CRM1-mediated nuclear export determines the cytoplasmic localization of the antiapoptotic protein Survivin

Survivin is a member of the inhibitor of apoptosis (IAP) family of negative regulators of programmed cell death that is frequently overexpressed in human tumors. Survivin is not only involved in the regulation of apoptosis, but is also known to play a role in the control of cell cycle progression at the G2/M phase. Survivin is a predominantly cytoplasmic protein expressed in a cell cycle-dependent manner, but the mechanism(s) that determine its nuclear-cytoplasmic localization have not been described. In this study, we report that Survivin is a nuclear shuttling protein that is actively exported from the nucleus via the CRM1-dependent pathway. Nuclear export of Survivin is independent of the export of other shuttling proteins that control the G2/M phase transition, such as cyclin B1 and cdc25. The carboxy-terminal domain of Survivin is both necessary and sufficient for its nuclear export, although this region does not contain a functional leucine-rich nuclear export signal. Differences in the amino acid sequence of this region determine the dramatically different localization of Survivin (in the cytoplasm) and its splicing variant Survivin-DeltaEx3 (in the nucleus). The carboxy-terminal end of Survivin-DeltaEx3 contains a bipartite nuclear localization signal, not present in Survivin, which mediates its strong nuclear accumulation. These data suggest that active transport between the nucleus and cytoplasm may constitute an important regulatory mechanism for Survivin function.     

A single amino acid change (Asp 53 --> Ala53) converts Survivin from anti-apoptotic to pro-apoptotic

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that has been implicated in both apoptosis inhibition and cell cycle control. Recently, Survivin has attracted growing attention because of its tumor-specific expression and potential applications in tumor therapy. However, its inhibitory mechanism and subcellular localization remain controversial. Here, we report a novel Survivin mutant Surv-D53A, which displays a function opposite to Survivin and a distinctive subcellular distribution compared with its wild-type counterpart. Surv-D53A was shown to induce apoptosis in a p53-independent manner, indicating that tumor suppressor p53 is not involved in its apoptosis pathway. Surv-D53A was shown to markedly sensitize apoptosis induced by TRAIL, doxorubicin, and RIP3. We also demonstrated that similar to wild-type Survivin, Surv-D53A was localized in cytoplasm in interphase and to midbody at telophase. However, it fails to colocalize in chromosomes with Aurora-B in metaphase as wt-Survivin. Surv-D53A mutant is less stable than wt-Survivin and is degraded more rapidly by ubiquitin-proteasome pathway. Additionally, we found that Surv-D53A interacts with wt-Survivin to form heterodimer or with itself to form mutant homodimer, which may account for the loss of its antiapoptotic function. Finally, unlike Survivin*Survivin, neither Surv-D53A*Survivin nor Surv-D53A*Surv-D53A is able to bind to Smac/DIABLO, which may explain the underlying mechanism for its abolishment of antiapoptotic activity of Survivin.     

Participation of Survivin in mitotic and apoptotic activities of normal and tumor-derived cells

Survivin is a member of the inhibitor of apoptosis (IAP) gene family, containing a single baculovirus IAP repeat (BIR) and no RING finger, that is expressed in many human cancers. Although it has been proposed to be involved in mitotic and cytokinetic processes, its functional subcellular distribution in the cytoplasm and nucleus, and its binding to centrosomes, spindle fibers, and centromeres in relation to these processes, is not fully resolved. We have analyzed the localization of Survivin in normal (Detroit 551, IMR-90) and tumor-derived (HeLa, Saos-2) cell lines, and found that it does colocalize with centrosomes in the cytoplasm during interphase, then moves to centromeres during mitosis, and finally localizes to the midbody spindle fibers during telophase. However, Taxol, a popular microtubule stabilizing agent that is frequently used in the study of these processes, severely disrupted the localization of Survivin. Taxol treatment of cells promoted extensive relocalization of Survivin with alpha-tubulin on microtubules during either interphase or mitosis. Survivin antisense oligonucleotide markedly sensitized HeLa cells to cell death induced by agents acting at the level of cell surface receptor (Fas pathway) or at the level of mitochondria (etoposide). HeLa cell death induced by Survivin antisense oligonucleotide could be partially complemented by Deterin, the Drosophila homolog of Survivin (Jones et al. [2000] J. Biol. Chem. 275:22157-22166). Reciprocally, a chimera of the Deterin BIR domain and Survivin C-terminus could rescue Drosophila Kc cells from death induced by transfection of a human caspase-7-expressing plasmid. These results indicate common components of Survivin and Deterin antiapoptotic action in the vertebrate and invertebrate phyla.     

