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Endocrinology, doi:10.1210/en.2006-1622
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Endocrinology Vol. 148, No. 5 2027-2036
Copyright © 2007 by The Endocrine Society

Role of Androgens in Fetal Testis Development and Dysgenesis

Hayley M. Scott, Gary R. Hutchison, I. Kim Mahood, Nina Hallmark, Michelle Welsh, Karel De Gendt, Guido Verhoeven, Peter O’Shaughnessy and Richard M. Sharpe

Medical Research Council Human Reproductive Sciences Unit (H.M.S., G.R.H., I.K.M., N.H., M.W., R.M.S.), Centre for Reproductive Biology, The Queen’s Medical Research Institute, Edinburgh EH16 4TJ, United Kingdom; Division of Biochemistry (K.D.G., G.V.), Catholic University of Leuven, B-300 Leuven, Belgium; and Institute of Comparative Medicine (P.O.), University of Glasgow Veterinary School, Glasgow G61 1QH, United Kingdom

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study sought to establish whether reduced androgen levels/action in the fetal rat testis induced by di(n-butyl) phthalate (DBP) contributes to dysgenetic features, namely reduced Sertoli cell number, occurrence of multinucleated gonocytes (MNG), and Leydig cell aggregation. Pregnant rats were administered treatments or cotreatments designed to manipulate testosterone levels [DBP, testosterone propionate (TP)] or action [flutamide, 7,12-dimethyl-benz[a]anthracene (DMBA)]. The aforementioned end points were analyzed and related to intratesticular testosterone (ITT) levels and peripheral androgen action (anogenital distance). Dysgenetic features were also evaluated in mice with inactivation of the androgen receptor (testicular feminized or ARKO mice). Exposure to DBP alone, or combined with flutamide, DMBA, or TP, resulted in reduced Sertoli cell number and ITT levels, as did exposure to TP alone; coadministration of DBP + TP caused the most severe reduction in both parameters. A positive correlation between ITT levels and Sertoli cell number was found (r = 0.791; P = 0.019). Similarly, exposure to DBP alone, or as a cotreatment, significantly increased occurrence of MNG and Leydig cell aggregation, and these were negatively correlated with ITT levels. Exposure to flutamide or DMBA alone had no significant effect on these dysgenetic end points. These findings suggest that reduced ITT decreases fetal Sertoli cell numbers and might be involved in Leydig cell aggregation and MNG. However, of these three end points, only Sertoli cell number was affected significantly in ARKO/testicular feminized mice with absent androgen action. Therefore, induction of MNG and Leydig cell aggregation might result from DBP-induced effects other than suppression of ITT levels.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DISORDERS OF MALE reproductive health that manifest at birth (cryptorchidism, hypospadias) or in young adulthood (testicular germ cell cancer and low sperm counts) are common and may be increasing in incidence in the West (1). Each of these disorders can originate in fetal life and they share risk factors (1, 2). Based on this and other evidence, it has been hypothesized that these disorders comprise a testicular dysgenesis syndrome (TDS), in which abnormal testis development (dysgenesis) leads secondarily to hormonal or other malfunctions of the Leydig and/or Sertoli cells during male sexual differentiation, leading in turn to increased risk of the aforementioned disorders (1, 2).

Testis formation and early development are hormone independent (3, 4), but impaired testosterone production/action resulting from dysgenesis may contribute to, or exacerbate, testicular maldevelopment. This is evident in patients with complete androgen insensitivity syndrome who may exhibit focal areas of testicular dysgenesis (5) and are at considerably increased risk of testicular germ cell cancer (6, 7). Similarly, testicular feminized mice (8) and mice with complete knockout of the androgen receptor (AR) (9), both equivalent to complete androgen insensitivity syndrome in the human, display a 32–75% decrease in Sertoli cell number, suggesting that androgens play an important role in Sertoli cell proliferation in perinatal life. Because the Sertoli cells do not express AR in fetal life in the human or rat (10, 11, 12), it is assumed that androgen effects on Sertoli cell number occur indirectly, via the AR-positive peritubular myoid cells (9, 10). Because Sertoli cell number in adulthood is the primary determinant of sperm count in humans (10), impaired androgen production/action within the fetal testis is one mechanism via which fetal events could lead to low sperm counts in adulthood. In this regard, studies have shown that maternal smoking results in a 20–49% reduction in testis size and sperm count in the resulting male offspring, probably via a reduction in Sertoli cell number (13, 14). The mechanism behind this is unclear, but cigarette smoke contains polycyclic aromatic hydrocarbons (PAHs), which bind to the aryl hydrocarbon receptor, and activation of the aryl hydrocarbon receptor can antagonize androgen action in cell transfection systems (15, 16). Others have suggested that maternal smoking could be a cause of TDS disorders in human males (17).

