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Testis |
Division of Endocrinology, Department of Medicine, Harbor-UCLA Medical Center and Research and Education Institute, Torrance, California 90509
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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apoptosis, male reproductive tract, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The extrinsic pathway for apoptosis involves ligation of the death receptor (such as Fas) to its ligand, Fas L. Binding of Fas L to Fas induces trimerization of Fas receptors, which recruit FADD (Fas-associated death domain) through shared death domains (DD). FADD also contains a death effector domain, or DED, in its N-terminal region. Fas/ FADD complex then binds to the initiator caspase 8 or 10 through interactions between DED of the FADD and these caspase molecules. Caspase 8 or 10 then activates the effector or executioner caspases 3, 6, and 7, resulting in cellular disassembly. Both pathways converge on caspase 3 and other executioner caspases and nucleases that drive the terminal events of programmed cell death.
Crosstalk between these pathways does occur at multiple levels [1, 2]. For example, in certain types of apoptosis, proper activation of effector caspases by Fas depends on caspase 8-mediated cleavage of the proapoptotic Bcl-2 member Bid and subsequent release of cytochrome c from mitochondria, which, in turn, results in caspase 9 activation via apoptosome formation [7, 8]. Supportive of this is the demonstration that Bid-deficient mice are resistant to Fas- induced hepatocyte apoptosis [9].
A growing body of evidence demonstrates that both spontaneous (during normal spermatogenesis) and increased germ cell death triggered by various regulatory stimuli, including suppression of gonadotropins and intratesticular testosterone (T) and increased scrotal temperature in rat, occurs via apoptosis [10–14]. The mechanisms by which these regulatory stimuli activate germ cell apoptosis are not well understood. In a recent study, using the rat model of testicular hyperthermia [12, 14], we have provided evidence for involvement of the mitochondria-dependent pathway for heat-induced germ cell death in the rat testis [15].
Here we report the involvement of the mitochondria- dependent apoptotic pathway in heat-induced germ cell apoptosis in gld (generalized lymphoproliferation disease) and the lprcg (lymphoproliferation complementing gld) mice as well as their respective wild types.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Seven to eight-wk-old male gld (B6Smn.C3H-FasLgld) and lprcg (MRL. CBAJms-Tnfrsf6lpr-cg formerly known as MRL/MpJ-Tnfrsf6lpr-cg) mice and their wild types (C57BL6J and MRL/MpJ, respectively) were obtained from the Jackson Laboratories (Bar Harbor, ME). Animals were housed in a standard animal facility under controlled temperature (22°C) and photoperiod (12L:12D) with food and water ad libitum. Heating of the scrota was performed as described previously [12, 14]. Briefly, after mice were anesthetized with an i.p. injection of sodium pentobarbital (40 mg/kg body weight [BW]), their scrota were immersed in a thermostatically controlled water bath at 43°C (treated) for 15 min. Mice were killed at 0 (control), 0.5, 2, or 6 h after heat exposure. Animal handling and experimentation were in accordance with the recommendation of the American Veterinary Medical Association and were approved by the Harbor-UCLA Research and Education Institute animal care and use review committee.
Tissue Preparation
Both control and experimental animals were injected with heparin (130 IU/100 g BW, i.p.) 15 min before a lethal injection of sodium pentobarbital (100 mg/kg BW, i.p.) to facilitate testicular perfusion using a whole-body perfusion technique [16]. After perfusion with saline, one testis was removed, decapsulated, weighed, snap frozen in liquid N2, and stored at –70°C for subsequent analysis. The contralateral testes were then fixed by vascular perfusion with either 5% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) or Bouin solution (Sigma Diagnostics, St. Louis, MO). The testes were removed and processed for routine paraffin embedding for either in situ detection of apoptosis or immunohistochemistry. Portions of glutaraldehyde-fixed testes were further diced into small pieces, postfixed into 1% osmium tetroxide, and embedded in Epon 812 (Polysciences, Warrington, PA). Thin sections from selected tissue blocks were cut with an LKB ultramicrotome (LKB Instruments, Rockville, MD), stained with uranyl acetate and lead citrate, and examined with a Hitachi 600 electron microscope (Hitachi Ltd., Tokyo, Japan).
