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Institut National de la Santé et de la Recherche Médicale (Unité 407; A.O., S.C., C.M., A.F., E.T., M.B.), Faculté de Médecine Lyon-Sud, 69921 Oullins, France; and BayerCropScience (F.C., R.B.) 06903 Sophia-Antipolis, 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.
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Abstract |
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Introduction |
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Because the number of cells in an organ is determined by the rates of cell migration, cell division, and cell death (10) in the present study, we investigated the possibility that the impairment of the spermatogenetic process resulting in germ cell loss in adult rats exposed in utero to flutamide might be related to a dysregulation of the apoptotic germ cell death process. Indeed, it is established that apoptotic cells undergo a regulated autodigestion, which involves the disruption of cytoskeletal integrity, cell shrinkage, nuclear condensation, and the activation of endonucleases. The chief effectors of the apoptotic cell death pathway are the caspase family of cysteine proteases. Caspases are synthesized as inactive precursors (procaspases) that are cleaved at specific aspartate residues to generate the active subunits. Procaspase cleavage can occur by several mechanisms including proximity-induced autoprocessing or cleavage by other caspases, revealing a caspase cascade with upstream initiator caspases such as caspase-8, -9, and -10 and downstream, effector caspases, such as caspase-3, -6, and -7 (11). The activity of these caspases is regulated by several families of both pro- and antiapoptotic cellular proteins (12, 13).
In the present study, we examined the apoptotic cell death process and expression and activation of two effector caspases, caspase-3 and -6 in terms of mRNA and protein (including the procaspases and cleaved active caspases) in the rat testis exposed in utero to flutamide. Because the apoptotic cell death process and caspase gene expression were analyzed in germ cells, doses of flutamide were chosen to minimize or avoid important germ cell loss that may confound the interpretation of effects of flutamide on testicular (germ cell) gene expression. We report here that in utero exposure to flutamide induced in adult rat germ cells a chronic apoptotic cell death process associated with a long-term increase in the expression and activation of caspase-3 and -6.
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Materials and Methods |
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Animals
Rats were exposed to flutamide according to two different protocols.
The first protocol was related to in utero exposure to flutamide. 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 (approximately 15 air changes per hour) and given free access to water and feed (certified rodent pellet diet, AO4C, UAR, Villemoisson-sur-orge, 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 or flutamide by daily gavage from GD 6 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·d (adjusted daily based on body weight). Dams were weighed daily from GD 6 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 then killed by CO2 inhalation.
The second protocol was related to exposure to flutamide during the adulthood. Adult (90-d-old) male Sprague Dawley rats from Charles River Laboratories, Inc. were individually housed in controlled conditions (see protocol 1) and acclimatized 1 wk before experimental procedure. Twelve rats (three rats per treatment condition) were administered vehicle control or flutamide (10 mg/kg·d) by daily gavage during 3 d. One group of vehicle control or flutamide exposed animals were killed just after treatment. One group of vehicle control or flutamide exposed animals were killed 1–2 wk after treatment. Treatments were repeated at least three times.
All studies on animals were conducted in accordance with current regulation and standards approved by Institut National de la Santé et de la Recherche Médicale animal care committee.
Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate-biotin nick end labeling (TUNEL)
Paraffin sections (5 µm) of Bouin-fixed testis were mounted on glass slides. The sections were deparaffinized and rehydrated (xylene 5 min; ethanol 100%, 95%, 70%, 1 min each) and then washed in distilled water before beginning the TUNEL reaction as described earlier (14).
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, Frelburg, Germany), deparaffinized, rehydrated, treated 20 min at 93–98 C in citric buffer (0.01 M, pH 6), rinsed in osmosed water (2 x 5 min), washed (2 x 5 min) in Tris-buffered saline. For antiprocaspase-3 and anticleaved caspase-3 antibodies, the Envision+ kit was used. For antiprocaspase-6 antibody, the UltraVision detection system (Lab Vision Corp., Fremont, CA) was used. Immunohistochemistry was conducted according to the manufacturer’s recommendations as described earlier (15), and antibodies were diluted as follows: 1/300 anticaspase-3, 1/50 anticleaved caspase-3, 1/200 antiprocaspase-6.
