Alteration of Transforming Growth Factor- Signaling System Expression in Adult Rat Germ Cells with a Chronic Apoptotic Cell Death Process after Fetal
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《内分泌学杂志》
Institut National de la Sante et de la Recherche Medicale, Unite 407 (M.M., A.F. K.K., D.R., P.C., E.T., C.M., M.B.), Faculte de Medecine Lyon-Sud, 69921 Oullins, France
BayerCropScience (R.B), Sophia-Antipolis F-06903, France
Abstract
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- 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- signaling members, such as the three isoforms of TGF- ligands (TGF-1–3), the two TGF- receptors (TGF-RI 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- 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- 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
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- pathway. The TGF-s 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- receptors and their unique intracellular mediators, but several reports show that TGF- and BMPs interact with the MAPK pathways. The three TGF- ligand isoforms and the two major receptors are expressed in fetal and adult rat testis (21, 22, 23, 24, 25). Specifically, TGF-1 is expressed in Sertoli cells and germ cells, with an increased expression in spermatocytes and round spermatids at stages VIII and IX (26). TGF-2 has been localized to Leydig cells and round spermatids but not to Sertoli cells during spermatogenesis (26). Recently, TGF-3 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- 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- 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- 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- 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- signaling system in the adult testis, which exhibits a chronic germ cell death process.
Materials and Methods
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 [-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-1 polyclonal antibody (V), rabbit anti-TGF-2 polyclonal antibody (V), rabbit anti-TGF-3 polyclonal antibody (V), rabbit anti-TGF-RII 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 Sante et de la Recherche Medicale 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 -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 [-33P]dATP, and 2 μl RT mixture 1/10 (cDNA). TGF-1, TGF-2, TGF-3, TGF- receptor type I (TGF-RI), TGF-RII, 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 -actin, and Smad 6 was coamplified with HPRT. Coamplification with -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 1 shows the primer sequences, the optimized number of cycles, and annealing temperatures of each target gene. Table 2 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.
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-1, Smad 1, and Smad 2; 10 μg for TGF-2; 3 μg for TGF-3; 20 μg for TGF-RII, 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-1, TGF-2, TGF-RII, 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
Immunolocalization of Smad 1 to Smad 7 to the different adult testicular cell types
Because TGF- 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. 1, 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. 1D). Smad 5 immunostaining was observed exclusively in Sertoli cells (Fig. 1E). Smad 6 was immunolocalized predominantly to pachytene spermatocytes in a proacrosomal position and also in the interstitial compartment (Fig. 1F). 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. 1G).
Effect of fetal exposure to flutamide on TGF- ligands, TGF- receptors, and Smad mRNA and protein levels in adult rat testis
The data in Fig. 2 show that the two TGF- 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-1 (Fig. 2A), 24% (P < 0.001) for TGF-2 (Fig. 2B), and 12% (P < 0.004) for TGF-3 (Fig. 2C). TGF-RI and TGF-RII mRNA levels were also reduced. TGF-RI mRNA levels were reduced at 0.4, 2, and 10 mg/kg·d of flutamide (24.3% decrease, P < 0.0001) (Fig. 2D). The maximal effect was already obtained with 0.4 mg/kg·d of flutamide (18.3% decrease, P < 0.0003). TGF-RII mRNA levels (Fig. 2E) 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. 2I) and Smad 6 (Fig. 2K), 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. 2F), Smad 2 (28.4%, P < 0.007) (Fig. 2G), Smad 3 (14.8%, P < 0.0003) (Fig. 2H), and Smad 5 (25.8%, P < 0.005) (Fig. 2J). 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. 2L), the maximal significant increase was obtained with 2 mg/kg·d of flutamide.
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-1, TGF-2, TGF-3, TGF-RII, 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-1 (Fig. 3A), 51% (P < 0,05) for TGF-2 (Fig. 3B), 46.5% (P < 0.02) for TGF-3 (Fig. 3C), 35% (P < 0.03) for TGF-RII (Fig. 3D), 49% (P < 0.02) for Smad 1 (Fig. 3E), 42% (P < 0.05) for Smad 2 (Fig. 3F), and 60% (P < 0.05) for Smad 3 (Fig. 3G) respectively. Because of the lack of a specific antibody against TGF-RI, we were not able to evaluate TGF-RI protein levels. Consistent with the changes in mRNA levels, Smad 4 (Fig. 3H) and Smad 6 (Fig. 3J) protein levels were not affected, whereas Smad 7 (Fig. 3K) protein levels (26%, P < 0.007) were increased. Table 3 synthesizes the results obtained in the present study (Figs. 2 and 3).
Effect of in utero exposure to flutamide on the expression of TGF- target gene TIMP-1 in the adult testis
Because TGF- ligands, TGF- receptors, and Co-Smads expressions were decreased in adult rat testis, the transcriptional activation of a TGF- target gene, TIMP-1, was analyzed. TIMP-1 mRNA levels (Fig. 4) 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- family members leading to an inhibition of the TGF- signaling is functional because the expression of a classical target gene, TIMP-1, is reduced.
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. 5 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. 5A), 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. 5B). 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. 5C). Among the genes whose expression is targeted by c-jun is Fas-L (for review, see Ref.49). The data in Fig. 5D 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.
