Protection of porcine oocytes against apoptotic cell death caused by oxidative stress during In vitro maturation: role of cumulus cells

The present study was conducted to examine the protective effect of cumulus cells on oocyte damage caused by reactive oxygen species (ROS), generated by the hypoxanthine-xanthine oxidase (XOD) system, during in vitro maturation of porcine oocytes. Cumulus-oocyte complexes (COCs) and cumulus-denuded oocytes (DOs) were cultured for 44 h in NCSU37 supplemented with cysteine, gonadotropins, 10% porcine follicular fluid, and hypoxanthine in the presence or absence of XOD. DNA cleavage and damage were analyzed using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and single cell microgel electrophoresis (comet) assay, respectively, and caspase-3 activity and glutathione (GSH) content were measured in each experimental group. Exposure of DOs to ROS resulted in meiotic arrest and the increase of degenerated oocytes. These degenerated DOs underwent apoptosis, as shown by the TUNEL-positive reaction within their germinal vesicles and the activation of caspase-3. The length of DNA migration in DOs treated with XOD was significantly longer than that of untreated DOs (P: < 0.05). However, irreparable cell damage caused by ROS was not observed in COCs, and no difference was observed in the caspase-3 activity of both COCs treated with and without XOD. A significantly (P: < 0.05) high level of GSH was found in COCs after 44 h of culture, compared with that of oocytes freshly isolated from their follicles, whereas GSH content in DOs markedly decreased after treatment with or without XOD. These findings suggest that cumulus cells have a critical role in protecting oocytes against oxidative stress-induced apoptosis through the enhancement of GSH content in oocytes.     

Preovulatory follicular fluid during in vitro maturation decreases polyspermic fertilization of cumulus-intact porcine oocytes in vitro maturation of porcine oocytes

Porcine follicular fluid (pFF), as a supplement of maturation media, has been shown several times to improve the in vitro production (IVP) of porcine embryos. As a transudate of serum, pFF contains locally produced factors in addition to the ones derived from serum. The objective of this study was to determine the additional positive effects of these pFF specific factors on the nuclear and cytoplasmic maturation of porcine oocytes. Follicular fluid and autologous serum were collected from sows in the preovulatory phase of the estrous cycle. Subsequently, oocytes from prepubertal gilts were matured in NCSU23 supplemented with either 10% pFF or 10% autologous serum derived from the same sow. Oocytes were then fertilized and the putative zygotes were cultured for 7 days. Nuclear maturation and cumulus expansion were assessed after the maturation culture. For evaluation of cytoplasmic maturation, oocyte glutathione (GSH) content, fertilization parameters and embryonic development were evaluated. After in vitro maturation (IVM) of the oocytes, both cumulus expansion rate and oocyte GSH content were increased for oocytes matured in pFF (P<0.05). More monospermic penetration was found when cumulus-intact oocytes had been matured in 10% pFF but this effect was lost after fertilization of cumulus denuded oocytes indicating that the pFF was acting through the cumulus. We speculate that the increased cumulus expansion and increased glutathione content, which were prevalent after IVM in pFF, are responsible for the positive effects on fertilization and the pre-implantation development of the embryos.     

Protection of Methanosarcina barkeri against oxidative stress: identification and characterization of an iron superoxide dismutase

Methanosarcina barkeri is a methanogenic archaeon that can only grow under strictly anoxic conditions but which can survive oxidative stress. We have recently reported that the organism contains a monofunctional catalase. We describe here that it also possesses an active iron superoxide dismutase. The enzyme was purified in three steps over 130-fold in a 14% yield to a specific activity of 1500 U/mg. SDS-PAGE revealed the presence of only one band, at an apparent molecular mass of 25 kDa. The primary structure determined from the cloned and sequenced gene revealed similarity to iron- and manganese superoxide dismutases. The highest similarity was to the iron superoxide dismutase from Methanobacterium thermoautotrophicum. The enzyme from M. barkeri was found to contain, per mol, 1 mol iron, but no manganese in agreement with the general observation that anaerobically growing organisms only contain iron superoxide dismutase. The enzyme was not inhibited by cyanide (10 mM), which is a property shared by all iron- and manganese superoxide dismutases. The presence of superoxide dismutase in M. barkeri is noteworthy since a gene encoding superoxide dismutase (sod) has not been found in Archaeoglobus fulgidus, a sulfate-reducing archaeon most closely related to the Methanosarcinaceae.     

Effects of oxygen tension and follicle cells on maturation and fertilization of porcine oocytes during in vitro culture in follicular fluid

The objective was to investigate the effects of oxygen tension and follicle cells (FCs) during in vitro maturation of porcine oocytes in only porcine (Sus scrofa domesticus) follicular fluid (pFF), using static and non-static (rotating) culture systems, on the nuclear maturation and subsequent in vitro fertilization of the oocytes. In the first experiment, cumulus-oocyte complexes (COCs) were matured for 48 h in pFF supplemented with (+) or without (−) FCs (5.2 × 10⁶ cells/mL), using the static (S) and rotating (R) culture systems (+FC/S, −FC/S, +FC/R, and −FC/R) under 5% or 20% O₂. Co-culture with FCs in the static culture system (+FC/S) had a detrimental effect on the meiotic competence of oocytes, whereas co-culture with FCs in the rotating culture system (+FC/R) increased maturation rates. In both culture systems, oxygen tension had no apparent effects on meiotic competence of oocytes, irrespective of culture system and FC addition. In the second experiment, COCs were matured under 5% or 20% O₂ using the −FC/S or +FC/R culture systems and then fertilized. Oxygen tension had no significant effects on fertilization parameters, irrespective of the culture system. The rotating culture system increased rates of sperm penetration and male pronuclear formation and decreased polyspermic fertilization compared with the static culture system (P < 0.05). In conclusion, both −FC/S and +FC/R culture systems supported meiotic competence, irrespective of oxygen tension. However, the +FC/R culture system may be superior to the −FC/S culture system for promoting fertilization.     

Dynamic changes of cumulus-oocyte cell communication during in vitro maturation of porcine oocytes

Oocyte maturation is a key issue of current animal biotechnology. This study was designed to examine the morphodynamics of the cumulus-oocyte association during oocyte maturation. Porcine cumulus-oocyte complexes were recovered from slaughterhouse ovaries; matured in vitro for 0, 24, 36, and 44 h; and evaluated by scanning electron microscopy either combined or not combined with the osmium-dimethyl sulfoxide-osmium maceration (ODO) method. The cytoskeleton distribution was also observed by fluorescence staining. Prior to maturation culture (0 h), the spherical cumulus cells were tightly clustered around the oocyte, with narrow intercellular spaces. They showed active secretion at 36 h and were fully expanded at 44 h of culture. The ODO methods revealed that the cumulus cells projected numerous long and thin transzonal projections at 0 h, but these were largely disconnected at 44 h. The outer surface of the zona pellucida showed a meshwork surface regardless of time of incubation, whereas the inner surface changed from a fine fibrous surface to a spongy surface that was coated with mucin. The vitelline surface changed from a sparse distribution of short microvilli (MV) to a dense distribution of well-developed MV. Fluorescence staining showed that the cumulus cell projections consisted mainly of microfilaments, which were abundant at the germinal vesicle and metaphase-I (M-I) stages (0-24 h) but which were decreased in number at the M-II stage (36-44 h). We conclude that the cumulus-oocyte transzonal projections became disconnected between the M-I and M-II stages as a result of cumulus expansion. The cumulus-cumulus communications, however, remained intact at these stages, although the biological functions of these communications were not clear.     

Mitochondria superoxide dismutase mimetic inhibits peroxide-induced oxidative damage and apoptosis: role of mitochondrial superoxide

