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Regular Article |
a Departments of Urology and
b Legal Medicine, Yamaguchi University School of Medicine, Ube, Japan
ABSTRACT
Testicular torsion requires emergent release of the twisted spermatic cord. Ischemia/reperfusion (I/R) plays an important role in its pathogenesis, and recent data suggest that germ cells undergo apoptosis during I/R. In a model of torsion/detorsion (i.e., I/R) of the rat testis, involvement of calpain and caspase in necrotic and apoptotic cell death was examined. After 1 h of ischemia followed by 0, 0.5, 1, 6, or 24 h of reperfusion, the germ cells positively stained with in situ TUNEL, and DNA fragmentation, activation of caspase-3, and proteolysis of caspase substrates increased with time of reperfusion, demonstrating apoptosis. In addition, m-calpain activation and proteolysis of
-fodrin were increased during reperfusion, and its activation is thought to be involved in the necrosis. A calpain inhibitor, acety-leucyl-leucyl-norleucinal, inhibited the phenomena associated with apoptosis and necrosis induced by I/R, although a caspase inhibitor, Z-Val-Ala-Asp-fluoromethlyketone, only inhibited apoptotic changes. The inhibition of calpain but not caspase ameliorated the injury after 60 days of reperfusion following 1 h of ischemia. The calpain inhibitor injected just before reperfusion effectively suppressed -fodrin proteolysis, suggesting its usefulness in the treatment of testicular torsion.
apoptosis, testes
INTRODUCTION
Testicular torsion is a urological emergency referred to as "acute scrotum," because early diagnosis and surgical intervention determine the prognosis of spermatogenesis. Misdiagnosis and inappropriate treatment lead to male factor infertility [1]. The main pathophysiology of testicular torsion is ischemia/reperfusion (I/R) injury of the testis caused by the twisted spermatic cord and its release [1]. Many investigations, including our own previous report, focused on injury of the contralateral testis in relation to male factor infertility [2, 3]. Until recently, the ischemic injury of the ipsilateral twisted testis was called "necrotic," being followed by the loss of its endocrine and exocrine function [4]. Although necrotic cell death has been thought to be the predominant type of cell death after I/R of the testis, recent evidence has shown the involvement of apoptotic cell death [5]. Apoptosis is characterized by plasma membrane blebbing, chromatin condensation, and fragmentation of chromosomal DNA [6]. The central component of this machinery is a proteolytic system involving a family of proteases called caspases, all of which are synthesized as inactive proenzymes that can be activated on apoptotic stimulation and cleave poly (ADP-ribose) polymerase (PARP), lamin B, fodrin, and various substrates [7].
On I/R, the intracellular Ca2+ concentration increases, which activates two types of calcium-dependent neutral protease: µ-calpain, and m-calpain. These two proteases require micromolar and millimolar Ca2+, respectively, require for activation. The calcium-dependent neutral protease calpain is ubiquitously present in the cytosol of all mammalian cells and is activated by Ca2+ [8]. -Fodrin, a membrane-cytoskeletal protein that maintains the integrity of the cell membrane, is a typical substrate of calpain. We have shown the involvement of calpain-mediated proteolysis of -fodrin during the I/R injury occurring in the heart and brain as well as its cleavage, followed by cell necrosis, in each organ [9–11]. On the other hand, calpain has been shown to be involved in apoptosis under various conditions and in various cells [12–17]. However, the role of calpain in apoptosis and necrosis has yet to be determined in testicular torsion.
In this study, we focused on the role of calpain and caspase in apoptotic and necrotic cell death in testicular torsion of the rat. Among many caspases, we focused on the activation of caspase-3, which is the common pathway on apoptotic stimulation [7, 18]. We also examined whether calpain and/or caspase inhibitor could alleviate apoptosis and improve spermatogenesis after testicular torsion followed by 60 days recovery.
MATERIALS AND METHODS
Animal Surgical Procedure
Eight-week-old adult male Wistar rats (
190–224 g) were obtained and maintained on a 12L:12D cycle. Rats were anesthetized with sodium pentobarbital (50 mg/kg body weight [BW] i.p.). All animal experiments followed a protocol approved by the Ethics Committee on Animal Experiments in Yamaguchi University School of Medicine and were controlled by the Guidelines for Animal Experiments of the Committee. As described elsewhere [2], we opened the left scrotum and rotated the left spermatic cord clockwise by 1080° to minimize individual deviation of blood flow and then sutured the tunica albuginea to the scrotal skin after confirming congestion of the testis in a sterile manner. After 1 or 6 h, the torsion (i.e., ischemia) was relieved (i.e., reperfusion), and the testes were returned to the scrotum. All surgical procedures were performed at a constant temperature (22 ± 2°C).
