Expression of superoxide dismutases in the bovine oviduct during the estrous cycle.

The superoxide dismutases (SODs) are first-line enzymatic antioxidants that dismute superoxide anion (O(2)(-)*) to produce hydrogen peroxide (H(2)O(2)). The primary objective was to characterize, by western blot analysis, the expression of two SODs, the cytosolic (Cu,ZnSOD or SOD1) and the mitochondrial (MnSOD or SOD2) forms in three sections of the oviduct, i.e. isthmus (I), ishtmic-ampullary junction (IA), and ampulla (A), during the estrous cycle. The Cu,ZnSOD and MnSOD proteins were mostly expressed in the ampulla (I

Increased placental phospholipase A2 gene expression and free F2-isoprostane levels in response to oxidative stress in preeclampsia.

Vasoactive eicosanoids such as thromboxane (TX) A2 and F2-isoprostanes (F2-isoPs) were shown to be increased in the preeclamptic placenta. Only one of the 64 possible isomers of F2-isoPs derived from the oxidation of arachidonic acid was investigated in the placenta so far. F2-isoPs are released from membrane phospholipids by phospholipases A2 (PLA2) and were shown to act on the TXA2 receptor (TBXA2R). However, the PLA2 deregulated in preeclampsia (PE) remains to be determined. In this study, we analyzed the concentrations of six isomers of F2-isoPs; 8-iso-PGF2α, 8-iso-15(R)-PGF2α, 15(R)-PGF2α, iPF2α-IV, iPF2α-VI, 5-iPF2α-VI and the concentrations of the stable metabolites of TXA2, TXB2, by high performance liquid chromatography coupled with tandem mass spectrometry in placentas of PE (n = 17) and normotensive (n = 15) pregnancies according to the biopsy site: peri-insertion or periphery. In the same biopsies, relative mRNA expression of PLA2G2A, PLA2G4A, PLA2G5, PLA2G7, the PLA2 receptor (PLA2R1), the TXA2 synthase and TBXAR2 were measured by quantitative RT-PCR. We observed similar concentrations of total F2-isoP isomers between groups whereas higher concentrations (>40%) of free F2-isoP were observed for all isomers (p ≤ 0.033) in PE than normotensive controls. As expected, we also observed higher placental concentrations of TXB2 in PE (p = 0.005). Interestingly, we concomitantly found higher mRNA expression of secretory PLA2G2A (p = 0.010), PLA2G5 (p = 0.038) and TBXA2R (p = 0.023) in PE than normotensive placentas. In sum, deregulated PLA2 could potentially be implicated in freeing F2-isoP which could participate in local hypertension observed in the PE placenta through the TX pathway.

Placental dimethyl acetal fatty acid derivatives are elevated in preeclampsia.

Preeclampsia (PE) was shown to affect the placental content and the transfer of polyunsaturated fatty acids (PUFA) to the fetus. Plasmalogens, a type of phospholipids with a vinyl-ether link at the sn-1 position, play an antioxidant role and are specifically enriched in PUFA at the sn-2 position. In this study, we characterized plasmalogen-derived dimethyl acetal (DMA) fatty acid derivatives, 16:0 DMA, 18:0 DMA, 9c-/11c-18:1 DMA and PUFA in the placenta of normotensive (n = 20) and PE (n = 20) pregnancies, according to the sampling site: peri-insertion or periphery. Phospholipid fatty acids from the placenta and maternal erythrocytes were identified by gas chromatography mass spectrometry and quantified by flame ionization detection. We found elevated total DMA in the PE placenta by 18% when compared to normotensive controls (p = 0.026). Moreover, the 16:0 DMA account for more than 55% of DMA fatty acids measured in the placenta, and its level is significantly higher in PE than controls (p = 0.018). Also, we found elevated placental PUFA, 20:5(n-3), 22:5(n-3) and a low level of 20:4(n-3) in PE compared to controls. Placental DMA was highly correlated with n-6 and n-3 PUFA in both, normotensive and PE pregnancies. In sum, elevated DMA fatty acids in the PE placenta could be an indirect defensive mechanism against oxidative stress and poor placental fatty acid transfer in PE.

Effects of nonsteroidal antiinflammatory drugs on oxidative pathways in A/J mice.

