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Testis; |
Medical Research Council Human Reproductive Sciences Unit, Centre for Reproductive Biology, The Queen's Medical Research Institute, Edinburgh EH16 4TJ, United Kingdom
ABSTRACT
Pregnancy exposure to di(n-butyl) phthalate (DBP) in rats induces a testicular dysgenesislike syndrome (TDS) in male offspring. Earlier studies suggested altered Sertoli cell development/maturation may result, especially in testes that become cryptorchid. This study quantitatively assessed Sertoli cell numerical and functional development in DBP-exposed rats and compared (unilaterally) cryptorchid and scrotal testes. Pregnant rats were gavaged with 500 mg/kg/day DBP or corn oil from embryonic (E) Days 13.5 to 21.5. Male offspring were sampled on E21.5 or Postnatal Day 6, 10, 15, 25, or 90. Sertoli cell number in DBP-exposed males was reduced by
50% at E21.5 but recovered to normal by Days 25–90, accompanied by significant changes in plasma inhibin B and testosterone levels. Sertoli cell maturational development in DBP-exposed males, assessed using five protein markers (anti-müllerian hormone, cytokeratin, androgen receptor, CDKN1B, and Nestin), was largely normal, with some evidence of delayed maturation. However, in adulthood, Sertoli cells (SC) in areas lacking germ cells (Sertoli cell-only [SCO] tubules) often exhibited immature features, especially in cryptorchid testes. Sertoli cells in DBP-exposed animals supported fewer germ cells during puberty, but this normalized in scrotal testes by adulthood. Scrotal and especially cryptorchid testes from DBP-exposed animals exhibited abnormalities (SCO tubules, focal dysgenetic areas) at all postnatal ages. Cryptorchid testes from DBP-exposed animals exhibited more Sertoli cell abnormalities at Day 25 compared with scrotal testes, perhaps indicating more severe underlying Sertoli cell malfunction in these testes. Our findings support the concept of altered Sertoli cell development in TDS, especially in cryptorchid testes, but show that maturational defects in Sertoli cells in adulthood most commonly reflect secondary dedifferentiation in absence of germ cells.
di(n-butyl) phthalate (DBP), rat, Sertoli cell number, testicular dysgenesis syndrome (TDS), testis development
Sertoli cells have two functionally separate roles. First, they play the lead role in the formation and development of the testis in fetal and early postnatal life [1, 2]. Second, in postpubertal life they provide the environment and support for germ cells during the process of spermatogenesis [3]. The transition of Sertoli cells from their "developmental" to their "adult" roles involves terminal differentiation of the Sertoli cells and is characterized by the loss of proliferative ability and the switching on of various functions necessary for the support of spermatogenesis, a process also referred to as Sertoli cell maturation [4]. It is unclear to what extent the adult functions of Sertoli cells are programmed by their development in fetal/perinatal life, but it is accepted that their proliferation in perinatal life is a key event in relation to future spermatogenesis, as the number of Sertoli cells per testis is the primary determinant of the number of sperm produced per day, and thus of testis size in adulthood [4, 5]. There is increasing evidence that maldevelopment of the fetal testis and of Sertoli and/or Leydig cells may be important factors in the risk of testicular disorders in humans, encapsulated in the hypothesis of a "testicular dysgenesis syndrome" (TDS) [6].
TDS is hypothesized to encompass disorders, such as testicular germ cell cancer, cryptorchidism, hypospadias, and some cases of low sperm counts [6, 7]. Although these disorders manifest at different ages, they are thought to have a common origin in fetal life as the result of abnormal function of Sertoli and/or Leydig cells [6]. If Sertoli cell proliferation and/or maturation is impaired in TDS, the resultant ability of Sertoli cells to support spermatogenesis might also be adversely affected. As reduced testosterone production/action in fetal life results in lower Sertoli cell numbers in mice [8, 9] and rats [10] and leads to increased risk of cryptorchidism, hypospadias, and (in humans) testicular germ cell cancer [6, 7], impaired Sertoli cell development perinatally would provide a logical explanation for some of the TDS disorders. It has also been hypothesized that the common occurrence of unilateral cryptorchidism in patients with presumptive TDS could be indicative of more severe malfunction of the Sertoli and/or Leydig cells during development in the cryptorchid than in the contralateral scrotal testis [6, 7].
