Mol. Hum. Reprod. Advance Access originally published online on August 6, 2004
Molecular Human Reproduction 2004 10(10):743-753; doi:10.1093/molehr/gah094
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Activator protein-1 in human male germ cell apoptosis
1Programme for Developmental and Reproductive Biology, Biomedicum Helsinki and Hospital for Children and Adolescents, University of Helsinki, FIN-00029, Helsinki, 2Molecular Cancer Biology Research Program, Biomedicum Helsinki, University of Helsinki, FIN-00014, Helsinki, 3Wihuri Research Institute, Helsinki, 4Department of Urology, Helsinki University Central Hospital, Helsinki, and 5Kuopio University Central Hospital, Kuopio, Finland
6 To whom correspondence should be addressed at: Hospital for Children and Adolescents, University of Helsinki, Biomedicum Helsinki, Haartmaninkatu 8, 5 krs B529b, PO Box 700, FIN-00029, Helsinki, Finland. Email: laura.suomalainen{at}hus.fi
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Abstract |
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Apoptosis limits germ cell number in the testis, and its dysregulation is associated with male infertility. Here, we evaluated the role of the transcription factor activator protein 1 (AP-1) in male germ cell apoptosis in a culture of human seminiferous tubules. AP-1 DNA-binding activity increased in cultured tubules within 2.5 h, which was earlier than the onset of apoptosis as detected by caspase 3 activation and apoptotic DNA fragmentation. The c-Jun, c-Fos and JunD proteins were detected in the Sertoli cell nuclei, whereas apoptosis occurred in the germ cells. Follicle-stimulating hormone (FSH), whose receptors are expressed in the Sertoli cells, inhibited germ cell apoptosis and concomitantly suppressed AP-1 DNA-binding activity, but had no effect on nuclear factor
B (NF-B) activation. These results suggest that AP-1 transcription factors are involved in the Sertoli cell-mediated control of germ cell apoptosis, and that inhibition of germ cell apoptosis by FSH appears to involve suppression of AP-1 activation. Key words: apoptosis/germ cells/Sertoli cells/signal transduction/spermatogenesis
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Introduction |
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Death of selected spermatocytes and spermatids is a common feature of normal spermatogenesis (Huckins, 1978
; Sinha Hikim et al., 1999; Print et al., 2000). The number of these proliferating and differentiating germ cells has to match the capacity of the non-proliferative somatic Sertoli cells, which provide structural support and biochemical factors essential for germ cell development (Griswold, 1998). Apoptotic cell death is therefore a prerequisite for continuous spermatogenesis in limiting the germ cell population in physiological conditions (Rodriguez et al., 1997; Sassone-Corsi 1997; Sinha Hikim et al., 1999; Print and Loveland, 2000) and under stress caused by external disturbances (Tapanainen et al., 1993; Blanchard et al., 1996; Henriksen et al., 1996).
The regulation of male germ cell apoptosis is a complex and still poorly understood process. An interesting candidate in the regulation of male germ cell death is activator protein-1 (AP-1), which is developmental-stage specifically expressed in the rodent testis (Wolfes et al., 1989; Alcivar et al., 1990) and which in some other cell types plays a role in the regulation of apoptosis (Shaulian et al., 2002). AP-1 is a collective term referring to dimeric transcription factor proteins belonging to the Jun (c-Jun, JunB, JunD), Fos (c-Fos, FosB, Fra-1, Fra-2) and ATF (ATF2, LRF1/2, B-ATF, JDP1, JDP2) subfamilies (Shaulian et al., 2001). The activity of AP-1 is induced by multiple environmental insults and physiological stimuli, which activate mitogen-activated protein kinase (MAPK) cascades. Three well-characterized subfamilies of MAPKs exist in multicellular organisms: the extracellular signal-regulated kinase (ERK1 and ERK2), the stress activated c-jun N-terminal kinases (JNK1, JNK2 and JNK3), and the p38 enzymes (p38
, p38ß, p38
, p38
) (Johnson et al., 2002; Shaulian and Karin, 2002).
