25 Apoptotic Signaling in Male Germ Cells

ABSTRACT

Programmed germ cell death (apoptosis) is conspicuous during normal spermatogenesis and serves as a quality control system for the production of normal sperm. Deregulation of germ cell death is associated with defective spermatogenesis and impaired fertility. Mitochondria-dependent intrinsic pathway signaling constitutes a critical component of apoptotic signaling in male germ cells across species. However, the regulation of germ cell apoptosis may vary depending on the nature of the apoptotic stimulus and can be triggered by more than one pathway. Activation of p38 mitogen-activated protein kinase (MAPK) and induction of inducible nitric oxide synthase are critical for activation of the intrinsic pathway signaling in male germ cells. In addition, there is increasing evidence that caspase-2 is an upstream activator of the p38 MAPK and nitric oxide–mediated intrinsic pathway signaling. This chapter focuses on the recent progress in our understanding of the regulation of germ cell apoptosis in the testis.

1. INTRODUCTION

maturity, and such spontaneous death of certain classes of germ cells by apoptosis appears to be a constant feature of normal spermatogenesis. Increased germ cell death by apoptosis can be triggered by various regulatory stimuli, including testicular hyperthermia or experimental male hormonal contraceptive (Sinha Hikim and Swerdloff, 1999; Sinha Hikim et al., 1999; Sinha Hikim et al., 2003a). Disruption of this orderly process of germ cell death is associated with several impairments of spermatogenesis and fertility (Dunkel et al., 1997; Yamamoto et al., 2001; Pentikaainen, Dunkel, and Erkkila, 2003; Shaha, 2007; Wang et al., 2007). This chapter focuses on unique aspects of genetic regulation of spermatogenesis and highlights the signal transduction pathways inducing male germ cell apoptosis across species.

2. TESTICULAR GERM CELL APOPTOSIS HAS MANY

UNIQUE REGULATORY GENES

Spermatogenesis is a dynamic process in which stem spermatogonia, through a series of events, become mature spermatozoa and occurs continuously during the reproductive lifetime of the individual (Russell et al., 1990). Stem spermatogonia undergo mitosis to produce two types of cells: additional stem cells and differentiating spermatogonia; the latter undergo rapid and successive mitotic divisions to form primary spermatocytes. The spermatocytes then enter a lengthy meiotic phase as preleptotene spermatocytes and proceed through two cell divisions (meiosis I and II) to give rise to haploid spermatids. These in turn undergo a complex process of morphological and functional differentiation resulting in the production of mature spermatozoa. All these phases are supported by and are dependent on an intimate interaction between germ cells and the Sertoli cells (Russell et al., 1990). Not all germ cells, however, achieve

The most important insight into the intracellular mechanisms that control male germ cell apoptosis comes from studies using genetically altered mice either overexpressing or harboring a null mutation of specific genes. Male germ cell apoptosis, like that of other cell systems, is a genetically driven process (reviewed in references Sinha Hikim and Swerdloff, 1999; Matzuk and Lamb, 2002; Baum, St. George, and McCall, 2005; Sinha Hikim, Swerdloff, and Wang, 2005). Null mutation of some genes, which are expressed in many tissues, including the testis, can accelerate germ cell apoptosis and cause specific defects in spermatogenesis. Many genetic alteration impair spermatogenesis without affecting other systems including oogenesis.

A number of examples exist whereby gene deletion results in specific defects in spermatogenesis. Elimination of Bcl-w leads to male sterility with no discernible