PCR coamplification
Total RNAs were extracted from rat testis tissues with TRIzol reagent. The amount of RNA was estimated by spectrophotometry at 260 nm.
The cDNAs were obtained from reverse transcription of 5 µg total RNAs using random hexanucleotides as primers (5 µM) in the presence of dNTPs (0.2 mM), dithiothreitol (10 mM), and Muloney murine leukemia virus (10 U/µl), 1 h at 37 C. For PCR analysis the target gene (caspase-3, -6) was coamplified with the standard gene (ß-actin). The stock reactions (20 µl) were prepared on ice and contained 0.02 U/µl Taq polymerase, 1.5 mM MgCl2, 200 µM dNTP, 1 µM caspase primers, 10 nM ß-actin primers, and 2 µl reverse transcription mixture (cDNA). The PCR conditions were 94 C for 5 min followed by 25 cycles of 94 C for 30 sec, 58 C for 30 sec, 72 C for 1 min, and then 72 C for 7 min. After amplification the PCR products were separated by electrophoresis on 2% agarose gels containing 0.005% of ethidium bromide, visualized by UV light. Band intensities were estimated by densitometric scanning using GelDoc scanner (Bio-Rad Laboratories, Inc., Marnes la coquette, France). Data are expressed as caspase/ß-actin mRNA ratios. Primer used for caspase-3 were: upstream primer 5'-ACGGTACGCGAAGAAA-AGTGAC-3', downstream primer 5'-TCCTGACTTCGTATTTCAGGGC-3' (282 bp); caspase-6: upstream primer 5'-AACCACATTTACGCATACGATG-3', downstream primer 5'-CGGTGAGAGTAATACCCTTCTG-3' (289 bp); ß-actin: upstream primer 5'-TTGCTGATCCACATCTGCTG-3', downstream primer 5'-GACAGGATGCAGAA-GGAGAT-3' (146 bp). PCR controls were conducted as described earlier (15).
Western blotting analysis
Testicular tissue was homogenized in 200 µl ice-cold hypotonic buffer (25 mM Tris-HCl, pH 7.4; and protease inhibitor cocktail). Tissues were further homogenized by sonication (10 sec). Protein concentration was determined by the Bradford assay.
Proteins (100 µg) were resolved on 10% sodium dodecyl sulfate/polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes using 3-(cyclohexylamino)-1-propanesulfonic acid buffer (x1, pH 11), containing 20% methanol at a constant voltage of 100 V for 60 min. Following transfer, Western blotting was conducted as described earlier (15) and antibodies were diluted as follows: 1/100 procaspase-3; 1/100 cleaved caspase-3; 1/250 caspase-6. The protein loading was checked by reprobing the blot with a rabbit IgG antiactin (1/500).
Data analysis
Data are expressed as the mean ± SD. At least three different male offspring (n = 3–7/condition) from different litters were used. For statistical analysis one-way ANOVA was performed to determine whether there were differences among all groups (P < 0.05), and then the Bonferroni/Dunn posttest was performed to determine the significance of the differences between the pair of groups. A P value less than 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).
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Results |
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) and not at lower doses (Fig. 1, B and C). Indeed, at 10 mg/kg·d, an arrest of germ cell maturation began to be observed (Fig. 1D) in about 10% of the seminiferous tubules. At this dose level of flutamide, which induces cryptorchidism in about 50% of the animals, the spermatogenetic process alterations were more pronounced in the undescended (cryptorchid) testes because the great majority if not all the tubules were empty and devoid of postmeiotic germ cells (data not shown). In the present study, we therefore studied the apoptotic cell death process and caspase expression and activation in adult rat testes (excluding cryptorchid testes) exposed in utero to 0.4, 2, and 10 mg/kg·d of flutamide.
