Induction of apoptosis involving multiple pathways is a primary response to cyclin A1-deficiency in male meiosis

Authors

  • Glicella Salazar,

    1. Departments of Genetics & Development and Obstetrics & Gynecology, Institute of Human Nutrition, Center for Reproductive Sciences, and Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, College of Physicians & Surgeons, New York, New York
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  • Ayesha Joshi,

    1. Departments of Genetics & Development and Obstetrics & Gynecology, Institute of Human Nutrition, Center for Reproductive Sciences, and Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, College of Physicians & Surgeons, New York, New York
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  • Dong Liu,

    1. Departments of Genetics & Development and Obstetrics & Gynecology, Institute of Human Nutrition, Center for Reproductive Sciences, and Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, College of Physicians & Surgeons, New York, New York
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  • Hongquan Wei,

    1. Departments of Genetics & Development and Obstetrics & Gynecology, Institute of Human Nutrition, Center for Reproductive Sciences, and Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, College of Physicians & Surgeons, New York, New York
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  • Jenny Liao Persson,

    1. Departments of Genetics & Development and Obstetrics & Gynecology, Institute of Human Nutrition, Center for Reproductive Sciences, and Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, College of Physicians & Surgeons, New York, New York
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  • Debra J. Wolgemuth

    Corresponding author
    1. Departments of Genetics & Development and Obstetrics & Gynecology, Institute of Human Nutrition, Center for Reproductive Sciences, and Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, College of Physicians & Surgeons, New York, New York
    • Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, College of Physicians & Surgeons, 630 West 168th Street, New York, NY 10032
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Abstract

The meiotic arrest in male mice null for the cyclin A1 gene (Ccna1) was associated with apoptosis of spermatocytes. To determine whether the apoptosis in spermatocytes was triggered in response to the arrest at G2/M phase, as opposed to being a secondary response to overall disruption of spermatogenesis, we examined testes during the first wave of spermatogenesis by terminal deoxynucleotidyl transferase–mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) staining. We observed enhanced apoptosis coinciding with the arrest point in postnatal day 22 tubules, with no overt degeneration. Along with activation of caspase-3, an increase in the levels and change of subcellular localization of Bax protein was observed in cyclin A1–deficient spermatocytes, which coincided with the detection of apoptosis. As p53 is implicated in the activation of Bax-mediated cell death, we generated mice lacking both cyclin A1 and p53. Although the absence of p53 did not rescue the meiotic arrest, there was a decrease in the number of apoptotic cells in the double-mutant testes. This finding suggested that p53 may be involved in the process by which the arrested germ cells are removed from the seminiferous tubules but that other pathways function as well to ensure removal of the arrested spermatocytes. Developmental Dynamics 234:114–123, 2005. © 2005 Wiley-Liss, Inc.

INTRODUCTION

Control of cell division cycles are key events to ensure normal development of all systems. During spermatogenesis, male germ cells undergo precisely controlled mitotic and meiotic division cycles. As in other systems, the cyclin–cyclin-dependent kinase (Cdk) complexes are the key regulators of the mitotic and meiotic divisions of male germ cells. The expression patterns of cyclins and Cdks in the testis suggest elaborate control of their timing of expression and function (reviewed in Wolgemuth et al., 1995; Handel et al., 1999; Wolgemuth, 2003).

Of particular interest with regard to spermatogenesis are the A-type cyclins. There are two mammalian A-type cyclins, cyclin A1 and A2. Cyclin A1 expression is limited to male germ cells (Sweeney et al., 1996) and is essential for passage of spermatocytes into meiosis I, as revealed in the phenotype of the cyclin A1–deficient mice (Liu et al., 1998). The late arrest in meiotic prophase was associated with desynapsis abnormalities, low activity of M-phase promoting factor (MPF) kinase, and a wave of apoptosis.

Loss of germ cells by apoptosis is a normal feature during spermatogenesis (Wolgemuth, 2003). Apoptosis is detected in the gonocytes as early as embryonic day 15.5 (Matsui et al., 2000). After birth, a wave of apoptosis of spermatogonia and spermatocytes has been observed during a critical period at around 2–4 weeks of age (Rodriguez et al., 1997) and is believed to be essential for establishing and maintaining an optimum ratio of Sertoli cells to germ cells. Apoptosis of spermatogonia and a few spermatocytes at Stage XII of adult spermatogenesis has also been observed (Koji, 2001).