AP-1 is involved in a broad range of biological responses such as proliferation, transformation, cell differentiation, cell migration and apoptosis (Leppä et al.,1999; Johnson and Lapadat, 2002; Shaulian and Karin, 2002). AP-1 activation can lead either to induction or prevention of apoptosis depending on the tissue involved and on the developmental stage of the animal (Colotta et al., 1992; Hilberg et al., 1993; Smeyne et al., 1993; Estus et al., 1994; Hafezi et al., 1997). Several studies suggest diverse functions of AP-1 in the testis. JunD –/– male mice exhibit multiple age-specific defects in reproduction, hormone imbalance and impaired spermatogenesis, with abnormalities in head and flagellum sperm structures (Thepot et al., 2000). Further, JNK participates in the regulation of mouse spermatogenesis (Phelan et al., 1999), whereas ERK1/2 is induced in sperm maturation (Lu et al., 1999) and capacitation (Luconi et al., 1998).
Follicle-stimulating hormone (FSH), a pituitary-derived /ß heterodimeric glycoprotein, is a key hormone involved in the initiation of pubertal spermatogenesis and regulation of spermatogenesis. The synthesis and release of FSH in the pituitary is triggered by gonadotropin-releasing hormone (GnRH), which appears to regulate FSH transcription via activation of AP-1 (Kraus et al., 2001). FSH in turn induces DNA synthesis in its target Sertoli cell, at least partly via the ERK 1/2 (Crepieux et al., 2001). The effect of FSH on the ERK 1/2 in rat Sertoli cells is age-specific; it activates this pathway in immature rat testis and inhibits it in mature rat testis (Crepieux et al., 2001).
Regarding the role of MAPK cascades in male germ cell apoptosis, vasectomization of rat testis that leads to germ cell apoptosis also leads to concomitant activation of all MAPKs (Shiraishi et al., 2002). It is not known, however, whether the activation of MAPKs is involved in the induction or prevention of germ cell death, and accordingly, these kinases are not necessarily needed for apoptosis induction. Interestingly, FSH inhibits germ cell apoptosis in the rodent testis (Yan et al., 2000) and in cultured segments of human seminiferous tubules (Tesarik et al., 2000). Whether this regulation involves MAPKs or AP-1 is unknown. The aims of our study were therefore to investigate the role of AP-1 in germ cell apoptosis in the human testis and to study whether hormonal modulation of AP-1 activation by endogenous AP-1 regulator FSH has any effect on male germ cell death.
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Patients
Testis tissue was obtained from 17 men aged 59–80 years undergoing orchidectomy as a treatment for prostate cancer. These patients had received no hormonal, chemotherapeutic or radiotherapeutic treatment before the operations. The operations were performed between January 2000 and May 2003 at the department of Urology, Helsinki University Central Hospital (Helsinki, Finland). The ethics committees of the Hospital for Children and Adolescents and the Department of Urology, University of Helsinki, approved the study protocol.
Tissue culture and treatments
Apoptosis of the human testicular germ cells was induced by incubating segments of seminiferous tubules under serum-free conditions. The physiological contact between the Sertoli cells and the germ cells was maintained by culturing the segments of seminiferous tubules rather than isolated testicular cells. The testicular tissue was microdissected on a Petri dish containing culture medium (Nutrient mixture, Ham's F10; Gibco, Paisley, UK) supplemented with 0.01% human serum albumin (Sigma Chemical Co., St Louis, MO) and 10 µg/ml gentamicin (Gibco). Segments of seminiferous tubules were transferred to a Petri dish containing the same serum-free culture medium and incubated for 0–10 h at 34°C in a humidified atmosphere containing 5% CO2. Induction of apoptosis by this culture model has been well validated and reported in several articles by this group (Erkkilä et al., 1997, 1998, 1999, 2002, 2003; Pentikäinen et al., 1999, 2000, 2001, 2002; Suomalainen et al., 2003).
Recombinant human FSH (Gonal F; Serono, MA) was dissolved in culture medium as a 100 IU/ml stock and used at 0.01, 0.1 and 1.0 IU/ml. N-acetyl-L-cysteine (NAC; Sigma) was prepared as a 1 M stock in distilled water, with the pH adjusted to 7.5 with NaOH, and used at 100 mmol/l. 17ß-estradiol (E2; Sigma) was prepared as a 103 stock in absolute ethanol and used at 10–10 mol/l (final concentration of ethanol: 0.0001
). The specific inhibitor of MEK (upstream regulator of ERK 1 and 2) (PD98059; Calbiochem; Merck, Darmstadt, Germany) and JNK (SP600125; Biomol, Research laboratories Inc., Plymouth, USA) were used at 10 µmol/l, 50 µmol/l, and 100µ µmol/l. For each treatment, the culture medium containing the compound under investigation was prepared just before the operation, and immediately after the operation, equal aliquots of the tissue were microdissected in treatment or control medium. Segments of seminiferous tubules were then transferred to Petri dishes containing the same medium.