Five rats were randomly assigned to one of the following groups: the acety-leucyl-leucyl-norleucinal group (ALLnaL; 1 mM in 1.0 ml of 1% dimethyl sulfoxide [DMSO]; Nacalai Tesque Co., Kyoto, Japan), the Z-Val-Ala-Asp-fluoromethlyketone group (Z-VAD; 1 mM in 1.0 ml of 1% DMSO; MBL Co., Nagoya, Japan,), the Z-Asp-Glu-Val-Asp-luoromethlyketone group (DEVD; 1 mM in 1.0 ml of 1% DMSO; MBL Co.), or the DMSO group (1.0 ml of 1% DMSO). These inhibitors were injected i.v. 10 min before the onset of ischemia. The rats underwent ischemia for either 1 or 6 h, followed by 0, 0.5, 1, 6, or 24 h of reperfusion in the absence or presence of the inhibitors (n = 5 in each group). The control rat underwent a sham operation with the left spermatic cord rotated 1080° and then immediately relieved. In another series, ALLnaL (1 mM in 1.0 ml of 1% DMSO), SJA6017 (N-[4-fluorophenylsulfonyl]-L-valyl-L-leucinal; 1 mM in 1.0 ml of 1% DMSO; a generous gift from Senju Pharmaceutical, Kobe, Japan), or DMSO (1.0 ml of 1% DMSO) was injected i.v. 10 min before the onset of reperfusion for 24 h following 1 h of ischemia.
We harvested the testes and stored the samples separately for each assay. To investigate the effect of these inhibitors on spermatogenesis, rats were assigned to one of five groups as follows: the sham-operated group (n = 5), the ALLnaL group (1 mM in 1.0 ml of 1% DMSO, injected 10 min before 1 h of ischemia with the testes harvested 60 days after the reperfusion, n = 9), the Z-VAD group (1 mM in 1.0 ml of 1% DMSO, injected in the same manner as previously described, n = 9), the DEVD group (1 mM in 1.0 ml of 1% DMSO, injected in the same manner as previously described, n = 9), or the DMSO group (1% DMSO, injected in the same manner as previously described, n = 5). After the rats were killed with an overdose treatment of sodium pentobarbital (25 mg per 100 g BW i.p.), the testes were removed and weighed, and each testis was dispersed with 0.1% collagenase for flow cytometric analysis as described elsewhere [2].
In Situ End-Labeling of Germ Cells
After 0, 0.5, 1, 6, and 24 h of reperfusion following 1 or 6 h of ischemia, the testes were harvested and divided longitudinally into three pieces. A part of the tissue was fixed in Bouin solution for 2 h. Four micrometer-thick, paraffin-embedded sections were mounted on silan-coated glass slides (Dako Japan, Kyoto, Japan), deparaffinized and hydrated to permeabilize the sections, and then either stained with hematoxylin-eosin (HE) or digested with 20 µg/ml of proteinase K (Nacalai Tesque, Kyoto, Japan) for 15 min at 37°C. Endogenous peroxidase was inactivated by 3% H2O2 for 5 min at room temperature. Apoptotic nuclei in tissue sections were identified with in situ TUNEL technique using an apoptosis in situ detection kit (Wako, Osaka, Japan.) according to the manufacturer's instructions. Fifty seminiferous tubules on circular cross-sections in each testis were evaluated in the five testes from each group. The number of TUNEL-positive nuclei per tubule was counted and expressed as the mean ± SEM for each group.
Analysis of DNA Fragmentation
The frozen testes were homogenized on ice and lysed with lysis buffer containing 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 100 mM NaCl, and 0.5 % SDS, and they were digested with 0.1 mg/ml of proteinase K at 55°C overnight. The genomic DNA was isolated with phenol and ethanol and dissolved in TE buffer (10 mM Tris and 1 mM EDTA). The DNA (10 µg) was electrophoresed on a 1.5% agarose gel with Hind digests of DNA as a molecular marker and then stained with ethidium bromide. The DNA was visualized with an ultraviolet transilluminator, and the content of fragmented DNA (<1000 base pairs) was quantified with an image analyzer (Densitograph AE-6900M; Atto Co., Tokyo, Japan). The data (mean ± SEM, n = 5 for each condition) are expressed in arbitrary units.