The purpose of this study was to test the hypothesis whether Mito-carboxy proxyl (Mito-CP), a mitochondria-targeted nitroxide, inhibits peroxide-induced oxidative stress and apoptosis in bovine aortic endothelial cells (BAEC). Glucose/glucose oxidase (Glu/GO)-induced oxidative stress was monitored by dichlorodihydrofluorescein oxidation catalyzed by intracellular H(2)O(2) and transferrin receptor-mediated iron transported into cells. Pretreatment of BAECs with Mito-CP significantly diminished H(2)O(2)- and lipid peroxide-induced intracellular formation of dichlorofluorescene and protein oxidation. Electron paramagnetic resonance (EPR) studies confirmed the selective accumulation of Mito-CP into the mitochondria. Mito-CP inhibited the cytochrome c release and caspase-3 activation in cells treated with peroxides. Mito-CP inhibited both H(2)O(2)- and lipid peroxide-induced inactivation of complex I and aconitase, overexpression of transferrin receptor (TfR), and mitochondrial uptake of (55)Fe, while restoring the mitochondrial membrane potential and proteasomal activity. In contrast, the "untargeted" carboxy proxyl (CP) nitroxide probe did not protect the cells from peroxide-induced oxidative stress and apoptosis. However, both CP and Mito-CP inhibited superoxide-induced cytochrome c reduction to the same extent in a xanthine/xanthine oxidase system. We conclude that selective uptake of Mito-CP into the mitochondria is responsible for inhibiting peroxide-mediated Tf-Fe uptake and apoptosis and restoration of the proteasomal function.     

Effects of porcine follicular fluid on male pronucleus formation in porcine oocytes matured in vitro

Porcine follicular oocytes, collected from antral follicles (2–5 mm in diameter) of gilt ovaries, were matured in vitro with or without porcine follicular fluid (pFF), gonadotrophins (GTH) or fetal calf serum (FCS) for 48 hours at 37°C under 5% CO₂ in air, and their ability of male pronucleus (mPN) formation was examined after in vitro fertilization. Formation of mPN was observed in 38.6% of penetrated oocytes matured in modified Krebs-Ringer bicarbonate solution (TYH) 18 hours after insemination. The addition of GTH into the maturation medium did not improve the proportion of mPN-formed oocytes (20–30%). In contrast, the mPN formation rate elevated significantly (59.5%) when the oocytes were cultured with pFF, and the addition of follicle-stimulating hormone (FSH) enhanced this pFF action (the rate became 81.0%). In the presence of FSH, significant pFF effect was observable at the concentration of 5%, and its efficiency was elevated with the increase of pFF concentration. When the oocytes were matured with FCS, the mPN formation rate was unchanged or decreased rather than improved (0–25%). These results suggest that pFF, but not FCS, have substance(s) stimulating the ability of mPN formation in porcine oocytes.     

The role of superoxide dismutase in combating oxidative stress in higher plants

Superoxide dismutase (SOD) isozymes are compartmentalized in higher plants and play a major role in combating oxygen radical mediated toxicity. In this review we evaluate the mode of action and effects of the SOD isoforms with respect to oxidative stress resistance, correlating age, species, and specificity of plants during development.     

Pure preovulatory follicular fluid promotes in vitro maturation of in vivo aspirated equine oocytes

In the mare, rates of fertilization and development are low in oocytes matured in vitro, and a closer imitation of in vivo conditions during oocyte maturation might be beneficial. The aims of the present study were, therefore, to investigate whether (1) equine oocytes can be matured in vitro in pure equine preovulatory follicular fluid, (2) priming of the follicular fluid donor with crude equine gonadotrophins (CEG) before aspiration of preovulatory follicular fluid promotes the in vitro maturation rate, (3) the in vitro maturation rate differs between oocytes aspirated during estrus and those aspirated again 8 days after the initial follicular aspiration, and (4) high follicular concentrations of meiosis activating sterols (MAS) are beneficial for in vitro maturation of equine oocytes. During estrus, 19 pony mares were treated with 25 mg CEG. After 24 h (Al) and again after 8 days (A2), all follicles >4mm were aspirated and incubated individually for 30 h in the following culture media: standard culture medium (SM), preovulatory follicular fluid collected before CEG containing low MAS concentrations (FF1), preovulatory follicular fluid collected before CEG containing high MAS concentrations (FF2) or preovulatory follicular fluid collected 35 h after administration of CEG containing low MAS concentrations (FF3). Cumulus expansion rate was significantly affected by culture medium. The overall nuclear maturation rate was significantly higher for oocytes collected at A1 (67%) than for oocytes collected at A2 (30%). For oocytes collected at A1, the maturation rates were 71% (FF1), 61% (FF2), 79% (FF3) and 56% (SM). An electrophoretic protein analysis of the culture media revealed the presence of a 200-kDa protein in FF3. The results demonstrate that (1) equine oocytes can be matured during culture in pure equine preovulatory follicular fluid, (2) preovulatory follicular fluid collected after gonadotrophin-priming seems superior in supporting in vitro maturation than standard culture medium, (3) oocytes aspirated 8 days after a previous aspiration are less competent for in vitro maturation than oocytes recovered during the initial aspiration, and (4) the regulation of meiotic resumption during in vitro culture of equine oocytes might be related to the presence of a 200-kDa protein.     

