mice lacking functional FAS, suggesting that the caspase- 8–mediated cleavage of BID is not responsible for the observed release of cytochrome c from the mitochondria (Vera et al., 2004).

In additional studies, we further examined whether the FasL-defective gld mice would confer resistance to apoptosis induced by hormone withdrawal. We found that germ cells from wild-type and FasL-defective mice are equally sensitive to apoptosis triggered by hormone deprivation. These findings reinforce our earlier hypothesis that the intrinsic pathway signaling is the key apoptotic pathway for male germ cell death (Vera et al., 2006).

Nair and Shaha (2003) showed the involvement of the mitochondria-dependent pathway, characterized by loss of mitochondrial membrane potential, BAX translocation to mitochondria, cytochrome c release from mitochondria and subsequent activation of the caspase-9 and caspase-3, and PARP cleavage in diethylstilbestrolinduced testicular germ cell apoptosis in the rat. One other important finding that comes out of this study is the involvement of the FAS-FASL system, characterized by upregulation of FASL and FAS and activation of caspase-8 in germ cells. Theas and colleagues (2006) have further demonstrated the involvement of both death receptor and mitochondrial pathways in germ cell apoptosis in an experimental model of autoimmune orchitis. It would be interesting to know whether the link between these two pathways is the caspase- 8–mediated cleavage of BID. Evidence exists that germ cells, in particular spermatocytes, are able to undergo tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–induced apoptosis and that pretreatment with anti-DR5 antibody can increase their sensitivity to TRAIL-mediated apoptosis (McKee, Ye, and Richburg, 2006). Taken together, these results indicate that regulation of testicular germ cell apoptosis varies depending on the nature of apoptotic stimulus and can be triggered by more than one pathway.

p38 MAPK and inducible nitric oxide synthase (iNOS) in apoptotic signaling of male germ cells in rats after hormone deprivation by a potent GnRH-A treatment. Activation of p38 MAPK, as evidenced by an increase in phospho-activating transcription factor-2 (ATF-2), was detected as early as 2 days after GnRH-A treatment and remained active thereafter throughout the treatment period (Figure 25-4A). Activation of p38 MAPK was also substantiated by immunohistochemistry and confocal microscopy. Compared with control, where no staining is detected, a strong phospho-p38 MAPK immunoreactivity was noted in the condensed nuclei of apoptotic germ cells after hormone withdrawal (Figure 25-4B, panels I–III). Co-staining for TUNEL and for phospho-p38 MAPK further confirmed activation of p38 MAPK only in those germ cells undergoing apoptosis (Fig. 25-4B, panels IV–VI). We found a similar profile in the induction of iNOS after GnRH-A treatment. Most importantly, p38 MAPK activation and iNOS induction within 2 days after GnRH-A treatment indicate that these events are indeed upstream of activation of apoptosis, which was first detected 5 days after GnRH-A treatment. p38 MAPK activation and iNOS induction were further accompanied by a marked perturbation of the BAX/BCL-2 rheostat, cytochrome c, and DIABLO release from mitochondria, caspase activation, and PARP cleavage (Vera et al., 2006). Concomitant administration of aminoguanidine (AG), a selective iNOS inhibitor, significantly prevented hormone deprivation-induced germ cell apoptosis (Vera et al., 2006). Relevant to this is the demonstration that such hormone deprivation-induced male germ cell apoptosis can be effectively prevented by minocycline (Castanares et al., 2005), which suppresses p38 MAPK activation, iNOS induction, and cytochrome c–mediated death pathway in other systems (Zhu et al., 2002; Teng et al., 2004; Wei et al., 2005). Induction of germ cell apoptosis after hormone withdrawal is independent of JNK or ERK (Castellanos, 2007).

