercle of rabbit fetuses during sexual organogenesis: effects of fetal decapitation. J Steroid Biochem 17:149–154[CrossRef][Medline]
  12. George FW, Wilson J 1994 Gonads and ducts in mammals. In: Knobil E, Neill JD, eds. The physiology of reproduction. 2nd ed. New York: Raven Press
  13. Warren DW, Haltmeyer GC, Eik-Nes KB 1972 Synthesis and metabolism of testosterone in the fetal rat testis. Biol Reprod 7:94–99[Abstract]
  14. Gray LE, Ostby J, Furr J, Wolf CJ, Lambright C, Parks L, Veeramachaneni DN, Wilson V, Price M, Hotchkiss A, Orlando E, Guillette L 2001 Effects of environmental antiandrogens on reproductive development in experimental animals. Hum Reprod Update 7:248–264[Abstract/Free Full Text]
  15. Yeh S, Tsai MY, Xu Q, Mu XM, Lardy H, Huang KE, Lin H, Yeh SD, Altuwaijri S, Zhou X, Xing L, Boyce BF, Hung MC, Zhang S, Gan L, Chang C 2002 Generation and characterization of androgen receptor knockout (ARKO) mice: an in vivo model for the study of androgen functions in selective tissues. Proc Natl Acad Sci USA 99:13498–13503[Abstract/Free Full Text]
  16. Lyon MF, Hawkes SG 1970 X-linked gene for testicular feminization in the mouse. Nature 227:1217–1219[CrossRef][Medline]
  17. Imperato-McGinley J, Sanchez RS, Spencer JR, Yee B, Vaughan ED 1992 Comparison of the effects of the 5 {alpha}-reductase inhibitor finasteride and the antiandrogen flutamide on prostate and genital differentiation: dose-response studies. Endocrinology 131:1149–1156[Abstract]
  18. Mylchreest E, Cattley RC, Foster PM 1998 Male reproductive tract malformations in rats following gestational and lactational exposure to di(n-butyl) phthalate: an antiandrogenic mechanism? Toxicol Sci 43:47–60[Abstract/Free Full Text]
  19. McIntyre BS, Barlow NJ, Wallace DG, Maness SC, Gaido KW, Foster PM 2000 Effects of in utero exposure to linuron on androgen-dependent reproductive development in the male Crl:CD(SD)BR rat. Toxicol Appl Pharmacol 167:87–99[CrossRef][Medline]
  20. Foster PM, Mylchreest E, Gaido KW, Sar M 2001 Effects of phthalate esters on the developing reproductive tract of male rats. Hum Reprod Update 7:231–235[Abstract/Free Full Text]
  21. McIntyre BS, Barlow NJ, Sar M, Wallace DG, Foster PM 2002 Effects of in utero linuron exposure on rat Wolffian duct development. Reprod Toxicol 16:131–139[CrossRef][Medline]
  22. Silversides DW, Price CA, Cooke GM 1995 Effects of short-term exposure to hydroxyflutamide in utero on the development of the reproductive tract in male mice. Can J Physiol Pharmacol 73:1582–1588[Medline]
  23. Foster PM, Harris MW 2005 Changes in androgen-mediated reproductive development in male rat offspring following exposure to a single oral dose of flutamide at different gestational ages. Toxicol Sci 85:1024–1032[Abstract/Free Full Text]
  24. Wolf CJ, LeBlanc GA, Ostby JS, Gray Jr LE 2000 Characterization of the period of sensitivity of fetal male sexual development to vinclozolin. Toxicol Sci 55:152–161[Abstract/Free Full Text]
  25. Edwards TM, Moore BC, Guillette Jr LJ 2006 Reproductive dysgenesis in wildlife: a comparative view. Int J Androl 29:109–121[CrossRef][Medline]
  26. McIntyre BS, Barlow NJ, Foster PM 2001 Androgen-mediated development in male rat offspring exposed to flutamide in utero: permanence and correlation of early postnatal changes in anogenital distance and nipple retention with malformations in androgen-dependent tissues. Toxicol Sci 62:236–249[Abstract/Free Full Text]
  27. Mylchreest E, Sar M, Cattley RC, Foster PM 1999 Disruption of androgen-regulated male reproductive development by di(n-butyl) phthalate during late gestation in rats is different from flutamide. Toxicol Appl Pharmacol 156:81–95[CrossRef][Medline]
  28. Singh S, Sadacharan S, Su S, Belldegrun A, Persad S, Singh G 2003 Overexpression of vimentin: role in the invasive phenotype in an androgen-independent model of prostate cancer. Cancer Res 63:2306–2311[Abstract/Free Full Text]
  29. Paranko J, Virtanen I 1986 Epithelial and mesenchymal cell differentiation in the fetal rat genital ducts: changes in the expression of cytokeratin and vimentin type of intermediate filaments and desmosomal plaque proteins. Dev Biol 117:135–145[CrossRef][Medline]
  30. Schlatt S, Weinbauer GF, Arslan M, Nieschlag E 1993 Appearance of {alpha}-smooth muscle actin in peritubular cells of monkey testes is induced by androgens, modulated by follicle-stimulating hormone, and maintained after hormonal withdrawal. J Androl 14:340–350[Abstract/Free Full Text]
  31. Bentvelsen FM, Brinkmann AO, van der Schoot P, van der Linden JE, van der Kwast TH, Boersma WJ, Schroder FH, Nijman JM 1995 Developmental pattern and regulation by androgens of androgen receptor expression in the urogenital tract of the rat. Mol Cell Endocrinol 113:245–253[CrossRef][Medline]
  32. Hotchkiss AK, Ostby JS, Vandenburgh JG, Gray Jr LE 2002 Androgens and environmental antiandrogens affect reproductive development and play behavior in the Sprague-Dawley rat. Environ Health Perspect 110 (Suppl 3):435–439
  33. Neri RO, Monahan M 1972 Effects of a novel nonsteroidal antiandrogen on canine prostatic hyperplasia. Invest Urol 10:123–130[Medline]
  34. Turner KJ, McIntyre BS, Phillips SL, Barlow NJ, Bowman CJ, Foster PM 2003 Altered gene expression during rat Wolffian duct development in response to in utero exposure to the antiandrogen linuron. Toxicol Sci 74:114–128[Abstract/Free Full Text]
  35. Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD 1994 Molecular biology of the cell. New York: Garland Publishing
  36. Aupperle H, Schoon D, Schoon HA 2004 Physiological and pathological expression of intermediate filaments in the equine endometrium. Res Vet Sci 76:249–255[CrossRef][Medline]
  37. Klymkowsky MW, Bachant JB, Domingo A 1989 Functions of intermediate filaments. Cell Motil Cytoskeleton 14:309–331[CrossRef][Medline]
  38. Habert R, Picon R 1984 Testosterone, dihydrotestosterone and estradiol-17ß levels in maternal and fetal plasma and in fetal testes in the rat. J Steroid Biochem 21:193–198[CrossRef][Medline]
  39. Tapanainen J, Kellokumpu-Lehtinen P, Pelliniemi L, Huhtaniemi I 1981 Age-related changes in endogenous steroids of human fetal testis during early and midpregnancy. J Clin Endocrinol Metab 52:98–102[Abstract]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/7/3185    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Welsh, M.
Right arrow Articles by Sharpe, R. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Welsh, M.
Right arrow Articles by Sharpe, R. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals