Ubiquitin dependent proteolysis is activated in apoptotic fibroblasts in culture

The ubiquitin mediated pathway constitutes an early response in cultured cells where apoptosis, assessed by internucleosomal specific DNA fragmentation, was induced by serum withdrawal. Data demonstrate that nuclear ubiquitin proteolytic system, but not cytoplasmic, is activated. This activation is paralleled by a substantial chromatin de-condensation. We suggest that chromatin relaxation is causative of the fragmentation since it exposes the DNA to nucleolytic attack. Finally, maintenance of homeostasis and induction of apoptosis seem to undergo a parallel contemporary pathway with a possible mutual feedback.     

Nitric oxide from both exogenous and endogenous sources activates mitochondria-dependent events and induces insults to human chondrocytes

During inflammation, overproduction of nitric oxide (NO) can damage chondrocytes. In this study, we separately evaluated the toxic effects of exogenous and endogenous NO on human chondrocytes and their possible mechanisms. Human chondrocytes were exposed to sodium nitroprusside (SNP), an NO donor, or a combination of lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) as the exogenous and endogenous sources of NO, respectively. Administration of SNP or a combination of LPS and IFN-gamma in human chondrocytes increased cellular NO levels but decreased cell viability. Exposure to exogenous or endogenous NO significantly induced apoptosis of human chondrocytes. When treated with exogenous or endogenous NO, the mitochondrial membrane potential time-dependently decreased. Exposure to exogenous or endogenous NO significantly enhanced cellular reactive oxygen species (ROS) and cytochrome c (Cyt c) levels. Administration of exogenous or endogenous NO increased caspase-3 activity and consequently induced DNA fragmentation. Suppression of caspase-3 activation by Z-DEVD-FMK decreased NO-induced DNA fragmentation and cell apoptosis. Similar to SNP, exposure of human chondrocytes to S-nitrosoglutathione (GSNO), another NO donor, caused significant increases in Cyt c levels, caspase-3 activity, and DNA fragmentation, and induced cell apoptosis. Pretreatment with N-monomethyl arginine (NMMA), an inhibitor of NO synthase, significantly decreased cellular NO levels, and lowered endogenous NO-induced alterations in cellular Cyt c amounts, caspase-3 activity, DNA fragmentation, and cell apoptosis. Results of this study show that NO from exogenous and endogenous sources can induce apoptotic insults to human chondrocytes via a mitochondria-dependent mechanism.     

Serum withdrawal and etoposide induce apoptosis in human lung carcinoma cell line A549 via distinct pathways

The molecular events associated with apoptosis induced by two distinct triggers (1) serum withdrawal and (2) etoposide treatment were investigated in the human lung carcinoma cell line A549. Although both serum withdrawal and etoposide treatment resulted in internucleosomal DNA fragmentation, the morphologic features were distinct. Serum deprived apoptotic cells appeared small, round and refractile, with little evidence of nuclear fragmentation; etoposide-induced apoptotic cells appeared enlarged and flattened and displayed prominent nuclear fragmentation. p53 and p21/waf1 protein levels were elevated in etoposide-treated cells, but not in cells subjected to serum with-drawal. Apoptosis induced by both treatments was accompanied by a significant reduction in Rb protein levels. However, etoposide treatment led to hypo-phosphorylation of Rb, while serum withdrawal did not alter the Rb phosphorylation pattern. Serum withdrawal-induced apoptosis was correlated with activation of JNK and suppression of ERK activities, while both JNK and ERK activities were slightly elevated during etoposid- induced apoptosis. Together, these results support the hypothesis that apoptosis induced by serum withdrawal and etoposide treatment occurs through different pathways and involves distinct mediators.     