Detection of apoptosis
Southern blot analysis of apoptotic DNA fragmentation
DNA was extracted with the apoptotic DNA Ladder Kit (Roche Molecular Biochemicals, Mannheim, Germany) as described (Pentikäinen et al., 2000). After DNA was quantified spectrophotometrically (absorbance at 260 nm), 1 µg of the total DNA from each sample was subjected to 3' end-labeling with digoxigenin-dideoxy-UTP (Dig-dd-UTP, Roche) by terminal transferase (Roche) reaction. The DNA samples were electrophoresed on 2% agarose gels, blotted onto nylon membranes, and crosslinked to the membranes by UV irradiation. The membranes were then washed and blocked with 1% blocking reagent (Roche) in maleic acid buffer (100 mmol/l maleic acid, 150 mmol/l NaCl, pH 7.5) for 30 min at room temperature. The 3' end-labeled DNA in the membranes was localized with alkaline phosphatase-conjugated anti-digoxigenin antibody (Anti-Digoxigenin-AP; Roche), and the bound antibody was detected by the chemiluminescense reaction (CSPD; Roche).
Caspase 3 activation
The activity of caspase 3 was measured by caspase 3 fluorometric assay kit (RD Systems, Minneapolis, USA) according to the manufacturer's instructions. Briefly, samples of human testis tissue were homogenized in lysis buffer (R&D Systems), centrifuged at 17 000 g for 20 min and the supernatants were collected for determination of protein concentration by the DC protein assay (Bio-Rad laboratories, Inc.). Thereafter, three parallel 100 µg aliquots of protein homogenate from each sample were added to 96-well plates in 50 µl lysis buffer (R&D Systems), 50 µl of reaction buffer 3 (R&D Systems), and 5 µl caspase 3 fluorogenic substrate (DEVD-AFC) and the plates were incubated at 37°C for 2 h. Finally, fluorescence was measured on a fluorescence microplate reader (Perkin Elmer HTS 7000 Plus Bio Assay Reader) using 405 nm excitation and 505 nm emission filters. For negative controls, fluorescence was measured from wells containing no substrate or no protein homogenate.
Nonradioactive in situ end-labeling of DNA (ISEL)
Small segments of seminiferous tubules (
1 mm in length) were squashed under coverslips to produce a monolayer of cells, and the preparations were fixed as previously described (Parvinen and Hecht, 1981). These preparations were then rehydrated and washed twice for 5 min in distilled water. After incubation for 10 min with terminal transferase reaction buffer (1 mol/l potassium cacodylate, 125 mmol/l Tris–HCl and 1.25 mg/ml BSA, pH 6.6) the apoptotic DNA was 3' end-labeled with Dig-dd-UTP (Roche) by the terminal transferase reaction for 1 h at 37°C. For the negative controls, the terminal transferase enzyme was replaced with the same volume of distilled water. Dig-dd-UTP was detected with the antidigoxigenin antibody conjugated to horseradish peroxidase (Anti-Digoxigenin-POD, Roche). For location of the antibody, 0.05% diaminobenzidine substrate (Sigma) was added. Light counterstaining was performed with hematoxylin, and the samples were dehydrated and mounted.
Nuclear protein extracts
Segments of seminiferous tubules were gently homogenized with a tight-fitting Potter–Elvehjelm homogenizer in ice-cold hypotonic buffer A (50 mmol/l HEPES, pH 7.4, 10 mmol/l KCl, 1 mmol/l ethylenediaminetetra-acetic acid, 1 mmol/l dithiothreitol, 0.3 mmol/l phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, 0.5% nonidet P-40) and nuclear protein extracts were prepared as previously described (Han and Brasier, 1997). Protein concentrations were determined by the DC protein assay (Bio-Rad, Hercules, CA) and the protein extracts were stored at –80°C until used in electrophoretic mobility shift assays (EMSAs).