Immunohistochemical and Immunofluorescence Analyses
Bouin fixed, paraffin-embedded testicular sections were immuno-stained as described previously [14, 15, 17, 18]. Briefly, testicular sections were deparaffinized, hydrated by successive series of ethanols, rinsed in phosphate buffered saline (PBS), and then incubated in 2% H2O2 to quench endogenous peroxidase. Sections were then blocked with a blocking serum containing 1 drop of normal goat serum in 1 ml of PBS to suppress nonspecific binding of IgG and subsequently incubated with rabbit polyclonal Bax (1:400; Santa Cruz Biotechnology, Santa Cruz, CA), Smac/DIABLO (1:1000; Calbiochem, San Diego, CA), caspase 9 (1:50; Cell Signaling Technology, Beverly, MA) that recognizes both full-length and the cleaved product of p39 and p37 subunits, and caspase 3 antibody (CM1; 1:1000; kindly provided by Dr. Ann Srinivasan, Idun Pharmaceuticals, San Diego, CA) that recognizes only the cleaved product of p18 and p12 subunits of active caspase 3 but not the inactive zymogen [19], for 1 h at room temperature. Testicular sections were then washed three times in PBS and subsequently incubated with biotinylated goat anti-rabbit IgG secondary antibody (1:1000) for 30 min at room temperature followed by a 30-min incubation with ready-to-use avidin-biotinylated horseradish peroxidase (HRP) complex (commercially available and prepared according to the manufacturer's instructions). The preformed avidin-biotinylated HRP complex binds to the biotin on the secondary antibody and a lattice is formed, which localizes the HRP to the areas where the primary antibody has specifically bound to its antigen, and is visualized with diaminobenzidine tetrahydrochloride as per the manufacturer's instructions (rabbit Unitect ABC Immunohistochemistry Detection System; Oncogene Science, Inc., San Diego, CA). Slides were counterstained with hematoxylin. Germ cell types and their stages of occurrence were identified according to the criteria proposed by Russell et al. [20] for paraffin sections.
Activation of the initiator caspase 9 and the executioner caspase 3 in germ cells undergoing apoptosis was also detected by the confocal microscopy using double immunostaining for caspase 9 (1:50) or caspase 3 (1:1000) and DNA fragmentation [15]. In situ detection of cells with DNA strand breaks was performed in Bouin-fixed, paraffin-embedded testicular sections by the terminal deoxynucleotidyl transferase (TdT)-mediated deoxy-UTP nick end labeling (TUNEL) using an ApopTag-fluorescein kit (Intergen, Purchase, NY). In brief, after deparaffinization and rehydration, tissue sections were incubated with proteinase K for 15 min at room temperature and washed in PBS for 5 min at room temperature. Sections were incubated with a mixture containing digoxigenin-conjugated nucleotide and TdT 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. For staining of caspases, slides were then incubated in a humidified chamber for 1 h with rabbit polyclonal active caspase 3 or 9 antibody followed by goat-anti- rabbit Texas Red-labeled secondary antibody for 45 min at room temperature. Slides were washed and then 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 TCS-SP-MP confocal microscope (Leica Microsystems, Wetzler, Germany) equipped with a 488-nm argon laser for excitation of green fluorophores such as FITC 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 a modification of the procedure described earlier [21]. 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 dithiothreitol) supplemented with protease inhibitors (Complete Protease Inhibitors; Roche, Indianapolis, IN). 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 following 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. Protein concentration was determined using Bradford method (DC Bio- Rad Assay; Bio-Rad, Hercules, CA).
Western blotting was performed using testicular lysates and subcellular fractions as described previously [14, 15, 17]. In brief, proteins were separated on a 4–12% SDS-polyacrylamide gel with 2-(4-morpholine)-ethane-sulfonic acid or MOPS buffer purchased from Invitrogen (Invitrogen, Carlsbad, CA) at 200 V. Gel was transferred on Immuno-blot polyvinylidene fluoride Membrane (Bio-Rad) overnight at 4°C. Membranes were blocked in blocking solution (0.05% Tween 20 in Tris-buffered saline and 10% nonfat dry milk) for 1 h at room temperature, then probed using a rabbit polyclonal antibody to cytochrome c (1:2000; Santa Cruz Biotechnology), Smac/DIABLO (1:5000), Bid (1:500; BD Biosciences, San Diego, CA) Caspase 9 (1:500), or Caspase 3 (1:1000) for 1 h at room temperature with constant shaking. Following 3x 10-min washes in TBS-T buffer, membranes were then incubated in anti-rabbit IgG-HRP (Amersham Biosciences, Piscataway, NJ) secondary antibodies at a 1:2000 dilution. All antibodies were diluted in blocking buffer. For immunodetection, membranes were washed three times in TBS-T wash buffer, incubated with ECL solutions per the manufacturer's specifications (Amersham Biosciences), and exposed to Fuji X-ray film (Fuji Medical Systems, Inc., Stamford, CT). Band intensities were determined using Quantity One software from Bio-Rad.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We first examined the changes in the in vivo expression of Bax in both wild-type and gld mice at stages XI–XII (Fig. 1). In untreated wild-type (Fig. 1A) and gld (Fig. 1B) mice, a strong cytosolic Bax immunostaining was found in the Sertoli cells, while weak to moderate Bax immunostaining was detected in late spermatocytes and spermatids. A redistribution of Bax from a cytoplasmic to paranuclear localization was clearly evident in heat-susceptible late spermatocytes 2 h after heat treatment in both wild-type (Fig. 1C) and gld (Fig. 1D) mice. Similar results were also obtained in the lprcg mice (data not shown).