Mechanism of apoptosis induced by a lysosomotropic agent, L-Leucyl-L-Leucine methyl ester

Lysosomes are fundamental for cell growth, and thus inhibition of the lysosomal function often leads to cell death. L-Leucyl-L-leucine methyl ester (LeuLeuOMe) is a lysosomotropic agent that induces apoptosis of certain immune cells. LeuLeuOMe is taken up through receptor-mediated endocytosis, and then converted into (LeuLeu)_n-OMe (n>3) by dipeptidyl peptidase I (DPPI) in lysosomes, which reportedly causes rupture of the lysosomes and DNA fragmentation. In this study we examined how lysosomal damage causes DNA fragmentation in LeuLeuOMe-treated HL-60 cells. When acridine orange was employed to monitor lysosomal membrane integrity, orange or red granular fluorescence was seen in normal cells. In contrast, LeuLeuOMe-treated cells showed orange, yellow or green cellular fluorescence all over the cytoplasm, suggesting that LeuLeuOMe induced a loss of lysosomal membrane integrity. The loss was inhibited by a DPPI inhibitor, GlyPheCHN₂ (GFCHN₂), but not by a caspase-3 inhibitor, Ac-DEVD-CHO, indicating that a condensation product was responsible for the loss. LeuLeuOMe also induced the activation of caspase-3-like protease and DNA fragmentation, both of which were inhibited by either GFCHN₂ or Ac-DEVD-CHO. Therefore, the activation of caspase-3-like protease links the loss of lysosomal membrane integrity to DNA fragmentation during apoptosis induced by LeuLeuOMe.     

Quercetin inhibited DNA synthesis and induced apoptosis associated with increase in c-fos mRNA level and the upregulation of p21WAF1CIP1 mRNA and protein expression during liver regeneration after partial hepatectomy

Quercetin, a widely distributed bioflavonoid, inhibited DNA synthesis in regenerating liver after partial hepatectomy. This inhibition was accompanied by apoptosis, evidenced by in situ end-labeling and gel electrophoresis of DNA fragmentation. Characteristic DNA fragmentation was detected as early as 2 h after injection. Northern blot analysis revealed that quercetin induced the increases in c-fos and p21WAF1CIP1 mRNA levels within 2 h. The expression of p21 protein was also enhanced, while p53 mRNA and protein levels were not affected by quercetin. These results suggest that quercetin-induced apoptosis is associated with the increase in c-fos mRNA level and the upregulation of p21 mRNA and protein expression, probably in a p53-independent pathway.     

Presence of DNA fragmentation and lack of neuroprotective effect in DFF45 knockout mice subjected to traumatic brain injury

BACKGROUND: Apoptosis plays an important pathophysiologic role in neuronal cell loss and associated neurologic deficits following traumatic brain injury (TBI). DNA fragmentation represents one of the characteristic biochemical features of neuronal apoptosis and is observed after experimental TBI. DFF45 and DFF40 are essential for DNA fragmentation in various models of apoptosis. MATERIALS AND METHODS: We used mice deficient in DFF45 and wild-type controls. Oligonucleosomal DNA fragmentation induced by TBI was analyzed using in vivo and in vitro assays. Expression and integrity of DFF45 and DFF40 proteins was assessed by Western analysis. Other outcome measurements included neurologic scoring, learning/memory tests, lesion volume measurements (MRI), and assessment of cell viability in vitro among others. RESULTS: We compared the effects of controlled cortical impact (CCI) trauma in DFF45 knockout mice and wild-type controls. Analysis of TBI-induced DNA fragmentation in brain cortex from wild-type and DFF45 knockout mice indicates that, although somewhat delayed, oligonucleosomal cleavage of DNA occurs after TBI in DFF45 knockout mice. DFF45 knockouts showed no significant differences in behavioral outcomes or lesion volumes after TBI as compared to wild-type controls. Using an in vitro reconstitution system, we also demonstrated that cleavage of DFF45 by caspase-3 is not sufficient for DNA fragmentation induced by protein extracts from rat brain cortex. We found that endonuclease activity induced in rat brain cortex following TBI depends on the presence of Mg2+ and Ca2+, but is not inhibited by Zn2+. Primary neuronal cultures from DFF45 knockouts failed to show DNA laddering in response to staurosporine, but did show prominent, albeit delayed, DNA fragmentation following treatment with etoposide. In contrast, primary neurons from wild-type animals demonstrated marked DNA fragmentation following treatment with staurosporine or etoposide. CONCLUSIONS: The results of this study suggest that, in addition to DFF45/40, other endonucleases may be essential for chromatin degradation during neuronal apoptosis in adult brain after TBI.     

