MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, The Chancellors Building, University of Edinburgh, 49 Little France Crescent, Edinburgh EH16 4SB, UK
1 To whom correspondence should be addressed. e-mail: r.sharpe{at}hrsu.mrc.ac.uk
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Abstract |
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Key words: animal model/dibutyl phthalate/rat/testicular dysgenesis syndrome
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Introduction |
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Testicular organogenesis involves a cascade of gene activation and differentiation of the component cell types. Sertoli cells organize themselves into testicular cords surrounded by peritubular myoid cells and enclosing fetal germ cells, whereas the interstitial Leydig cells differentiate and produce testosterone to induce masculinization. When cell organization is abnormal, the testis is described as dysgenetic, and can result from chromosomal abnormalities or androgen insensitivity, or for unknown reasons (Skakkebaek et al., 2001). Recently, testicular dysgenesis has been described in three different knock-out mice. The Desert hedgehog (dhh) signalling protein is secreted by Sertoli cells and interacts with the patched-1 receptor on Leydig and peritubular myoid cells. Testes from dhh/ mice show dysgenetic features such as anastomotic seminiferous cords and extra-cordal gonocytes (Clark et al., 2000
; Pierucci-Alves et al., 2001
). When assessed in adulthood only 7.5% of dhh/ males were masculinized, and they were infertile (Bitgood et al., 1996
; Clark et al., 2000
). Postnatally, the testes of Dax1/ mice display foci of dysgenesis associated with an abnormal peritubular cell layer, Leydig cell hyperplasia and intratubular Leydig cells (Jeffs et al., 2001
). Fgf9 stimulates mesonephric cell migration and Sertoli cell differentiation and Fgf9/ mice show male-to-female sex reversal (Colvin et al., 2001
). Testes of Fgf9/ mice have disorganized testicular cords and extra-cordal clusters of germ and somatic cells, which lack peritubular myoid cells (Colvin et al., 2001
). Although each of these knock-out mice shows testicular dysgenesis, none displays the full spectrum of disorders characteristic of human TDS.
Recent toxicological studies have shown that in-utero exposure of pregnant rats to certain phthalate esters, such as dibutyl phthalate (DBP), results in a range of reproductive abnormalities in the male offspring (Mylchreest et al., 1998; 1999
; Gray et al., 1999
; Parks et al., 2000
; McIntyre et al., 2001
). The effects in rodents are associated with suppression of fetal androgen levels, and induction of Leydig cell hyperplasia and multinucleated gonocytes (Mylchreest et al., 1998
; 1999
; Gray et al., 1999
; Parks et al., 2000
; McIntyre et al., 2001
). Taken at face value, the gross endpoints examined in the published studies are suggestive of a TDS-like syndrome, but no studies to date have evaluated the effect of DBP administration on the underlying physiology of the developing testis. We have repeated the method of published studies using a single dose of DBP, and have complemented the gross observations by the use of a battery of cell-specific markers to chart the development of each of the cellular components of the testis. These show that DBP-exposure induces foci of testicular dysgenesis coincident with failure of Sertoli cell maturation and the generation of abnormal fetal germ cells. These changes are associated with a high (60100%) incidence of (mainly unilateral) cryptorchidism, hypospadias and infertility. We suggest that this may be a useful model with which to study the cellular ontogeny of human TDS. As human exposure to phthalates is considerable (Blount et al., 2000
), such studies would also aid assessment of the risk that such exposure poses to the human fetus, although this issue is not addressed in the present study.
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Materials and methods |
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On GD 15, 17 and 19, dams were killed by inhalation of carbon dioxide. Fetuses were removed, decapitated and the torso fixed for 24 h in Bouins, and then transferred into 70% ethanol before the gonads were removed via microdissection. Testes from other GD 19 rats were snap frozen and stored at 70°C until analysed for testosterone. Male rats killed at 4, 10, 15, 25 and 90 days (adults) were anaesthetized via flurothane inhalation, blood was collected by cardiac puncture, and then they were killed by cervical dislocation. All age groups contained five to 10 rats. Tissues were fixed for 56 h in Bouins, then transferred to 70% ethanol (adult testes were halved during fixation to aid penetration of the fixative). Tissue was embedded in paraffin using an automated tissue processor.
