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Division of Endocrinology (Y.V., K.E., C.W., C.N., Y.L., R.S.S., A.P.S.H.), Department of Medicine, Harbor-University of California at Los Angeles (UCLA) Medical Center and Los Angeles Biomedical Research Institute, David Geffen School of Medicine at UCLA, Torrance, California 90509; Program for Developmental and Reproductive Biology (K.E., S.K., L.D.), Biomedicum Helsinki, and Hospital for Children and Adolescents, University of Helsinki, FIN-00029, Helsinki, Finland; and Department of Pediatrics (L.D.), Kuopio University Hospital, FIN-70211, Kuopio, Finland
Address all correspondence and requests for reprints to: Amiya P. Sinha Hikim, Division of Endocrinology, Harbor-University of California at Los Angeles Medical Center and Los Angeles Biomedical Research Institute, Box 446, 1000 West Carson Street, Torrance, California 90509. E-mail: hikim{at}labiomed.org.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The signaling events leading to apoptosis can be divided into two major pathways, involving other mitochondria (intrinsic) or death receptors (extrinsic). Recently, using murine models of testicular hyperthermia, we have demonstrated the involvement of the mitochondria-dependent (intrinsic) apoptotic pathway, characterized by BAX translocation, cytochrome c release, and activation of the initiator caspase 9 and the executioner caspases 3, 6, and 7, and poly(ADP-ribose) polymerase (PARP) cleavage, in heat-induced male germ cell apoptosis (2, 5). In additional studies, using the generalized lymphoproliferation disease (gld) and lymphoproliferation complementing gld (lprcg) mice, which harbor loss-of-function mutations in Fas ligand (FasL) and Fas, respectively (6), we have shown that the Fas signaling system had little, if any, role in male germ cell death triggered by mild testicular hyperthermia (7). We further emphasize the functional role of caspases in heat-induced male germ cell apoptosis (8). These studies indicate that the mitochondria-dependent intrinsic pathway signaling is the key apoptotic pathway for heat-induced male germ cell apoptosis. However, we do not know whether the involvement of the mitochondria-dependent apoptotic pathway is a common phenomenon during germ cell apoptosis in general or a specific event during heat-induced germ cell apoptosis, or its upstream activators.
MAPKs comprise a family of serine/threonine kinases that function as critical mediators of a variety of extracellular signals (9, 10, 11). Members of the MAPK superfamily include the ERKs, the c-Jun NH2-terminal kinases (JNKs), also known as stress-activated protein kinases, and the p38 MAPKs. Available data from various cell systems other than male germ cells suggest that ERK1 and ERK2 are activated in response to growth stimuli and promote cell growth, whereas both JNKs and p38 MAPKs are activated in response to a variety of environmental stresses and inflammatory signals and promote apoptosis and growth inhibition (9, 10, 11). However, the role of JNK/p38 MAPK signaling in the apoptotic responses of testicular germ cells is not known.
A role for p38 MAPK-mediated signaling in the transcription control of inducible nitric oxide synthase (iNOS) gene expression has been suggested in glial cells (12, 13). DNA sequence analysis indicates that promoter regions of iNOS contain consensus binding sites for numerous transcription factors, including activating transcription factor-2 (ATF-2), nuclear factor 
B, activator protein 1, cAMP response element, and cAMP response element binding protein, that act as substrates of p38 MAPK or its downstream kinases (11, 13, 14). Some of these are also targets for JNK (15).
Several lines of evidence indicate that increased nitric oxide (NO) synthesis through up-regulation of iNOS plays a major role in the induction of apoptosis in various cell systems (16, 17). A number of recent in vitro studies have shown the involvement of mitochondria-dependent intrinsic pathway signaling in NO-mediated cell death (18, 19, 20). Thus, one possible mechanism by which p38 MAPK can induce apoptosis is through the induction of iNOS resulting in increased NO output, which perturbs the BAX/BCL-2 rheostat and triggers the cytochrome c-mediated death pathway.
The objectives of the present study were 3-fold. The first was to examine whether the mitochondria-dependent apoptotic pathway, as noted after heat treatment, is also the key pathway for induction of apoptosis in male germ cells after hormone deprivation. The second was to examine the possible role of p38 MAPK and iNOS in activating such death pathway. The final aim of the present study was to investigate whether p38 MAPK and iNOS could also play a role in human testicular germ cell death. Our results indicate that p38 MAPK and NO-mediated intrinsic pathway signaling constitutes a critical component of apoptotic signaling in male germ cells across species.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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. Testicular weight showed no appreciable differences from control at 2 or 5 d after GnRH-A treatment. GnRH-A treatment for 14 d, however, suppressed testicular weight to approximately 47.0% of the control values. Within 2 d of GnRH-A treatment, testicular T levels declined markedly to 17.1% of control values and plasma T levels fell below detectable limits. Within the study paradigm, the maximum reduction (4.7% of the control values) in testicular T levels was achieved by d 14.
