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Medical Research Council Human Reproductive Sciences Unit, Centre for Reproductive Biology, The Queen’s Medical Research Institute, Edinburgh EH16 4TJ, Scotland, United Kingdom
Address all correspondence and requests for reprints to: Richard M. Sharpe, Medical Research Council Human Reproductive Sciences Unit, Centre for Reproductive Biology, The Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, Scotland, United Kingdom. E-mail: r.sharpe{at}hrsu.mrc.ac.uk.
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Introduction |
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Among the TDS disorders, TGCT is of particular importance for several reasons. First, it is the most severe manifestation of TDS. Second, it has increased progressively in incidence for at least the past 50 yr in Caucasian men across the world (2, 3), which may indicate that other TDS disorders could also be increasing in prevalence. Third, it originates from premalignant cells, known as carcinoma in situ (CIS) cells, which are thought to originate from primordial germ cells/gonocytes in utero (5, 6, 7). The evidence for this is that CIS cells express a range of specific proteins that are normally expressed by gonocytes during fetal life but that are not expressed by any of the normal adult germ cells (GCs) present during the process of spermatogenesis (6, 8). These markers includes octamer-binding transcription factor (OCT) 3/4 (9, 10), known to be crucial for early embryonic development and involved in regulation of pluripotency (11). During normal perinatal maturation of GCs, OCT3/4 disappears (12), as do other markers of CIS cells such as placental/germ cell alkaline phosphatase and stem cell factor receptor KIT (13, 14). Based on this evidence, the CIS cells have been hypothesized to result from a block in maturational differentiation of gonocytes into prespermatogonia (5, 15).
The hypothesis of a common fetal origin of TDS disorders has been recently supported by an animal model. Thus, administration of di(n-butyl) phthalate (DBP) to female rats during pregnancy leads to a variety of reproductive malformations in the male offspring, including three of the four TDS end points (16, 17, 18) as well as focal testicular dysgenesis (16) and profound impairment of fetal Leydig cell function (19, 20, 21). Despite appearing to be a good model for TDS based on most features (22, 23), DBP exposure of rats does not lead to the formation of recognizable CIS cells or the development of TGCT in adulthood. Nevertheless, DBP exposure in utero does exert effects on gonocytes in rats because it leads to widespread appearance of abnormal multinucleated gonocytes (MNGs) and abnormal aggregation of gonocytes within the center of the seminiferous cords (21, 24, 25). Whether these changes have any relationship to the origin of CIS cells in the human fetal testis is unclear. However, prompted by these findings, we investigated whether there is any evidence for altered timing of GC development in the rat DBP model that might have analogies to the failure of GC differentiation proposed to result in CIS cells. Our results show that DBP exposure of rats does result in altered development/timing of differentiation of fetal gonocytes and that, in turn, this leads to major consequences for postnatal/pubertal GC development.
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Materials and Methods |
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75%) of focal dysgenetic areas in postnatal testes of our animals (16). The DBP administered was 99% pure according to the supplier. In one series of experiments, the dosing protocol for DBP was modified such that treatment was performed only on e19.5 and e20.5 (referred to as short-term dosing) to allow discrimination between early (e13.5–e17.5) and late (e19.5–e20.5) fetal effects on GCs. To label proliferating cells in postnatal d (d) 6 and d25 animals, 5-bromo-2'deoxyuridine-5'-monophosphate (BrdU; Sigma-Aldrich) was administered by ip injection at 100 mg/kg body weight in saline [0.9% NaCl (wt/vol)] at a dose volume of 2 ml/kg body weight 1 h before the animals were scheduled to be killed.
Sample collection and processing
Fetal samples.