Fertility in adulthood
To test the fertility of adult rats exposed to vehicle or DBP in utero, each male was housed singly with a female of proven fertility for a total of 8 days (two cycles). The female was then removed and monitored until the birth of her litter.
Immunohistochemistry
Specific proteins were detected by immunohistochemistry and double immunofluorescence. Sections of 5 µm were mounted onto coated slides (BDH Chemicals, Poole, UK), dewaxed and rehydrated. To block endogenous peroxidase activity, slides were incubated in 3% (v/v) hydrogen peroxide in methanol. Immunohistochemistry for p27kip used antigen retrieval by pressure-cooking slides for 5 min in 0.01 mol/l citrate buffer. Slides were washed in Tris-buffered saline (TBS) 0.05 mol/l (pH 7.4) and 0.85% NaCl, and blocked in TBS containing 5% bovine serum albumin (Sigma) prior to addition of the primary antibody, and incubated overnight at 4°C. The primary antibodies used, their dilutions and source are listed in Table I.
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Double immunohistochemistry used combinations of SMA/ 3-HSD, SMA/DAZL, 3
-HSD/vimentin and WT-1/p27kip. In each case the primary antibodies were raised in different species (mouse or rabbit). Fluorescent immunohistochemistry was performed as described above until prior to addition of the primary antibody (antigen retrieval was only required for p27kip). Both primary antibodies were added simultaneously and incubated overnight at 4°C. After washing in phosphate-buffered saline (PBS tablets; Sigma), both secondary antibodies were added simultaneously (goat anti-rabbit peroxidase; 1:100; DAKO) and goat anti-mouse biotinylated (1:500; Sigma) and incubated for 1 h at room temperature. To produce blue fluorescence (SMA), tyramide Cy5 (TSA plus cyanine 5 system; Perkin Elmer Life Science, Inc., Boston, MA, USA) was added to the slides for 10 min using a 1:50 dilution of Cy5 in the buffer supplied. Fluorescent green staining (SMA and 3
-HSD) was achieved by incubation with streptavidin alexa 488 (Molecular Probes, Poort Gebouw, The Netherlands) diluted 1:200 with PBS and incubated for 2 h at room temperature. Fluorescent red staining (DAZL and vimentin) was achieved by incubation with streptavidin alexa 546 (Molecular Probes) diluted 1:200 with PBS and incubated for 2 h at room temperature. Where a red counterstain was used, this was achieved via incubation with propidium iodide (1:2000 in PBS; Sigma) for 2 min before washing slides in PBS. The slides were mounted in an aqueous mounting medium (Permaflour; Beckman Coulter, High Wycombe, UK).
Microscopy
Non-fluorescent images were captured using an Olympus Provis microscope (Olympus Optical, London, UK) and a Kodak DCS330 digital camera (Eastman Kodak, Rochester, NY, USA). Fluorescent images were captured using a Zeiss LSM 510 Axiovert 100M confocal microscope (Carl Zeiss Ltd, Welwyn Garden City, UK). Images were compiled using Photoshop 5.0 (Adobe Systems, Inc., Mountain View, CA, USA).
Histological analysis of adult testis
To assess testicular abnormalities in adult testes after in-utero exposure to vehicle or DBP, one section from each paraffin block (two to six blocks per testis) was stained with haematoxylin and eosin and a list of abnormalities compiled.
Plasma and testicular testosterone analysis
Plasma testosterone levels were measured using an enzyme-linked immunosorbent assay adapted from a radioimmunoassay method, as detailed elsewhere (Atanassova et al., 1999). The detection limit for this assay was
12 pg/ml, and all samples were assayed together.
Statistical analysis
Testis weights and testicular and plasma testosterone levels in control and DBP-exposed animals were compared using Students t-test (two-tailed), whereas fertility rates in adult animals from the two groups were compared using Fishers exact test.
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Results |
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Prior to adulthood, mean testis weights in control animals were significantly greater than DBP-exposed males (Table II). As all DBP-exposed adult males exhibited unilateral or bilateral cryptorchidism (Table III), weights of scrotal and cryptorchid testes were evaluated separately. There was no difference in scrotal testis weights in control and DBP-exposed adult males, but cryptorchid testes from the latter were significantly reduced in weight (Table II).