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, the mean incidence of apoptotic germ cells (expressed as numbers per 100 Sertoli cells) exclusively at stages VII–VIII increased significantly (108.1 ± 7.4; P < 0.05) by d 5, and increased another 1.9-fold (over the 5-d treatment value) on d 14 after GnRH-A treatment when compared with controls, where no apoptosis was detected.
Withdrawal of Gonadotropins and Intratesticular T Results in p38 MAPK Activation and iNOS Induction
Several independent lines of investigation have now converged to suggest that stress-activated MAPKs such as p38 MAPK plays an important role in the regulation of apoptosis in various extragonadal cell systems (18, 22, 23, 24). Accordingly, as a first step, we examined the time course of p38 MAPK activation after withdrawal of gonadotropins and testicular T. Activation of p38 MAPK, as evidenced by an increase in phospho-ATF-2 in testis lysates, was detected as early as 2 d after GnRH-A treatment and remained active thereafter throughout the treatment period (Fig. 1A). Activation of p38 MAPK in the testis was also ascertained by immunocytochemistry and confocal microscopy using a phosphospecific antibody, which detects p38 MAPK only when dually phosphorylated at threonine 180 and tyrosine 182 (Fig. 1B, panels I–VI). Compared with control, where no staining was detected (Fig. 1B, panel I), a strong phospho-p38 MAPK immunoreactivity was noted in the condensed nuclei of apoptotic germ cells after hormone withdrawal (Fig. 1B, panel II). No such immunostaining was noted when primary antibody was substituted by the same volume of rabbit IgG (Fig. 1B, panel III). Costaining for TUNEL and for phospho-p38 MAPK further confirmed activation of p38 MAPK only in those germ cells undergoing apoptosis (Fig. 1B, panels IV–VI). Because p38 MAPK can transcriptionally activate iNOS gene (12, 13) and plays an integral role in NO-mediated apoptosis in various cell systems (18, 22, 25, 26), we then examined the time course of iNOS induction after withdrawal of gonadotropins and testicular T. We found a similar profile in the induction of iNOS after GnRH-A treatment, as shown by immunoblotting (Fig. 1C). To substantiate our Western blot data, we further examined the GnRH-A-induced changes in the in vivo pattern of iNOS expression by immunocytochemistry and confocal microscopy (Fig. 1D, panels I–VI). In the control testes, using a rabbit polyclonal antibody, we detected an intense iNOS expression in the Leydig cells with little or no immunoreactivity in the Sertoli and germ cells (Fig. 1D, panel I). In contrast, we found a strong iNOS staining in susceptible germ cells after induction of apoptosis by GnRH-A treatment (Fig. 1D, panel II). No such immunostaining was noted when primary antibody was substituted by the same volume of rabbit IgG (Fig. 1D, panel III). No changes in iNOS immunoreactivity were noted in the somatic cells as wells as in the nonsusceptible germ cells that do not undergo apoptosis after hormonal deprivation. Costaining for terminal deoxynucleotidyl transferase-mediated biotinylated uridine triphosphate nick end labeling (TUNEL) and for iNOS further confirmed induction of iNOS only in those germ cells undergoing apoptosis (Fig. 1D, panels IV–VI). In addition, to complement the data on iNOS expression, we further compared the GnRH-A-induced changes in the iNOS expression using the same mouse monoclonal antibody used in the Western blot assay. We found a similar increase in iNOS immunoreactivity again only in those susceptible germ cells after hormone withdrawal (data not shown). Most importantly, p38 MAPK activation and iNOS induction coincided with the dramatic fall in testicular T levels, which was first detected 2 d after GnRH-A treatment (Table 1).
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, a robust accumulation of cytochrome c was detected in cytosolic fractions of testicular lysates at 5 and 14 d after GnRH-A treatment. Like cytochrome c, cytosolic accumulation of increased DIABLO was readily detected at 2, 5, and 14 d after withdrawal of gonadotropins and testicular T by GnRH-A treatment. We also visualized DIABLO release by immunofluorescence microscopy. As shown in Fig. 2B, the susceptible pachytene spermatocytes from a control rat exhibited punctate perinuclear staining characteristic of its mitochondrial localization. After apoptosis induction, these cells from a rat treated with GnRH-A for 5 d exhibited mostly diffuse cytoplasmic staining of DIABLO, which is consistent with its translocation from mitochondria to cytoplasm.