We and others have shown that exposure of rats in utero to the ubiquitous environmental chemical di(n-butyl) phthalate (DBP) can induce a TDS-like syndrome in the male offspring [4, 11–13]. This model system has the potential to provide insights into the mechanisms via which TDS and its disorders may arise in fetal life. Findings from this model point clearly to hormonal dysfunction of the fetal Leydig cells as an important etiological change [14–16], but one study [4] suggested that failure of Sertoli cell maturation might be indicated by the focal occurrence of Sertoli cell-only (SCO) tubules or regions within otherwise normal tubules in DBP-exposed males in adulthood [4, 17]; such regions are found also in testis biopsies from TDS patients [18–20]. Other changes in the fetal testis of DBP-exposed rats, such as reduced Sertoli cell number [10], abnormal aggregation of fetal germ cells [21, 22], occurrence of multinucleated germ cells [15, 23], reduced fetal germ cell numbers, and delayed development of fetal and postnatal germ cells [20], could also reflect impaired Sertoli cell function. The aim of the present study was, therefore, to assess Sertoli cell numerical and functional development in rats exposed in utero to DBP using stereological techniques and a battery of maturational markers as well as to evaluate the capacity of Sertoli cells to support germ cells postnatally; these changes were related to dysgenetic features (the occurrence of SCO tubules and focal dysgenetic areas). As DBP induces a high incidence of unilateral cryptorchidism in Wistar rats [4], comparison of the same Sertoli cell parameters in scrotal and cryptorchid testes was also undertaken. Our findings show that Sertoli cell numerical and functional development is impaired to a variable extent according to age and testis position, although a number of the findings in adulthood may be attributable to secondary changes resulting from germ cell loss.
Animals, Treatments, Sample Collection, and Processing
Wistar rats were maintained according to UK Home Office guidelines and were fed a soy-free breeding diet (SDS, Dundee, Scotland). Animals were housed in rooms on 12L:12D cycles with lights on 0700 h to 1900 h. Time-mated females were treated from embryonic (E) Days 13.5 to E21.5 with either 0 (control) or 500 mg/kg DBP (Sigma-Aldrich Co. Ltd., Dorset, UK) in 1 ml/kg corn oil administered daily by oral gavage. The DBP was 99% pure according to the supplier. Male offspring were subsequently sampled on E21.5 or on Postnatal Day 4, 6, 10, 15, 25, or 90 (adults). Animals were killed by CO2 inhalation followed by cervical dislocation. The main studies focused on Days 15, 25, and 90, as these ages span puberty, in which Sertoli cells should functionally differentiate/mature and adopt key functions to support spermatogenesis. At necropsy, testicular position was classified as high abdominal (at level of the kidney), midabdominal, inguinal, or scrotal, which enabled classification of testes in Day 25 and Day 90 males as cryptorchid or scrotal. Testes were carefully inspected for normality of the epididymis and vas deferens and then dissected, weighed, and fixed for 2–6 h in Bouin, depending on age, before being transferred into 70% ethanol. Tissue was subsequently processed into paraffin wax using standard methods [17].
Seminiferous Tubule Morphology
Seminiferous tubule morphology was evaluated in testes from animals at Days 15, 25, and 90. Tubules were evaluated for normality of overall morphology and complement of germ cells. All tubules (N = 300–750) in one complete testis cross section per animal were evaluated and classed as normal or abnormal. Certain tubule abnormalities were specifically recorded, namely, SCO tubules, in which germ cells were absent from all or most of the tubule cross section, or "focal dysgenetic areas," in which aberrant and often anastomotic tubules appeared in a focal region. Tubules in these dysgenetic regions were invariably SCO but were not included in the tubule morphology calculations.
