Human Reproduction, Vol. 18, No. 7, 1383-1394,
July 2003
© 2003 European Society of Human Reproduction and Embryology
Human ‘testicular dysgenesis syndrome’: a possible model using in-utero exposure of the rat to dibutyl phthalate
MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, The Chancellor’s 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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BACKGROUND: The disorders comprising human ‘testicular dysgenesis syndrome’ (TDS) may be increasing in incidence. TDS originates in fetal life but the mechanisms are not known, and discerning them requires an animal model. METHODS AND RESULTS: The study investigated whether male rats exposed in utero to dibutyl phthalate [DBP; 500 mg/kg on gestational days (GD) 13–21] would provide a suitable model for human TDS. DBP induced a high rate (>60%) of cryptorchidism (mainly unilateral), hypospadias, infertility and testis abnormalities, similar to those in human TDS. Cell-specific immunohistochemistry and confocal microscopy were used to track development of Sertoli [anti-Müllerian hormone (AMH), Wilm’s tumour (WT-1) protein, p27kip], Leydig [3
-hydroxysteroid dehydrogenase (3-HSD)], germ (DAZL protein) and peritubular myoid (smooth muscle actin) cells from fetal life to adulthood. In scrotal and cryptorchid testes of DBP-exposed males, areas of focal dysgenesis were found that contained Sertoli and Leydig cells, and gonocytes and partially formed testicular cords; these dysgenetic areas were associated with Leydig cell hyperplasia at all ages. Suppression (
90%) of testicular testosterone levels on GD 19 in DBP-exposed males, coincident with delayed peritubular myoid cell differentiation, may have contributed to the dysgenesis. Double immunohistochemistry using WT-1 (expressed in all Sertoli cells) and p27kip (expressed only in mature Sertoli cells) revealed immature Sertoli cells in dysgenetic areas. DBP-exposed animals also exhibited Sertoli cell-only (SCO) tubules, sporadically in scrotal and predominantly in cryptorchid, testes, or foci of SCO within normal tubules in scrotal testes. In all SCO areas the Sertoli cells were immature. Intratubular Leydig cells were evident in DBP-exposed animals and, where these occurred, Sertoli cells were immature and spermatogenesis was absent. Abnormal Sertoli cell–gonocyte interaction was evident at GD 19 in DBP-exposed rats coincident with appearance of multinucleated gonocytes, although these disappeared by postnatal day 10 during widespread loss of germ cells. CONCLUSIONS: Abnormal development of Sertoli cells, leading to abnormalities in other cell types, is our hypothesized explanation for the abnormal changes in DBP-exposed animals. As the testicular and other changes in DBP-exposed rats have all been reported in human TDS, DBP exposure in utero may provide a useful model for defining the cellular pathways in TDS.
Key words: animal model/dibutyl phthalate/rat/testicular dysgenesis syndrome
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Introduction |
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Disorders of human male reproductive development are common and may be increasing in incidence in Western countries (Toppari et al., 1996
; Sharpe and Skakkebaek, 2003). For example, cryptorchidism and hypospadias are the two most common congenital malformations in children, and in adulthood 6–8% of men have subnormal sperm counts; the lifetime risk of testicular germ cell cancer is 0.3–0.8% in Caucasian men (SEER, 2002; Sharpe and Skakkebaek, 2003). Although the latter two disorders manifest in adulthood, it is generally accepted that testis germ cell cancers arise from abnormal fetal germ cells (Rajpert-De Meyts et al., 1998; Rorth et al., 2000) and disorders of sperm production may also arise at this time (Sharpe and Franks, 2002; Sharpe and Skakkebaek, 2003). Each of the above disorders are risk factors for each other, and share other pregnancy-related risk factors (Skakkebaek et al., 2001; Sharpe and Skakkebaek, 2003). Based on these observations, it has been hypothesized that together they comprise a ‘testicular dysgenesis syndrome’ (TDS), which arises in fetal life due to abnormal development of Sertoli and Leydig cells (Skakkebaek et al., 2001). Support for this concept comes from analysis of the contralateral testis of men with testis cancer, which reveals areas of dysgenesis (poorly formed seminiferous tubules), transformed fetal germ cells, areas with immature Sertoli cells lacking germ cells and foci of Leydig cell hyperplasia (Berthelsen and Skakkebaek, 1983; Hoei-Hansen et al., 2003). The mechanisms that give rise to TDS are unknown and are impossible to study in the human fetus. Therefore, an appropriate animal model is required to enable the mechanistic pathways to be defined.
