help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2008-0413
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
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 HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Scott, H. M.
Right arrow Articles by Sharpe, R. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Scott, H. M.
Right arrow Articles by Sharpe, R. M.
Endocrinology Vol. 149, No. 10 5280-5287
Copyright © 2008 by The Endocrine Society

Relationship between Androgen Action in the "Male Programming Window," Fetal Sertoli Cell Number, and Adult Testis Size in the Rat

Hayley M. Scott, Gary R. Hutchison, Matthew S. Jobling, Chris McKinnell, Amanda J. Drake and Richard M. Sharpe

Medical Research Council Human Reproductive Sciences Unit, Centres for Reproductive Biology (H.M.S., G.R.H., M.S.J., C.M., R.M.S.) and Cardiovascular Science (A.J.D.), Queen’s Medical Research Institute, Edinburgh EH16 4TJ, 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
 
Fetal androgen action is an important determinant of Sertoli cell (SC) number at birth. Androgens "program" reproductive tract development in rats between embryonic d (e) 15.5 and e17.5 ("male programming window"), and this is reflected for life by anogenital distance (AGD). We investigated if androgen regulation of SC number/proliferation was also programmed by androgens in this window. Pregnant rats were treated in various fetal time windows with vehicle (control) or 500 mg/kg·d di(n-butyl) phthalate (DBP), which suppresses fetal intratesticular testosterone (ITT). ITT and SC number/proliferation index were determined at e17.5 or e21.5; AGD was also determined at e21.5. In controls, SC number increased 11-fold and ITT by 10-fold from e17.5–e21.5. In animals exposed daily to DBP from e13.5, SC number was reduced by approximately 50% at e21.5, but increased 6-fold, as did ITT, from e17.5–e21.5; DBP had no effect on ITT at e15.5, reduced ITT by 50% at e17.5, and by more than 75% at e19.5–21.5. DBP exposure just in the male programming window did not alter SC number at e17.5 or 21.5 but reduced AGD. DBP treatment beyond e19.5 caused major reductions in SC number/proliferation index and ITT at e21.5. Only DBP treatments that included the male programming window led to reduced AGD at e21.5, but SC number was clearly not programmed in this window. Nevertheless, testis weight correlated highly (P < 0.001) with AGD at e21.5, and postnatal d 25 and 90 in animals exposed in utero to vehicle or DBP (e13.5–e21.5). Thus, AGD may predict adult testis size but probably not through a direct relationship with SC number.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DISORDERS OF human male reproductive health that manifest at birth (cryptorchidism, hypospadias) or in young adulthood (low sperm counts, testicular germ cell cancer) are common and/or increasing progressively in incidence (1, 2, 3). These disorders are risk factors for each other and share other pregnancy related risk factors (2, 3). Based on this and other information, these disorders have been hypothesized to comprise a testicular dysgenesis syndrome (TDS) with a common origin in fetal life (2). Of the TDS disorders, the most prevalent is low sperm counts, with recent data for young men in several European countries suggesting that 20% or more may have an abnormally low sperm count, relative to World Health Organization criteria (4). The most straightforward explanation for low sperm counts in men is a lower number of Sertoli cells because this is the primary determinant of sperm production capacity in adulthood, and both Sertoli cell number and sperm count vary widely in adult men (5). A central component of the TDS hypothesis is that deficient testosterone production (or action) may be responsible for some/all of the downstream disorders mentioned previously (2, 6), but there is little direct data to support this idea. Moreover, until recently there was no obvious mechanism to account for how reduced testosterone action in fetal life might lead to reduced sperm counts in adulthood. However, we and others have recently demonstrated that reduced fetal testosterone levels/action can lead to halving of Sertoli cell numbers in perinatal life in rodents (7, 8, 9). These findings provide strong support for the TDS hypothesis (6).

Another recent development has been the demonstration that androgen-mediated development of a normal male reproductive system occurs via androgen "programming" within a specific fetal time window [embryonic d (e) 15.5–18.5] in the rat, a time window that precedes morphological differentiation and development of the relevant tissues (10). Importantly, the same study showed that cryptorchidism and hypospadias can only occur due to deficient androgen action within this "male programming window." This being the case, an obvious question raised is whether or not androgen regulation of Sertoli cell proliferation/numbers in fetal life is also "programmed" by androgen action within the same time window in which cryptorchidism and hypospadias occur, because if this was the case, it would provide a clear (fetal) mechanistic explanation for the interrelationships between these disorders and low sperm counts in young adulthood as part of TDS. The primary aim of the present studies was to test this hypothesis. To do this we used a rat model of TDS based on in utero exposure to di(n-butyl) phthalate (DBP) because this causes a 40–50% reduction in Sertoli cell numbers at the end of gestation (e21.5) in association with suppression of intratesticular testosterone levels (9), and leads to an increased incidence of cryptorchidism and hypospadias in the male offspring postnatally (11, 12). In the present studies, we varied the fetal time window of exposure to DBP and evaluated the impact on Sertoli cell number and intratesticular testosterone levels at e21.5, as well as on testis size at this age, in puberty and adulthood.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and treatments
Wistar rats were maintained in our own animal facility under standard conditions according to United Kingdom Home Office guidelines, which includes review by an ethical committee. Animals had access ad libitum to water and a soy-free breeding diet (SDS, Dundee, Scotland, UK). Time-mated females were gavaged daily with either 500 mg/kg DBP (Sigma-Aldrich Co. Ltd., Dorset, UK) in 1 ml/kg corn oil or with the vehicle alone (controls) according to the schedules listed below. The dose of DBP administered results in a high incidence of TDS-like disorders (11, 12) together with an approximate 50% reduction in Sertoli cell number at e21.5 after treatment from e13.5-e20.5 (9). The DBP was 99% pure according to the supplier, and treatment was administered between 0900 and 1030 h.