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Mitochondria Are Sequestered into Paranuclear Areas of Apoptotic Germ Cells
Given the observation that Bax is redistributed from a cytoplasmic to paranuclear localization, we performed electron microscopy to characterize the sequestration of organelles, if any, into such areas of the heat-susceptible germ cells. The occurrence of germ cell apoptosis, characterized by nuclear condensation, chromatin fragmentation, and cytoplasmic shrinkage, was readily detected within 2 h of heating (Fig. 2). After induction of apoptosis, late spermatocytes exhibited sequestration of small ultracondensed mitochondria into a crescent-shaped area close to the nuclear periphery (Fig. 2B), whereas, in the untreated cells, mitochondria are sparsely distributed and often aggregated in groups of two (Fig. 2A). Conglomeration of ultracondensed mitochondria was also noted in the paranuclear areas in apoptotic late spermatocytes in lprcg mice (data not shown).
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Cytochrome c and DIABLO Are Released from Mitochondria into Cytosol During Heat-Induced Germ Cell Death
We then examined the cytochrome c and DIABLO release during heat-induced programmed germ cell death in both wild-type and mutant mice. Testicular lysates were fractionated into cytosolic and mitochondrial fractions and analyzed by Western blotting. As shown in Figure 3, no cytochrome c was detected in cytosol from untreated testes. In contrast, cytosolic accumulation of cytochrome c was clearly evident in both wild-type (Fig. 3A) and gld mice (Fig. 3B) 2 and 6 h after heat treatment. Like cytochrome c, cytosolic accumulation of DIABLO was readily detected in both wild-type (Fig. 3A) and gld mice (Fig. 3B) at 2 and 6 h after heating. Similar results were also obtained in the lprcg mice, where cytosolic accumulation of cytochrome c and DIABLO was detected during both spontaneous and heat-induced germ cell apoptosis (Fig. 4). We also visualized DIABLO release by immunocytochemistry. A strong cytosolic DIABLO immunostaining was found in primary and secondary spermatocytes and in round and elongated spermatids. In contrast, no DIABLO immunoreactivity was noted in earlier germ cells or in the somatic cells. In both untreated gld (Fig. 5) and lprcg mice (data not shown), the heat-susceptible late spermatocytes exhibited punctate perinuclear staining of DIABLO characteristic of its mitochondrial localization (Fig. 5, A and B). After apoptosis induction, these cells exhibited mostly diffuse cytoplasmic staining of DIABLO, which is consistent with its translocation from mitochondria to cytoplasm (Fig. 5, C and D).
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Cytochrome c release from Mitochondria into Cytosol During Heat-Induced Germ Cell Death Is Independent of Bid
We next tested the apparent role of Bid in heat-induced germ cell apoptosis. We did not observe the appearance of truncated Bid (p15) in either the cytosolic or mitochondrial fractions of testes lysates in both wild-type and lprcg mice after heat treatment (Fig. 6), suggesting that Bid was not cleaved, nor did the truncated Bid translocate to mitochondria during heat-induced germ cell death. Similar studies were not done in gld mice.
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Activation of the Initiator Caspase 9 and the Executioner Caspase 3 During Heat-Induced Programmed Germ Cell Death
Because the release of cytochrome c from mitochondria into the cytosol triggers caspase activation, we then examined the activation of the initiator caspase 9 and the executioner caspases 3. After apoptosis induction by heat treatment, the initiator caspase 9 was activated in both gld and lprcg mice as evidence by immunofluorescence staining of testis sections (Fig. 7A) for caspase 9 and immunoblotting (Fig. 7B). We also found activation of caspase 3 in germ cells undergoing apoptosis, as evidenced by double immunofluorescence staining of active caspases 3 and DNA fragmentation during heat-induced programmed germ cell death in both groups of mice (Fig. 8).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Bcl-2 family of proteins governs the mitochondria- dependent pathway for apoptosis [1–3, 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 Bid, translocate from cytosol to mitochondria early during apoptosis [29, 30]. 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 [31–33]. Thus, it is conceivable that the signal for cytochrome c release from mitochondria in heat-induced testicular germ cell apoptosis emanates from relocation of Bax to mitochondria. It is pertinent to note here that we did not observe any appearance of the truncated Bid in either cytosolic or mitochondrial fractions of heat-treated testicular lysates of both wild-type and mice lacking functional Fas, suggesting that the caspase 8-mediated cleavage of Bid is not responsible for the observed release of cytochrome c from mitochondria.
