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Biology of Reproduction 63, 1465-1472 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular Article

Molecular Pathway of Germ Cell Apoptosis Following Ischemia/Reperfusion of the Rat Testis1

Jeffrey J. Lysiak2,a, Stephen D. Turnera, and Terry T. Turnera

a Departments of Urology and Cell Biology, b The University of Virginia Health Science Center, Charlottesville, Virginia 22908

ABSTRACT

The present study investigates the molecular apoptotic pathway in germ cells following acute ischemia of the rat testis. Rats were subjected to ischemia-inducing torsion and testes were harvested after reperfusion. Apoptotic cells were identified with an antibody to single-stranded DNA. Seminiferous tubule RNA was examined by RNase protection assay or by reverse transcriptase-polymerase chain reaction (RT-PCR) for the presence and regulation of apoptotic molecules. Proteins from seminiferous tubules were used for Western blot analysis of cytochrome c. Germ cell apoptosis was maximal at 24 h after repair of torsion. Germ cells in stages II–III of the seminiferous epithelium cycle were the predominant early responders. The RNase protection assays revealed that Bcl-XL was the prominent mRNA species. Caspases 1, 2, 3, and Bax mRNA were consistently upregulated; however, the time of upregulation after torsion was variable. The Bcl-XL and Bcl-XS mRNAs were less consistently upregulated and there was no evidence for upregulation of Fas or Bcl-2. Fas ligand (FasL) was not detected by RNase protection assay, but RT-PCR revealed a significant increase in FasL expression 4 h after the repair of torsion. Western blot analysis for cytochrome c release demonstrated a significant increase 4 h after the repair of torsion. Results suggest that germ cell apoptosis following ischemia/reperfusion of the rat testis is initiated through the mitochondria-associated molecule Bax as well as Fas-FasL interactions.

apoptosis, sperm, spermatogenesis

INTRODUCTION

Testicular torsion, or more specifically, torsion of the spermatic cord results in an impedence of blood flow to the testis that can render the testis ischemic. Testicular torsion is a medical emergency in humans [1] that usually requires surgical intervention to allow reperfusion of the affected testis. In the rat it has been demonstrated that a 720° torsion induces ischemia and that a 1 h duration of this torsion followed by repair of torsion results in the permanent loss of spermatogenesis [2]. It has been demonstrated that this loss of spermatogenesis is not due to the primary disruption of Leydig cells [3] or Sertoli cells [4, 5] nor to the permanent loss of blood flow [2]. Recently, it was shown that a 1-h 720° testicular torsion in the rat model results in germ cell-specific apoptosis as assessed by in situ TUNEL and by DNA laddering [6]. The increase in apoptosis was first noted 4 h after torsion repair. An increase in adhesion of polymorphonuclear (PMN) cells to subtunical venules also occurred at 4 h after repair of torsion, coinciding with an increase in reactive oxygen species (ROS) as indicated by lipid peroxidation. Finally, oxygen radical scavengers were infused into animals prior to the repair of torsion, and testicular weight and daily sperm production were examined 30 days after surgery. Results revealed that ROS scavengers provided significant rescue of testis function as assessed by daily sperm production and testicular weight [6]. These results suggest that testicular torsion and repair has the features of a classical ischemia-reperfusion (IR) injury, and ROS released from recruited PMNs is at least partially responsible for the germ cell-specific apoptosis observed after torsion repair.

Apoptotic cell death may follow one of at least two broad and convergent apoptotic pathways [7]. Upon ligation of death receptors, e.g., Fas or tumor necrosis factor receptors, caspase 8, an initiator caspase, is recruited and activated. In turn, caspase 8 activates downstream effector caspases that leads to cell death [8–10]. The other pathway involves the activation of caspase 9, another initiator caspase, which in turn also activates the cascade leading to cell death [11]. Activation of caspase 9 requires the release of cytochrome c from the mitochondria, which interacts with Apaf-1 [12–14]. Members of the Bcl-2 family of proteins are important regulators of this pathway [15–17]. Activation of either caspase 8 or 9 leads directly to the activation of caspase 3, which in turn will activate caspases 1 and 2 [7, 9]. Thus, the activation of caspases 1, 2, and 3 results from the stimulation of either apoptotic pathway and is a broad indicator of apoptotic cell death.