The tobacco-specific N-nitrosamine, NNK, is a potent carcinogen in laboratory animals. The authors have shown previously that NNK-induced lung tumorigenesis in A/J mice can be reduced significantly by certain nonsteroidal antiinflammatory drugs (NSAIDs), such as sulindac, ibuprofen, or piroxicam treatments. In this study, the authors investigated whether NSAIDs could reduce NNK-induced oxidative, DNA damage and/or inhibit endogenous lipid peroxidation, or prostaglandin E2 (PGE2) synthesis in A/J mice. In the first experiment, A/J mice were gavaged with NNK (112 mumol/kg b.w.) three times a week while being maintained on a diet to which either ibuprofen (263 mg/kg diet), naproxen (230 mg/kg), sulindac (123 mg/kg), piroxicam (25 mg/kg), indomethacin (5 mg/kg), or no NSAIDs had been added. Levels of 8-OH-dG in the DNA of lung and liver were measured by high-performance liquid chromatography with electron capture detector. Treatment with NSAIDs had no significant effects on the endogenous or NNK-induced formation of 8-OH-dG in the lung of the mice. In a second experiment, after treatment of A/J mice with NSAIDs for 2 weeks, lipid peroxidation was assayed by determining thiobarbituric acid-reactive substances (TBA-RS) in lung tissues, and prostaglandin E2 levels were measured in plasma by an enzyme immunoassay. Treatments with some NSAIDs lowered the levels of lipid peroxidation and plasma levels of PGE2 below basal levels. Taken together, these results suggest that the inhibition of NNK-induced lung tumorigenesis by NSAIDs is more likely related to an inhibition of prostaglandin synthesis than to a direct inhibition of lipid peroxidation or oxidative DNA damage induced by NNK.

Thiols prevent H2O2-mediated loss of sperm motility in cryopreserved bull semen.

We previously showed that cryopreservation of bull spermatozoa in egg yolk Tris extender (EYTG) significantly reduced the intracellular level of thiols. Other studies showed the beneficial effects of adding antioxidants to cryopreserved bull spermatozoa. These studies led us to investigate the effects of various thiols, an important class of antioxidants, on sperm motility of cryopreserved bull semen in a commonly used extender, EYTG. Sperm motility was analyzed by computer-assisted semen analysis (CASA). After thawing, a diluted pool of bull semen was incubated at 38.5 degrees C in airtight tubes with the following thiols for 6 hours: glutathione (GSH/GSSG), cysteine, N-acetyl-L-cysteine (NAC) and 2-mercaptoethanol in the presence or absence of oxidative stress. The oxidative stress was caused by adding H2O2 (100 microM) to diluted semen. Incubation of diluted bull semen in EYTG at 38.5 degrees C over a period of 6 h decreased sperm motility by approximately 9 fold from the start (72 +/- 3, mean +/- SEM, n=4) to the end (9 +/- 4, n=4) of the incubation. We found that all thiols to a concentration above 0.5 mM maintained high sperm motility for 6 h in the absence of an external source of oxidative stress (52 +/- 4, for 4 thiols). However, one mM of each thiol was required to efficiently protect sperm motility in the presence of 100 microM of H2O2 for 6 h. We also found that the GSH concentration in diluted semen was too low (microM) to adequately supply exogenous addition of 72 U/mL of glutathione peroxidase (GPx), an enzyme that detoxifies H2O2 and hydroperoxides using GSH as a cofactor. In fact, a better protection of sperm motility could be achieved with only 5 U/mL of GPx and 0.1 mM of GSH added to diluted semen. Our results also demonstrated that added GSSG (0.5 mM) in diluted semen was not regenerated efficiently to GSH over 6 h. The latter result indicated in the extender that the glutathione redox-cycle was deficient. Therefore, deleterious effects sperm motility after cryopreservation in EYTG can be counteracted by adding various thiols at mM concentration.

Antioxidant requirements for bovine oocytes varies during in vitro maturation, fertilization and development.