Electrophoretic mobility shift assay (EMSA)
AP-1 and NF-B DNA binding activities were assayed by DNA probe containing the consensus AP-1 site 5'-GATCTATCTGAGTCAGCAG-3' (Eriksson et al., 2002) or the consensus B enhancer element 5'-AGTTGAGGGGACTTTCCCAGGC-3' (Santa Cruz sc-2505; Santa Cruz Biotechnology, Santa Cruz, CA). The probes were 5' end-labeled with [-32P]ATP using polynucleotide kinase (Promega corp., Madison, WI). Testicular protein nuclear extracts (10 µg) were incubated on ice for 10 min with 2 µg poly (dIdC)(di-dC) (Amersham Pharmacia Biotech, Piscataway, NJ) in 50 mmol/l HEPES, pH 7.6, 10% glycerol v/v, 225 mmol/l KCl, 1 mmol/l ethylenediaminetetra-acetic acid, 2.5 mmol/l dithiothreitol, 1 mmol/l MgCl2, 0.75 mmol/l phenylmethylsulfonyl fluoride and 1.5 µmol/l leupepetin A 5' end-labeled probe (15 000–30 000 c.p.m.) was then added, and incubation was continued at room temperature for 30 min. In the competition experiments, a 100-fold molar excess of unlabeled probe was added before the labeled probe. Reaction products were separated on 4% polyacrylamide gels run in 22.5 mmol/l of Tris-borate and 0.5 mmol/l of ethylenediaminetetra-acetic acid at 200 V at room temperature. After electrophoresis, the gels were dried and visualized by autoradiography. In the supershift assays, 2 µg of an affinity-purified polyclonal antibody was added after binding reactions, and incubation was further continued for 1 h at room temperature. The antibodies were purchased from Santa Cruz (p-c-Jun, sc-822X; c-Fos, sc-52X; JunD, sc-74 X).
Immunohistochemistry
Immunostainings of c-Jun, JunD and c-Fos were performed on paraffin embedded sections of formalin-fixed adult testicular tissues. Paraffin sections were incubated at 60°C for 30 min and deparaffinized in xylene. The sections were then rehydrated, microwaved at high power for 5 min in citrate buffer (10 mmol/l citrate pH 6.0) for antigen retrieval, washed, and then blocked with blocking solution [phosphate-buffered saline (PBS) containing 5% goat normal serum, 3% bovine serum albumin (BSA) and 0.1% Tween] for at least 30 min at room temperature. The JunD and c-Fos proteins, and the phosphorylated, i.e. active, form of the c-Jun protein (p-c-Jun) were detected with affinity-purified polyclonal antibodies to human JunD (sc-74; Santa Cruz), c-Fos (sc-52; Santa Cruz) and a monoclonal antibody to human p-c-Jun (sc-822; Santa Cruz). Antibodies were used at concentrations of 0.5–0.7 µg/ml. The primary antibodies were added to the samples and incubated overnight at 4°C. After incubation, the slides were washed in PBS. The primary antibodies were detected by use of biotin-conjugated goat anti-rabbit or rabbit anti-mouse IgG from the ABC-Elite kit (Vector laboratories Inc., Burlingame, CA) followed by incubation with ABC solution. For location of the secondary antibody, 0.05% diaminobenzidine substrate (Sigma) was added. For the negative controls, the primary antibodies were replaced with non-immune rabbit or mouse IgG (Sigma). After the staining protocols, light counterstaining was performed with hematoxylin, and the samples were dehydrated and mounted. Double immunofluorescence was used to co-localize p-c-Jun androgen receptor, a Sertoli cell-specific protein. Slides were prepared as described and incubated overnight at 4°C with primary antibodies against both p-c-Jun and human androgen receptor (N20; sc-816; Santa Cruz) at concentrations of 0.5 µg/ml. After washing with PBS, the primary antibodies were detected by use of Alexa-conjugated goat anti-mouse IgG1 (Alexa Fluor 488; Molecular Probes Inc., Leiden, The Nederlands; dilution 1:200) and goat anti-rabbit IgG (Alexa Fluor 548; Molecular probes; dilution 1:200) secondary antibodies. The nuclei were counterstained with DAPI (Molecular probes). After washing with PBS, the sections were mounted using SlowFade (Molecular probes). For the negative controls, the primary antibodies were replaced with blocking solution. The samples were viewed with a Nikon E600 fluorescent microscope equipped with appropriate fluorescent filters and photographed with a cooled CCD camera (Spot RT, Diagnostic instruments, MI).
Electron microscopy
Segments of the seminiferous tubules were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, dehydrated, and embedded in epoxy resin. The tissue blocks were sectioned at 50 nm with an ultramicrotome (Reichert Jung, Vienna, Austria) and stained with uranyl acetate and lead citrate by the Leica EMstain apparatus (Leica, Vienna, Austria). The samples were examined with a JEOL JEM 1200 EX transmission electron microscope (JEOL, Tokyo, Japan) at the Institute of Biotechnology, electron microscopy unit, Finland.