The death of cells following IR injury is of clinical importance in many pathological conditions including stroke [18], transplantation [19], and cardiac infarction [20]. Numerous studies have implicated Fas ligand (FasL) [21, 22] as well as ROS [23] in the induction of apoptosis following IR injury. The aim of the present study was to investigate the molecular mechanism of germ cell-specific apoptotic cell death in the rat testis following acute IR. Characterizing the apoptotic pathway in this tissue and condition may lead to new insights for therapies to protect spermatogenesis as well as to a better understanding of germ cell survival and death in both the normal and pathological testis.

MATERIALS AND METHODS

Animals

This work was conducted in accordance with the Guiding Principals of the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction. Male adult, Sprague Dawley rats (450–550 g) were maintained on a 12L:12D cycle with ad libitum food and water.

Experimental Testicular Torsion

Animals were anesthetized with an intraperitoneal injection of 50 mg/kg sodium pentobarbital and the testis was rotated as described by Turner et al. [6]. Briefly, the testis was exteriorized through a low midline laparotomy, the gubernaculum was divided, and the testis was freed from the epididymotesticular membrane. The testis was rotated 720° for 1 h, during which time it remained in the abdomen with a closed incision. At the appropriate time the incision was reopened, the testis was counter-rotated back to the natural position, the gubernaculum was rejoined, and the testis was reinserted into the scrotum via the inguinal canal. Animals remained anesthetized throughout the surgical procedure and the body temperature was maintained by placing the animal under a heat lamp. At the time of repair testes were examined and scored for apparent degree of ischemia and of reperfusion, respectively. Scoring of the testis was subjective, based on color of the testis during torsion and reperfusion, and was done intraoperatively only to ensure consistency of the treatment. Testes were collected at 1, 2, 4, and 24 h after the repair of torsion for use in various assays. Sham-operated animals were treated identically except that the testis was immediately counter-rotated after completion of the torsion maneuver.

Evaluation of Germ Cell Apoptosis

Germ cell apoptosis was examined immunohistochemically with the monoclonal antibody F7-26 (Apostain; Alexis Corporation, San Diego, CA) directed against single-stranded DNA (ssDNA) as well as by the TUNEL technique. At the specified time points after repair of torsion, testes were removed from the scrotum, rinsed in saline, cut into halves, immersed in Bouin fixative for 6 h, paraffin-embedded, and coronal cross sections of the entire testis were mounted on glass slides. This procedure presented approximately 400 seminiferous tubule profiles per testis cross section for examination. The Apostain technique was performed according to the manufacture's protocol. Briefly, sections were deparaffinized, rehydrated, rinsed in 5 mM MgCl2, in PBS, rinsed in dH2O, and incubated for 15 min in ice-cold 0.1 N HCl. Subsequently, sections were rinsed in dH2O and incubated for 5 min in 5 mM MgCl2, 0.2% Triton X-100, in PBS. The slides were then placed into 50-ml centrifuge tubes containing 30 ml of 5 mM MgCl2 in PBS, and the tubes were immersed in water, preheated to 99°C, for 5 min. After heating, the slides were immediately removed and transferred into ice-cold PBS for 10 min. Next, slides were immersed in 3% H2O2 to block endogenous peroxidases, immersed in 0.1% BSA, 1% nonfat dry milk to block nonspecific binding of antibody, rinsed in PBS, and incubated for 1 h with a 1:100 dilution of F7-26. Slides were then washed, incubated for 1 h with biotinylated rat anti-mouse antibody (Zymed, San Francisco, CA), and washed. The biotinylated secondary antibody was visualized with avidin-biotin-peroxidase complex (Elite ABC Kit; Vector Laboratories, Burlingame, CA) and diaminobenzidine (DAB; Sigma, St. Louis, MO) as the chromogen. Sections were counterstained with hematoxylin, dehydrated, and mounted.

The TUNEL technique was performed according to the manufacture's protocol (Trevigen, Gaithersburg, MD). Briefly, sections were treated with proteinase K, endogenous peroxidase activity was quenched with H2O2, and incubated for 1 h at 37°C with TdT, dUTP-biotin, dNTPs, and reaction buffer. The biotinylated dUTP was detected with avidin-biotin-peroxidase complex and visualized with DAB. Sections were lightly counterstained with methyl green dye.

