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Research Article |
Departments of Urology3
and Cell Biology,4 University of Virginia Health Science System, Charlottesville, Virginia 22908
ABSTRACT
Early in postnatal life the first phase of spermatogenesis is accompanied by an initial wave of germ cell apoptosis. This wave of germ cell death is thought to reflect an adjustment of germ cell numbers that can be adequately maintained by Sertoli cells. Caspase 2 is an initiator caspase whose activation has been found to stimulate apoptosis through the mitochondria. The present study investigates if germ cell apoptosis during the first phase of spermatogenesis involves activation of caspase 2. Germ cell apoptosis was found to peak at Postnatal Days (pnds) 15 and 16 in male C57BL/6 mice. Western blot analysis revealed that caspase 2 also increased in the testes at pnd 16. Immunolocalization of total caspase 2 showed staining of germ cells in the periphery of the seminiferous tubules as well as germ cells more centrally located in an area where apoptotic germ cells were observed. Cytoplasmic as well as nuclear staining was observed. Western blot analysis of cytoplasmic and nuclear proteins from pnd 16 testis revealed pro-caspase 2 in both fractions. Further Western blot analysis for caspase 2 detected an increase in the activation of caspase 2 at pnd 16 in proteins isolated from the cytoplasm but not from the nucleus. Proteins isolated from mitochondria from pnd 16 testes revealed an increase in pro-caspase 2 as well as activated caspase 2 corresponding with an increase in cytochrome c in cytoplasmic fractions. Injection of the caspase 2-specific inhibitor z-VDVAD-fmk directly into the testis significantly reduced the observed germ cell apoptosis at pnds 15 and 16. These results suggest that caspase 2 is present in germ cells in the murine testis in early postnatal life and increases in expression in correspondence to the initial wave of germ cell apoptosis. Caspase 2 also localizes to mitochondria, where it is correlated with a release of cytochrome c and germ cell apoptosis. Blockade of caspase 2 activation reduced the number of apoptotic germ cells in the initial wave of germ cell apoptosis, indicating that caspase 2 plays an important role upstream of the mitochondria in germ cell apoptosis during the first phase of spermatogenesis.
apoptosis, gamete biology, spermatogenesis, testis
INTRODUCTION
Spermatogenesis in mammals is a dynamic process consisting of a series of highly ordered differentiation steps with each step being dependent on the previous one. Spermatogonia proliferate via mitosis and give rise to spermatocytes. Spermatocytes undergo meiotic divisions to give rise to spermatids that differentiate and become spermatozoa, the functional male gamete. This process is reliant on many factors, but perhaps the most important is the intimate relationship between the germ cells and Sertoli cells. Germ cells and Sertoli cells make up the seminiferous epithelium of the seminiferous tubules. Postnatal development of the seminiferous epithelium is fundamental to adult spermatogenesis and thus male fertility. Early in postnatal life the first phase of spermatogenesis is accompanied by an initial wave of germ cell apoptosis in both the mouse [1–3] and rat seminiferous epithelium [4]. This transient increase in germ cell apoptosis is thought to reflect the adjustment of germ cell numbers to those that can be adequately maintained by Sertoli cells for normal spermatogenesis [5].
Specific Bcl2 family members have been implicated in this initial wave of germ cell death during development of the seminiferous epithelium. Recent studies by Jahnukainen et al. [4] in the rat testis have reported an increase in the expression of BAX and cleaved caspase 3 at Postnatal Day (pnd) 18, a time when peak germ cell apoptosis was observed. BAX was detected in pachytene spermatocytes, the same cells that are undergoing apoptosis, from pnds 18 to 26, and it was suggested that BAX regulates apoptosis in midpachytene spermatocytes during the first phase of spermatogenesis [4]. Previous studies employing BAX-deficient mice have demonstrated that the absence of BAX leads to prolonged survival of premeiotic germ cells and a lack of the first wave of germ cell apoptosis [6]. This premeiotic survival, however, ultimately leads to cell death, possibly because of the subsequent Sertoli cell overload [2, 6]. Thus, a delicate balance exists between germ cells and Sertoli cells. In fact, there is evidence that Sertoli cells can support only so many germ cells [7].