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Testicular and plasma testosterone
Testicular testosterone levels at GD 19 were reduced by 90% in DBP-exposed males compared with controls (Figure 1). Postnatally, plasma testosterone levels displayed no significant difference at pnd 4 and 10 or in adulthood in DBP-exposed males compared with controls, but the former exhibited a significant reduction in plasma testosterone level at day 25 (Figure 1).
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Evaluation of gross testicular histology in adulthood
Areas of Leydig cell hyperplasia, either focal (in scrotal testes) or more general (in cryptorchid testes), were frequent in the testes of DBP-exposed males, but were not observed in controls (Table III). Spermatogenesis was grossly normal in control testes (Figure 4A) and in the scrotal testes of DBP-exposed males (not shown). However, the latter exhibited subtle abnormalities that were not evident in controls. The most common abnormality was the presence of dysgenetic areas (Table III), as already described. Equally common was the focal occurrence of Sertoli cell-only (SCO) tubules (Table III; Figure 4B, C). Two types of SCO tubules were distinguished. Type I contained immature spindle-shaped Sertoli cells and no evidence of lumen formation, and were confined to cryptorchid testes (Figure 4C). Type II appeared more developed than type I, with signs of lumen formation (Figure 4B), and were found in scrotal and cryptorchid testes. Another common finding in scrotal testes of DBP-exposed males was tubules that exhibited incomplete spermatogenesis (Figure 4D; Table III) or which had complete spermatogenesis but with focal SCO areas (Figure 4E; Table III). A curious finding in occasional tubules in scrotal testes of three DBP-exposed males was the occurrence of two layers of Sertoli cells that bisected (Figure 4F), or protruded into (Figure 4G), an otherwise normal seminiferous tubule (Figure 4F, G). There was no peritubular layer between these two Sertoli cell layers, as evidenced by lack of SMA immunostaining (Figure 4F). Immunostaining for WT-1 protein confirmed the layers were composed of Sertoli cells (not shown).
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Evaluation of Sertoli cell maturation
Some Sertoli cells in DBP-exposed rats exhibited immature features, so maturational status was evaluated using three protein markers: AMH, WT-1 protein and p27kip (see Sharpe et al., 2003). Immature Sertoli cells in control testes immunoexpressed AMH at GD 1519 (Figure 5A) with expression decreasing through to pnd 10 (not shown). A similar pattern was exhibited by DBP-exposed animals (Figure 5B). In the latter, AMH was also immunoexpressed in cells scattered throughout dysgenetic areas at pnd 4 (Figure 5C), but this expression was not detectable beyond pnd 10 (not shown). AMH immunostaining highlighted the location of gonocytes on GD 19, and indicated differences in their location and interaction with Sertoli cells in DBP-exposed animals compared with controls (Figure 5A, B). This is detailed below.
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Leydig cell development and localization
The abnormal hyperplastic areas of 3-HSD-immunopositive (Leydig) cells in the fetal testes of DBP-exposed animals were still evident postnatally (Figure 6B), and were commonly associated with dysgenetic areas, as illustrated using immunofluorescence for 3
-HSD (green) and SMA (blue) (Figure 6C). In contrast, fetal Leydig cells in control testes were in small clumps, and 3
-HSD and SMA immunostaining was always demarcated (Figure 6A). At later ages, abnormal aggregations of Leydig cells in scrotal testes of DBP-exposed animals were confined to dysgenetic areas, whereas aggregations of Leydig cells were present in numerous regions of cryptorchid testes (not shown). Double immunostaining for 3
-HSD and SMA also revealed the occasional presence of 3
-HSD immunopositive cells located basally within seminiferous tubules of DBP-exposed males at all postnatal ages (e.g. 25 days; Figure 6D). Double immunostaining for 3
-HSD (green) with vimentin (red cytoplasmic staining), which is a Sertoli cell marker, ruled out the possibility that these were aberrant Sertoli cells (Figure 6E, F). Intratubular Leydig cells were evident in scrotal (Figure 6F) and cryptorchid (Figure 6G) testes of adult DBP-exposed animals, but were never observed in controls (e.g. Figure 6H). Intratubular Leydig cells were always found in partially or completely formed tubules adjacent to dysgenetic regions (e.g. Figure 6C, D, E), but occasional single cells were found in tubules remote from dysgenetic areas (Figure 6I). Where intratubular Leydig cells were found, spermatogenesis was absent, even though Sertoli cells were present (Figure 6DG). In DBP-exposed animals at all ages, the majority of Leydig cells were located normally in the interstitium (e.g. Figure 6E, G).