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) Indeed, whereas in control untreated animals, very few if any TUNEL-positive cells were observed in the testis (Fig. 2A), TUNEL-positive (apoptotic) cells were identified in adult rat testis exposed in utero to 10 mg/kg·d flutamide (Fig. 2A). These TUNEL-positive cells were mainly detected in postmeiotic germ cells. No TUNEL-positive cells were detected in somatic Leydig and Sertoli cells or spermatogonia in adult rat testes exposed in utero to 10 mg/kg·d of flutamide. The number of apoptotic germ cells in adult rat testes increased after in utero flutamide exposure in a dose-dependent manner (Fig. 2B). A significant increase (in TUNEL-positive germ cells) was observed in the adult rats exposed in utero to the different doses of the antiandrogen including 0.4 (P < 0.0001), 2 (P < 0.0001), and 10 (P < 0.0001) mg/kg·d of flutamide. The highest increase was observed in testes exposed in utero to 10 mg/kg·d of flutamide.
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Effect of in utero exposure to flutamide on caspase-3 and -6 immunostaining in the adult rat testis
These experiments aimed to immunolocalize caspases-3 (procaspase and cleaved active caspase) and -6 in the adult rat testis exposed in utero to flutamide.
For the immunolocalization of caspase-3, two different antibodies were used. The first one immunodetected exclusively procaspase-3 (Fig. 3, A–D), whereas the second antibody detected exclusively cleaved (active) caspase-3 (Fig. 3, E–H). In the adult rat testes, procaspase-3 immunostaining was specifically detected in germ cells and Leydig cells. Procaspase-3 immunostaining was predominantly observed in pachytene spermatocytes and to a lesser extent the more mature germ cells both from untreated control (Fig. 3A) or from in utero-exposed rats to different doses of flutamide including 0.4 (Fig. 3B), 2 (Fig. 3C), and 10 (Fig. 3D) mg/kg·d. In utero exposure to the antiandrogen also induced activation of caspase-3 in germ cells. Indeed, active cleaved caspase-3 immunostaining was not detected in control untreated rat testes (Fig. 3E), whereas it appeared exclusively in postmeiotic germ cells particularly in round spermatids in rat testes exposed in utero to the antiandrogen at the different doses including 0.4 mg/kg·d (Fig. 3F), 2 mg/kg·d (Fig. 3G), and 10 (Fig. 3H) mg/kg·d. Although procaspase-3 immunostaining was observed in all the tubules (Fig. 3, A–D, inset), cleaved caspase-3 was predominantly immunodetected in the tubules at stages VII, VIII, and very weakly at stage IX (Fig. 3, G–H, inset). No cleaved active caspase-3 immunostaining was observed in Leydig cells (Fig. 3, E–H), clearly suggesting that in the adult rats exposed in utero to flutamide, the activation of caspase-3 occurs in germ cells but not in Leydig cells.
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, I–L). Caspase-6 immunostaining appeared in its particular punctiform aspect as reported in other tissues (16). Both in control untreated or treated rat testes, procaspase-6 was predominantly immunolocalized to pachytene spermatocytes and to a lesser extent in more mature germ cells rat testes (Fig. 3, I–L). Procaspase-6 was also immunolocalized to Leydig cells (Fig. 3, I–L). As for procaspase-3, procaspase-6 immunostaining was observed in germ cells from all the tubules (Fig. 3, I–L, inset). In the present study, we were not able to immunolocalize cleaved activated caspase-6 to the different testicular cell types mainly because of the lack of a specific antibody to be used in immunohistochemical experiments. In the following experiments, we have examined whether in utero exposure to flutamide could induce changes in caspase-3 and -6 mRNA and protein levels in the adult rat testes.
