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1 Inserm, U407, Oullins F-69921, France 2 University Claude Bernard Lyon 1, Lyon F-69921, France 3 Hospices Civils de Lyon, Hôpital Lyon Sud, Service Anatomie Pathologique, Lyon F-69921, France
(Correspondence should be addressed to M Benahmed who is now at INSERM, U895, équipe 5, Bâtiment Archimed, Centre de Médecine Moléculaire, 151 route Saint-Antoine Ginestière, 06200 Nice, France; Email: benahmed.m{at}chu-nice.fr)
* (L Benbrahim-Tallaa, B Siddeek and A Bozec contributed equally to this work) 
(M Benahmed and C Mauduit are the senior co-authors)
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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). In conclusion, fetal androgen disruption may induce alterations of Sertoli cell activity probably related to Sertoli cell maturation, which potentially leads to increased adult germ cell apoptosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The aim of the present study was to determine the potential role of Sertoli cells in the adult germ cell death through the evaluation of plasma hormones that target Sertoli cells, AR expression, Sertoli cell number, and functions via the evaluation of the levels of specific transcript expressed in these adult somatic cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All studies on animals were conducted in accordance with the current regulation and standards approved by the INSERM (French Institute for Health and Medical Research) Animal Care Committee.
Materials
Flutamide obtained from Sigma was dissolved in an aqueous solution of 0.5% w/v methylcellulose 400 (Fluka, Mulhouse, France) and stored for a maximum of 1 week at 
5 °C (±3 °C). Dulbecco's Modified Eagle (DME)/Ham's F12 (1:1) medium, reverse transcriptase (Moloney-murine leukemia virus (M-MLV)), polyacrylamide 37.5:1 solution, Ponceau Red, and TRIzol were obtained from Life Technologies. Collagenase/dispase and protease cocktail inhibitor were obtained from Roche. Gentamicin, 3-cyclohexylamino-1-propanesulfonic acid (CAPS), Kodak XOMAT films, antibody raised against actin, diaminobenzidine (DAB), sodium bicarbonate, HEPES, DNase I, 3-N-morpholinopropane-sulfonic acid (MOPS), SDS, random primers, and BSA were purchased from Sigma. Rabbit polyclonal anti-human Glutathione-S-transferase (GST) antibody (Mehta et al. 1994, Murray et al. 1995) was purchased from Novocastra-Tebu (Le Perray en Yvelines, France). Peroxidase-conjugated goat anti-rabbit immunoglobulin G and Covalight chemiluminescence detection kit were obtained from CovalAb (Lyon, France). AR rabbit polyclonal antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody diluent, Harris hematoxylin, and Envision+ kit were obtained from Dako (Copenhagen, Denmark). Bradford reagents and Pierce detection kit were purchased from Bio-Rad Laboratories. Hybond-C membranes [-33P] dATP and [-32P] dCTP were purchased from Amersham Biosciences. Proligo SA (Paris, France) was the source for oligonucleotide primers. Random priming DNA labeling kit and Taq polymerase were purchased from Promega Life Science, and Schleicher & Schuell polyvinyl difluoride (PVDF) membrane from Merck Eurolab (Strasbourg, France).
Methods
Animals: in utero exposure to anti-androgens Fifteen pregnant rats (Sprague–Dawley rats from Charles River Laboratories, St Aubin les Elbeuf, France) per group were administered vehicle (methylcellulose) or flutamide by daily gavage from day 10 of gestation (GD 10) up to the day before delivery (GD 21 or 22). This period includes the period of male internal and external sex differentiation. The experimental protocol used was based on the previous data reported by McIntyre et al. (2001). However, lower doses of the anti-androgen were chosen to avoid or minimize important germ cell loss, i.e., before the occurrence of major histological lesions. Indeed, alterations in testicular cellularity (i.e., the ratio of somatic cells to germ cells) may confound the interpretation of the effects of the anti-androgen on gene expression in the different testicular cell types (for reference see Ivell & Spiess 2002). Consistently, at the doses used here, there were no changes in adult testicular weight and epididymis sperm counts (untreated: 472.4±186.9 vs 10 mg/kg d flutamide: 352.9±263.4; P>0.05). At the maximal dose (10 mg/kg per day) used for flutamide, anti-androgenic effects of the compound were observed including i) increased cryptorchid testes rate (Bozec et al. 2004) ii) reduced ventral prostate weight (dose 0: 0.683±0.119 versus dose 10: 0.07±0.053; P<0.0001) and iii) increased malformations of vas deferens (dose 0: 0% versus dose 10: 18.75%; P<0.05). Animals were given flutamide at doses of 0, 0.4, 2, and 10 mg/kg body weight per day (mg/kg d) adjusted daily based on body weight. Dams were weighed daily from GD 10 up to the day of delivery. After birth, male pups were grown without treatment until they were killed at 15 or 90 days by CO2 inhalation. Each testis was weighted before being fixed, used for Sertoli cell isolation, or frozen. The position of the testes was carefully observed to detect cryptorchidism. Cryptorchid testes were most often located ectopically in the peritoneal cavity or inguinal area. Fully descended testes are the ones positioned in the scrotum. In the present study, we have used only descended testes. At least seven rats from three different litters were used per condition. The experiment was repeated at least three times.