Control and DBP-treated pregnant dams were killed by inhalation of carbon dioxide on e15.5 (n = 5 control, n = 5 DBP), e17.5 (n = 5, 5), e19.5 (n = 4, 4), or e21.5 (n = 6, 6; n = 3, 3 short-term DBP treatment). Fetuses were removed, decapitated and placed in ice-cold PBS (Sigma-Aldrich). Testes were removed via microdissection and either fixed for 1 h in Bouins or transferred to an Eppendorf tube and frozen immediately on dry ice for subsequent protein extraction. Fixed testes were transferred to 70% ethanol and then weighed before being processed for 17.5 h in an automated Leica TP1050 processor and embedded in paraffin wax. Representative fetuses from three to six of the aforementioned litters were subsequently used for the immunohistochemical and quantitative studies detailed below.
Postnatal samples.
Male rats aged d4 (n = 6 control, n = 6 DBP; n = 5, 5 short-term DBP treatment), d6 (n = 6, 4), d8 (n = 6, 6), d15 (n = 6, 6), d25 (n = 6, 5), or d90 (n = 5, 5) were anesthetized via Fluothane inhalation and then killed by cervical dislocation. Testes were carefully inspected for normality of the epididymis and vas deferens and then removed, weighed, fixed for 5–6 h in Bouins, and transferred into 70% ethanol. Testes were embedded in paraffin as described above. The results reported in the present studies derive from males from at least three separate litters per age group, with the exception of data for GC proliferation index on d6 and d25 for DBP-exposed animals, in which data derive from males from only two litters in both cases. At autopsy, testicular position was classified as high abdominal (at level of the kidney), midabdominal, inguinal, or scrotal, which enabled classification of testes from d25 and d90 males into cryptorchid or scrotal groups.
Immunohistochemistry
Sections of 5 µm were mounted onto coated slides (VWR, Poole, UK), dewaxed, and rehydrated. Antigen retrieval was performed by pressure cooking slides for 5 min in 0.01 M citrate buffer (pH 6.0). Slides were incubated for 30 min in 3% (vol/vol) hydrogen peroxide in methanol to block endogenous peroxidase activity and then washed in Tris-buffered saline [TBS; 0.05 M Tris, 0.85% (wt/vol) NaCl (pH 7.4)]. Nonspecific binding sites were blocked with an appropriate normal serum diluted 1:5 in TBS containing 5% (wt/vol) BSA before the addition of the primary antibody and overnight incubation at 4 C. The primary antibodies used in the present study, their dilution, and sources are listed in Table 1
. After washing in TBS, slides were incubated for 30 min with the appropriate secondary antibody conjugated to biotin (rabbit antigoat, swine antirabbit, rabbit antimouse; Dako, Cambridgeshire, UK) diluted 1:500 in the blocking mixture. This was followed by 30 min incubation with horseradish peroxidase-labeled avidin-biotin complex (Dako). Immunostaining was developed by application of diaminobenzidine (liquid DAB+; Dako), and slides were counterstained with hematoxylin, dehydrated, and mounted using Pertex mounting medium (Cell Path, Hemel Hempstead, UK). For negative controls, tissue sections were incubated either with primary antibody preabsorbed with the immunizing peptide (OCT4) or without addition of primary antibody.
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Stereological analyses
Stereological analyses were performed using 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).
MNG analysis
For the purposes of clarity and simplicity, we refer in the present study to fetal GCs as gonocytes. MNGs were identified on sections stained with toluidine blue. Sections of 5 µm were mounted onto coated slides, dewaxed, and rehydrated. Toulidine blue stain was filtered and used at 50% dilution in distilled water. The stain was applied to each section until staining was optimal. The slides were then immersed in distilled water to arrest staining and slides then mounted as described above. One complete cross-section of each fetal testis was evaluated systematically at e17.5, e19.5, e21.5, and d4 and the percent of seminiferous cords containing MNGs was scored; no account was taken of the number of MNGs per cord cross-section or the number of nuclei present in individual MNGs.