IGF-1 regulates apoptosis of cardiac myocyte induced by osmotic-stress

Insulin-like growth factor-1 (IGF-1) is a natural protectant of cardiac myocytes that has been shown to improve cardiac function. The role of IGF-1 in attenuating apoptosis induced by osmotic stress (sorbitol, SOR) or by other known apoptotic stimuli (doxorubicin, angiotensin II, and serum withdrawal) was determined in cultured cardiac myocytes. After 6 h of exposure to SOR, apoptosis was initiated, concomitant with a decrease in cell survival and increases in poly-[ADP-ribose] polymerase (PARP) degradation and DNA fragmentation. These effects were maximal after 24 h. IGF-1 partially attenuated apoptosis induced by sorbitol but not that induced by angiotensin II, doxorubicin, or serum withdrawal. In cells preincubated with IGF-1 before the addition of SOR, we detected an increase in the number of viable cells, a decrease in the generation of DNA fragments on agarose gel electrophoresis and in the percentage of positive TUNEL cells, and a reduction on PARP levels. These results suggest that IGF-1 prevents apoptosis induced by osmotic stress in cardiac myocytes but not apoptosis induced by doxorubicin and angiotensin II.     

Antioxidants Inhibit the Human Cortical Neuron Apoptosis Induced by Hydrogen Peroxide,Tumor Necrosis Factor Alpha,Dopamine and Beta-amyloid Peptide 1-42

Several substances related to the neurodegenerative diseases of Alzheimer and Parkinson, such as hydrogen peroxide, tumor necrosis factor alpha, dopamine and beta-amyloid peptide 1-42, have been shown to induce apoptosis in tumoral cell lines and rat neurons but not in human neurons. Moreover, the role of mitochondria (membrane potential) during neuronal apoptosis is still a matter of debate. We present here, for the first time, in cultured human cortical neurons that the DNA fragmentation induced by these substances was preceded by a decrease of the mitochondrial membrane potential. We have also examined the antiapoptotic effect of the antioxidants glutathione, N -acetyl-cysteine and ascorbic acid. All these antioxidants inhibited the apoptosis induced by hydrogen peroxide, tumor necrosis factor alpha, dopamine and beta-amyloid peptide 1-42, since they were able to inhibit completely the mitochondrial membrane potential depolarization and the DNA fragmentation.     

Antioxidants inhibit the human cortical neuron apoptosis induced by hydrogen peroxide,tumor necrosis factor alpha,dopamine and beta-amyloid peptide 1-42

Several substances related to the neurodegenerative diseases of Alzheimer and Parkinson, such as hydrogen peroxide, tumor necrosis factor alpha, dopamine and beta-amyloid peptide 1-42, have been shown to induce apoptosis in tumoral cell lines and rat neurons but not in human neurons. Moreover, the role of mitochondria (membrane potential) during neuronal apoptosis is still a matter of debate. We present here, for the first time, in cultured human cortical neurons that the DNA fragmentation induced by these substances was preceded by a decrease of the mitochondrial membrane potential. We have also examined the antiapoptotic effect of the antioxidants glutathione, N -acetyl-cysteine and ascorbic acid. All these antioxidants inhibited the apoptosis induced by hydrogen peroxide, tumor necrosis factor alpha, dopamine and beta-amyloid peptide 1-42, since they were able to inhibit completely the mitochondrial membrane potential depolarization and the DNA fragmentation.     

Glutathione depletion-induced chromosomal DNA fragmentation associated with apoptosis and necrosis

Chromosomal DNA and mitochondrial dysfunctions play a role on mammalian cell death induced by oxidative stress. The major biochemical dysfunction of chromosome is the presence of an ordered cleavage of the DNA backborn, which is separated and visualized as an electrophoretic pattern of fragments. Oxidative stress provides chromatin dysfunction such as single strand and double strand DNA fragmentation leading to cell death. More than 1 Mb of giant DNA, 200-800 kb or 50-300 kb high molecular weight (HMW) DNA and internucleosomal DNA fragments are produced during apoptosis or necrosis induced by oxidative stress such as glutathione (GSH) depletion in several types of mammalian cells. Reactive oxygen species (ROS)-mediated DNA fragmentation is enhanced by polyunsaturated fatty acids including arachidonic acid or their hydroperoxides, leading to necrosis. Mitochondrial dysfunction on decrease of trans membrane potential, accumulation of ROS, membrane permeability transition and release of apoptotic factors during apoptosis or necrosis has been implicated. This review refers to the correlation of chromosomal DNA fragmentation and apoptosis or necrosis induced by GSH depletion, and the possible mechanisms of oxidative stress-induced cell death.     