Caspases are a family of cysteine proteases that are involved in apoptotic cell death. Caspases can be broadly grouped into two main groups: initiator caspases and effector caspases [8]. Previous findings suggest that caspase 2 is an initiator caspase that lies upstream of the mitochondrial pathway to apoptosis [9–12]. Studies by Lassus et al. [9] have demonstrated that caspase 2 is required for the translocation of the proapoptotic protein BAX from the cytosol to the mitochondria and the subsequent release of cytochrome c and DIABLO (second mitochondria-derived activator of caspases) from the mitochondria [9]. Other studies report that caspase 2 can cleave BID, another proapoptotic protein that can associate with the mitochondria and facilitate the release of cyotchrome c [10]. An even more upstream role for caspase 2 has also been described whereby caspase 2 directly induces the release of cytochrome c and apoptosis-inducing factor (PDCD8) from the mitochondria [11]. Caspase 2 has also been reported in the nucleus, where it can signal the mitochondrial release of cytochrome c and start the apoptotic process [12]. Thus, these recent studies point to a pivotal role of caspase 2 in initiating an intrinsic pathway to apoptosis. What triggers the activation of caspase 2 has not been established; however, it is thought that cellular stress is one such trigger [9].
Caspase 2 mRNA expression has been detected in the testis [13]; however, its specific cellular localization in the testis has not been described. Caspase 2-deficient mice are viable, but females display excess numbers of germ cells in the ovaries, and the oocytes are resistant to apoptotic cell death [14]. It remains to be determined whether germ cells in the testes of male caspase 2-deficient mice display a similar phenotype. The present study examines whether caspase 2 is involved in the first wave of germ cell apoptosis in the developing seminiferous epithelium.
MATERIALS AND METHODS
Reagents and Antibodies
A protease inhibitor cocktail was obtained from Sigma (St. Louis, MO). Caspase 2-specific antibody (Ab) was purchased from Cell Signaling Technology (Beverly, MA). The germ cell-specific Ab TRA 98 was a kind gift from Dr. B. Maier, Department of Neuroscience, University of Virginia (originally obtained from Dr. Y. Nishimune, Osaka University, Japan). Anti-ß-actin Ab was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The caspase 2-specific inhibitor benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethyl ketone (z-VAVAD-fmk; molecular weight 695 Da) was obtained from Enzyme Systems Products (Dublin, CA).
Detection of Apoptotic Cells
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 C57BL/6 mice were killed at specific days postnatal, and testes were removed, fixed in Bouin fixative, paraffin embedded, and sectioned. Apoptotic cells were detected immunohistochemically with the monoclonal Ab F7–26 (Apostain; Alexis Corporation, San Diego, CA) directed against single-stranded DNA as previously employed in our lab [15, 16]. 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 50% formamide and the tubes immersed in water, preheated to 56°C, for 20 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, rinsed in PBS, and incubated overnight at 4°C with a 1:100 dilution of F7–26. The next day, slides were washed, incubated for 1 h with biotinylated rat anti-mouse antibody (Zymed), and washed. The biotinylated secondary antibody was visualized with avidin-biotin-peroxidase complex (Elite ABC Kit; Vector Laboratories, Burlingame, CA) and DAB (Sigma) as the chromogen. Sections were counterstained with hematoxylin, dehydrated, and mounted. The number of apoptotic cells was evaluated by counting the positively stained nuclei in 30 random, circular seminiferous tubule cross sections per testis section. Data were averaged for each testis and expressed as apoptotic cells per tubule cross section.