通过核质转运调节细胞凋亡的蛋白质

凋亡,也称Ⅰ型程序性细胞死亡,是细胞在面临严重威胁时发起的保护性主动死亡机制. 凋亡对于个体的生长发育及各种生理功能具有不可或缺的作用. 作为涉及整个细胞的复杂过程,凋亡的顺利进行有赖于众多凋亡相关因子的协调合作与精确调控. 细胞受到凋亡刺激后,核内的某些蛋白质转运出核,将凋亡信号传递到核外,胞质内的多种蛋白质则转运入核,在细胞核这一信息整合的大本营直接发挥作用. 这种双向交流机制在胞核与胞质间建立起密切的联系,同时使得相关蛋白质在特定场所发挥促进或抑制凋亡的作用,确保凋亡信号及时、通畅、有序地传递. 因此,蛋白质的核质转运作为介导胞核与胞质物质交换、信号交流的关键机制,在凋亡过程中就显得尤为重要. 本文主要就核质转运的机制、通过核质转运调节凋亡的蛋白质及其作用机理作一综述.     

Survivin is essential for fertile egg production and female fertility in mice

Survivin is the smallest member of the inhibitor of apoptosis protein (IAP) family and acts as a bifunctional protein involved in mitosis regulation and apoptosis inhibition. To identify the physiological role of Survivin in female reproduction, we selectively disrupted Survivin expression in oocytes and granulosa cells (GCs), two major cell types in the ovary, by two different Cre-Loxp conditional knockout systems, and found that both led to defective female fertility. Survivin deletion in oocytes did not affect oocyte growth, viability and ovulation, but caused tetraploid egg production and thus female infertility. Further exploration revealed that Survivin was essential for regulating proper meiotic spindle organization, spindle assembly checkpoint activity, timely metaphase-to-anaphase transition and cytokinesis. Mutant mice with Survivin depleted in GCs showed reduced ovulation and subfertility, caused by defective follicular growth, increased follicular atresia and impaired luteinization. These findings suggest that Survivin has an important role in regulating folliculogenesis and oogenesis in the adult mouse ovary.     

Survivin regulates Plk1 localization to kinetochore in mouse oocyte meiosis

Survivin is a member of inhibitors of apoptosis proteins (IAPs), and also belongs to be a member of the chromosomal passenger complex (CPC) which has multiple functions including inhibition of apoptosis and regulation of cell division and SAC activity. Plk1 (polo-like kinase 1) associates with the spindle poles and also distributes to the kinetochores and is shown to involve in spindle organization, APC/C activation and cytokinesis in many models. Our recent work has shown that Survivin is a critical regulator of chromosome segregation and spindle assembly checkpoint (SAC) in meiosis. In the present study, we found that Plk1 co-localized with Survivin at metaphase I (MI) and telophase I (TI) stage after GVBD. Plk1 dispersed into the oocyte cytoplasm or accumulated near the chromosomes after the depletion of Survivin by morpholino (MO) injection. Our results showed that the localization of Plk1 to kinetochores required the involvement of Survivin.     

Promyelocytic leukemia protein 4 induces apoptosis by inhibition of survivin expression

The promyelocytic leukemia protein (PML) plays an essential role in multiple pathways of apoptosis. Our previous study showed that PML enhances tumor necrosis factor-induced apoptosis by inhibiting the NFkappaB survival pathway. To continue exploring the mechanism of PML-induced apoptosis, we performed a DNA microarray screening of PML target genes using a PML-inducible stable cell line. We found that Survivin was one of the downstream target genes of PML. Cotransfection experiments demonstrated that PML4 repressed transactivation of the Survivin promoter in an isoform-specific manner. Western blot analysis demonstrated that induced PML expression down-regulated Survivin. Inversely, PML knockdown by siRNA up-regulated Survivin expression. A substantial increase in Survivin expression was found in PML-deficient cells. Re-expression of PML in PML-/- mouse embryo fibroblasts down-regulated the expression of Survivin. Furthermore, cells arrested at the G2/M cell cycle phase expressed a high level of Survivin and a significantly lower level of PML. Overexpression of PML in A549 cells reduced Survivin expression leading to massive apoptotic cell death associated with activation of procaspase 9, caspase 3, and caspase 7. Together, our results demonstrate a novel mechanism of PML-induced apoptosis by down-regulation of Survivin.     