Induction of Apoptotic Cell Death in a Neuroblastoma Cell Line by Dibucaine

Dibucaine, a local anesthetic known to interact with cell membranes, induced apoptosis in SK-N-MC human neuroblastoma cells in a dose-dependent manner. Apoptosis was demonstrated by direct visualization of morphological nuclear changes using a DAPI staining technique and confirmed by the production of characteristic ladder patterns of DNA fragmentation on gel electrophoresis. At concentrations which induced apoptosis, dibucaine significantly altered membrane fluidity, indicating that fluidity may be a major target for the cytotoxic action of dibucaine. Also, dibucaine increased intracellular calcium levels more effectively in calcium-containing Krebs–Ringer buffer than in calcium-free Krebs–Ringer buffer. Removal of extracellular calcium or addition of antioxidants or protein synthesis inhibitor effectively blocked dibucaine-induced apoptosis. These results suggest that membrane damage, intracellular calcium levels, and oxygen free radicals may be involved in the apoptosis induced by dibucaine.     

Brefeldin A Is a Potent Inducer of Apoptosis in Human Cancer Cells Independently of p53

Brefeldin A (BFA) is a natural product that affects the structure and function of the Golgi apparatus and is in development for cancer chemotherapy. We observed that a wide range of cancer cells could undergo DNA fragmentation associated with apoptosis after BFA treatment. This DNA fragmentation was induced within 15 h in HL60 leukemia cells and after 48 h in K562 leukemia and HT-29 colon carcinoma cells with BFA concentrations as low as 0.1 μM.The DNA fragmentation had the typical internucleosomal pattern in HL60 and HT-29 cells. Apoptotic cells were also detected by microscopy. BFA-induced apoptosis is p53-independent as HL60 and K562 cells are p53 null and HT-29 are p53 mutant cells. BFA could potentiate UCN-01 and staurosporine-induced DNA fragmentation in HL60 cells. Cyclin B1/Cdc2 kinase activity decreased after BFA treatment in HL60 cells, indicating that BFA-induced DNA fragmentation was independent of a cyclin B1/Cdc2 kinase upregulation pathway. Cycloheximide could not prevent BFA-induced DNA fragmentation in HL60 cells, suggesting that protein synthesis is not needed for HL60 cells to undergo apoptosis. On the contrary, cycloheximide blocked BFA-induced DNA fragmentation in HT-29 cells, indicating that apoptosis in HT-29 cells requires macromolecular synthesis. Cell-free system experiments suggested that cytosolic proteins play an important role in triggering DNA fragmentation during apoptosis induced by BFA. Our results show that transduction signaling pathways play central roles in apoptotic regulation.     

Herpes simplex virus 1 blocks caspase-3-independent and caspase-dependent pathways to cell death

Earlier reports have shown that herpes simplex virus 1 (HSV-1) mutants induce programmed cell death and that wild-type HSV blocks the execution of the cell death program triggered by viral gene products, by the effectors of the immune system such as the Fas and tumor necrosis factor pathways, or by nonspecific stress agents such as either osmotic shock induced by sorbitol or thermal shock. A report from this laboratory showed that caspase inhibitors do not block DNA fragmentation induced by infection with the HSV-1 d120 mutant. To identify the events in programmed cell death induced and blocked by HSV-1, we examined cells infected with wild-type virus or the d120 mutant or cells infected and exposed to sorbitol. We report that: (i) the HSV-1 d120 mutant induced apoptosis by a caspase-3-independent pathway inasmuch as caspase 3 was not activated and DNA fragmentation was not blocked by caspase inhibitors even though the virus caused cytochrome c release and depolarization of the inner mitochondrial membrane. (ii) Cells infected with wild-type HSV-1 exhibited none of the manifestations associated with programmed cell death assayed in these studies. (iii) Uninfected cells exposed to osmotic shock succumbed to caspase-dependent apoptosis inasmuch as cytochrome c was released, the inner mitochondrial potential was lost, caspase-3 was activated, and chromosomal DNA was fragmented. (iv) Although caspase-3 was activated in cells infected with wild-type HSV-1 and exposed to sorbitol, cytochrome c outflow, depolarization of the inner mitochondrial membrane, and DNA fragmentation were blocked. We conclude that although d120 induces apoptosis by a caspase-3-independent pathway, the wild-type virus blocks apoptosis induced by this pathway and also blocks the caspase-dependent pathway induced by osmotic shock. The block in the caspase-dependent pathway may occur downstream of caspase-3 activation.     