Effect of sheep and human follicular fluid on the maturation of sheep oocytes in vitro

This study examines the effect of sheep and human follicular fluid on the in vitro maturation (IVM) of sheep follicular oocytes. Oocyte cumulus complexes recovered post mortem were matured for 24 to 26 h at 38.6 degrees C, 5% CO(2) in air, in TCM-199 bicarbonate medium supplemented with 20% fetal calf serum (FCS) and, where stated, with maturation hormones, including FSH (5.0 ug/ml), LH (5.0 ug/ml) and estradiol (1 ug/ml), or with sheep follicular fluid recovered from large (>5mm) or small (2 to 5mm) ovarian follicles post mortem, or with human periovular follicular fluid obtained during routine IVF procedures. The matured oocytes were then denuded, and their maturation stage and developmental capacity were assessed by in vitro fertilization (IVF) and culture (IVC). It was found that inclusion of sheep or human follicular fluid or hormone supplements in the IVM media more than doubled the number of oocytes completing maturation (FCS alone 33%, compared with 76.2% for maturation hormones, 84.2% for fluid from large and 69.6% for fluid from small sheep follicles and 82.6% for human follicular fluid), and significantly increased fertilization rates (FCS alone 51.6%, compared with 71.9% for maturation hormones, 78.4% for fluid from the large and 75.7% for fluid from small sheep follicles and 73.1% for human follicular fluid) without discernible adverse effects on the development of the cleaving embryos to the morula or blastocyst stage in culture. Omission of FCS and supplements from the IVM medium resulted in a marked reduction (56%) in the number of oocytes maturing. This reduction could be offset to a large part, but not completely, by inclusion of human follicular fluid or human follicular fluid plus LH (5 ug/ml) in the medium. The results of this study show that addition of sheep or human follicular fluid to maturation medium can enhance rather than inhibit the maturation and fertilizability of sheep follicular oocytes in vitro.     

Effects of ovarian cortex cell co-culture during in vitro maturation on porcine oocytes maturation, fertilization and embryo development

The objective of the experiments was to evaluate the effects of porcine ovarian cortex cells (pOCCs) during in vitro maturation (IVM) of porcine oocytes on IVM of porcine oocytes, in vitro fertilization (IVF) parameters and subsequent embryo development. The pOCCs was cultured in the 500 microl TCM199 without hormone until the confluence, and then cultured in 500 microl TCM199 supplemented with hormone for 12 h before the oocytes added. Porcine oocytes were co-cultured with the pOCCs monolayers in the co-culture system for 44 h, following fertilized in the mTBM for 6 h. Finally, the presumptive zygotes were cultured for 144 h in the NCSU-23 supplemented with 0.4% BSA. The results showed that matured M II oocytes in the co-culture group were higher than that in the control group (P<0.05). Although penetration did not differ between the co-culture and control groups (P=0.481), polyspermy declined in the co-culture group (P<0.05), whereas male pronucleus (MPN) formation was improved in the co-culture group compared with the control group (P<0.05). More blastocysts developed in the co-culture group than that in the control group (P<0.05); however, the cleavage rates and the mean number cells per blastocyst showed no significant difference between the treated group and the control group (P=0.560 and 0.873, respectively). In conclusion, the presence of the pOCCs monolayers during IVM enhanced the maturation quality of the porcine oocytes, reduced the polyspermy, increased the percentages of MPN formation and blastocyst, but the blastocyst quality was not improved.     