Immunohistochemistry and Evaluation of Sertoli Cell Maturation
Immunohistochemistry was used to identify cells for enumeration (WT1, DAZL) or to evaluate the functional maturation of Sertoli cells, namely, CDKN1B (also known as p27kip1), androgen receptor (AR), anti-müllerian hormone (AMH), Nestin and cytokeratin, and histone H3 as a proliferation indicator. The proteins used to assess maturational status were chosen so as to include some that are normally switched off during maturation (AMH, Nestin, cytokeratin, and histone H3) and others that are switched on as the Sertoli cells functionally mature (AR and CDKN1B). Specific proteins were detected by immunohistochemistry using methods that have been detailed previously [4, 17]. Table 1 lists the primary antibodies used, their dilutions, the need for antigen retrieval, and their sources. Fluorescence immunohistochemistry and double immunohistochemistry for WT1/CDKN1B and confocal microscopy used the antibodies listed in Table 1 and methods detailed elsewhere [4].
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Quantification of Sertoli Cell Maturation Markers
Tubules were evaluated for normal expression of maturation markers (excluding AR) in animals at Postnatal Days 15, 25, and 90 (scrotal and cryptorchid). All tubules (N = 300–750) in one complete testis cross section per animal (five animals from at least three litters) were evaluated. Cords expressing markers correctly for animal age were counted and scored as a percentage of the total tubule number.
Nonfluorescent images were examined and photographed using a Provis AX70 microscope (Olympus Optical, London, UK) fitted with a Canon DS6031 digital camera (Canon Europe, Amsterdam, The Netherlands). Fluorescent images were captured using a Zeiss LSM 510 Meta Axiovert 100M confocal microscope (Carl Zeiss Ltd., Welwyn Garden City, UK). Images were compiled using Photoshop 7.0 (Adobe Systems Inc., Mountain View, CA).
Determination of Sertoli and Germ Cell Numbers
Sertoli and germ cell numbers per testis were determined using standard stereological techniques as described previously [24, 25]. Complete transverse sections of testes were immunostained for WT1 (Sertoli cell counts) or Dazl (germ cell counts). Stereological analyses used Image-Pro Plus 4.5.1 with Stereologer-Pro 5 plug-in software (Media Cybernetics UK, Wokingham, Berkshire, UK) and used an Olympus BH-2 microscope fitted with a Prior automatic stage (Prior Scientific Instruments Ltd., Cambridge, UK). In brief, the software was used to select random fields for counting and to place a grid over the tissue, and points falling over Sertoli or germ cell nuclei were counted and expressed as a percentage of the total points counted. Germ cells were distinguished as being spermatogonia, spermatocytes, or round spermatids based on their morphology and position; elongate spermatids were not counted because of the elongated shape of their nucleus. The number of fields counted per animal (10–75 fields per section, one section per animal) was dependent on obtaining a standard error value of <5% for cell counts. The data so obtained were converted to absolute cell volumes per testis by multiplying by testis weight (equivalent to volume), as shrinkage was minimal. These data were then converted to cell numbers per testis after determination of the mean nuclear volume of Sertoli cells and the specific germ cell classes in each animal (average of 70–100 nuclei) using the selector function of the Stereologer-Pro 5 software. Cell nuclei were drawn around on the screen, and the program then determined the average length of several diameters measured at two degree intervals, which passed through the center of the nucleus.
DBP-exposed male Wistar rats exhibit a high incidence of (mainly unilateral) cryptorchidism [4, 17], which may indicate that the cryptorchid testis was more severely affected by the DBP treatment in fetal life than was the contralateral scrotal testis. Therefore, in the present studies, five DBP-exposed males aged 25 and 90 days that were unilaterally cryptorchid were selected for stereological assessment. Sertoli cell number was determined for scrotal and cryptorchid testes at both ages, whereas germ cell numbers were determined in scrotal and cryptorchid testis at Day 25 but only in scrotal testes at Day 90, as cryptorchid testes at this age lacked most germ cells. None of the testes used for germ cell analyses at Day 25 and Day 90 exhibited any obvious abnormalities of the epididymis, which can occur after DBP exposure.