As noted above, Ccna1−/− mice are sterile due to arrest of germ cells at the pachytene-diplotene stage of meiosis. Neither the downstream targets nor the signaling pathways affected by the lack of cyclin A1 have yet been identified. However, the arrested germ cells subsequently underwent apoptosis as demonstrated by terminal deoxynucleotidyl transferase–mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) labeling (Liu et al., 1998). The tight correlation of cell cycle arrest and the induction of apoptosis in late pachytene to diplotene spermatocytes of the Ccna1−/− mice led us to investigate whether apoptosis was a primary response to loss of cyclin A1 function and to investigate the apoptotic pathway(s) that were activated in response to the absence of cyclin A1. As most apoptotic pathways lead to the activation of caspases (Cory and Adams, 2002; Orrenius et al., 2003), we specifically examined the activation of the executioner caspase, caspase 3. We further examined regulators upstream of the caspases, specifically the pro-apoptotic protein Bax. Finally, as the role of p53 in apoptosis is well established (Haupt et al., 2003) and p53 is expressed in spermatocytes at stages XI to VIII (Almon et al., 1993; Beumer et al., 1998), we examined the effect on apoptosis of depleting p53 by generating mice that were doubly deficient for both p53 and cyclin A1.

Our results demonstrate that apoptosis in spermatocytes is a primary response to the absence of cyclin A1. The apoptosis is mediated by up-regulation of Bax and subsequent activation of caspase 3. Also, absence of p53 only partly alleviated the cell death phenotype but did not rescue the cell cycle arrest. Cyclin A1, therefore, may regulate divergent signal transduction pathways, one leading to progression of meiosis and the other leading to inhibition of apoptosis, possibly involving p53.

RESULTS

Examination of Apoptosis During the First Wave of Spermatogenesis

With time, some tubules in adult cyclin A1–deficient testes exhibited severe degeneration, such that they were almost devoid of germ cells, whereas others exhibited the typical TUNEL-positive spermatocytes (Liu et al., 1998; and data not shown). To determine whether the apoptosis of late-stage spermatocytes in the adult testis was a direct response to the cell cycle arrest or a secondary effect to a general disruption of spermatogenesis, the onset of apoptosis in the first wave of spermatogenesis was evaluated. The TUNEL assay was performed on testicular sections of 17-, 22-, and 28-day-old control and Ccna1−/− animals. At day 17 of postnatal development, the TUNEL assay detected occasional apoptotic cells in both Ccna1+/+ and Ccna1−/− testes, with spermatocytes being the most frequent TUNEL-positive cell type (Fig. 1A,a,d).

Figure 1.

Apoptosis in the Ccna1−/− animals results as a direct consequence of cell cycle arrest due to lack of cyclin A1. A: Terminal deoxynucleotidyl transferase–mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) staining on testes sections of 17-day-old (a,d), 22-day-old (b,e) and 28-day-old (c,f) wild-type and Ccna1−/− animals, respectively; original magnification, ×20. B: Quantification of TUNEL staining in A. The apoptotic index (AI) was calculated by multiplying the percentage of tubules containing apoptotic germ cells by the number of apoptotic germ cells per tubule at each time point (Woolveridge et al., 1999; Yu et al., 2001).

The TUNEL labeling of cells at day 22 increased in both the control as well as the cyclin A1–deficient testes, although more obviously in the latter (Fig. 1A,b,e). An increase in the number of apoptotic spermatocytes as well as spermatogonia is seen as a normal feature of development at this stage (Rodriguez et al., 1997). However, as this corresponds to the time when the spermatocytes in the first wave of spermatogenesis should have just completed the first meiotic prophase, the higher number of apoptotic cells observed in the cyclin A1–deficient testes reflected the arrested cells. At day 28, apoptosis of germ cells in the control testis decreased significantly when compared with days 17 and 22 (Fig. 1A,c), whereas the number of apoptotic cells in the mutant was strikingly higher (Fig. 1A,f).

As described in other studies, an apoptotic index (AI) was calculated to quantitatively assess the apoptotic process (Woolveridge et al., 1999; Yu et al., 2001). At 17 days, there appeared to be no difference among the AI of the Ccna1+/+, Ccna1+/−, and Ccna1−/− testes (Fig. 1B), but by day 22 of age, the AI of the Ccna1−/− tubules was significantly higher (107.33 ± 7.45; P < 0.01) compared with the control values (62.33 ± 2.33 and 50.67 ± 2.33 for wild-type and heterozygous testes, respectively). This increase was even more evident at day 28 of age, where the AI rose significantly (148.33 ± 5.29; P < 0.01) in the Ccna1−/− mice compared with the wild-type and heterozygous control mice (9.0 ± 1 and 17.0 ± 1). However, there were no obviously degenerating or germ cell–deficient tubules observed. These results suggest that cell death is a primary consequence of cell cycle arrest in the first wave of spermatogenesis.

