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BOR - Papers in Press, published online ahead of print October 27, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.034520
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BIOLOGY OF REPRODUCTION 72, 516–522 (2005)
DOI: 10.1095/biolreprod.104.034520
© 2005 by the Society for the Study of Reproduction, Inc.

Functional Role of Caspases in Heat-Induced Testicular Germ Cell Apoptosis1

Yanira Vera, Susana Rodriguez, Mark Castanares, Yanhe Lue, Vince Atienza, Christina Wang, Ronald S. Swerdloff, and Amiya P. Sinha Hikim2

Division of Endocrinology, Department of Medicine, Harbor-UCLA Medical Center and Los Angeles Biomedical Research Institute, David Geffen School of Medicine at UCLA, Torrance, California 90509


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, we determined whether a pan caspase inhibitor could prevent or attenuate heat-induced germ cell apoptosis. Groups of five adult (8 wk old) C57BL/6 mice pretreated with vehicle (DMSO) or Quinoline-Val-Asp (Ome)-CH2-O-Ph (Q-VD-OPH), a new generation broad-spectrum caspase inhibitor, were exposed once to local testicular heating (43°C for 15 min) and killed 6 h later. The inhibitor (40 mg/kg body weight) or vehicle was administered intraperitoneally (i.p.) 1 h before local testicular heating. Germ cell apoptosis was detected by TUNEL assay and quantitated as number of apoptotic germ cells per 100 Sertoli cells at stages XI–XII. Compared with controls (16.8 ± 3.1), mild testicular hyperthermia within 6 h resulted in a marked activation (277.3 ± 21.6) of germ cell apoptosis, as previously reported by us. Q-VD-OPH at this dose markedly inhibited caspase 3 activation and significantly prevented (by 67.0%) heat-induced germ cell apoptosis. Q-VD-OPH-mediated rescue of germ cells was independent of cytosolic translocation of mitochondrial cytochrome c and DIABLO. Electron microscopy further revealed normal appearance of these rescued cells. Similar protection from heat-induced germ cell apoptosis was also noted after pretreatment with minocycline, a second-generation tetracycline that effectively inhibits cytochrome c release and, in turn, caspase activation. Collectively, the present study emphasizes the role of caspases in heat-induced germ cell apoptosis.

apoptosis, caspase inhibitors, gamete biology, germ cells, minocycline, spermatogenesis, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Caspases are key effectors of apoptosis and function in both cell disassembly (executioners or effectors, such as caspases 3, 6, and 7) and in initiating this disassembly (initiators, such as caspases 8 and 9) in response to proapoptotic signals [1, 2]. Two central pathways, intrinsic and extrinsic, are involved in the process of caspase activation and apoptosis in various cell systems that converge on the activation of the downstream executioner caspases [3–5]. The intrinsic pathway for apoptosis involves the release of cytochrome c from mitochondria into the cytosol, where it binds to apoptotic protease activating factor 1 (Apaf 1), resulting in the activation of the initiator caspase 9 and the subsequent proteolytic activation of the executioner caspases 3, 6, and 7. Members of the BCL2 family of proteins play a major role in governing this mitochondria-dependent apoptotic pathway, with proteins such as BAX functioning as inducers of apoptosis and proteins such as BCL2 as suppressors of cell death [6].

The extrinsic pathway for apoptosis involves ligation of the death receptor (such as FAS) to its ligand, FASL. Binding of FASL 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 (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.

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 [7–9]. In earlier studies, using murine models of testicular hyperthermia, we have provided evidence for the involvement of the mitochondria-dependent pathway for caspase activation and apoptosis in heat-induced male germ cell death [10–12]. In the present study, we determine whether a pan caspase inhibitor could prevent or attenuate such heat-induced germ cell apoptosis in mice.

In light of the fact that minocycline, a second-generation tetracycline, effectively inhibits cytochrome c release and caspase activation [13, 14], we further evaluated the efficacy of this compound in preventing or attenuating such heat-induced germ cell apoptosis. Minocycline has been shown to have robust neuroprotective effects in rodent models of neurodegeneration, cerebral ischemia, and traumatic brain injury [13–16]. Questions remain. Is the protection of minocycline for nervous system only or does it also protect male germ cells? Finally, if so, what is the molecular basis of its protection in heat-induced germ cell death?