effects on most of the systems, including the female reproductive function (Ross et al., 1998; Print et al., 1998). Mutant animals have a block in the later phases of spermatogenesis and exhibit progressive depletion of germ cells through accelerated apoptosis to a Sertoli- cell-only phenotype by approximately 6 months of age followed by loss of Sertoli cells. Given that BCLW is expressed in the elongated spermatids and in Sertoli cells (Ross et al., 1998), it is likely that death of late spermatids is due to the absence of BCLW function in those germ cells, whereas depletion of the entire germline in adults reflects the loss of BCLW function in the Sertoli cells. Targeted gene disruption of Hsp 70–2 results in failed meiosis and increased germ cell apoptosis in males (Dix et al., 1996); however, neither meiosis nor fertility is affected in female Hsp 70–2–/– mice. Likewise, targeted disruption of the mouse homolog of Drosophila Vasa (Mvh) results in male sterility as a result of massive increase in germ cell apoptosis involving meiotic and postmeiotic germ cells, with no adverse effects on female fertility (Tanaka et al., 2000). Inactivation of HR6B ubiquitin-conjugating DNA repair enzyme causes male sterility due to increased germ cell loss with no effect on female fertility (Roest et al., 1996). In contrast to a lethal phenotype of TATA-binding protein-related factor 2 (TRF2) inactivation in Caenorhabditis elegans and Xenopus laevis, TRAF2-deficient mice are viable and show no apparent abnormalities in major organs (Zhang et al., 2001). However, TRAF2–/– male mice are sterile because of a severe defect in spermatogenesis, whereas female TRAF2-deficient mice are fertile and produce normal average litter size (Zhang et al., 2001). Female transgenic mice with selective over-expression of BCL- 2 in the somatic cells of the ovary exhibit decreased follicle apoptosis, enhanced folliculogenesis, larger litter size, and increased susceptibility to germ cell tumorigenesis (Hsu et al., 1996). In striking contrast, selective over-expression of BCL-2 in the somatic cells of the testis exhibits variable impairment of spermatogenesis (Yamamoto et al., 2001). Apaf-1 deficient mice closely resemble caspase-3 and caspase-9 knockout mice and die perinatally with severe craniofacial abnormalities, brain overgrowth, and reduced apoptosis in the central nervous system (Cecconi et al., 1998; Yoshida et al., 1998; Honarpour et al., 2000). Of further interest, approximately 5% of the Apaf-1 knockouts survive to adulthood, and, in contrast to the nonsurviving mutants, the survivors lack brain pathology but exhibit profound defects in spermatogenesis (Honarpour et al., 2000). The ablation of the Bax gene by homologous recombination also results in male sterility due to accumulation of atypical premeiotic germ cells but with accelerated

apoptosis of mature germ cells, leading to complete cessation of sperm production (Knudson et al., 1995). Conversely, thymocytes and B cells display hyperplasia, and Bax–/– ovaries accumulate an excess of granulosa cells and primary as well as primordial follicles as compared with those of wild-type mice (Knudson et al., 1995; Perez et al., 1999). Thus the loss of Bax results in hyperplasia or hypoplasia, depending on the cellular context. Taken together, these studies suggest that even the same molecule can play different roles in regulation of male germ cell apoptosis. More detailed analysis of the available mutant models with additional manipulation (using different apoptotic inducers) are needed to understand the precise signal transduction pathways regulating testicular germ cell apoptosis.

3. MODELS TO STUDY TESTICULAR GERM

CELL APOPTOSIS

3.1. Murine models

The issue of experimental models is important from a clinical standpoint because many aspects of signal transduction pathways regulating human testicular germ cell apoptosis are not amenable to study directly. Accordingly, we took advantage of various wellcharacterized model systems. Programmed germ cell death can be triggered by a variety of apoptotic stimuli, such as mild testicular hyperthermia, and deprivation of gonadotropins and testicular testosterone (T) by gonadotropin-releasing hormone antagonist (GnRH-A) or by exogenous administration T, Sertoli cell toxicant, and chemotherapeutic agents (reviewed in Sinha Hikim et al., 2003a). We previously reported that selective deprivation of gonadotropins and testicular T is followed by a stage-specific apoptosis of germ cells involving preleptotene and pachytene spermatocytes and round and elongated spermatids at mid (VII and VIII) stages (Sinha Hikim et al., 1995; Sinha Hikim et al., 1997). In subsequent studies, we demonstrated that transient heat exposure also induces stage-specific activation of apoptosis, but at different stages of spermatogenic cycle (Lue et al., 1999; Lue et al., 2000; Sinha Hikim et al., 2003b). In striking contrast to hormone deprivation model, a transient exposure of the testes to heat (43◦ C for 15 minutes) induces germ cell apoptosis exclusively at early (I– IV) and late (XII–XIV) stages. Pachytene spermatocytes and early spermatids (steps 1–4) at stages I through IV and pachytene, diplotene, and dividing spermatocytes at stages XII through XIV are most susceptible to heat. As depicted in Figure 25-1, the vulnerability of germ cells to apoptosis in these two paradigms is different. Similar

stage-specific activation of germ cell apoptosis triggered by testicular hyperthermia or hormone deprivation has also been validated in mouse models (Sinha Hikim et al., 2003a; Sinha Hikim et al., 2003b; Sinha Hikim et al., 2005; Vera et al., 2006). However, we had to add flutamide to GnRH-A for effective induction of male germ cell apoptosis in mice after hormone deprivation (Sinha Hikim et al., 2005; Vera et al., 2006).