Increase in Caspase 3 Activity

Having established that spermatocytes die as a direct consequence of the absence of cyclin A1, we wished to investigate the apoptotic pathways that operate in the spermatocytes. Because most apoptotic signaling pathways culminate in the activation of caspase 3, including those important in the reproductive system (Kuida et al., 1996; Kim et al., 2000, 2001; Carambula et al., 2002), we investigated whether caspase 3 activation was enhanced in the cyclin A1–deficient testes. Induction of apoptosis is accompanied by cleavage of the pro-form of caspase 3 into subunits of approximately 17–19 and 10–12 kDa. We determined the activation of caspase 3 by immunohistochemistry, with an antibody that detects the activated 17- to 19-kDa subunit of caspase 3. The expression of activated caspase 3 was detected at significant levels only in the spermatocytes in the cyclin A1–deficient testis (Fig. 2A,c). Activated caspase 3 in the Ccna1−/− spermatocytes was detected in both the cytoplasm and nucleus (Fig. 2A,c inset).

Figure 2.

Apoptotic pathways in the Ccna1−/− spermatocytes lead to activation of caspase 3. A: Immunohistochemical localization of activated caspase 3 in adult testis sections of wild-type (a, b) and Ccna1−/− (c,d) mice. Activated caspase 3 subunit is detected only in the spermatocytes of the Ccna1−/− testis sections, in the nucleus, as well as the cytoplasm (arrow; inset in c, original magnification, ×100); original magnification, ×40. Panels b and d represent no primary antibody controls. B: Immunoblot analysis of nuclear and cytoplasmic preparations from testis of wild-type and Ccna1−/− animals using antibodies to pro-caspase 3 and activated caspase 3. Elevated levels of pro- and activated caspase 3 in the nuclear and cytoplasmic extracts from the Ccna1−/− testis, confirmed the immunohistochemical staining. Lamin B and copper–zinc superoxide dismutase (Cu/Zn SOD) were used as markers for purity of the nuclear and cytoplasmic preparations, respectively.

To further confirm the subcellular localization of the activated caspase 3, nuclear and cytoplasmic extracts from testes of Ccna1+/+ and Ccna1−/− mice were prepared and analyzed by immunoblot analysis. Pro-caspase 3 was evident in the cytoplasm of both the wild-type and mutant testes but was seen to also be present in the nuclear fraction of the mutant testicular cells (Fig. 2B). The 17- to 19-kDa activated subunit was detected at low levels in the cytoplasm and nuclei of the wild-type testes but was up-regulated in both subcellular compartments in the Ccna1−/− testes (Fig. 2B). Lamin B and copper–zinc superoxide dismutase (Cu/Zn SOD) served as markers to determine relative purity of the nuclear and cytoplasmic preparations, respectively (Fig. 2B). The induction of apoptosis in the Ccna1−/− testes, thus, was accompanied by elevated levels and activation of caspase 3 in the cytoplasm and its localization to the nucleus.

Involvement of Bax in the Apoptosis of Cyclin A1–Deficient Spermatocytes

We next investigated the role of the pro-apoptotic protein Bax, known to be highly expressed in spermatocytes (Beumer et al., 2000; Yan et al., 2000; Jahnukainen et al., 2004) and to be essential for the normal apoptosis that occurs during the first wave of spermatogenesis (Knudson et al., 1995). Levels of Bax protein were up-regulated in extracts from testes of the cyclin A1–deficient mice compared with the control testes (Fig. 3A). To precisely localize the cell type in which Bax was up-regulated, immunohistochemistry was performed (Fig. 3B). In the control testes (Fig. 3B,a), Bax expression was cytoplasmic and was detected in spermatogonia, spermatocytes, and Sertoli cells. In testes of mice lacking cyclin A1, elevated expression of Bax was observed in the arrested spermatocytes (Fig. 3B,c and inset), which were also TUNEL-positive (Fig. 3C). Therefore, the increase in Bax observed in total testis extracts was due to an increase of Bax in the arrested and apoptosing spermatocytes. We also noted a redistribution of Bax from a cytoplasmic to a nuclear and perinuclear localization in spermatocytes of Ccna1−/− mice (Fig 3B,c inset), similar to observations by Yamamoto et al. (2000) and Vera et al. (2004) in models of hyperthermia in the rat and mouse testis, respectively. Additionally, colocalization of 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI) staining and Bax immunofluorescence (Fig. 3C) in the mutant testis further confirmed that Bax was present in the nucleus. Finally, we prepared nuclear and cytoplasmic extracts from testes of wild-type and Ccna1−/− animals (Fig. 3D). Bax was predominantly cytoplasmic in the wild-type testes. In the Ccna1−/− testes, there was increased Bax expression, which was localized in both the cytoplasm as well as the nucleus.