Intracellular nucleotides act as critical prosurvival factors by binding to cytochrome C and inhibiting apoptosome

Cytochrome c (CC)-initiated Apaf-1 apoptosome formation represents a key initiating event in apoptosis. This process can be reconstituted in vitro with the addition of CC and ATP or dATP to cell lysates. How physiological levels of nucleotides, normally at high mM concentrations, affect apoptosome activation remains unclear. Here we show that physiological levels of nucleotides inhibit the CC-initiated apoptosome formation and caspase-9 activation by directly binding to CC on several key lysine residues and thus preventing CC interaction with Apaf-1. We show that in various apoptotic systems caspase activation is preceded or accompanied by decreases in overall intracellular NTP pools. Microinjection of nucleotides inhibits whereas experimentally reducing NTP pools enhances both CC and apoptotic stimuli-induced cell death. Our results thus suggest that the intracellular nucleotides represent critical prosurvival factors by functioning as natural inhibitors of apoptosome formation and a barrier that cells must overcome the nucleotide barrier to undergo apoptosis cell death.     

Cleavage of Survivin by Granzyme M Triggers Degradation of the Survivin-X-linked Inhibitor of Apoptosis Protein (XIAP) Complex to Free Caspase Activity Leading to Cytolysis of Target Tumor Cells

Granzyme M (GzmM) is a chymotrypsin-like serine protease that preferentially cuts its substrates after Met or Leu. GzmM is constitutively expressed in activated innate effector natural killer (NK) cells. GzmM-induced cell death is consistent with the kinetics of cytotoxicity of NK cells. These suggest that GzmM may play an important role in innate immunity. Our previous work demonstrated that GzmM induces caspase-dependent apoptosis. However, it is unknown about how GzmM causes caspase activation. Here, we showed that the inhibitor of the apoptosis gene family member Survivin is a physiological substrate for GzmM. GzmM hydrolyzes Survivin at Leu-138 to remove the last four C-terminal residues. The truncated form (sur-TF) is more rapidly hydrolyzed through proteasome-mediated degradation. In addition, Survivin is in complex with X-linked inhibitor of apoptosis protein (XIAP) to inhibit caspase activation as an endogenous inhibitor. Survivin cleavage by GzmM abolishes the stability of the Survivin-XIAP complex and enhances XIAP hydrolysis, which amplifies caspase-9 and 3 activation of target tumor cells. The noncleavable L138A Survivin overexpression can significantly inhibit GzmM-mediated XIAP degradation, caspase activation, and GzmM- and NK cell-induced cytotoxicity. Moreover, Survivin silencing promotes XIAP degradation and enhances GzmM-induced caspase activation as well as GzmM- and NK cell-induced cytolysis of target tumor cells.     

The mitotic regulator Survivin binds as a monomer to its functional interactor Borealin

Survivin is a member of the IAP (inhibitor of apoptosis) protein family, defined in part by the presence of a zinc-binding baculoviral inhibitory repeat (BIR) domain. Most BIR domains bind short sequences beginning with alanine, and in this manner, they recognize and block the action of key targets in apoptotic pathways. However, Survivin binds only very weakly to typical IAP ligands. Unique features of Survivin are the long C-terminal helix following the BIR domain and a short segment (linking the helix and BIR domains) that mediates Survivin homodimerization. Despite this detailed knowledge of the structure of Survivin itself, there is a current lack of understanding about how Survivin recognizes cellular binding partners, and consequently, many questions about Survivin function remain unanswered. We determined two co-crystal structures of Survivin and a minimal binding fragment from the chromosomal passenger protein Borealin, a well validated functional interactor. The interaction between Survivin and Borealin involves extensive packing between the long C-terminal helix of Survivin and a long Borealin helix. Surprisingly, an additional important interaction occurs between the Survivin homodimerization interface and a short segment of Borealin. This segment both structurally mimics and displaces one Survivin monomer. The relevance of this unexpected interaction was tested by mutagenesis of two key Borealin residues. Mutant Borealin introduced into HeLa cells failed to localize properly during mitosis and also caused mislocalization of other chromosomal passenger proteins. This suggests that the mutant is dominant-negative and confirms the functional importance of the interaction surface identified in the crystal structures.     

Structural changes of mitochondria related to apoptosis: swelling and megamitochondria formation.