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Discussion |
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Our studies have expanded earlier toxicological studies that had shown that in-utero exposure to DBP or to diethylhexyl phthalate could induce cryptorchidism, hypospadias and decreased testis weights in the exposed male offspring (Mylchreest et al., 1998). These studies had also described suppression of fetal testosterone levels and Leydig cell hyperplasia, as well as the occurrence of multinucleated gonocytes (Parks et al., 2000
; Mylchreest et al., 2002
). Our findings have confirmed these observations, although in our experiments a consistently higher rate of abnormalities (e.g. 100% crytptorchidism versus
8%; Mylchreest et al., 1998
) was achieved when using the same dose and treatment regimen for DBP, and this extended also to lower doses of DBP (our unpublished data).
Our studies are the first to illustrate that the earlier described areas of Leydig cell hyperplasia, are, in most cases, areas of testicular dysgenesis where incomplete seminiferous cord formation has occurred and in which Sertoli, peritubular and germ cells are intermixed with the abundant Leydig cells. These dysgenetic areas persisted throughout life, although only the somatic cells survived beyond pnd 10 within these areas. The dysgenetic areas in testes from DBP-exposed animals are indicative of abnormal seminiferous cord formation. Cord formation involves co-operation between Sertoli and peritubular myoid cells to secrete the basal lamina (Skinner et al., 1985). A transient delay in differentiation of peritubular myoid cells on GD 19, as evidenced by reduced immunoexpression of SMA (Palombi et al., 1992
), was evident in DBP-exposed rats. This coincided with suppressed intratesticular testosterone levels, and once these normalized (in plasma) by pnd 4, peritubular cell expression of SMA appeared normal, consistent with testosterone inducing peritubular cell differentiation (Schlatt et al., 1993
). Delayed maturation of peritubular myoid cells and impaired Sertoli cell development (see below) probably underlies the development of dysgenetic areas in DBP-exposed males. Similar abnormalities in cord formation have been described in mice lacking the dhh gene, and within these regions Sertoli and Leydig cells were intermixed (Clark et al., 2000
) as shown presently in DBP-exposed rats. In men with testicular germ cell cancer, biopsies of the cancer-containing or contralateral (non-cancerous) testis show areas of testicular dysgenesis (Sohval, 1956
; Berthelsen and Skakkebaek, 1983
; Hoei-Hansen et al., 2003
) similar to those described here in DBP-exposed animals.
Suppression of testosterone production is presumed to explain the occurrence of hypospadias in DBP-exposed animals, as blockade of androgen action by the anti-androgen flutamide in fetal life results in hypospadias (Imperato-McGinley et al., 1985; Imperato-McGinley et al., 1992
). Androgen suppression might also explain the occurrence of cryptorchidism, as testicular descent is partly androgen dependent (Hutson et al., 1997
). However, in-utero exposure to flutamide does not induce any of the other abnormalities described in the present study, despite inducing 100% hypospadias (our unpublished data). Moreover, failure of Sertoli cell maturation (see below) cannot be accounted for by reduced testosterone exposure, as these cells do not express androgen receptors during fetal life in either the rat or the human (Majdic et al., 1995
; Williams et al., 2001
; Sharpe et al., 2003
). Indeed, our findings, and those of Shono et al. (2000)
, suggest that DBP-induced cryptorchidism occurs because of impaired transabdominal testis descent, which is not androgen dependent (Hutson et al., 1997
; Shono et al., 2000
). Interference with production/action of AMH by Sertoli cells (Kubota et al., 2002
) or insulin-like growth factor 3 (Insl3) by fetal Leydig cells (Nef and Parada, 1999
; Sharpe, 2001
) can impair transabdominal testis descent. However, in the present study AMH immunoexpression was normal in Sertoli cells in DBP-exposed males, although it remains to be shown whether production or action of Insl3 are impaired.