Figure 3.

Elevated levels and changes in subcellular localization of Bax in Ccna1−/− spermatocytes. A: Immunoblot analysis, using Bax antibody, of total testis extracts from wild-type and Ccna1−/− animals. Bax was up-regulated in the Ccna1−/− testis. B: Immunohistochemical localization of Bax in testis sections from adult wild-type (a,b) and Ccna1−/− (c,d) animals, demonstrating up-regulation of Bax in the cytoplasm (arrow) and movement to nuclear regions (arrowheads; inset in c, original magnification, ×100); original magnification, ×40. In the wild-type animals, faint Bax expression was detected in Sertoli cells (asterisk), spermatogonia (arrow), and spermatocytes (equation image; inset in a, original magnification, ×100). Panels b and d represent no primary antibody controls. C: Colocalization of terminal deoxynucleotidyl transferase–mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) staining and Bax staining. Bax is up-regulated in TUNEL positive spermatocytes. Colocalization of Bax immunofluorescence and 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI) staining demonstrates nuclear localization of Bax in the spermatocytes. D: Western blot analysis of nuclear and cytoplasmic preparations from testis of wild-type and Ccna1−/− animals using Bax antibody. Apoptosis in the Ccna1−/− testis leads to up-regulation in the cytoplasm as well as redistribution to the nuclear compartment.

Upstream Regulators of Bax and Caspase 3 in the Spermatocytes

The role of p53 in apoptosis is well established (Haupt et al., 2003). p53 is normally expressed in spermatocytes at stages XI to VIII and radiation treatment leads to up-regulation of p53 in pre-leptotene spermatocytes in stage VII (Almon et al., 1993; Beumer et al., 1998) Furthermore, it has been reported that a deficiency of p53 rescues germ cell degeneration in Wv/Wv mice (Jordan et al., 1999) and that a partial rescue of the prophase I defects of Atm-deficient mice was observed in Atm/p53 and Atm/p21 double-mutant mice (Barlow et al., 1997). Therefore, to determine whether the lack of p53 function would prevent apoptosis and alleviate the germ cell degeneration phenotype in Ccna1−/− male mice, we bred our cyclin A1–deficient mice with p53-deficient mice to generate double mutants.

Morphological analysis along with TUNEL staining was undertaken on four resulting genotypes: (1) Ccna1+/+; Trp53+/+, (2) Ccna1+/+;Trp53−/−, (3) Ccna1−/−;Trp53−/−, and (4) Ccna1−/−;Trp53+/+. The double-mutant Ccna1−/−;p53−/− testes revealed no haploid spermatids, which suggested that in the absence of p53, and the cells were still arrested at the G2/M transition (Fig. 4A,d). Specimens from adult single versus double-mutant testes were then examined by TUNEL staining (Fig. 4A). A significant number of TUNEL-positive spermatogenic cells were observed in both Ccna1−/−;Trp53+/+ and Ccna1−/−;Trp53−/− testes (Fig. 4A,c,d). The cells appeared to be pachytene–diplotene spermatocytes in both genotypes. Very few TUNEL-positive germ cells were observed in Ccna1+/+;Trp53+/+ or Ccna1+/+;Trp53−/− tubules (Fig. 4A,a,b). Apoptosis did occur in the absence of p53, mainly in spermatogonia.

Figure 4.

The apoptosis phenotype in the Ccna1−/− animals is partially rescued by p53. A: Terminal deoxynucleotidyl transferase–mediated deoxyuridinetriphosphate nick end-labeling (TUNEL) staining on adult testis sections of mice with Ccna1+/+;Trp53+/+ (a), Ccna1+/+;Trp53−/− (b), Ccna1−/−;Trp53+/+ (c), and Ccna1−/−;Trp53−/− (d) genotypes, original magnification, ×40. B: Quantification of the TUNEL staining in A. The apoptotic index (AI) was calculated by multiplying the percentage of tubules containing apoptotic germ cells by the number of apoptotic germ cells per tubule at each time point (Woolveridge et al., 1999; Yu et al., 2001).

The AI was then determined among all four allelic combinations, as described for the analysis of the postnatal testes. The AIs in Ccna1−/−;Trp53+/+ and Ccna1−/−;Trp53−/− testes were significantly (approximately sevenfold and twofold, respectively) increased over the Ccna1+/+;Trp53+/+ control testis (Fig. 4B) but the AI of Ccna1+/+;Trp53−/− testes was not statistically different from the control. In addition, the apoptotic indices were also significantly different (P < 0.01) between the cyclin A1–deficient mice versus the double-mutant mice (Fig. 4B). These results suggested that p53 may be partially involved in the process of removing the arrested spermatocytes, but that other, non–p53-dependent pathways function.