Effect of in utero exposure to flutamide on caspase-3 and -6 mRNA and protein levels in adult rat germ cells
In the adult rat testes exposed in utero to flutamide, caspase-3 mRNA levels were significantly increased at all dose levels of the antiandrogen. The minimal significant (P < 0.0001) increase (3-fold) in caspase-3 mRNA levels was observed already with 0.4 mg/kg·d (Fig. 4A). A maximal increase (4-fold) in mRNA levels was observed at 2 (P < 0.0001, Fig. 4A) and 10 (P < 0.0001, Fig. 4A) mg/kg·d of flutamide. Furthermore, procaspase-3 protein levels were increased in adult rat testes exposed in utero to flutamide. As for caspase-3 mRNA levels, a significant (P < 0.0003) increase (2-fold) in procaspase-3 protein levels was detected from 0.4 mg/kg·d of flutamide (Fig. 4B). A further significant (P < 0.0001) maximal increase (2.5-fold) in procaspase-3 levels was observed in rat testes exposed to 2 and 10 mg/kg·d of flutamide (Fig. 4B). In Western blotting experiments, active cleaved caspase-3 was not detected in the testes from control rats, whereas it was clearly detected in testes from adult rats exposed to 0.4 mg/kg·d of flutamide (Fig. 4C). Active cleaved caspase-3 levels further increased in the testes from rats exposed to 2 and 10 mg/kg·d of the antiandrogen (Fig. 4C).
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). A significant (P < 0.0001) increase in mRNA levels was observed from 0.4 mg/kg·d (Fig. 5A). A further maximal significant (P < 0.0001) increase (2.5-fold) in mRNA levels was observed at 2 and 10 mg/kg·d (Fig. 5A). To detect and quantify procaspase and cleaved active caspase-6 protein levels (Fig. 5, B and C), two different antibodies were used. The first one that was previously used in the immunohistochemical experiments (Fig. 3) detects exclusively the procaspase-6 protein (34 kDa) (Fig. 5B), whereas the second antibody detects both procaspase-6 and cleaved caspase-6 (14 kDa) (Fig. 5C). Procaspase-6 protein levels were increased in adult rat testes exposed in utero to flutamide. Indeed, as for procaspase-3 protein levels, a significant (P < 0.0002) increase (3-fold) in procaspase-6 protein levels was already detected at 0.4 mg/kg·d. A further significant (P < 0.0001) increase in procaspase-6 levels was observed in rat testes exposed to 2 and 10 mg/kg·d of flutamide (Fig. 5B). In utero exposure to the antiandrogen flutamide also induced an activation of procaspase-6. Indeed, whereas active cleaved caspase-6 (14 kDa) was absent in the testes from control rats, it was clearly detected in testes exposed to 0.4 mg/kg·d of flutamide and its levels were further increased in the gonads exposed to 2 and 10 mg/kg·d of the antiandrogen (Fig. 5C).
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indicate that in the adult rat testes exposed in utero to flutamide, there is a long-term (irreversible) increase in caspase-3 and -6 expression and activation. In the following experiments, we have tested the reversibility of the increase in the two effector caspase expression and activation in adult rat testes when exposure to flutamide occurred during adulthood.
Effect of an exposure to flutamide during adulthood on caspase-3 and -6 mRNA and protein levels in adult rat germ cells
To test the reversibility of the increase in effector caspase expression and activation in adult rat testes exposed to flutamide during adulthood, animals were exposed during adulthood to the highest dose of antiandrogen (10 mg/kg·d, 3 d). TUNEL-positive (apoptotic) cell number was evaluated, caspase-3 and -6 immunostaining was performed, mRNA and protein levels were measured after 3 d of exposure to flutamide and 1 or 2 wk after the cessation of exposure to the antiandrogen.
The data in Fig. 6A show that TUNEL-positive (apoptotic) cells were essentially detected at the level of meiotic and postmeiotic germ cells in adult rat testis exposed to flutamide. No TUNEL was observed at the level of somatic Leydig and Sertoli cells. The data in Fig. 6B show that after flutamide exposure, there was a significant (P < 0.0001) increase in the apoptotic cell number, which was back to control level 1 wk after the cessation of exposure to the antiandrogen.