Cell isolation and culture
Seminiferous tubules were isolated from testes of adult (90-day-old) rats exposed in utero to methylcellulose (control) or flutamide (0, 0.4, 2, and 10 mg/kg d). Once the albuginea was removed, the testicular tissues were mechanically dispersed with forceps in DME/F12 medium (supplemented with 1.2 mg/ml sodium bicarbonate, 15 mM HEPES, and 20 µg/ml gentamicin) containing DNase I (0.05 mg/ml). Testicular tissues were then dispersed by collagenase/dispase treatment (0.5 mg/ml) in DME/F12 medium through mild stirring. After enzymatic dissociation, testicular cells were washed three times by gravity sedimentation (3–5 min). The seminiferous tubules were washed at least three times to remove potential contaminating Leydig cells. Seminiferous tubules were dry frozen and kept at –70 °C until use for GST expression.
In the co-culture Sertoli cell–germ cell model, Sertoli cells were isolated, by collagenase dissociation, from pre-pubertal (15-day-old) testes of rats exposed in utero to methylcellulose (control) or the anti-androgen flutamide (10 mg/kg d). Mature germ cells (spermatocytes and spermatids) were isolated and purified from adult untreated rat testes by the centrifugation–elutriation method as reported previously (Boussouar et al. 2003). The Sertoli cells were seeded onto Petri dishes (Falcon, Los Angeles, CA, USA) 6 cm in diameter, at a density of 500 000 cells/cm2 and cultured in standard conditions (Florin et al. 2005). Two days after Sertoli cell isolation and culture, germ cells were added to Petri dishes generating three groups of cultured cells: Sertoli cells cultured (alone) without germ cells, Sertoli cells co-cultured with spermatocytes (500 000 cells/cm2), and Sertoli cells co-cultured with spermatids (500 000 cells/cm2). After 72 h of co-culture, the cells were either collected in an ice-cold lysis buffer (25 mM Tris, 0.1% SDS, 1% vol/vol protease inhibitor cocktail) for western blotting analysis or in 1 ml TRIzol for RT-PCR analysis. The purity of the isolated pre-pubertal Sertoli cell was checked by western blotting (tubulin-β3 and GATA-6). The resulting Sertoli cells were free of Leydig (assessed by 3-β-hydroxysteroid dehydrogenase) and germ cells (assessed by c-kit for spermatogonia, poly (ADP-ribose) polymerase (PARP)-1 for spermatocytes, protamine-1 for round spermatids, and MCT2 for elongated spermatids) and contained between 2% and 5% peritubular myoid cells, as evaluated by fibronectin and alkaline phosphatase immunostaining (data not shown). The purity of spermatocytes and round spermatids was assessed by the presence of PARP-1 and protamine-1 respectively. The absence of somatic cells was assessed by the markers mentioned above.