We and others have shown that in utero exposure of rats to certain phthalate esters, such as di(n-butyl) phthalate (DBP), can induce a TDS-like spectrum of disorders in the male offspring (18, 19, 20, 21, 22, 23, 24). In this model, DBP induces a marked reduction in Leydig cell hormone (testosterone and insulin-like factor 3) production, widespread occurrence of multinucleated fetal germ cells (21, 25, 26, 27, 28), and focal dysgenesis of seminiferous cords/tubules due to abnormal migration/aggregation of fetal Leydig cells (21, 29, 30). Whether reduced androgen levels contribute causally to the latter two end points and/or cause a reduction in Sertoli cell proliferation/numbers in fetal life in DBP-exposed animals is unknown. The primary aim of the present studies was to address this question together with the wider issue of the role that androgens might play in disorders of testis development relevant to TDS. To achieve this, pregnant rats were treated with the AR antagonist flutamide; a candidate PAH [7,12-dimethyl-benz[a]anthracene (DMBA)]; DBP, alone or in combination with flutamide or DMBA; or DBP plus testosterone or testosterone alone. Fetal testicular end points relevant to the origins of TDS (listed above) were evaluated and related to testicular testosterone levels and peripheral androgen action (anogenital distance). Where possible, AR knockout (ARKO) (d 2) mice and testicular feminized mice (tfm) [embryonic day (E) 18.5] were analyzed as positive controls.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals, treatments, sample collection, and processing
Wistar rats were maintained in our own animal facility according to U.K. Home Office guidelines and were fed a soy-free breeding diet (SDS, Dundee, Scotland, UK). Time-mated females were subjected to daily oral gavage (unless otherwise stated) with one of the treatments described below. Treatments were administered between 0900 and 1030 h.

  1. DBP (Sigma-Aldrich Co. Ltd., Dorset, UK) at a dose of 500 mg/kg in 1 ml/kg corn oil from E13.5 to E20.5.
  2. Flutamide (Sigma, Poole, UK) at a dose of 100 mg/kg dissolved in 2.5% dimethyl sulfoxide (Sigma) in 1 ml/kg corn oil from E15.5 to E20.5.
  3. A combination of treatments 1 and 2.
  4. DMBA (Alfa Aesar, Heysham, UK) at a dose of 400 µg/kg in 1 ml/kg corn oil from E15.5 to E20.5.
  5. A combination of treatments 1 and 4.
  6. Testosterone propionate (TP; Sigma) at a dose of 20 mg/kg in 1 ml/kg corn oil by sc injection from E14.5 to E20.5.
  7. A combination of treatments 1 and 6.
  8. A dose of 1 ml/kg of corn oil alone (control) from E13.5 to E20.5.

The chosen dose of DBP was shown previously to result in a high incidence of TDS-like disorders together with changes in Leydig cell aggregation in fetal testes/dysgenetic areas in adulthood and to induce widespread occurrence of multinucleated gonocytes (MNG) in the fetal testis (21, 22, 29, 30). The DBP was 99% pure according to the supplier and treatment commenced at E13.5, around the time of seminiferous cord formation. Administration of more than 24 mg/kg flutamide, to pregnant rats, has been shown to induce major reproductive tract abnormalities in the male including complete prevention of masculinization of the external genitalia and anogenital distance (31, 32). We chose a dose of 100 mg/kg with the aim of maximally antagonizing testosterone action within the testis, without producing a toxic effect in the dam. Flutamide treatment commenced at E15.5, the point at which androgens are first produced by the fetal rat testis (4). Previous publications have shown that a single dose of 1–20 mg/kg DMBA induces DNA damage and mammary tumors in adult rats (33, 34); consequently, we chose a lower dose to avoid inducing overt DNA damage but to reflect the chronic lower level exposure that would typify PAH exposure from smoking. Based on the evidence that suggests that DMBA has antiandrogenic properties, treatment started at the onset of steroidogenesis, at E15.5. Exposure to 20 mg/kg TP, from E14.5–20.5 (in accordance with Ref. 35) has been shown to rescue the Wolffian ducts in 100% of female pups, with minimal toxic effects (our unpublished data). In preliminary studies lower doses of flutamide (50 mg/kg), DMBA (200 µg/kg), and TP (5 mg/kg) were used, but all were found to have no significant effect on Sertoli cell number. Consequently, we increased the doses to those administered in the present study to increase the likelihood of inducing effects.

Control and treated pregnant dams were killed by inhalation of carbon dioxide on E21.5. Fetuses were removed, weighed, decapitated, and then placed in ice-cold PBS (Sigma-Aldrich). Anogenital distance (AGD) was measured using digital calipers. Testes were removed via microdissection and either snap frozen and stored at –70 C or fixed for 1 h in Bouin’s before being transferred to 70% ethanol. Testes were weighed before processing into paraffin wax using standard methods. Representative fetuses from the aforementioned litters were subsequently used for the quantitative and immunohistochemical studies detailed below. A minimum of three litters was analyzed for each of the treatment groups.