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) was mainly detected in Leydig and germ cells both following flutamide exposure (Fig. 7B) and 1 wk after the cessation of exposure to the antiandrogen (Fig. 7C), confirming previous observations (Fig. 3). Exposure to the antiandrogen resulted in the activation of procaspase-3. Indeed, whereas no immunostaining was detected in control untreated rat testes (Fig. 7D), it appeared in round spermatids from adult rat testes exposed to flutamide (Fig. 7E). Such an immunostaining was again seminiferous tubule stage dependent and was observed predominantly in tubules at stages VII, VIII, and IX (Fig. 7E, inset). One week after the interruption of the antiandrogen administration, cleaved caspase-3 immunostaining was no more observed in the rat testis (Fig. 7F), clearly suggesting that caspase-3 activation was reversible at the cessation of exposure to flutamide.
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, G–I) was mainly detected in Leydig and germ cells both following flutamide exposure (Fig. 7 H) and 1 wk after the cessation of exposure to the antiandrogen (Fig. 7I).
The data in Fig. 8 show that after 3 d of exposure to flutamide (10 mg/kg·d), a significant (P < 0.0001) increase (2.3-fold) in caspase-3 mRNA levels was observed (Fig. 8A). One or two weeks after the interruption of flutamide exposure, caspase-3 mRNA levels were back to control (Fig. 8A). Parallel changes in procaspase-3 protein levels were also observed with an increase (P < 0.0001) after flutamide exposure (Fig. 8B), which was then followed by a decrease in the proenzyme levels, which were back to control 1 or 2 wk after the cessation of the exposure to the antiandrogen (Fig. 8B). Procaspase-3 was also activated after exposure to the antiandrogen. Indeed, cleaved active caspase-3 was not detected in control untreated rat testes, whereas it was clearly detected in adult rat testes following administration of flutamide (Fig. 8C). Activation of procaspase-3 was interrupted following the cessation of exposure to the antiandrogen because active caspase-3 was no more detected 1 or 2 wk after the interruption of flutamide administration (Fig. 8C).
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). Testicular procaspase-6 protein levels increased (P < 0.0001) in adult animals exposed to the antiandrogen (Fig. 9B) and were back to control 2 wk following the cessation of exposure to the antiandrogen (Fig. 9B). Finally, cleaved caspase-6 (12 kDa) was absent in adult untreated rat testes, whereas it was present in adult testes exposed to flutamide. The cessation of exposure to flutamide was followed 1 wk later by the disappearance of testicular activated caspase-6 (Fig. 9C).
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Discussion |
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The TUNEL method shows that in adult rat testes exposed in utero to flutamide, the apoptotic process affects exclusively meiotic and postmeiotic germ cells, whereas the somatic Leydig and Sertoli cells were not affected by the cell death process. The number of apoptotic germ cells in the adult testis increased with the dose of flutamide received, indicating that in utero exposure to the antiandrogen resulted in a chronic activated germ cell apoptotic process. A significant TUNEL-positive cell number was observed with the lowest dose of flutamide tested, i.e. 0.4 mg/kg·d.
We show here that the apoptotic germ cells in adult rats exposed in utero to flutamide could be related to an increase in the expression and activation of two caspases, caspases-3 and -6. Caspases are a family of cysteine proteases that are a central component of the apoptotic machinery (17, 18). Caspases are expressed as inactive precursors (procaspases) that are activated on proteolytic cleavage. According to the current model, two classes of caspases, initiators and executors, are involved in apoptosis (19). Once activated, the initiator caspases (caspases-2, -8, and -9) cleave and activate the executor caspases (caspases-3, -6, and -7), which cause cellular collapse by cleaving a specific set of protein substrates. Within the execution phase, caspase-3 appears to be upstream of caspase-6 and -7; therefore, its activation represents a critical point in transmission of the apoptotic signal. In apoptotic cells, the cleavage of more than 70 proteins by caspases has been reported (20). Among the mechanisms and molecules underlying the germ cell death process in adult rats exposed in utero to flutamide, we identified a long-term increase in the expression of two effector caspases, i.e. caspases-3 and -6 in germ cells. The immunohistochemical experiments allowed their detection predominantly in premeiotic germ cells and somatic Leydig but not in peritubular myoid cells and Sertoli cells. Procaspases-3 and -6 were immunodetected in all the seminiferous tubules, predominantly in pachytene spermatocytes although to a lesser extent, they were also present in more mature germ cells.
