Mitochondria-dependent pathway is involved in heat-induced male germ cell death: lessons from mutant mice

The signaling events leading to apoptosis can be divided into two major pathways, involving either mitochondria (intrinsic) or death receptors (extrinsic). In a recent study, we have shown the involvement of the mitochondria-dependent apoptotic pathway in heat-induced male germ cell apoptosis in the rat. In additional studies, using the gld (generalized lymphoproliferation disease) and lprcg (lymphoproliferation complementing gld) mice, which harbor loss-of-function mutations in Fas L and Fas, respectively, we have shown that heat-induced germ cell apoptosis is not blocked, thus providing evidence that the Fas signaling system is not required for heat-induced germ cell apoptosis in the testis. In the present study, we have found that the initiation of apoptosis in wild-type mice was preceded by a redistribution of Bax from a cytoplasmic to paranuclear localization in heat-susceptible germ cells. The relocation of Bax is accompanied by sequestration of ultracondensed mitochondria into paranuclear areas of apoptotic germ cells, cytosolic translocation of mitochondrial cytochrome c and DIABLO, and is associated with activation of the initiator caspase 9 and the executioner caspase 3. Similar events were also noted in both gld and lprcg mice. Taken together, these results indicate that the mitochondria-dependent pathway is the key apoptotic pathway for heat-induced male germ cell death in mice.     

Functional role of caspases in heat-induced testicular germ cell apoptosis

In the present study, we determined whether a pan caspase inhibitor could prevent or attenuate heat-induced germ cell apoptosis. Groups of five adult (8 wk old) C57BL/6 mice pretreated with vehicle (DMSO) or Quinoline-Val-Asp (Ome)-CH2-O-Ph (Q-VD-OPH), a new generation broad-spectrum caspase inhibitor, were exposed once to local testicular heating (43 degrees C for 15 min) and killed 6 h later. The inhibitor (40 mg/kg body weight) or vehicle was administered intraperitoneally (i.p.) 1 h before local testicular heating. Germ cell apoptosis was detected by TUNEL assay and quantitated as number of apoptotic germ cells per 100 Sertoli cells at stages XI-XII. Compared with controls (16.8 +/- 3.1), mild testicular hyperthermia within 6 h resulted in a marked activation (277.3 +/- 21.6) of germ cell apoptosis, as previously reported by us. Q-VD-OPH at this dose markedly inhibited caspase 3 activation and significantly prevented (by 67.0%) heat-induced germ cell apoptosis. Q-VD-OPH-mediated rescue of germ cells was independent of cytosolic translocation of mitochondrial cytochrome c and DIABLO. Electron microscopy further revealed normal appearance of these rescued cells. Similar protection from heat-induced germ cell apoptosis was also noted after pretreatment with minocycline, a second-generation tetracycline that effectively inhibits cytochrome c release and, in turn, caspase activation. Collectively, the present study emphasizes the role of caspases in heat-induced germ cell apoptosis.     

Role of caspase 2 in apoptotic signaling in primate and murine germ cells

This study investigates the role of caspase 2 in apoptotic signaling of nonhuman primate male germ cells triggered by mild testicular hyperthermia, testosterone (T(e)) implants, or by combined interventions. Mean incidence of germ cell apoptosis increased significantly by Day 3 in the heat (H(e)) alone group and by Day 8 in the Te alone group but peaked at Day 3 in H(e) + T(e) group. We found activation of caspase 2 in both germ cells and Sertoli cells after induction of apoptosis. Most notably, active caspase 2 immunoreactivity was detected only in those germ cells susceptible to apoptosis compared with controls, where little or no such staining is detected. To further explore the role of caspase 2 in regulating male germ cell death, we next evaluated the efficacy of caspase 2 inhibition in preventing or attenuating heat-induced germ cell apoptosis in rats. Caspase 2 inhibition significantly (P < 0.05) prevented such heat-induced germ cell apoptosis. The protection offered by the caspase 2 inhibitor occurred upstream of mitochondria, involving suppression of mitogen-activated protein kinase (MAPK) 14 activation and inducible nitric oxide synthase (NOS2) induction and, in turn, suppression of cytochrome c-mediated death pathway. Together, our results show that caspase 2 is activated in male germ cells undergoing apoptosis in nonhuman primates after heat stress, hormonal deprivation, or after combined interventions. Blockade of caspase 2 activation prevents heat-induced germ cell apoptosis in rats by suppressing the MAPK14- and NO-mediated intrinsic pathway signaling.     