1) Dosing from e13.5–e20.5 (standard/full window), which reduces intratesticular testosterone and Sertoli cell number by 50% or more (9).

2) Dosing from e11.5–e20.5 (extended window), investigated to determine whether increasing the length of DBP exposure before seminiferous cord formation and testosterone production, induced a greater reduction in Sertoli cell number.

3) Dosing from e13.5–e15.5 (early window), restricting DBP exposure to before the "male programming window" (10) and the onset of testosterone production.

4) Dosing from e15.5–e17.5 (middle window), encompassing the "male programming window" (10).

5) Dosing from e19.5–e20.5 (late window), encompassing the period after the "male programming window" but a period within which DBP induces the largest reduction in intratesticular testosterone levels (13), and that also encompasses the onset of FSH secretion and its potential stimulatory effect on Sertoli cell proliferation (14, 15).

For each of the aforementioned DBP treatment regimens, three to eight pregnant dams were used. A preliminary study compared control animals treated with vehicle during the standard/full window with those treated during the other time windows, and because no differences were identified for any parameter investigated (data not shown), control animals used in these studies were all treated with 1 ml/kg corn oil (vehicle alone) during the standard/full window. At 1 h before sampling in fetal life, dams were injected ip with 100 mg/kg 5-bromo-2'-deoxyuridine (BrdU) (Sigma-Aldrich) to label proliferating Sertoli cells.

Animals exposed to DBP or corn oil during one of the treatment windows described above were killed on e21.5 for determination of Sertoli cell number or measurement of intratesticular testosterone, as detailed below. In addition, to determine the effects of DBP treatment on intratesticular testosterone concentrations at earlier ages during the standard/full treatment window, other control or DBP-exposed fetuses were killed on e15.5, e17.5, e19.5, or e21.5, after treatment until 24 h before death. Finally, to evaluate the consequences of standard window DBP treatment (e13.5–e21.5) on postnatal testis size, other animals were killed on postnatal d (pnd) 25 or as adults (~3 months of age), when testis weight and anogenital distance (AGD) (see below) were determined. In addition, in vehicle- and DBP-exposed animals sampled at pnd 25, Sertoli cell number was determined (see below); at this age, final Sertoli cell number has been reached (5). For each of the sampling times referred to, animals originated from at least three, and in most cases five to eight, separate litters. Testes from one or more fetuses from all litters were used for the quantitative and immunohistochemical studies detailed below.

Fetal testis collection and processing
Control and DBP-treated pregnant dams were killed by inhalation of carbon dioxide. Fetuses were removed, weighed, decapitated, and then placed in ice-cold PBS (Sigma-Aldrich). AGD was measured in e21.5 males using digital calipers (Faithfull Tools, Kent, UK) (10). Testes were removed via microdissection, and either snap frozen and stored at –80 C for subsequent testosterone assay (see below) or fixed for 1 h in Bouin’s before transfer to 70% ethanol, followed by processing into paraffin blocks using standard techniques. Testis weights at e21.5 were determined before processing into paraffin wax, but e17.5 testes were too small to weigh, so total testis volume was determined by measuring the length and width of testes, and then calculating their volume using the formula for a prolate spheroid (cigar shaped). A minimum of eight testes from three control and three DBP-exposed litters was microdissected at x40 under an MZ6 dissecting microscope (Leica, Nusslcoh, Germany), and images were captured using a Leica ICA camera. These images were then viewed using Image Pro 6.2 (Media Cybernetics, Wokingham, Berkshire, UK), and the length and width of each testis were measured. Height was assumed to be the same as the width as defined for a prolate spheroid. Volume was then determined using the formula:

Formula
To validate this approach, we compared actual testis weight (= volume) with derived testis volumes for e19.5 fetuses using this approach and found good, consistent agreement.

Postnatal animals
Male offspring of dams that had been treated with vehicle or 500 mg/kg·d DBP from e13.5–e21.5 were killed on pnd 25 or 90 (adulthood) by inhalation of carbon dioxide and subsequent cervical dislocation. Testes were removed and weighed, and AGD was measured as described previously.

Testicular testosterone measurement
Testicular testosterone content was measured by RIA as described previously (11). Testes from control and DBP-exposed animals at e15.5 (n = 6, five litters for control and DBP, respectively), e17.5 (n = 7, 10 litters), e19.5 (n = 6, six rats from four, four litters for control and DBP, respectively), and e21.5 (n = 17, 12 rats from 10, eight litters, respectively) were analyzed to determine normal changes in intratesticular testosterone content during the last week of gestation and the impact of DBP exposure on this. Testes at e15.5 and e17.5 were too small to be analyzed individually, so they were pooled within litters. For the different DBP time window treatments, testes were analyzed individually from each treatment window group at e21.5 with testes (n = 3–20) being derived from animals from a minimum of three different litters. The limit of detection of the testosterone assay was 40 pg/testis.