DISCUSSION

We have shown previously that the cell cycle regulator cyclin A1 is essential in male germ cells for the progression from G2 to M phase in the meiotic cycle (Liu et al., 1998). One of the features of the Ccna1−/− phenotype was that arrested late-pachytene to diplotene spermatocytes underwent apoptosis. The induction of an apparent apoptotic response (TUNEL-positive) has been seen in various other gene knockout studies that resulted in impaired meiosis, such as A-myb (Toscani et al., 1997), Hsp 70.2 (Dix et al., 1997), Mlh1 (Edelmann et al., 1996), Spo11 (Baudat et al., 2000; Romanienko and Camerini-Otero, 2000), Atm (Xu et al., 1996), and so on (reviewed in Salazar et al., 2003). It would appear that the induction of cell death is critical for ensuring not only that the proper number of germ cells is produced during normal spermatogenesis but also that gametes that have not gone through meiosis properly are not formed.

In the present study, the temporal appearance of cell death coincident with the G2/M cell cycle arrest that occurred in cyclin A1–deficient, late pachytene to diplotene spermatocytes suggested that apoptosis was a primary response to the arrest. At 17 days of age, there were few apoptotic pachytene spermatocytes with an AI not significantly different among the different genotypes. However, at the time when the first wave of differentiating cells should be completing the first and second meiotic divisions and forming haploid spermatids, significantly higher numbers (by almost twofold; P < 0.01) of TUNEL-positive pachytene spermatocytes were observed in the cyclin A1–deficient testes as compared with the control testes. That there was a peak of germ cell death at day 22 of age in the normal developing testis was in agreement with a previous study by Rodriguez et al. (1997). By 28 days of postnatal development, a reduction of AI was seen in the control samples. In contrast, in the Ccna1−/− mice, the AI actually increased further and was significantly higher (P < 0.01) than the controls. The predominant TUNEL-positive nuclei were pachytene–diplotene spermatocytes. These results demonstrate that apoptosis occurred as a direct consequence of the cell cycle arrest at G2/M stage in the absence of cyclin A1. The absence of cyclin A1, therefore, either actively potentiated apoptotic pathways or inactivated survival pathways.

The most downstream effector of the apoptotic pathway examined in the Ccna1−/− spermatocytes was caspase 3. Caspase 3 was clearly involved in the cyclin A1–deficiency mediated apoptosis, as increases in the amount of the pro-caspase protein and changes in the subcellular distribution of the activated form were observed. The activated form of caspase 3 was seen in the nucleus as well as cytoplasm of the spermatocytes of the Ccna1−/− mice. The apoptotic cascade, therefore, might result in transport of the activated form into the nucleus or activation of the nuclear pro-caspase form. In the Ramos Burkit lymphoma cell line, An et al. (2003) observed similar localization patterns for pro-caspase 3; after induction of apoptosis, activated caspase 3 was demonstrated to be present in the nucleus as well as in the cytoplasm. In another model system, pro-caspase 3 was demonstrated to be constitutively nuclear in Jurkat cells (Ramuz et al., 2003). However, upon stimulation with Fas, active caspase 3 first appeared in the cytoplasm and then was subsequently detected in the nucleus. The targets of activated caspase 3 are cellular proteins found in the nucleus as well as cytoplasm and include transcription factors (HSPs, GATA proteins, NF-kB), cell cycle regulators (Cdc 27, p21, Rb), and cytoskeletal proteins (actin, lamins, beta-catenins), and so on (Chang and Yang, 2000).

Apoptosis in the testis can involve both cell-intrinsic (Bcl-2 family-mediated) and cell-extrinsic Fas-mediated pathways (Lee et al., 1997; Beumer et al., 2000). Some proteins of the cell-intrinsic pathway, the Bcl-2 family of proteins, are essential for normal spermatogenesis. The expression of Bax, a pro-apoptotic protein, peaks during the time of the massive germ cell death observed at day 22 during postnatal development of the testis in mice (Rodriguez et al., 1997) and is still expressed in pachytene spermatocytes in the adults (Yan et al., 2000; Meehan et al., 2001). In testes of Bax-deficient mice, there was an accumulation of spermatogonia, a phenotype consistent with the failure of germ cell death during the first wave of spermatogenesis (Knudson et al., 1995). The preleptotene spermatocytes failed to undergo meiosis, presumably because of the resulting aberrant Sertoli cell to spermatocyte ratio. Similar phenotypes were observed in transgenic animals overexpressing the anti-apoptotic proteins Bcl-2 and Bcl-X (Furuchi et al., 1996; Rodriguez et al., 1997; Sugiyama et al., 2001). It appears that a fine balance of pro- and anti-apoptotic proteins is essential for the establishment of spermatogenesis and the normal progression of spermatocytes into meiosis (Print and Loveland, 2000). Bcl6−/− mice have reduced sperm counts compared with the wild-type due to increased apoptosis of spermatocytes in MI (Kojima et al., 2001). The spermatocytes also exhibited elevated levels of p38 MAP kinase; therefore, Bcl6 might play a role in protecting spermatocytes from stress-induced apoptosis.