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Experimental Protocol

Seven- to eight-week-old male C57BL/6 mice 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. Groups of five mice pretreated with vehicle (DMSO) or Quinoline-Val-Asp (Ome)-CH2-O-Ph (Q-VD-OPH), obtained from Enzyme Systems Products-A Division of ICN Biomedicals (Livermore, CA) were exposed once to local testicular heating (43°C for 15 min) and killed 6 h later. The inhibitor (40 mg/kg body weight [BW]) or vehicle was administered intraperitoneally (i.p.) 1 h before local testicular heating. The rationale for using Q-VD-OPH is that it is a broad-spectrum inhibitor of caspases 1, 3, 8, and 9 with improved potency and reduced toxicity [17]. A dose of 20 mg/kg has been used most frequently. It is nontoxic in mice even when used in a very high dose (1000 mg/kg BW). Available data from extragonadal cell systems indicate that Q-VD-OPH, compared with the benchmark caspase inhibitors, is effective in preventing apoptosis mediated by the major apoptotic pathways [18]. The dosage of 40 mg/kg BW of Q-VD-OPH was chosen to constitute a minimum effective dose of this compound capable of providing maximum protection against heat-induced germ cell apoptosis. Additional groups of five mice received an i.p. injection of vehicle or Q-VD-OPH (40 mg/kg BW) and served as controls. Heating of the scrota was performed as described previously [8, 19].

To determine the therapeutic efficacy of minocycline treatment in preventing heat-induced germ cell apoptosis, groups of five adult (8 wk old) C57BL/6 mice pretreated with vehicle (saline) or minocycline (45 mg/kg BW) were exposed once to local testicular heating (43°C for 15 min) and killed 6 h later. The minocycline dose is based on results of a previous study, which showed that a single i.p. injection of minocycline (45 mg/kg BW) either immediately before or after hypoxic brain injury in neonatal rats provided nearly complete neuroprotection [20]. Minocycline hydrochloride powder (Sigma Chemicals) was prepared in normal saline. The vehicle or minocycline was administered i.p. 1 h before local testicular heating. Additional groups of five mice received an i.p. injection of vehicle or minocycline and served as controls.

Animal handling and experimentation were in accordance with the recommendation of the American Veterinary Medical Association and were approved by the Harbor-UCLA Medical Center and Los Angeles Biomedical Research 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 [21]. 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, stained with uranyl acetate and lead citrate, and examined with a Hitachi 600 electron microscope.

Assessment of Apoptosis

In situ detection of cells with DNA strand breaks was performed in glutaraldehyde-fixed, paraffin-embedded testicular sections by the terminal deoxynucleotidyl transferase (TdT)-mediated deoxy-UTP nick end-labeling (TUNEL) technique [7–12] using an ApopTag-peroxidase kit (Chemicon International, San Francisco, CA). Enumeration of the nonapoptotic Sertoli nuclei with distinct nucleoli and apoptotic germ cell population was carried out at stages XI–XII using an Olympus BH-2 microscope (New Hyde Park, NY) with a 100x oil immersion objective. For each mouse, at least 10 tubules were used. Stages were identified according to the criteria proposed by Russell et al. [22] for paraffin sections. The rate of germ cell apoptosis or apoptotic index (AI) was expressed as the number of apoptotic germ cells per 100 Sertoli cells [8–10, 19, 23].