The present study demonstrates widespread failure of Sertoli cell maturation in DBP-exposed animals. Functionally mature Sertoli cells can be identified by their expression of cell cycle proteins that inhibit proliferation, i.e. p27kip, a marker of terminally differentiated Sertoli cells in rats and humans (Beumer et al., 1999; Sharpe et al., 2003
). Co-localization studies using p27kip and WT-1 (a protein expressed in all Sertoli cells regardless of maturational status; Sharpe et al., 2003
), enabled discrimination between immature (expressing only WT-1) and mature (co-expressing WT-1 and p27kip) Sertoli cells. The latter were evident in controls in all seminiferous tubules at puberty (day 25) and in adulthood. At the same ages in DBP-exposed animals, co-expression of WT-1 and p27kip was evident in all tubules displaying normal spermatogenesis. However, p27kip was not expressed in Sertoli cells within cryptorchid testes, nor in dysgenetic areas of scrotal testes of DBP-exposed rats at any age. Furthermore, wherever foci of SCO were found in the scrotal testes of DBP-exposed animals, including within otherwise normal seminiferous tubules, no p27kip expression was detected in Sertoli cells. This suggests that exposure to DBP led to changes in a proportion of Sertoli cells that rendered them incapable of functionally maturing, and consequently unable to support spermatogenesis. The fact that cryptorchid testes contained only immature Sertoli cells may also indicate that the cryptorchidism occurred because of abnormal development of Sertoli cells, rather than the converse. Our studies have not yet identified any specific functional abnormality of the fetal Sertoli cells induced by exposure to DBP, but certain phthalates can adversely affect Sertoli cell function neonatally (Dostal et al., 1988
) and prepubertally (Gray and Beamand, 1984
; Lloyd and Foster, 1988
), although the mechanisms are unclear. In humans, immature Sertoli cells have been associated with SCO tubules, infertility and carcinoma in situ (CIS) of the testis, based on morphology (Nistal et al., 1982
; Regadera et al., 2001
), androgen receptor expression (Suarez-Quain et al., 1999
) or persistent cytokeratin 18 expression (Stosiek et al., 1990
; Bergmann and Kliesch, 1994
; Kliesch et al., 1998
; Maymon et al., 2000
).
In the present study, abnormalities in gonocytes provides indirect evidence for DBP-induced Sertoli cell dysfunction during fetal life. During normal testicular differentiation, the gonocytes are enveloped by Sertoli cells to form testicular cords (GD 13) and gonocyte mitosis is blocked. During late fetal/early postnatal life gonocytes resume mitosis and migrate from the centre of the cord towards the basal lamina (Orth et al., 2000
). This was evident in controls in the present study at GD 19, when gonocytes interdigitated with the Sertoli cells during their migration. However, in DBP-exposed animals at this age, most gonocytes were not interdigitated with Sertoli cells but remained centrally within the cords, suggestive of impaired gonocyteSertoli cell adhesion. Co-culture of Sertoli cells and gonocytes from neonatal rats have shown that addition of mono (2-ethylhexyl)-phthalate induces detachment of gonocytes from Sertoli cells (Li et al., 1998
). Two mechanisms might underlie such effects. First, Sertoli cells secrete stem cell factor that binds to the Kit receptor on gonocytes, an interaction vital for gonocyte migration, proliferation and survival (Orth et al., 2000
). Expression of c-kit and genes involved in its downstream signalling are reduced in fetal rat testes exposed in utero to DBP (Shultz et al., 2001
). Secondly, Sertoli cells and gonocytes make contact via neural cell adhesion molecule in fetal and early neonatal life (Orth and Jester, 1995
), but whether this is disrupted by exposure to phthalates is not known.