Generation of ARKO mice and testicular feminized mice
The ARKO mice were generated using Cre recombinase (Cre)/loxP technology. ARflox/+ female animals (129/Swiss) with exon 2 of the AR floxed were crossed with phosphoglycerate kinase-1-Cre+/+ male animals (C57BL/6) expressing Cre ubiquitously. Full details are provided elsewhere (36). These mice were killed on postnatal d 2. All animals were treated according to the National Institutes of Health guide for the care and use of laboratory animals, and all experiments were approved by the local ethical committee of Leuven University. The testicular feminized male (tfm) mice were bred at the University of Glasgow on a C3H/HeH-101/H genetic background from stock animals obtained originally from the Medical Research Council Radiobiology Unit (now the Medical Research Council, Mammalian Genetics Unit, Harwell, UK). These mice were killed on E18.5. Appropriate wild-type (WT) controls for ARKO and tfm mice were included in the various analyses.

Testicular testosterone analysis
Testicular testosterone levels in E21.5 control pups (n = 15) and pups in each of the treatment groups [DBP (n = 14), flutamide (n = 5), DBP + flutamide (n = 6), DMBA (n = 5), DBP + DMBA (n = 5), TP (n = 5), and DBP + TP (n = 5)] were measured by RIA, as described previously (21). The limit of detection of the assay was 40 pg/testis. Testicular extracts were not all assayed together. To avoid consequent interassay errors, testosterone levels for testes from treated animals were expressed as a percentage of the mean value for control testes run in the same assay. A similar magnitude of reduction in testicular testosterone levels in DBP-exposed animals was found in each assay run.

Immunohistochemistry
Specific proteins were detected by immunohistochemistry using methods that have been detailed previously (21, 29). Sections of 5 µm were mounted onto coated slides (BDH Chemicals, Poole, UK), dewaxed, and rehydrated. Slides were incubated in 3% (vol/vol) hydrogen peroxidase in methanol to block endogenous peroxidase activity and washed in Tris-buffered saline [0.05 M Tris, 0.85% NaCl (pH 7.4)]. Immunohistochemistry for Wilm’s tumor gene 1 (WT-1; Dako, Cambridgeshire, UK) but not 3ß-hydroxysteroid dehydrogenase (3ß-HSD; gift from Ian Mason) required antigen retrieval by pressure cooking slides for 5 min in 0.01 m citrate buffer (pH 6.0). Nonspecific binding sites were blocked with an appropriate normal serum diluted 1:5 in Tris-buffered saline containing 5% BSA (Sigma) before the addition of the primary antibody (WT-1 used at 1:500, 3ß-HSD used at 1:4000) and overnight incubation at 4 C.

Slides were incubated for 30 min with the appropriate secondary antibody conjugated to biotin at a dilution of 1:500 (rabbit antimouse for WT-1; goat antirabbit for 3ß-HSD; Dako). The biotinylated antibody was linked to horseradish peroxidase by 30 min incubation with avidin-biotin-horseradish peroxidase complex (Dako). Antibody localization was determined by application of diaminobenzidine (liquid DAB+; Dako) until staining in control sections was optimal, and the reaction was stopped by immersing slides in distilled water. Slides were counterstained with hematoxylin, dehydrated, and mounted using Pertex mounting medium (Cell Path, Hemel Hampstead, UK).

Determination of Sertoli number per testis
Standard stereological methods were used to determine Sertoli cell nuclear volume and number per testis at E21.5 in control males (n = 19) and males exposed in utero to DBP (n = 9), flutamide (n = 6), DBP + flutamide (n = 6), DMBA (n = 6), DBP + DMBA (n = 6), TP (n = 8), and DBP + TP (n = 6). Testes from d 2 postnatal WT and ARKO males (n = 5) were also assessed. Animals in each group were derived from at least three litters and were analyzed using methods detailed elsewhere (37, 38, 39). In brief, cross-sections of testes immunostained for WT-1 to stain Sertoli cell nuclei were examined under oil immersion using a x63 plan apo objective fitted to an Olympus BH-2 microscope (Olympus, London, UK) and a 121-point eyepiece graticule. Fifteen fields were selected and counted using a systematic clock-face sampling pattern from a random starting point; points falling over Sertoli cell nuclei were scored and expressed as a percentage of the total points counted. For each animal, the values for percentage nuclear volume were converted to absolute nuclear volumes per testis by multiplying by testis weight (equivalent to volume) because shrinkage was minimal. Sertoli cell nuclear volume in each animal was also determined using methods similar to those described previously (37, 39). Briefly, images were captured from an BH2 microscope (Olympus, Tokyo, Japan) fitted with an automatic stage (Prior Scientific Instruments Ltd., Cambridge, UK) using a video camera (HV-C20; Hitachi, Tokyo, Japan) and were analyzed with Image-Pro Plus 4.5.1 software with a Stereology 5.0 plug-in (Media Cybernetics, Wokingham, Berkshire, UK). An area of interest was created by drawing around the Sertoli cell nucleus, within which the computer program then determined the average length of several diameters measured at 2° intervals, which passed through the center of the nucleus. This was measured for a minimum of 90 Sertoli cell nuclei per testis and mean nuclear volume then determined. Data for Sertoli cell nuclear volume per testis was then converted to absolute numbers of Sertoli cell per testis by dividing by the average Sertoli cell nuclear volume.