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 (60–100%) 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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Animals, treatments, sample collection and processing
Wistar rats were maintained in our own animal facility according to UK Home Office guidelines, and were fed a soy-free breeding diet (SDS, Dundee, UK). In our study, the day after mating is designated as gestational day (GD) 1 and the day of birth is postnatal day (pnd) 1. Time-mated females were treated from GD 13–21 with either 0 or 500 mg/kg DBP (Sigma–Aldrich Company Ltd, Dorset, UK) in 1 ml/kg corn oil, administered daily by oral gavage. The dose of DBP administered was towards the upper range of doses shown to increase the prevalence of cryptorchidism and hypospadias (Mylchreest et al., 1998
). 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 5–6 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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For non-fluorescent immunohistochemistry [anti-Müllerian hormone (AMH), 3
hydroxysteroid dehydrogenase (3-HSD), smooth muscle actin (SMA) and Wilm’s tumour (WT-1) protein] the slides were incubated for 30 min with the appropriate secondary antibody conjugated to biotin, at a dilution of 1:500 (swine-anti rabbit, DAKO Ltd., Cambridge, UK; rabbit anti-goat, Vector Labs, Burlingame, CA, USA; rabbit anti-mouse, DAKO). The biotinylated antibody was linked to horseradish peroxidase (HRP) by a 30 min incubation with an avidin–biotin–HRP complex (ABC-HRP; DAKO). Antibody localization was determined by application of diaminobenzidine (liquid DAB; DAKO). Development of the control slides was timed and all other slides were developed accordingly and the reaction stopped by immersing slides in water. Slides were counterstained with haematoxylin, dehydrated and mounted using Pertex mounting media (Cell Path, Hemel Hempstead, UK). Negative controls, omitting the primary antisera, were included in each experiment and the results were negative (not shown).
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 Student’s t-test (two-tailed), whereas fertility rates in adult animals from the two groups were compared using Fisher’s exact test.
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Results |
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Bodyweight and testis weight
As reported previously (Mylchreest et al., 1998
; Parks et al., 2000), DBP treatment had no discernible adverse effect on maternal well-being or weight gain during pregnancy. Similarly, other than the changes reported below, there were no discernible adverse effects in the offspring. Bodyweights of control and DBP-exposed males were largely comparable at all ages, although there was a small but significant decrement in bodyweight at day 25 in DBP-exposed males, and in adulthood DBP-exposed males were significantly heavier (13%) than vehicle-exposed controls (not shown). 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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Abnormalities of reproductive development and function
Exposure to DBP in utero resulted in cryptorchidism and hypospadias in the male offspring (Table III). However, a higher prevalence of both disorders was apparent in our study (e.g. 100% incidence of cyrptorchidism) compared with earlier studies (
8% of cryptorchidism; Mylchreest et al., 1998). Evidence of abnormal testis descent in DBP-exposed animals was observed at earlier ages, with one or both testes remaining higher in the abdomen, rather than descending to the inguinal ring. Cryptorchid testes in DBP-exposed males were frequently on the contralateral side of the abdomen, and in one animal the right testis had descended into the left side of the scrotum, while the left testis remained abdominal. DBP-exposed males exhibited subfertility and only two out of 10 males impregnated females, whereas nine out of 10 controls achieved a pregnancy (Table III; P < 0.05). Both DBP-exposed males that impregnated females were unilaterally cryptorchid. As in controls, a grossly normal epididymis and vas deferens was present in all DBP-exposed males, irrespective of testicular position (not shown).
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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Leydig cell hyperplasia
Leydig cell hyperplasia was evident in the testes of fetuses from DBP-treated mothers. The earliest alteration (Figure 2A, B), observed at GD 17 (but not GD15) in DBP-exposed males (Figure 2C, D), was a tendency towards Leydig cell ‘clumping’ in DBP-exposed males (compare Figure 2A, C), often leading to uneven distribution of Leydig cells throughout the testis (not shown). After double immunofluorescence staining for SMA (blue) and 3
-HSD (green), the clumping and Leydig cell hyperplasia were more apparent (Figure 2B, D), but revealed that the hyperplastic areas contained numerous cells that were 3-HSD negative, and which were therefore presumed to be non-Leydig cells (see below).