Immunohistochemistry
Specific proteins were detected by immunohistochemistry using general methods that have been detailed previously (9, 11, 16). Testis 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 and 0.85% NaCl (pH 7.4)]. Immunohistochemistry for Wilms’ tumor gene 1 (WT-1) (Dako, Cambridgeshire, UK), androgen receptor (AR) (Novocastra, Newcastle-upon-Tyne, UK), VASA (Abcam, Cambridge, UK), and BrdU (Roche Diagnostics Corp., Indianapolis, IN) required antigen retrieval by pressure cooking slides for 5 min in 0.01 M citrate buffer (pH 6.0). Immunohistochemistry for 3β-hydroxysteroid dehydrogenase (3β-HSD) used a rabbit antihuman antiserum gifted by Professor J. I. Mason (Edinburgh, UK), and methods detailed elsewhere (16). Nonspecific binding sites were blocked with an appropriate normal serum diluted 1:5 in Tris-buffered saline containing 5% BSA (Sigma-Aldrich) before the addition of the primary antibody (WT-1 used at 1:500, AR used at 1:20, VASA used at 1:200, 3β-HSD used at 1:2000, and BrdU used at 1:2000) 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 and BrdU, porcine antirabbit for VASA, and goat antirabbit for AR and 3β-HSD; all from Dako). The biotinylated antibody was linked to horseradish peroxidase by 30 min incubation with streptavidin-horseradish peroxidase (Dako). Antibody localization was determined by application of liquid diaminobenzidine (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 Hempstead, UK).

Determination of Sertoli cell number per testis
Standard stereological methods were used to determine Sertoli cell nuclear volume and number per testis at e17.5 and e21.5 as described previously (9, 17). In brief, cross-sections of testes immunostained for WT-1 to stain Sertoli cell nuclei were examined under oil immersion using a Leitz x63 plan apo objective fitted to a Leitz Laborlux microscope and a 121-point eyepiece graticule. There were 15 fields 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 percent 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 determined as follows. Images were captured from an Olympus BH2 microscope (Olympus, Hamburg, Germany) fitted with a Prior 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). 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 two-degree intervals, which passed through the center of the nucleus. This approach takes account of the considerable natural variation in shape of the Sertoli cell nucleus and any effects that DBP treatment may have on shape. A minimum of 90 Sertoli cell nuclei per testis was measured for determination of mean nuclear volume. Data for Sertoli cell nuclear volume per testis were then converted to absolute numbers of Sertoli cell per testis by dividing by the average Sertoli cell nuclear volume.

Determination of numbers of peritubular myoid cells (PTMCs), germ cells, and Leydig cells per testis at e21.5
Similar stereological methods as those used to determine Sertoli cell nuclear volume and number per testis were used to determine the number of PTMCs, germ cells, and Leydig cells per testis. Germ cells were identified for counting by immunostaining with the cell-specific marker VASA, Leydig cells by immunostaining for 3β-HSD, and PTMCs by immunostaining for the AR. PTMCs were then identified as AR-immunopositive spindle-shaped cells that were immediately proximal to the seminiferous cords. Cell volumes per testis for each of these components were determined and then converted to cell numbers per testis as described for Sertoli cells. For determination of PTMC nuclear volume, the width and length of at least 90 cell nuclei per animal were measured, and nuclear volume was then calculated on the basis that the nucleus was an oblate ellipsoid.

Determination of the proliferation index (PI) of Sertoli cells
To determine what percentage of Sertoli cells were proliferating at e17.5 and e21.5, cross-sections of testes were immunostained for BrdU to stain proliferating cell nuclei. Sections were examined under the microscope as described previously. There were 15 fields selected and counted using a systematic clock-face sampling pattern from a random starting point. For each field all Sertoli cell nuclei were counted and recorded as either positive for BrdU (proliferating) or negative for BrdU (nonproliferating). The PI was then calculated using the following formula: PI = number of Sertoli cells expressing BrdU x 100/total number of Sertoli cells.

Statistical analysis
Values are expressed as means ± SEM, and data were analyzed using the 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). Data for testicular testosterone levels were log transformed before statistical analysis to normalize variances. Because many of the present analyses involved evaluation of correlations between parameters (e.g. AGD vs. testis weight), statistical analysis used data from individual animals because this is the appropriate comparison unit. For some measurements, such as Sertoli cell number, occasional duplicate animals were used from the same litter, although for most treatment groups/analyses, animals from five to eight litters were used. Reanalysis of data (other than correlations) using litter means did not alter any of the major findings and had only minor effects on P values.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sertoli cell number and proliferation at e17.5 vs. e21.5 and effect of DBP exposure
In controls, Sertoli cell number increased 11.4-fold between e17.5 and 21.5 (Fig. 1AGo), and a similar PI (23–27%) was found at both ages (Fig. 1BGo). In DBP-exposed animals, mean Sertoli cell number was unchanged from controls at e17.5, whereas a highly significant decrease (48%) in cell numbers was evident by e21.5 when it was associated with a significant reduction (64% of control) in the Sertoli cell PI (Fig. 1BGo). However, Sertoli cell number in DBP-exposed animals still increased by 6-fold between e17.5 and e21.5 (Fig. 1AGo).


Figure 1
View larger version (12K):
[in this window]
[in a new window]

  		FIG. 1. Sertoli cell number (A), the Sertoli cell PI (B), and testicular testosterone content (C) at specific ages in fetuses from mothers treated daily since e13.5 with vehicle or 500 mg/kg DBP. The timing of the "male programming window" is shown in C; suppression of testosterone levels after this period has minimal impact on reproductive tract development (10 ). Values are means ± SEM for five to 19 (A), four to six (B), or five to 17 (C) animals at each age, which were derived from three to eight litters. *, P < 0.05; **, P < 0.01; ***, P < 0.001, in comparison with respective control; aP < 0.001, in comparison with respective control value at e17.5; bP < 0.001, in comparison with respective DBP value at e17.5.