The number of apoptotic cells was evaluated by counting the positively stained nuclei in 30 circular seminiferous tubule cross sections per testis section. Data were averaged for each testis and expressed as apoptotic cells per tubule cross section. Statistical evaluations were by ANOVA followed by Tukey's range test (P < 0.05) after evaluation of each data set criterion for homogeneity by Chauvenet's criterion [24].

To determine the stage(s) of the cycle of the seminiferous epithelium most sensitive to torsion-induced apoptosis, specific stage evaluation was performed according to the criteria of Russell et al. [25]. The proportion of each stage showing induced apoptosis was adjusted for the proportion of that stage in the normal testis.

RNase Protection Assays

The RNase protection assays were carried out using the Multi-Probe RNase Protection Assay System with rAPO-1 multiprobe template set (PharMingen, San Diego, CA) specific for rat-associated mRNA molecules. Apoptotic associated mRNA molecules probed for were Fas, FasL, Bcl-2, Bcl-XL, Bcl-XS, Bax, and caspases 1, 2, and 3. Phosphoglyceraldehyde dehydrogenase (GAPDH) and L32, two housekeeping genes, were included as load controls. At described times after the repair of torsion, seminiferous tubule RNA was collected using TRIzol reagent (Life Technologies, Grand Island, NY) as stated in the manufacturer's instructions (n = 7). The RNA was stored in RNase-free water at -70°C until use. For each time point after torsion, 20 µg of RNA was precipitated and resuspended in 8 µl of hybridization buffer. Riboprobes were synthesized using the In Vitro Transcription Kit (PharMingen). Briefly, riboprobes were synthesized and labeled from 1 µl of the rAPO-1 template set, 32P-UTP (New England Nuclear, Boston, MA), T7 RNA polymerase, transcription buffer, dithiothreitol, RNasin, and GACU pool at 37°C for 1 h. The reaction was terminated by the addition of 2 µl DNase (1 U/µl) and incubated at 37°C for 30 min. The labeled probes were phenol:chloroform extracted and washed in ice-cold 90% ethanol. The resultant pellet was air dried prior to solubilization in 50 µl of hybridization buffer. One-microliter samples were counted in a scintillation counter (Beckman, Fullerton, CA) in the absence of scintillation fluid, to obtain Cherenkov counts. The probe mixture was diluted to a concentration of 3.6 x 105 cpm/µl with hybridization buffer.

Two microliters of diluted probe were added to the RNA samples, overlayed with mineral oil, and placed in a heat block at 90°C. The temperature was slowly decreased to 56°C and samples were incubated for 16 h. The temperature was lowered to 37°C for 15 min and treated with RNase A for 45 min at 30°C followed by proteinase K for 5 min at 37°C. A phenol:chloroform extraction was performed, the RNA was precipitated, and the precipitate was washed in ethanol. The pellet was air dried and resuspended in 1x loading buffer. Sample radioactivity was determined in a scintillation counter and equal counts were loaded into the wells of a 5% acrylamide sequencing gel. A sample of labeled undigested-probes was also loaded onto the gel as a marker lane. The gel was run at 50 W constant power until the dye front migrated 30 cm, after which time the gel was dried on a vacuum gel drier at 80°C for 1 h and placed on a phoshorimager screen for subsquent PhosphorImager (Molecular Dynamics, Sunnyvale, CA) analysis.

Upregulation of mRNA was assessed after standardizing phosphoimagery data (PD; cpm/band) for each identified band to the load control for that lane of the separating gel. An mRNA was considered upregulated if its PD was greater than 20% of the respective value in the sham control testis. Small increases were considered important because only a minority of total cells in the testis are participating in apoptosis at any given time. To be considered consistently upregulated after torsion, an mRNA had to show increases in band density over controls in five out of the seven testes examined per group. To be considered less consistently upregulated, mRNA had to show increases in band density in three to four of the seven testes. The mRNA exhibiting increases in band density in less than three of seven testes were considered not to be upregulated consistently.