Antioxidants may be beneficial additives to synthetic culture media because these well defined media lack serum or other macromolecules that serve as reactive oxygen species scavengers. In this study, three separate experiments were performed to determine the effects of antioxidants on the development of oocytes to the morula and blastocyst stage when added during in vitro maturation (IVM) of bovine oocytes, during in vitro fertilization (IVF), and during embryo culture for the first 72 h of the development period. Bovine oocytes were matured, fertilized (under 20% O(2)), and embryos were cultured (under 7% O(2)) in defined conditioned medium in vitro with or without supplementation with the antioxidant cysteine, N-acetyl-L-cysteine (NAC), catalase and superoxide dismutase (SOD). Significant improvements in the proportion of oocytes undergoing morula and blastocyst development (33.3% versus 20.3%, P<0.05) were achieved when cysteine (0.6 mM) was added to the maturation medium as compared to control medium without antioxidant supplementation. However, the addition of NAC (0.6mM), catalase (5 or 127 U/ml) or SOD (10 or 1000 U/ml) to the maturation medium did not improve the proportion of oocytes undergoing morula and blastocyst development. During the IVF period, addition of antioxidants (cysteine or NAC 0.6mM, catalase 127U/ml, SOD 100U/ml) significantly reduced the subsequent rate of bovine embryo development to the morula and blastocyst stage (P<0.05). In a defined medium for embryo culture (7% O(2)), the addition of cysteine improved the development of bovine embryos while NAC, catalase and SOD had no positive effect on embryonic development. Our study showed that medium supplementation with cysteine during IVM and in vitro culture (IVC) improved the rate of bovine embryo development, in contrast to extracellular antioxidants like catalase and SOD that caused no improvement.

Epithelial and stromal uterine cells cultured in vitro protect bovine sperm from hydrogen peroxide.

It is known that large amounts of leukocytes colonize the uterus, and that these leukocytes can produce considerable quantities of hydrogen peroxide (H2O2) and other reactive oxygen species that are toxic to sperm. It has been shown recently that oviductal fluid has a catalase that helps to maintain sperm motility. Therefore, the current experiment was performed to determine if a similar mechanism of protection exists against peroxides within uterine cells. Sperm motility and velocity were recorded after a 6h incubation in 1) conditioned media in the presence of endometrial cells, 2) conditioned media without endometrial cells, 3) control media (48h without cells) over endometrial cells, or 4) control media alone. All these treatments were performed in the presence or absence of added catalase. Conditioned media, endometrial cells and catalase had a significant positive effect on the maintenance of sperm motility and velocity. Addition of anti-catalase antibodies did not neutralize the beneficial effect of the conditioned media. However, the concentrations of aromatic amino acids, known substrates for sperm amino acid oxidase, were significantly lower in uterine conditioned media as compared to control medium. This reduction of aromatic amino acids was in correlation with reduced H2O2 production by sperm as estimated by chemiluminescence. These results suggest that epithelial and stromal uterine cells do not maintain sperm motility by secreting catalase in the conditioned media, but rather by reducing the levels of aromatic amino acids and thus of peroxides generated in the presence of spermatozoa.

Review: maternal and placental antioxidant response to preeclampsia - impact on vasoactive eicosanoids.

The abnormally developed placenta is believed to be the pathophysiological cause of preeclampsia (PE). The resulting malperfusion of the placenta in PE can be associated with fluctuations in oxygen levels, leading to oxidative stress. How then do the placenta and the circulatory system of the mother adapt and respond to the increased oxidative challenge associated with PE? Many antioxidant systems have been shown to be upregulated or downregulated in the placenta and/or the maternal circulation during PE. Such altered antioxidant response can lead to increased lipid peroxidation. Oxidation of arachidonoyl residues in phospholipids generates bioactive lipids such as F2-isoprostanes, which are known vasoconstrictors. The consequences of changes in antioxidant status can also affect signal transduction and enzymatic pathways related to eicosanoid synthesis.

Effect of dietary organic and inorganic selenium on antioxidant status, embryo development, and reproductive performance in hyperovulatory first-parity gilts.

This project aimed to determine the effect of Se as inorganic Na-selenite (MSe) or organic Se-yeast (OSe) on antioxidant status, hormonal profile, reproductive performance, and embryo development in first-parity gilts. Forty-nine gilts were allocated to 1 of the 3 dietary treatments starting at first pubertal estrus and lasting up to 30 d after AI: control [CONT: basal diet (Se = 0.2 mg/kg) without added Se; n = 16], MSe (CONT + 0.3 mg/kg of MSe; n = 16), and OSe (CONT + 0.3 mg/kg of OSe; n = 17). Blood was collected from all gilts on the day after each onset of estrus and on d 30 after AI. Blood was also collected daily from d -4 to d +4 of the third onset of estrus (d 0) in 8 CONT, 9 MSe, and 8 OSe cannulated gilts. Gilts had received, after d 14 and 15 of their third estrus, a hormonal challenge to induce super-ovulation. At slaughter, embryos and corpora lutea (CL) were weighed and measured. Blood Se was less (P < 0.01) in CONT than in Se gilts and greater in OSe than in MSe (P < 0.01) from the first estrus until d 30 of gestation. At the same time, blood Se-dependent glutathione peroxidase (GSH-Px) decreased for CONT gilts, whereas it increased for both Se groups. The increase was greater in MSe than in OSe gilts (treatment × time, P = 0.02). Plasma 3,3',5-triiodothyronine and thyroxine concentrations for MSe tended to be less than for OSe gilts (P < 0.06). In cannulated gilts, plasma FSH tended to change among treatments (treatment × time, P = 0.06), and plasma estradiol-17ß (E(2)) was less (P = 0.01) for MSe than for OSe. There was no treatment effect on mean litter size or embryonic antioxidant status. The Se content of individual embryos was greater for Se-treated than for CONT gilts (P = 0.03), and Se content of individual embryos and total litter was greater for OSe than for MSe gilts (P < 0.01). The length, weight, and protein content of embryos were greater in OSe than in MSe gilts (P < 0.05). There was no treatment effect on weight, length, Se content, and ferric reducing antioxidant power of CL, but GSH-Px in CL was greater for Se than for CONT gilts (P = 0.02). In summary, the Se status response of gilts to dietary Se was affected by both the quantity and the source of Se dietary supplements. Moreover, the uterine transfer of Se to embryos was improved with OSe as compared with MSe, and this was concomitant with an enhanced development of embryos.