 
Effect of DBP exposure on testicular testosterone content between e15.5 and e21.5
In controls, testosterone content per testis increased more than 10-fold from e15.5 to e19.5–e21.5, whereas in DBP-exposed animals, this increase was only approximately 6-fold (Fig. 1CGo). In DBP-exposed animals, mean testosterone content per testis was reduced significantly at e17.5–e21.5, this reduction ranging from 50% at e17.5 to more than 75% at the two later ages.

Effect of DBP exposure in different fetal time windows
AGD was measured as an index of systemic androgen exposure within the "male programming window" (10). As expected, this revealed that DBP exposure during any time window that included the main portion of the male programming window (i.e. e15.5–e17.5) resulted in a significant approximate 20% reduction in AGD in male fetuses at e21.5, whereas restricting exposure to DBP either before (e13.3–e15.5) or after (e19.5–e21.5) this time window had no effect on AGD (Fig. 2AGo). In contrast, only DBP exposure late in gestation and including the day immediately before sampling on e21.5 resulted in a significant reduction in testosterone content per testis on e21.5 when compared with control (Fig. 2CGo). Similarly, Sertoli cell number at e21.5 was only reduced significantly in DBP treatment groups in which testis testosterone levels were reduced at this age, though the trend was for the magnitude of the decrease in Sertoli cell number to get larger with duration of DBP exposure (Fig. 2BGo). DBP exposure just within the male programming window (e15.5–e17.5) had no effect on Sertoli cell number at e21.5, even though this treatment had significantly reduced testis testosterone content at e17.5 (Fig. 1CGo) as well as reducing AGD when measured at e21.5 (Fig. 2AGo).


Figure 2
View larger version (15K):
[in this window]
[in a new window]

  		FIG. 2. AGD (A), Sertoli cell number (B), and testicular testosterone content (C) at e21.5 in fetuses from mothers treated daily with vehicle or 500 mg/kg DBP in specific time windows. AGD values for e21.5 control females are shown in the top panel for comparison with the males. Values are means ± SEM for five to 25 (A), five to 19 (B), or three to 20 (C) animals at each age, which were derived from three to eight litters. *, P < 0.05; **, P < 0.01; ***, P < 0.001, in comparison with respective control males. n/a, Not applicable.

 
Relationship between Sertoli cell number or AGD and testis weight at different ages after fetal exposure to vehicle or DBP
It is established that Sertoli cell number is a key, but indirect, determinant of adult testis size (5). To establish whether Sertoli cells were also an important determinant of testis size at e21.5, we determined the relative numbers of the key cell types in the testis at this age, and also determined the correlation between testis weight and Sertoli cell number. This showed that Sertoli cells are numerically the most abundant cell type in the testis at e21.5, and their numbers correlated significantly with testis weight using data for control and DBP-exposed animals in the e13.5–e20.5 treatment groups (Fig. 3Go). It was not possible to determine whether Sertoli cell number also correlated significantly with AGD at e21.5 because AGD had not been measured in most animals in which Sertoli cell number was determined. Because the effect of DBP exposure to reduce testicular testosterone levels and Sertoli cell number/proliferation did not appear to be restricted to the male programming window (Fig. 2Go), it was expected that AGD, which reflects androgen action only within the male programming window (Fig. 2AGo), would therefore not correlate with testis weight, which will largely reflect Sertoli cell number. However, it was found that testis weight was significantly correlated with AGD at the end of fetal life (e21.5; Fig. 4AGo), in early puberty (pnd 25; Fig. 4BGo), and in adulthood (Fig. 4CGo) when data from vehicle- and DBP-exposed (e13.5–e21.5) animals were assessed together.


Figure 3
View larger version (12K):
[in this window]
[in a new window]

  		FIG. 3. Relative cellular composition of the e21.5 testis in control rats (A) and the correlation between Sertoli cell number and testis weight in control and DBP-exposed rats (B). The proportions shown in the pie chart are based on the percent contribution of each cell type to the overall mean number of cells that were counted and, therefore, does not include non-Leydig interstitial cells or endothelial/perivascular cells, which were not counted. Values for absolute cell numbers (A) (x10–3) are means ± SEM for five to 19 animals from a minimum of five different litters.

 

Figure 4
View larger version (11K):
[in this window]
[in a new window]

  		FIG. 4. Correlation between AGD and testis weight at e21.5 (A), pnd 25 (B), and pnd 90 (C) in males from mothers treated daily from e13.5–e20.5 (A) or e13.5–e21.5 (B and C) with vehicle or 500 mg/kg DBP. Testis weight is based on the mean weight of left and right testes for postnatal animals, some of which were unilaterally or bilaterally cryptorchid (DBP exposed). The depicted lines of best fit were determined by linear regression.

 
In view of this surprising finding, the interrelationship between AGD, testis weight, and Sertoli cell number was determined at pnd 25 in a small sample of controls and rats exposed in utero to DBP from e13.5–e21.5. pnd 25 was chosen because final Sertoli cell number has been fixed (5), and any differences due to testis position are minimal at this age (18). This analysis showed that at pnd 25, there was no significant correlation between Sertoli cell number and AGD (Fig. 5CGo), whereas there was a significant correlation of AGD with testis weight (Fig. 5BGo), and confirmed that AGD was significantly reduced in DBP-exposed animals (Fig. 5AGo). This analysis also confirmed our earlier report (18) that Sertoli cell number per testis in DBP-exposed animals had normalized by pnd 25 (control 44.7 ± 2.1 x 106 cells; DBP 42.9 ± 2.8 x 106; P > 0.5; n = 6 and 9, respectively).