Relative-Quantitative Reverse Transcription-Polymerase Chain Reaction

To examine further mRNA expression levels of FasL following the repair of testicular torsion, reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on total RNA isolated from seminiferous tubules at 4 h after the repair of torsion or after sham operations. Four micrograms of seminiferous tubule RNA was reverse transcribed using a SuperScript Preamplification System (Life Technologies) according to manufacturer's instructions. The RT product was diluted 1:10 and PCR was performed with primers specific for rat FasL and primers and competimers for the 18S rRNA (Ambion, Austin, TX). Initial pilot experiments were performed to determine the ratio of competimers to primers for the 18S rRNA so the PCR product would be in the same linear range as that for FasL. The FasL primers were designed with the aid of Gene Runner (Hastings Software, Inc., Hastings, NY) and purchased from GIBCO BRL Custom Primers (Life Technologies). The FasL forward primer was 5'-GTCCTGTCCTTGACACTTCAGTCTCC-3' and the FasL reverse primer was 5'-TCAACTTCTTCTCCTCCATTAGCACC-3'. The PCR was performed in 35 cycles with an annealing temperature of 55°C and an elongation temperature of 68°C, with Platinum Taq DNA polymerase High Fidelity (Life Technologies) in a thermal cycler (Perkin Elmer, Foster City, CA) yielding an expected product of 750 base pairs (bp) for FasL and 488 bp for 18S rRNA. Gel images were subsquently captured using a GelPrint 2000i (Genomic Solutions, Ann Arbor, MI) and the reaction products analyzed using ImageQuant software (Molecular Dynamics Inc.).

Western Blot Analysis

Western blot analysis of cytochrome c released from the mitochondrial inner membrane was performed by isolating proteins from seminiferous tubules 4 h after the repair of torsion or after sham operations. Briefly, approximately 200 µg of seminiferous tubules were homogenized on ice with four strokes of a teflon pestle in a 1.5-ml eppendorf tube in the presence of Hepes buffer, 2 µg/ml aprotinin (Sigma), 100 µM leupeptin (Sigma), 10 µM E-64 (Sigma), and 1 mM PMSF (Sigma). Tubes were centrifuged at 4°C, 1000 x g for 2 min to remove large tissue fragments and then spun at 4°C at 128 000 x g for 30 min to pellet the mitochondria. The protein concentration in the supernatant was determined using the bicinchoninic acid assay kit (Sigma), and 100 µg of protein per lane was subjected to SDS-PAGE. The gel contents were electrotransferred to nitrocellulose membranes (Bio-Rad, Hercules, CA), blocked with 5% nonfat dried milk, 0.1% Tween 20 in PBS, incubated overnight at 4°C with monoclonal anti-cytochrome c antibody (PharMingen), washed, and incubated for 1 h at room temperature with peroxidase-conjugated horse anti-mouse antiserum and detected with enhanced chemiluminescence (SuperSignal West Pico; Pierce, Rockford, IL). To ensure equal loading of protein samples, membranes were simultaneouly incubated with monoclonal anti-tubulin antibody (Sigma). Densitometry and Image-Quant analysis were subsequently performed. Statistical analysis was by Student's t-test ({alpha} = 0.05).

RESULTS

Evaluation of Germ Cell Apoptosis

Tissue sections from testis from sham-operated animals stained by either the TUNEL technique or via Apostain revealed very few stained nuclei (Fig. 1, A and C). However, sections taken 24 h after the repair of torsion showed increased numbers of apoptotic germ cells detected by both TUNEL and Apostain (Fig. 1, B and D). Because similar results were obtained with each technique and corresponded well with previous data, the Apostain technique was used in subsequent experiments to document the induction of germ cell-specific apoptosis following IR of the rat testis. A trend toward increased apoptosis began at 4 h after torsion repair (Fig. 2) and reached statistical significance (P < 0.05) by 24 h after repair of torsion (Fig. 2).



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  		FIG. 1. Photomicrographs of sections from rat testis from sham-operated animals (A and C) or from animals 24 h after the repair of a 1-h 720° rotation of the testis (B and D). A, B) Sections stained with Apostain and counterstained with hematoxylin. C, D) Sections stained with the TUNEL technique and lightly counterstained with methyl green dye. Note the increase in apoptotic germ cells (arrows) in B and D. Magnification x219



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  		FIG. 2. Histogram of Apostain-positive germ cells from rat testis from sham-operated animals (n = 8) and those killed at 1 (n = 3), 2 (n = 3), 4 (n = 8), and 24 h (n = 6) after the repair of torsion. A significant increase in apoptotic germ cells has occurred by 24 h after the repair of torsion (mean ± SEM)

At 4 h after repair of torsion, stages II–III of the cycle of the seminiferous epithelium were most sensitive to induction of apoptosis (a 4.5-fold increase in apoptotic germ cells versus no increase in stages XII and XIII, for example). Twenty-four hours after torsion repair stages XII–XIII showed the largest increase in apoptotic nuclei compared to controls (a 2.6-fold increase in apoptotic germ cells in those stages versus a 1.8-fold increase at stages II–III, for example).

