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Endocrinology Vol. 146, No. 12 5135-5143
Copyright © 2005 by The Endocrine Society

Alteration of Transforming Growth Factor-ß Signaling System Expression in Adult Rat Germ Cells with a Chronic Apoptotic Cell Death Process after Fetal Androgen Disruption

Magali Maire, Anne Florin, Krisztian Kaszas, Daniel Regnier, Pierre Contard, Eric Tabone, Claire Mauduit, Remi Bars and Mohamed Benahmed

Institut National de la Santé et de la Recherche Médicale, Unité 407 (M.M., A.F. K.K., D.R., P.C., E.T., C.M., M.B.), Faculté de Médecine Lyon-Sud, 69921 Oullins, France; and BayerCropScience (R.B), Sophia-Antipolis F-06903, France

Address all correspondence and requests for reprints to: Mohamed Benahmed, Institut National de la Santé et de la Recherche Médicale Unité 407, Faculté de Médecine Lyon-Sud, BP 12, 69921 Oullins Cedex-France. E-mail: benahmed{at}grisn.univ-lyon1.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In utero exposure to chemicals with antiandrogen activity induces undescended testis, hypospadias, and sub- or infertility. The hypospermatogenesis observed in the adult rat testis exposed in utero to the antiandrogen flutamide has been reported to be a result of a long-term apoptotic cell death process in mature germ cells. However, little if anything is known about the upstream signaling mechanisms controlling this apoptosis. In the present study, we have investigated the possibility that the TGF-ß signaling pathway may be at play in this control of the apoptotic germ cell death process. By using a model of adult rat exposed in utero to 0, 0.4, 2, or 10 mg/kg·d flutamide, we observed that pro-TGF-ß signaling members, such as the three isoforms of TGF-ß ligands (TGF-ß1–3), the two TGF-ß receptors (TGF-ßRI and -RII) and the R-Smads Smad 1, Smad 2, Smad 3, and Smad 5 were inhibited at the mRNA and protein levels, whereas the anti-TGF-ß signaling member Smad 7 was overexpressed. Furthermore, we report that the overexpression of Smad 7 mRNA could induce an activation of c-Jun N-terminal kinase, because of the observed c-Jun overexpression, activation, and nuclear translocation leading to an increase in the transcription of the proapoptotic factor Fas-L. Together, the alterations of TGF-ß signaling may represent upstream mechanisms underlying the adult germ cell apoptotic process evidenced in adult rat testis exposed in utero to antiandrogenic compounds such as flutamide.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE FETAL HORMONAL (androgen) disruption induced by exposure to various environmental compounds gives rise to a wide range of reproductive system abnormalities including undescended testis, hypospadias, and subfertility (1, 2, 3, 4, 5, 6). By using as a model adult rats exposed in utero to an antiandrogenic compound such as flutamide, we have reported that such a hypospermatogenesis could be related to a chronic apoptotic process (7, 8) occurring in adult germ cells. This apoptotic process was related to a long-term increase in the expression and activation of effector caspases-3 and -6 (7), probably resulting from changes in the ratio of Bcl-2 family peptides in favor of the proapoptotic members, with a specific decrease in (antiapoptotic) Bcl-2 and Bclw and an increase in (proapoptotic) Bax protein levels (8). More recently, such a germ cell death process was also observed in adult rat testes exposed in utero to other compounds with antiandrogen activity such as methoxychlor and vinclozolin (9). However, the upstream mechanisms leading to this chronic germ cell apoptosis process after in utero androgen disruption still remains to be investigated.

In the present study, we have examined the possibility that such a chronic germ cell apoptosis might be related to upstream signaling system alterations. Among the key intratesticular signaling systems is the TGF-ß pathway. The TGF-ßs are a family of potent multifunctional cytokines that modulate a wide variety of cellular activities. Members of this superfamily include bone morphogenic protein (BMPs), activin, decapentaplegic Vg-related proteins, dorsalin, nodal, Müllerian-inhibiting substance, inhibin, growth and differentiation factors , and glial-cell-derived neutropic factor (for review, see Ref.10). These cytokines bind to two different types of serine/threonine kinase receptors (type I and type II) (11, 12) and activate intracellular substrates, e.g. Smad proteins (for review, see Ref.13). Smads are subdivided in three subclasses based on their structure and function (for review, see Ref.14). R-Smads (Smads 1, 2, 3, and 5) are directly activated by the type I receptor and then form a complex with the common partner (Co-Smad), Smad4/DPC4, to be internalized into the nucleus (15). Inhibitory Smads, Smad 6 and 7 (I-Smads) (16), are induced by ligand stimulation (17) and interfere with the receptor activation (18, 19) or complex formation of R-Smads/Co-Smad (20). Smads originally were believed to be the only substrates of TGF-ß receptors and their unique intracellular mediators, but several reports show that TGF-ß and BMPs interact with the MAPK pathways. The three TGF-ß ligand isoforms and the two major receptors are expressed in fetal and adult rat testis (21, 22, 23, 24, 25). Specifically, TGF-ß1 is expressed in Sertoli cells and germ cells, with an increased expression in spermatocytes and round spermatids at stages VIII and IX (26). TGF-ß2 has been localized to Leydig cells and round spermatids but not to Sertoli cells during spermatogenesis (26). Recently, TGF-ß3 has been localized to spermatocytes and round spermatids, but its expression seems to be stage dependent, with an increase at stages V–VIII (27). Types I and II TGF-ß receptors are present in spermatogonia and spermatocytes and weakly in round spermatids (28). In contrast, Smad expression has been poorly studied in the testis. TGF-ß family peptides have been shown to exert different functions in the testis as they control steroidogenesis in fetal and adult Leydig cells (29, 30), germ cell number (31, 32, 33), extracellular matrix synthesis (34), Sertoli cell tight junction formation (35, 36), and cell-to-cell interactions between Sertoli cells and peritubular myoid cells (37). TGF-ß family peptides have been shown to be involved in the apoptotic cell death process, although the molecular and cellular mechanisms still remain to be investigated. In addition, they have been shown to promote (38, 39, 40) or inhibit (41, 42, 43) apoptosis, depending on the cellular and molecular context.