Origin of bovine follicular fluid and its effect during in vitro maturation on the developmental competence of bovine oocytes

Protein supplementation during in vitro maturation can profoundly affect both the rate and overall efficiency of the maturation procedure. The present study was conducted to assess the ability of different concentrations (1, 5, and 10%) of bovine follicular fluid (bFF) to support in vitro maturation of oocytes and subsequent developmental capacity. The bFF was derived either from competent follicles ( > 8 mm) obtained by transvaginal recovery following superovulation or from a pool of small follicles (2-5 mm) from abbatoir-derived ovaries. Bovine oocytes were cultured for 24 h in synthetic oviduct fluid medium (m-SOF) supplemented with polyvinylpyrrolidone. Following fertilization and embryo culture, more oocytes (P < 0.05) reached the blastocyst stage when oocytes were cultured with 5% bFF from competent follicles (41 +/- 3.7%) compared with bFF derived from small follicles (16 +/- 2.9%). Estradiol and recombinant human follicle stimulating hormone added to the competent bFF during maturation acted in synergy to increase blastocyst production rate (P < 0.05); this blastocyst production rate (57 +/- 1.2%) was higher than those obtained with the addition of these two hormones to bFF derived from small follicles (26 +/- 2.9%). The quality of blastocysts obtained was reflected by inner cell mass (51.30 +/- 3.5 and 25.50 +/- 3.7) and trophectoderm cell numbers (99.72 +/- 2.5 and 94.80 +/- 4.7) for bFF from competent and small follicles, respectively. In conclusion, follicular fluid originating from competent follicles increased the developmental competence of abbatoir-derived oocytes.     

In vitro maturation and fertilization of follicular oocytes in cattle

This work aims towards developing research concerning the improvement of animal reproduction, embryo development and genetic engineering. In our laboratory, an attempt has been made to standardize in vitro conditions able to optimally support bovine oocyte maturation and fertilization in order to yield viable embryos. Ovaries from cows and heifers, obtained from local slaughter-house, were used for recovery of oocytes from antral follicles. Cumulus-oocyte complexes were statically cultured for 24h at 39 degrees C in medium TCM 199 supplemented with fetal calf serum inactivated, hormones, glucose and granulosa cells under a 5% CO2 and 95% humidity atmosphere. A first group of oocytes was used for fixing and staining procedure for evidence of in vitro maturation. After culture 69.4% (77/111) of oocytes reached full maturation showing cumulus expansion, first polar body extrusion and the 2nd metaphase plate. A 2nd group was used for in vitro fertilization. In vitro semen capacitation was obtained with swim-up system (8.9) with separation of high motility fraction in Talp Hepes medium. Oocytes and spermatozoa were coincubated for 18-20h in Talp medium at 39 degrees C with 5% CO2 and 95% humidity. At the end of culture stereoscope and microscope observations were made for evidence of fertilization. After IVF 67.4% (58/86) resulted fertilized. Most of them showed two pronuclei and residual sperm tail. In few cases oocytes with 1 pronucleus and the swollen sperm head or with syngamy or polyspermic were found. In these experiments high percentages of in vitro matured and in vitro fertilized oocytes have been obtained. These bovine zygotes can be considered an essential step to develop new technologies in cattle breeding.     

Luteinizing hormone receptor formation in cumulus cells surrounding porcine oocytes and its role during meiotic maturation of porcine oocytes

We investigated the formation of LH receptor (LHR) in cumulus cells surrounding porcine oocytes and the role of LHR in meiotic maturation of oocytes. At least three splice variants of LHR mRNA were detected in cumulus cells, in addition to the full-length form. Low levels of three types of products were seen in cumulus cells from cumulus oocytes complexes (COCs), whereas the full-length form was significantly increased by 12-h cultivation with FSH. The addition of FSH also significantly increased the binding level of biotinylated hCG to COCs. The formation of LHR in FSH-stimulated cumulus cells was not affected by additional 0.5 mM phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), and the oocytes were synchronized to the germinal vesicle (GV) II stage by exposure to 0.5 mM IBMX and FSH for 20 h. The binding of LH to its receptor induced a further increase in cAMP level and progesterone production and acceleration of meiotic progression to the metaphase I stage. The oocytes cultured with LH for 24 h following cultivation with FSH and IBMX were used for in vitro fertilization. At 6 days after in vitro fertilization, blastocyst rate in oocytes matured under these conditions was significantly higher than that of oocytes cultured in the absence of LH. Treatment of oocytes with FSH and 0.5 mM IBMX to express LH receptor in cumulus cells while holding oocytes at the GV II stage is a very beneficial way to produce in vitro-matured oocytes, which have high developmental competence.     