Figure 5
View larger version (9K):
[in this window]
[in a new window]

  		FIG. 5. Correlation between AGD and either testis weight (B) or Sertoli cell number (C) at pnd 25 in control rats (n = 6) and animals exposed in utero to DBP from e13.5–e21.5 (n = 9). Panel A shows that in this subset of animals, DBP causes a significant reduction in AGD (means ± SEM). ***, P < 0.001, in comparison with control value.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The primary goal of the present studies was to establish whether androgen-mediated regulation of fetal Sertoli cell number/proliferation in the rat is programmed by testosterone action only within the recently established "male programming window." In this early fetal time window (within e15.5–e18.5), the development of all of the male reproductive tract structures (internal and external genitalia) that occurs later in gestation and after birth is programmed by androgen action (10). If Sertoli cell number/proliferation was programmed in a similar manner, it would provide an important new insight into how fetal events might affect adult testis size and sperm production, as well as accounting for how low sperm counts in adulthood might be part of the same syndrome of disorders (TDS) as cryptorchidism and hypospadias because these two disorders can only occur via deficient androgen action within the male programming window. However, using our DBP rat model of TDS and varying time windows of DBP exposure (and associated reduction in testicular levels of testosterone), our findings show that impaired androgen action later in gestation, rather than during the male programming window, has the greatest impact on Sertoli cell number at e21.5; the implication is that androgen regulation of Sertoli cell proliferation extends well beyond the male programming window. This would fit with other evidence from rats (19) and monkeys (20, 21) that androgens can also positively regulate Sertoli cell number after birth. As a consequence of this finding, our expectation was that AGD, which reflects androgen action only within the male programming window (10), would not correlate with testis weight, which in fetal life (this study) as in adulthood (5), is largely determined by Sertoli cell number. Paradoxically, we found that testis weight was significantly correlated with AGD at all ages examined from fetal life through to adulthood, an observation for which we have no clear explanation but which has implications regarding the fetal origins of low sperm counts in young adulthood as part of TDS.

We and others have established that deficient androgen action in the fetal rodent testis results in approximately a halving of Sertoli cell number at birth (7, 8, 9). The effect of androgens on Sertoli cell number is indirect and presumed to be mediated via the strongly AR-positive PTMCs (9) because fetal Sertoli cells do not express AR (22), and targeted knockout of AR in Sertoli cells does not affect Sertoli cell number at birth (8). A proportion of fetal Leydig cells expresses AR as do some non-Leydig interstitial cells (unpublished data), so it is also possible that these cells may somehow signal to Sertoli cells to affect proliferation. We had reported earlier that exposure of rats to DBP in utero, leading to 50–90% reductions in intratesticular levels of testosterone, results in a similar reduction (~50%) in Sertoli cell number at birth in rats as does complete knockout of the AR in mice (9). Because deficiency in fetal androgen action is implicated in the common fetal origin of TDS disorders in humans (6), including cryptorchidism, hypospadias, and (in adulthood) low sperm counts, these findings in rodents have given new insight into TDS origins as well as providing a rational explanation as to how fetal events might lead to low sperm counts in adulthood because this would be an inevitable consequence of reduced Sertoli cell number (5). In the present studies, we have confirmed our earlier findings regarding DBP-induced reduction in fetal Sertoli cell numbers at e21.5 but have shown additionally that this effect occurs mainly in the period between e17.5 and e21.5, and especially after e19.5, and is associated with a significant reduction in the Sertoli cell PI. This corresponds to what we have termed the "male differentiation window," which is when male reproductive structures differentiate and grow in the rat (10). In contrast, our present findings show that DBP exposure does not cause any detectable reduction in Sertoli cell number or the PI before e17.5, which is during the male programming window. This was despite the fact that DBP clearly reduced intratesticular testosterone content at e17.5 (but not at e15.5), and clearly affected AGD, which reflects androgen action only within the male programming window (10); these effects confirm earlier studies for both intratesticular testosterone content and AGD (13, 23, 24). More importantly, the present findings show that exposure to DBP just within the male programming window (e15.5–e17.5) does not result in any later (programmed) reduction in Sertoli cell number at e21.5, during the male differentiation window. These observations appear to exclude the possibility that androgen-mediated Sertoli cell proliferation in fetal life is programmed by androgen action at the same time as is the development of the male reproductive tract (10).

One factor not considered in our time window experiments with DBP is the possibility that different levels of exposure occur at different fetal ages because of differing ability to metabolize DBP to monobutyl phthalate (MBP), which is considered to be the active compound, or to inactivate MBP via glucoronidation. The limited studies published (25, 26) suggest that availability of MBP to the rat fetus might be higher at e18.5 than e20.5, so greater exposure to MBP may occur at the earlier time for the same administered dose. In contrast, our findings suggest that suppression of testosterone production and Sertoli cell number/proliferation is more marked later (>e19.5) than earlier (e15.5–e17.5) in gestation. This suggests that age-dependent differences in the availability of MBP to the fetal testis are unlikely to provide a straightforward explanation for the current findings.