5. P38 MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) AND NITRIC OXIDE (NO)–MEDIATED INTRINSIC PATHWAY SIGNALING CONSTITUTES A CRITICAL COMPONENT OF APOPTOTIC SIGNALING IN MALE GERM CELLS AFTER HORMONE DEPRIVATION

MAPKs comprise a family of serine/threonine kinases that function as critical mediators of a variety of extracellular signals. These kinases include the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK; also known as stress-activated protein kinase), and the p38 MAPK. To provide some insight into the upstream signaling pathways, we examined the role of

6. P38 MAPK PATHWAY IS ALSO THE KEY PATHWAY FOR

HEAT-INDUCED MALE GERM CELL APOPTOSIS

To characterize the upstream signaling pathways by which heat stress triggers male germ cell apoptosis, the contributions of the ERK, JNK, and p38 MAPK to stage-specific activation of germ cell apoptosis triggered by testicular hyperthermia were examined (Castellanos, 2007). Our data constitute the first demonstration that testicular hyperthermia results in stageand cell-specific activation of both p38 MAPK and ERK, but not JNK. Activation of p38 MAPK, as evidenced by a significant (P < 0.05) increase (by 5.3-fold) in phospho-p38 MAPK

levels in testis lysates, was detected within one-half hour of heating and remained active thereafter throughout the treatment period. Because the phosphorylation status of BCL-2 plays an important role in its prosurvival activity (Halder, Basu, and Croce, 1998; Fan et al., 2000; Rajah, Lee, and Cohen, 2002; Bu et al., 2006), and this can be induced by p38 MAPK (Bu et al., 2006; Shimada et al., 2003), we next examined whether the increased germ cell apoptosis after heat stress is associated with BCL-2 phosphorylation. Compared with control, in which no staining was detected, we found marked increase in the serine-phosphorylated form of inactive BCL-2 only in heat-susceptible germ cells (Figure 25-5, panels I and II). Co-staining for TUNEL and phospho-BCL-2 further confirmed phosphorylation of BCL-2 only in those germ cells undergoing apoptosis (Figure 25-5, panels III through V). Most importantly, we further show that SB203580, a selective inhibitor of p38 MAPK, effectively suppressed BCL-2 phosphorylation and cytochrome c release and significantly (P < 0.05) prevented heat-induced germ cell apoptosis (Jia

et al., unpublished data). It is thus conceivable that the signal for activating mitochondria-dependent pathway during heat-induced male germ cell apoptosis emanates from p38 MAPK-mediated inactivation of BCL-2 through phosphorylation, thereby resulting in the perturbation of the BAX/BCL-2 rheostat in the mitochondria and the subsequent activation of the mitochondria-dependent death pathway.

Unlike p38 MAPK, we found activation of ERK within one-half hour of heating in the Sertoli cells at heatsusceptible stages (Figure 25-6). Thus the activation of ERK in the Sertoli cells is indeed upstream of activation of germ cell apoptosis, which was first detected 6 hours after heating (Yamamoto et al., 2001; Sinha Hikim et al., 2003b). Inhibition of ERK by U0126 had no effect on the incidence of heat-induced germ cell apoptosis, suggesting that ERK signaling may be dispensable for heatinduced germ cell apoptosis in the testis (Castellanos, 2007). At present, we do not know the possible significance of our findings. These observations, however, do suggest that not only germ cells, but also Sertoli cells

involving suppression of p38 MAPK activation and iNOS induction and, in turn, suppression of the cytochrome c–mediated death pathway (Johnson et al., 2008). We found an almost identical level of protection (by 67.0%) of testicular germ cells from heat-induced apoptosis in mice pretreated with a Quinoline-Val-asp (Ome)- CH2-O-ph (Q-VD-OPH), a broad-spectrum pan caspase inhibitor (Vera et al., 2005). However, compared with Z-VDAVDK, the protection offered by Q-VD-OPH was independent of mitochondrial cytochrome c release and occurred by inhibiting caspase activation (Vera et al., 2005). Together, these studies indicate that caspase-2 activation is needed to fuel cytochrome c or DIABLO release from mitochondria.