Considering 1) the potential roles of TGF-ß family peptides in the testis formation and function and 2) the critical role of the endocrine (androgen) system in spermatogenesis, we have examined in the present study the possibility as to whether a fetal hormonal (androgen) disruption may affect TGF-ß signaling system in the adult testis, which exhibits a chronic germ cell death process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
TRIzol, deoxy-NTPs (dNTPs), oligonucleotide primers, and Moloney murine leukemia virus reverse transcriptase kit were obtained from Invitrogen Life Technologies, Inc. (Eragny, France). Taq polymerase was obtained from Promega Corp. (Lyon, France). Amersham Bioscience (Little Chalfont, UK) was the supplier of [{alpha}-33P]dATP. Flutamide was obtained from Sigma-Aldrich Corp. (Meylan, France) and was dissolved in an aqueous solution of methylcellulose 400 (Fluka, Mulhouse, France) at 0.5% (wt/vol) and stored for a maximum of 1 wk at approximately 5 C (±3 C). Sigma-Aldrich was also the supplier for BSA and hexanucleotide primers and Biomax MR films. Protease inhibitors cocktail was obtained from Roche Molecular Biochemicals (Mannheim, Germany). Rabbit antiactin polyclonal antibody (H-300), goat anti-TGF-ß1 polyclonal antibody (V), rabbit anti-TGF-ß2 polyclonal antibody (V), rabbit anti-TGF-ß3 polyclonal antibody (V), rabbit anti-TGF-ßRII polyclonal antibody (C-16), goat anti-Smad1 polyclonal antibody (T-20), goat anti-Smad2 polyclonal antibody (S-20), goat anti-Smad3 polyclonal antibody (I-20), rabbit anti-Smad4 polyclonal antibody (H-552), goat anti-Smad5 polyclonal antibody (D-20), goat anti-Smad6 polyclonal antibody (S-20), goat anti-Smad7 polyclonal antibody (N-19), and mouse anti-p-c-jun monoclonal antibody (KM-1) were obtained from Santa Cruz Biotechnology (Le Perray en Yvelines, France). Taq polymerase was purchased from Promega Corp. (Madison, WI). Peroxidase-conjugated AffiniPure goat antirabbit IgG (H+L) and peroxidase-conjugated AffiniPure goat antimouse IgG (H+L) were purchased from Jackson ImmunoResearch (West Grove, PA). Covalight kit was obtained from CovalAb (Lyon, France). Dako Corp. (Trappes, France) was the source for Faramount, the CSA kit (K1500), antibody diluent, and hematoxylin.

Animals
Rats were exposed to flutamide exclusively during the prenatal period. Virgin female Sprague-Dawley rats from Charles River laboratories, Inc. (St. Aubin les Elbeuf, France) were individually housed in controlled conditions of lighting (12 h light, 12 h dark), temperature (22 ± 2 C), humidity (55 ± 15%), and ventilation (~15 air changes per hour) and given free access to water and feed (certified rodent pellet diet, AO4C; UAR, Villemoisson-sur-Orges, France). Females were mated on a one-to-one basis with males of the same strain from the same supplier. Day 0 of gestation (GD 0) was considered when a vaginal sperm plug was noted. Before mating and during gestation, dams were housed in suspended stainless steel wire mesh cages. Shortly before parturition and during lactation, dams were housed in Makrolon cages with soft wood bedding. Pregnant rats were administered vehicle control (methylcellulose) or flutamide by daily gavage from GD 11 up to the day before delivery (GD 21 or 22). Animals were administered flutamide at doses of 0, 0.4, 2, or 10 mg/kg body weight per day (adjusted daily based on body weight). Dams were weighed daily from GD 10 up to day of delivery. At birth, each pup was sexed, weighed, and identified. The male rats were then left with no flutamide treatment until postnatal d 90 and were killed by CO2 inhalation. The position of each testis was carefully noted. Only bilateral descended testes were studied in the present report. Ten different animals were used in control and in each treated group. This study was conducted in accordance with current regulations and standards approved by Institut National de la Santé et de la Recherche Médicale Animal Care Committee.

Immunohistochemistry
Paraffin sections of Bouin-fixed testis were sectioned at 5 µm. The sections were mounted on positively charged glass slides (SuperFrost plus; Menzel-Glaser, Freiburg, Germany), deparaffinized, rehydrated, treated 20 min at 93–98 C in citric buffer (0.01 M, pH 6), rinsed in osmosed water (twice for 5 min each), and washed (twice for 5 min each) in Tris-buffered saline (TBS). The CSA kit was used and immunohistochemistry was conducted according to the manufacturer’s instructions. Antibodies were diluted at 1:800 for Smad 1, Smad 2, Smad 3, and Smad 7; 1:1000 for Smad 2; and 1:400 for Smad 4, Smad 5, and Smad 6.

RT-PCR coamplification with an endogenous control
Total RNAs were extracted from rat testis tissues using TRIzol, following an improvement of the single-step RNA isolation method (44). The final amount of RNA was estimated by spectrometry at 260 nm. Coamplification RT-PCR (45) was performed to determine the mRNA levels of the different genes studied. The cDNAs were obtained from RT of 2 µg total RNAs using random hexanucleotides as primers (5 µM) in the presence of dNTPs (250 µM), dithiothreitol (10 µM), and Moloney murine leukemia virus (10 U/µl), 1 h at 37 C in 1x first-strand buffer. For PCR analysis, the target genes (using the appropriate sense- and antisense-specific primers) were coamplified with ß-actin or hypoxanthine phosphoribosyltransferase (HPRT) as the standard genes. The stock reactions (20 µl) were prepared on ice and contained 0.5 U/µl Taq polymerase, 2.5 µM MgCl2, 200 nM dNTP, 0.75 µCi [{alpha}-33P]dATP, and 2 µl RT mixture 1/10 (cDNA). TGF-ß1, TGF-ß2, TGF-ß3, TGF-ß receptor type I (TGF-ßRI), TGF-ßRII, Smad 1, Smad 2, Smad 3, Smad 4, Smad 5, Smad 7, c-jun, Fas-L, and tissue inhibitor of metalloproteases-1 (TIMP-1) were coamplified with ß-actin, and Smad 6 was coamplified with HPRT. Coamplification with ß-actin or HPRT was performed to check that equal amounts of cDNAs were amplified in each reaction tube. The PCR conditions were 94 C for 5 min followed by denaturing at 94 C for 30 sec, annealing for 30 sec, and extension at 72 C for 30 sec for the required number of cycles, followed by a final extension step at 72 C for 5 min. Table 1Go shows the primer sequences, the optimized number of cycles, and annealing temperatures of each target gene. Table 2Go shows primer concentrations used for each PCR. After amplification, the PCR products were separated by electrophoresis on an 8% polyacrylamide gel. Dried gels were exposed to storage phosphor screen (Packard Instrument Co., Inc., Meriden, CT) for 20 min at room temperature. Band intensities were estimated by densitometric scanning using the Packard Optiquant software. Data are expressed as target gene/standard gene mRNA ratios. Primers were designed inside separate exons to avoid any bias caused by a residual genomic contamination.