Quantitative analysis of X-ray films
The X-ray films exposed to chemiluminescence (Southern blots) or autoradiography (EMSA) were scanned with a tabletop scanner (Hewlett Packard Scan Jet 6300C) and the digital image was analyzed with the gel plot 2 macro for Scion image ß 4.0.2. (Scion Corp., Frederic, MD) analysis software. For the Southern blots, the digitized quantification of the low molecular weight DNA fragments (<1.3 kb) in the sample cultured for 10 h (in the time-course analysis of nuclear apoptosis; Figure 1A) or for 5 h (in the FSH, NAC and E2-treated samples; Figures 4A and 5A and C Figures 4A and 5A and C) without treatments was taken as 1.0 (100% apoptosis), and the amounts of low molecular weight DNA fragments in the other samples were expressed in relation to this. For time-course analysis of AP-1 activation (EMSA; Figure 1B), the digitized quantification of the specific AP-1 band in the sample cultured for 5 h was set as 1.0 and the intensities of the bands in other samples were expressed in relation to this.
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Statistics
Cultures for studying Southern blot analysis of the effects of FSH on low molecular weight DNA fragmentation were repeated on 11 independent occasions, and the effects of NAC, E2, SP600125 and PD98059 were repeated on three independent occasions. Quantitative data represent integrated optical density from scanned X-ray films. Data obtained from 3-11 cultures (mean±SEM) were analyzed by one-way ANOVA, and if significant differences existed, this was followed by comparison of the groups with two-tailed unpaired Student's t-test. P<0.05 was considered statistically significant. The cultures for time-course analyses of Southern blot analysis of apoptotic DNA fragmentation were repeated three times independently. The EMSAs for studying the cultures for time-course analyses were repeated five times independently. The time-course analyses for caspase 3 activation were repeated twice. The supershift assay was performed using samples from four independent cultures. The EMSAs for studying the effect of FSH on AP-1 DNA-binding activity were performed with samples from five independent cultures, and those for studying the effects of NAC and E2 with samples from three independent cultures. The EMSAs for studying the effects of SP600125 or PD98059 on AP-1 DNA-binding activity and the effects of FSH or E2 on NF-
B DNA-binding activity were studied by EMSAs using samples from two independent cultures. The immunohistochemical analysis for each antibody was performed with three independent samples and three parallel slides. Immunofluorescent double-staining analysis was performed using samples from four independent cultures and two to three parallel slides. |
Results |
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AP-1 DNA binding activity during in vitro induced human testicular apoptosis
Culturing the segments of seminiferous tubules under serum-free conditions resulted in induction of apoptotic DNA fragmentation within 5 h, and DNA fragmentation was further increased at 10 h (Figure 1A). The AP-1 DNA binding activity, as measured by EMSA with nuclear protein extracts from human seminiferous tubules, was clearly increased at the 2.5 h time-point and was further elevated at the 5 h time-point, after which it started to decrease (Figure 1B). To further clarify the kinetics of apoptosis induction, we measured the activation of caspase 3 from samples incubated in serum-free conditions for 0 h, 2.5 h, 5 h and 10 h. Caspase 3 activity did not start to increase until after 2.5 h (Figure 1C) and steadily increased up to the 10 h time-point.
Protein composition of the inducible AP-1 complex
As demonstrated by the supershift assays, the AP-1 proteins c-Jun, JunD and c-Fos participated in formation of the DNA-protein complexes. The nuclear extracts from 5 h cultured samples were pre-incubated with antibodies against AP-1 proteins prior to EMSA. Incubation of the nuclear extracts with the antibodies against c-Jun, JunD and c-Fos, but not with the antibody against JunB, resulted in the supershifts of the basal DNA-protein complexes (Figure 2).
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AP-1 activation occurs in Sertoli cells but apoptotic cell death in germ cells
We next studied the localization of the p-c-Jun, JunD and c-Fos proteins by immunohistochemistry in paraffin-embedded sections of formalin-fixed human seminiferous tubules at different time-points (0 h, 2.5 h and 5 h). All these antibodies produced a similar nuclear staining pattern. In non-cultured samples, we observed no nuclear expression of AP-1. In contrast, after 2.5 h (data not shown) or 5 h culture in serum-free conditions, the Sertoli cells in most of the seminiferous tubules expressed nuclear AP-1 (Figure 3). The positively staining nuclei were identified as Sertoli cell nuclei on the account of their basal location in the seminiferous tubules and their characteristic morphology and nucleolus (Figure 3Ba). In addition to the nuclear staining, in non-cultured seminiferous tubules, some spermatogonia and early meiotic spermatocytes expressed cytoplasmic immunostainings for JunD and c-Fos. When the primary antibodies were replaced with a similar concentration of non-specific rabbit or mouse IgG, we detected no specific immunostaining. The localization of p-c-Jun was confirmed by immunofluorescence double-staining method in paraffin-embedded sections of formalin-fixed seminiferous tubules cultured for 5 h. The expressions of p-c-Jun (Figure 4A) and Sertoli cell-specific androgen receptor (AR) (Figure 4B) were co-localized in the Sertoli cell nuclei (Figure 4C).