Immunohistochemical and Immunofluorescence Analyses

Bouin-fixed, paraffin-embedded testicular sections were immunostained as described previously [10, 12, 19, 24]. 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 one drop of normal goat serum in 1 ml of PBS to suppress nonspecific binding of IgG and subsequently incubated with rabbit polyclonal caspase 9 (1:50; Cell Signaling Technology, Beverly, MA), which recognizes both full-length and the cleaved product of p 39 and p 37 subunits and caspase 3 antibody (1:1000; kindly provided by Dr. Annu Srinivasan, Idun Pharmaceuticals, San Diego, CA), which recognizes only the cleaved product of p 18 and p 12 subunits of active caspase 3 but not the inactive zymogen [25], for overnight at 4°C. 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 visualized with diaminobenzidine tetrahydrochloride (DAB) as per the manufacturer's instructions (rabbit Unitect ABC Immunohistochemistry Detection System; Calbiochem, 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. [22] for paraffin sections. Enumeration of the nonapoptotic Sertoli nuclei with distinct nucleoli and caspase 9- or 3-positive cells was carried out at stages XI–XII using an Olympus BH-2 microscope with a 100x oil immersion objective. For each mouse, at least 10 tubules were used. Cell counts were finally expressed as the number of caspase 9- or 3-positive cells per 100 Sertoli cells.

Subcellular Fractionation and Western Blotting

Cytosolic and mitochondrial fractions were prepared as a modification of the procedure described earlier [26]. 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). The purity of the cytosolic and mitochondrial fractions was assessed by Western blotting using antibodies to cytochrome c oxidase subunit IV (COX IV; 1:500; Molecular Probes, Inc., Eugene, OR) and porin 31HL (1:500, Calbiochem-Novabiochem Corp., San Diego, CA), as described by us before [10]. The absence of COX IV or porin in cytosolic extracts confirmed that the cytosolic preparations were free of mitochondrial contamination (data not shown).

Western blotting was performed using testicular lysates and subcellular fractions as described previously [10, 12, 24]. In brief, proteins were separated on a 4%–12% SDS-polyacrylamide gel with 2-(4-morpholino)-ethane-sulfonic acid or MOPS buffer purchased from Invitrogen (Carlsbad, CA) at 200 V. Gel was transferred on Immuno-blot PVDF Membrane (Bio-Rad) 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, then probed using a rabbit polyclonal cytochrome c (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA), smac/DIABLO (1: 5000; Calbiochem), caspase 3 (1:1000), and a mouse-specific poly (ADP) ribose polymerase (PARP; 1:1000; Cell Signaling Technology, Inc., Beverly, MA), which recognizes only the cleaved (89-kDa) PARP antibodies for 1 h at room temperature with constant shaking. Following three 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 Hyper film ECL. Band intensities were determined using Quantity One software from Bio-Rad.

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 Student-Newman-Keuls method test after one-way repeated-measures ANOVA. Linear regression analysis was performed to examine the relationship between reduction in the number of apoptotic germ cells and caspase 3- or 9-positive germ cells. Differences were considered significant if P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Q-VD-OPH Markedly Inhibits Caspase 9 and Caspase 3 Activation, PARP Cleavage, and Attenuates Heat-Induced Germ Cell Apoptosis

Given that caspase 9 and caspase 3 are activated during heat-induced germ cell apoptosis in both rats [10] and mice [12], we first examined whether Q-VD-OPH inhibits such caspase activation (Fig. 1). In vehicle-treated mice, immunostaining of caspase 9 in heat-susceptible germ cells other than a few spermatocytes late in meiosis were rarely detected at stages XI–XII (Fig. 1A). After induction of apoptosis by heat treatment, the initiator caspase 9 was activated in a large majority of heat-susceptible late spermatocytes (Fig. 1B). Mice pretreated with Q-VD-OPH had markedly fewer caspase 9-positive germ cells compared with the heat alone group (Fig. 1C). Morphometric analysis further confirmed the histological findings and revealed a significant (P < 0.05) decrease (52.5%) in the number of caspase 9-positive germ cells at stages XI–XII 6 h after heating when compared with heat alone (Fig. 1D). Q-VD-OPH markedly inhibited caspase 3 activation, as evidenced by immunoblotting (Fig. 2A) and immunocytochemical staining of testicular sections for active caspase 3 (Fig. 2, B–D). We also found almost a similar decrease (54.2%) in number of active caspase 3-positive germ cells at stages XI–XII 6 h after heating in mice pretreated with caspase inhibitor when compared with heat alone (Fig. 2E). We then examined the effect of Q-VD-OPH on poly-(ADP) ribose polymerase (PARP) cleavage, a downstream substrate of the effector caspases, by Western blotting. As shown in Figure 3, Q-VD-OPH almost fully prevented PARP cleavage.