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  		TABLE 1. Primers used and experimental conditions for coamplification PCR

 

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  		TABLE 2. Primer concentrations used for each PCR

 
Western blotting analysis
Testicular protein extracts were prepared by direct addition of 5 vol cold lysis buffer to the samples and mechanical homogenization of the tissues. Lysis buffer consisted of 50 mM Tris (pH 7.4), 250 mM NaCl, 5 mM EDTA, 50 mM NaF, and 0.001% Triton X-100 and was supplemented immediately before use with a cocktail of protease inhibitors (Sigma, Isle d’Abeau, France). The protein concentration of the tissue lysates was determined using a colorimetric Bradford method. Optimized quantities of each protein sample were resolved on a 10% sodium dodecyl phosphor-polyacrylamide gel (8 µg for TGF-ß1, Smad 1, and Smad 2; 10 µg for TGF-ß2; 3 µg for TGF-ß3; 20 µg for TGF-ßRII, Smad 5, and Smad 6; 5 µg for Smad 3; and 15 µg for Smad 4, Smad 7, and p-c-jun) and electroblotted onto a nitrocellulose membrane for 30 min at constant voltage of 10 V (semidried transfer). The membrane was blocked in TBS with 0.05% Tween 20 (TBST), 5% nonfat dried milk (or 5% BSA for p-c-jun Western blotting detection) for 2 h, incubated with the diluted primary antibody (dilution 1:500 for TGF-ß1, TGF-ß2, TGF-ßRII, Smad 1, Smad 2, and Smad 3; 1:200 for Smad 4; 1:250 for Smad 5, Smad 6, and Smad 7; and 1:100 for p-c-jun) overnight at room temperature, washed (three times for 5 min each) in TBST, incubated with diluted goat antigoat, antirabbit (1:2000), or antimouse (1:10,000) antibody for 2 h, and washed (three times for 5 min each) in TBST and then rinsed (three times for 5 min each) in TBS. Protein loading was checked by reprobing the blot with the rabbit IgG antiactin (1:2000) and the diluted goat antirabbit antibody. Bound antibodies were detected using the chemiluminescence detection kit according to the manufacturer’s recommendations and Kodak Biomax films.

Data analysis
Data are expressed as the mean ± SEM. Ten different male offspring from different litters were used. For statistical analysis, one-way ANOVA was performed to determine whether there were differences among all groups, and then the Bonferroni/Dunn posttest was performed to determine the significance of the differences between the pairs of groups. P < 0.05 was considered significant. The statistical tests were performed on StatView software (version 5.0; SAS Institute Inc., Cary, NC) on a Macintosh computer (Cupertino, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunolocalization of Smad 1 to Smad 7 to the different adult testicular cell types
Because TGF-ß ligands and receptors have been previously immunolocalized to the different testicular cell types by different laboratories (21, 22, 23, 24, 25, 26, 28, 36, 46), in the present study, we have focused on Smad 1 to Smad 7 immunolocalization in the adult rat testis. Smad 1, 2, and 3 immunostainings were detected only in the seminiferous tubules, specifically in germ cells, in a juxtanuclear position from the pachytene spermatocytes (Fig. 1Go, A–C, respectively) to the more mature germ cells. Smad 4 immunostaining appeared to be more diffuse in the whole testis because it was predominantly detected both in the seminiferous tubules (Sertoli cells) and in the interstitium (particularly in Leydig cells) (Fig. 1DGo). Smad 5 immunostaining was observed exclusively in Sertoli cells (Fig. 1EGo). Smad 6 was immunolocalized predominantly to pachytene spermatocytes in a proacrosomal position and also in the interstitial compartment (Fig. 1FGo). Smad 7 immunostaining was observed, as for Smad 1, 2, and 3, in all germ cell types in a juxtanuclear position from the pachytene spermatocytes to the elongated spermatids (Fig. 1GGo).



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  		FIG. 1. TGF-ß signaling system protein immunostaining in the adult rat testis. The figure shows testes obtained from normal adult rats. Testes were fixed, sectioned, and treated with Smad 1 antibody (A), Smad 2 antibody (B), Smad 3 antibody (C), Smad 4 antibody (D), Smad 5 antibody (E), Smad 6 antibody (F), Smad 7 antibody (G), or with no antibody (H) for negative control.

 
Effect of fetal exposure to flutamide on TGF-ß ligands, TGF-ß receptors, and Smad mRNA and protein levels in adult rat testis
The data in Fig. 2Go show that the two TGF-ß ligand mRNA levels were significantly reduced in the adult testes exposed in utero to 10 mg/kg·d of flutamide, by 30% (P < 0.008) for TGF-ß1 (Fig. 2AGo), 24% (P < 0.001) for TGF-ß2 (Fig. 2BGo), and 12% (P < 0.004) for TGF-ß3 (Fig. 2CGo). TGF-ßRI and TGF-ßRII mRNA levels were also reduced. TGF-ßRI mRNA levels were reduced at 0.4, 2, and 10 mg/kg·d of flutamide (24.3% decrease, P < 0.0001) (Fig. 2DGo). The maximal effect was already obtained with 0.4 mg/kg·d of flutamide (18.3% decrease, P < 0.0003). TGF-ßRII mRNA levels (Fig. 2EGo) were significantly reduced at 10 mg/kg·d (20% decrease, P < 0.001) of the antiandrogen. In contrast, Smad 1 to Smad 7 mRNA levels in the adult rat testis were affected in a different manner by the in utero exposure to the antiandrogen. Indeed, although for some of them such as Smad 4 (Fig. 2IGo) and Smad 6 (Fig. 2KGo), their mRNA levels were not affected, for others their mRNA levels were decreased. Specifically, in utero exposure to 10 mg/kg·d of flutamide induced a decrease in Smad 1 (32%, P < 0.004) (Fig. 2FGo), Smad 2 (28.4%, P < 0.007) (Fig. 2GGo), Smad 3 (14.8%, P < 0.0003) (Fig. 2HGo), and Smad 5 (25.8%, P < 0.005) (Fig. 2JGo). In contrast, Smad 7 mRNA levels were increased both at 2 mg/kg·d (31%, P < 0.005) and 10 mg/kg·d (29%, P < 0.008) of flutamide (Fig. 2LGo), the maximal significant increase was obtained with 2 mg/kg·d of flutamide.