Sertoli cell number is an important determinant of testis size (5). At e21.5 the Sertoli cells are numerically the predominant cell type present in the testis, as demonstrated in the present studies. In contrast, in adulthood they are a relatively minor component because germ cells comprise the majority of the volume of the testis (18, 27). However, because germ cell number per testis in adulthood is determined by Sertoli cell number (27, 28, 29), it is accepted that Sertoli cells indirectly determine adult testis size/volume, at least for scrotal testes (5, 27, 29). Because the present findings found no evidence for androgens determining Sertoli cell number within the male programming window, within which AGD is determined (10), our expectation was that AGD would show no obvious correlation with testis size at any age. This expectation was reinforced by our earlier demonstration (and confirmed in the present study) that male rats with a 50% reduction in Sertoli cell numbers at birth, as a consequence of in utero exposure to DBP from e13.5–e21.5, apparently compensate for this deficit and restore normal numbers of Sertoli cells by pnd 25 (18). In stark contrast to these expectations, the present findings show that AGD is a highly significant predictor (P < 0.001) of testis size at e21.5, as well as at puberty and in adulthood. In searching for an explanation for this paradox, we first considered that because AGD is a predictor of cryptorchidism (10), and the latter will obviously reduce testis size in adulthood, the correlation between AGD and testis size might be a consequence of both parameters being correlated independently with cryptorchidism. However, this would fail to explain the correlation between AGD at pnd 25 [when cryptorchid testes differ little in weight from scrotal testes (25)] and particularly the correlation at e21.5, when all testes are still located within the abdomen.

All of the AGD vs. testis weight correlation analyses in the present studies involved vehicle- and DBP-exposed animals that had been treated from e13.5–e21.5, and it is possible that the combination of DBP exposure in the male programming window (which will result in reduced AGD due to reduced androgen action) with continued exposure in later gestation (which will result in reduced Sertoli cell number due to reduced androgen action) results in a correlation between the two effects because of the common denominator of reduced androgen levels/action, even though the effects of the latter on the two different endpoints appear to be entirely separated in time. This might provide a logical explanation for our findings, at least at e21.5, although as normal Sertoli cell numbers are then restored postnatally in DBP-exposed animals, as shown in the present and a previous study (18), it is difficult to explain why AGD remains similarly correlated with testis weight during puberty and in adulthood. Indeed, by pnd 25, when final Sertoli cell number has been determined (5), we could find no significant correlation between AGD and Sertoli cell number, even though there was a significant correlation of AGD with testis weight. This suggests that some other factor(s), programmed by androgen action within the male programming window along with AGD, is able to affect final testis size, but it is not clear what this might be.

One drawback of the present experimental approach is that, although DBP exposure clearly reduces testosterone levels within the fetal testis, we cannot exclude the possibility that some, or all, of the reduction in Sertoli cell numbers induced by DBP-exposure could result from a direct effect on the Sertoli cells (18) rather than stemming indirectly from the reduction in testosterone levels. It is established that DBP and certain other phthalates can directly affect Sertoli cells in the rat postnatally (30), and it is also clear that in fetal life, several of the reported effects of DBP exposure on the testis (e.g. germ cell effects) are unrelated to testosterone suppression (31, 32). Nevertheless, in our present studies, the magnitude of increase in testicular testosterone content and Sertoli cell numbers paralleled each other in both controls and DBP-exposed animals, and, in the latter, the period of maximum suppression of testosterone levels (e19.5–e21.5) coincided with the period when maximum reduction in Sertoli cell numbers could be induced. Our attempts to use alternative methods of reducing intratesticular testosterone levels/action using flutamide (9) or a steroidogenesis inhibitor (unpublished data) have proved unsuccessful for technical reasons. Therefore, the only alternative approach is transgenic manipulation of AR expression, but even though cell-specific knockouts of the AR are available and have considerable utility in addressing androgen regulation of fetal Sertoli cell proliferation (8), what would really be needed is a time window-selective, inducible, cell-specific AR knockout model, if the issues raised in the present studies are to be addressed effectively.

In conclusion, the present studies have shown unequivocally that DBP-induced reduction in Sertoli cell numbers in the fetal rat testis, which is thought to occur secondary to suppression of intratesticular testosterone levels, is not confined to the recently identified male programming window. Instead, the most pronounced effect of DBP exposure on Sertoli cell proliferation/numbers and on intratesticular testosterone levels occurs late in gestation. Despite this finding, our results show that AGD, which reflects (systemic) androgen action only within the male programming window, remains strongly correlated with testis weight from fetal life through to adulthood. Therefore, in the context of the TDS hypothesis, our results provide support for the suggestion that deficiencies in fetal androgen action are likely to be reflected by reduced testis size and, therefore, sperm production/sperm counts in adulthood. However, this does not appear to involve a straightforward relationship to Sertoli cell number, as had been hypothesized, and the explanation for the relationship between AGD and testis weight remains to be established.


   Footnotes
 
Disclosure Summary: The authors have nothing to disclose.

First Published Online June 19, 2008

Abbreviations: AGD, Anogenital distance; AR, androgen receptor; BrdU, 5-bromo-2'-deoxyuridine; DBP, di(n-butyl) phthalate; 3β-HSD, hydroxysteroid dehydrogenase; e, embryonic day; ITT, intratesticular testosterone; MBP, monobutyl phthalate; PI, proliferation index; pnd, postnatal d; PTMC, peritubular myoid cell; SC, Sertoli cell; TDS, testicular dysgenesis syndrome; WT-1, Wilms’ tumor gene 1.