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  		FIG. 1. Reduction in the number of caspase 9-positive germ cells after testicular hyperthermia in mice pretreated with Q-VD-OPH. A) A stage-XI tubule from a vehicle-treated mouse shows no caspase 9 immunoreactivity in germ cells. B) In contrast, the same stage tubule from a heat-treated mouse shows a marked increase in the number of caspase 9-positive germ cells 6 h after heat treatment. Mice pretreated with Q-VD-OPH before induction of hyperthermia exhibit a marked reduction in the number of caspase 9-positive germ cells 6 h after heat treatment (C) when compared with the heat-alone group (B). D) Quantitation of caspase 9-positive germ cells at late (XI–XII) stage tubules in heat-treated mice with or without Q-VD-OPH treatment. Note a significant reduction in the number of caspase 9-positive germ cells in Q-VD-OPH + heat-treated mice compared with heat alone. Bar = 40 µm A–C



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  		FIG. 2. Lack of caspase 3 activation and reduction in the number of caspase 3-positive germ cells after testicular hyperthermia in mice pretreated with Q-VD-OPH. A) Lack of caspase 3 activation, as detected by immunoblotting using a caspase 3 antibody that recognizes only the active caspase 3 but not the inactive zymogen in Q-VD-OPH-pretreated mice in response to heat exposure. B) A late-stage tubule from a vehicle-treated mouse shows a lack of caspase 3 immunoreactivity in germ cells. Stage-XII tubules from mice pretreated with Q-VD-OPH before induction of hyperthermia exhibit a marked reduction in the number of caspase 3-positive germ cells 6 h after heat treatment (D) when compared with the heat-alone group (C). E) Quantitation of caspase 3-positive germ cells at late (XI–XII) stage tubules in heat-treated mice with or without Q-VD-OPH treatment. Note a significant reduction in the number of caspase 3-positive germ cells in Q-VD-OPH + heat-treated mice compared with heat alone. Bar = 40 µm B–D



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  		FIG. 3. Q-VD-OPH treatment started 1 h before local testicular heating inhibits heat-induced PARP cleavage. Total testicular lysates from vehicle (DMSO), heat (H), and Q-VD-OPH + heat (Q-VD-OPH + H)-treated mice were analyzed by SDS-PAGE and immunoblotted with a mouse-specific rabbit polyclonal antibody, which specifically recognizes the cleaved PARP. The same blot was reprobed with an actin antibody and used as a control for equal loading. The gels are representative of two animals in each treatment group from one of three separate experiments

To evaluate the effects of caspase inhibition on heat-induced germ cell apoptosis, we analyzed the changes in the incidence of germ cell apoptosis (expressed as numbers per 100 Sertoli cells) at stages XI–XII in mice pretreated with or without Q-VD-OPH after heat stress. As summarized in Table 1, the apoptotic index was very low in controls (16.8 ± 3.1). No significant changes in the apoptotic index were noted between controls and mice treated with Q-VD-OPH without heat (8.6 ± 2.2). Compared with both controls, mild testicular hyperthermia resulted in a marked activation (277.3 ± 21.6) of germ cell apoptosis. Q-VD-OPH at this dose level significantly (P < 0.05) attenuated heat-induced germ cell apoptosis by 67.0%.


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  		TABLE 1. Q-VD-OPH and minocycline prevent heat-induced germ cell apoptosis in mice.a

Linear regression analysis of data further demonstrated a high degree of correlation between reduction in the number of apoptotic germ cells and attenuation of caspase 9- (R = 0.91; P < 0.001) or caspase 3- (R = 0.94; P < 0.001) positive germ cells after Q-VD-OPH treatment. A highly significant correlation (R = 0.83; P < 0.001) was also noted between reduction in the number of caspase 9- and caspase 3-positive germ cells after Q-VD-OPH treatment.