Presence of Apoptotic Molecules

The RNase protection assays were performed on total RNA isolated from the testis from sham-operated animals and from the spleen. Because the spleen contains numerous cell types at various stages of apoptosis, RNA isolated from the spleen was used as a positive control for the RNA probes in the RNase protection assay kit. Rat spleen RNA contains the mRNA species for all the apoptotic-specific probes present, indicating that all 10 probes hybridize specifically to their target mRNA molecules (Fig. 3). The RNA isolated from a control testis revealed the presence of Fas, Bcl-XL, Bcl-XS, caspases 1, 2, and 3, and Bax. The L32 and GAPDH are load controls. Messenger RNA for Bcl-2 was not detected in the testis RNA, and similarly, a specific hybridization product for FasL was not readily detectable. Messenger RNA for the anti-apoptotic molecule Bcl-XL was the most abundant apoptosis-associated mRNA detected in the rat testis (Fig. 3).



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  		FIG. 3. An RNase protection assay from total RNA isolated from rat spleen and a sham-operated testis. Total spleen RNA was used for a positive control to ensure that the 10 radiolabeled RNA probes hybridized to their specific target mRNA molecules

Torsion-Induced Modulation of Apoptotic-Associated mRNA Molecules

Figure 4 displays the results from a representative RNase protection assay performed on RNA isolated from the testes at 1, 4, and 24 h after the repair of torsion. In this particular assay all mRNA products detected 1 h after repair of torsion were similar to those from sham-operated animals (Fig. 4). Modest upregulation of caspases 1, 2, and 3, and Bcl-XS occurred by 4 h and Bax was increased by 24 h when standardized against load controls. Examination of all seven assays revealed that the proapoptotic molecules caspases 1, 2, and 3, and Bax were consistently upregulated after torsion, though the timing and extent of their upregulation varied. Bcl-XL and Bcl-XS were less consistently upregulated after torsion, and Fas, FasL, and Bcl-2 did not show evidence of upregulation (Table 1).



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  		FIG. 4. Representative results from RNase protection assays on total RNA isolated from testis from sham-operated animals or those sacrificed at 1, 4, and 24 h after the repair of torsion


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  		TABLE 1. Peak expression of apoptosis-associated mRNA molecules in the rat testis when upregulated after repair of 1 h, 720° torsion

A specific hybridization product for FasL was not readily observable in the RNase protection assays; thus, a further examination for the presence and regulation of FasL was made using relative-quantitative RT-PCR. Total RNA isolated from sham control testes and testes taken 4 h after the repair of torsion did reveal the presence of FasL mRNA. Relative-quantitative RT-PCR demonstrated a significant increase (P <= 0.01) in FasL 4 h after the repair of torsion (Fig. 5).



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  		FIG. 5. A) Representative results of relative-quantitative RT-PCR for rat FasL. Total RNA from testis from sham-operated (n = 5) and from 4 h after the repair of torsion (n = 7) were evaluated for changes in mRNA expression levels of FasL (750-bp PCR product). The 18S RNA (488-bp PCR product) was used as the load control standard. B) Histogram of data shows a significant upregulation of FasL (P <= 0.01) (mean ± SEM)

Mitochondrial Cytochrome c Release

Western blot analysis for cytochrome c on proteins isolated from testis 4 h after the repair of torsion revealed a significant increase (P < 0.01) in cytoplasmic cytochrome c compared to testes from sham-operated animals (Fig. 6).