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Considering the potential role of AP-1 in the regulation of testicular apoptosis, we demonstrated that AP-1 is activated in the Sertoli cells. In contrast, by ISEL analysis of DNA fragmentation, we detected apoptotic DNA fragmentation in late meiotic germ cells and in occasional spermatogonia (Figure 3Bb). In addition, in electron microscopy, the most common type of cell death was found to be apoptotic and the morphological signs of apoptosis occurred most frequently in germ cells, particularly in spermatocytes (Figure 3Bc and d).
Inhibition of the testicular AP-1 activity by FSH, NAC and E2
To evaluate the effect of AP-1 on male germ cell survival, we first tested the ability of endogenous AP-1 regulator, FSH, to modulate AP-1 activity and apoptosis. The concentrations used were 0.1 IU/ml, 0.01 IU/ml and 1 IU/ml. The 0.1 IU/ml and 0.01 IU/ml concentrations of FSH showed no significant inhibitory effect on human male germ cell apoptosis (data not shown), whereas the 1.0 IU/ml concentration of FSH (n=11) inhibited testicular apoptosis by 35%, (P<0.05) (Figure 5A) and concomitantly suppressed AP-1 DNA-binding activity (n=5) (Figure 5B). To evaluate the specificity of the effect of FSH on AP-1 DNA binding activity, we tested the effect of FSH on NF-B, which is also activated during male germ cell apoptosis (Pentikäinen et al., 2002). In samples from two independent cultures, we observed that although FSH suppressed AP-1 DNA binding activity, it had no effect on NF-B DNA-binding activity (Figure 4C). To further evaluate the effect of AP-1 on male germ cell survival, we tested the effects of two known inhibitors of human male germ cell apoptosis: NAC (Erkkilä et al., 1998) and 17ß-estradiol (E2) (Pentikäinen et al., 2000) on AP-1 activation. NAC (n=3) at a concentration of 100 mmol/l inhibited apoptosis by 78% and concomitantly suppressed AP-1 DNA binding activity (n=3) almost to the basal level (Figure 6A). E2 at a concentration of 10–10 mol/l inhibited apoptosis by 56% but did not reduce the AP-1 DNA-binding activity (n=3) below the level detected in control samples (testicular tissue cultured for 5 h without treatment) (Figure 6B). E2 (n=2) had no effect on NF-B DNA-binding activity, which was measured as a control for AP-1 DNA binding (data not shown). Similarly, we have previously shown that NAC has no effect on NF-B DNA-binding activity (Pentikäinen et al., 2002).
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To further specify the role of AP-1 and MAPK kinases in germ cell death we attempted to study the effects of the specific inhibitors of MEK and JNK, PD98059 and SP600125, respectively, on the induction of testicular apoptosis and AP-1 DNA binding activity. As measured by Southern blot analysis of DNA fragmentation (n=3) and by EMSA of AP-1 DNA-binding activity (n=2), neither PD98059 nor SP600125 had any effects on male germ cell apoptosis or AP-1 DNA-binding activity (data not shown).
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Discussion |
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AP-1 activation is involved in both the induction and prevention of apoptosis (Leppä and Bohmann, 1999
; Shaulian and Karin, 2001; Johnson and Lapadat, 2002; Shaulian and Karin, 2002). Continuous c-Fos expression precedes kainic acid-induced neuronal cell apoptosis, and c-Jun induction precedes induction of apoptosis of cells exposed to alkylating agents or UV irradiation (Smeyne et al., 1993). Furthermore, overexpression of c-Jun and c-Fos induces apoptosis in various cell lines, and antisense nucleotides against c-Fos and c-Jun mRNAs enhance the survival of growth factor-deprived lymphoid cells (Colotta et al., 1992). Expression of dominant-negative c-jun mutant or injection of neutralizing antibodies against c-jun protect neuronal cells from NGF withdrawal or chronic depolarization-induced apoptosis (Estus et al., 1994; Ham et al., 1995; Xia et al., 1995; Le-Niculescu et al., 1999; Whitfield et al., 2001). Finally, light-induced apoptosis of retinal photoreceptors is impaired in c-Fos-deficient mice (Hafezi et al., 1997). Evidence of apoptosis suppression by AP-1 transcription factors, however, also exists. Hepatocytes derived from c-jun –/– mouse embryos undergo massive apoptosis (15). Fibroblasts of these mice are resistant to UV-induced apoptosis but exhibit increased spontaneous apoptosis (Hilberg et al., 1993; Eferl et al., 1999). Some studies also suggest that JNK inhibits both TNF- and Fas-induced apoptosis, yet the mechanism remains unknown (De Smaele et al., 2001; Shaulian and Karin 2001). Thus the effect of AP-1 transcription factors on apoptosis appears to be highly tissue- and developmental-stage specific.