Altered placental glutathione peroxidase mRNA expression in preeclampsia according to the presence or absence of labor.

Increased placental oxidative stress in preeclampsia (PE) has been associated in part to a decrease in glutathione peroxidase (GPX) antioxidant activity. However, it is not clear if GPX mRNA expression is affected in PE, and how the presence of labor may impair this expression. In this study, we characterized by quantitative PCR, in situ hybridization and immunohistochemistry, the expression of four GPX (GPX1 to 4) in the placenta of normotensive (NP; n = 23) and PE pregnancies (n = 25) according to mode of delivery: vaginal delivery (with labor) or cesarean (without labor); the tissue layer: amnion-chorion (AC) and villi; and the sampling site: peri-insertion or peripheral. Concomitantly, oxidative stress markers mRNA expression, HSP70 and HO-1 were measured. All GPX mRNA and protein were detected in all layers of the placenta and sampling sites. In absence of labor, GPX1 is more expressed near the umbilical cord than at the periphery of the villi (p = 0.037). At the periphery of AC membranes, GPX2 was more expressed in PE than in controls in presence of labor (p = 0.037). Interestingly, GPX4 mRNA level was clearly deficient in the PE villi in presence or absence of labor (p < 0.0473). Also, the GPX4 expression in PE was lower than controls in AC membranes in presence of labor (p = 0.0007). Oxidative stress markers, HSP70 and HO-1, were higher in PE placental membranes than in controls in absence of labor (p < 0.011). HSP70 was also upregulated in PE placental membranes in presence of labor (p = 0.034). Correlations between stress markers and GPX mRNA expression were mostly present in AC membranes in presence of labor in NP. Most of the latter correlations were lost in PE. In conclusion, our results suggest that the reported decrease in GPx activity and increased oxidative stress in PE placental villi may be attributed in part to GPX4 independently of the presence or absence of labor.

Expression of prostaglandin E synthases in the bovine oviduct.

The oviduct is a specialized organ responsible for the storage and the transport of male and female gametes. It also provides an optimal environment for final gamete maturation, fertilization, and early embryo development. Prostaglandin (PG) E(2) is involved in many female reproductive functions, including ovulation, fertilization, implantation, and parturition. However, the control of its synthesis in the oviduct is not fully understood. Cyclooxygenases (COXs) are involved in the first step of the transformation of arachidonic acid to PGH(2.) The prostaglandin E synthases (PGESs) constitute a family of enzymes that catalyze the conversion of PGH(2) to PGE(2), the terminal step in the formation of this bioactive prostaglandin. Quantitative real-time PCR was used to determine the expression of COX-1, COX-2, microsomal prostaglandin E synthase-1 (mPGES-1), microsomal prostaglandin E synthase-2 (mPGES-2), and cytosolic prostaglandin E synthase (cPGES) mRNA in various sections of the oviduct, both ipsilateral and contralateral (to the ovary on which ovulation occurred) at various stages of the estrous cycle. Furthermore, protein expression and localization of cPGES, mPGES-1, and mPGES-2 were determined by Western blot and immunohistochemistry. All three PGESs were detected at both mRNA and protein levels in the oviduct. These PGESs were mostly concentrated in the oviductal epithelial layer and primarily expressed in the ampulla section of the oviduct and to a lesser extent in the isthmus and the isthmic-ampullary junction. The mPGES-1 protein was highly expressed in the contralateral oviduct, which contrasted with mPGES-2 mostly expressed in the ipsilateral oviduct. This is apparently the first report documenting that the three PGESs involved in PGE(2) production were present in the Bos taurus oviduct.