PAP modulations in Daudi cells and Molt-3 cells treated with etoposide are mutually associated with morphological evidence of apoptosis

Daudi (B-cell line) and Molt-3 (T-cell line) cells provide a model for the study of apoptosis, the induction of which is often accompanied by concominant modulations of proteins involved in mRNA maturation. One of these proteins is poly(A) polymerase (PAP), which is responsible for mRNA cleavage and polyadenylation. A number of recent reports also suggest involvement of mRNA maturation and stability in the induction of specific pathways of cell apoptosis. In this study we identified PAP activity levels and isoform modulations in two different cell lines (Daudi and Molt-3) and related them to DNA fragmentation (a hallmark of apoptosis) and cell cycle phase specificity in terms of the temporal sequence of events and the time that elapsed between administration of the apoptosis inducer (the widely used anticancer drug etoposide) and the observed effects. Treatment of both cell lines with 20 microg/mL etoposide induced apoptosis after four hours in Molt-3 cells and only after 24 hours in Daudi cells, as revealed by two independent methods. In Daudi cells the PAP activity levels and isoforms were downregulated prior to deltapsim reduction, DNA fragmentation and the morphological changes of the nucleus, whereas in Molt-3 cells no PAP activity and isoform modulations were observed prior to the early hallmarks of apoptosis.     

Glutamate-Treated Rat Cortical Neuronal Cultures Die in a Way Different from the Classical Apoptosis Induced by Staurosporine

The alkaloid protein kinase inhibitor staurosporine induced neuronal cell death with both the morphological and the biochemical characteristics of apoptosis. The punctate chromatin associated with apoptosis with retention of plasma membrane integrity was observed in neurons identified by colocalization of NeuN staining. Such cells had DNA fragmentation visualized byin situend-labeling which was seen as a laddered pattern upon gel electrophoresis. In contrast cells treated with glutamate did not exhibit either of these morphological or biochemical hallmarks of apoptosis. Instead a much smaller and more compact pyknotic structure was observed associated with smeared DNA fragmentation patterns. A confocal time-lapse study of the appearance of the morphological changes in individual nuclei after staurosporine treatment showed collapse into punctate chromatin over a period of 10 min. In contrast, the collapse into small pyknotic nuclei after glutamate treatment was at least 10 times slower. It is concluded that excitotoxicity produced by glutamate did not induce cell death by an apoptotic mechanism in cultured cortical neurons.     

The cellular apoptosis of murine BW5147 thymoma during cold shock]

The mode of T-lymphoma cell death induced by cold shock was studied. The rewarming of cells at 37 degrees C following a brief period of cold (0 degrees C) resulted in internucleosomal DNA fragmentation. The cells underwent cold shock-mediated apoptosis only at a reduced (2%) serum concentration. The apoptosis was not blocked by macromolecular synthesis inhibitors such as cycloheximide and antinomycin D, or by Quin-2. EGTA per se was responsible for the initiation of cell death. Colchicine also induced internucleosomal fragmentation of DNA. Our findings suggest that cold shock induced apoptosis is associated with low temperature mediated disruption of microtubules. The role of Ca2+ and growth factors in cold shock induced cell death is discussed.     

Hepatitis B virus X protein induces apoptosis by enhancing translocation of Bax to mitochondria

Hepatitis B virus X protein (HBx) is essential for viral replication and plays an important role in viral pathogenesis. HBx transactivates many viral and cellular genes and participates in cellular signal transduction pathways, proliferation, and apoptosis. In the present study, we report that HBx induces apoptosis by enhancing the translocation of Bax to mitochondria, followed by inducing the loss of mitochondrial membrane potential and release of cytochrome C. In addition, Bcl-2, inhibitor of Bax, rescues the disruption of mitochondrial membrane potential and DNA fragmentation induced by serum starvation in HepG2-X cells expressing HBx. We also found that HBx binds directly to Bax and interferes with the interaction between Bax and 14-3-3epsilon to enhance the translocation of Bax to mitochondria. Taken together, our data suggest that HBx induces apoptosis by interacting with Bax and enhancing its translocation to mitochondria.     