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Abnormal seminiferous cord/tubule formation (‘dysgenetic areas’)
The progression of seminiferous cord formation is most easily observed using SMA, a marker of differentiated peritubular myoid cells. These cells form a ring around the developing cords (Figure 3). No difference between control and DBP-exposed males was observed in the intensity of SMA immunolocalization at GD 15 or 17 when immunoexpression was weak (data not shown). However, at GD 19 a reduction in the intensity of SMA staining around the seminiferous cords was seen in DBP-exposed males compared with controls (Figure 3A, B). This reduction coincided with suppression of intratesticular testosterone levels (Figure 1), but was no longer apparent by pnd 4 (Figure 3C), when (plasma) testosterone levels had normalized (Figure 1).
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Some areas of apparent Leydig cell hyperplasia in DBP-exposed males also contained SMA-positive cells (Figure 3D). It was also apparent that seminiferous cord formation had been disrupted in some areas, resulting in partially formed and/or anastomotic cords (Figure 3E, F), and these ‘dysgenetic areas’ contained gonocytes (both multinucleated and mononucleated; Figure 3E, F) and Sertoli cells (see below). Dysgenetic areas (usually one to three areas) were a permanent feature of the postnatal testis in DBP-exposed males and, in adulthood, >50% of scrotal (Figure 3H) and cryptorchid (Figure 3I) testes exhibited these areas (Table III). No such areas were evident in controls (e.g. Figure 3G; Table III). The size and complexity of dysgenetic areas varied considerably in DBP-exposed males, but overall they were a minor component of the testis, particularly in adulthood. We therefore sought evidence of more widespread changes in the adult testes of DBP-exposed males and, using cell-specific markers, we evaluated whether the cell types of the testis had developed normally.
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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In cryptorchid testes of DBP-exposed animals, most seminiferous tubules were SCO (Table III), although isolated tubules had some spermatogonia and some exhibited patchy spermatogenesis up to early spermatocytes (Figure 4H). Notably, where dysgenetic areas occurred in cryptorchid testes they were intermixed with type I SCO tubules (Figure 4I).
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 15–19 (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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WT-1 is a nuclear protein expressed by Sertoli cells at all ages (Sharpe et al., 2003
), as confirmed in the present studies for control and DBP-exposed males (Figure 5D–I). In the latter, WT-1 immunostaining was evident within Sertoli cells in dysgenetic areas (Figure 5E, H) and in both normal (Figure 5H) and SCO tubules (Figure 5I). In contrast, p27kip is expressed only by differentiated Sertoli cells (Beumer et al., 1999; Sharpe et al., 2003), and was first detected in the nucleus of Sertoli cells of control rats at age 25 days, when it co-localized with WT-1 (Figure 5F); this co-localization persisted in all tubules in controls in adulthood (Figure 5G). Co-localization of WT-1 and p27kip occurred in Sertoli cells of normal tubules in scrotal testes of DBP-exposed rats (Figure 5H), but not in SCO tubules or abnormal areas, including focal SCO regions of otherwise normal tubules. Only WT-1 was expressed in Sertoli cell nuclei within abnormal regions (Figure 5E, H) showing that Sertoli cell maturation was incomplete. Furthermore, all Sertoli cells within SCO tubules of cryptorchid testes from DBP-exposed animals expressed WT-1 only (Figure 5I).
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 6D–G). 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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Gonocyte/germ cell development
DAZL protein was used as a specific cytoplasmic marker of germ cells (Ruggiu et al., 2000
), and co-localization studies with SMA confirmed that gonocytes were present within dysgenetic areas in testes from DBP-exposed animals at pnd 4 (Figure 7B), as they had been at GD 19 (see Figure 5C). However, at all later ages, germ cells were not detected in dysgenetic areas (not shown). This disappearance coincided with generalized loss of germ cells from testes of DBP-exposed animals by pnd 10 (Figure 7E versus F). However, in adult DBP-exposed animals, the majority of tubules in scrotal testes displayed normal spermatogenesis, with DAZL staining of spermatogonia and spermatocytes, whereas cryptorchid testes showed no or few spermatogonia and the majority of tubules were SCO, as already described.