Discussion
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- signaling system (including TGF- 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- 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- 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-1 expression observed in our model. Indeed, several reports show that TGF-1 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-1 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- 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- 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 Sante et de la Recherche Medicale, Universite 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-RI, TGF- receptor type I; TIMP-1, tissue inhibitor of metalloproteases-1.
Accepted for publication September 6, 2005.
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BayerCropScience (R.B), Sophia-Antipolis F-06903, France
Abstract
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- 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- signaling members, such as the three isoforms of TGF- ligands (TGF-1–3), the two TGF- receptors (TGF-RI 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- 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- 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
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- pathway. The TGF-s 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- receptors and their unique intracellular mediators, but several reports show that TGF- and BMPs interact with the MAPK pathways. The three TGF- ligand isoforms and the two major receptors are expressed in fetal and adult rat testis (21, 22, 23, 24, 25). Specifically, TGF-1 is expressed in Sertoli cells and germ cells, with an increased expression in spermatocytes and round spermatids at stages VIII and IX (26). TGF-2 has been localized to Leydig cells and round spermatids but not to Sertoli cells during spermatogenesis (26). Recently, TGF-3 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- 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- 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- 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- 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- signaling system in the adult testis, which exhibits a chronic germ cell death process.
Materials and Methods
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 [-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-1 polyclonal antibody (V), rabbit anti-TGF-2 polyclonal antibody (V), rabbit anti-TGF-3 polyclonal antibody (V), rabbit anti-TGF-RII 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 Sante et de la Recherche Medicale 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 -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 [-33P]dATP, and 2 μl RT mixture 1/10 (cDNA). TGF-1, TGF-2, TGF-3, TGF- receptor type I (TGF-RI), TGF-RII, 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 -actin, and Smad 6 was coamplified with HPRT. Coamplification with -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 1 shows the primer sequences, the optimized number of cycles, and annealing temperatures of each target gene. Table 2 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.
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-1, Smad 1, and Smad 2; 10 μg for TGF-2; 3 μg for TGF-3; 20 μg for TGF-RII, 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-1, TGF-2, TGF-RII, 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
Immunolocalization of Smad 1 to Smad 7 to the different adult testicular cell types
Because TGF- 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. 1, 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. 1D). Smad 5 immunostaining was observed exclusively in Sertoli cells (Fig. 1E). Smad 6 was immunolocalized predominantly to pachytene spermatocytes in a proacrosomal position and also in the interstitial compartment (Fig. 1F). 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. 1G).
Effect of fetal exposure to flutamide on TGF- ligands, TGF- receptors, and Smad mRNA and protein levels in adult rat testis
The data in Fig. 2 show that the two TGF- 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-1 (Fig. 2A), 24% (P < 0.001) for TGF-2 (Fig. 2B), and 12% (P < 0.004) for TGF-3 (Fig. 2C). TGF-RI and TGF-RII mRNA levels were also reduced. TGF-RI mRNA levels were reduced at 0.4, 2, and 10 mg/kg·d of flutamide (24.3% decrease, P < 0.0001) (Fig. 2D). The maximal effect was already obtained with 0.4 mg/kg·d of flutamide (18.3% decrease, P < 0.0003). TGF-RII mRNA levels (Fig. 2E) 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. 2I) and Smad 6 (Fig. 2K), 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. 2F), Smad 2 (28.4%, P < 0.007) (Fig. 2G), Smad 3 (14.8%, P < 0.0003) (Fig. 2H), and Smad 5 (25.8%, P < 0.005) (Fig. 2J). 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. 2L), the maximal significant increase was obtained with 2 mg/kg·d of flutamide.
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-1, TGF-2, TGF-3, TGF-RII, 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-1 (Fig. 3A), 51% (P < 0,05) for TGF-2 (Fig. 3B), 46.5% (P < 0.02) for TGF-3 (Fig. 3C), 35% (P < 0.03) for TGF-RII (Fig. 3D), 49% (P < 0.02) for Smad 1 (Fig. 3E), 42% (P < 0.05) for Smad 2 (Fig. 3F), and 60% (P < 0.05) for Smad 3 (Fig. 3G) respectively. Because of the lack of a specific antibody against TGF-RI, we were not able to evaluate TGF-RI protein levels. Consistent with the changes in mRNA levels, Smad 4 (Fig. 3H) and Smad 6 (Fig. 3J) protein levels were not affected, whereas Smad 7 (Fig. 3K) protein levels (26%, P < 0.007) were increased. Table 3 synthesizes the results obtained in the present study (Figs. 2 and 3).
Effect of in utero exposure to flutamide on the expression of TGF- target gene TIMP-1 in the adult testis
Because TGF- ligands, TGF- receptors, and Co-Smads expressions were decreased in adult rat testis, the transcriptional activation of a TGF- target gene, TIMP-1, was analyzed. TIMP-1 mRNA levels (Fig. 4) 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- family members leading to an inhibition of the TGF- signaling is functional because the expression of a classical target gene, TIMP-1, is reduced.
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. 5 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. 5A), 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. 5B). 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. 5C). Among the genes whose expression is targeted by c-jun is Fas-L (for review, see Ref.49). The data in Fig. 5D 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.
Discussion
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- signaling system (including TGF- 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- 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- 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-1 expression observed in our model. Indeed, several reports show that TGF-1 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-1 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- 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- 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 Sante et de la Recherche Medicale, Universite 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-RI, TGF- receptor type I; TIMP-1, tissue inhibitor of metalloproteases-1.
Accepted for publication September 6, 2005.
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