Immunohistochemical Study
A part of the tissue was immersed in O.C.T. compounds (Miles Inc., Elkhart, IN), rapidly frozen in liquid nitrogen, and stored at -80°C. Frozen sections of 6 µm were obtained using a cryostat, and the specimens were fixed on silan-coated glass slides with 4% neutralized formalin for 10 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS (0.3 M NaCl, 20 mM Tris-HCl [pH 7.8], and 0.05% Tween-20) for 10 min, treated with cold 3% H2O2 in distilled water for 5 min, biotin-blocked with the kit from Dako, and then blocked with 2% normal horse serum in PBS for 1 h. The sections were incubated with anti--fodrin antibody (1:500 dilution; Genex, Helsinki, Finland) overnight at 4°C and then immunostained by the avidin-biotin-peroxidase complex (ABC) method using an ABC kit (Dako).
SDS-PAGE and Immunoblotting
The frozen testes were homogenized in 10 volumes of sucrose-Tris-EGTA buffer, comprising 0.34 M sucrose, 20 mM Tris-HCl (pH 7.4), 1 mM EGTA, 5 mM NaN2, 10 mM ß-mercaptoethanol, 0.2 mM phenylmethylsulfonyl fluoride, 150 nM pepstatin A, and 50 M leupeptin with a homogenizer three times each for 30 sec at the maximum speed. Protein concentrations were determined according to the method described by Lowry et al. [19] using BSA as a standard. The SDS-PAGE and immunoblotting were performed by using antibody to -fodrin (1:2500 dilution; Genex), m, µ-calpain (1:2000 dilution, monoclonal antibody, specific for the 80-kDa proform of calpain, generous gift from Dr. M. Kunimatsu, Nagoya City University of Medicine, Nagoya, Japan), caspase-3, and PARP (1:2000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) according to the method described by Laemeli [20] and Towbin et al. [21], respectively, with modifications. Equal amounts of protein were applied to the gels (6.5% gels for -fodrin, 7.5% gels for m, µ-calpain and PARP, 12.5% gels for caspase-3) and then electrophoresed and blotted to nitrocellulose membranes (-fodrin) or polyvinylidene difluoride membranes (m, µ-calpain, caspase-3, and PARP). These membranes were blocked with 5% nonfat dried milk in Tris-buffered saline (TBS) containing 150 mM NaCl, 10 mM Tris-HCl (pH 7.4), and 0.05% Tween 20 for 1 h. The membranes were incubated with primary antibody in 1% BSA overnight at 4°C and then washed three times each for 10 min with TBS. Next, the membranes were reacted with secondary antibody (mouse immunoglobulin [Ig] G from Amersham Pharmacia Biotech, UK, for -fodrin; rabbit IgG from Amersham Pharmacia Biotech for calpains; and goat IgG from Santa Cruz Biotech for caspase-3 and PARP) at room temperature for 1 h. The antigens were visualized with an enhanced chemiluminescence Western blotting detection kit (Amersham Pharmacia Biotech). The native molecule or its proteolytic fragments were quantified using an image analyzer (Densitograph AE-6900M; Atto Co., Tokyo, Japan). The data (mean ± SEM, n = 5 for each group) are expressed in arbitrary units.
Flow Cytometry
The DNA histograms of the testicular cells were obtained by flow cytometry as reported elsewhere [2]. Briefly, the testis was dispersed with 0.1% collagenase (type Ia; Sigma, St. Louis, MO) at 37°C for 1 h, fixed in 70% ethanol, and stored at 4°C until the assay. The samples were treated with ribonuclease (type IIIa; Sigma) in Dulbecco PBS (-) (Nissui, Tokyo, Japan) at 37°C for 30 min and 0.5% pepsin A (Sigma) at 37°C for 15 min before being stained with 2.5 µg/ml of propidium iodide (Sigma) for flow cytometry (EPICS XL; Coulter Electronics, Inc., Tokyo, Japan). The relative proportions of haploid (N), diploid (2N), and tetraploid (4N) cells were calculated, and the percentage of haploid cells was used as an index of spermatogenesis [2].
Data Analysis
Data were expressed as mean ± SEM. The significance of differences was evaluated by Mann-Whitney U-test. A P value of less than 0.05 was considered to be statistically significant.