Leydig cell aggregation analysis
To determine whether the distribution of Leydig cells throughout the fetal testis was altered by treatment, Leydig cell aggregation was quantified by analyzing the number and size of Leydig cell clusters using methods previously described (29). Testes from at least five pups from each of the treatment groups [control (n = 5), DBP (n = 5), flutamide (n = 5), DBP + flutamide (n = 5), DMBA (n = 5), DBP + DMBA (n = 5), TP (n = 6), DBP + TP (n = 8), and WT and tfm mice (n = 3) for each] were serially sectioned and three representative sections from each testis then selected and immunostained for 3ß-HSD. The three sections chosen were those corresponding to approximately 25, 50, and 75% intervals through the serially sectioned testis; at E21.5 in rats this corresponded to sections that were 20–30 sections apart from each other. Sections from at least three separate litters from each treatment group were used for analysis. Quantification of Leydig cell cluster number and area in these sections was undertaken using Image-Pro Plus 4.5.1 software and equipment as described above. Specimens immunostained for 3ß-HSD were of sufficient homogeneity, high contrast, and low background to allow computer-assisted thresholding and subsequent computer-assisted counting of Leydig cell (3ß-HSD immunopositive) clusters and determination of Leydig cell cluster area. Digital images of complete testis sections were captured at x4 magnification. The software was used to trace around each section, creating an area of interest, allowing the area of each section to be calculated. Computer-assisted thresholding was then used to identify and analyze clusters of 3ß-HSD-immunopositive cells, generating data on cluster number, area, and the proportion of each section occupied by each Leydig cell cluster. Each cluster was then expressed as a percentage of the total Leydig cell cluster area in that animal and was then assigned to one of three groups: small clusters, which accounted for 5% or less of the total Leydig cell cluster area per testis; medium clusters, which accounted for 5.1–14.9%; and large clusters, which accounted for 15% or more of the total Leydig cell cluster area per testis. This approach was necessary to take account of the reduction in total Leydig cell area per testis in DBP-exposed animals due to reduction in Leydig cell size (29).

Multinucleated gonocyte analysis
Testes from control (n = 24), DBP (n = 10), flutamide (n = 9), DBP + flutamide (n = 15), DMBA (n = 7), DBP + DMBA (n = 9), TP (n = 6), and DBP + TP (n = 8) exposed animals were assessed (animals were from at least three separate litters) as well as WT and tfm mice (n = 3). They were sectioned and a representative section stained with toluidine blue to enable clear visualization of the gonocytes. Slides were dewaxed and rehydrated as outlined above. The toluidine blue stain (BDH Chemicals) was filtered and applied to slides at a 50% dilution with distilled water. Once staining was optimal, the slides were immersed in distilled water and then dehydrated and mounted as normal. Stereological analysis of the occurrence of MNG was performed using Image-Pro Plus software and equipment as described above. Each testis section was analyzed to determine the percentage of tubules containing one or more MNG.

Image capture
Images were examined and photographed using a Provis microscope (Olympus Optical, London, UK) fitted with a DCS330 digital camera (Eastman Kodak, Rochester, NY). Images were compiled using Photoshop 7.0 (Adobe Systems Inc., Mountain View, CA).