Received March 26, 2008.

Accepted for publication June 11, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Toppari J, Larsen JC, Christiansen P, Giwercman A, Grandjean P, Guillette Jr LJ, Jégou B, Jensen TK, Jouannet P, Keiding N, Leffers H, McLachlan JA, Meyer O, Müller J, Rajpert-De Meyts E, Scheike T, Sharpe RM, Sumpter JS, Skakkebaek NE 1996 Male reproductive health and environmental xenoestrogens. Environ Health Perspect 104(Suppl 4):741–803
  2. Skakkebaek NE, Rajpert-De Meyts E, Main KM 2001 Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects. Hum Reprod 16:972–978[Abstract/Free Full Text]
  3. Skakkebaek NE, Rajpert-De Meyts E, Jorgensen N, Main KM, Leffers H, Andersson A-M, Juul A, Jensen TK, Toppari J 2007 Testicular cancer trends as ‘whistle blowers’ of testicular developmental problems in populations. Int J Androl 30:198–205[CrossRef][Medline]
  4. Jorgensen N, Asklund C, Carlsen E, Skakkebaek NE 2006 Coordinated European investigations of semen quality: results from studies of Scandinavian young men is a matter of concern. Int J Androl 29:54–61[CrossRef][Medline]
  5. Sharpe RM, McKinnell C, Kivlin C, Fisher JS 2003 Proliferation and functional maturation of Sertoli cells, and their relevance to disorders of testis function in adulthood. Reproduction 125:769–784[Abstract]
  6. Sharpe RM, Skakkebaek NE 2008 Testicular dysgenesis syndrome: mechanistic insights and potential new downstream effects. Fertil Steril 89(Suppl 1):e33–e38
  7. Johnston H, Baker PJ, Abel M, Charlton HM, Jackson G, Fleming L, Kumar TR, O’Shaughnessy PJ 2004 Regulation of Sertoli cell number and activity by follicle-stimulating hormone and androgen during postnatal development in the mouse. Endocrinology 145:318–329[Abstract/Free Full Text]
  8. Tan KA, De Gendt K, Atanassova N, Walker M, Sharpe RM, Saunders PT, Denolet E, Verhoeven G 2005 The role of androgens in Sertoli cell proliferation and functional maturation: studies in mice with total or Sertoli cell-selective ablation of the androgen receptor. Endocrinology 146:2674–2683[Abstract/Free Full Text]
  9. Scott HM, Hutchison GR, Mahood IK, Hallmark N, Welsh M, de Gendt K, Verhoeven G, O’Shaughnessy PJ, Sharpe RM 2007 Role of androgens in fetal testis development and dysgenesis. Endocrinology 148:2027–2036[Abstract/Free Full Text]
  10. Welsh M, Saunders PT, Fisken M, Scott HM, Hutchison GR, Smith LB, Sharpe RM 2008 Identification in rats of a programming window for reproductive tract masculinization, disruption of which leads to hypospadias and cryptorchidism. J Clin Invest 118:1479–1480[CrossRef][Medline]
  11. Fisher JS, Macpherson S, Marchetti N, Sharpe RM 2003 Human ‘testicular dysgenesis syndrome’: a possible model using in-utero exposure of the rat to dibutyl phthalate. Hum Reprod 18:1383–1394[Abstract/Free Full Text]
  12. Mahood IK, Scott HM, Brown R, Hallmark N, Walker M, Sharpe RM 2007 In utero exposure to di(n-butyl) phthalate and testicular dysgenesis: comparison of fetal and adult end points and their dose sensitivity. Environ Health Perspect 115(Suppl 1):55–61
  13. Shultz VD, Phillips S, Sar M, Foster PM, Gaido KW 2001 Altered gene profiles in fetal rat testes after in utero exposure to di(n-butyl) phthalate. Toxicol Sci 64:233–242[Abstract/Free Full Text]
  14. El-Gehani F, Zhang FP, Pakarinen P, Rannikko A, Huhtaniemi I 1998 Gonadotropin-independent regulation of steroidogenesis in the fetal rat testis. Biol Reprod 58:116–123[Abstract/Free Full Text]
  15. Habert R, Picon R 1982 Control of testicular steroidogenesis in foetal rat: effect of decapitation on testosterone and plasma luteinizing hormone-like activity. Acta Endocrinol (Copenh) 99:466–473[Abstract/Free Full Text]
  16. Mahood IK, Hallmark N, McKinnell C, Walker M, Fisher JS, Sharpe RM 2005 Abnormal Leydig cell aggregation in the fetal testis of rats exposed to di (n-butyl) phthalate and its possible role in testicular dysgenesis. Endocrinology 146:613–623[Abstract/Free Full Text]
  17. Atanassova N, McKinnell C, Walker M, Turner KJ, Fisher JS, Morley M, Millar MR, Groome NP, Sharpe RM 1999 Permanent effects of neonatal estrogen exposure in rats on reproductive hormone levels, Sertoli cell number, and the efficiency of spermatogenesis in adulthood. Endocrinology 140:5364–5373[Abstract/Free Full Text]
  18. Hutchison G, Scott HM, Walker M, McKinnell C, Mahood IK, Ferrara D, Sharpe RM 2008 Sertoli cell development and function in an animal model of testicular dysgenesis syndrome. Biol Reprod 78:352–360[Abstract/Free Full Text]
  19. Atanassova NN, Walker M, McKinnell C, Fisher JS, Sharpe RM 2005 Evidence that androgens and oestrogens, as well as follicle-stimulating hormone, can alter Sertoli cell number in the neonatal rat. J Endocrinol 184:107–117[Abstract/Free Full Text]