RESULTS
TUNEL Staining of Testes after I/R Injury
In the control testes (Fig. 1A), TUNEL-positive nuclei per seminiferous tubule numbered 0.2 ± 0.1 (Fig. 1C). The number of TUNEL-positive nuclei per seminiferous tubule increased significantly with time of reperfusion after 1 h (0.2 ± 0.1, 0.4 ± 0.2, 0.8 ± 0.2, 1.4 ± 0.3, 5.1 ± 0.7, and 19.2 ± 1.9 per seminiferous tubule, respectively; Fig. 1C) and 6 h (0.2 ± 0.1, 0.6 ± 0.2, 4.5 ± 0.6, 6.1 ± 0.8, 13.0 ± 1.3, and 22.7 ± 1.6 per seminiferous tubule, respectively; Fig. 1D) of ischemia, and the extent of increase was greater after 6 h of ischemia than that after 1 h of ischemia. The TUNEL-positive nuclei appeared mainly in germ cells in the seminiferous tubules (Fig. 1B). No significant increase was found in the number of TUNEL-positive nuclei after 48 h of reperfusion compared with that after 24 h (data not shown). The final numbers of TUNEL-positive nuclei after 24 h of reperfusion were not significantly different after 1 or 6 h of ischemia. Ischemia alone of either 1- or 6-h duration did not statistically increase the number of TUNEL-positive nuclei. There seemed to be no association between the TUNEL positivity, the germinal stages, and the kind of germ cells (data not shown).
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Agarose Gel Electrophoresis Showing DNA Fragmentation and Its Quantification
Less than 1000 base pair DNA fragments were evident at 24 h of reperfusion after 1 h of ischemia (Fig. 2A) and from 1 h of reperfusion after 6 h of ischemia (Fig. 2B). The densitometric analysis confirmed a significant increase in the DNA fragmentation with time of reperfusion after 1 h (Fig. 2C) and 6 h (Fig. 2D) of ischemia, which is consistent with the results of TUNEL staining (Fig. 1, C and D).
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Immunohistochemical Staining for -Fodrin
-Fodrin was distributed beneath the plasma membranes in the germ cells and basement membrane of seminiferous tubules in the control testis (Fig. 3A). After ischemia for 1 h and reperfusion for 6 h, the immunoreactivity for -fodrin in the plasma membrane, but not in the basement membrane, decreased (Fig. 3B) compared with that in the control testis.
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Time Course of Calpain Activation During I/R and Its Inhibition by Calpain Inhibitor
To examine calpain activation during I/R, we evaluated the 150- and 145-kDa fragments of -fodrin by Western blotting as reported in our previous studies [9–11]. The control testis continued to have a substantial amount of 150-kDa fragment being observed, suggesting a high basal level of -fodrin proteolysis in the testis. Figure 4A shows that the 150- and 145-kDa fragments of -fodrin increased significantly with time of reperfusion for 0.5, 6, and 24 h after 1 h of ischemia compared with the control (146.7% ± 13.4%, 213.9% ± 31.8%, and 323.8% ± 72.0%, respectively; Fig. 4A). m-Calpain was localized predominantly in the cytosolic fraction as an inactive proform (80 kDa) and underwent limited proteolysis with time of reperfusion following ischemia (37.3% ± 7.2% and 21.0% ± 5.6%, respectively; Fig. 4B). The activation of m-calpain, as demonstrated by the limited proteolysis, closely correlated with the cleavage of -fodrin. Preischemic infusion of a calpain inhibitor, ALLnaL, inhibited the proteolysis of -fodrin significantly after 6 and 24 h of reperfusion (137.1% ± 26.0% and 168.1% ± 30.0%, respectively, Fig. 4A) and m-calpain autolysis after 24 h of reperfusion following 1 h of ischemia (48.1% ± 7.6%; Fig. 4B). Preischemic ALLnaL treatment had no effect on the -fodrin and m-calpain proteolysis in the reperfused testes after 6 h of ischemia (data not shown).