Determination of GC numbers
GC volume per testis was determined using paraffin sections of testis that had been immunostained for Dazl, as described above, and stereological analysis (26) with modifications as described elsewhere (27). In brief, the software was used to select random fields for counting and place a grid over the tissue. The number of fields counted per animal (10–75 fields/section, one section per animal) was dependent on obtaining a percentage SE value less than 5%. The data so obtained were converted to absolute volumes per testis by multiplying by testis weight (equivalent to volume) because shrinkage was minimal. These data were then converted to GC number per testis after determination of mean germ cell nuclear diameter and volume (average of 70–100 nuclei) using the selector function of the Stereologer-Pro 5 software, which calculates volume from an average of three nuclear diameter measurements of the same GC nucleus. In animals at e21.5 and on d4–15, all GCs were classified as being the same, whereas at d25, spermatogonia and spermatocytes were identified and counted separately, and additionally, at d90 round spermatids were also counted separately from other GC types; at the latter age, no attempt was made to count elongate spermatids due to the extreme variation in shape of the nucleus.
Determination of the germ cell proliferation index at d6 and d25
The GC proliferation index was determined by counting the number of BrdU-immunopositive (Sigma-Aldrich) and -immunonegative cells (28). At d25, this analysis was straightforward because only GCs were proliferating. However, at d6 Sertoli cells were also proliferating, so to avoid errors in cell identification at this age, double-nonfluorescent immunohistochemistry was performed on testis sections combining staining for Dazl (a GC-specific marker) and BrdU. Dazl localization was performed first using Fast Blue detection and BrdU was then detected using DAB+ (Dako). The number of BrdU-immunopositive and BrdU-immunonegative GC was counted in one complete testicular cross-section (d6) or randomly selected fields of one cross-section using the stereologer system (d25) and was based on a minimum of 180 GCs. The proliferation index was calculated as the number of positively stained GC nuclei divided by the number of positively + negatively stained GC nuclei x 100.
Determination of GC apoptosis
Apoptotic GCs were determined using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) as described previously (26, 29). Complete testis cross-sections (three per animal at e17.5, two per animal at all other ages) were evaluated systematically and the average number of apoptotic GC per testis cross-section computed.
Statistical analysis
In the present studies, the data presented and analyzed derive from individual animals irrespective of their litter of origin. In most instances, data derived from three to six different litters (usually one to three animals per litter), and analysis using litter mean data rather than individual mean data did not affect the outcome or interpretation of the results other than to reduce the level of significance in some instances. In two instances (GC proliferation index at d6 and d25 in DBP-exposed animals), data derived only from two litters, but even in this instance, analysis of litter means still indicated a significant change (P < 0.05) in the same direction as for data analysis based on individual animals. Comparison of data points for control and DBP-exposed animals at each age used the Student’s t test. However, for evaluation of the prevalence of GC apoptosis, nonparametric methods (Mann-Whitney U test, Fisher’s exact test) were used.
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, A and B). At this stage, approximately 35% of seminiferous cords contained MNGs. At d4, MNGs were still observed, even though DBP treatment had ceased. The prevalence of MNGs was significantly elevated above that found in control animals, in which less than 2% of seminiferous cords contained MNGs, at e19.5, e21.5, and d4 (Fig. 1C). MNGs were no longer detectable in testes of control or DBP-exposed animals at and beyond d15.
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, A–C). In DBP-exposed animals, OCT4 immunostaining was consistently more intense at e15.5, compared with the corresponding controls, as well as at e17.5, when considerably more gonocytes were immunopositive for OCT4. However, by e19.5 OCT4 immunostaining appeared not to be detectable in gonocytes of DBP-exposed animals, as in controls (Fig. 2, D–F). For comparison and because DBP is not reported to affect female reproductive development (31, 32), we also analyzed OCT4 immunoexpression in ovaries of control and DBP-exposed rats. At e17.5, OCT4 was expressed in gonocytes of control females, but in ovaries of DBP-exposed animals, the intensity of OCT4 immunoexpression was similar to controls and did not show evidence of any increase as in the testis (Fig. 2, B and E).