Rescued Germ Cells Exhibit Normal Ultrastructure

Given the observation that inhibition of caspases does not prevent apoptosis-associated changes in overall cellular morphology but does prevent PARP cleavage in chemotherapy-induced apoptosis of lymphoid cells [27], we performed electron microscopy to evaluate cellular morphology at stages XI–XII. As expected from our recent study [12], the occurrence of germ cell apoptosis, characterized by chromatin condensation and fragmentation, and cell shrinkage, involving the majority of late spermatocytes, was readily detected within 6 h of heating. Mice pretreated with Q-VD-OPH had fewer apoptotic germ cells at these stages. The overall morphology of the rescued germ cells in Q-VD-OPH pretreated mice after heat stress was essentially similar to that of untreated mice, showing no ultrastructural evidence of apoptosis (data not shown).

Q-VD-OPH Does Not Affect Cytosolic Translocation of Cytochrome c and DIABLO after Heat Treatment

To prove that the upstream events, such as the release of cytochrome c and DIABLO from mitochondria to cytosol during heat-induced male germ cell apoptosis, are not impaired by caspase inhibition, we further examined the effects of Q-VD-OPH on cytosolic translocation of these proteins. Testicular lysates were fractionated into cytosolic and mitochondrial fractions and analyzed by Western blotting. As shown in Figure 4, neither cytochrome c nor DIABLO was detected in cytosol from control testes. In contrast, cytosolic accumulation of both cytochrome c and DIABLO was clearly evident after heat treatment. Q-VD-OPH had no effect on heat-induced release of cytochrome c and DIABLO from mitochondria (Fig. 4).



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  		FIG. 4. Q-VD-OPH treatment started 1 h before local testicular heating does not affect the release of mitochondrial DIABLO and cytochrome c into the cytosol during heat-induced germ cell apoptosis. Representative Western blots of cytosolic (C) and mitochondrial (M) fractions of testicular lysates from vehicle (DMSO), heat (H), and Q-VD-OPH + heat (Q-VD-OPH + H)-treated animals show accumulation of DIABLO and cytochrome c in the cytosol in heat-treated mice with or without Q-VD-OPH treatment. No DIABLO or cytochrome c is detected in the cytosol from vehicle-treated testes. Data are representative of four animals at each time point from one of three separate experiments

Minocycline Confers Partial Resistance to Heat-Induced Apoptosis by Inhibiting Cytochrome c and DIABLO Release from Mitochondria and Caspase Activation

We next evaluated the efficacy of minocycline in preventing or attenuating such heat-induced germ cell apoptosis. As summarized in Table 1, the apoptotic index (expressed as numbers per 100 Sertoli cells) measured at stages XI–XII was very low in controls (15.4 ± 3.6). No significant changes in the apoptotic index were noted between control and minocycline-treated (13.6 ± 1.03) mice. In contrast, a massive increase (344.1 ± 59.9) in the number of apoptotic germ cells was readily detected 6 h after heat exposure. Minocycline significantly (P < 0.05) prevented such heat-induced germ cell apoptosis by 60.2%.

We next investigated the mechanism by which minocycline protects heat-induced germ cell apoptosis. Because of the involvement of the mitochondria-dependent apoptotic pathway in heat-induced male germ cell apoptosis [10, 12], we examined the effect of minocycline on cytochrome c and DIABLO release from mitochondria. As shown in Figure 5, cytosolic translocation of both cytochrome c and DIABLO was readily detected 6 h after heat treatment and minocycline effectively prevented such heat-induced release of cytochrome c and DIABLO from mitochondria. Because the release of cytochrome c and DIABLO plays an important role during intrinsic pathway signaling in heat-induced male germ cell death [10, 12], we further examined the effect of minocycline on PARP cleavage, a downstream substrate of the effector caspases, by Western blotting. As shown in Figure 6, minocycline also effectively prevented PARP cleavage.