Western Blot Analysis
Proteins were isolated from C57BL/6 mice testis from pnds 10 to 22, n = 5 for each. Proteins were extracted on ice with 0.1% Triton X-100, 0.25% sodium deoxycholate, 50 mM Tris-HCl, 150 mM NaCl, 1 mM EGTA, protease inhibitors, and 1 mM phenylmethylsulfonyl fluoride (extraction buffer; chemicals from Sigma). The protein suspension was vortexed, incubated on ice for 15 min, and centrifuged at 14 000 x g for 15 min at 4°C, and the supernatant was collected. The protein concentration in the supernatant was determined using the bicinchoninic assay (BCA) kit (Pierce, Rockford, IL), and 50 µg of protein per lane were subjected to SDS-PAGE. Depending on the experiment, either 8% or 10% acrylamide gels were used. The gel contents were electrotransferred to nitrocellulose membranes (Bio-Rad, Hercules, CA), blocked with 5% nonfat dried milk, 0.1% Tween 20 in PBS, and incubated with anti-caspase 2 Ab overnight at 4°C. Blots were then washed and incubated with peroxidase-conjugated secondary Ab, and immunocomplexes were detected with enhanced chemiluminescence (SuperSignal West Pico; Pierce). Densitometry and ImageQuant (Molecular Dynamics, Sunnyvale, CA) analysis of the resultant bands were subsequently performed.
Immunohistochemistry
Testes from mice from pnds 10 to 22 (n = 3–4 each) were fixed in 4% paraformaldehyde and paraffin embedded, and 5-µm-thick sections were cut. Sections were deparaffinized, rehydrated, incubated in 3% H2O2 in methanol to block endogenous peroxidases, and incubated in 1% nonfat dried milk in PBS to block nonspecific binding. Sections were then incubated overnight at 4°C with anti-caspase-2 Ab. The pnd 16 sections were also incubated overnight with TRA 98. The next day, sections were washed in PBS, incubated for 1 h at room temperature with biotinylated horse anti-mouse secondary Ab, and subsequently washed. The immunocomplexes were visualized with avidin-biotin-peroxidase complex (Elite ABC Kit; Vector) with diaminobenzidene (DAB) as the chromagen. All slides were lightly counterstained with hematoxylin. Specificity controls included incubation without primary antibody.
Isolation of Nuclear and Cytoplasmic Proteins
Testes were retrieved from mice and rinsed with PBS, and the tunica was removed. The resultant seminiferous tubules were added to a buffer consisting of 10 mM Tris-HCl pH 7.5, 1.5 mM MgCl2, and 10 mM KCl and homogenized. The solution was incubated on ice for 10 min and then centrifuged at 3500 x g for 5 min at 4°C. The resultant supernatant containing the cytoplasmic proteins was saved in a new tube, and the pellet containing the nuclear proteins was resuspended in 0.42 M KCl, 20 mM Tris-HCl pH 7.5, 1.5 mM MgCl2, and 20% glycerol and incubated at 4°C for 30 min with constant rotation. The solution was then centrifuged at 13 500 x g for 30 min at 4°C. The supernatant was dialyzed in 20 mM Tris-HCl pH 7.5, 0.1 M KCl, 0.2 mM EDTA, and 20% glycerol for 3 h at 4°C with one buffer change. The dialysate was centrifuged at 13 500 x g for 10 min at 4°C and the supernatant retrieved. Protein concentration in both the cytoplasmic and the nuclear preparations was determined by BCA (Pierce). To ensure specificity of the cytoplasmic and nuclear protein preparations, Western blot analysis for the cytoplasmic protein ß-actin was performed on all samples.