The data presented here also indicate that in utero exposure to flutamide induces in adult rat germ cells an increase in caspase-3 and -6 mRNA and protein levels. The increase in caspase-3 and -6 mRNA levels in the adult rat testes was flutamide dose dependent. Although this is, to our knowledge, the first report demonstrating a positive regulatory action of flutamide (and thus a negative action of androgens) on caspase mRNA levels, it remains also to be determined whether such a regulatory action occurs at a transcriptional level and/or results from a stabilizing effect on caspase mRNA. Furthermore, concerning the parallel increase in procaspase protein levels in adult rat testes, it remains also to be clarified whether flutamide exerted its effects at the level of the translational/turnover of proteins. Besides the effects of flutamide on caspase-3 and -6 mRNA and procaspase-3 and -6 protein levels, our data indicate that the in utero exposure to the antiandrogen also triggers the activation of caspases-3 and -6 in adult germ cells. The availability of an antibody that recognizes exclusively activated caspase-3 allowed the immunolocalization of this activated caspase to postmeiotic germ cells, specifically to round spermatids. Furthermore, activated caspase-3 was detected in round spermatids in seminiferous tubules at stages VII, VIII, and IX, i.e. in androgeno-dependent stages during spermatogenesis. Taken together, these observations would suggest that although procaspase-3 was already and predominantly immunodetected in meiotic (pachytene spermatocytes) germ cells, its activation occurs later in the course of germ cell development in more mature germ cells, i.e. in postmeiotic (round spermatids) germ cells under androgen control as it was observed in the androgen-dependent seminiferous tubules. It is of interest to note that although pro-caspase-3 was also immunodetected in Leydig cells, its activation did not occur in this cell type as active cleaved caspase-3 was not detected in Leydig cells from adult rat testes exposed in utero to flutamide. Together, these findings support the concept that in utero exposure to the antiandrogen triggers, in the adult rat testis, a long-term activation of caspase-3 exclusively in postmeiotic germ cells. Although it appears that such an activation of this effector caspase occurs under (anti-) androgen control, the molecular mechanisms involved in caspase-3 activation under hormonal control remain to be investigated.
In the present study, it is also clearly shown that in utero exposure to flutamide induces comparable if not similar effects on both caspase-3 and caspase-6. Indeed, the antiandrogen induced an increase in caspase-6 mRNA, procaspase-6 and activated caspase-6 protein levels. However, although, as for procaspase-3, procaspase-6 was predominantly immunodetected in premeiotic (and to a lesser extent in meiotic and postmeiotic) germ cells, we do not know in which germ cells activated caspase-6 is immunoexpressed. Indeed, as the antibody used detects both pro- and active caspase-6, it was not possible to immunolocalize specifically active caspase-6 to the different testicular cell types. Additional experiments are required to immunolocalize this activated caspase-6 in germ cells. With regard to caspase-7, which is known as the third effector caspase, it is probably not at play in the germ cell apoptotic process in the adult rat testes exposed in utero to flutamide in that it was predominantly immunodetected in somatic (but not in germ) testicular cells, and its expression appeared not to be affected in adult rat testes exposed in utero to the antiandrogen (our unpublished data). Together, the data related to the dose effects of flutamide clearly indicate that caspase expression and activation were affected before any histological alterations could be observed in the seminiferous tubules. For example, at 0.4 and 2 mg/kg·d of flutamide, caspase expression and activation were maximally increased, whereas the adult rat testis histology appears not to be affected.