Statistical analysis
Values are expressed as means ± SEM, and data were analyzed using Student’s unpaired t test or one-way ANOVA followed by the Bonferroni post-test, comparing all single and combined treatments with controls and with each other, using GraphPad Prism (version 4; GraphPad Software Inc., San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Body weight and testis weight
Fetal body weight was not affected by treatment with DBP, flutamide, or DMBA alone but was reduced significantly, and to a variable degree, by treatment with DBP + flutamide, DBP + DMBA, DBP + TP, and TP (Table 1Go). Treatment with TP ± DBP caused the most marked decrease (25–34%) in body weight. Exposure to DBP resulted in a 27% decrease in testis weight, compared with control animals, and in males exposed to DBP + TP, testis weight was decreased by a further 14%, although this was not significantly different from values for DBP alone (Fig. 1AGo). Flutamide exposure on its own did not induce a reduction in testis weight but, when combined with DBP treatment, resulted in a 37% reduction in testis weight, which was greater than the reduction in animals exposed to DBP alone, although this was not statistically significant (Fig. 1AGo). DMBA exposure did not cause a significant reduction in testis weight but, when combined with DBP, resulted in a significant 23% decrease (Fig. 1AGo). Testis weight in ARKO males (postnatal d 2) was reduced by 29% (0.53 ± 0.04; mean ± SEM, n = 12) in comparison with WT littermates (0.75 ± 0.06; mean ± SEM, n = 10). Some of the aforementioned differences in testis weight between treatment groups were not evident when testis weight was expressed relative to body weight, but overall there was no consistent relationship between treatment-induced reduction in body weight and testis weight (Table 1Go) or between body weight and the other testicular parameters reported below, including for Sertoli cell number (P = 0.146).


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  		TABLE 1. Body weights and testis weights per gram of body weight in rats at E21.5 in all treatment groups and at d 2 in mice

 

Figure 1
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  		FIG. 1. Testis weights (A), intratesticular testosterone levels (B), and anogenital distance (C) at E21.5 in male rats exposed in utero to flutamide, DMBA, TP, DBP or DBP + flutamide, DBP + DMBA, or DBP + TP. Values are means ± SEM from a minimum of three litters (n = 5–28 animals/treatment group). *, P < 0.05, **, P < 0.001 in comparison with respective control value. Dashed line shows the mean control level. None of the treatments combined with DBP induced effects significantly different from treatment with DBP alone.

 
Testicular testosterone and anogenital distance (E21.5)
Testicular testosterone levels were significantly reduced in animals exposed to DBP (48% reduction), DBP + flutamide (48%), DBP + DMBA (69%), and DBP + TP (89%), compared with controls; none of the testosterone values for combined treatments, including for DBP + TP, differed significantly (P > 0.05) from treatment with DBP alone. Exposure to either DMBA or TP alone caused a 42% reduction in testicular testosterone, but this was not statistically significant due to high between-animal variation (Fig. 1BGo). In male pups exposed to flutamide alone or flutamide + DBP, AGD was significantly decreased such that values were similar to those found in female control animals (Fig. 1CGo). Exposure to DBP alone caused an average 11% reduction in AGD, but this did not reach statistical significance, and similar results were found for DBP + DMBA exposed males (Fig. 1CGo). Exposure to TP alone increased AGD by 22% on average and, when administered with DBP, reversed the reduction in AGD caused by DBP, so there was an overall increase of 14% in AGD when compared with the average control male.

Sertoli cell number per testis
Sertoli cell nuclei were identified by immunostaining for WT-1 (Fig. 2AGo) and then enumerated using standard stereological techniques. ARKO mice exhibited a 50% reduction in Sertoli cell number when compared with phosphoglycerate kinase-1-Cre (control) mice (Fig. 2BGo), and tfm mice have previously been shown to exhibit a reduction of similar magnitude at birth (8). Sertoli cell number per testis was reduced by 49% in animals exposed to DBP alone (Fig. 2BGo). Animals exposed to TP alone also exhibited a significant reduction (33%) in Sertoli cell number, and when DBP + TP treatments were combined, a 59% reduction in Sertoli cell number was evident. Exposure to flutamide alone had no effect on Sertoli cell number, but animals exposed to both DBP + flutamide exhibited a slightly larger reduction in Sertoli cell number (56%) than did DBP treatment alone (49% reduction; Fig. 2BGo). Exposure to DMBA alone had no significant effect on Sertoli cell number and combined treatment with DBP + DMBA did not result in any greater reduction in Sertoli cell number than after treatment with DBP alone (Fig. 2BGo).


Figure 2
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  		FIG. 2. A, Photomicrograph demonstrating the Sertoli cells (WT-1-positive, brown) in the testis of an E21.5 animal exposed in utero to corn oil (control). Scale bar, 50 µm. B, Effect of in utero exposure to flutamide, DMBA, TP, DBP or DBP + flutamide, DBP + DMBA, or DBP + TP on Sertoli cell numbers in E21.5 rat testes. Values are means ± SEM from a minimum of three litters (n = 6–19 animals/group). For comparison, data are also shown for WT and ARKO mice (n = 5 each) at postnatal d 2. *, P < 0.05, **, P < 0.001 in comparison with respective control value. ***, P = 0.0004 in comparison with WT. Dashed line shows the mean control level. None of the treatments combined with DBP induced effects significantly different from treatment with DBP alone.