Assessment of Mitochondrial Cytochrome c Release
Mitochondria and cytoplasmic proteins from seminiferous tubules were isolated for Western blot analysis of cytochrome c. Briefly, testes were removed from mice at pnds 12, 14, 16, and 18, and the tunica was removed and discarded. The seminiferous tubules were homogenized on ice with a Teflon pestle in a 1.5-ml Eppendorf tube in the presence of Hepes buffer plus protease inhibitors (Sigma). Tubes were first centrifuged 1000 x g for 2 min at 4°C to remove large tissue fragments then again centrifuged at 128 000 x g for 30 min at 4°C to pellet the mitochondria. The supernatant containing cytoplasmic proteins was collected. The protein concentration of the supernatant was determined using the BCA kit (Pierce), and 100 µg of protein per lane were subjected to SDS-PAGE. Proteins were then electrotransferred to nitrocellulose, and Western blot analysis for cytochrome c was performed using a monoclonal Ab to cytochrome c (BD PharMingen, San Diego, CA). Western blot analysis for the cytoplasmic protein ß-actin was performed on all samples to assess the purity of the mitochondrial and cytoplasmic protein preparations.
Intratesticular Injections
On pnd 13, mice were anesthetized with an intraperitoneal injection of 0.01 mg/g sodium pentobarbital, and a midline incision was performed. The testes were exteriorized through a low midline laparotomy and the testes exposed. The specific caspase inhibitor z-DVDAD-fmk, 60 µM in DMSO, was infused via a glass micropuncture pipette (50-µm tip diameter) inserted through the tunica albugenia with the tip resting in the testicular interstitium. Approximately 5 µl of the drug were delivered. Interstitial fluid flow aided in distribution of the drug throughout the microinfused testis, and the infusion volume allowed simple replacement of the native interstitial fluid volume. Following drug delivery, the testes were returned to the scrotum, and the incision was closed. Control injections were made with vehicle, DMSO, alone. Mice were then killed at pnds 14, 15, 16, and 17 for the assessment of germ cell apoptosis.
Statistical Analysis
All statistical evaluations were either by ANOVA followed by the Tukey range test or by the Student t-test (P < 0.05) after evaluation of each data set by the Chauvenet criterion for homogeneity.
RESULTS
Initial Wave of Germ Cell Apoptosis
To determine if caspase 2 plays a role in the wave of germ cell apoptosis observed during the first round of spermatogenesis, we first had to determine the precise time of the wave of germ cell apoptosis in our mouse model, the C57BL/6 strain. Staining for apoptotic cells in testes from mice killed from pnds 10 to 22 revealed an increase in the number of apoptotic cells starting at pnd 14 and peaking at pnds 15, 16, and 17 (Fig. 1). After pnd 17, the incidence of apoptotic cells decreased until pnd 19, where it remained stable until the end of the time course at pnd 22. Examination of tissue sections not only revealed this pattern of germ cell apoptosis (Fig. 2) but also demonstrated that apoptotic cells were commonly not the most basal germ cell layer. This is consistent with those cells being spermatocytes.
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Localization and Activation of Caspase 2 During the Initial Wave of Germ Cell Apoptosis
Western blot analysis for caspase 2 on proteins isolated from testes from pnds 12 to 22 revealed an increase in pro-caspase 2 (48 kDa) at pnds 15 and 16 relative to pnd 12 (Fig. 3, A and B). As observed in the Western blot (Fig. 3B), a protein band slightly larger than 50 kDa is present; this may represent caspase 2 in complex with its binding protein, termed proapoptotic caspase adaptor protein (PACAP), which is highly expressed in the testis [17]. Immunolocalization of caspase 2 in testes from pnds 12 to 18 demonstrated that both spermatogonia and spermatocytes were stained for caspase 2 (Fig. 4). To aid in the confirmation that the caspase 2-positive cells are germ cells, sections were also stained with TRA 98, an antibody that is germ cell specific. As can be seen in Figure 4E, many germ cells are positive and in a radial pattern in the seminferous tubule. We cannot, however, rule out that some caspase 2-positive cells may be Sertoli cells. The incidence of caspase 2-positive cells also increased at pnds 15 and 16, but since the antibody used for these studies recognizes both the pro- and active forms of caspase 2, it was not possible to distinguish between cells that had the proenzyme and those that had the active enzyme. Western blot analysis of proteins isolated from nuclear and cytoplasmic preparations confirmed the presence of caspase 2 in the nuclei (Fig. 5).