3.2. Primate models

We have extended our experimental paradigm from rodents to monkeys (Lue et al., 2006; Jia et al., 2007) and more recently in humans (Wang et al., 2007) and demonstrated that, indeed, germ cell apoptosis plays an important role in the organized regression of spermatogenesis after hormone deprivation and/or testicular hyperthermia. We have further initiated in vitro studies in humans to elucidate the molecular mechanism of germ cell apoptosis (Vera et al., 2006). Induction of germ cell

apoptosis can be readily achieved by culturing segments of seminiferous tubules under serum-free conditions.

We took advantage of these different but complementary models for induction of testicular germ cell apoptosis to elucidate the key signal transduction pathways for male germ cell apoptosis across species.

3.3. Pathways of caspase activation and apoptosis

As depicted in Figure 25-2, the signaling events leading to apoptosis can be divided into two major pathways, involving either mitochondria or death receptors (Tilly 2001; Danial and Korsmeyer, 2004; Youle and Srasser, 2008). The mitochondria or the intrinsic pathway for apoptosis involves the release of cytochrome c into the cytosol, where it binds to apoptotic protease activating factor-1 (Apaf-1), resulting in the activation of the initiator caspase-9 and the subsequent proteolytic activation of the executioner caspases-3, -6, and -7. Members of the BCL-2 family of proteins play a major role in governing

this mitochondria-dependent apoptotic pathway, with proteins such as BAX functioning as inducer and proteins such as BCL-2 as suppressor of cell death. Additionally, SMAC (second mitochondria-derived activator of caspases), also known as DIABLO, is released from mitochondria into the cytosol after apoptotic stimuli and promotes apoptosis by antagonizing inhibitor of apoptosis proteins (IAPs). The death receptor or the extrinsic pathway for apoptosis involves ligation of the death receptor (such as FAS) to its ligand, FASL. Binding of FASL to FAS induces trimerization of FAS receptors, which recruit Fas-associated death domain (FADD) through shared death domains (DD). FADD also contain a death effector domain (DED) in its N-terminal region. FAS/FADD complex then binds to the initiator caspase-8 or -10, through interactions between DED of the FADD and these caspase molecules. Cross-talk between these pathways does occur at some levels. In certain cells, caspase- 8 through cleavage of BID, a proapoptotic BCL-2 family member, can induce cytochrome c release from mitochondria in FAS-mediated death signaling. Both these pathways converge on caspase-3 and other executioner caspases and nucleases that drive the terminal events of programmed cell death.

3.4. Apoptotic signaling in male germ cells

Mitochondria-dependent intrinsic pathway signaling is a key pathway for male germ cell apoptosis.

In earlier studies, using a rat model of testicular hyperthermia, we characterized the key molecular components of the effector pathways leading to caspase activation and increased germ cell death in the testis (Sinha Hikim et al., 2003b). Short-term exposure (43◦ C for 15 minutes) of the rat testis to mild heat results, within 6 hours, in stageand cell-specific activation of germ cell apoptosis. Initiation of apoptosis was preceded by a redistribution of BAX from a cytoplasmic to a paranuclear localization in heat-susceptible germ cells and elevated levels of BCL-2 in the mitochondria. The relocation of BAX is accompanied by cytosolic translocation of cytochrome c and is associated with activation of the initiator caspase-9 and the executioner caspases-3, -6, and -7 and cleavage of poly (ADP-ribose) polymerase (PARP). Collectively, these data suggest the involvement of the mitochondria-dependent pathway for heat-induced male germ cell apoptosis.