Plasma levels of inhibin B were measured using a two-site enzyme-linked immunoassay that uses a capture antibody directed against the C-terminal portion of the human ßB-subunit and the F(ab) fraction of a mouse monoclonal antibody (R1) to the N-terminal portion of the inhibin subunit conjugated to alkaline phosphatase [26]. The assay has been validated previously for measurement of inhibin B in the rat [27, 28].
Plasma levels of testosterone were measured using an ELISA adapted from an earlier radioimmunoassay (RIA), as described previously [4]. The limit of detection was 12 pg/ml, and interassay and intra-assay coefficients of variation were <15%.
Plasma levels of FSH were measured by RIA using materials supplied by the National Institute of Diabetes and Digestive and Kidney Diseases, as previously described [29].
Values are expressed as mean ± SEM, and data were analyzed using Student unpaired t-test or one-way ANOVA followed by Tukey post-hoc test using GraphPad Prism (version 4; GraphPad Software Inc., San Diego, CA). The paired t-test was used to compare values for scrotal and cryptorchid testes from the same DBP-exposed animals at Days 25 and 90.
DBP exposure resulted in a 31% reduction in testis weight at E21.5 (Table 2); there was no significant effect of DBP exposure on body weight (data not shown). At Day 15, testis weight was still reduced (by 28%) in DBP-exposed animals when compared with controls and also at Day 25, at which age there was no significant difference between the weight of scrotal and cryptorchid testes from DBP-exposed animals (Table 2). At Day 90, scrotal testes from DBP-exposed animals were comparable in weight to controls, whereas cryptorchid testes were reduced in weight by 75% (Table 2).
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Sertoli Cell Number and Nuclear Volume
At E21.5, DBP-exposed males exhibited a 50% reduction in Sertoli cell number in comparison to controls (Fig. 1A). However, by Day 25, Sertoli cell number in scrotal testes from DBP-exposed animals had normalized, and remained so in adulthood (Fig. 1A). Sertoli cell number at Day 25 showed a small but significant decrease in cryptorchid testes from DBP-exposed animals compared with controls, but this difference could be incidental, as no reduction in Sertoli cell number was evident in adult cryptorchid testes, and numbers did not differ significantly between cryptorchid and scrotal testes of DBP-exposed animals at Day 25 or 90 (Fig. 1A).
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Sertoli cell mean nuclear volume (MNV) was assessed as a measure of Sertoli cell functionality, as it increases considerably in size as the cell matures and adopts new functions. Thus, in controls, Sertoli cell MNV doubled from E21.5 to Postnatal Day 25, and further increased nearly 3-fold between Days 25 and 90 (Fig. 1B). Similar age-related changes in Sertoli cell MNVs were evident in the scrotal testes of DBP-exposed animals. In contrast, Sertoli cell MNV in cryptorchid testes from DBP-exposed animals at Day 25 was already significantly reduced compared with both the contralateral scrotal testis and scrotal testes from controls, and it showed no increase at all between Days 25 and 90 (Fig. 1B).
Plasma Testosterone, Inhibin, and FSH
Plasma inhibin B levels reflect both Sertoli cell number and the status of spermatogenesis [28, 30]. Plasma inhibin B levels in controls showed a characteristic increase between Days 4 and 15, reflecting the increase in Sertoli cell numbers during this period [28], before decreasing gradually to reach adult levels (Fig. 2A). Although DBP-exposed animals showed a similar pattern of change, there were notable significant differences from control levels. Thus, at Day 15, inhibin B levels in DBP-exposed animals were significantly increased above control values. This, however, was not a result of increased FSH stimulation, as at each time point, FSH levels were not significantly different from controls (Fig. 2A). In contrast, at both Days 25 and 90, inhibin B levels in DBP-exposed animals were significantly decreased relative to controls, especially at Day 90 (Fig. 2A), probably reflecting the major decrease in germ cell numbers per paired testes at these ages [30]. Testosterone levels were significantly decreased in DBP-exposed animals relative to controls from Day 15 through to adulthood (Fig. 2B).