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  		FIG. 6. A) Representative Western blot for the detection of cytochrome c release into the cytoplasm. Proteins were isolated from testis of sham-operated animals (n = 5) and from animals 4 h after the repair of torsion (n = 5). A specific cytochrome c reaction product at approximately 18 kDa is observed in the torsion lane. {alpha}-Tubulin was used as a load control. B) Histogram of data from cytochrome c Western blot analysis standardized for {alpha}-tubulin. A significant increase (P < 0.01) in cytochrome c release is observed in testis 4 h after the repair of torsion (mean ± SEM)

DISCUSSION

Acute testicular torsion and repair in the rat results in germ cell-specific apoptosis [6] with the retention of functional Leydig [3] and Sertoli cell [4, 5] populations. Germ cell apoptosis is stimulated contemporaneously with the margination and diapedesis of leukocytes and an increase in intratesticular oxidative damage [6]; thus, it has been hypothesized that the pathology seen after testicular torsion/torsion repair is the result of classical IR injury.

In the present study, germ cell-specific apoptosis was reassessed using both the TUNEL and Apostain techniques (Fig. 1). Both techniques showed an increase in the number of apoptotic cells after the repair of torsion, though the values obtained via TUNEL were consistently higher than those seen with Apostain (data not shown). This most likely reflects the fact that the TUNEL technique can also label necrotic cells [26], whereas Apostain is specific for apoptotic cells only [27, 28]. This is the first report we are aware of verifying the utility of the Apostain technique in nonsomatic cells.

The trend toward an increase in germ cell apoptosis 4 h after torsion repair (Fig. 2) supports the previous data from Turner et al. [6] who detected significant increases in germ cell apoptosis by 4 h after torsion repair. In both studies the much larger increases seen at 24 h after torsion repair were statistically significant (P < 0.05). In the present study staging of the cycle of the seminiferous epithelium in tubule cross sections revealed that stages II–III are the most sensitive stages to initial induction of apoptosis. This is in contrast to gonadotropin withdrawal that induces apoptosis first in stages VII–VIII then stages I–IV at later time points [29]. Thus, different cellular insults may selectively target cells of a specific stage of the seminiferous epithelium.

The rAPO-1 multiprobe RNase protection assays allowed for the identification and regulation of eight different apoptosis-associated mRNA molecules, of which Bcl-XL, Bcl-XS, Bax, Fas, and caspases 1, 2, and 3 are present in the rat testes (Fig. 3). Previous studies have noted the presence of Bcl-XL [30] and Bax [31] in the rat testis; however, to our knowledge this study is the first demonstration of Bcl-XS mRNA in the rat testes. Messenger RNA for Bcl-2 was not observed in the total RNA isolated from rat testes even though a specific reaction product was observed in RNA isolated from the spleen, used as a control. The presence of Bcl-2 was also not observed in the testes of mice [31, 32]. Recently, Woolveridge et al. [30] have reported the presence of Bcl-2 protein by Western blot analysis in extracts from rat testes. This discrepancy in Bcl-2 expression could be explained if the mRNA for Bcl-2 has a very short half life; however, these exact same procedures did reveal Bcl-2 mRNA in the spleen. It is also possible that the antibody used by Woolveridge et al. [30] cross-reacted with other members of the Bcl-2 family. The Bcl-XL mRNA was among those not consistently upregulated after the repair of torsion. The fact that Bcl-XL was upregulated in some cases is not unexpected. The expression of Bcl-XL may represent an attempt of some cells to overcome the apoptotic insult.

Recent studies have examined the induction of apoptosis in germ cells following chemical insult [33], androgen withdrawal [30, 34], or cryptorchidism [35]. Rats treated with mono-(2-ethylhexyl)phthalate (MEHP) were found to have increased germ cell apoptosis associated with an increase in FasL [33], whereas, germ cells undergoing apoptosis following androgen withdrawal exhibited increased levels of Bax and Bcl-2 [30]. Thus, different cellular insults to the testis may trigger more than one possible apoptotic pathway. The importance of apoptotic molecules in spermatogenesis has also been documented in gene knockout mice [31, 36] or in mice that overexpress an apoptosis-associated gene [37, 38]. Male mice that have either proapoptotic Bax [31] or antiapoptotic Bcl-W [36] knockout deletions are infertile due to abnormal spermatogenesis. Other transgenic mice overexpressing human Bcl-2 also exhibit abnormal spermatogenesis and are subfertile [37] or sterile [38].