 
Leydig cell aggregation
Changes in Leydig cell distribution were clearly visible in all DBP-exposed treatment groups when compared with controls (Fig. 3Go), with the result that abnormally large Leydig cell clusters were present in the center of the testes and there was a corresponding decrease in the number of small Leydig cell clusters. Leydig cell distribution in testes from animals exposed to flutamide, DMBA or TP alone were not obviously different from control sections (not shown). Quantification of Leydig cell aggregation by cluster analysis revealed a significant reduction in the percentage of total Leydig cell cluster area accounted for by small Leydig cell clusters in animals exposed to DBP alone or DBP combined with flutamide, DMBA, or TP (Fig. 4AGo). Leydig cell clusters of medium size were evident in all treatment groups (not shown). Large Leydig cell clusters were not detected in controls but were detected in all treatment groups. However, the induction of large Leydig cell clusters was only significant in animals exposed to DBP (32%), DBP + DMBA (34%), or DBP + TP (31%) (Fig. 4BGo). The distribution of Leydig cells in tfm mice at E18.5 did not differ from that in the controls (Fig. 4CGo). The same analysis was not undertaken for the ARKO mice because tissue was available only at postnatal day 2, but close inspection of 3ß-HSD-immunostained sections showed no evidence of Leydig cell aggregation.


Figure 3
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  		FIG. 3. Representative photomicrographs illustrating the distribution of Leydig cells (3ß-HSD positive, brown) in control (A) and DBP-exposed (B) testes from E21.5 rats. Note the reduced numbers of Leydig cell clusters, especially small ones, in the testis of the DBP-exposed animal and the presence of several large Leydig cell clusters that are not evident in the control.

 

Figure 4
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  		FIG. 4. The occurrence of small (A) and large (B) Leydig cell clusters in the testes of E21.5 rats exposed in utero to corn oil (control), flutamide, DMBA, TP, DBP or DBP + flutamide, DBP + DMBA, or DBP + TP. Values are means ± SEM for n = 5–8 animals/treatment group from a minimum of three litters. **, P < 0.001 in comparison with respective control value. C, The occurrence of small, medium, and large Leydig cell clusters in the testes of wild-type and tfm mice at E18.5. Values are means ± SEM for three animals from three litters. Dashed line shows mean control level. None of the treatments combined with DBP induced effects significantly different from treatment with DBP alone.

 
Occurrence of multinucleated gonocytes
All treatment groups, including the control group, exhibited MNG within the seminiferous cords (Fig. 5Go). In utero exposure to DBP resulted in a 44-fold increase in the percentage of cords exhibiting MNG (Fig. 5Go), and combined treatment with DBP plus either flutamide, DMBA, or TP caused a similar level of increase in MNG (Fig. 5Go). Exposure to flutamide, DMBA, or TP alone did not produce a significant increase in the occurrence of MNG, compared with controls (Fig. 5Go). The tfm mice did not show an increase in MNG occurrence when compared with controls (Fig. 5Go), and similar analysis of the ARKO testes at postnatal day 2 showed no evidence for increased MNG (data not shown).


Figure 5
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  		FIG. 5. Occurrence of MNG at E21.5 in testes of rats exposed in utero to corn oil (control), flutamide, DMBA, TP, DBP or DBP + flutamide, DBP + DMBA, or DBP + TP. The top panel illustrates the occurrence of MNGs (arrows) in males exposed to corn oil (A), flutamide (B), DBP (C), or DBP + flutamide (D). Scale bar shows 50 µm. The graphs (E and F) in the lower panel show the percentage of seminiferous cords exhibiting MNGs in the different treatment groups. Values are means ± SEM for seven to 21 animals per group from at least three litters per group. **, P < 0.001 in comparison with control values. For comparison, data are also shown for wild-type and tfm mice (means ± SEM for three animals from three litters). None of the treatments combined with DBP induced effects significantly different from treatment with DBP alone.

 
Androgen dependence of the evaluated end points
Potential relationships/correlations between the intratesticular levels of testosterone and the various endpoints of testicular dysgenesis were evaluated by plotting the mean intratesticular testosterone values (expressed as a percentage of intraassay control) vs. each of the evaluated endpoints. Because the latter were not measured in the same animals as those in which the testosterone levels were evaluated, only group mean values were used for analysis. There was no clear relationship between intratesticular testosterone levels and AGD (Fig. 6AGo). In contrast, there was a clear positive correlation between intratesticular testosterone levels and Sertoli cell number (Fig. 6BGo). Conversely, there was a negative correlation (Fig. 6CGo) between intratesticular testosterone levels and the percentage of large Leydig cell clusters as well as between intratesticular testosterone levels and the occurrence of MNG (Fig. 6DGo).