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  		FIG. 2. Effects of in utero exposure to flutamide on TGF-ß ligands, receptors, and Smads mRNA levels in the adult rat testis. mRNA of TGF-ß1 (A), TGF-ß2 (B), TGF-ß3 (C), TGF-ßRI (D), TGF-ßRII (E), Smad 1 (F), Smad 2 (G), Smad 3 (H), Smad 4 (I), Smad 5 (J), Smad 6 (K), and Smad 7 (L) expression was analyzed in 90-d-old rat testes exposed in utero to flutamide. mRNAs were analyzed by coamplification RT-PCR. The upper panels represent the band intensities, and the lower panelsrepresent data yielded by the quantification of the band intensities, expressed as a percentage of the ratio (molecule of interest/ß-actin mRNA) detected in the control (vehicle-treated) animals. Ten animals were used for each experimental condition (i.e. control and flutamide doses of 0.4, 2, and 10 mg/kg·d).

 
At the protein levels, the data showed similar results as those obtained at the messenger levels, except that the amplitude of the effects seemed to be more pronounced. Indeed, TGF-ß1, TGF-ß2, TGF-ß3, TGF-ßRII, Smad 1, Smad 2, and Smad 3 protein levels were decreased at the dose of 10 mg/kg·d of flutamide, by 41.5% (P < 0.03) for TGF-ß1 (Fig. 3AGo), 51% (P < 0,05) for TGF-ß2 (Fig. 3BGo), 46.5% (P < 0.02) for TGF-ß3 (Fig. 3CGo), 35% (P < 0.03) for TGF-ßRII (Fig. 3DGo), 49% (P < 0.02) for Smad 1 (Fig. 3EGo), 42% (P < 0.05) for Smad 2 (Fig. 3FGo), and 60% (P < 0.05) for Smad 3 (Fig. 3GGo) respectively. Because of the lack of a specific antibody against TGF-ßRI, we were not able to evaluate TGF-ßRI protein levels. Consistent with the changes in mRNA levels, Smad 4 (Fig. 3HGo) and Smad 6 (Fig. 3JGo) protein levels were not affected, whereas Smad 7 (Fig. 3KGo) protein levels (26%, P < 0.007) were increased. Table 3Go synthesizes the results obtained in the present study (Figs. 2Go and 3Go).



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  		FIG. 3. Effects of in utero exposure to flutamide on TGF-ß ligands, receptors, and Smads protein levels in the adult rat testis. Protein levels of TGF-ß1 (A), TGF-ß2 (B), TGF-ß3 (C), TGF-ßRII (D), Smad 1 (E), Smad 2 (F), Smad 3 (G), Smad 4 (H), Smad 5 (I), Smad 6 (J), and Smad 7 (K) were analyzed by Western blotting. The upper panelsshow representative autoradiographs, and lower panels represent quantification of the autoradiographs, expressed as a percentage of the ratio (molecule of interest/ß-actin protein) detected in the control (vehicle-treated) animals. Ten animals were used for each experimental condition (i.e. control and flutamide dose of 10 mg/kg·d).

 

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  		TABLE 3. Summary of mRNA and protein level changes of TGF-ß ligands, receptors, and Smads in 90-d-old rat testes exposed in utero to 10 mg/kg·d of flutamide

 
Effect of in utero exposure to flutamide on the expression of TGF-ß target gene TIMP-1 in the adult testis
Because TGF-ß ligands, TGF-ß receptors, and Co-Smads expressions were decreased in adult rat testis, the transcriptional activation of a TGF-ß target gene, TIMP-1, was analyzed. TIMP-1 mRNA levels (Fig. 4Go) were decreased at the dose of 2 mg/kg·d of flutamide by 37.2% (P < 0.0003) and at the dose of 10 mg/kg·d of flutamide by 25.4% (P < 0.007). These observations suggest that the decrease in the expression of the different TGF-ß family members leading to an inhibition of the TGF-ß signaling is functional because the expression of a classical target gene, TIMP-1, is reduced.



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  		FIG. 4. Effects of an exposure to flutamide during fetal life on TIMP-1 mRNA levels in the adult rat testis. TIMP-1 mRNA levels in 90-d-old rat testes exposed in utero to flutamide were analyzed by coamplification RT-PCR. The upper panel represents the band intensities, and the lower panel represents data yielded by the quantification of the band intensities, expressed as a percentage of the ratio (molecule of interest/ß-actin mRNA) detected in the control (vehicle-treated) animals. Ten animals were used for each experimental condition (i.e. control and flutamide doses of 0.4, 2, and 10 mg/kg·d).

 
Effect of in utero exposure to flutamide on c-Jun and Fas-L expression in the adult rat testis
Because it has been reported that Smad 7 could enhance c-Jun N-terminal kinase (JNK) activation (47), which promotes the phosphorylation of c-jun (to p-c-jun) leading to an increased transcriptional activity (48), we have also examined whether in utero exposure to flutamide may affect the expression of c-jun, as well as of a c-Jun target gene, Fas-L, in the adult rat testis, together with increased expression in Smad 7. The data in Fig. 5Go show that whereas the fetal exposure to the antiandrogen slightly increased c-Jun mRNA levels at 0.4 (14.9%, P < 0.008), 2 (17.51%, P < 0.001), and 10 (13.93%, P < 0.005) mg/kg·d of flutamide (Fig. 5AGo), it augmented more severely the phosphorylated c-jun protein level in the adult testis. Indeed, such an increase in phosphorylated c-jun protein levels was observed at both 2 (2.5-fold increase, P < 0.03) and 10 (2.3-fold, P < 0.001) mg/kg·d of the antiandrogen (Fig. 5BGo). Furthermore, the quantification of p-c-jun protein levels in the cytoplasm plus nucleus compartments vs. the cytoplasmic compartment clearly indicates a strong increase (5.3-fold, P < 0.008) in the nucleus when compared with the cytoplasmic localization at the dose of 10 mg/kg·d (Fig. 5CGo). Among the genes whose expression is targeted by c-jun is Fas-L (for review, see Ref.49). The data in Fig. 5DGo show an increase in Fas-L mRNA from 2 mg/kg·d (47%, P < 0.007) to 10 mg/kg·d of flutamide (56%, P < 0.006) in the adult rat testis, potentially supporting a functional efficiency of the increase in c-Jun levels.