In the present study, we have examined the possible involvement of Bax in inducing apoptosis in cyclin A1–deficient spermatocytes. Immunohistochemical analysis revealed that Bax was localized in the cytoplasm of Sertoli cells, spermatogonia, and spermatocytes during the normal seminiferous epithelial cycle. In contrast, the cyclin A1–deficient testis showed an increase of Bax-positive spermatocytes and redistribution from a cytoplasmic to perinuclear and nuclear localization. Furthermore, the association of Bax expression and apoptotic cells was confirmed by localization of Bax protein in TUNEL-positive cells. Induction of apoptosis is accompanied by change in conformation of Bax, its movement to the mitochondrial outer membrane, and subsequent oligomerization (Hsu and Youle, 1998; Nechushtan et al., 1999, 2001; Antonsson et al., 2001). However, Bax translocation from the cytosol to either close proximity of the nucleus or into the nucleus has also been observed in various cell lines upon induction of apoptosis (Mandal et al., 1998; Yamamoto et al., 2000). Our observation of the presence of Bax in the nucleus indicated that, in the absence of cyclin A1, Bax-mediated apoptosis might involve its movement to the nucleus, although translocation to the mitochondria needs to be investigated. These findings together indicate that up-regulation and redistribution of Bax may play a crucial role in the initiation of apoptosis in cyclin A1–deficient spermatocytes.

We have also taken advantage of the Ccna1 and Trp53 mutant mouse strains to dissect the role of p53 in regulating cell death in the absence of cyclin A1 in vivo by producing mice that were both cyclin A1- and p53-deficient. Our results clearly demonstrated that the loss of p53 gene function could not rescue the cell cycle arrest in Ccna1−/− mutant testis. However, we did observe that the AI of the doubly mutant testes was significantly reduced compared with the Ccna1−/− mice alone. Therefore, the absence of p53 partially rescues the apoptosis occurring in the absence of cyclin A1. This observation suggests a dual role for cyclin A1 in spermatocytes in regulating two signaling cascades—one leading to progression through meiosis and the other, possibly mediated in part by p53, preventing apoptosis in spermatocytes. In the doubly mutant adult mice; however, we still observed apoptotic cells, suggesting that cell death in the mutant tubules also involved a p53-independent pathway(s).

In other models of testicular germ cell apoptosis, p53-dependent as well as p53-independent apoptotic pathways have been observed. Yin et al. (1998) found p53-dependent and p53-independent apoptosis in experimental cryptorchidism. In their studies, apoptosis in the cryptorchid testis of Trp53−/− animals occurred after a 3-day lag compared with the wild-type cryptorchid testis. Therefore, they suggested that p53 is necessary for the initial phases of apoptosis, and subsequently, other biochemical events are the cause for the delayed cell death. Even in the Wv/Wv;Trp53−/− mice, where the absence of p53 was able to partially rescue spermatogenic defects of the Wv/Wv phenotype, some apoptotic cells were still detected in the double mutant animals, also suggesting the presence of p53-independent pathways (Jordan et al., 1999).

As mentioned previously, the role of p53 in DNA damage-induced apoptosis in other systems has been well established. In the testis as well, p53 is thought to play an important role in heat-induced and radiation induced apoptosis of germ cells (Beumer et al., 1998). Many of the Bcl-2 family genes (Bax, Noxa, Puma, and Bid) have been demonstrated to be up-regulated by p53 due to the presence of p53-responsive elements in their promoters (Bax and Noxa) as well as in the intronic sequences (Puma) (Oda et al., 2000; Nakano and Vousden, 2001; Thornborrow et al., 2002; Haupt et al., 2003). Other than the Bcl-2 family members, p53 has also been demonstrated to up-regulate Apaf-1 gene expression (Kannan et al., 2000; Moroni et al., 2001; Robles et al., 2001).