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). Unlike our hyperthermia model (5), we found an increase in BAX and a decrease in BCL-2 levels in mitochondrial fractions of testicular lysates (Fig. 3A). Immunocytochemical analysis further revealed a cell type-specific increase in the BAX expression involving only those germ cells undergoing apoptosis (Fig. 3B, panels I and II). Costaining for TUNEL and for BAX further confirmed a noticeable increase in BAX expression only in those germ cells undergoing apoptosis after hormone deprivation (Fig. 3B, panels III–V). Together, these data indicate a perturbation of the BCL-2/BAX rheostat during hormone deprivation.
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, A and B). Active caspase 9 immunostaining was detected in only in those germ cells undergoing apoptosis, compared with controls, where no such staining is detected. Immunostaining for active caspase 3 was not detected in the somatic cells as well as in nonsusceptible germ cells that do not undergo apoptosis after hormone deprivation (data not shown). To further explore the potential involvement of these effector caspases during germ cell apoptosis induced by hormone deprivation, we then examined the cleavage of their downstream substrate PARP. As shown in Fig. 4C, no PARP cleavage product was detected in cytosol from control testis. In contrast, PARP cleavage product was clearly detected at 2, 5, and 14 d after GnRH-A treatment.
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, GnRH-A treatment for 5 d led to a significant (P < 0.05) increase (100.4 ± 10.2) in the mean incidence of germ cell apoptosis exclusively at stages VII–VIII, when compared with controls, where no apoptosis was detected. Concomitant administration of AG at all dose levels (100, 250, and 500 mg/kg BW) effectively prevented hormone deprivation-induced germ cell apoptosis by 61.7, 58.6, and 67.9%, respectively.
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, there was an apparent reduction of BCL-2 levels in the mitochondrial fractions after GnRH-A treatment. AG addition to GnRH-A treatment effectively prevented such alteration of BCL-2 levels in the mitochondrial fractions.
Both iNOS and p38 MAPK Inhibitors Attenuate Male Germ Cell Apoptosis in Humans Induced by Culturing Seminiferous Tubules under Hormone-Free Conditions
Given that p38 MAPK and NO-mediated intrinsic pathway signaling constitutes a critical component of apoptotic signaling in male germ cells in rats, we next evaluated the efficacy of iNOS as well as p38 MAPK inhibitors in preventing or attenuating human male germ cell apoptosis induced by deprivation of survival factors. Apoptosis of the human testicular germ cells was induced by culturing segments of human seminiferous tubules under serum-free conditions. Consistent with our previous results (34, 35, 36), culturing seminiferous tubules for 4 h resulted in clear apoptotic DNA laddering, as detected by Southern blot analysis of DNA fragmentation (Figs. 6 and 7). Like our murine models (21, 37), the majority of the cells undergoing apoptosis in this in vitro tissue culture model were found to be spermatocytes and spermatids as detected by both TUNEL and electron microscopy (34, 35, 36). Concomitant treatments with SB203580 (Fig. 6, A and B), a p38 MAPK inhibitor, significantly suppressed low molecular DNA fragmentation induced by culturing segments of human seminiferous tubules under hormone-free conditions. Very similar results were obtained with ML3403, a different p38 MAPK inhibitor (data not shown). We further examined the induction of iNOS during human male germ cell apoptosis by Western blotting from samples cultured under hormone-free conditions. As shown in Fig. 6C, little or no iNOS expression was detected in the noncultured seminiferous tubule fragments (0 h). In contrast, culturing seminiferous tubules for 4 h resulted in induction of iNOS and that could be effectively suppressed by SB203580 (Fig. 7C), indicating that p38 MAPK is an upstream activator of iNOS during human male germ cell apoptosis. Like our rat model, AG (Fig. 7, A and B) also significantly suppressed low molecular DNA fragmentation induced by culturing segments of human seminiferous tubules under hormone-free conditions. Together, these data indicate that p38 MAPK and NO-mediated intrinsic pathway signaling also constitutes a critical component of apoptotic signaling in germ cells in men.