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  		FIG. 5. Effect of flutamide in utero exposure on c-Jun mRNA expression, c-jun phosphorylation, and Fas-L mRNA expression in adult rat testis. c-Jun mRNA (A), Fas-L mRNA (D) levels in 90-d-old rat testes exposed in utero to flutamide were analyzed by coamplification RT-PCR. Protein levels of p-c-Jun (B) were analyzed by Western blotting. The upper panels represent the band intensities, and the lower panels represent data yielded by the quantification of the band intensities, expressed as a percentage of the ratio (molecule of interest/ß-actin mRNA) detected in the control (vehicle-treated) animals. The ratio obtained between the total vs. the cytoplasmic protein fraction of the p-c-jun form is shown in C. Ten animals were used for each experimental condition (i.e. control and flutamide doses of 0.4, 2, and 10 mg/kg·d.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is reported in the present study that in utero exposure to the antiandrogen flutamide results in alterations in the expression of the molecular components of the TGF-ß signaling system (including TGF-ß ligands, receptors, and Smads 1, 2, 3, 5, and 7) in adult rat testes shown to exhibit a chronic increased germ cell death process (7, 8, 50). More specifically, an increase in Smad 7 and a decrease in TGF-ß ligands, receptors, and Smads 1, 2, 3, and 5 were evidenced.

In adult rat testes exposed in utero to flutamide, a long-term apoptotic cell death has been observed in germ cells (7) with an increase in the caspases-3 and -6 expression and activation and an increase of the Bax/Bcl-w protein ratio (8). Because in testes from adult rats exposed in utero to the antiandrogen, the apoptotic process occurs in germ cells, low doses of flutamide (0.4, 2, and 10 mg/kg·d) were used to minimize or avoid an important germ cell loss (observed at higher doses such as 25 mg/kg·d of flutamide) that may confound the interpretation of the effects of the antiandrogen on TGF-ß ligand, receptor, and Smad protein levels in the germ cells. Interestingly, the flutamide doses used here are comparable to the low doses of the fungicide vinclozolin reported to affect rat sexual androgen-dependent tissue development (51). It is assumed that both vinclozolin and its metabolites (8) and flutamide (6) exert their effects by blocking the androgen action at the level of the androgen receptor (AR). However, it has been also recently reported that flutamide may have influences on apoptotic pathways independent of its antiandrogen effects (52).

The data in the present report support the hypothesis that a possible signaling pathway leading to the apoptosis evidenced in adult rat germ cells exposed in utero to antiandrogens could involve an increase in the expression of Smad 7. Indeed, Smad 7 is predominantly localized to pachytene spermatocytes and spermatids, germ cells reported to undergo the death process (7, 8, 50, 53). Because it has been reported that Smad 7 may increase the apoptotic process via JNK activation of activator protein 1 (AP-1) (homo or heterodimer of the proteins c-fos and c-jun) (54), we have examined c-Jun mRNA and protein levels in the adult rat testes with fetal androgen disruption. The data obtained indicated an increase in c-Jun mRNA as well as its active protein form (p-c-Jun) in rat testis exposed in utero to flutamide. An increased translocation of p-c-jun to the nuclear compartment was also observed, indicating that JNK is activated (48). Such a process could contribute to the apoptotic germ cell death observed in adult rat testis exposed in utero to flutamide. Indeed, several reports have shown that JNK may activate apoptosis through the activation of c-jun (for review, see Ref.49), leading to AP-1 transcriptional complex activation, which has been reported to induce germ cell apoptosis (55). This AP-1 activation of transcriptional activity can induce Fas-L transcription (56), which activates the death receptor pathway of apoptosis (for review, see Ref.57). Our present data are in favor of the existence of a death receptor pathway leading to germ cell apoptosis, as we report here that Fas-L mRNA levels were increased in adult rat testis exposed in utero to flutamide. Moreover, it has been shown that this pathway, via AP-1 activation, induces an activation of caspase-3 in human apoptotic germ cells (55), an activation we previously reported in our experimental model (7). Together, our present findings support that 1) JNK activation could be involved in the onset of apoptosis in germ cells of rats exposed to flutamide in utero and 2) this activation might be linked to the increased Smad 7 amounts in these testes. Besides Smad 7 increase, JNK activation could also be a result of the decreased TGF-ß1 expression observed in our model. Indeed, several reports show that TGF-ß1 could suppress apoptosis in human lung carcinoma cells and murine cultured macrophages (41, 42) by inhibiting JNK activation (41). Together, the present data support the hypothesis that increased Smad 7 and decreased TGF-ß1 expressions could be involved in the germ cell apoptotic process in rat testis exposed in utero to flutamide with a subsequent JNK activation.

Inhibition of R-Smads known to be involved in BMP signaling (Smads 1 and 5) could also be implicated in the abrogated cell protection against germ cell apoptosis observed in the experimental model used here. Indeed, it has been shown that in homozygote Bmp8b (58) and Bmp8atm1 (59) mutant testes, pachytene spermatocytes exhibit an increased apoptosis. These observations could be related to the massive apoptosis detected in mouse embryo cells lacking functional Smad 5 (60). As Smad 5 and Smad 1 are the intracellular mediator of BMPs, inhibition of these proteins could abrogate the BMP signaling leading to apoptosis (yet through an unknown mechanism). Together, the decreased expressions of Smad 1 and Smad 5 at the mRNA and protein levels may, at least in part, explain the apoptosis of pachytene spermatocytes in adult rat testis exposed in utero to flutamide.