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Sertoli Cell Functional Maturation Based on Expression of Protein Markers
Seven Sertoli cell protein markers (Table 1) were evaluated as indicators of normal maturational changes. These included proteins that are switched off as the Sertoli cell matures (AMH, cytokeratin, Nestin, and the proliferation marker histone H3) and others that are switched on as the Sertoli cell becomes functionally mature (AR and CDKN1B). Testicular sections from control and DBP-exposed animals were evaluated on Postnatal Days 15, 25, and 90, as they encompass the periods during and after functional maturation. Examination of an earlier time point (Day 6) showed no consistent differences between control and DBP-exposed animals for these maturation markers (data not shown), with the exception of AMH (see below).
Anti-müllerian hormone. AMH is expressed in immature Sertoli cells and is switched off as functional maturation occurs [31]. AMH immunostaining was evident in all seminiferous cords at E21.5 and Day 6 (data not shown) in the controls, had reduced to barely detectable levels by Day 10, and was undetectable by Day 15 (not shown). In DBP-exposed animals, a similar pattern was observed, with AMH immunoexpression being absent from both scrotal and cryptorchid testes beyond Day 15. However, at Days 6 and 10, there was evidence of more intense immunoexpression of AMH than in controls in most, but not all, DBP-exposed animals (data not shown), perhaps suggesting a slight delay in its maturational downregulation.
Cytokeratin. Cytokeratin expression is switched off between Days 15 and 25 as the Sertoli cell matures [31]. Almost 100% of seminiferous cords in both control and DBP-exposed animals at Day 15 contained Sertoli cells that immunostained positively for cytokeratin. At Days 25 and 90, control testes were immunonegative for cytokeratin, as were scrotal testes from DBP-exposed animals. However, cryptorchid testes from DBP-exposed animals did show positive staining for cytokeratin in 2% of tubules at Day 25 only (data not shown).
Nestin. Nestin is an intermediate filament protein with an expression pattern similar to cytokeratin that is located in the basal compartment of Sertoli cells and is switched off between Days 15 and 25 as the Sertoli cell matures [31, 32]. Almost 90% of seminiferous cords at Day 15 in control animals contained Sertoli cells that immunostained positively for Nestin (Fig. 3). DBP-exposed animals at the same age displayed almost 100% positive staining in Sertoli cells. At Days 25 and 90, control testes were immunonegative for Nestin, as were scrotal and cryptorchid testes from DBP-exposed animals, except in focal dysgenetic areas, in which Nestin immunoexpression was often still evident at low levels (Fig. 3). In adulthood, SCO tubules and focal dysgenetic areas frequently immunoexpressed Nestin, and in some cases the expression was intense (Fig. 3).
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Histone H3.
As loss of proliferative ability by Day 15 is another maturational feature of Sertoli cells in the rat [24, 26], the occurrence of Sertoli cell mitosis was assessed using histone H3 expression at Day 15. No proliferating Sertoli cells were detectable in either control or DBP-exposed animals, including in cryptorchid testes (data not shown).
CDKN1B. This is a marker of mature Sertoli cells and switches on coincident with cessation of proliferation [31, 33]. Sertoli cells in controls were immunonegative for CDKN1B at E21.5 through to Day 10 but were immunopositive at 15, 25, and 90 days of age (data not shown). A similar pattern was observed in scrotal testes of DBP-exposed animals, with the exception of SCO seminiferous tubules or SCO areas within tubules and dysgenetic areas in adulthood, in which CDKN1B immunoexpression was usually absent (Figs. 4 and 5). This was more evident in cryptorchid testes of DBP-exposed adult animals, which had significantly fewer (45%) tubules staining positive for CDKN1B compared with controls, and these were characteristically SCO (Figs. 4 and 5). Notably, in tubules in which germ cells were present only in one area, immunostaining for CDKN1B was only evident in regions in which germ cells were present (data not shown). In contrast, at Day 25 in DBP-exposed animals, Sertoli cells in SCO tubules were uniformly immunopositive for CDKN1B (Fig. 5), suggesting that loss of CDKN1B immunoexpression in SCO areas in adulthood was secondary to the absence of germ cells at this age rather than due to the failure of these cells to switch on CDKN1B earlier during maturation.