The investigation of the apoptosis in the cyclin A1–deficient spermatocytes has helped to elucidate at least partly, the role of cyclin A1 in spermatogenesis. Cyclin A1 is essential for the progression of the meiotic division (Liu et al., 1998); in its absence, apoptosis is initiated in spermatocytes by both p53-dependent and p53-independent pathways. Phosphorylation has been demonstrated to stabilize and activate p53 during DNA damage-induced responses (Burns and El-Deiry, 1999). The absence of cyclin A1 kinase activity in the spermatocytes, therefore, may act as a “sensor,” which initiates an apoptotic response. The mechanism by which this response is initiated is unclear, but the absence of cyclin A1/Cdk kinase activity, through a yet unknown signaling pathway, may promote phosphorylation of p53.

EXPERIMENTAL PROCEDURES

Experimental Animals

All animals were housed in a pathogen-free facility, and all manipulations were performed in strict adherence to IACUC guidelines. Male mice of the inbred 129 Sv/Ev and C57Bl/6 lines were purchased from The Jackson labs (Bar Harbor, ME) and used for mating with our existing Ccna1−/− female mice (Liu et al., 1998) to produce Ccna1+/− male mice and, ultimately, Ccna1−/− male mice. To generate Ccna1−/−;Trp53−/− mice, Trp53−/− mice were obtained from The Jackson Labs and were mated with female Ccna1−/− mice. Genotyping for mutant Ccna1 and Trp53 alleles was done by polymerase chain reaction (PCR) -based analysis according to Liu et al. (1998) and Ludwig et al. (1997), respectively.

Tissue Preparation

Testes of mice at 17, 22, 28 days, and adult (8+ weeks) were dissected from anesthetized animals that had been perfused through the heart with phosphate buffered saline (PBS) and then with 4% paraformaldehyde in PBS. Tissues were fixed overnight at 4°C in 4% paraformaldehyde in PBS. Perfused, fixed tissues were embedded in paraffin, sectioned at 5-μm-thick, and mounted on slides.

Antibodies

For immunostaining, antibodies to activated caspase 3 (Cell Signaling Technology, Inc., Beverly, MA) and Bax (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were used at a dilution of 1:100 and 1:200, respectively. For immunoblot analysis, the Bax antibody was used at a dilution of 1:500. Other antibodies used for immunoblot analysis were anti–pro-caspase3, anti–Cu/Zn SOD (Upstate Biotechnology, Lake Placid, NY, 1:1,000), anti-lamin B (Santa Cruz Biotechnology, 1:400), and anti-glycerladehyde-3-phosphate dehydrogenase (GAPDH; BD-Pharmingen, 1:200). All secondary antibodies conjugated to horseradish peroxidase (HPO) were purchased from Amersham Life Science, Inc. (Arlington Heights, IL) and used at a dilution of 1:6,000.

TUNEL Staining of Apoptotic Cells and Calculation of the AI

TUNEL staining was performed on tissues sections using the in situ cell death detection kit (Roche Diagnostics, Indianapolis, IN), according to the manufacturer's instructions. Briefly, histological sections were deparaffinized in Histoclear, hydrated through an alcohol series, washed with distilled water, and treated with 20 μg/ml proteinase K in 10 mM Tris/HCl (pH 8). Endogenous peroxidase was blocked in 0.3% H2O2 for 30 min, and the tailing reaction was carried out in the terminal deoxynucleotidyl transferase (TdT) buffer with fluorescein labeled UTP. After incubation with anti-fluorescein Fab fragments conjugated to HPO, staining was visualized with diaminobenzidine (DAB)/H2O2. Sections were counterstained with hematoxylin. Only clearly stained cells were scored as apoptotic and only tubules cut perpendicular to the length of the tubule (yielding round tubules in section) were evaluated. At least 100 tubules per section from three different animals from each genotype and time point were counted. To quantitatively assess apoptosis, the AI was determined as described by other investigators (Woolveridge et al., 1999; Yu et al., 2001). Tubules containing three or more TUNEL-positive cells were considered apoptotic. The AI was assessed by multiplying the percentage of apoptotic tubules by the mean number of TUNEL-positive cells per tubule. Significant differences (P < 0.01) between groups were assessed by statistical analysis using one way-analysis of variance (ANOVA) followed by the Tukey honest significant difference multiple comparison test. The software used was VassarStats Web page from Vassar College (Poughkeepsie, NY). The results of individual counts were compared with counts within the same group as well as between groups.