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Multinucleated gonocytes were frequent in testes from DBP-exposed rats between GD 19 and pnd 4 (Figure 7C, D), whereas only mononucleated gonocytes were evident in controls (Figure 7A). Multinucleated gonocytes were detected in normal cords (Figure 7C) and in dysgenetic areas (see Figure 3E) of DBP-exposed animals, but all had disappeared by pnd 10. A clue to the cause of the multinucleated gonocytes was that at GD 19, when mainly mononucleated cells were evident, gonocytes in DBP-exposed testes formed clusters in the centre of the seminiferous cords with few showing migration or interdigitation with Sertoli cells (Figure 5B), as was the case in controls (Figure 5A).
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Discussion |
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The present study assessed whether exposing male rat pups to DBP in utero might provide a model system to study TDS in humans. There is currently no animal model for human TDS, and no means via by which to study the changes in testicular development that may account for the incidence of congenital abnormalities or the origins of adult disease (cryptorchidism, hypospadias, infertility and testis abnormalities in adulthood). Our findings have established that, in male rats exposed to DBP in utero, all of the major cell types in the testis show abnormal changes in distribution and/or function during development, and these changes provide a logical explanation for the phenotype of animals in adulthood. Although these studies have not established the precise cascade of events that connect DBP exposure to the various abnormalities, the pervasive failure of Sertoli cell maturation and the association of such immature cells with the other abnormal changes suggests that failure of a fundamental aspect of Sertoli cell development in fetal life is likely to be the major event, although fetal androgen deprivation is clearly important. This conclusion is consistent with the current understanding of human TDS, and the spectrum of changes observed in DBP-exposed males, ranging from dysgenesis to foci of immature Sertoli cells in otherwise normal seminiferous tubules, bears a remarkable resemblance to human TDS.
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 gonocyte–Sertoli 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 Sertoli–Leydig 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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We thank Catrina Kivlin and Arantza Esnal for technical assistance, and Denis Doogan and Jim MacDonald for expert animal husbandry. This study was supported in part by contracts QLK4-1999-01422 and QLK4-CT-2002-00603 from the European Union.
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Submitted on January 14, 2003; accepted on March 12, 2003.
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R.T. Mitchell, G. Cowan, K.D. Morris, R.A. Anderson, H.M. Fraser, K.J. Mckenzie, W.H.B. Wallace, C.J.H. Kelnar, P.T.K. Saunders, and R.M. Sharpe Germ cell differentiation in the marmoset (Callithrix jacchus) during fetal and neonatal life closely parallels that in the human Hum. Reprod., December 1, 2008; 23(12): 2755 - 2765. [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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 A. Boyer, L. Hermo, M. Paquet, B. Robaire, and D. Boerboom Seminiferous Tubule Degeneration and Infertility in Mice with Sustained Activation of WNT/CTNNB1 Signaling in Sertoli Cells Biol Reprod, September 1, 2008; 79(3): 475 - 485. [Abstract] [Full Text] [PDF]
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F. Brucker-Davis, K. Wagner-Mahler, I. Delattre, B. Ducot, P. Ferrari, A. Bongain, J.-Y. Kurzenne, J.-C. Mas, P. Fenichel, and and the Cryptorchidism Study Group from Nice Area Cryptorchidism at birth in Nice area (France) is associated with higher prenatal exposure to PCBs and DDE, as assessed by colostrum concentrations Hum. Reprod., August 1, 2008; 23(8): 1708 - 1718. [Abstract] [Full Text] [PDF]