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Androgen Receptor. AR immunoexpression in Sertoli cells is absent at E21.5 and then switches on progressively between Days 4 and 15 and becomes stage dependent in adulthood [34], as was found in controls in the present study (data not shown). This pattern was unaffected in DBP-exposed animals (Fig. 5, H and J), although in adulthood in cryptorchid testes there were sporadic tubules, or areas within tubules, that lacked Sertoli cell AR immunostaining, and these were usually SCO areas (Fig. 5J).
Sertoli Cell Functional Capacity to Support Germ Cells
Germ cell numbers were determined and expressed relative to Sertoli cell numbers for each animal on Days 25 and 90 to indicate the functional capacity of each Sertoli cell. This showed that at Day 25, Sertoli cells in DBP-exposed animals supported numbers of spermatogonia similar to controls, regardless of whether the testis was scrotal or cryptorchid in position (Fig. 6). In contrast, the numbers of spermatocytes supported by each Sertoli cell at Day 25 in DBP-exposed animals was reduced by 35% in scrotal testes and by 55% in cryptorchid testes compared with controls (Fig. 6); the difference between cryptorchid and scrotal testes was not statistically significant. By adulthood, numbers of spermatogonia, spermatocytes, and round spermatids supported per Sertoli cell were not different between controls and scrotal testes in DBP-exposed animals (Fig. 6); in adult cryptorchid testes of DBP-exposed animals, there was major and variable depletion of germ cells and the widespread occurrence of SCO tubules (not shown).
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Seminiferous Tubule Abnormalities
Testicular sections were assessed at Postnatal Days 15, 25, and 90 (scrotal and cryptorchid) for incidence of SCO tubules and focal dysgenetic areas (Fig. 7). None of these abnormalities were detected in control testes at any age, whereas DBP-exposed animals showed occurrence of both abnormalities at all ages (Fig. 7), although not in every animal. The prevalence of focal dysgenetic areas in DBP-exposed animals showed no major difference according to age or testis position (scrotal versus cryptorchid), although these areas always were more extensive in cryptorchid than in scrotal testes. The prevelance of SCO tubules increased with age, at least in cryptorchid testes (Fig. 7). It was notable at Day 25 that cryptorchid testes already showed an increased incidence of abnormal seminiferous tubules compared with controls or with contralateral testes from DBP-exposed animals (Fig. 7). Another abnormality observed at Day 25, but not at Day 90, in testes from DBP-exposed animals was the occurrence of Sertoli cell nuclei adjacent to the lumen of seminiferous tubules, and this was again observed more frequently in cryptrochid than in scrotal testes (Fig. 7).
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The primary objective of this study was to establish whether DBP exposure in utero, which induces a TDS-like syndrome in the male offspring, had any transient or long-term effects on Sertoli cell development and function, as evidenced by changes in Sertoli cell number, maturation, and postnatal function. A related objective was to evaluate whether any observed changes were more pronounced in cryptorchid than in scrotal testes of DBP-exposed animals at an age (Day 25) when secondary effects of failed testis descent would be minimal. Our results show that exposure to DBP during the last week of gestation results in a major but transient decrease in Sertoli cell number at the end of gestation, as recently reported [10], with some evidence for delayed Sertoli cell maturation postnatally, based on the pattern, timing, and level of expression of five specific markers of Sertoli cell maturation. However, overall, Sertoli cell maturation appeared to occur relatively normally based on these protein markers. From a functional point of view, Sertoli cells in DBP-exposed animals were shown to support fewer germ cells during puberty, although this normalized by adulthood in scrotal testes. However, tubules lacking germ cells (SCO) as well as focal dysgenetic areas were found in most scrotal as well as cryptorchid testes from DBP-exposed animals in adulthood, and such tubules were also evident at 15–25 days of age, especially in cryptorchid testes. Significant changes in Sertoli cell secretion of inhibin B were also found postnatally in DBP-exposed animals in comparison to controls, changes which are probably explained by the overall reduction in germ cell complement [25]. Although our results provide evidence to suggest more severe impairment of Sertoli cell development in cryptorchid versus scrotal testes of DBP-exposed animals, our overall interpretation is that Sertoli cell development and function proceed relatively normally in the postnatal period, and that most of the changes observed reflect recovery from the effects of DBP exposure in fetal life or are secondary to absence of germ cells.