Cell type counting For Sertoli and Leydig cell counting, ten 90-day-old rats (originating from three different litters) treated in utero with flutamide (0 or 10 mg/kg d) were used for each experimental condition. After the animals were killed by CO2 inhalation, the testes were rapidly dissected in order to remove the connective tissue and the epididymis and weighed. The left testis of each animal was fixed overnight in Bouin's solution by immersion. These were weighed and then sampled in a random systematic manner. The testes were sliced transversally into six pieces and the slices 1, 3, and 5 or 2, 4, and 6 were randomly processed in graded ethanol and infiltrated with JB4 resin (TAAB, Berkshire, UK). After full polymerization, 25 µm sections were cut on an Ultracut microtome (Reichert-Jung Inc., Wien, Austria) using glass knives, mounted onto adhesive glass slides, and stained with Harris hematoxylin. Sertoli and Leydig cells were then counted using the optical dissector method as described by Wreford (1995) and Sharpe et al. (1998), (2000). The thick sections were viewed at high magnification and optically sectioned with a microscope equipped with a microcator to measure stage movement in the z-axis. The top of the section was focused and a small guard area of 3 µm was traversed, the microcator was zeroed, and the section was then viewed at a series of 1.5 µm intervals for a further 15 µm. The images of each plane were acquired using a video camera coupled with a Leitz Quantimet 570 image analyzer system (Milton Keynes, UK). Sertoli and Leydig cell nuclei were then counted in an unbiased counting frame of 6400 µm2 area giving a 96 000 µm3 volume for each field. More than 100 Sertoli or Leydig cells were counted for each animal representing at least 1000 counted nuclei for each experimental condition. The counting results gave a direct estimation of the number of Sertoli or Leydig cells per testis.
mRNA quantification Co-amplification RT-PCR with an endogenous control: total RNA (2 µg), from whole testis, was reverse transcribed into cDNA. PCR was then performed on 1 µl reverse transcription product as described previously (Bozec et al. 2004). PCR mixtures were submitted to an initial denaturing step at 95 °C, followed by n cycles consisting of 30 s at 95 °C, 30 s at hybridization temperature, and 30 s at 72 °C, and the reaction ended with a final extension step of 5 min at 72 °C (Table 1). PCR products were subsequently resolved on 8% polyacrylamide gel, which were then exposed to a Storage Phosphor Screen and the signals analyzed using the Cyclone OptiQuant Software (Packard, Meriden, CT, USA). Results from at least three separate experiments were used for statistical analysis. PCR analyses were carried out from the logarithmic phase of amplification. PCRs with different cycle numbers were realized for each primer pair to determine the minimum number of cycles necessary to detect the PCR product. Primers were designed inside separate exons to avoid any bias caused by residual genomic contamination. Moreover, for all primers, no amplification was observed when PCR was performed on RNA preparations.
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probe was prepared by PCR and labeled as described previously (Benbrahim-Tallaa et al. 2002a). Total RNAs from adult rat seminiferous tubules were size fractionated by electrophoresis on 1.2% agarose and 2.2 M formaldehyde gels. The northern blotting analysis was performed as described previously (Benbrahim-Tallaa et al. 2002a). The intensities of the autoradiographic bands were estimated by densitometric scanning using Intelligent Quantifier Software (Bio-Image, Cheshire, UK). The data are expressed as the ratio of GST to 18S mRNA.
Western blotting analysis
Total proteins were extracted from rat whole testes, seminiferous tubules, or cultured testicular cells treated under different conditions. Protein extracts (80 µg/well for GST, 100 µg/well for AR) were size fractionated on SDS-polyacrylamide gel (Benbrahim-Tallaa et al. 2002b). The membranes were incubated with the rabbit polyclonal anti-GST antibody diluted 1:1000 or the rabbit polyclonal anti-AR antibody diluted 1:100 in a solution of tris buffered saline (TBS) with 0.5% nonfat dry milk for 2 h at room temperature. After washing with TBS, the membranes were then incubated with the goat anti-rabbit peroxidase-conjugated antibody diluted at 1:2000 in TBS buffer with 0.5% nonfat dry milk. The antibody–antigen complexes were detected by chemiluminescence using CovalAb detection kit. The protein loading was checked by reprobing the blot with a rabbit IgG anti-actin antibody (1:500).
Data analysis
Data are expressed as the mean±S.D. Three to seven animals from different litters were used. For statistical analysis of data generated in in vivo and in vitro models, one-way ANOVA was performed to determine whether there were differences between all groups (P<0.05), and then the Bonferroni post-test was performed to determine the significance of the differences between the pair of groups. P<0.05 was considered significant. The statistical tests were performed on StatView software version 5.0 (SAS institute Inc., Cary, NC, USA).