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DISCUSSION |
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The p38 MAPK signaling pathway has been implicated in the apoptotic response of various cell systems exposed to a variety of environmental stresses and inflammatory signals (18, 22, 23, 24, 38). Indeed, in the present study, we found activation of p38 MAPK within 2 d after GnRH-A treatment, coincident with the drastic fall in testicular T content to 17.1% of the control values. It is well established that T is a major regulator of germ cell survival (39). Thus, the signal for p38 MAPK activation most likely emanates from the stress generated by the dramatic loss of T production in the testis after GnRH-A treatment. These data indicate that p38 MAPK plays a role in testicular germ cell apoptosis induced by deprivation of survival factors. Consistent with a role for p38 MAPK in male germ cell apoptosis, in the present study, we show that p38 MAPK inhibitors significantly suppressed apoptotic DNA induced by culturing human seminiferous tubules under serum-free conditions. This concept is supported by another line of evidence showing that androgen, through inhibition of JNK/p38 MAPK activation, strongly suppresses 2-methoxyestradiol-induced apoptosis in a human prostate cancer cell line, LNCaP (40).
The downstream signaling events that couple p38 MAPK activation with male germ cell death have not been previously identified. One intriguing possibility is that p38 MAPK could induce apoptosis through induction of iNOS, resulting in increased NO production and subsequent activation of the cytochrome c-mediated death pathway. Indeed, there have studies indicating a role for p38 MAPK-mediated signaling in the transcriptional activation of iNOS gene in rat glial cells (12, 13). The rat iNOS promoter contains several transcription binding sites, including ATF-2, nuclear factor B, and cAMP response element binding protein, that act as substrates of p38 MAPK or its downstream kinases (13, 14). Relevant to this is the demonstration that lipopolysaccharide-induced iNOS gene expression and NO production in neuron-glia mixed cultures can be significantly reduced by the p38 MAPK inhibitor SB202190 (22). We show in the present study that the time course of p38 MAPK activation after hormone withdrawal paralleled the temporal increase in iNOS levels in the rat model. Most importantly, we further show that p38 MAPK inhibitor effectively diminished iNOS induction and significantly suppressed apoptotic DNA induced by culturing human seminiferous tubules under serum-free conditions.
Increased NO synthesis through up-regulation of iNOS has been implicated in cellular injury and apoptosis in various cell systems (16, 19, 41). This concept is further supported by another line of evidence showing that up-regulation of testicular iNOS after treatment with lipopolysaccharide causes significant germ cell loss (42, 43). Decisive evidence that iNOS plays an important role in testicular germ cell apoptosis derives from our recent studies of iNOS knockout animals (44). These mice have enlarged testis and increased sperm number and exhibit stage-specific suppression of spontaneous germ cell apoptosis. These mice also confer partial resistance to heat-induced male germ cell apoptosis. The results of the present study confirm and extend those findings by demonstrating that AG, a selective inhibitor of iNOS, is capable of preventing testicular germ cell apoptosis induced by withdrawal of survival factors in both rat as well as in men. Together, these results indicate that iNOS plays an important role in the regulation male germ cell apoptosis.
One mechanism by which NO can induce apoptosis is through stimulation of BAX translocation to mitochondria, resulting in the activation of the cytochrome c-mediated death pathway (18, 19, 20, 45). Indeed, in the present study, we found cytosolic translocation of mitochondrial cytochrome c and DIABLO during germ cell apoptosis induced by withdrawal of gonadotropins and intratesticular T. The release of cytochrome c is further associated with the activation of the initiator caspase 9 and the executioner caspase 3, and PARP cleavage. This is consistent with our previous works indicating the involvement of the mitochondria-dependent (intrinsic) pathway signaling in testicular germ cell death triggered by heat stress (2, 5, 7).
The BCL-2 family of proteins governs the mitochondria-dependent pathway for apoptosis (27, 28). One of the intriguing aspects of apoptosis regulation by members of this family is their subcellular localization and translocation. Some BCL-2 family members such as BCL-2 and BAK constitutively localize to the mitochondrial membrane, whereas others such as BAX and BAD translocates from cytosol to mitochondria early during apoptosis. Furthermore, insertion of BAX into mitochondrial membranes has been shown to play an essential role in releasing cytochrome c from the mitochondrial membrane space to the cytosol in various cell systems (46, 47, 48). Several lines of evidence suggest that the BCL-2 family members also regulate the NO-mediated apoptosis in certain cell lines. BCL-2 or BCL-XL has been shown to prevent apoptosis, whereas BAX has been hypothesized to mediate NO-induced apoptosis (18, 19, 20). Our Western blot data clearly show that induction of male germ cell apoptosis is associated with an increase in BAX and a decrease in BCL-2 levels in the mitochondrial fractions of testicular lysates. We further show that AG apparently restored BCl-2 levels in the mitochondrial fractions of testicular lysates. This implies a perturbation of the BCL-2/BAX rheostat during NO-mediated apoptosis triggered by withdrawal of gonadotropins and intratesticular T. Thus, it is conceivable that the signal for cytochrome c and DIABLO release from mitochondria into cytosol during germ cell apoptosis induced by hormone withdrawal emanates from NO-mediated changes in the BCL-2/BAX ratio in the mitochondria.