Response of porcine oocytes to electrical and chemical activation during maturation in vitro

These studies were conducted to examine activation of in vitro-matured porcine oocytes in response to an electrical stimulus or to an ionophore. Cumulus-enclosed porcine oocytes were incubated in maturation medium supplemented with either FSH and LH (MM:Exp.1) or pregnant mare serum gonadotropin (PMSG; MM-P: experiments 2-4) at 39 degrees C in 5% CO2:95% air with high humidity. In experiment 1, groups of oocytes were stripped of cumulus and then shampulsed (control) or electrically pulsed with a Zimmerman Cell Fusion unit at 24, 31, 41, 48, and 65 h of incubation. Control oocytes were exposed to the activation medium for 20 sec, whereas oocytes to be pulsed were subjected to a single activation pulse (120 V, 30 microseconds). Oocytes were cultured for an additional 24 h and then fixed and examined. For oocytes pulsed at 24, 31, 41, 48, and 65 h, the proportions which activated were 0, 0, 87, 88, and 83%, respectively. In experiment 2, oocytes were electrically or sham-pulsed with a BTX 200 Embryomanipulation System at 24, 30, and 40 h of incubation and respective proportions of oocytes activating were 27%, 39%, and 72%. In experiment 3, oocytes were subjected to 0, 1, or 2 activation pulses after 41 h of incubation in MM-P. Double-pulsing halved the proportion of activated oocytes (P less than .0001). In experiment 4, oocytes were subjected to 0, 25, 50, or 100 microM ionophore at 48 h of incubation. Proportions of oocytes activated by ionophore were greater than for control (P less than .05), but activation was not increased by increasing dose of ionophore.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycosaminoglycans in porcine follicular fluid promoting viability of oocytes in culture

The viability of oocytes cultured in vitro was determined by the trypan blue exclusion test. Isolated porcine oocytes with or without cumulus cells cultured in modified Krebs-Ringer medium undergo cell death after 48 h. The addition of glycosaminoglycans (GAGs) prepared from porcine follicular fluid (pFF) to the medium delayed or prevented the onset of cell death in vitro. GAGs at concentrations of 0.25 mg/ml or greater prevented cell death in a dose-dependent manner. To identify the active factor, GAGs were purified from pFF by ethanol precipitation, chromatography on Dowex 1-x2, and high performance liquid chromatography (HPLC) on TSK gel DEAE-2 SW column. The fraction with a retention time nearly coincident with that of hyaluronic acid possessed high oocyte viability promoting activity. The present results suggest that the viability of oocytes in vitro is influenced by the presence of specific GAGs separated from follicular fluid.     

Ovarian epidermal growth factor-like activity. Concentrations in porcine follicular fluid during follicular enlargement

Numerous data indicate that epidermal growth factor has important effects on cultured granulosa cells. However, most of the few attempts to detect epidermal growth factor in ovarian tissue have been unrevealing. In this study, ovarian epidermal growth factor-like activity was easily detected by a radioreceptor assay based on the A431 cell line but not by an immunoassay for mouse epidermal growth factor. The concentration of this activity in follicular fluid from small porcine ovarian follicles was higher than that in fluid from medium or large follicles or serum (p less than 0.01), but lower than that in salivary gland extracts. Receptor-active epidermal growth factor-like peptides could function as local ovarian regulators.     

Prolonged hypoxia during cell development protects mature manganese superoxide dismutase-deficient astrocytes from damage by oxidative stress.

Mouse astrocytes deficient in the mitochondrial form of superoxide dismutase do not grow in culture under 20% atmospheric O2 levels. By flow cytometry, immunocytochemistry, and enzymatic analysis we have shown that the oxygen block of cell division is due to a decrease in the number of cells entering the S phase of the cell cycle and is concomitant with higher DNA oxidation and impairment of mitochondrial functions. Seeding the cells under 5% O2 until the cultures become confluent can circumvent this problem. An initial hypoxic environment increases the resistance of manganese superoxide dismutase-deficient astrocytes to superoxide radicals artificially produced by paraquat treatment, preserves respiratory activity, and allows normoxic division during a subsequent passage. DNA oxidation is then not higher than in wild-type control cells. However, the adaptation of the cells is not due to compensation by other enzymes of the antioxidant defense system and is specific to cells totally lacking manganese superoxide dismutase. Alteration of the phenotype by prior hypoxia exposure in the SOD2-deficient mutant provide a unique model to study adaptative mechanisms of cellular resistance to oxygen toxicity.