In the present study, male germ cell death was induced in a culture model, in which the interactions of somatic Sertoli cells and germ cells are maintained in their physiological environment, and the apoptotic pathways involving important cell to cell interactions and signaling between different types of cells can be identified. It is not likely that the model is capable of supporting the process of spermatogenesis. However, despite its limitations, this in vitro model enables us to evaluate apoptotic mechanisms in the human testis, which may differ from those of other species. In our previous studies we have discovered that in the present culture model, apoptosis occurs mainly in the germ cells, whereas Sertoli cells are rather resistant to apoptosis (Erkkilä et al., 1997, 1998; Pentikäinen et al., 2000, 2001). Here we found that AP-1 activity began to increase in testicular Sertoli cells earlier than the beginning of caspase 3 activation and apoptotic DNA fragmentation. On the other hand, AP-1 was activated later than NF-B, which in this model activates strongly and rapidly in the Sertoli cells within 30 min (Pentikäinen et al., 2002). Considering the relatively slow beginning of AP-1 activity as compared with the rapid activation of immediate mediators such as NF-B, the role of AP-1 activation in germ cell apoptosis is not fully clear. Many parallel apoptotic pathways appear to be involved in the regulation of human male germ cell death: rapid pathways leading to initial activation of effector caspases such as caspase 3 (Suomalainen et al., 2003) and slower ones involving changes in expression of apoptosis-related proteins such as FasL (Pentikäinen et al., 2001). Activation of AP-1 before caspase 3 and low molecular-weight DNA fragmentation suggests that AP-1 activation is associated with induction of apoptosis. Most likely, AP-1 is involved in apoptotic pathways that are induced in the present model after the initial rapid pathways. ISEL analysis of apoptotic DNA fragmentation revealed that apoptosis was induced in late meiotic and postmeiotic germ cells, whereas positive nuclear immunostaining of AP-1 proteins appeared in the Sertoli cell nuclei. These findings suggest that the effect of AP-1 is mediated by somatic Sertoli cells and involves cell to cell signaling. During severe stress, AP-1 in somatic Sertoli cells may thus induce transcription of Sertoli cell gene(s) that are able to mediate germ cell death.
High levels of c-Jun, JunB and JunD exist in type B spermatogonia and interstitial cells of immature mouse testis (Alcivar et al., 1991). In adult mouse testis, c-Jun and c-Fos exist in type B spermatogonia and early spermatocytes, whereas JunD exists in post-meiotic round spermatids and in pachytene spermatocytes (Alcivar et al., 1991). Although c-Fos is considered to be an unstable nuclear protein difficult to detect (Acquaviva et al., 2002), we observed weak positive immunostaining against c-Fos and JunD in the cytoplasm instead of nuclei of some spermatogonia and early meioitic spermatocytes in non-cultured seminiferous tubules, where Sertoli cells remained negative. Accordingly, c-Fos has been found in both nuclear and cytosolic extracts of the frog (Rana esculenta) testis (Cobellis et al., 1999, 2002) suggesting that c-Fos may be stored in the cytoplasm before entry into the germ cell nuclei (Cobellis et al., 2002). Our results together with previous findings that the expression of AP-1 proteins exist in type B spermatogonia, which represent the last mitotic cell division before entry into meiotic prophase, and in the meiotic germ cells (Alcivar et al., 1991), suggests a role for AP-1 also in regulating meiosis of germ cells.