Figure 6
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  		FIG. 6. Correlations on E21.5 between intratesticular testosterone levels and AGD (A), Sertoli cell number (B), the percentage of large Leydig cell clusters (C), and the occurrence of multinucleated gonocytes (D). Each data point represents the mean value for each treatment group. Intratesticular testosterone levels have been expressed as the percentage of the mean control value in the same testosterone assay (see Materials and Methods).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The aim of this study was to try to establish what role, if any, intratesticular testosterone action may play in the origins of testicular dysgenesis/maldevelopment, using an animal model. Specifically, the present study sought to establish whether altered androgen levels/action within the fetal testis contributed causally to the reduction in Sertoli cell proliferation/numbers, the occurrence of MNG, and the abnormal aggregation of fetal Leydig cells seen in animals exposed in utero to DBP. To investigate this, pregnant dams were exposed to a variety of treatments and cotreatments, designed to manipulate testosterone levels (DBP, TP) or action (flutamide and DMBA) in the testis during fetal development. The end points selected for investigation have been shown previously to be induced by DBP treatment, coincident with a major reduction in intratesticular testosterone levels (21, 25, 26, 27, 28, 29, 30). The role of androgens in regulating Sertoli cell proliferation/number was of particular interest because evidence from ARKO and tfm mice have both shown a significant reduction in Sertoli cell number or volume per testis, in the absence of a functioning AR (8, 9). The present data demonstrate that exposure to DBP alone, or after cotreatment with flutamide, DMBA or TP, causes a reduction in both Sertoli cell number and intratesticular testosterone levels and, unexpectedly, that the coadministration of DBP + TP resulted in the most severe reduction in both of these parameters; TP treatment alone also reduced both parameters to some extent. It was also surprising that flutamide exposure had no significant effect on either of the parameters despite completely preventing masculinization of AGD. DMBA had no significant effect on either Sertoli cell number or intratesticular testosterone levels. At the treatment group level, the results clearly demonstrate a positive correlation between intratesticular testosterone and Sertoli cell number, supporting the view that androgens are involved in regulating Sertoli cell proliferation/number in fetal life. A similar comparison also suggested a possible relationship between suppression of intratesticular testosterone and the occurrence of MNG and the formation of fetal Leydig cell clusters, as seen in each of the treatment groups in which DBP was administered alone or together with other treatments. However, analysis of tfm and/or ARKO mice showed that, in the absence of androgen action, these animals did not exhibit an abnormal frequency of MNG or evidence for large Leydig cell clusters. If androgen action on the fetal testis is comparable in mice and rats, this suggests it is more likely that the latter two endpoints are not androgen dependent but are instead induced via some other effect(s) of the DBP treatment.

It was initially hoped that exposing animals to flutamide would be the most straightforward way of determining what role, if any, androgens play in the intratesticular changes seen in DBP-exposed animals, and in particular their involvement in Sertoli cell proliferation/number. It became apparent, however, that although flutamide had very pronounced effects at peripheral sites of androgen action, namely reduction of the male AGD to that of a typical female, it had no obvious effect on the testis when administered on its own. There are two possible, and opposing, explanations for this. The first is that flutamide does not cause any changes to the end points evaluated because none of them are androgen dependent. The second is that levels of testosterone within the testis are too high for flutamide to effectively antagonize them. Whereas the former possibility cannot be ruled out, there are two pieces of data that support the latter interpretation. First, this dose of flutamide fails to induce regression of the Wolffian duct in exposed males (40), consistent with the higher local testosterone levels in this duct, compared with peripheral sites (e.g. prostate, perineum), and testosterone levels within the testis are presumably higher than in the Wolffian duct. Second, combined exposure to DBP + flutamide induced a 17% larger decrease in Sertoli cell number than did exposure to DBP alone, which may mean that flutamide is marginally effective when intratesticular testosterone levels are markedly subnormal due to DBP treatment. An additional possibility is that flutamide exposure elevates endogenous FSH levels in male fetuses, as it does in neonatal male rats (38), which would increase Sertoli cell proliferation and thus counteract any antiandrogenic effect on this parameter.

It has already been well established that exposure to DBP results in a reduction in intratesticular testosterone (21, 22, 23, 24) and, in postnatal life, a reduced AGD (41), and our present data confirmed this, although the reduction in AGD evident at E21.5 in the present studies was slight and nonsignificant. All of the groups exposed to either DBP alone, or with a cotreatment, showed a reduction in intratesticular testosterone and a concurrent and equivalent reduction in Sertoli cell number. It cannot be excluded that DBP gave rise to this reduction in Sertoli cell number via a non-testosterone-dependent mechanism. However, if this were the case, it would fail to account for the reduction in Sertoli cell number observed in ARKO and tfm mice (this study and Refs. 8 , 9), and there is also evidence supporting a role for androgens in increasing Sertoli cell number in immature monkeys (42, 43, 44) and neonatal rats (38). Consequently, the present findings on changes in Sertoli cell number in rats are most plausibly explained by the change in intratesticular testosterone levels. Studies involving DBP administration to normal and ARKO mice could clarify this more definitively, but this may be difficult in practice because it has been shown previously that mice are less sensitive to DBP exposure, at least postnatally (45).