8. SIGNALING PATHWAYS FOR TESTICULAR GERM CELL

DEATH IN NONHUMAN PRIMATES

Our group has also characterized the signaling pathways in inducing accelerated apoptosis after testicular hyperthermia, hormonal deprivation, or combined interventions in a nonhuman primate model (Lue et al., 2006). Treatment with T, heat, or both in adult cynomolgus monkeys led to sustained activation of both ERK and p38 MAPK. Activation of both these kinases were accompanied by an increase in BCL-2 levels in both cytosolic and mitochondrial fractions of testicular lysates (BAX levels remained unaffected) and cytochrome c and DIABLO release from mitochondria. These treatments also resulted in inactivation of BCL-2 through phosphorylation at serine 70, thereby favoring the death pathway. We conclude that the serine phosphorylation of BCL-2 and activation of the p38 MAPK-mediated mitochondriadependent pathway are critical for male germ cell death in monkeys (Jia et al., 2007).

9. SIGNALING PATHWAYS FOR TESTICULAR GERM CELL

DEATH IN HUMAN

Having established that p38 MAPK-mediated intrinsic pathway signaling constitutes a critical component of apoptotic signaling in male germ cells in rats (Vera et al., 2006) and monkeys (Jia et al., 2007), we next evaluated the efficacy of iNOS as well as p38 MAPK inhibitors in preventing or attenuating human male germ cell apoptosis induced by deprivation of survival factors. As expected, culturing seminiferous tubules for 4 hours resulted in clear apoptotic DNA laddering, as detected by Southern blot analysis of DNA fragmentation. Concomitant treatments with SB 203580, a p38 MAPK inhibitor, and AG, a selective iNOS inhibitor, significantly suppressed low molecular DNA

fragmentation induced by culturing segments of human seminiferous tubules under hormone-free conditions. We further examined the induction of iNOS during human male germ cell apoptosis by immunoblotting from tubular samples cultured under hormone-free conditions. No iNOS expression was detected in the noncultured seminiferous tubule fragments. In contrast, culturing seminiferous tubules for 4 hours resulted in induction of iNOS and that could be effectively suppressed by SB 203580 treatment, indicating that p38 MAPK is an upstream activator of iNOS during human male germ cell apoptosis.

Together, these results establish a new signal transduction pathway involving p38 MAPK and iNOS that, through activation of the intrinsic pathway signaling, promotes male germ cell death in response to a lack of hormonal stimulation across species (Vera et al., 2006; Jia et al., 2007).

10. COMPLETE REVERSIBILITY OF SPERMATOGENESIS

AFTER DISCONTINUATION OF SUPPRESSION OF

GONADOTROPINS BY EXPERIMENTAL CONTRACEPTIVES

Increased germ cell apoptosis plays an important role in organized regression of spermatogenesis after hormonal suppression, including human (Wang et al., 2007). With this hormonal suppression, azoospermia (no sperm in ejaculate) or severe oligozoospermia (< 3 million sperm per mL of semen) sufficient for contraceptive purposes can be achieved (Wang and Swerdloff, 2004). Thus it is important to investigate the reversibility of spermatogenesis after cessation of hormonal contraceptive treatment. In a recent study, we undertook an integrated analysis of data from 1,549 participants in hormonal contraceptive studies (T with or without progestagen) in 20 centers across the globe (Liu et al., 2006). The data show full reversibility within a predictable time course (Liu et al., 2006). This finding provides a strong foundation on which a safe, reliable, and reversible contraception based on hormonal suppression of spermatogenesis could soon become available.

11. CONCLUSIONS AND PERSPECTIVES

It is now widely accepted that male germ cell apoptosis is a genetically driven form of cell death and is a critical prerequisite for functional spermatogenesis. There is increasing evidence that null mutations of a number of genes in mice result in severe spermatogenic disruption and infertility through accelerated germ cell apoptosis. Most notably, it appears that null mutation of some genes, expressed in many tissues, including the testis,