Having established that p38 MAPK and NO-mediated intrinsic pathway signaling constitute a critical component of apoptotic signaling in male germ cell apoptosis, we next evaluated the requirement for Fas signaling during male germ cell apoptosis induced by hormone withdrawal. To this end, we examined whether the gld mice that express a nonfunctional form of FasL would confer resistance to apoptosis upon hormone deprivation. We found that germ cells from wild-type and FasL-defective gld mice are equally sensitive to apoptosis induced by hormone withdrawal (data not shown). These findings reinforce our earlier hypothesis (2, 5, 7) that the intrinsic pathway signaling is the key apoptotic pathway for male germ cell apoptosis.
In summary, we have demonstrated a new signal transduction pathway involving p38 MAPK and iNOS that, through activation of the intrinsic pathway signaling, promotes male germ cell apoptosis in response to a lack of hormonal stimulation. Targeting the p38 MAPK and interrupting NO production may have a protective role in acute testicular injury associated with increased germ cell apoptosis. Future efforts toward improved fertility control and clinical management of infertility associated with reduced sperm production in men are hampered by incomplete understanding of the processes responsible for normal germ cell homeostasis. Elucidation of the mechanisms by which various environmental stresses regulates germ cell death will fill a major gap in our knowledge of this fundamental biological process.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Experimental Protocol
To investigate the contributions of p38 MAPK, iNOS, and the intrinsic pathway signaling to testicular germ cell death after suppression of intratesticular T, groups of six male rats were given a daily injections of vehicle (distilled water) for 14 d or a GnRH-A, acyline (1.6 mg/kg BW), for 2, 5, and 14 d. Acyline was kindly provided by Dr. Richard P. Blye (Contraceptive and Reproductive Health Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD).
To further explore the role of iNOS in regulating testicular germ cell apoptosis, we examined whether AG, a selective iNOS inhibitor (31, 32, 33), could prevent or attenuate hormone deprivation-induced germ cell death. Groups of six adult male Sprague Dawley rats received one of the following treatments daily for 5 d: 1) a sc injection of GnRH-A, acyline (1.6 mg/kg BW), 2) an ip injection of saline as a vehicle control, and 3) GnRH-A plus ip injections of 100, 250, or 500 mg/kg BW of AG. Rats were killed 5 d after treatment.
To evaluate the requirement for Fas signaling during male germ cell apoptosis induced by hormone withdrawal, we examined the incidence of germ cell apoptosis in the Fas ligand-defective gld mice (6) after deprivation of gonadotropins and intratesticular T. In these mice, there is a point mutation near the COOH terminus of the coding region. This mutation results in the replacement of phenylalanine with leucine, and abolishes the ability of Fas ligand to bind to Fas. Groups of six 7- to 8-wk-old male wild-type (C57BL/6J) and gld (B6Smn.C3H-FasLgld) mice received a single sc injection of vehicle (distilled water) or GnRH-A, acyline (20 mg/kg BW) plus flutamide (in the form of sc pellets of 25 mg; Innovative Research, Sarasota, FL). The rationale for using a combined treatment of acyline and flutamide is that, unlike rats, a total abolition of androgen action is required for adequate suppression of spermatogenesis through increased germ cell apoptosis in mice (37, 49). Consistent with this is the demonstration that LH receptor knockout (LuRKO) mice at the age of 12 months show completion of qualitatively normal spermatogenesis even in the presence of dramatically reduced (2% of control level) intratesticular T levels due to lack of LH stimulation (50). However, the completion of spermatogenesis up to the elongated spermatids of late steps 13–16 was blocked by flutamide (50), indicating that very low endogenous T levels that are produced without LH stimulation are sufficient to maintain spermatogenesis in mice.
Animal handling and experimentation were in accordance with the recommendation of the American Veterinary Medical Association and were approved by the Harbor-University of California at Los Angeles and Los Angeles Biomedical Research Institute Animal Care and Use Review Committee.
Blood Collection and Tissue Preparation
Both control and experimental animals were injected with heparin (130 IU/100 g BW, ip) 15 min before a lethal injection of sodium pentobarbital (100 mg/kg BW, ip) to facilitate testicular perfusion using a whole-body perfusion technique (51). Blood samples were collected from each animal by cardiac puncture immediately after death, and plasma was separated and stored at –20 C for subsequent T assays. After perfusion with saline, one testis was removed, decapsulated, and weighed. Portions of testicular parenchyma were snap frozen in liquid N2, and stored at –70 C for subsequent analysis of testicular T. The remaining portions of testicular parenchyma were used for subcellular fractionation and Western blotting. The contralateral testes were then fixed by vascular perfusion with either 5% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) or Bouin’s solution (Sigma-Aldrich, St. Louis, MO). The testes were removed and processed for routine paraffin embedding for either in situ detection of apoptosis or immunohistochemistry.