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, the phosphorylated form of Rb at Ser 807/811 was present in fetal rat testes in both Sertoli cell (SC) and gonocyte nuclei. In gonocytes, phospho-Rb immunostaining was evident in control animals at e15.5, when gonocytes are cycling, but was absent at e17.5 and e19.5, when the gonocytes arrest in G1 (Fig. 2, G–I). In testes of DBP-exposed animals, phospho-Rb immunostaining in gonocyte nuclei was more intense than in controls at e15.5, and expression was still present in some gonocyte nuclei at e17.5, in contrast to its absence in controls; however, by e19.5 phospho-Rb expression in gonocytes was no longer evident in DBP-exposed males (Fig. 2, J–L). The effect of DBP exposure on cell cycling in gonocytes was confirmed by immunohistochemistry for Ki67, an established marker of proliferation. At e17.5 in control animals, only occasional gonocytes were weakly immunopositive for Ki67, whereas in DBP-exposed animals, Ki67 expression was more intense and evident in many more gonocytes than controls (Fig. 3, A and B). These results demonstrate prolongation/increase in expression of both phospho-Rb and Ki67 similar to that observed for OCT4 in DBP-exposed animals at e15.5–e17.5. In contrast to GCs, SCs from DBP-exposed animals showed reduced prevalence of staining for phospho-Rb at e19.5 (Fig. 2L) in comparison with controls (Fig. 2I), suggesting they may be less proliferative.
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, C and D, and Table 2); no apoptotic gonocytes were detected at e19.5 or e21.5 in either control or DBP-exposed animals (data not shown). The DBP-induced increase in GC apoptosis at e15.5–e17.5 resulted in a 37% decrease (P = 0.004) in GC numbers per testis by e21.5 (Fig. 4A).
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In males exposed in utero to DBP, GC number was decreased by approximately 50% at d4, and by d8 and by d15, this decrease had magnified to approximately 80% (Fig. 4B). As already reported, in utero exposure of Wistar rats to 500 mg/kg DBP induces a high rate of cryptorchidism, which is usually unilateral (16). For this reason, at d25, when testis descent in controls is usually complete, we classified testes of DBP-exposed animals as scrotal or cryptorchid according to their position registered at autopsy. The prevalence of unilateral and bilateral cryptorchidism in DBP-exposed males was 53 and 33%, respectively, at d25 and 50 and 40% at d90, whereas in controls, all testes were scrotal in position at d25 and d90. For both scrotal and cryptorchid testes in DBP-exposed animals as well as testes of control animals, the number of spermatogonia (SPG) and spermatocytes (SPCs) was determined separately. As shown in Fig. 4C, no significant difference was observed in SPG number at d25 between control and DBP-exposed animals. In contrast, DBP exposure was associated with a marked decrease in SPC number of approximately 50% for scrotal testes and approximately 60% for cryptorchid testes, when compared with controls. In adult animals, GC numbers were determined only for the scrotal testes because most tubules in cryptorchid testes from DBP-exposed animals generally lacked most GCs apart from SPG. In scrotal testes of adults, numbers of SPG, SPCs, and round spermatids were determined separately and revealed no difference between control and DBP-exposed animals (Fig. 4D).