Exposure to DBP during pregnancy results in a number of reproductive abnormalities in the male offspring, including hypospadias, cryptorchidism, focal abnormalities of spermatogenesis, and reduced fertility [4, 11–13, 17, 35]. Although some of these changes are explained by impairment of hormone production in fetal life [14–16], the effects on fertility and spermatogenesis are largely unexplained, but could be indicative of Sertoli cell malfunction [4]. Our earlier study [4] had shown that in adult rats exposed in utero to the same dose of DBP as used in the present study, there was focal occurrence of tubules that lacked germ cells partially or completely and that the Sertoli cells in such tubules failed to normally express CDKN1B. As the latter protein is normally switched on in Sertoli cells coincident with cessation of their proliferation and terminal differentiation [31, 33], its absence suggested possible failure of normal maturation and, thus, of the capacity of such Sertoli cells to support spermatogenesis. This was a primary motivating factor for the present studies. The present study has confirmed these earlier findings with regard to absence of CDKN1B expression in SCO areas in adult rats. However, investigation of CDKN1B expression during the period of Sertoli cell maturation (Days 10–25) in the present study has shown that it is switched on normally in Sertoli cells of DBP-exposed animals, even in tubules that are SCO at these ages. This suggests that the absence of immunoexpression of CDKN1B in some Sertoli cells in adulthood is most likely a secondary effect due to the absence of germ cells. The fact that disappearance of CDKN1B expression in such SCO tubules only occurs with age implies that it is the prolonged absence of germ cells that causes this secondary "de-differentiation" [7, 36]. A similar change might also explain the sporadic absence of AR immunoexpression and persistence of Nestin and cytokeratin immunoexpression in some adult Sertoli cells in SCO areas in DBP-exposed animals. Nevertheless, our studies do not allow us to completely exclude deficiencies in Sertoli cell maturation, but the overall impression gained is that if this is the case, it is not a generalized phenomenon.
Despite the DBP-induced halving of Sertoli cell numbers at the end of gestation, by Postnatal Day 25, normal numbers of Sertoli cells had been restored. This must mean that "compensatory mechanisms" were activated to restore normal cell numbers. However, the mechanism(s) responsible for this recovery are unclear. Follicle-stimulating hormone levels remained normal and testosterone levels subnormal at 6–15 days of age in DBP-exposed animals, so it seems unlikely that either of these hormones could have been involved in promoting supranormal Sertoli cell proliferation postnatally. Additionally, analysis of cell proliferation at Day 15 found no evidence that cessation of Sertoli cell proliferation was delayed in DBP-exposed animals, ruling this out as an explanation for the recovery; the latter finding is another indicator of normal Sertoli cell maturation in DBP-exposed animals.
A more robust indicator that Sertoli cells have functionally matured is their capacity to support germ cells through spermatogenesis [3, 31]. In DBP-exposed animals, the number of germ cells per Sertoli cell at age 25 days was significantly reduced compared with controls, although no difference was evident in adulthood. However, we have shown previously that numbers of spermatogonia are reduced, and their resumption of proliferation delayed, in DBP-exposed rats at Days 4–6 postnatally [20], suggesting that the reduction in germ cell numbers per Sertoli cell observed at age 25 days in the present study might reflect this delay rather than a decreased ability of Sertoli cells to support germ cells. This conclusion may not be so straightforwardly applicable to cryptorchid testes in DBP-exposed animals which, at Day 25, exhibited a significant reduction in Sertoli cell nuclear volume compared with contralateral scrotal testes (or with control testes) in association with the largest deficit in germ cells per Sertoli cell and with a high incidence of abnormal (germ cell-depleted) tubules. These changes could reflect early effects of abnormal testis position, as descent is normally complete by 21–23 days of age. However, as weights between descended and nondescended testes were not different at Day 25 in DBP-exposed animals, an alternative explanation is that the Sertoli cells in cryptorchid testes are more severely affected in utero by the DBP treatment than are Sertoli cells in testes destined to descend normally, and that this results both in cryptorchidism and in a reduced ability to support germ cells. The present data do not allow us to distinguish conclusively between the two aforementioned possibilities, but the consistently higher prevalence of tubule abnormalities and focal evidence of Sertoli cell maturation delay in cryptorchid testes at Day 25 give some support to the idea that the cryptorchidism is partly a reflection of more pronounced Sertoli cell malfunction during development.