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  		FIG. 5. Minocycline treatment started 1 h before local testicular heating inhibits the release of mitochondrial DIABLO and cytochrome c into the cytosol during heat-induced germ cell apoptosis. Representative Western blots of cytosolic (C) and mitochondrial (M) fractions of testicular lysates from minocycline (Mino), heat (H), and minocycline + heat (Mino + H)-treated animals show inhibition of DIABLO and cytochrome c release from mitochondria into the cytosol. No DIABLO or cytochrome c is detected in the cytosol from vehicle-treated testes. Data are representative of four animals at each time point from one of three separate experiments



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  		FIG. 6. Minocycline treatment started 1 h before local testicular heating inhibits heat-induced PARP cleavage. Testicular lysates from minocycline (Mino), heat (H), and minocycline + heat (Mino + H)-treated mice were analyzed by SDS-PAGE and immunoblotted with a mouse-specific rabbit polyclonal antibody, which specifically recognizes the cleaved PARP. The same blot was reprobed with an actin antibody and used as a control for equal loading. The gels are representative of two animals in each treatment group from one of three separate experiments


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Given that caspases are essential in the final execution of cell death, they were probably one of the first obvious therapeutic targets for modulating apoptosis in various disease processes. As a result, a variety of caspase inhibitors have been developed and used as valuable tools to study the mechanism of action of these enzymes and to define their role in apoptotic-modulating therapies [14, 28, 29]. In earlier studies using murine models of testicular hyperthermia, we have provided evidence for the involvement of the mitochondria-dependent apoptotic pathway leading to the activation of the initiator caspase 9 and the executioner caspase 3 in heat-induced germ cell death [10–12]. In the present study, using the mouse models of testicular hyperthermia, we determine whether Q-VD-OPH, a newly developed broad-spectrum caspase inhibitor could prevent or attenuate heat-induced male germ cell apoptosis. We find that Q-VD-OPH markedly diminished caspase 3 activation and significantly prevented heat-induced germ cell apoptosis.

Evidence exists that, in cell systems such as fibroblasts or lymphoid cells, caspase inhibitors will prevent several aspects of cell death, such as PARP cleavage, membrane blebbing, and some degree of chromatin condensation, but they do not inhibit ultimate cell death induced by disparate signals [27, 30]. Contrary to those earlier observations, we show that Q-VD-OPH prevents heat-induced male germ cell death as shown by biochemical assay and by essentially the normal ultrastructural appearance of germ cells in mice after heat stress. Such a cell type-specific effect of a caspase inhibitor is not unexpected. Earlier studies have provided evidence for a protective role of a pan caspase inhibitor or a selective inhibitor of caspase 3 in various cell types, including hepatocytes [31, 32], lung endothelial and epithelial cells [33], and neurons [34].

In a preliminary study, we previously showed that Q-VD-OPH at a dosage of 20 mg/kg BW, the most frequently used dose of this compound [17], effectively prevented such heat-induced germ cell apoptosis in mice by 55.5% (unpublished data). The dosage of 40 mg/kg BW of Q-VD-OPH was chosen to constitute a minimum effective dose of the inhibitor because, at higher concentrations, such agents could have inhibitory effects on other proteases not involved in apoptosis [35], capable of providing maximum protection against heat-induced germ cell apoptosis. No significant difference in the degree of protection against heat-induced germ cell apoptosis was noted between low (20 mg) and high (40 mg) doses of Q-VD-OPH. The reasons for our inability to achieve complete protection of germ cell apoptosis triggered by heat stress remain unknown. One obvious possibility is that the dose of Q-VD-OPH is insufficient. Consistent with this is the demonstration that Q-VD-OPH at this dose level caused the number of caspase 9-positive cells to decline to 52.5% and the number of caspase 3-positive cells to 54.2% of the values measured in the heat-treated group. The present findings of significant correlations between reduction in the number of apoptotic germ cells and reduction in the number of germ cells expressing caspase 9 or 3 are also in accord with this notion. Of note, Q-VD-OPH has been shown to inhibit recombinant caspases 1, 3, 8, and 9 [17]. Thus, it remains possible that other caspases such as 2, 11, and 12 are involved in heat-induced germ cell apoptosis. Indeed, several lines of evidence from cell systems other than spermatogenesis demonstrate the role of these caspases in apoptosis [36–39]. The possibility that a caspase-independent mechanism is involved in induced germ cell apoptosis can also not be excluded on the basis of the data presently available. Nevertheless, the present study clearly demonstrates a protective role of Q-VD-OPH in heat-induced testicular germ cell apoptosis. At present, we do not know how this compound crosses the blood-testis barrier. In this context, however, it is important to note that, compared with the benchmark caspase inhibitor Z-Val-Ala-Asp (Ome)-CH2F (Z-VAD-FMK), the Q-VD-OPH has significant differences in structural design. These changes include replacement of the carbobenzoxy blocking group (Z) with a quinoline derivative (Q), modification of the tripeptide sequence from VAD to VD, and replacement of the putatively toxic fluromethyl ketone (FMK) with the nontoxic 2,6-difluorophenoxy (OPH) group [17, 40]. The OPH group is primarily responsible for nontoxicity, increased potency, and increased membrane permeability. Thus, based on the structural design, it is conceivable that increased membrane permeability most likely makes Q-VD-OPH permeable through the blood-testis barrier.