At birth GCs are still blocked in G1, but reportedly at around d3–d4, they resume proliferation (33) and migrate toward the basal lamina of the cords, in which they differentiate into SPG. The progressive decrease in GC number in testes of DBP-exposed animals between d4 and d25 could be indicative of an increased rate of apoptosis or a reduced rate of proliferation. Identification of apoptotic GCs by immunostaining for TUNEL revealed only occasional stained cells at d4–d8, with no evidence of any difference between control and DBP-exposed animals (data not shown). Therefore, we investigated whether DBP treatment affected the exit of GCs from the nonproliferative phase, initially by examining the expression pattern of the phosphorylated form of Rb. At d4, the testes of both control and DBP-exposed animals revealed that GCs were generally phospho-Rb immunonegative and still resided centrally in the cords (Fig. 5, A and B). At d6 in controls, phospho-Rb immunostaining was present in most GCs, which had also now migrated to the basal lamina (Fig. 5C), whereas in DBP-exposed animals, many GCs were still phospho-Rb negative and resided in the center of cords (Fig. 5D). Interestingly, in DBP-exposed animals, the abnormal MNGs followed the same pattern as normal GCs, with some nuclei appearing immunopositive and others immunonegative. At d8, phospho-Rb immunostaining was present in all GCs in both control and DBP-exposed animals (Fig. 5, E and F). To confirm that DBP exposure had affected GC proliferation postnatally, the GC proliferation index (BrdU incorporation) was determined at d6 and d25. As shown in Fig. 6, the GC proliferation index on d6 was significantly decreased by approximately 30% in testes of DBP-exposed rats when compared with controls, whereas the converse was found at d25 when the GC proliferation index in DBP-exposed animals was increased by 84% (P < 0.02) relative to controls.
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). In contrast, the short-term DBP treatment had no effect at all on GC number at d4, whereas the longer-term DBP treatment had induced a 50% decrease in GC number (Fig. 7B).
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Discussion |
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In this study, for the first time, we demonstrated that DBP exposure affects the testicular expression of OCT4 by fetal rat gonocytes, also known as a sensitive and specific biomarker for human CIS cells (37). This transcription factor plays an important role in the establishment and maintenance of mammalian totipotent cell populations (38) and is expressed in totipotent embryonic stem and germ cells (39). In the human fetal testis, gonocytes, intermediate GCs, and pre-SPG have been described as three different subpopulations of GCs, with OCT4 being specifically expressed only in the first of these (30). Because the gonocytes differentiate into intermediate GCs, they lose OCT4 expression, and failure of this normal differentiation process and retention of OCT4 expression is thought to lead to formation of CIS cells (9, 10). As a consequence, in adulthood only some spermatogonial stem cells may express OCT4, whereas all other normal adult GCs do not (40). In contrast, CIS cells and TGCT cells invariably express OCT4 (10, 41). Our results show that in the rat testis, OCT4 expression gradually declines during the proliferative phase of gonocytes within newly formed seminiferous cords (e15.5–e17.5) and that DBP is able to enhance/prolong this temporal expression pattern.
This promising result led us to investigate further the effect of DBP on the transition between proliferative and nonproliferative gonocyte phases in the fetal rat testis. Because gonocytes arrest in G1 during this period, we focused on Rb, which is a crucial regulator of the cell cycle. Rb is thought to impose a block on G1 progression, but when it is phosphorylated by the cyclin-dependent kinases 2, -4, and -6, it loses its function and releases its target, the E2F family transcription factors, resulting in initiation of DNA replication (41, 42). In the present paper, we demonstrate that in rat testicular gonocytes, Rb phosphorylated at Ser 807/811 follows the same temporal expression pattern as does OCT4 and that DBP is able to induce a change in this pattern similar to its effect on OCT4.
The evidence for an effect of DBP on gonocytes during a critical phase of their development was further confirmed by our results for the proliferation marker Ki67 and apoptosis. At the transition period, Ki67 expression was enhanced in animals exposed to DBP, consistent with prolongation of gonocyte proliferation. At the same time, DBP also caused a notable increase in gonocyte apoptosis. It is established that there is a natural peak of gonocyte apoptosis during their proliferative phase (36), but its physiological significance is uncertain, and it is unclear why DBP exposure should have exacerbated it. The latter presumably accounts for the marked decrease in GC numbers that was evident at e21.5 and on d4 in DBP-exposed animals because there was no evidence for gonocyte proliferation or apoptosis in controls or DBP-exposed animals in the period between e19.5 and d4. Nevertheless, between d4 and d8, there was a further massive increase in the GC deficit in DBP-exposed animals when compared with controls. This was not due to any change in apoptosis but was most likely the result of the major decrease in GC proliferation index at d6; this was confirmed by a lower prevalence of phospho-Rb expression during this period. Because phospho-Rb expression had normalized in GC by d8 in DBP-exposed animals, our interpretation is that fetal DBP exposure has caused a delay in the exit of GC from their quiescent period and therefore in the resumption of proliferation at d6.