One particular aspect of the observed defective spermatogenesis in adult rats exposed to flutamide in utero is that these alterations are observed at adulthood with plasma testosterone and testis AR levels not altered (8, 50, 61). These observations suggest that the alterations in the androgen signal are specifically related to androgen action as it might be located at post-AR levels. Although the mechanisms of such alterations remain to be investigated, the decrease in the levels of Smad 3, which is known as a cofactor of AR, may represent a possible explanation. Indeed, Smad 3 has been shown to enhance AR-mediated transactivation in prostate cancer cells (62) by direct interaction with the AR, in addition to its role as a TGF-ß transcription mediator. Nevertheless, the apparent AR activity dysregulation does not seem to be attributable to only Smad 3 inhibition because transcriptional activity of AR is dependent on the Smad3/Smad 4 ratio, and in the case of a high ratio, Smad 3 would rather enhance AR transactivation (63). Moreover, it has also been suggested that the ratio of AR to c-Jun is critical in AR transactivation control because overexpression of AP-1 (c-Jun/c-Fos) inhibits androgen-induced promoter activity in the LNCaP cell line (64) because of direct interaction between AR and c-Jun. An inhibitory effect of a c-Jun increased level as well of its phosphorylated form on AR-regulated promoter efficiency has also been noted in prostate cancer cells (65). Together, we suggest herein two possible explanations for the potential AR dysfunctional activity in adult rat testis exposed to flutamide in utero: 1) the Smad 3 mRNA and protein decreased level and 2) the increase of c-Jun mRNA and protein level and activation.

In summary, by using as a model adult rats exposed in utero to the antiandrogen flutamide it is reported herein an alteration in the expression of several members of the TGF-ß signaling system. Such alterations may represent one of the potential upstream mechanisms leading to the long-term apoptotic germ cell death process observed in adult rat testis with fetal androgen disruption.


   Footnotes
 
This work was supported by Institut National de la Santé et de la Recherche Médicale, Université Claude Bernard Lyon 1, a grant and a doctoral fellowship (to M.M.) from the commission of the European Communities, specific research and technology development program Quality of Life and Management of Living Resources (ENDISRUPT QLK4-2000-00684).

First Published Online September 15, 2005

Abbreviations: AP-1, Activator protein 1; AR, androgen receptor; BMP, bone morphogenic protein; dNTP, deoxy-NTP; GD 0; gestation d 0; HPRT, hypoxanthine phosphoribosyltransferase; JNK, c-Jun N-terminal kinase; TBS, Tris-buffered saline; TBST, TBS with 0.05% Tween 20; TGF-ßRI, TGF-ß receptor type I; TIMP-1, tissue inhibitor of metalloproteases-1.

Received May 16, 2005.