In certain types of apoptosis involving extrinsic pathway signaling, proper activation of effector caspases depends on caspase 8-mediated cleavage of a proapoptotic Bcl-2 family member Bid and subsequent release of cytochrome c from mitochondria, which, in turn, results in caspase 9 activation via apoptosome formation [41, 42]. There have been studies in nongonadal cell systems indicating that the release of cytochrome c and DIABLO from mitochondria to cytosol is impaired by caspase inhibition [43, 44]. Accordingly, we examined the effects of Q-VD-OPH on cytosolic translocation of cytochrome c and DIABLO. We show that heat-induced release of cytochrome c and DIABLO from mitochondria into cytosol was not affected by caspase inhibition, suggesting that caspase activation is not required to fuel cytochrome c or DIABLO release, at least in our hyperthermia model. In this context, it is interesting to note that, in a recent study, we did not observe any appearance of the truncated Bid in either cytosolic or mitochondrial fraction in heat-treated testicular lysates in mice, suggesting that caspase 8-mediated cleavage of Bid is not responsible for the observed release of cytochrome c from mitochondria [12]. Data presented herein are in accord with our earlier observation that the release of mitochondrial cytochrome c and DIABLO is clearly upstream of caspase activation and apoptosis [12].

A growing body of evidence demonstrates that minocycline, a second-generation tetracycline, has a robust neuroprotective effect in rodent models of neurodegeneration, cerebral ischemia, and traumatic brain injury through blocking cytochrome c release and caspase activation [3, 14, 16]. Here we show that minocycline is also effective in protecting male germ cells from heat-induced apoptosis. However, unlike Q-VD-OPH, the protection offered by minocycline occurred at mitochondria, involving suppression of cytochrome c release and subsequent inhibition of caspase 9 and caspase 3. Several lines of evidence indicate that, 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 inhibitor of apoptosis proteins (IAPs) on caspase activation [4, 12, 45]. Interestingly, minocycline-mediated protection of germ cell death is associated with suppression of DIABLO release. Clearly, one implication of this observation is that minocycline can protect cells, not only by inhibiting caspase activation by suppressing cytochrome c release, but also by augmenting the inhibitory effect of IAPs on caspase activation by suppressing the DIABLO release.

In summary, the present study further emphasizes the terminal role of caspases in heat-induced germ cell apoptosis and suggests that inhibition of caspase activity may have a protective role in acute testicular injury associated with increased germ cell apoptosis.


   FOOTNOTES
 
1 Supported by NIH grants HD 39293 to A.P.S.H., HD 39293-02S1 to Y.V., and through the U*STAR (S.R., M.C.) GM 08683 program. Back

2 Correspondence: Amiya P. Sinha Hikim, Division of Endocrinology, Harbor-UCLA Medical Center and Los Angeles Biomedical Research Institute, Box 446, 1000 West Carson Street, Torrance, CA 90509. FAX: 310 533 0627; hikim{at}gcrc.rei.edu Back

Received: 22 July 2004.

First decision: 27 August 2004.

Accepted: 13 October 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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