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R. A. Clewell, J. J. Kremer, C. C. Williams, J. L. Campbell Jr, M. E. Andersen, and S. J. Borghoff Tissue Exposures to Free and Glucuronidated Monobutylyphthalate in the Pregnant and Fetal Rat following Exposure to Di-n-butylphthalate: Evaluation with a PBPK Model Toxicol. Sci., June 1, 2008; 103(2): 241 - 259. [Abstract] [Full Text] [PDF]
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 D. Gunnarsson, P. Leffler, E. Ekwurtzel, G. Martinsson, K. Liu, and G. Selstam Mono-(2-ethylhexyl) phthalate stimulates basal steroidogenesis by a cAMP-independent mechanism in mouse gonadal cells of both sexes Reproduction, May 1, 2008; 135(5): 693 - 703. [Abstract] [Full Text] [PDF]
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G. R Hutchison, H. M Scott, M. Walker, C. McKinnell, D. Ferrara, I. K. Mahood, and R. M Sharpe Sertoli Cell Development and Function in an Animal Model of Testicular Dysgenesis Syndrome Biol Reprod, February 1, 2008; 78(2): 352 - 360. [Abstract] [Full Text] [PDF]
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K. J Johnson, J. B Hensley, M. D Kelso, D. G Wallace, and K. W Gaido Mapping Gene Expression Changes in the Fetal Rat Testis Following Acute Dibutyl Phthalate Exposure Defines a Complex Temporal Cascade of Responding Cell Types Biol Reprod, December 1, 2007; 77(6): 978 - 989. [Abstract] [Full Text] [PDF]
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 W. Dhooge, N. Van Larebeke, F. Comhaire, and J.-M. Kaufman Reproductive Parameters of Community-Dwelling Men From 2 Regions in Flanders Are Associated With the Consumption of Self-Grown Vegetables J Androl, November 1, 2007; 28(6): 836 - 846. [Abstract] [Full Text] [PDF]
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L. Ritter and T. E. Arbuckle Can Exposure Characterization Explain Concurrence or Discordance between Toxicology and Epidemiology? Toxicol. Sci., June 1, 2007; 97(2): 241 - 252. [Abstract] [Full Text] [PDF]
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S. Plummer, R. M. Sharpe, N. Hallmark, I. K. Mahood, and C. Elcombe Time-Dependent and Compartment-Specific Effects of In Utero Exposure to Di(n-butyl) Phthalate on Gene/Protein Expression in the Fetal Rat Testis as Revealed by Transcription Profiling and Laser Capture Microdissection Toxicol. Sci., June 1, 2007; 97(2): 520 - 532. [Abstract] [Full Text] [PDF]
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H. M. Scott, G. R. Hutchison, I. K. Mahood, N. Hallmark, M. Welsh, K. De Gendt, G. Verhoeven, P. O'Shaughnessy, and R. M. Sharpe Role of Androgens in Fetal Testis Development and Dysgenesis Endocrinology, May 1, 2007; 148(5): 2027 - 2036. [Abstract] [Full Text] [PDF]
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 K. M Main, J. Toppari, and N. E Skakkebaek Gonadal development and reproductive hormones in infant boys Eur. J. Endocrinol., November 1, 2006; 155(suppl_1): S51 - S57. [Abstract] [Full Text] [PDF]
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M. Welsh, P. T. K. Saunders, N. I. Marchetti, and R. M. Sharpe Androgen-Dependent Mechanisms of Wolffian Duct Development and Their Perturbation by Flutamide Endocrinology, October 1, 2006; 147(10): 4820 - 4830. [Abstract] [Full Text] [PDF]
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 C L Acerini and I A Hughes Endocrine disrupting chemicals: a new and emerging public health problem? Arch. Dis. Child., August 1, 2006; 91(8): 633 - 641. [Full Text] [PDF]
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 L. Aksglaede, A. Juul, H. Leffers, N. E. Skakkebaek, and A.-M. Andersson The sensitivity of the child to sex steroids: possible impact of exogenous estrogens Hum. Reprod. Update, July 1, 2006; 12(4): 341 - 349. [Abstract] [Full Text] [PDF]
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 P R Dalsenter, G M Santana, S W Grande, A J. Andrade, and S L Araujo Phthalate affect the reproductive function and sexual behavior of male Wistar rats Human and Experimental Toxicology, June 1, 2006; 25(6): 297 - 303. [Abstract] [PDF]