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Effects of Calpain and Caspase Inhibitors on TUNEL Staining
To examine the role of caspase in I/R-induced apoptosis of the testis, we evaluated the inhibitory effect of the caspase inhibitor: broad-spectrum caspase inhibitor (z-VAD-fmk), and caspase-3-specific inhibitor (DEVD-fmk). We infused the same amount of the inhibitors (1 mM in 1.0 ml of 1% DMSO) that was reported to effectively inhibit these proteases among in vivo models in previous studies [22–25]. The number of TUNEL-positive nuclei of the germ cells after 24 h of reperfusion following 1 h (12.1 ± 1.6 versus 18.2 ± 1.9) but not 6 h (19.6 ± 1.5 versus 22.7 ± 1.6) of ischemia was significantly reduced by z-VAD-fmk compared to the group without the inhibitor after I/R (Fig. 5). In addition, ALLnaL significantly, but to a lesser extent, decreased the number of TUNEL-positive nuclei after 1 h of ischemia (13.5 ± 1.3 per seminiferous tubule), but DEVD-fmk was not effective after 1 h (17.5 ± 1.8) or 6 h of ischemia (23.9 ± 1.7) (Fig. 5).
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Effects of Calpain and Caspase Inhibitors on DNA Fragmentation
We also examined DNA fragmentation in the testis after 1 h (Fig. 6A) and 6 h (Fig. 6B) of ischemia followed by 24 h of reperfusion by densitometric analysis of DNA ladder. Consistent with the data from TUNEL staining, z-VAD-fmk inhibited DNA ladder formation the most significantly (41.2% of the no-inhibitor group; Fig. 6C), although ALLnaL also inhibited DNA fragmentation, significantly, but to a lesser extent (71.0% of the no-inhibitor group). However, DEVD-fmk was not effective. These inhibitors had no effect on DNA fragmentation after 24 h of reperfusion following 6 h of ischemia (Fig. 6C).
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Effects of Calpain and Caspase Inhibitors on the Substrates of Calpain and Caspase Family
To evaluate the mechanism of the contribution of calpain and caspase to apoptosis and necrosis after I/R of the testis, we examined the proteolysis of the substrates of the two proteases. Preischemic treatment with ALLnaL inhibited the cleavages of -fodrin (Figs. 4A and 7A) and m-calpain (Figs. 4B and 7B), but caspase inhibitors, z-VAD-fmk, and DEVD-fmk did not affect the proteolysis of -fodrin or m-calpain (Fig. 7, A and B). As for caspase-3, a broad-spectrum inhibitor, z-VAD-fmk significantly inhibited its proteolysis after reperfusion for 24 h following ischemia for 1 h compared with the nontreatment group (53.6 ± 7.9 versus 11.5 ± 3.8), whereas a caspase-3-specific inhibitor, DEVD-fmk, did not (Fig. 7C). In addition, z-VAD-fmk inhibited cleavage of PARP compared with the nontreatment group (73.4 ± 7.7 versus 18.8 ± 2.8; Fig. 7D), and a calpain inhibitor, ALLnaL, also significantly inhibited the proteolysis, but to a lesser extent (57.8 ± 5.4). However, DEVD-fmk had no effect (Fig. 7, C and D).
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Effects of Protease Inhibitors on Testicular Weight
We adopted both testicular weight and percentage of haploid cells as indices of spermatogenesis in our previous study [2]. In this study, after ischemia for 1 h followed by reperfusion for 60 days, the weight of testes significantly decreased compared with that of sham-operated testes (1.6 ± 0.1 g and 0.4 ± 0.1 g, respectively; Fig. 8). However, z-VAD-fmk, which most effectively inhibited apoptosis after 24 h of reperfusion (Figs. 5 and 6), had no effect on the testicular weight (0.4 ± 0.1 g). In contrast, the calpain inhibitor ALLnaL significantly inhibited the atrophy of the injured testis (0.8 ± 0.1 g; Fig. 8), but DEVD-fmk and DMSO had no effect on testicular weight.
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Effects of Protease Inhibitors on Percentage of Haploid Cells
We reported previously that flow cytometric determination of the percentage of haploid cells, including spermatozoon and spermatids, is useful in analyzing germ cell differentiation. The normal testis contains approximately 70% haploid cells [2]. The present study confirmed that haploid cells accounted for 67.8% ± 2.8% in the sham-operation group (Fig. 9, A and D), decreasing to 17.9% ± 2.5% after 1 h of torsion followed by 60 days of reperfusion (Fig. 9, B and D). Although z-VAD-fmk had no effect on the decrease in haploid cells (21.5% ± 2.5%), ALLnaL significantly increased haploid cells (32.1% ± 2.3%; Fig. 9, C and D), whereas DEVD-fmk and DMSO were not effective. Histological examination of HE staining from the testis both with (Fig. 9E) and without (Fig. 9F) preischemic treatment with ALLnaL revealed few infiltrating cells, such as neutrophils or macrophages, that would interfere with precise evaluation of spermatogenesis.