In DBP-exposed males, numerous multinucleated gonocytes were evident, as described in earlier studies (Parks et al., 2000; Mylchreest et al., 2002
). In the present study, multinucleated gonocytes were first observed on GD19 in DBP-exposed rats, and by pnd 4 many gonocytes were multinucleated, corresponding to the period of mitotic activity in gonocytes (McGuinness and Orth, 1992
). Most multinucleated gonocytes disappeared by pnd 10, and none were observed at pnd 25. It is presumed they degenerated because they did not contact the basal lamina of the seminiferous cord. How DBP treatment interferes with the re-initiation of mitosis, and in particular with gonocyte cytokinesis, is not understood. Interestingly, in Caenorhabditis elegans, ptc-1 (a homologue of mammalian patched receptors that is essential for hedgehog signalling) is confined to the germ line, and null mutants are sterile with multinucleated germ cells (Kuwabara et al., 2000
). In humans, testicular germ cell cancers and their precursors, CIS, are thought to originate from gonocytes that have failed to differentiate normally (Rajpert-De Meyts et al., 1998
; Rorth et al., 2000
). Abnormal gonocyte development in testes of DBP-exposed rats therefore provides intriguing parallels to CIS induction, although the abnormal gonocytes did not survive beyond pnd 25. It is also relevant that multinucleated spermatogonia have been reported in biopsies from cryptorchid prepubertal boys (Cortes et al., 2002
).
In agreement with earlier studies (Parks et al., 2000; Mylchreest et al., 2002
), we observed that Leydig cell hyperplasia was evident from GD 17 in DBP-exposed animals, and persisted thereafter, particularly, but not exclusively, around dysgenetic areas. Our preliminary cell counts indicate that this hyperplasia is real (unpublished data). As the differentiation and function of fetal Leydig cells is regulated by factors probably emanating from Sertoli cells (Koopman, 2001
), disturbance of SertoliLeydig cell signalling would provide a plausible explanation for the suppression of fetal testosterone production, and the observed hyperplasia could represent activation of local compensatory mechanisms (Mylchreest et al., 2002
). Hyperplasia was most pronounced in fetal life within dysgenetic areas and in adult cryptorchid testes, and immature Sertoli cells populated both locations. Leydig cells abnormally located inside the seminiferous cords (and tubules) were also a feature of DBP-exposed rats. These cells were identified by their expression of 3
-HSD and their lack of co-localization with vimentin (a marker of Sertoli cell cytoplasm); these displaced cells were evident from pnd 4 through to adulthood and are presumed to be fetal-type Leydig cells. Similar intratubular fetal-like Leydig cells have been reported in DAX-1-deficient mice in regions adjacent to peritubular myoid cell disruption (Jeffs, 2001
). In the present study, most intratubular Leydig cells were found within, or bordering, dysgenetic areas, but regardless of where they were found, the adjacent Sertoli cells were always immature and no spermatogenesis occurred in their immediate vicinity. The presence of intratubular Leydig cells therefore appears to be an indication of abnormal cord formation and cell segregation in fetal life. Intratubular Leydig cells have been reported in patients with infertility or cryptorchidism and were implicated in the impaired spermatogenesis in these patients (Mori et al., 1978
; 1987
). These various findings suggest that intratubular Leydig cells are another indicator of testicular dysgenesis.
The main aim of the present study was to evaluate whether DBP-treatment is a useful model to delineate the mechanisms underlying human TDS. In humans, the conditions comprising TDS are detected after birth or in adulthood, but are thought to arise from abnormal differentiation of Sertoli and Leydig cells during fetal development (Skakkebaek et al., 2001). In men with testis cancer, areas of testicular dysgenesis akin to those described presently in DBP-exposed animals, are often found adjacent to testicular tumours (Sohval, 1956
) or in biopsies of the contralateral (normal) testis (Berthelsen and Skakkebaek, 1983
; Hoei-Hansen et al., 2003
). In the same testes, foci of SCO tubules with immature Sertoli cells and/or incomplete spermatogenesis (Berthelsen and Skakkebaek, 1983
; Hoei-Hansen et al., 2003
) also occurred. Similarly, studies of men with cryptorchidism have reported foci of tubules with undifferentiated Sertoli cells (Regadera et al., 2001
), multinucleated spermatogonia (Warren and Nelson, 1950
; Cortes et al., 2002
) and intratubular Leydig cells (Mori et al., 1978
; 1987
). Similar abnormalities have been shown in this study after in-utero exposure of rats to DBP, and coincided with a high rate of cryptorchidism, hypospadias and infertility. We consider that a detailed characterization of the pathways via which DBP exposure induces these abnormalities are probably of direct relevance to studies of human TDS.
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Acknowledgements |
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Submitted on January 14, 2003; accepted on March 12, 2003.