Assessment of Apoptosis
In situ detection of cells with DNA strand breaks was performed in glutaraldehyde-fixed, paraffin-embedded testicular sections by the TUNEL technique (1, 2, 3, 4, 5, 7, 21) using an ApopTag-peroxidase kit (Chemicon International, San Francisco, CA). Enumeration of the nonapoptotic Sertoli cell nuclei with distinct nucleoli and apoptotic germ cell population was carried out at stages VII–VIII of the seminiferous epithelial cycle using an Olympus BH-2 microscope (New Hyde Park, NY) with a x100 oil immersion objective. For each rat, at least 10 tubules were used. In the rat, of the 14 stages in the seminiferous epithelial cycle, stages VII–VIII are the most sensitive to acute withdrawal of gonadotropins after GnRH-A treatment as evidenced by the presence of apoptotic germ cells (21, 52). Unlike in rats, in both wild-type and gld mice, enumeration of the nonapoptotic Sertoli cell nuclei with distinct nucleoli and apoptotic germ cell population was carried out at stages I–IV, V–VI, VII–VIII, IX–X, and XI–XII using an Olympus BH-2 microscope with a x100 oil immersion objective. For each mouse, at least 10 tubules per stage group were used. These stages were chosen not only to examine the whole seminiferous cycle but also to characterize the stage-related susceptibility of germ cells to apoptosis after hormone deprivation in mice. Stages in both species were identified according to the criteria proposed by Russell et al. (53) for paraffin sections. The rate of germ cell apoptosis (apoptotic index) was expressed as the number of apoptotic germ cells per 100 Sertoli cells (2, 5, 8, 44).
Immunohistochemical and Immunofluorescence Analyses
Bouin’s fixed, paraffin-embedded testicular sections were immunostained as described previously (2, 4, 5, 7, 8). Primary antibodies included rabbit polyclonal BAX (1:200), BCL-2 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), iNOS (1:100; BD Transduction Laboratories, San Diego, CA), cleaved caspase 9 (1:50; this antibody detects only the cleaved product p38 and p17 of active caspase 9), caspase 6 (1:50; this antibody recognizes only the p18 subunit of the active caspase 6), caspase 7 (1:50; this antibody recognizes only the p20 subunit of active caspases 7), a mouse monoclonal iNOS (1:100; BD Transduction Laboratories), and a rabbit monoclonal phospho-p38 MAPK, which detects p38 MAPK only when dually phosphorylated at threonine 180 and tyrosine 182 (1:50) antibodies (Cell Signaling Technology, Beverly, MA). Immunoreactivity was detected using biotinylated goat antirabbit IgG secondary antibody followed by avidin-biotinylated horseradish peroxidase complex visualized with diaminobenzidine tetrahydrochloride as per the manufacturer’s instructions (rabbit Unitect Immunohistochemistry Detection System; Oncogene Science, San Diego, CA). Slides were counterstained with hematoxylin. Negative and positive controls were run for every assay. The negative controls were processed in an identical manner, except the primary antibody was substituted by the rabbit IgG. Heat-treated testicular sections were used as a positive control (4, 5).
p38 MAPK activation, iNOS induction, and changes in the BAX expression in germ cells undergoing apoptosis was detected by the confocal microscopy using double immunostaining as previously described (2, 5, 7). The same primary antibodies used in immunohistochemistry were used. Activation of the executioner caspase 3 in germ cells undergoing apoptosis and cytosolic translocation of mitochondrial DIABLO was also detected by the confocal microscopy using double immunostaining. In situ detection of cells with DNA strand breaks was performed in Bouin’s fixed, paraffin- embedded testicular sections using an ApopTag-fluorescein kit (Chemicon International). In brief, after deparaffinization and rehydration, tissue sections were incubated with proteinase K for 15 min at room temperature, washed in distilled water, and then treated with 2% hydrogen peroxide in PBS for 5 min in room temperature to quench endogenous peroxidase activity. Sections were incubated with a mixture containing digoxigenin-conjugated nucleotide and terminal deoxynucleotidyltransferase in a humidified chamber at 37 C for 1 h and subsequently treated with antidigoxigenin- fluorescein for 30 min in the dark. After fluorescein staining, slides were washed in PBS and incubated with blocking serum for 20 min to reduce nonspecific antibody binding. Slides were then incubated in a humidified chamber for overnight at 4 C with a rabbit monoclonal phospho-p38 MAPK (1:50), rabbit polyclonal BAX (1:200), iNOS (1:100), active caspase 3 (1:1000; kindly provided by Dr. Annu Srinivasan, Idun Pharmaceuticals, San Diego, CA), which recognizes only the cleaved product of p18 and p12 subunits of active caspase 3 but not the inactive zymogen (54), or smac/DIABLO (1:1000; Calbiochem, San Diego, CA) antibody followed by goat antirabbit Texas Red-labeled secondary antibody for 45 min at room temperature, washed, and mounted in ProLong Antifade (Molecular Probes, Eugene, OR). For controls, sections were treated only with secondary antibody, and no signals were detected. Confocal imaging was performed using a Leica (Deerfield, IL) TCS-SP-MP confocal microscope equipped with a 488-nm argon laser for excitation of green fluorophores such as fluorescein isothiocyanate and a 543-nm helium-neon laser for excitation of red flurophores such as Texas Red.