Existence of compensatory defense mechanisms against oxidative stress and hypertension in preeclampsia.

OBJECTIVE: Preeclampsia is a complex obstetrical syndrome characterized by hypertension and proteinuria. This syndrome is associated with oxidative stress, antioxidant imbalance and impaired production of vasoactive eicosanoids such as thromboxane A(2) (TXA(2)), a potent vasoconstrictor, and prostacyclin (PGI(2)), a well-known vasodilator. We hypothesized that there was a relationship between antioxidant vitamins, such as vitamin E and coenzyme Q(10) (CoQ(10)), and the production of vasoactive eicosanoids- PGI(2) and TXA(2)-potentially regulated by pro-oxidants and antioxidants in preeclampsia. METHODS: Therefore, the plasma levels of vitamin E, CoQ(10), TXA(2) and PGI(2) in normotensive (n = 30) and preeclamptic (n = 29) pregnancies were evaluated. Reduced and oxidized forms of vitamin E and CoQ(10) in blood were measured using a HPLC coupled to electrochemical detection. The levels of TXB(2) and 6-keto-PGF(1alpha), stable metabolites of TXA(2) and PGI(2) respectively, were measured by ELISA. RESULTS: The CoQ(10) oxidized/reduced ratio was significantly higher in preeclamptic compared to normotensive pregnancies (p = 0.04). A strong correlation between plasma levels of reduced vitamin E and CoQ(10), corrected for apolipoprotein B, was observed only in preeclampsia (r = 0.69, p < 0.0001). The 6-keto-PGF(1alpha)/TXB(2) ratio was higher in preeclampsia than in controls (p = 0.02), and this ratio was correlated to the oxidized/reduced ratio of both, vitamin E and CoQ(10) in all pregnancies (p <0.023). CONCLUSION: The data indicated that CoQ(10) is a sensitive marker of oxidative stress in preeclampsia. The correlation between vitamin E and CoQ(10) suggested a coordinated defense mechanism against oxidation. Furthermore, the higher 6-keto-PGF(1alpha)/TXB(2) ratio that strongly correlated with oxidative stress markers, suggests a mechanism developed by the maternal cardiovascular system to counteract hypertension during preeclampsia.

Effects of labor on placental expression of superoxide dismutases in preeclampsia.

A decreased antioxidant activity for superoxide dismutases (SODs) in the placenta was reported in preeclampsia (PE). However, it is unclear if this reduced enzymatic activity can be attributed to a specific SOD isoform. Moreover, the specific spatial SOD expression in the placenta and the impact of the mode of delivery on the latter are still lacking. There are three known SOD isoforms: SOD1 (cytosolic), SOD2 (mitochondrial) and SOD3 (extracellular). Our main objective was to characterize by RT-PCR, western blot and immunolocalization, the expression of SOD1, SOD2, and SOD3 in placentas of normotensive (n = 23) and PE pregnancies (n = 25) according to the presence or absence of labor, the sampling site (peri-insertion, mid-disc and periphery) and the placental layer: amnion-chorion, villi, and maternal side layer (MS). In absence of labor (cesarean), SOD1 expression in the placental villi and MS was lower in PE than in controls (p < 0.049). In presence of labor (vaginal deliveries), SOD1 expression in the amnion-chorion only was higher in PE than controls (p = 0.014). Additionally, SOD2 and SOD3 expression in presence of labor were higher in all three layers in PE than controls, with a strong positive correlation between these two SODs (mRNA; r > 0.65, p < 0.008). The sampling site and gestational age had no effect on SOD expression within the placenta. In this study, we showed that the reported decrease for SOD activity in PE may be attributed to SOD1 in absence of labor. Also, this is the first study characterizing specific SOD isoforms according to the mode of delivery. We demonstrated in PE that labor upregulates SOD1 in fetal membranes as well as SOD2 and SOD3 in the whole placenta.

Evaluation of tolerability and ability to increase immunosuppression in renal transplant patients converted from mycophenolate mofetil to enteric-coated mycophenolate sodium.