Recently we have found that the formation of megamitochondria in culture cells of various sources, induced by chemicals capable of generating free radicals, is followed by apoptotic changes of the cell. Detailed analysis on functional and morphological aspects of megamitochondria has enabled us to speculate that the formation of megamitochondria may be a prerequisite for free radical-mediated apoptosis: free radicals modify the mitochondrial membranes resulting in the fusion of adjacent mitochondria (megamitochondria formation). If the intracellular level of free radicals is continuously kept high, the permeability transition pores of the megamitochondria membranes are opened and megamitochondria become swollen. Oxygen consumption and the ability to synthesise ATP by swollen megamitochondria decrease distinctly. At the same time, cytochrome c is released from swollen megamitochondria into the cytoplasm. If lowered rates of the generation of reactive oxygen species from swollen megamitochondria, possibly due to decrease in their oxygen consumption, are effective enough to lower the intracellular level of free radicals, megamitochondria may return to normal. If not, decrease in the membrane potential of megamitochondria membranes causes the release of apoptosis-inducing factor into the cytoplasm. Cytochrome c and apoptosis-inducing factor thus released into the cytoplasm may cause cytoplasmic and nuclear apoptotic changes. Experimental data to support this hypothesis are presented.     

Separating the anti-apoptotic and mitotic roles of survivin

Survivin is a bifunctional protein that acts as a suppressor of apoptosis and has an essential role in mitosis. To date whether these two functions can be divorced has not been addressed. Here we show that the linker region between the BIR (baculovirus inhibitor of apoptosis repeat) domain of survivin and COOH-terminal alpha helix may be the key to separating its roles. When overexpressed survivin is present in interphase cells and shuttles between the cytoplasm and nucleus. Here we identify a rev-like nuclear exportation signal (NES) in the central domain of survivin and demonstrate that point mutations within this region cause accumulation of survivin in the nucleus. Interestingly cells expressing NES mutants exhibit reduced survival after X-irradiation. Moreover, cells expressing survivin(L98A)-green fluorescent protein (GFP) showed increased poly(ADP-ribose) polymerase-cleavage and caspase-3 activity after tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment compared with cells expressing full-length survivin-green fluorescent protein. These data suggest a direct link between the interphase localization of survivin and cellular responsiveness to apoptotic stimuli. Using a cell proliferation assay, we also found that ectopic expression of NES mutants can complement for depletion of endogenous survivin, indicating that they can execute the mitotic duties of survivin. Thus we demonstrate for the first time that 1) survivin has a functional NES; 2) nuclear accumulation of overexpressed survivin correlates with increased sensitivity of cells to ionising radiation; and 3) the anti-apoptotic and mitotic roles of survivin can be separated through mutation of its NES. Separating these two functions of survivin could open up new possibilities for therapeutic strategies aimed at eliminating cancer cells yet preserving normal cell viability.     

Differential Cellular Localization of Galectin-1 and Galectin-3 in the Regressing Corpus Luteum of Mice and Their Possible Contribution to Luteal Cell Elimination

Galectin-1 and galectin-3, β-galactoside–binding lectins, are predominantly expressed in the regressing corpus luteum (CL) of mouse ovary. This study revealed the expression patterns and cellular localizations of galectins during CL formation and regression by ISH and IHC. Galectin-1 mRNA expression temporarily increased in active CL, preceding the expression of progesterone degradation enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD), which represents functional luteolysis. The expressions of both galectin-1 and galectin-3 remarkably increased in the structurally regressing CL, which vigorously expressed 20α-HSD and contained abundant apoptotic luteal cells. Ultrastructurally, galectin-1– and galectin-3–immunoreactive cells were identified as fibroblasts and infiltrating macrophages, respectively. In addition, some populations of luteal cells themselves expressed galectin-3 in regressing CL and formed unique demarcation membranes in the cytoplasm, showing a non-typical apoptotic feature. Ovary of adult mice with repeated estrus cycles contained CL of three different generations. Among them, the old CL formed during previous estrus cycles consisted of galectin-3–positive luteal cells. The galectin-3–positive old CL was resistant to apoptosis and seemed to be eliminated by a mechanism different from apoptosis. The stage- and cell-specific expression of galectin in CL suggests its differential contribution to luteolysis, and this expression may be mediated by major regulatory molecules of CL function, prolactin and/or prostaglandin F2α. (J Histochem Cytochem 58:741–749, 2010)     