Subcellular Fractionation and Western Blotting
Cytosolic and mitochondrial fractions were prepared as described earlier (5, 7, 8). Briefly, saline-perfused testes were homogenized using a Dounce homogenizer in 3 ml buffer A (0.25 M sucrose, 50 mM HEPES, 10 mM NaCl, 10 mM EDTA, 2 mM DTT) supplemented with protease inhibitors (Complete Protease Inhibitors; Roche, Basel, Switzerland). The crude homogenates were centrifuged at 1000 x g for 10 min at 4 C, and the resultant supernatant centrifuged at 10,000 x g for 15 min at 4 C to sediment the low-speed fraction containing mainly mitochondria. The mitochondria were washed two times in buffer A and pelleted. The cytosolic and high-speed fractions were isolated after centrifugation of the 10,000 x g supernatant fraction at 100,000 x g for 60 min at 4 C. The resulting supernatant was the cytosolic fraction. The purity of the cytosolic and mitochondrial fractions was assessed by Western blotting using antibodies to actin (1:2000; Sigma-Aldrich) and cytochrome c oxidase subunit IV (COX IV; 1:500; Molecular Probes), respectively.
Western blotting was performed using rat testicular lysates and subcellular fractions as described previously (5, 7, 8). In brief, proteins (50–80 µg) were separated on a 4–12% sodium dodecyl sulfate-polyacrylamide gel with 2-(N-morpholino)ethanesulfonic acid or 4-morpholinepropanesulfonic acid buffer purchased from Invitrogen (Carlsbad, CA) at 200 V. Gel was transferred on a Immuno-blot PVDF Membrane (Bio-Rad, Hercules, CA) overnight at 4 C. Membranes were blocked in blocking solution (0.3% Tween 20 in Tris-buffered saline and 10% nonfat dry milk) for 1 h at room temperature, and then probed using a mouse monoclonal iNOS (1:1000; BD Transduction Laboratories), rabbit polyclonal BAX (1:500), BCL-2 (1:500), cytochrome c (1:2000; Santa Cruz Biotechnology), smac/DIABLO (1:5000; Calbiochem), and a rat-specific PARP (1:1000; Cell Signaling Technology, Beverly, MA), which recognizes only the cleaved (89-kDa) PARP antibodies for 1 h at room temperature or overnight at 4 C with constant shaking. After three 10-min washes in 0.3% Tween 20 in Tris-buffered saline, membranes were then incubated in antirabbit (Amersham Biosciences, Piscataway, NJ), antigoat, or antimouse IgG-HRP (Santa Cruz Biotechnology) secondary antibodies at a 1:2000 dilution. All antibodies were diluted in blocking buffer. For immunodetection, membranes were washed three times in 0.3% Tween 20 in Tris-buffered saline wash buffer, incubated with enhanced chemiluminescence solutions per the manufacturer’s specifications (Amersham Biosciences), and exposed to Hyperfilm ECL. The membranes were stripped and reprobed with a goat polyclonal actin (1:2000) or a rabbit polyclonal COX IV (1:500) for normalization of the loading. Band intensities were determined using Quantity One software from Bio-Rad.
Measurements of p38 MAPK Activation
Activation of p38 MAPK was measured using a p38 MAPK assay kit (Cell Signaling Technology). In brief, a monoclonal phosphospecific antibody to p38 MAPK (Thr180/Tyr182) was used to selectively immunoprecipitate active p38 MAPK from testis lysates. The resulting immunoprecipitate was then incubated with ATF-2 fusion proteins in the presence of ATF and kinase buffer, which allows immunoprecipitated active p38 MAPK to phosphorylate ATF-2. Phosphorylation of ATF-2 at Thr71 was measured by Western blotting as described before using a rabbit polyclonal phospho-ATF2 (Thr71) antibody.