Accepted for publication September 6, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. McIntyre BS, Barlow NJ, Foster PM 2001 Androgen-mediated development in male rat offspring exposed to flutamide in utero: permanence and correlation of early postnatal changes in anogenital distance and nipple retention with malformations in androgen-dependent tissues. Toxicol Sci 62:236–249[Abstract/Free Full Text]
  2. Imperato-McGinley J, Sanchez RS, Spencer JR, Yee B, Vaughan ED 1992 Comparison of the effects of the 5{alpha}-reductase inhibitor finasteride and the antiandrogen flutamide on prostate and genital differentiation: dose-response studies. Endocrinology 131:1149–1156[Abstract]
  3. van der Schoot P 1992 Disturbed testicular descent in the rat after prenatal exposure to the antiandrogen flutamide. J Reprod Fertil 96:483–496
  4. Kassim NM, McDonald SW, Reid O, Bennett NK, Gilmore DP, Payne AP1997 The effects of pre- and postnatal exposure to the nonsteroidal antiandrogen flutamide on testis descent and morphology in the Albino Swiss rat. J Anat 190:577–588
  5. Miyata K, Yabushita S, Sukata T, Sano M, Yoshino H, Nakanishi T, Okuno Y, Matsuo M 2002 Effects of perinatal exposure to flutamide on sex hormones and androgen-dependent organs in F1 male rats. J Toxicol Sci 27:19–33[CrossRef][Medline]
  6. Peets EA, Henson MF, Neri R 1974 On the mechanism of the anti-androgenic action of flutamide ({alpha}-{alpha}-{alpha}-trifluoro-2-methyl-4'-nitro-m-propionotoluidide) in the rat. Endocrinology 94:532–540[Medline]
  7. Omezzine A, Chater S, Mauduit C, Florin A, Tabone E, Chuzel F, Bars R, Benahmed M 2003 Long-term apoptotic cell death process with increased expression and activation of caspase-3 and -6 in adult rat germ cells exposed in utero to flutamide. Endocrinology 144:648–661[Abstract/Free Full Text]
  8. Bozec A, Chuzel F, Chater S, Paulin C, Bars R, Benahmed M, Mauduit C 2004 The mitochondrial-dependent pathway is chronically affected in testicular germ cell death in adult rats exposed in utero to anti-androgens. J Endocrinol 183:79–90[Abstract/Free Full Text]
  9. Anway MD, Cupp AS, Uzumcu M, Skinner MK 2005 Epigenetic transgenerational actions of endocrine disruptors and male fertility. Science 308:1466–1469[Abstract/Free Full Text]
  10. Massague J, Wotton D 2000 Transcriptional control by the TGF-ß/Smad signaling system. EMBO J 19:1745–1754[CrossRef][Medline]
  11. Wrana JL, Attisano L, Wieser R, Ventura F, Massague J 1994 Mechanism of activation of the TGF-ß receptor. Nature 370:341–347[CrossRef][Medline]
  12. Heldin CH, Miyazono K, ten Dijke P 1997 TGF-ß signalling from cell membrane to nucleus through SMAD proteins. Nature 390:465–471[CrossRef][Medline]
  13. Miyazawa K, Shinozaki M, Hara T, Furuya T, Miyazono K 2002 Two major Smad pathways in TGF-ß superfamily signalling. Genes Cells 7:1191–1204[Abstract]
  14. Miyazono K, ten Dijke P, Heldin CH 2000 TGF-ß signaling by Smad proteins. Adv Immunol 75:115–157[Medline]
  15. Hahn SA, Schutte M, Hoque AT, Moskaluk CA, da Costa LT, Rozenblum E, Weinstein CL, Fischer A, Yeo CJ, Hruban RH, Kern SE 1996 DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science 271:350–353[Abstract]
  16. Topper JN, Cai J, Qiu Y, Anderson KR, Xu YY, Deeds JD, Feeley R, Gimeno CJ, Woolf EA, Tayber O, Mays GG, Sampson BA, Schoen FJ, Gimbrone Jr MA, Falb D 1997 Vascular MADs: two novel MAD-related genes selectively inducible by flow in human vascular endothelium. Proc Natl Acad Sci USA 94:9314–9319[Abstract/Free Full Text]
  17. Afrakhte M, Moren A, Jossan S, Itoh S, Sampath K, Westermark B, Heldin CH, Heldin NE, ten Dijke P 1998 Induction of inhibitory Smad6 and Smad7 mRNA by TGF-ß family members. Biochem Biophys Res Commun 249:505–511[CrossRef][Medline]
  18. Imamura T, Takase M, Nishihara A, Oeda E, Hanai J, Kawabata M, Miyazono K 1997 Smad6 inhibits signalling by the TGF-ß superfamily. Nature 389:622–626[CrossRef][Medline]
  19. Hayashi H, Abdollah S, Qiu Y, Cai J, Xu YY, Grinnell BW, Richardson MA, Topper JN, Gimbrone Jr MA, Wrana JL, Falb D 1997 The MAD-related protein Smad7 associates with the TGFß receptor and functions as an antagonist of TGFß signaling. Cell 89:1165–1173[CrossRef][Medline]
  20. Hata A, Lagna G, Massague J, Hemmati-Brivanlou A 1998 Smad6 inhibits BMP/Smad1 signaling by specifically competing with the Smad4 tumor suppressor. Genes Dev 12:186–197[Abstract/Free Full Text]
  21. Watrin F, Scotto L, Assoian RK, Wolgemuth DJ 1991 Cell lineage specificity of expression of the murine transforming growth factor ß3 and transforming growth factor ß1 genes. Cell Growth Differ 2:77–83[Abstract]
  22. Mullaney BP, Skinner MK 1993 Transforming growth factor-ß (ß1, ß2, and ß3) gene expression and action during pubertal development of the seminiferous tubule: potential role at the onset of spermatogenesis. Mol Endocrinol 7:67–76[Abstract]
  23. Le Magueresse-Battistoni B, Morera AM, Goddard I, Benahmed M 1995 Expression of mRNAs for transforming growth factor-ß receptors in the rat testis. Endocrinology 136:2788–2791[Abstract]
  24. Gautier C, Levacher C, Saez JM, Habert R 1997 Expression and regulation of transforming growth factor ß1 mRNA and protein in rat fetal testis in vitro. Biochem Biophys Res Commun 236:135–139[CrossRef][Medline]
  25. Olaso R, Gautier C, Levacher C, Durand P, Saez J, Habert R 1997 The immunohistochemical localization of transforming growth factor-ß2 in the fetal and neonatal rat testis. Mol Cell Endocrinol 126:165–172[CrossRef][Medline]
  26. Teerds KJ, Dorrington JH 1993 Localization of transforming growth factor ß1 and ß2 during testicular development in the rat. Biol Reprod 48:40–45[Abstract]
  27. Lui WY, Lee WM, Cheng CY 2003 Transforming growth factor ß3 regulates the dynamics of Sertoli cell tight junctions via the p38 mitogen-activated protein kinase pathway. Biol Reprod 68:1597–1612[Abstract/Free Full Text]
  28. Olaso R, Pairault C, Habert R 1998 Expression of type I and II receptors for transforming growth factor ß in the adult rat testis. Histochem Cell Biol 110:613–618[CrossRef][Medline]
  29. Benahmed M, Sordoillet C, Chauvin MA, de Peretti E, Morera AM 1989 On the mechanisms involved in the inhibitory and stimulating actions of transforming growth factor-ß on porcine testicular steroidogenesis: an in vitro study. Mol Cell Endocrinol 67:155–164[CrossRef][Medline]
  30. Gautier C, Levacher C, Saez JM, Habert R 1997 Transforming growth factor ß1 inhibits steroidogenesis in dispersed fetal testicular cells in culture. Mol Cell Endocrinol 131:21–30[CrossRef][Medline]
  31. Olaso R, Pairault C, Boulogne B, Durand P, Habert R 1998 Transforming growth factor ß1 and ß2 reduce the number of gonocytes by increasing apoptosis. Endocrinology 139:733–740[Abstract/Free Full Text]
  32. Prepin J, Le Vigouroux P 1997 Inhibition by TGF-ß1 of the in vitro thymulin-stimulated proliferation of gonocytes from fetal rat testes. Reprod Nutr Dev 37:203–206
  33. Donovan PJ 1994 Growth factor regulation of mouse primordial germ cell development. Curr Top Dev Biol 29:189–225[Medline]