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Effects of Calpain Inhibitor Injection Before Reperfusion
The effect of ALLnaL on the inhibition of -fodrin proteolysis was less when it was given during the prereperfusion period than when it was injected before the preischemia period (223.5% ± 28.1% versus 138.2% ± 20.2%; Fig. 10). However, even after administration before reperfusion, both ALLnaL (223.5% ± 28.1%) and SJA6017 (213.9% ± 25.9%) significantly reduced the proteolysis of -fodrin after I/R (324.1% ± 32.8%).
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DISCUSSION
To our knowledge, this study is the first systematic demonstration of the role played by two types of proteases, calpain and caspase, in the pathogenesis of testicular torsion. In the two types of cell death, necrosis and apoptosis, calpains and caspases play important roles [26]. In the rat testicular torsion model, I/R induced both apoptosis and necrosis. A caspase inhibitor, z-VAD-fmk, inhibited the germ cell apoptosis after I/R, but it did not improve spermatogenesis after 60 days of recovery following the testicular torsion. However, a calpain inhibitor, ALLnaL, greatly improved spermatogenesis, whereas it inhibited proteolysis of -fodrin after I/R. Thus, calpain inhibition may open a therapeutic window against the disease.
The number of TUNEL-positive nuclei were few in control testis (Fig. 1, A and C), quite likely because of phagocytosis by Sertoli cells [27]. That I/R, but not ischemia per se, increased the number of TUNEL-positive nuclei in the germ cells in the seminiferous tubules (Fig. 1) is consistent with previous results reported by Turner et al. [5]. The number of TUNEL-positive nuclei in the testis was much higher than in that in the heart or brain [9–11], reflecting the higher proliferation rate of germ cells in the testes. The single-stranded DNA fragments increase during meiosis [28], which may increase the number of TUNEL-positive cells. However, we demonstrated fragmentation of the genomic DNA of the testes by agarose gel electrophoresis. After 1 or 6 h of ischemia, DNA laddering increased with the time of reperfusion, in correlation of the number of TUNEL-positive nuclei (Fig. 2). The high-molecular-weight DNA cleavage [29, 30] was not identified by conventional agarose gel electrophoresis under the present condition.
One line of evidence shows the activation of the caspase pathway during apoptosis in the testis after I/R, in that I/R induced proteolysis of caspase-3 and PARP (Fig. 7). A broad-spectrum caspase inhibitor, z-VAD-fmk, but not a caspase-3-specific inhibitor, DEVD-fmk, reduced the number of TUNEL-positive nuclei (Fig. 5), DNA fragmentation (Fig. 6), and proteolysis of PARP (Fig. 7). These data suggest that some yet-to-be-identified caspase other than caspase-3 is involved in the evolution of apoptosis during I/R.
A major membrane-cytoskeletal protein, -fodrin is a substrate for calpain and caspase during necrosis and apoptosis, respectively [26]. We showed previously that postischemic reperfusion, but not ischemia alone, induces proteolysis of -fodrin by calpain to generate the 150- and 145-kDa fragments, thereby disrupting the integrity of the membrane and causing necrosis in the heart and brain [9–11]. By contrast, Nath et al. [14] reported that caspase-3 proteolyzes -fodrin to generate a 120-kDa fragment in the cultured cells during apoptosis. In the present study, we showed that I/R, but not ischemia alone, proteolyzed -fodrin to generate 150- and 145-kDa, but not 120-kDa fragments, whereas ALLnaL, but not z-VAD-fmk, inhibited the -fodrin proteolysis. These data support the involvement of calpain, but not caspase, in the -fodrin proteolysis after I/R (Fig. 7). In addition, I/R reduced the immunostaining of -fodrin in the cell membrane and induced cell swelling and membrane disruption (Fig. 3). Thus, I/R likely induces membrane disruption through the -fodrin proteolysis in the germ cell by calpain and causes necrosis.
We showed that calpain is also involved in apoptotic germ cell death after I/R, as reported for various cells with various stimuli [12–17], on the basis of the observation that a calpain inhibitor, ALLnaL, reduced the number of TUNEL-positive nuclei (Fig. 5) and DNA fragmentation (Fig. 6) after 24 h of reperfusion following 1 h of ischemia. A more selective calpain inhibitor, SJA6017, was also effective (Fig. 10). That ALLnaL inhibited PARP proteolysis (Fig. 7D) and DNA fragmentation (Fig. 6) after reperfusion following 1 h of ischemia suggests that calpain lies upstream of the caspase pathway and is involved in the evolution of apoptosis during I/R.