Our primary intention in treating with TP was to ascertain which, if any, of the DBP effects could be prevented if the DBP-induced reduction in intratesticular testosterone levels were attenuated, thus confirming that they were testosterone dependent. Surprisingly, TP did not reverse any of the DBP-induced effects, although it was able to successfully masculinize AGD, induce prostate formation, and stabilize the Wolffian duct in females, and to some extent increase AGD in males, when administered alone or with DBP. Therefore, whereas this dose of TP was clearly biologically active at more peripheral sites, it was unable to restore intratesticular testosterone levels in DBP-exposed animals to normal. In fact, animals exposed to DBP + TP had lower intratesticular testosterone levels than did those exposed to DBP alone and treatment with TP alone also resulted in a (nonsignificant) reduction in intratesticular testosterone levels. The most logical interpretation of these findings is that TP treatment caused a reduction in LH levels via increased negative feedback at the hypothalamic-pituitary axis (46, 47, 48), and thus reduced LH drive to testosterone production, and consequently this exacerbated the reduction in intratesticular testosterone caused by DBP alone. The reduction in Sertoli cell number observed in animals exposed to TP alone could be explained by this suppression of androgen action, or it could be due to a reduction in FSH stimulation because neonatal rats treated with testosterone have been shown to have reduced levels of FSH and reduced Sertoli cell number (38). Exposure to DBP + TP resulted in the greatest reduction in Sertoli cell number, the biggest increase in occurrence of MNG and one of the largest increases in large Leydig cell clusters. However, because treatment with TP ± DBP also caused quite marked decreases in fetal body weight, consistent with previous reports (35), it is also possible that growth restriction may have contributed to the effect on Sertoli cell number.

DMBA treatment was undertaken in response to earlier in vitro evidence that suggested that it could antagonize testosterone action distal to AR binding, thus providing a credible explanation for the presumed reduction in Sertoli cell number in men whose mothers smoked during pregnancy and who display a pronounced decrease in testis size and sperm count (13, 14). However, in the present studies, DMBA exposure did not induce any significant effects on measured parameters, including Sertoli cell number, when administered on its own, and cotreatment with DBP + DMBA did not obviously exacerbate any of these parameters, compared with DBP treatment alone. Therefore, at the dose used in this study, no evidence was obtained to support the hypothesis that DMBA antagonizes androgen action either peripherally or intratesticularly.

The data from this study have provided strong evidence that androgens are involved in the regulation of fetal Sertoli cell proliferation, consistent with present and earlier observations in ARKO mice (9). Our findings also provide evidence that reduced intratesticular testosterone levels could be involved in the etiology of MNG formation and fetal Leydig cell aggregation, although this evidence is not as convincing as the data for Sertoli cell number because it is not supported by parallel findings from the ARKO and tfm mice because neither of these end points were identified as being affected in these animals. Irrespective of which of the measured effects are testosterone dependent, they probably all occur via indirect mechanisms because neither the Sertoli cells nor germ cells express AR during fetal life (10, 11) and only a proportion of the fetal Leydig cells express AR. These indirect pathways have yet to be explored, but any effects that androgens have on Sertoli cells are expected to involve the AR-positive peritubular myoid cells (10, 11). It is important to note that any studies undertaken to identify these pathways will have to take into account lessons from the present study on how best to manipulate intratesticular androgen action without compromising steroid production/action essential to pregnancy and other fetal functions. For certain, our findings emphasize that the intratesticular and peripheral effects of androgen action are differentially susceptible to treatments that are designed to manipulate androgen levels/action, and the present study demonstrates that it is vital to measure intratesticular testosterone levels to make sense of results regarding testicular changes.


   Acknowledgments
 
We thank Professor Ian Mason for antiserum to 3ß-HSD and Mark Fisken for expert animal husbandry.


   Footnotes
 
This work was partly supported by the U.K. Medical Research Council and partly by Grant QLK4-CT-200-00603 from the European Union.

Disclosure Summary: The authors have nothing to disclose.

First Published Online February 8, 2007

Abbreviations: AGD, Anogenital distance; AR, androgen receptor; ARKO, AR knockout; Cre, Cre recombinase; DBP, di(n-butyl) phthalate; DMBA, 7,12-dimethyl-benz[a]anthracene; E, embryonic day; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase; MNG, multinucleated gonocytes; PAH, polycyclic aromatic hydrocarbon; TDS, testicular dysgenesis syndrome; tfm, testicular feminized male; TP, testosterone propionate; WT, wild type.

Received December 5, 2006.

Accepted for publication January 26, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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