Immunohistochemistry

Immunohistochemical analyses were performed using a Vectastin ABC kit (Vector Laboratories, Burlingame, CA) as previously described (Liu et al., 1998). Briefly, histological sections were deparaffinized in Histoclear, hydrated through an alcohol series, and washed with distilled water. Antigen unmasking was performed by boiling slides in a microwave for 10 min in 0.01M citrate buffer, pH6 (Shi et al., 1991). After inactivation of endogenous peroxidase activity, nonspecific binding was blocked by incubation with 2.5% goat serum for 1 hr at room temperature. The sections were then treated with primary antibodies at the dilutions described above overnight at 4°C. After extensive washing in PBST (PBS with 0.1% Triton X-100), subsequent steps were performed according to the Vectastain ABC kit manufacturer's instructions. For visualization of immunostaining, the sections were incubated with DAB solution for 2 min, washed 3 times with distilled water, and counterstained with hematoxylin. The slides were viewed on a Nikon photomicroscope under brightfield optics and the images were captured with a Spot digital camera using Adobe Photoshop 5.0 software (Adobe, San Jose, CA).

Immunofluorescent Staining With Bax Antibody

To demonstrate nuclear localization of Bax in the Ccna1−/− spermatocytes, immunofluorescence staining with Bax antibody on Ccna1−/− testis sections was performed along with DAPI staining. Histological sections were deparaffinized in Histoclear, hydrated through an alcohol series, and washed with distilled water. After antigen unmasking, performed as described for immunohistochemistry, nonspecific binding was blocked by incubation with 4% goat serum for 1 hr at room temperature. Bax antibody (1:200) was then applied onto the sections and incubated at 4°C overnight. The sections were washed three times with PBS, and then incubated with goat anti-rabbit, Texas Red–labeled secondary antibody for 30 min at room temperature. After two washes in PBS, the slides were counterstained with 300 nM DAPI solution and diluted in PBS for 10 min. The slides were washed in PBS twice and mounted using mounting medium.

Immunohistochemistry and DNA Fragmentation Double Labeling

To demonstrate that spermatocytes with increased expression of Bax were also TUNEL positive, concomitant TUNEL and Bax immunofluorescence staining were performed as described in Ahuja et al. (1997). Briefly, sections were washed twice in PBS, post-fixed in ethanol:acetic acid (2:1) for 5 min at −20°C, and again washed twice with PBS. Sections were equilibrated with terminal deoxynucleotidyl transferase (TdT) buffer for 5 min; the solution was changed for one containing TdT, and the incubation was continued for 1 hr at 37°C. The sections were treated with prewarmed stop-wash buffer for 30 min, rinsed three times with PBS, and exposed to anti-digoxigenin FITC fluorescein for 30 min. After a wash with PBS containing 0.1% Tween-20 (PBST), sections were incubated for 20 min in 0.3% H2O2, followed by two washes with PBST. Rabbit polyclonal anti-Bax (1:200) antibody was added to blocking solution and incubated overnight at 4°C. The sections were washed three times with PBST, and then incubated with goat anti-rabbit, Texas Red–labeled secondary antibody for 30 min at room temperature. The slides were mounted with mounting medium and observed under a fluorescence microscope.

Immunoblot Analyses

Proteins were extracted from minced fragments of adult testes for 15 min at 4°C with RIPA buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulfate [SDS]) containing aprotinin and protease inhibitors (Mammalian Protease Inhibitor Cocktail, Sigma). After centrifugation at 15,000 × g, the amount of protein in the supernatants was quantified by the Bradford method. Tissue lysates (50 μg/lane) were subjected to electrophoresis on SDS-polyacrylamide gel electrophoresis (PAGE) gels. Proteins were transferred to polyvinylidene fluoride (PDVF) membranes and blocked with 5% nonfat milk in TBST (0.9% NaCl, 0.1% Tween 20, 100 mM Tris-HCl, pH 7.5) and the membranes were incubated with primary antibodies at dilutions mentioned previously for 1 hr at room temperature. After three washes with TBST, the membranes were incubated with appropriate secondary antibodies for 1 hr. After three washes with TBST, HPO activity was visualized with the Amersham ECL Western Blotting Detection kit per manufacturer's instructions. GAPDH antibody was used to normalize for protein loading. For preparing nuclear and cytoplasmic extracts, subcellular fractionation was performed using the nuclear and cytoplasmic extract kit (Active Motif, Carlsbad, CA). Nuclear and cytoplasmic preparations were resolved by SDS-PAGE and transferred to PVDF membrane. Immunoblot analysis was performed with the anti-Bax or anti–caspase 3 antibodies as described above. Rabbit polyclonal anti–Cu/Zn SOD and anti-lamin B were used as markers to determine relative purity of the cytosolic and nuclear preparations, respectively.

Acknowledgements

D.J.W. was funded by a grant from the NIH, HD34915.

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