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E. Rajpert-De Meyts Developmental model for the pathogenesis of testicular carcinoma in situ: genetic and environmental aspects Hum. Reprod. Update, May 1, 2006; 12(3): 303 - 323. [Abstract] [Full Text] [PDF]
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 R. M. David Proposed Mode of Action for In Utero Effects of Some Phthalate Esters on the Developing Male Reproductive Tract Toxicol Pathol, April 1, 2006; 34(3): 209 - 219. [Abstract] [Full Text] [PDF]
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E. Kleymenova, C. Swanson, K. Boekelheide, and K. W. Gaido Exposure In Utero to Di(n-Butyl) Phthalate Alters the Vimentin Cytoskeleton of Fetal Rat Sertoli Cells and Disrupts Sertoli Cell-Gonocyte Contact Biol Reprod, September 1, 2005; 73(3): 482 - 490. [Abstract] [Full Text] [PDF]
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 K. A. Boisen, M. Chellakooty, I. M. Schmidt, C. M. Kai, I. N. Damgaard, A.-M. Suomi, J. Toppari, N. E. Skakkebaek, and K. M. Main Hypospadias in a Cohort of 1072 Danish Newborn Boys: Prevalence and Relationship to Placental Weight, Anthropometrical Measurements at Birth, and Reproductive Hormone Levels at Three Months of Age J. Clin. Endocrinol. Metab., July 1, 2005; 90(7): 4041 - 4046. [Abstract] [Full Text] [PDF]
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K. Liu, K. P. Lehmann, M. Sar, S. S. Young, and K. W. Gaido Gene Expression Profiling Following In Utero Exposure to Phthalate Esters Reveals New Gene Targets in the Etiology of Testicular Dysgenesis Biol Reprod, July 1, 2005; 73(1): 180 - 192. [Abstract] [Full Text] [PDF]
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D. R. Holsberger, G. M. Buchold, M. C. Leal, S. E. Kiesewetter, D. A. O'Brien, R. A. Hess, L. R. Franca, H. Kiyokawa, and P. S. Cooke Cell-Cycle Inhibitors p27Kip1 and p21Cip1 Regulate Murine Sertoli Cell Proliferation Biol Reprod, June 1, 2005; 72(6): 1429 - 1436. [Abstract] [Full Text] [PDF]
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S. K. Lee and D. N. R. Veeramachaneni Subchronic Exposure to Low Concentrations of Di-n-Butyl Phthalate Disrupts Spermatogenesis in Xenopus laevis Frogs Toxicol. Sci., April 1, 2005; 84(2): 394 - 407. [Abstract] [Full Text] [PDF]
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J. C. Corton and P. J. Lapinskas Peroxisome Proliferator-Activated Receptors: Mediators of Phthalate Ester-Induced Effects in the Male Reproductive Tract? Toxicol. Sci., January 1, 2005; 83(1): 4 - 17. [Abstract] [Full Text] [PDF]
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K. L. Britt, P. K. Saunders, S. J. McPherson, M. L. Misso, E. R. Simpson, and J. K. Findlay Estrogen Actions on Follicle Formation and Early Follicle Development Biol Reprod, November 1, 2004; 71(5): 1712 - 1723. [Abstract] [Full Text] [PDF]
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A.-M. Andersson, N. Jorgensen, L. Frydelund-Larsen, E. Rajpert-De Meyts, and N. E. Skakkebaek Impaired Leydig Cell Function in Infertile Men: A Study of 357 Idiopathic Infertile Men and 318 Proven Fertile Controls J. Clin. Endocrinol. Metab., July 1, 2004; 89(7): 3161 - 3167. [Abstract] [Full Text] [PDF]
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J. S Fisher Environmental anti-androgens and male reproductive health: focus on phthalates and testicular dysgenesis syndrome Reproduction, March 1, 2004; 127(3): 305 - 315. [Abstract] [Full Text] [PDF]
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C. J. Thompson, S. M. Ross, and K. W. Gaido Di(n-Butyl) Phthalate Impairs Cholesterol Transport and Steroidogenesis in the Fetal Rat Testis through a Rapid and Reversible Mechanism Endocrinology, March 1, 2004; 145(3): 1227 - 1237. [Abstract] [Full Text] [PDF]
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H. Li and K. H. Kim Effects of Mono-(2-Ethylhexyl) Phthalate on Fetal and Neonatal Rat Testis Organ Cultures Biol Reprod, December 1, 2003; 69(6): 1964 - 1972. [Abstract] [Full Text] [PDF]
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