Isolation of Membrane Attack Complex of Complement from Myelin Membranes Treated with Serum Complement

Abstract: The interaction between complement and myelin membranes and its possible role in myelin damage and in the disposal of damaged myelin in vivo is of interest because activation of complement generates both opsonin(s) and membrane attack complex of complement. In our studies on the role of complement in demyelin-ation, we have shown that isolated myelin activates serum complement in the absence of myelin-specific antibody and that membrane attack complex of complement is the required factor in antibody-mediated demyelination of mouse cerebellar expiant cultures. In the present study, we examined whether activation of serum complement by myelin is associated with the formation of membrane attack complex of complement in myelin membranes. Extracts of myelin-associated proteins following incubation of myelin with fresh serum were studied by ultracentrifugation on a sucrose density gradient for detection of C5b-9 neoantigen. The subunit structure of C5b-9 was determined by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis, electroblotting, and immunostaining. Results indicate that the macromolecular complex consisting of late-acting complement components, C5-C9, was assembled in the target myelin membranes.     

Anti-NeuGcGM3 antibodies, actively elicited by idiotypic vaccination in nonsmall cell lung cancer patients, induce tumor cell death by an oncosis-like mechanism

1E10 is a murine anti-idiotypic mAb specific for an idiotypic mAb that reacts with NeuGc-containing gangliosides, sulfatides, and Ags expressed in some human tumors. In melanoma, breast, and lung cancer patients, this anti-idiotypic Ab was able to induce a specific Ab response against N-glycosylated gangliosides, attractive targets for cancer immunotherapy as these glycolipids are not naturally expressed in humans. A clinical study with nonsmall cell lung cancer patients showed encouraging clinical benefits. Immunological studies performed in 20 of these patients suggested a correlation between the induction of Abs against NeuGcGM3 and longer survival times. The induced anti-NeuGcGM3 Abs recognized and directly killed tumor cells expressing the Ag, by a mechanism independent of complement activation. In the present work, we show that this cytotoxicity differs from apoptosis because it is temperature independent, no chromatin condensation or caspase 3 induction are detected, and the DNA fragmentation induced has a different pattern than the one characteristic for apoptosis. It is a very quick process and involves cytosqeleton reorganization. The Abs induce cellular swelling and the formation of big membrane lesions that allow the leakage of cytoplasm and the loss of the cell membrane integrity. All of these characteristics resemble a process of oncotic necrosis. To our knowledge, this is the first report of the active induction in cancer patients of NeuGcGM3-specific Abs able to induce complement independent oncotic necrosis to tumor cells. These results contribute to reinforcing the therapeutic potential of anti-idiotypic vaccines and the importance of NeuGcGM3 ganglioside as antitumor target.     

Cryopreservation and thawing is associated with varying extent of activation of apoptotic machinery in subsets of ejaculated human spermatozoa

We investigated the impact of cryopreservation and thawing on levels of caspases-3, -8, and -9 activity, intact mitochondrial membrane potential (Deltapsim), and DNA fragmentation in human spermatozoa. Eleven pools of cryopreserved and eight pools of fresh semen samples were examined. Mature and immature fractions were separated on a two-layer density gradient (47% and 90%) and further subdivided based on the externalization of phosphatidylserine and its binding to annexin V-labeled superparamagnetic microbeads (ANMB). Levels of activated caspases were assessed using fluorescein-labeled inhibitors of caspases (FLICA), Deltapsim using a lipophilic cationic dye, and DNA fragmentation by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Cryopreservation was significantly associated with activation of caspases-3, -8, and -9, as well as disruption of the mitochondrial membrane potential but no significant changes were observed in DNA fragmentation. In mature sperm, caspase activation was only detected in the ANMB+ fraction, whereas in immature sperm, both ANMB+ and ANMB- fractions showed activated caspase levels. In ANMB+ immature sperm, apoptosis seemed to be triggered by a surface ligand-receptor mechanism as well as by disruption of mitochondria, whereas in ANMB- immature sperm, apoptosis was induced by activation of caspase-9 following loss of intact Deltapsim. These results demonstrate that selection of annexin V-negative mature spermatozoa might be of clinical relevance for fertility preservation, as this sperm fraction shows no activated apoptosis during the cryopreservation process.