Our hypothesis that AP-1 plays a role in the induction of germ cell apoptosis is supported by the finding that FSH inhibits male germ cell death in cultured human seminiferous tubules concomitantly with inhibition of AP-1 activation. Consistently with nuclear localization of the AP-1 proteins in Sertoli cells, the FSH receptors are expressed in the same cells (Simoni et al., 1997). Similarly, male germ cell apoptosis has recently been inhibited by FSH and the pro-survival effect of FSH mediated through the Sertoli cell-derived stem cell factor in the rat (Yan et al., 2000). Moreover, c-Fos, induced by cAMP or FSH, has been shown to bind to the AP-1 site in the promoter region of the FSH receptor gene in vitro and in vivo (Griswold et al., 2001). Thus, the apoptosis-inhibiting effect of FSH may be related to the inhibition of AP-1 DNA-binding activity in the Sertoli cells.
To evaluate the role of AP-1 further in germ cell death, we studied the effects of NAC and E2, which are known to inhibit human male germ cell apoptosis (Erkkilä et al., 1998; Pentikäinen et al., 2000) and are potential regulators of AP-1 (Paech et al., 1997; Sawada et al., 2000; Wu et al., 2002; Yang et al., 2002) on apoptotic DNA laddering and on AP-1 activation. NAC effectively inhibited apoptosis and suppressed the AP-1 DNA binding activity almost to the basal (0 h) level. Neither NAC (Pentikäinen et al., 2002) nor FSH had, however, any effect on NF-B DNA-binding activity. These findings support the role of AP-1 in controlling human male germ cell apoptosis and suggest that these compounds specifically inhibit AP-1, and not a wider variety of transcription factors. On the other hand, on the basis of these results, activation of AP-1 could also be interpreted as only a secondary event that follows apoptosis induction and does not occur if apoptosis is prevented. This is not likely, since E2 also effectively inhibited germ cell apoptosis but not the AP-1 DNA-binding activity. Taken together, these findings suggest that AP-1 is not a universal regulator of human male germ cell apoptosis, since although FSH, NAC and E2 inhibited male germ cell apoptosis, the effects of each of them on AP-1 activity differed. Yet, AP-1 activation is not a secondary phenomenon which always accompanies apoptosis, since not all of these inhibitors of apoptosis also blocked the AP-1 activity. Thus, in the human testis, many parallel apoptotic cascades may exist, and the activation of AP-1 appears to be specifically regulated, depending on stimulus.
We found that the AP-1 proteins c-Jun, c-Fos and JunD participated in the formation of AP-1 protein complexes that are induced during germ cell apoptosis. As the previous literature suggests that the AP-1 complexes formed by these proteins are most likely regulated by MAPKs JNK and ERK (Leppä et al., 1999), we aimed to evaluate the role of these kinases in male germ cell apoptosis. For this purpose, we tested the effects of specific inhibitors of ERK and JNK on germ cell death. These inhibitors had, however, no effect on apoptosis or on AP-1 DNA-binding activity, suggesting that the inhibitors may not have been active in the present culture conditions, or that these kinases play no role in induction of germ cell apoptosis. There is evidence that p38 MAPK plays an important role in the regulation of mitochondria-dependent apoptosis (Park et al., 2003), which appears crucial in heat-induced rat male germ cell death (Vera et al., 2004). Whether p38 MAPK plays a role in the regulation of male germ cell death also in humans, remains to be elucidated in future studies.
In conclusion, we have shown that with induction of male germ cell apoptosis, activation of the transcription factor AP-1 occurs before the activation of effector caspases such as caspase 3. AP-1 activity increases in Sertoli cells, whereas apoptosis occurs in germ cells, suggesting that AP-1 regulates Sertoli cell genes that participate in the paracrine control of germ cell death. Furthermore, germ cell apoptosis can be suppressed by FSH, and this effect appears to be associated with inhibition of the AP-1 DNA-binding activity. Since AP-1 is a menagerie of proteins involved in a wide range of cellular processes, our results on only three AP-1 proteins do not necessarily provide unequivocal support to the role of AP-1 in the human testis. Further studies are therefore needed to elucidate the function of other AP-1-forming proteins in the regulation of germ cell death and spermatogenesis. However, our results may be helpful when aiming to protect male gonads from apoptosis and subsequent infertility caused by external stress, such as cancer therapy.
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We gratefully thank Sauli Kyttänen, Virpi Ahokas and Kaisa Alasalmi for their skillful technical assistance and Professor Jorma Toppari and Dr Markku O.Pentikäinen for their valuable comments. We also thank the staff of the Department of Surgery, Helsinki University Central Hospital, for providing orchidectomy samples. This study was supported by the Foundation for Pediatric Research, Finland and the Sigrid Juselius Foundation, Finland.
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