Among the changes in caspase gene expression and activation, which may represent early molecular events leading to the hypospermatogenesis observed at higher doses of flutamide (9), changes of active caspase-3 are of a particular interest. Indeed, active caspase-3 could represent an interesting (sensitive) biomarker in that it is absent in control testes, whereas it appears in testes exposed to the lowest dose of flutamide. Finally, although the first level of caspase regulation is achieved by controlling procaspase activation, there is also a second level, equally important, which involves the direct inhibition of nascent active caspases. In mammals, this is achieved by inhibitors of apoptosis (IAPs) (21). We do not know at the present time whether IAPs are involved in the germ cell death process triggered by exposure to flutamide. However, that in the adult rat testes exposed in utero to the antiandrogen, caspase activation was at a maximum at 2 mg/kg·d, whereas the number of apoptotic germ cells (evaluated through the TUNEL approach) is not maximal until 10 mg/kg·d suggests that there are probably, besides caspase activation, other processes in motion to trigger the germ cell death process. In this context, IAPs appear as interesting candidates to play such a role. We are currently investigating such a possibility.
It is also shown in this study that the increase in the expression and activation of caspases-3 and -6 in adult germ cells is chronic when the exposure occurs in utero but transient when the exposure occurs during the adulthood. Indeed, following exposure to flutamide at the highest (10 mg/kg·d) of adult rats during adulthood, we observed an increase in caspase-3 and -6 mRNA levels as well as procaspase and activated caspase protein levels. However, 1–2 wk after the cessation of flutamide treatment, caspase-3 and -6 mRNA and (procaspase and active caspase) protein levels were back to control levels. Together, these data imply that the effector caspase expression and activation are under the negative control of the androgens and the increase in the two effector caspase expression and activation in the adult rat germ cells is reversible at cessation of the exposure to the antiandrogen when it occurs during the adulthood. The androgen disruption appears to affect at least three different levels, i.e. 1) a transcriptional level leading to an increase in the effector caspase mRNA levels, 2) a translational level resulting in increased procaspase protein amounts, and 3) ultimately, caspase activation as shown by the appearance of activated caspases in germ cells. This is the first report showing that both expression and activation of the two effector caspases in adult germ cells are negatively regulated by testosterone in the testis. To our knowledge, there is in the literature only one report (22) that shows that caspase-3 and caspase-activated deoxyribonuclease are associated with testicular germ cell apoptosis resulting from reduced intratesticular testosterone following an LH-suppressive treatment (administration of testosterone and 17ß-estradiol). Finally, the regulatory mechanisms underlying the long-term increase in caspase expression and activation in adult rat germ cells when the exposure to the antiandrogen occurs during the fetal life remain to be investigated. However, the possibility exists that the in utero exposure to the antiandrogen could alter the epigenetic control of some specific testicular genes expressed under androgen control. Such a possibility has been suggested for yet another endocrine disrupter (23).
Based on the observation demonstrating the dependency of the effector caspase expression and activation on testosterone activity in the adult rat testis as shown here, one could speculate that in adult rats exposed in utero to flutamide, the increase in the expression of testicular caspase-3 and -6 expression could be related to an androgen deficiency and/or action. However, there are at least two observations arguing against such a possibility. First, circulating levels of testosterone in adult animals exposed in utero to antiandrogens such as flutamide (our unpublished data) and vinclozolin whose metabolites also prevent testosterone binding to its receptor (24) were not significantly different from those found in untreated control animals. Moreover, in utero exposure to flutamide has been also reported not to affect intratesticular testosterone levels in adult rat testes (8). Second, in the seminiferous tubules, androgen receptor is normally immunoexpressed in Sertoli cells, and its mRNA and protein levels as well as the number of Sertoli cells that are the targets of androgen action were not affected (our unpublished data).
One interesting point relates to the role of Sertoli cells in the apoptotic process that affects germ cells in the adult rat testis exposed to the antiandrogen. Indeed, although apoptosis occurred in germ cells, it is Sertoli cells (and peritubular myoid cells) that are the direct target cells of testosterone action because they are the cells that express androgen receptor in the seminiferous tubules. These observations would suggest that Sertoli cells (in cooperation with peritubular myoid cells?) may produce factors that control the apoptotic process in germ cells. These factors could potentially act as pro- or antisurvival factors for germ cells. The antiapoptotic role of testosterone reported here and in numerous previous studies (25) may therefore result from an inhibition of the production of the antisurvival factors and/or the stimulation of the prosurvival factors. In this context, in a coculture or transwell system, Fujioka et al. (26) have reported, by using a TUNEL approach, that Sertoli cells prevent apoptosis in pachytene spermatocytes and round spermatids. However, both the nature and the mode of action of these Sertoli cell pro- and antisurvival factors for germ cells are at the present time unknown and remain to be investigated.