  20. Arslan M, Weinbauer GF, Schlatt S, Shahab M, Nieschlag E 1993 FSH and testosterone, alone or in combination, initiate testicular growth and increase the number of spermatogonia and Sertoli cells in a juvenile non-human primate (Macaca mulatta). J Endocrinol 136:235–243[Abstract/Free Full Text]
  21. Ramaswamy S, Plant TM, Marshall GR 2000 Pulsatile stimulation with recombinant single chain human luteinizing hormone elicits precocious Sertoli cell proliferation in the juvenile male rhesus monkey (Macaca mulatta). Biol Reprod 63:82–88[Abstract/Free Full Text]
  22. Sharpe RM 2005 Sertoli cell endocrinology and signal transduction: androgen regulation. In: Skinner MK, Griswold MD, eds. Sertoli cell biology. London: Elsevier Academic Press; 199–216
  23. Thompson CJ, Ross SM, Gaido K 2004 Di(n-butyl) phthalate impairs cholesterol transport and steroidogenesis in the fetal rat testis through a rapid and reversible mechanisms. Endocrinology 145:1227–1237[Abstract/Free Full Text]
  24. Ema M, Miyawaki E, Kawashima K 2000 Critical period for adverse effects on development of reproductive system in male offspring of rats given di-n-butyl phthalate during late pregnancy. Toxicol Lett 111:271–278[CrossRef][Medline]
  25. Calafat AM, Brock JW, Silva MJ, Gray Jr LE, Reidy JA, Barr DB, Needham LL 2006 Urinary and amniotic fluid levels of phthalate monoesters in rats after the oral administration of di(2-ethylhexyl) phthalate and di-n-butyl phthalate. Toxicology 217:22–30[CrossRef][Medline]
  26. Fennell TR, Krol WL, Sumner SC, Snyder RW 2004 Pharmacokinetics of dibutyl phthalate in pregnant rats. Toxicol Sci 82:407–418[Abstract/Free Full Text]
  27. Sharpe RM 1994 Regulation of spermatogenesis. In: Knobil E, Neill JD, eds. The physiology of reproduction. 2nd ed. New York: Raven Press; 1363–1434
  28. Johnson L, Zane RS, Petty CS, Neaves WB 1984 Quantification of the human Sertoli cell population: its distribution, relation to germ cell numbers and age-related decline. Biol Reprod 31:785–795[Abstract]
  29. Orth JM, Gunsalus GM, Lamperti AA 1988 Evidence from Sertoli cell-depleted rats indicates that spermatid numbers in adults depends on numbers of Sertoli cells produced during perinatal development. Endocrinology 122:787–794[Abstract/Free Full Text]
  30. Lloyd SC, Foster PM 1988 Effect of mono-(2-ethylhexyl)phthalate on follicle-stimulating hormone responsiveness of cultured rat Sertoli cells. Toxicol Appl Pharmacol 95:484–489[CrossRef][Medline]
  31. Ferrara D, Hallmark N, Scott H, Brown R, McKinnell C, Mahood IK, Sharpe RM 2006 Acute and long-term effects of in utero exposure of rats to di(n-butyl) phthalate on testicular germ cell development and proliferation. Endocrinology 147:5352–5362[Abstract/Free Full Text]
  32. Gaido K, Hensley JB, Liu D, Wallace DG, Borghoff S, Johnson KJ, Hall SJ, Boekelheide K 2007 Fetal mouse phthalate exposure shows that gonocyte multinucleation is not associated with decreased testicular testosterone. Toxicol Sci 97:491–503[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
A. J. Drake, S. van den Driesche, H. M. Scott, G. R. Hutchison, J. R. Seckl, and R. M. Sharpe
Glucocorticoids Amplify Dibutyl Phthalate-Induced Disruption of Testosterone Production and Male Reproductive Development
Endocrinology, November 1, 2009; 150(11): 5055 - 5064.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
C. McKinnell, R. T. Mitchell, M. Walker, K. Morris, C. J.H. Kelnar, W. H. Wallace, and R. M. Sharpe
Effect of fetal or neonatal exposure to monobutyl phthalate (MBP) on testicular development and function in the marmoset
Hum. Reprod., September 1, 2009; 24(9): 2244 - 2254.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol SciHome page
L. E. Gray Jr, N. J. Barlow, K. L. Howdeshell, J. S. Ostby, J. R. Furr, and C. L. Gray
Transgenerational Effects of Di (2-Ethylhexyl) Phthalate in the Male CRL:CD(SD) Rat: Added Value of Assessing Multiple Offspring per Litter
Toxicol. Sci., August 1, 2009; 110(2): 411 - 425.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M. Welsh, R. M. Sharpe, M. Walker, L. B. Smith, and P. T. K. Saunders
New Insights into the Role of Androgens in Wolffian Duct Stabilization in Male and Female Rodents
Endocrinology, May 1, 2009; 150(5): 2472 - 2480.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
L. J. S. Brokken, A. Adamsson, J. Paranko, and J. Toppari
Antiandrogen Exposure in Utero Disrupts Expression of Desert Hedgehog and Insulin-Like Factor 3 in the Developing Fetal Rat Testis
Endocrinology, January 1, 2009; 150(1): 445 - 451.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
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 HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Scott, H. M.
Right arrow Articles by Sharpe, R. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Scott, H. 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