Our finding that by d25, the GC proliferation index in DBP-exposed animals was substantially increased over the control rate indicates recovery/compensation of GC proliferation and fits with the gradual restoration of normal GC numbers in the testis between d15 and adulthood, with the numbers of SPG normalizing (by d25) before the numbers of SPCs (between d25 and d90). Although this finding suggests that there is complete recovery of normal GC function/numbers by adulthood in DBP-exposed animals, at least in normally descended (scrotal) testes, this may not be entirely the case. Our analysis of fertility by mating studies in adult Wistar rats exposed in utero to DBP has shown an infertility rate of 75% (16), confirming an earlier study (44). Lesions of the epididymis (17, 45, 46) and/or cryptorchidism (16) can probably only partially explain this infertility (16). It is therefore possible that the present demonstration of altered development of GCs in fetal and early postnatal life, as a consequence of fetal exposure to DBP, could compromise the normality/function of GC in adulthood and thus affect fertility. This requires further investigation.
Taken together, the above findings represent good evidence that DBP induces a slight but significant delay of the early phase of gonocyte development in the fetal rat testis. The mechanism via which DBP causes this effect remains to be investigated, but could result from effects on the gonocytes themselves or via effects on the SCs or perhaps Leydig cells. Interestingly, during mouse early development (47) as well as in mouse embryonic stem cells (48) and human carcinoma cells (49), OCT4 expression is regulated by methylation. This epigenetic modification also plays a crucial role in GC development: after demethylation of the genome during primordial GC migration into the genital ridge (50), gonocytes undergo remethylation in a sex-specific manner during gonadal sex determination (51). This remethylation event appears to be dependent on their association with SCs in the testis (52) and could potentially be affected by phthalates. A recent study demonstrated that DBP or butyl benzyl phthalate induces demethylation of the estrogen receptor-
promoter in MCF7 cells (53); DBP has also been reported to cause hypomethylation of the c-myc protooncogene in mouse liver (54). These various findings suggest that alteration of methylation state could be a possible mechanism via which DBP regulates OCT4 expression in gonocytes and, more generally, affects gonocyte development in early stages of fetal rat testis development. This merits further investigation.
Until the present study, the only gonocyte abnormalities described in fetal testes of DBP-exposed animals were the induction of MNG and the aggregation of gonocytes within the center of seminiferous cords (21, 24, 25), findings that we confirm. In the present studies, we established the time course and quantified the prevalence of MNGs, demonstrating that, although sporadically present in control rat testes, they are considerably increased by DBP from e19.5 onward, reaching a maximum at e21.5. Interestingly, this event happens during the gonocyte nonproliferative phase, which occurs immediately after the time frame of DBP-induced changes described above. This appears to reflect a time-specific susceptibility because restriction of DBP exposure of rats to e19.5–e20.5 was able to induce MNGs at e21.5 with a frequency indistinguishable from that induced by daily DBP treatment from e13.5. This suggests strongly that there is no connection between the earlier effects of DBP on gonocyte development described above and the induction of MNGs. Although the origin of MNG and abnormal gonocyte aggregation in DBP-exposed rat remains unclear, the underlying mechanism most probably involves disruption of the interactions between gonocytes and SCs. This is suggested by reported alteration of the vimentin cytoskeleton (24) and changes in expression of factors such as gap junction protein connexin43, fibroblast growth factor, or c-KIT (55, 56).