  34. Panthou P, Barbey P, Thiebot B, Bocquet J 1994 Effects of transforming growth factor-ß1, interleukin-1{alpha} and interleukin-6 on rat Sertoli cell proteoglycan synthesis. Biochem Mol Biol Int 34:603–612[Medline]
  35. Lui WY, Lee WM, Cheng CY 2001 Transforming growth factor-ß3 perturbs the inter-Sertoli tight junction permeability barrier in vitro possibly mediated via its effects on occludin, zonula occludens-1, and claudin-11. Endocrinology 142:1865–1877[Abstract/Free Full Text]
  36. Lui WY, Lee WM, Cheng CY 2003 TGF-ßs: their role in testicular function and Sertoli cell tight junction dynamics. Int J Androl 26:147–160[CrossRef][Medline]
  37. Ailenberg M, Tung PS, Fritz IB 1990 Transforming growth factor-ß elicits shape changes and increases contractility of testicular peritubular cells. Biol Reprod 42:499–509[Abstract]
  38. Tachibana I, Imoto M, Adjei PN, Gores GJ, Subramaniam M, Spelsberg TC, Urrutia R 1997 Overexpression of the TGFß-regulated zinc finger encoding gene, TIEG, induces apoptosis in pancreatic epithelial cells. J Clin Invest 99:2365–2374[Medline]
  39. Jang CW, Chen CH, Chen CC, Chen JY, Su YH, Chen RH 2002 TGF-ß induces apoptosis through Smad-mediated expression of DAP-kinase. Nat Cell Biol 4:51–58[CrossRef][Medline]
  40. Valderrama-Carvajal H, Cocolakis E, Lacerte A, Lee EH, Krystal G, Ali S, Lebrun JJ 2002 Activin/TGF-ß induce apoptosis through Smad-dependent expression of the lipid phosphatase SHIP. Nat Cell Biol 4:963–969[CrossRef][Medline]
  41. Huang Y, Hutter D, Liu Y, Wang X, Sheikh MS, Chan AM, Holbrook NJ 2000 Transforming growth factor-ß1 suppresses serum deprivation-induced death of A549 cells through differential effects on c-Jun and JNK activities. J Biol Chem 275:18234–18242[Abstract/Free Full Text]
  42. Chin BY, Petrache I, Choi AM, Choi ME 1999 Transforming growth factor ß1 rescues serum deprivation-induced apoptosis via the mitogen-activated protein kinase (MAPK) pathway in macrophages. J Biol Chem 274:11362–11368[Abstract/Free Full Text]
  43. Shin I, Bakin AV, Rodeck U, Brunet A, Arteaga CL 2001 Transforming growth factor ß enhances epithelial cell survival via Akt-dependent regulation of FKHRL1. Mol Biol Cell 12:3328–3339[Abstract/Free Full Text]
  44. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  45. Foley KP, Leonard MW, Engel JD 1993 Quantitation of RNA using the polymerase chain reaction. Trends Genet 9:380–385[CrossRef][Medline]
  46. Caussanel V, Tabone E, Hendrick JC, Dacheux F, Benahmed M 1997 Cellular distribution of transforming growth factor ß1, ß2, and ß3 and their types I and II receptors during postnatal development and spermatogenesis in the boar testis. Biol Reprod 56:357–367[Abstract]
  47. Mazars A, Lallemand F, Prunier C, Marais J, Ferrand N, Pessah M, Cherqui G, Atfi A 2001 Evidence for a role of the JNK cascade in Smad7-mediated apoptosis. J Biol Chem 276:36797–36803[Abstract/Free Full Text]
  48. Derijard B, Hibi M, Wu IH, Barrett T, Su B, Deng T, Karin M, Davis RJ 1994 JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell 76:1025–1037[CrossRef][Medline]
  49. Dunn C, Wiltshire C, MacLaren A, Gillespie DA 2002 Molecular mechanism and biological functions of c-Jun N-terminal kinase signalling via the c-Jun transcription factor. Cell Signal 14:585–593[CrossRef][Medline]
  50. Florin A, Maire M, Bozec A, Hellani A, Chater S, Bars R, Chuzel F, Benahmed M 2005 Androgens and postmeiotic germ cells regulate claudin-11 expression in rat Sertoli cells. Endocrinology 146:1532–1540[Abstract/Free Full Text]
  51. Gray Jr LE, Ostby J, Monosson E, Kelce WR 1999 Environmental antiandrogens: low doses of the fungicide vinclozolin alter sexual differentiation of the male rat. Toxicol. Ind. Health 15:48–64
  52. Zhang M, Latham DE, Delaney MA, Chakravarti A 2005 Survivin mediates resistance to antiandrogen therapy in prostate cancer. Oncogene 24:2474–2482[CrossRef][Medline]
  53. Uzumcu M, Suzuki H, Skinner MK 2004 Effect of the anti-androgenic endocrine disruptor vinclozolin on embryonic testis cord formation and postnatal testis development and function. Reprod Toxicol 18:765–774[CrossRef][Medline]
  54. Chastel C, Jiricny J, Jaussi R 2004 Activation of stress-responsive promoters by ionizing radiation for deployment in targeted gene therapy. DNA Repair (Amst) 3:201–215
  55. Suomalainen L, Dunkel L, Ketola I, Eriksson M, Erkkila K, Oksjoki R, Taari K, Heikinheimo M, Pentikainen V 2004 Activator protein-1 in human male germ cell apoptosis. Mol Hum Reprod 10:743–753[Abstract/Free Full Text]
  56. Le-Niculescu H, Bonfoco E, Kasuya Y, Claret FX, Green DR, Karin M 1999 Withdrawal of survival factors results in activation of the JNK pathway in neuronal cells leading to Fas ligand induction and cell death. Mol Cell Biol 19:751–763[Abstract/Free Full Text]
  57. Hill MM, Adrain C, Martin SJ 2003 Portrait of a killer: the mitochondrial apoptosome emerges from the shadows. Mol Interv 3:19–26[Abstract/Free Full Text]
  58. Zhao GQ, Hogan BL 1997 Evidence that Mothers-against-dpp-related 1 (Madr1) plays a role in the initiation and maintenance of spermatogenesis in the mouse. Mech Dev 61:63–73[CrossRef][Medline]
  59. Zhao GQ, Liaw L, Hogan BL 1998 Bone morphogenetic protein 8A plays a role in the maintenance of spermatogenesis and the integrity of the epididymis. Development 125:1103–1112[Abstract]
  60. Yang X, Castilla LH, Xu X, Li C, Gotay J, Weinstein M, Liu PP, Deng CX 1999 Angiogenesis defects and mesenchymal apoptosis in mice lacking SMAD5. Development 126:1571–1580[Abstract]
  61. Goddard I, Florin A, Mauduit C, Tabone E, Contard P, Bars R, Chuzel F, Benahmed M 2003 Alteration of lactate production and transport in the adult rat testis exposed in utero to flutamide. Mol Cell Endocrinol 206:137–146[CrossRef][Medline]
  62. Kang HY, Lin HK, Hu YC, Yeh S, Huang KE, Chang C 2001 From transforming growth factor-ß signaling to androgen action: identification of Smad3 as an androgen receptor coregulator in prostate cancer cells. Proc Natl Acad Sci USA 98:3018–3023[Abstract/Free Full Text]
  63. Kang HY, Huang KE, Chang SY, Ma WL, Lin WJ, Chang C 2002 Differential modulation of androgen receptor-mediated transactivation by Smad3 and tumor suppressor Smad4. J Biol Chem 277:43749–43756[Abstract/Free Full Text]
  64. Sato N, Sadar MD, Bruchovsky N, Saatcioglu F, Rennie PS, Sato S, Lange PH, Gleave ME 1997 Androgenic induction of prostate-specific antigen gene is repressed by protein-protein interaction between the androgen receptor and AP-1/c-Jun in the human prostate cancer cell line LNCaP. J Biol Chem 272:17485–17494[Abstract/Free Full Text]
  65. Yuan H, Pan Y, Young CY 2004 Overexpression of c-Jun induced by quercetin and resverol inhibits the expression and function of the androgen receptor in human prostate cancer cells. Cancer Lett 213:155–163[CrossRef][Medline]



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L. Benbrahim-Tallaa, B. Siddeek, A. Bozec, V. Tronchon, A. Florin, C. Friry, E. Tabone, C. Mauduit, and M. Benahmed
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