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To determine if activation of caspase 2 occurs in the testis during this initial wave of germ cell apoptosis, Western blots were performed and examined for the lower-molecular-weight bands representing the processed or active enzyme [18]. Bands at approximately 34 and 18 kDa representing the active enzyme appear from pnds 14 to 18 (Fig. 3B). In nuclear and cytoplasmic protein preparations from pnds 12 to 18, only the cytoplasmic proteins of pnd 16 testes show the lower-molecular-weight bands indicating active caspase 2 (Fig. 5).
Caspase 2 Translocates to the Mitochondria
Isolation of mitochondrial proteins from testes from pnds 12 to 18 was performed to determine if caspase 2 translocates to the mitochondria (Fig. 6). Procaspase 2 increases in the cytoplasm at pnd 16 as previously shown; however, in the cytoplasmic fractions absent the mitochondrial proteins, a small amount of active enzyme was observed (Fig. 6A). In proteins isolated from mitochondrial fractions, procaspase 2, as well as the active enzyme, increases at pnd 16 (Fig. 6A). Corresponding with the detection of pro- and active caspase 2 in the mitochondria is an increase in cytochrome c detected in the cytoplasmic fraction of proteins (Fig. 6B).
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Inhibition of Caspase 2 Abrogates the Initial Wave of Germ Cell Apoptosis
To further identify the role of caspase 2 in this early phase of spermatogenesis, the specific caspase 2 inhibitor z-VDVAD-fmk was microinjected into the testis at pnd 13 and the incidence of apoptotic cells scored 1–4 days later (Fig. 7). The number of apoptotic germ cells was reduced by approximately 50% at pnds 15 and 16 after injection of the caspase 2 inhibitor (Fig. 8). Western blot analysis for caspase 2 performed on control and z-VAVAD-fmk-injected testes revealed a decrease in the appearance of caspase 2-activation intermediate products (Fig. 9).
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A reduction in germ cell apoptosis during the first phase of spermatogenesis has been associated with subsequent abnormal spermatogenesis in the long term [2, 6]; therefore, mice were injected with the caspase 2-specific inhibitor on pnd 13 and left to survive until 6 wk of age. Histological examination of testis sections revealed no difference between testes injected with inhibitor or vehicle only (data not shown).
DISCUSSION
In both the mouse [1–3] and the rat [4], the first phase of spermatogenesis is accompanied by a wave of germ cell apoptosis. This wave of apoptosis is thought to involve an adjustment of germ cells that can adequately be maintained by Sertoli cells [2, 5], and the proapoptotic protein BAX has been implicated in this response [2, 6]. Since recent data have now classified caspase 2 as an initiator caspase upstream of the mitochondria and since a potential association between BAX and caspase 2 has been described, the present study was undertaken to determine if caspase 2 is expressed in the testis and if it plays a role in the initial wave of germ cell apoptosis. Results show that caspase 2 is expressed by a subpopulation of murine germ cells in the testis and is increased in expression and activated at a time coinciding with the initial wave of germ cell apoptosis. Microinjection of a specific caspase 2 inhibitor abrogated the wave of germ cell apoptosis. To our knowledge this is the first reported role of caspase 2 in the murine testis and its involvement in germ cell apoptosis.