Because our findings indicated that the early and late effects of DBP on gonocytes were separate events, we also investigated which of these effects was likely to underlie the profound changes in GC development that we observed postnatally. Our results show that, in rats exposed to DBP only in the window e19.5–e21.5, there is no decrease in GC number postnatally as occurs in animals exposed to DBP during the window e13.5–e21.5, although this observation is based only on analysis at a single age (d4). In contrast, both the short and long DBP treatment regimens induced a similar prevalence of MNGs (present study) and a similar reduction in testicular testosterone levels at e21.5 (Hallmark, N., R. Bayne, I. K. Mahood, H. Scott, C. McKinnell, M. Walker, R. A. Anderson, I. Greig, K. Morris, and R. M. Sharpe, unpublished data). This result suggests that the changes observed in GCs of postnatal testes in DBP-exposed rats are directly dependent on the events induced in the early phase of fetal testis development. An earlier study (43) also suggested that there are time-specific windows of susceptibility to some of the effects of DBP treatment.
In summary, the present study demonstrates that in utero exposure to DBP has major generalized effects on GC development during rat perinatal life. In particular, our findings show that DBP alters the timing of gonocyte development/differentiation, with direct consequences for postnatal GC development/proliferation. The transient delay in differentiation of gonocytes in DBP-exposed rats bears a superficial resemblance to the more complete failure of this process that is hypothesized to result in the origin of human CIS cells; in this regard, further studies of whether OCT4+ fetal-like GCs might occasionally persist in the postnatal testis after fetal exposure to DBP would provide a more convincing parallel. Further studies of the normality of GC development and function in adulthood in DBP-exposed animals are also warranted to establish whether the high prevalence of infertility in such animals stems from changes to the GC induced in fetal life.
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Acknowledgments |
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Footnotes |
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Disclosure statement: the authors have nothing to disclose.
First Published Online August 17, 2006
Abbreviations: BrdU, 5-Bromo-2'deoxyuridine-5'-monophosphate; CIS, carcinoma in situ; d, postnatal day; DAB, diaminobenzidine; DBP, di(n-butyl) phthalate; e, embryonic day; GC, germ cell; MNG, multinucleated gonocyte; OCT, octamer-binding transcription factor; Rb, retinoblastoma; SC, Sertoli cell; SPC, spermatocyte; SPG, spermatogonia; TBS, Tris-buffered saline; TDS, testicular dysgenesis syndrome; TGCT, testicular germ cell tumor; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling.
Received April 25, 2006.
Accepted for publication August 9, 2006.
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-receptor 1.5 kb transcript, OCT-4, and c-KIT in human normal and malignant tissues. Implications for the early diagnosis of testicular germ cell tumours and for our understanding of regulatory mechanisms. J Pathol 196:467–477[CrossRef][Medline]
-irradiated rats during testicular development. Biol Reprod 64:1422–1431 gene by phthalates. J Toxicol Environ Health A 68:1995–2003[Medline]This article has been cited by other articles:
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  A. Lehraiki, C. Racine, A. Krust, R. Habert, and C. Levacher Phthalates Impair Germ Cell Number in the Mouse Fetal Testis by an Androgen- and Estrogen-Independent Mechanism Toxicol. Sci., October 1, 2009; 111(2): 372 - 382. [Abstract] [Full Text] [PDF]
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 H. Lin, Q.-Q. Lian, G.-X. Hu, Y. Jin, Y. Zhang, D. O. Hardy, G.-R. Chen, Z.-Q. Lu, C. M. Sottas, M. P. Hardy, et al. In Utero and Lactational Exposures to Diethylhexyl-Phthalate Affect Two Populations of Leydig Cells in Male Long-Evans Rats Biol Reprod, May 1, 2009; 80(5): 882 - 888. [Abstract] [Full Text] [PDF]
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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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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. W. Gaido, J. B. Hensley, D. Liu, D. G. Wallace, S. Borghoff, K. J. Johnson, S. J. Hall, and K. Boekelheide Fetal Mouse Phthalate Exposure Shows that Gonocyte Multinucleation is Not Associated with Decreased Testicular Testosterone Toxicol. Sci., June 1, 2007; 97(2): 491 - 503. [Abstract] [Full Text] [PDF]
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