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Results |
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As fetal androgen disruption induced a long-term apoptosis in adult germ cell characterized by caspase-3 activation in spermatocytes and spermatids (Omezzine et al. 2003) and Sertoli cells are targeted by androgens in the adult seminiferous tubules, we addressed the question as to whether the increased adult germ cell death might be related to altered Sertoli cell activity. For this purpose, purified post-natal (PND 15) Sertoli cells from rat testes exposed in utero to 10 mg/kg d of flutamide were co-cultured with adult purified germ cells (i.e., spermatocytes or spermatids) not treated with flutamide. While active caspase-3 levels were not affected in Sertoli cells isolated from testes treated in utero with flutamide and cultured alone compared with Sertoli cells originating from sham-treated (methylcellulose) testes (Fig. 1A), increased active caspase-3 levels were detected in spermatocytes (Fig. 1B; P<0.0001) or spermatids (Fig. 1C; P=0.037) when co-cultured with Sertoli cells treated in utero with the anti-hormone (10 mg/kg per day). Together, these observations would suggest a possible alteration in the functions of (flutamide-treated) Sertoli cells related to their role in promoting adult germ cell survival.
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As the germ cell survival has been linked to androgen action, which is mainly exerted through Sertoli cells (De Gendt et al. 2004, Tan et al. 2005), we addressed the question as to whether the long-term apoptosis observed in germ cells might be related to alterations in the androgen signal in the adult animals. The androgen signal could be evaluated, at least, through three components including the plasma levels of testosterone, its metabolite estradiol, and the number of Sertoli cells that are targeted by androgens in the adult seminiferous tubules and the AR expression. First, fetal androgen disruption following an in utero treatment with flutamide (0.4, 2, and 10 mg/kg per day) was shown to have no effect on the plasma levels of hormones including testosterone, estradiol, gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in adult animals (Table 2). Secondly, with regard to the number of somatic cells targeted by androgens, no change in Leydig cells (Fig. 2A) and Sertoli cell number (Fig. 2B) was observed in adult rat testes treated in utero with flutamide. It is noteworthy that the testicular weight is not affected by the anti-androgen treatment at the doses used here (Fig. 2D; see also Bozec et al. 2004). Thirdly, in terms of AR expression, immunohistochemical experiments revealed that AR nuclear staining occurred, as expected, in Sertoli, peritubular myoid, and Leydig cells from adult rat testes (Fig. 3A). Fetal treatment by the anti-hormone flutamide (10 mg/kg d) did not affect AR immunolocalization in the adult rat testes (Fig. 3B). Interestingly, the AR expression remained unaffected in the adult testis fetally treated by the anti-androgen, as evaluated through mRNA (Fig. 3C) and protein levels (Fig. 3D). Altogether, the data obtained suggest that the altered Sertoli cell activity leading to increased adult germ cell apoptotic process (Fig. 1) appears not to be linked to the androgen signal when evaluated through plasma hormone levels, Sertoli cell number, and AR expression.
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As the germ cell death process specifically affects adult germ cells (spermatocytes and spermatids), we hypothesized that the potential alterations in Sertoli cell functions in terms of protecting adult germ cells against death might be related to a possible lack of Sertoli cell maturation. To test such a hypothesis, we evaluated the levels of several transcripts reported to be specifically present in adult Sertoli cells (O'Shaughnessy et al. 2003): AMH type II receptor, Cox-1, cathepsin L, cystatin SC and cystatin TE, cyclin D2, and GST. In utero flutamide treatment (10 mg/kg d) resulted in a decrease in AMH type II receptor (Fig. 4A; P=0.0021), cathepsin L (Fig. 4B; P=0.0029), Cox-1 (Fig. 4C; P=0.0017), cyclin D2 (Fig. 4D; P=0.0055) mRNA levels. In contrast, cystatin TE (Fig. 4E) and cystatin SC (Fig. 4F) mRNA levels were not affected.