Homodimerization antagonizes nuclear export of survivin

Survivin plays separate roles during different phases of the cell cycle. In mitosis, Survivin is a key regulator of cell division, while in interphase, Survivin is able to protect cells from apoptosis. Survivin shuttles between nucleus and cytoplasm under the influence of one or more nuclear export signals (NESs). Paradoxically, our data show that Survivin poorly binds CRM1 in vitro because hydrophobic residues of the NES are occupied in homodimer contacts. We show that NES-preserving dimerization mutants behave as monomers in solution, show dramatically increased CRM1 binding and are more efficiently exported in vivo than wild-type Survivin. These data indicate that Survivin contains a monomer-specific NES and that dimerization modulates cytoplasmic access of the protein. Our findings have implications for both the mitotic and interphase roles of survivin.     

TOM20-mediated transfer of Bcl2 from ER to MAM and mitochondria upon induction of apoptosis

In this work, we have explored the subcellular localization of Bcl2, a major antiapoptotic protein. In U251 glioma cells, we found that Bcl2 is localized mainly in the ER and is translocated to MAM and mitochondria upon induction of apoptosis; this mitochondrial transfer was not restricted to the demonstrator cell line, even if cell-specific modulations exist. We found that the Bcl2/mitochondria interaction is controlled by TOM20, a protein that belongs to the protein import machinery of the mitochondrial outer membrane. The expression of a small domain of interaction of TOM20 with Bcl2 potentiates its anti-apoptotic properties, which suggests that the Bcl2–TOM20 interaction is proapoptotic. The role of MAM and TOM20 in Bcl2 apoptotic mitochondrial localization and function has been confirmed in a yeast model in which the ER–mitochondria encounter structure (ERMES) complex (required for MAM stability in yeast) has been disrupted. Bcl2–TOM20 interaction is thus an additional player in the control of apoptosis.Subject terms: Cancer, Apoptosis, Mitochondria     

Rearrangement of nuclear ribonucleoproteins and extrusion of nucleolus-like bodies during apoptosis induced by hypertonic stress

Short-term hypertonic (HT) stress induces apoptotic cell death in human EUE cells in culture, as observed by electron microscopy, agarose-gel electrophoresis of low-molecular-weight DNA, DNA flow cytometry and annexin-V-propidium iodide double-staining. During HT-induced apoptosis, nuclear ribonucleoprotein (RNP)-containing structures undergo rearrangement, with the formation of Heterogeneous Ectopic RNP-Derived Structures (HERDS) which pass into the cytoplasm, as already reported for other examples of spontaneous and drug-induced apoptosis. Of special interest was the observation that nucleolus-like bodies (NLBs) which resemble morphologically nuclear functional nucleoli may be extruded into the cytoplasm of apoptotic cells and are observed inside the cytoplasmic fragments blebbing-out at the cell surface; these NLBs still contain immunodetectable nucleolar proteins (such as fibrillarin). This is an additional example of RNP-containing structures of nuclear origin which are extruded from the nucleus, in an almost "native" form, during apoptosis.     

Survivin as a target for new anticancer interventions

Survivin is a member of the inhibitor of apoptosis protein (IAP) family, that has been implicated in both control of cell division and inhibition of apoptosis. Specifically, its anti-apoptotic function seems to be related to the ability to directly or indirectly inhibit caspases. Survivin is selectively expressed in the most common human neoplasms and appears to be involved in tumor cell resistance to some anticancer agents and ionizing radiation. On the basis of these findings survivin has been proposed as an attractive target for new anticancer interventions. Several preclinical studies have demonstrated that down-regulation of survivin expression/function, accomplished through the use of antisense oligonucleotides, dominant negative mutants, ribozymes, small interfering RNAs and cyclin-dependent kinase inhibitors, increased the apoptotic rate, reduced tumor-growth potential and sensitized tumor cells to chemotherapeutic drugs with different action mechanisms and gamma-irradiation in in vitro and in vivo models of different human tumor types.     