The RNase protection assays demonstrated that IR injury of the testis results in consistently increased mRNA expression levels of Bax and caspases 1, 2, and 3 between 2 to 4 h after the repair of torsion. These early time points were examined based on previous data showing a significant increase in rat germ cell apoptosis 4 h after the repair of torsion [6]; thus, critical molecular events would be engaged by this time. Increased expression of the proapoptotic molecule Bax suggests that torsion may induce an apoptotic pathway initiating at the mitochondria. Indeed, the release of cytochrome c from the mitochondria is also observed contemporaneously with increases in Bax mRNA at 4 h after the repair of torsion (Fig. 6). Cytochrome c may also be released from the mitochondria at some time point after stimulation of Fas [7].

The increased mRNA expression levels of Bax and caspases 1, 2, and 3 were modest (1.2–2-fold sham levels) throughout the study. This is not unexpected because total seminiferous tubule RNA was isolated and the number of apoptotic germ cells at any given time is only a small fraction (~1%; data not shown) of the total cells in the tubules. Also, caspases exist as proenzymes that become activated upon apoptotic stimuli; therefore, cells may initiate apoptosis with endogenous procaspases and without a requirement for large increases in caspase mRNA expression.

The FasL mRNA was not detected in any of the RNase protection assays performed in this study. However, RT-PCR, a more sensitive method for examining mRNA expression, did detect a FasL product (Fig. 5). Expression of FasL mRNA was significantly upregulated 4 h after reperfusion, a time by which upregulation of other molecules as detected by the RNase protection assays was also observed.

Interestingly, Richburg et al. [39] recently reported that gld mice that lack a functional Fas-signalling pathway actually have a small increase in the spontaneous incidence of germ cell apoptosis and are as sensitive to radiation-induced germ cell apoptosis as wild-type mice. Upon exposure to the Sertoli cell toxicant, MEHP, however, gld mice did show a reduced testicular apoptotic response compared to wild-type animals [39]. These results indicate that Fas signalling is important in some pathways for murine germ cell apoptosis but not in others.

The present study in the rat confirms and emphasizes these findings by Richburg et al. [39]. Rat germ cell apoptosis following torsion and torsion repair is likely initiated through two separate apoptotic pathways. One pathway involves Bax and its ability to cause cytochrome c release. This activates caspase 9 and the subsequent caspase cascade, leading to DNA degradation. The second pathway involves FasL binding to Fas, its cell surface receptor, and the initiation of the apoptosis cascade via caspase 8.

Testicular torsion followed by torsion repair causes all the hallmarks of classical reperfusion injury [6]. Apoptotic cell death following IR has been observed in several pathological conditions [18–20]. Reactive oxygen species are thought to play a key role in this apoptotic cell death because ROS increases in IR injury [23] and ROS can impair mitochondrial function directly and stimulate apoptosis [40, 41].

An IR injury resembles an inflammatory response in that it results in the activation of endothelial cells and subsequent recruitment of leukocytes to the affected site [42, 43]. Thus, the elevated ROS levels commonly detected in IR injury may be the result of the invading leukocytes. Activated leukocytes also have the ability to induce apoptosis by the secretion of FasL [44, 45]. In a previous study from this laboratory [6], torsion of the testis followed by torsion repair led to an increase in leukocyte margination and diapedesis in subtunical venules and an elevation in testicular ROS. Both of these parameters were positively correlated with the induction of germ cell apoptosis. Additionally, ROS scavengers partially restored testicular daily sperm production and testis weight. Thus, in the present study increased mRNA levels of the apoptotic stimulating molecules, Bax and FasL, could be the result of leukocyte diapedesis following IR injury of the rat testes. The secretion of FasL by leukocytes could directly stimulate apoptosis in germ cells expressing Fas. The generation of ROS, on the other hand, may increase Bax levels and stimulate a mitochondrial-driven apoptotic pathway indiscriminately.

ACKNOWLEDGMENTS

The authors thank the Molecular Biology Core and the Cell Science Core of the Center for Research in Reproduction at the University of Virginia. We also gratefully acknowledge Dr. R. John Lye and Jennifer Kirby for helpful advise on semiquantitative RT-PCR.

FOOTNOTES

First decision: 28 March 2000.

1 Supported by NIH R01-DK-53072. Back

2 Correspondence: Jeffrey J. Lysiak, Department of Urology, Box 422, University of Virginia Health Science Center, Charlottesville, VA 22908. FAX: 804 924 8311; jl6n{at}virginia.edu Back

Accepted: June 21, 2000.

Received: February 21, 2000.

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