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expression. This gene is expressed in both Leydig and Sertoli cells (Benbrahim-Tallaa et al. 2002b) and was reported to be under the control of androgens in Sertoli cells (Benbrahim-Tallaa et al. 2002b). In utero flutamide treatment resulted in a decrease in GST mRNA levels in seminiferous tubules with a significant decrease observed at 2 mg/kg d (P=0.01) and 10 mg/kg d (P=0.001) flutamide (Fig. 5A). The decrease in GST mRNA levels was associated with a parallel decrease in GST protein levels in the seminiferous tubules (Fig. 5B). Such a decrease in GST protein levels was further confirmed in cultured Sertoli cells isolated from rats exposed in utero to flutamide (Fig. 5C; P=0.0006).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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was found to be down-regulated in adult testes exposed in utero to the anti-androgen flutamide. This altered pattern of expression may suggest that following fetal exposure to the anti-androgen, Sertoli cells remained in a relative immature state. Such a relative lack of Sertoli cell maturation may explain the loss of their protective role in terms of adult germ cell survival. However, there are still two points that need more clarification. First, the relative alteration of Sertoli cell maturation needs still to be confirmed by the approaches related to ‘omic’ genome-wide approaches. Secondly, while we suggest that the decrease in adult Sertoli cell gene expression leading to this potential lack of Sertoli cell maturation may result in increased germ cell death, it still remains to identify adult Sertoli cell androgen-targeted genes whose altered expression may directly affect germ cell survival. Among these Sertoli cell genes, GST is a detoxification enzyme that protects the adult testicular germ cells against apoptosis (Rao & Shaha 2000) by decreasing the cytotoxic effects of oxidants (Yang et al. 2001). As such, the decrease in GST levels in adult Sertoli cells could represent also one of the possible mechanisms supporting the long-term apoptosis in germ cells from animals exposed in utero to anti-androgen. This could impair the Sertoli–germ cell interactions leading to a long-term apoptosis of germ cells. One of the major questions raised by the present findings is related to the causal link between the fetal exposure to the anti-androgen flutamide and the adult testicular phenotype characterized by increased germ cell death process probably related, as suggested here, to alteration in Sertoli cell functions. First, although we are still lacking in parameters (or molecular biomarkers) to identify the anti-androgenic effects of flutamide in the fetal testis (i.e., in the AR-expressing fetal testicular cells such as Leydig, peritubular myoid cells (You & Sar 1998), and gonocytes (Merlet et al. 2007), there are extratesticular parameters that clearly indicate the androgen action disruption after fetal exposure to flutamide at the doses used (maximum 10 mg/kg per day). These parameters include decreased prostate weight, increased malformations in vas deferens, and increased cryptorchid testis number. One should note that the recently reported fetal testicular parameters to be affected following fetal exposure to environmental endocrine disruptors such as phthalates appear to be more related to their toxic rather than to their anti-androgenic activities (Scott et al. 2007). One of the possible approach to identify parameters (or molecular biomarkers) AR-targeted genes related to androgen action disruption could be based on the use of omic tools in isolated and purified fetal testicular cells expressing AR. Secondly, in the adult testes, among the arguments supporting that the testicular phenotype, i.e., the increased germ cell death process, might be linked to androgen action disruption is that the death process occurred in spermatocytes and spermatids at androgen-dependent VI–VIII stages (Bozec et al. 2004). Such a germ cell death process occurred despite normal plasma testosterone levels, normal (androgen targeted) Sertoli cell number, and normal testicular AR protein and mRNA levels. It is possible that the androgen action disruption occurred at post-AR steps, as yet mentioned, in terms of AR activity, fetal exposure to flutamide may induce an alteration in AR cofactor balance in the adult testes (Maire et al. 2005). With regard to alterations of androgen-targeted gene expression, it is quite possible that these alterations might be related to epigenetic changes involving, for example, the methylation/acetylation pattern of the androgen-dependent gene promoters, as yet suggested, for example, for lactotransferrin (Li et al. 1997) and c-fos (Li et al. 2003) in adult uterine mice tissues from neonatally treated animals with diethylstilbestrol (Ruden et al. 2005) and more recently for phosphodiesterase type 4 in adult rat prostate neonatally exposed to estradiol (Ho et al. 2006).
In summary, we showed here that fetal androgen disruption induced a long-term alteration of Sertoli cell functions, which may result in a long-term increased adult germ cell death process in the context of Sertoli cell–germ cell interactions.
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Acknowledgements |
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References |
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Received in final form 7 September 2007
Accepted 9 October 2007
Made available online as an Accepted Preprint 10 October 2007
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