The release of cytochrome c from mitochondria initiates caspase activation by binding to the caspase activating protein Apaf-1 [reviewed in 34]. Indeed, in the present study, we found activation of the initiator caspase 9 and the executioner caspase 3. It is of interest that, with regard to caspase 9, the increase in cleaved fragments was not associated with a concomitant decrease in the pro-caspase 9 as revealed by Western blotting. In fact, there was an apparent increase in the amount of pro-caspase 9. In this context, it is important to note that, unlike other caspases, proteolytic processing of caspase 9 has only a minor effect on the enzyme's catalytic activity [2, 35]. Rather, the key requirement for caspase 9 activation is its association with a dedicated protein cofactor, Apaf-1, in the apoptosome. Evidence exists indicating that pro-caspase 9 can be activated without proteolytic processing and removal of the prodomain actually inactivates the enzyme, as it can no longer associate with the apoptosome [2, 35]. A wide variety of experimental evidence, including gene ablation experiments in mice, has demonstrated that caspase 9 serves as the initiator caspase in mitochondria-dependent apoptotic pathways [reviewed in 3, 34]. Results from these gene ablation studies further underscore the importance and linearity of each component of the mitochondria-dependent apoptotic pathway. For example, as compared with cell lines established from wild-type embryos, the Apaf-1 protein in cytochrome c knockout cells remained in the monomeric state in the presence of apoptotic stimuli [36]. Also, in Apaf-1 or caspase 9-deficient cells, no caspase 3 activation was detected in response to apoptotic stimuli even though cytochrome c was released into the cytosol [37–39].
The IAPs and their counteraction by the mitochondrial protein DIABLO have also emerged as important regulators of caspase activation and apoptosis in various cell systems [reviewed in 3]. Like cytochrome c, DIABLO is located in mitochondria and is released into the cytosol when cells undergo apoptosis [5, 6, 40]. Of note, we also found accumulation of mitochondrial cytochrome c and DIABLO in the cytosol from untreated testes of both MRL/Mpg wild- type and lprcg mice. These findings would be consistent with increased spontaneous germ cell apoptosis in these mice. Indeed, in an earlier study, we did find considerably higher incidence of spontaneous germ cell apoptosis in both untreated MRL/MpJ wild-type and lprcg mice [26]. Evidence exists that DIABLO, a key component in the mitochondria-dependent apoptotic pathway, promotes caspase 9 activation by inhibition of IAPs, which can be modulated by the Bcl-2 family members [40–42]. Of particular importance, the expression of DIABLO mRNA [5, 6] as well as the protein [43] has been found to be the most abundant in the adult testis. Western blot data reported herein clearly show that the release of DIABLO together with cytochrome c from mitochondria into the cytosol in both wild-type and mutant mice is clearly upstream of apoptosis, which was first detected by TUNEL assay at 6 h after heating [26]. Immunocytochemistry of heat-treated testes revealed diffused staining of DIABLO in those heat-susceptible germ cells before their eventual apoptosis at later time intervals, whereas in the untreated cells, staining is mostly punctate, suggesting cytosolic translocation of mitochondrial DIABLO, as reported for various mitochondrial proteins, including DIABLO in various extragonadal cell systems [5, 6, 40, 43–45]. Furthermore, because, after heat-stress, both cytochrome c and DIABLO were simultaneously released from mitochondria and accumulated in the cytosol, it is possible that the same mechanisms responsible for release of cytochrome c from the mitochondria are responsible for the translocation of DIABLO. Thus, one could assume that, while cytochrome c initiates, through interaction with Apaf- 1, caspase activation [34], DIABLO could bind and inhibit the cellular IAPs and, in turn, promote apoptosis. Future studies are clearly warranted to characterize the upstream signals promoting the release of DIABLO early in apoptosis and the mechanisms whereby DIABLO acts to promote male germ cell apoptosis.
In summary, the present study further underscores the importance of the mitochondria-dependent apoptotic pathway in heat-induced germ cell apoptosis in mice. In response to apoptotic stimuli, mitochondria not only release cytochrome c to induce the formation of caspase 9-activating apoptosome, but also release DIABLO to counter the inhibitory activity of IAPs on caspase activation.
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FOOTNOTES |
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2 Correspondence: Amiya P. Sinha Hikim, Division of Endocrinology, Harbor-UCLA Medical Center, Box 446, 1000 West Carson Street, Torrance, California 90509. FAX: 310 533 0627; hikim{at}gcrc.rei.edu
Received: 24 October 2003.
First decision: 16 November 2003.
Accepted: 22 January 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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