Reperfusion after 1 or 6 h of ischemia increased TUNEL staining (Fig. 1) and DNA laddering (Fig. 2), whereas ischemia per se for 6 h (Figs. 1D and 2D) or even 24 h (data not shown) did not. In addition, z-VAD-fmk ameliorated the apoptotic changes induced by reperfusion for 1 h, but not for 6 h, of ischemia (Figs. 5 and 6). On the other hand, ALLnaL inhibited the apoptotic change after 1 h of ischemia followed by reperfusion. Ischemia for 6 h induced necrotic changes such as cell swelling, membrane disruption, and eosinophilic change of cytosol, but apoptotic changes such as nuclear condensation and fragmentation were induced only after reperfusion following ischemia (Figs. 1 and 2). Taken together, it is tempting to speculate that I/R induces calpain-mediated -fodrin proteolysis that causes membrane disruption and necrosis during the early phase, and then caspase-mediated proteolysis of PARP and other apoptotic changes during the later phase.
Calpain inhibition may be a hopeful treatment in combination with early release of the twisted testis. The calpain inhibitor ALLnaL, but not the caspase inhibitor z-VAD-fmk, improved spermatogenesis after 60 days of reperfusion following 1 h of ischemia, but z-VAD-fmk suppressed apoptosis after 24 h of reperfusion (Figs. 8 and 9). It seems better not to inhibit apoptosis of the germ cells, because genomic defects may be inherited from the injured cells, with the DNA defect evoked by I/R. In other words, apoptosis physiologically eliminates the genetic defects of the germ cells after I/R [31]. By contrast, this study disclosed the importance of controlling necrosis with calpain inhibitors. Although ALLnaL also inhibits proteasome [32], a more selective calpain inhibitor, SJA6017, also inhibited -fodrin proteolysis [33]. The effectiveness of ALLnaL administration just before the release (i.e., reperfusion) of the twisted testis was supported by the observation that ALLnaL given before reperfusion inhibited -fodrin proteolysis (Fig. 10). In a long-term follow-up study of patients with testicular torsion, 68% of "saved" testes underwent atrophy [34], whereas only 50% of the cases escaped permanent injury, even when the twisted testis was released within 4 h after the onset of symptoms [35]. Treatment with calpain inhibition is hopeful under these situations. Previously, superoxide dismutase, catalase, verapamil [36], and hyperbaric oxygen were shown to be effective in the treatment of experimental testicular torsion [37]. A calpain inhibitor would be more effective than these, however, because calpain is involved in the final step of cell injury during I/R. Based on the data obtained during the present study, we recommend an infusion of calpain inhibitor within 6 h of the onset of testicular torsion and before the detorsion procedure, if the inhibitor can be used in clinical applications.
In conclusion, calpain is involved in the pathogenesis of experimental testicular torsion in rats. Although I/R of the testis induces the germ cell apoptosis and necrosis through calpain-mediated proteolysis of -fodrin, the necrosis seems to be the primary cause of I/R injury in testicular torsion followed by surgical release. The infusion of calpain inhibitor after the onset of torsion but before its release opens a therapeutic window to the treatment of testicular torsion.
ACKNOWLEDGMENTS
We thank Dr. M. Kunimatsu for the generous gift of the antibodies to m, µ-calpains and Senju Pharmaceutical Co. for providing the newly synthesized calpain inhibitor SJA6017. We also thank Dr. K. Kamada (Department of Urology, Yamaguchi Rosai Hospital) for helpful advice regarding the surgical procedure and Drs. K. Harada and T. Aki (Department of Legal Medicine, Yamaguchi University School of Medicine) for technical advice.
FOOTNOTES
First decision: 18 February 2000.
1 Correspondence: Koji Shiraishi, Department of Urology, Yamaguchi University School of Medicine, Minami-Kogushi 1-1-1, Ube, Yamaguchi 755-8505, Japan. FAX: 81 836 22 2276; urol{at}po.cc.yamaguchi-u.ac.jp 
Accepted: July 5, 2000.
Received: December 31, 1999.
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, P < 0.05 between the two groups