Classically, there are two major apoptotic pathways leading to effector caspase activation that have been identified in mammalian cells, the Fas/TNF-R1 death receptors, and the mitochondrial pathway. For the first pathway, the engagement of the death receptors by soluble ligand results in recruitment of upstream signaling caspases-2, -8, and -10 through specific death complexes formed by the binding of a death receptor to a death domain-containing protein with the death effector domains of caspases-8 and -10, which when activated, lead ultimately to effector caspase activation. The mitochondrial pathway is thought to be triggered by translocation into the mitochondria of a proapoptotic Bcl-2 family member such as Bax. The Bcl-2 family of proteins consists of proapoptotic and antiapoptotic proteins that compete through dimerization and regulate apoptosis mainly by controlling the release of cytochrome c and other mitochondrial apoptotic events (27). The death receptor pathway involvement in the testicular cell death apoptotic process is supported by the fact that an increase in testicular Fas content has been associated with germ cell apoptosis following the reduction of intratesticular testosterone levels (28). In addition, the colocalization of Fas-L and Fas-R in pachytene spermatocytes (29) supports a potential involvement of the Fas pathway in this germ cell apoptosis. Because caspase-8 mRNA and protein levels were not affected in germ cells from adult rat testes exposed in utero to flutamide (our unpublished data), this pathway could appear not to be at play.
It is also assumed that the death receptor pathway involves caspase-8 or caspase-10 and that caspase-10 can function independently of caspase-8 in initiating Fas and TNF-related apoptosis-inducing ligand receptor-mediated apoptosis (30). It remains therefore to be determined whether caspase-10 expression and activation is modified in the present model before excluding the possibility of the involvement of the death receptor pathway in germ cell apoptosis in adult rats exposed to flutamide. We are currently investigating such a possibility. Concerning the mitochondrial pathway, different studies have suggested that both the pro- and antiapoptotic members of the Bcl-2 family could be involved in germ cell death. Specifically, in rodent testes, the expression of Bax, Bad, Bcl-xl, and Bcl-2 has been demonstrated (31, 32, 33, 34). Studies using knockout and transgenic mice suggest that members of the Bcl-2 family are important regulators of apoptosis in the testis. For example, 1) proapoptotic Bax is expressed in pachytene spermatocytes (Ref. 35 and our unpublished data); 2) Bax knockout mice are infertile as a result of accumulation of premeiotic germ cells and an absence of mature haploid sperm (33); and 3) following androgen withdrawal in adult animals, the apoptotic process affecting germ cells may be related to an increased Bax (29). Whether the mitochondrial pathway is involved in caspase-3 and -6 activation in adult rat germ cells exposed in utero to flutamide is currently being investigated.
In summary, the data presented in the present study show that in utero exposure to flutamide induced in the adult rat testes a chronic apoptotic germ cell death process associated with a long-term increase in the expression and activation of two effector caspases, caspases-3 and -6, two major components in the apoptotic cell death machinery. These alterations may represent early molecular events leading to the hypospermatogenesis observed at higher doses of the antiandrogen.
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This study has been carried out with financial support from the Institut National de la Santé et de la Recherche Médicale (Unité 407), the Commission of the European Communities, specific RTD programme and "Quality of Life and Management of Living Resources," QLK4-2000-00684 ENDISRUPT, and University Claude Bernard Lyon I.
Abbreviations: dNTP, Deoxynucleotide triphosphate; GD, day of gestation; IAP, inhibitor of apoptosis; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate-biotin nick end labeling.
Received October 25, 2002.
Accepted for publication October 31, 2002.
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