Caspase 2 activity contributes to the initial wave of germ cell apoptosis during the first round of spermatogenesis

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.     

Role of death receptor,mitochondrial and endoplasmic reticulum pathways in different stages of degenerative human lumbar disc

Intervertebral disc (IVD) cell apoptosis has been suggested to play an important role in promoting the degeneration process. It has been demonstrated that IVD cell apoptosis occurs through either death receptor, mitochondrial or endoplasmic reticulum (ER) pathway. Our study aimed to explore the relationship among these three pathways and grade of IVD degeneration (IVDD). IVDs were collected from patients with lumbar fracture, vertebral tumor, disc herniation or spondylolisthesis. IVDs were distinguished by MRI and histomorphological examination, cell apoptosis was detected by TUNEL staining. Biomarkers of these three apoptosis pathways were detected by RT-PCR and Western blot. Furthermore, the correlation between apoptosis pathways biomarkers and disc pathology were analyzed. Nucleus pulposus cell density decreased with degeneration process, and increased apoptotic ratio. ER pathway was predominant in mild stage of IVDD (GRP78, GADD153 upregulation and caspase-4 activation), death receptor pathway was predominant in mild and moderate stages (Fas, FasL up-regulation and caspase-8 activation) and mitochondrial pathway was predominant in moderate and severe stages (Bcl-2 down-regulation, Bax up-regulation, cytochrome-c accumulation in cytoplasm and caspase-9 activation). There were significant differences in the expressions of Fas, FasL, Bax, GADD153, cytochrome-c and cleaved caspase-8/9/3 between contained and non-contained discs. In conclusion, apoptosis occurs via these three apoptosis pathways together in IVDD. ER pathway plays a more critical role in the mild compared to moderate and severe stages, death receptor pathway in mild and moderate, and mitochondrial pathway in moderate and severe stages of IVDD. Disc cells apoptosis may progress rapidly after herniation, and may depend on the type of herniation.     

BI-1 regulates an apoptosis pathway linked to endoplasmic reticulum stress

Bax inhibitor-1 (BI-1) is an evolutionarily conserved endoplasmic reticulum (ER) protein that suppresses cell death in both animal and plant cells. We characterized mice in which the bi-1 gene was ablated. Cells from BI-1-deficient mice, including fibroblasts, hepatocytes, and neurons, display selective hypersensitivity to apoptosis induced by ER stress agents (thapsigargin, tunicamycin, brefeldin A), but not to stimulators of mitochondrial or TNF/Fas-death receptor apoptosis pathways. Conversely, BI-1 overexpression protects against apoptosis induced by ER stress. BI-1-mediated protection from apoptosis induced by ER stress correlated with inhibition of Bax activation and translocation to mitochondria, preservation of mitochondrial membrane potential, and suppression of caspase activation. BI-1 overexpression also reduces releasable Ca(2+) from the ER. In vivo, bi-1(-/-) mice exhibit increased sensitivity to tissue damage induced by stimuli that trigger ER stress, including stroke and tunicamycin injection. Thus, BI-1 regulates a cell death pathway important for cytopreservation during ER stress.     

Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis

Bax and Bak play a redundant but essential role in apoptosis initiated by the mitochondrial release of apoptogenic factors. In addition to their presence at the mitochondrial outer membrane, Bax and Bak can also localize to the ER. Agents that initiate ER stress responses can induce conformational changes and oligomerization of Bax on the ER as well as on mitochondria. In wild-type cells, this is associated with caspase 12 cleavage that is abolished in bax-/-bak-/- cells. In bax-/-bak-/- cells, introduction of Bak mutants selectively targeted to either mitochondria or the ER can induce apoptosis. However, ER-targeted, but not mitochondria-targeted, Bak leads to progressive depletion of ER Ca2+ and induces caspase 12 cleavage. In contrast, mitochondria-targeted Bak leads to enhanced caspase 7 and PARP cleavage in comparison with the ER-targeted Bak. These findings demonstrate that in addition to their functions at mitochondria, Bax and Bak also localize to the ER and function to initiate a parallel pathway of caspase activation and apoptosis.     

Trichosanthin induced apoptosis in HL-60 cells via mitochondrial and endoplasmic reticulum stress signaling pathways

Trichosanthin (TCS), a traditional Chinese medicine, exerts antitumor activities by inducing apoptosis in many different tumor cell lines. However, the mechanisms remain obscure. The present study focused on various caspase pathways that may be involved in TCS-induced apoptosis in leukemia HL-60 cells. Key caspases in both intrinsic and extrinsic pathways including caspase-8, -9 and -3 were activated upon TCS treatment. Additionally, TCS treatment induced upregulation of BiP and CHOP and also activated caspase-4, which for the first time strongly supported the involvement of endoplasmic reticulum stress pathway in TCS-induced apoptosis. Interestingly, although caspase-8 was activated, Fas/Fas ligand pathway was not involved as evidenced by a lack of induction of Fas or Fas ligand and a lack of inhibitory effect of anti-Fas blocking antibody on TCS-induced apoptosis. Instead, caspase-8 was activated in a caspase-9 and -4 dependent manner. The involvement of mitochondria was demonstrated by the reduction of mitochondrial membrane potential and release of cytochrome c and Smac besides the activation of caspase-9. Further investigation confirmed that caspase-3 was the major executioner caspase downstream to caspase-9, -4 and -8. Taken together, our results suggested that TCS-induced apoptosis in HL-60 cells was mainly mediated by mitochondrial and ER stress signaling pathways via caspase-3.     

Germ cell apoptosis in the testes of Sprague Dawley rats following testosterone withdrawal by ethane 1,2-dimethanesulfonate administration: relationship to Fas?

Germ cell apoptosis, which occurs normally during spermatogenesis, increases after testosterone withdrawal from the testis. The molecular mechanism by which this occurs remains uncertain. The Fas system has been implicated as a possible key regulator of apoptosis in various cells: binding of Fas ligand (FasL), a type II transmembrane protein, to Fas, a type I transmembrane receptor protein, triggers apoptosis in cells expressing Fas. Recently, Fas has been localized to germ cells, and FasL to Sertoli cells, within the rat testis. We hypothesized that Fas protein content would rise in response to reduced levels of testosterone as part of a suicide pathway that would result in germ cell apoptosis. To test this hypothesis, ethane 1,2-dimethanesulfonate (EDS), a Leydig cell toxicant, was used to kill Leydig cells and thus reduce intratesticular testosterone levels in Sprague Dawley rats. Apoptosis was examined in situ and biochemically, and Fas protein content in the testis was monitored by Western blot analysis. We show that EDS injection results in the following sequence of events: apoptotic death of Leydig cells by a mechanism that does not involve Fas; reduced testosterone; increased testicular Fas content; and germ cell apoptosis. These results suggest that Fas may play a role in the apoptotic death of germ cells that results from reduced intratesticular testosterone levels, and that testosterone may play a role in germ cell survival via its suppression of Fas.     