Previous studies have shown that DBP exposure in utero can induce SCO tubules and focal dysgenetic areas in adulthood [4, 37–39], as confirmed presently. The present study is the first to have quantified these changes in relation to age and to have shown that they are evident at 15–90 days in both scrotal and cryptorchid testes. An additional finding in the present study that has not been reported previously, was the frequent occurrence at Day 25 of tubules containing Sertoli cell nuclei clumped together toward the center of the tubules where the lumen should be; this was not found in adulthood. This abnormal positioning of Sertoli cell nuclei suggests that tight junctions may not have formed normally in these tubules. This could be another indicator of delayed Sertoli cell development and/or may have relevance to the formation of SCO tubules.
The changes in tubule morphology and Sertoli cells identified in DBP-exposed animals in adulthood in the present study bear some striking similarities to those reported in the testes of patients with testicular germ cell cancer [18, 19, 40], the most severe manifestation of TDS; this includes focal areas of Sertoli cells that exhibit immature morphological or immunohistochemical features [41, 42]. The present findings suggest that such changes could be a secondary consequence of germ cell absence [36] rather than a reflection of a primary maturational defect in the Sertoli cells [31], at least in normally descended testes. One of the hypotheses to emerge from the TDS concept in humans is that the increased prevalence of cryptorchidism could reflect more severe malfunction of the developing somatic (Sertoli and Leydig) cells [6, 7]. Our present findings provide some support for this view by showing that at Day 25, shortly after failure of testis descent was identifiable definitively in DBP-exposed animals, a significant reduction in Sertoli cell nuclear volume was already evident (and was maintained through to adulthood), as was a significant increase in both abnormal (subnormal germ cell complement) and germ cell-deficient tubules, with both of these parameters being more prevalent than in the contralateral scrotal testes. Although this is consistent with inherently more severe malfunction of the Sertoli cells in cryptorchid testes of DBP-exposed animals, our studies cannot exclude an effect of altered testis position itself. Our results emphasize the importance of using multiple ages and multiple functional measures in order to separate the primary malfunction of Sertoli cells leading to absence of germ cells from the secondary changes in Sertoli cell function due to loss or absence of germ cells for other reasons (e.g., testis nondescent).
ACKNOWLEDGMENTS
We thank Mark Fisken for providing expert animal husbandry and technical assistance during this study.
FOOTNOTES
1This work was funded in part by grant QLK4-CT-200-00603 from the European Union. 
Correspondence: 2FAX: 44 0131 242 6231; e-mail: r.sharpe{at}hrsu.mrc.ac.uk
Received: 4 July 2007.
First decision: 7 August 2007.
Accepted: 28 September 2007.
REFERENCES
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  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., June 2, 2009; (2009) dep200v1. [Abstract] [Full Text] [PDF]
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 H. M. Scott, G. R. Hutchison, M. S. Jobling, C. McKinnell, A. J. Drake, and R. M. Sharpe Relationship between Androgen Action in the "Male Programming Window," Fetal Sertoli Cell Number, and Adult Testis Size in the Rat Endocrinology, October 1, 2008; 149(10): 5280 - 5287. [Abstract] [Full Text] [PDF]
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 K. J. Johnson, S. M. McCahan, X. Si, L. Campion, R. Herrmann, and J. S. Barthold The orl Rat with Inherited Cryptorchidism Has Increased Susceptibility to the Testicular Effects of In Utero Dibutyl Phthalate Exposure Toxicol. Sci., October 1, 2008; 105(2): 360 - 367. [Abstract] [Full Text] [PDF]
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