While results from other investigators using various strains of mice [1–3] or rats [4] have reported the first wave of germ cell apoptosis to occur between 2 and 3 wk of age, it was important to accurately determine the wave of germ cell apoptosis in C57BL/6 mice in our lab. Our results show a peak in germ cell apoptosis at pnds 15 and 16 (Fig. 1), results closely matching those of Kwon et al. [19], who demonstrated a significant increase in germ cell apoptosis at pnd 14 in the CBA strain of mice. Studies by Mori et al. [3] also observed an increase in germ cell apoptosis from pnds 8 to 22 in the ICR strain of mice. Using the rat model, Jahnukainen et al. [4] recently reported an increase in germ cell apoptosis as well as an increase in a number of apoptotic markers, such as active caspase 3, BAX, and cleaved PARP at pnd 18; thus, the results described here are within the window of rodent germ cell apoptosis reported by others during early postnatal life. As stated earlier, this initial wave of germ cell apoptosis is thought to establish the proper ratio of germ cells to Sertoli cells [2, 5]. In many species, Sertoli cells can support only a relatively fixed number of germ cells [7]. In fact, an increase in the ratio of germ cells to Sertoli cells has never been reported, and the only known way to increase the number of germ cells while still maintaining normal spermatogenesis is to increase the number of Sertoli cells [20, 21].
Caspase 2 was one of the earliest identified caspases, and only recently has its role in the induction of apoptotic cell death been elucidated. Caspase 2 has a long prodomain, which is characteristic of initiator caspases [9–12], and is highly expressed during embryonic development at periods of extensive cell death [13]. RT-PCR analysis of various mouse tissues for caspase 2 revealed the presence of caspase 2 mRNA in the testis [13]; however, it was not determined whether caspase 2 protein was present. To determine if caspase 2 protein is present in the murine testis and if it correlates with the initial wave of germ cell apoptosis, Western blot analysis of total testicular proteins from pnds 12 to 22 was performed. Results revealed an increase in expression of caspase 2 pro-enzyme that peaked at pnds 15 and 16 (Fig. 3). This peak in expression correlated in time with the observed peak of germ cell apoptosis, thus adding another piece of evidence that caspase 2 expression is correlated with periods of extensive cell death.
Two specific bands for caspase 2 were observed at approximately 48 kDa, suggesting the possible presence of another isoform of the protein. Caspase 2 was immunolocalized to germ cells, most likely spermatocytes and some spermatogonia (Fig. 4). The incidence of caspase 2-positive germ cells also increased at pnd 16 as also evident by Western blot analysis for it, but the antibody used for the immunolocalization recognizes all forms of caspase 2, and it was not possible to identify which germ cells had the active versus the pro-form of the enzyme. Within the germ cells expressing caspase 2, the enzyme may become activated only in a subpopulation of the cells, which would be destined to die. Another possibility is that caspase 2 is activated in all germ cells that express it, and it may be a very early marker for apoptotic cell death before detection of nicked DNA.
Germ cells that expressed caspase 2 appeared to be immunoreactive in both the cytoplasm and the nucleus, and Western blot analysis on cytoplasmic and nuclear extracts confirmed this localization (Figs. 4 and 5). Indeed, both Colussi et al. [22] and Paroni [12] have found caspase 2 localized to the nucleus of cells. Colussi et al. [22] demonstrated that the nuclear localization of caspase 2 is mediated via its prodomain. Paroni et al. [12] further demonstrated two nuclear localization signals in caspase 2 and showed that caspase 2 can trigger mitochondrial dysfunction without exiting the nucleus. This led these authors to suggest a nuclear/mitochondrial apoptotic pathway [12].
Definitive evidence of activation of caspase 2 requires the cleavage of the pro-enzyme into its active subunits of approximately 18 and 16 kDa. During this cleavage process, intermediate forms of caspase 2 may also be recognized at approximately 34–32 kDa [18]. To determine if this was occurring in the present study, Western blot analysis was performed on total testicular proteins. The results demonstrated the appearance of the cleaved caspase 2 products at pnds 15 and 16 (Fig. 3). These data demonstrate that procaspase 2 not only increases at pnds 15 and 16 in the murine testis but also becomes activated on those days. Further, the activation occurs in the cytoplasm, not in the nucleus (Fig. 5), and in the cytoplasm the activated caspase 2 is associated with the mitochondria (Fig. 6). This suggests that activation of caspase 2 occurs in the cytoplasm of germ cells, where it may lead to apoptotic cell death, and leaves open the interesting possibility that caspase 2 in the nucleus may have a unique role or require a different stimulus for activation.