Patients
Testicular tissues for human studies were obtained from eight adult men aged 57–82 yr undergoing orchidectomy as a treatment for prostate cancer. The operations were performed between December 2004 and April 2005 at the Department of Urology, Helsinki University Central Hospital, Helsinki, Finland. The patients had received neither hormonal nor chemotherapeutic medication, nor had they received radiotherapy before the operation, and none of them had suffered from cryptorchidism. The Ethics Committees of the Departments of Urology and of Children and Adolescents, University of Helsinki, approved the human study protocol (no. 14/95).
Tubule Culture and Treatments
The testicular tissues were microdissected in petri dishes containing tissue culture medium (Nutrient Mixture Ham’s F10; Invitrogen, Paisley, UK), supplemented with 0.1% of human serum albumin (Sigma-Aldrich) and 10 µg/ml gentamicin (Invitrogen). For induction of germ cell apoptosis, segments of seminiferous tubules (3–5 mm in length) were isolated and transferred to culture plates containing the hormone-free culture medium described above and incubated for 4 h at 34 C in a humidified room air with CO2 adjusted to 5% (34, 35, 36). AG was added to the cultures at final concentrations of 5 and 10 mM. Two different p38 MAPK inhibitors (SB203580 and ML3403; Calbiochem) were used at final concentrations of 100 µM. The stock solutions of p38 MAPK inhibitors were prepared in dimethylsulfoxide.
Detection of Apoptosis and Western Blotting
Segments of human seminiferous tubules were snap-frozen in liquid nitrogen and stored at –80 C until isolation of DNA. DNA was extracted using an Apoptotic DNA Ladder kit (Roche Molecular Biochemicals, Mannheim, Germany), as previously described (55). After DNA was quantified by spectrophotometry (absorbance at 260), 1 µg of the total DNA from each sample was 3'-end-labeled with digoxigenin-dideoxy-UTP (Roche) using the terminal-transferase (terminal deoxynucleotidyltransferase; Roche) reaction. The DNA samples were electrophoresed on 2% agarose gels, blotted onto nylon membranes, and cross-linked to the membranes by UV irradiation. The membranes were then washed and blocked with 1% blocking reagent (Roche) in maleic buffer [100 mmol/liter maleic acid, 150 mmol/liter NaCl (pH 7.5)]. The 3'-end-labeled DNA on the membranes was localized with alkaline phosphatase-conjugated antidigoxigenin antibody (Anti-Digoxigenin-AP; Roche) as previously described (34), and the bound antibody was detected by the chemiluminescence reaction (CSPD; Roche) at room temperature for 5 min, and enhanced at 37 C for 15 min. The x-ray films were exposed to the chemiluminescence, scanned, and the digitized information (optical density) was analyzed with Scion Image analysis software. Low-molecular-weight DNA fractions (<1.3 kb) of the 0-h sample were set at 1.0 (100%), to which the other settings were compared. Thus, the results are expressed in relation to the starting (0-h) time point.
Western blotting was performed using total seminiferous tubule lysates as described previously (5, 7, 8). In brief, 80 µg of proteins per sample were subjected to 7% sodium dodecyl sulfate-polyacrylamide gel with 2-(N-morpholino)ethanesulfonic acid or 4-morpholinepropanesulfonic acid buffer purchased from Invitrogen at 200 V.
Statistical Analysis
Statistical analyses were performed using the SigmaStat 2.0 Program (Jandel Cooperation, San Rafael, CA). Results were tested for statistical significance using the Tukey or Student-Newman-Keuls method test after one-way ANOVA. Differences were considered significant if P < 0.05.
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FOOTNOTES |
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Y.V., K.E., C.W., C.N., S.K., Y.L., L.D., R.S.S., and A.P.S.H. have nothing to declare.
First Published Online February 9, 2006
Abbreviations: AG, Aminoguanidine; ATF-2, activating transcription factor-2; BW, body weight; COX IV, cytochrome c oxidase subunit IV; FasL, Fas ligand; GnRH-A, GnRH antagonist; iNOS, inducible nitric oxide synthase; JNK, c-Jun NH2-terminal kinase; NO, nitric oxide; PARP, poly(ADP-ribose) polymerase; T, testosterone; TUNEL, terminal deoxynucleotidyl transferase-mediated biotinylated uridine triphosphate nick end labeling.
Received for publication September 27, 2005. Accepted for publication January 30, 2006.
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