Heat-induced testicular apoptosis occurs independently of hormonal depletion

Previous studies have demonstrated that testicular germ cell apoptosis can be induced both by heat stress and by withdrawal of androgens and gonadotrophins. To investigate whether heat-induced germ cell apoptosis occurs independently of the altered levels of hormones that occur with heat exposure, mouse testicular apoptosis was studied using an in vitro system with controlled levels of testosterone, FSH and LH. It was observed that cells underwent apoptosis sooner in the absence of hormones at the same temperature. Apoptosis also occurred earlier at abdominal temperature compared to scrotal temperature with the same hormonal levels. No somatic tissues studied underwent apoptosis at 37°C under the same culture conditions. These results suggest that heat stress may independently activate an apoptotic pathway in the testis, and that hormone deprivation may induce apoptosis via a separate mechanism.     

Caspase-independent Cell Killing by Fas-associated Protein with Death Domain

The binding of Fas ligand to Fas recruits caspase 8 to Fas via an adaptor, FADD/MORT1, and activates a caspase cascade leading to apoptosis. Here, we describe a human Jurkat-derived cell line (JB-6) that is deficient in caspase 8. This cell line was resistant to the apoptosis triggered by Fas engagement. However, the multimerization of Fas-associated protein with death domain, through the use of a dimerizing system, killed the JB-6 cells. This killing process was not accompanied by the activation of caspases or DNA fragmentation. The dying cells showed neither condensation nor fragmentation of cells and nuclei, but the cells and nuclei swelled in a manner similar to that seen in necrosis. These results suggested that Fas-associated protein with death domain can kill the cells via two pathways, one mediated by caspases and another that does not involve them.     

Bovine ephemeral fever virus-induced apoptosis requires virus gene expression and activation of Fas and mitochondrial signaling pathway

Although induction of apoptosis by bovine ephemeral fever virus (BEFV) in several cell lines has been previously demonstrated by our laboratory, less information is available on the process of BEFV-induced apoptosis in terms of cellular pathways and specific proteins involved. In order to determine the step in viral life cycle at which apoptosis of infected cells is triggered, chemical and physical agents were used to block viral infection. Treatment of BHK-21 infected cells with ammonium chloride (NH₄Cl) or cells infected with UV-inactivated BEFV was seen to abrogate virus apoptosis induction, suggesting that virus uncoating and gene expression are required for the induction of apoptosis. Using soluble death receptors Fc:Fas chimera to block Fas signaling, BEFV-induced apoptosis was inhibited in cells. BEFV infection of BHK-21 cells results in the Fas-dependent activation of caspase 8 and cleavage of Bid. This initiated the dissipation of the membrane potential and the release of cytochrome c but not AIF or Smac/DIABLO from mitochondrial into cytoplasm leading to activation of caspase 9. Combined activation of the death receptor and mitochondrial pathways results in activation of the downstream effecter caspase 3 leading to cleavage of PARP. Fas-mediated BEFV-induced apoptosis could be suppressed by the overexpression of Bcl-2 or by treatment with caspase inhibitors and soluble death receptors Fc:Fas chimera. Taken together, this study provided first evidence demonstrating that BEFV-induced apoptosis requires viral gene expression and occurs through the activation of Fas and mitochondrion-mediated caspase-dependent pathways. An erratum to this article can be found at     

Bax and the mitochondrial permeability transition cooperate in the release of cytochrome c during endoplasmic reticulum-stress-induced apoptosis

Endoplasmic reticulum (ER) stress induces apoptosis by mechanisms that are not fully clear. Here we show that ER stress induced by the Ca(2+)-ATPase inhibitor thapsigargin (THG) activates cytochrome c-dependent apoptosis through cooperation between Bax and the mitochondrial permeability transition (MPT) in human leukemic CEM cells. Pharmacological inhibition of the MPT as well as small interfering RNA (siRNA) knockdown of the MPT core component cyclophilin D blocked cytochrome c release and caspase-dependent apoptosis but did not prevent Bax activation, translocation or N-terminal exposure in mitochondria. siRNA knockdown of Bax also blocked THG-mediated cytochrome c release and apoptosis, but did not prevent MPT activation and resulted in caspase-independent cell death. Our results show that ER-stress-induced cell death involves a caspase and Bax-dependent pathway as well as a caspase-independent MPT-directed pathway.