Caspase 2 has previously been shown to cause mitochondrial dysfunction [9–11], but it remains unclear from the present studies whether pro-caspase 2 relocates to the mitochondria and becomes activated or whether both the pro- and the activated forms of caspase 2 can localize to the mitochondria. A direct correlation was observed between the presence of caspase 2 in the mitochondrial preparations and an increase in cytochrome c in the cytoplasmic protein preparations (Fig. 6), suggesting that caspase 2 causes the release of mitochondrial cytochrome c. Indeed, Guo et al. [11] have demonstrated that caspase 2 can directly cause the release of cytochrome c as well as PDCD8 (apoptosis-inducing factor) and DIABLO (second mitochondria-derived activator of caspases protein) from purified mitochondria.
To make a more direct determination of the role of caspase 2 in the initial wave of germ cell apoptosis, testes were injected on pnd 13 with the specific caspase 2 inhibitor, and apoptosis was examined at pnds 14, 15, 16, and 17. Injection of the caspase 2 inhibitor significantly reduced germ cell apoptosis observed at pnds 15 and 16 in the testis (Figs. 7 and 8) and the appearance of specific activation intermediates (Fig. 9). However, we must consider the possibility that the pnd 15 and 16 reductions in germ cell apoptosis are not directly due to caspase 2 inhibition. While it is possible that effects secondary to the apoptosis inhibition seen on pnd 14 could be in play by pnds 15 and 16, a rationale for such apoptosis-inhibiting effects is not at all evident. It is more likely that the prepubertal mouse testis with its low blood and lymphatic flow still retains effective doses of the inhibitor and that it is, in fact, the inhibitor that prevents caspase 2 activity and the subsequent apoptosis. Interestingly, injection of the caspase 2-specific inhibitor did not eliminate all germ cell apoptosis, only the wave of increased germ cell apoptosis observed at pnds 15 and 16. This suggests that the wave of germ cell apoptosis during the initial phase of spermatogenesis is caspase 2 dependent but that other apoptotic pathways are also operational and may be under different molecular control.
When mice were left to survive to 6 wk of age after injection of the caspase 2 inhibitor on pnd 13, the testicular histology appeared normal. Since it is known that Sertoli cells can support only a limited number of germ cells [2, 5] and that this initial wave of germ cell apoptosis is thought to establish this correct ratio of germ cells to Sertoli cells, it is possible that other compensatory mechanisms occurred to ensure successful spermatogenesis. The proapoptotic protein BAX is a most likely candidate. Functional BAX may also compensate for the lack of caspase 2; therefore, caspase 2-deficient male mice may appear normal [14]. In the caspase 2-deficient mice, excess numbers of germ cells in the female ovaries were observed, and oocytes were more resistant to apoptotic cell death following exposure to chemotherapeutic drugs, whereas the males were described as normal [14]. Thus, it appears the male germ cells may have evolved multiple compensatory mechanisms to ensure spermatogenesis and male fertility.
To our knowledge, this is the first study to demonstrate a role for caspase 2 in the maturation of the seminiferous epithelium. Future studies will be aimed at determining if caspase 2 interacts with BAX and investigating the mechanisms of how caspase 2 is activated during the onset of spermatogenesis.
FOOTNOTES
1 Supported by P50 DK45179 (J.J.L.), R01 DK53072 (T.T.T.), and a UVA Research and Development Grant (J.J.L.). 
2 Correspondence: FAX: 434 924 8311; jl6n{at}virginia.edu
Received: 14 June 2005.
First decision: 6 July 2005.
Accepted: 25 January 2006.
REFERENCES
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0.05) in apoptotic cells is seen at pnds 15, 16, and 17 (mean ± SEM). Data points are significantly different from pnd 10 (P 