Two Modes of Cell Death Caused by Exposure to Nanosecond Pulsed Electric Field

High-amplitude electric pulses of nanosecond duration, also known as nanosecond pulsed electric field (nsPEF), are a novel modality with promising applications for cell stimulation and tissue ablation. However, key mechanisms responsible for the cytotoxicity of nsPEF have not been established. We show that the principal cause of cell death induced by 60- or 300-ns pulses in U937 cells is the loss of the plasma membrane integrity (“nanoelectroporation”), leading to water uptake, cell swelling, and eventual membrane rupture. Most of this early necrotic death occurs within 1–2 hr after nsPEF exposure. The uptake of water is driven by the presence of pore-impermeable solutes inside the cell, and can be counterbalanced by the presence of a pore-impermeable solute such as sucrose in the medium. Sucrose blocks swelling and prevents the early necrotic death; however the long-term cell survival (24 and 48 hr) does not significantly change. Cells protected with sucrose demonstrate higher incidence of the delayed death (6–24 hr post nsPEF). These cells are more often positive for the uptake of an early apoptotic marker dye YO-PRO-1 while remaining impermeable to propidium iodide. Instead of swelling, these cells often develop apoptotic fragmentation of the cytoplasm. Caspase 3/7 activity increases already in 1 hr after nsPEF and poly-ADP ribose polymerase (PARP) cleavage is detected in 2 hr. Staurosporin-treated positive control cells develop these apoptotic signs only in 3 and 4 hr, respectively. We conclude that nsPEF exposure triggers both necrotic and apoptotic pathways. The early necrotic death prevails under standard cell culture conditions, but cells rescued from the necrosis nonetheless die later on by apoptosis. The balance between the two modes of cell death can be controlled by enabling or blocking cell swelling.     

Targeting Survivin expression induces cell proliferation defect and subsequent cell death involving mitochondrial pathway in myeloid leukemic cells

Survivin, a member of inhibitor of apoptosis family of proteins, plays important roles in both cell proliferation and cell death. We previously observed that Survivin is overexpressed in leukemic cell lines and blasts from patients with acute myelogenous leukemia (AML). To understand the roles of Survivin in AML and search for new approaches to the treatment of AML, we inhibited Survivin expression in HL-60 cells with a Survivin anti-sense oligonucleotide (sur-AS-ODN) (ISIS 23722). This blocked significant numbers of HL-60 cells in G2/M phase, and halted cell proliferation at 24 hrs and progressing over time. There was only a slight increase in the number of apoptotic cells at 24 hrs compared with cells treated with nonsense oligonucleotide (NS-ODN). At 48 hrs, however, there were significant increases in sub-G1 phase and annexin V+ cells, suggesting that cell division defects caused cell death. This was supported by the finding that a reduction in the Survivin protein by sur-AS-ODN in cells under serum-free medium did not induce G2/M block and cell death compared to cells treated with NS-ODN. The formation of polyploid cells was observed 48 hrs after sur-AS-ODN treatment, as was the activation of caspase 3, which suggested that apoptotic cell death had occurred. The mitochondrial release of cytochrome C and Smac and the nuclear translocation of the apoptosis-inducing factor were also detected. Our results suggest that Survivin is essential for cell cycle progression in leukemic cells. Reduced Survivin expression causes a cell-cycle defect that leads to cell death through a mitochondrial pathway. This finding has potential utility for therapy of patients with AML.     

Long term testicular ischemia-reperfusion injury-induced apoptosis: Involvement of survivin down-regulation

Testicular torsion is associated with damage to the testicular tissue as a result of ischemia-reperfusion injury (IRI) and induction of apoptosis leading to progressive damage to spermatogenesis. Survivin is suggested to be an important regulator in the control of the mitochondrial apoptotic pathway, although its role in torsion-induced IRI is unknown. Therefore, we sought to evaluate testicular survivin expression after long term IRI induced by testicular torsion. Survivin expression was measured by real-time PCR in 6-12 month old New Zealand white rabbits divided into three groups (4 animals/group): group (A) sham control, group (B) ischemia alone for 60 min and group (C) ischemia for 60 min followed by reperfusion for 6 months. Germ cell apoptosis was evaluated by TUNEL assay, Bax/Bcl-2 ratio and DNA fragmentation. The Johnsen score was used to assess testicular morphological damage, while lipid peroxidation was used as an indicator for oxidative stress. Survivin expression was detected in all testicular tissue samples. The rate of survivin expression after IRI was significantly higher (p < 0.05) compared with ischemic only and sham control testes. Its expression in IRI samples was inversely correlated with the significant increase (p < 0.05) in apoptosis, oxidative levels and spermatogenic damage. In conclusion, down-regulation of testicular survivin expression after long term IRI to the testis and its association with apoptosis induction suggests its involvement in the regulation of this apoptotic pathway. These findings also identify survivin as a potential new target for the prevention of germ cell death during testicular torsion.