To characterize the involvement of the intrinsic pathway signaling for induction of apoptosis in our hormone deprivation model, groups of adult male rats were given a daily injection of vehicle for 14 days or GnRH-A acyline at a dose of 1.6 mg/kg of body weight for 2, 5, and

Pathways of Caspase Activation and Apoptosis

Figure 25-2. Intrinsic and extrinsic pathways for caspase activation and apoptosis. The mitochondria or the intrinsic pathway of apoptosis involves release of cytochrome c (Cyt c) from mitochondria into the cytosol, where it binds to Apaf 1, resulting in the activation of caspase-9 and the subsequent activation of the executioner caspases- 3, -6, and -7. The BCL-2 family of proteins usually governs the intrinsic pathway for apoptosis. The extrinsic or the death receptor pathway involves ligation of FAS to FASL, resulting in the activation of a di erent set of initiator caspases, namely caspase-8 and -10, through interactions between death domains and death e ector domains of an adopter molecule such as FADD and these caspases. Both these pathways eventually converge on caspase-3 and other executioner caspases that drive the terminal events of programmed cell death. Cross-talk between these pathways is mediated by BID, a proapoptotic BCL-2 family member. BID exists in the cytosolic fraction of living cells that becomes activated upon cleavage by caspase-8. The truncated cleavage product (tBID) then translocates to mitochondria and induces cytochrome c release. Interestingly, the functions of these caspases are inhibited by the inhibitor of apoptosis proteins (IAPs). The mitochondrial protein such as SMAC or DIABLO is released from mitochondria into the cytosol after apoptotic stimuli and promotes apoptosis by antagonizing IAPs.

14 days. Within 2 days of GnRH-A treatment, testicular concentrations of T declined markedly to 17.1% of control values and plasma T levels fell below detectable limits. Germ cell apoptosis, involving exclusively stages VII through VIII, was achieved by day 5. Within the study paradigm, the highest number of dying cells occurred by day 14, at which time a modest but significant increase in the incidence of apoptosis was also noted at stages other than VII through VIII. As shown in Figure 25-3, unlike our hyperthermia model, we found an increase in BAX and a decrease in BCL-2 expression in the mitochondrial fraction of testicular lysates after hormone withdrawal (Figure 25-3A). Such alteration in the BAX and BCL-2 ratio is accompanied by cytosolic translocation of mitochondrial cytochrome C and DIABLO (Figure 25-3B), activation of caspase-9 (Figure 25-3C) and caspase-3 (Figure 25-3D), and PARP cleavage (Figure 25-3E). These results indicate that withdrawal of gonadotropins and

consequently intratesticular T induces germ cell apoptosis in the testis also by stimulating the intrinsic pathway signaling (Vera et al., 2006). Together, these results demonstrate that the mitochondria-dependent pathway appears to be the key apoptotic pathway for germ cell death in the testis.

4. THE FAS SIGNALING SYSTEM DOES NOT CONTRIBUTE

TO HEATOR HORMONE DEPRIVATION–INDUCED MALE

GERM CELL APOPTOSIS

To evaluate the involvement of the Fas signaling system in male germ cell apoptosis, in this study, we examined whether gld and lprcg mice, which harbor loss-of- function mutations in FasL and Fas, respectively (Nagata and Golstein, 1995), would confer resistance to heatinduced germ cell apoptosis. Similar to our rat model, scrota of gld and lprcg mice and their wild types (C57BL6J and MRL/Mpj, respectively) were exposed once to 22◦ C (control) or 43◦ C (heat-treated) for 15 minutes, and the

animals were killed at 0.5, 2, or 6 hours after heating. The incidence of germ cell apoptosis before and after heat treatment was similar in both wild-type and mutant mice, suggesting that germ cells from wildtype and mutant mice with loss-of-function mutations in Fas ligand and Fas, respectively, are equally sensitive to heat-induced apoptosis (Sinha Hikim et al., 2003a; Sinha Hikim et al., 2003b). Of note, the initiation of apoptosis was preceded by a redistribution of BAX from a cytoplasmic to perinuclear localization in heat-susceptible germ cells (Vera et al., 2004). The relocation of BAX is further accompanied by sequestration of ultra-condensed mitochondria into perinuclear areas of apoptotic germ cells and cytosolic translocation of mitochondrial cytochrome c and DIABLO and is associated with activation of the initiator caspase-9 and the executioner caspase-3 (Vera et al., 2004). Furthermore, we did not observe the presence of the truncated BID in either cytosolic or mitochondrial fractions of heat-treated testicular lysates of both wild-type and