BACKGROUND: Dose reductions or discontinuations of mycophenolate mofetil (MMF) result in higher incidences of acute rejection and graft loss. Converting renal transplant patients experiencing MMF-related gastrointestinal (GI) side effects to equimolar enteric-coated mycophenolate sodium (EC-MPS) may relieve GI symptoms. METHODS: In this prospective 12-month study, renal transplant patients maintained on suboptimal MMF doses (<1500 mg/d) due to GI intolerance were converted to equimolar EC-MPS followed by incremental EC-MPS dose increases (180 mg/d) every 7 weeks to an established maximum, if well tolerated. Changes in GI symptoms were assessed by physician judgment and Gastrointestinal Symptom Rating Scale (GSRS). RESULTS: Twenty-five patients (mean age: 52.0 +/- 13.6 years) were converted from MMF (930.0 +/- 153.4 mg/d) to equimolar EC-MPS (669.6 +/- 110.5 mg/d) at day 0. Twenty-three of 25 patients tolerated equimolar dose conversion and one or more EC-MPS dose increments at week 28. Compared to baseline, patients received significantly more EC-MPS at week 28 and week 49 (mean dose: 1033.0 +/- 164.8 mg/d, P < .0001 and 1001.7 +/- 209.0 mg/d, P < .0001, respectively). Two patients dropped out by week 7 for reasons unrelated to EC-MPS. The mean serum creatinine remained stable and no clinical acute rejection episodes occurred over 12 months. Mean GSRS total score remained stable through month 12 when compared to day 0 despite increases in EC-MPS dose. CONCLUSION: In renal transplant patients receiving suboptimal MMF doses due to GI symptoms, conversion to EC-MPS enabled equimolar prescription and subsequent dose increase without increased GI intolerance.

Increased resistance of GPx-1 transgenic mice to tumor promoter-induced loss of glutathione peroxidase activity in skin.

Reactive oxygen species formation is strongly suspected to play a role in multistep carcinogenesis, notably in tumor promotion. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces peroxide production, oxidative damage to DNA and inflammation in mouse skin. TPA is also known to cause a decrease in the activity of several antioxidant enzymes including glutathione peroxidases (GPx). The observation that several anti-oxidants can inhibit TPA-mediated tumor promotion suggests that a decline in GPx activity could contribute to tumor promotion. We report here the effects of TPA on GPx activity in the skin of transgenic GPx mice that contain human GPx-1 transgenes under the regulation of a metallothionein IIA promoter. As expected, no significant difference in basal level of skin GPx activity was detected in the 3 lines of tg-MT-GPx mice investigated compared with non-transgenic controls. A single topical application of TPA induced gradually, over 20 hr, a small but detectable increase in GPx mRNA and protein levels in skin of non-transgenic mice and a contrasting decrease in both selenium-dependent and selenium-independent GPx activity. The extent of GPx induction was more pronounced in transgenic mice, and in contrast with non-transgenic mice, no significant loss of GPx activity was observed in the TPA-treated skin of these mice. Transgenic mice may, therefore, offer a novel model suitable to assess the role of GPx-1 in skin carcinogenesis, without the potential disadvantage of abnormally high levels of GPx activity produced constitutively in other transgenic models.

Levels of antioxidant defenses are decreased in bovine spermatozoa after a cycle of freezing and thawing.

Growing evidence suggests that the generation of reactive oxygen species (ROS) and their detoxification by antioxidants plays a very important role in fertility. However, the relationship between the level of antioxidants in spermatozoa and the decreased fecundity following a freeze/thaw cycle remains poorly understood. We assessed the activities of antioxidant enzymes such as catalase, glutathione peroxidase (GPx), superoxide dismutase (SOD), and levels of reduced/oxidized glutathione (GSH/GSSG) in bovine semen. Sperm cells were isolated using a Percoll gradient to avoid contamination from seminal plasma, cellular debris, and other cell types. We found that bovine spermatozoa are poorly adapted to metabolize the toxic hydrogen peroxide (H(2)O(2)). Indeed, very low levels of GPx and an absence of catalase were observed. We also studied the effect of freezing and thawing bovine spermatozoa in a egg yolk-Tris-glycerol extender (EYTG). Cryopreservation significantly reduced sperm GSH levels by 78% and SOD activity by 50%. We also investigated whether the decrease in GSH level could be linked to oxidative metabolism and found that a greater reduction in intracellular GSH level occurred when fresh sperm cells were incubated in EYTG for 6 hr at 38.5 degrees C under aerobic conditions than when incubated under restricted oxygen availability. Our results strongly suggest the involvement of an oxidative stress during a freeze/thaw cycle and are consistent with the hypothesis that ROS generated during such a cycle are detrimental to sperm function.

Hyperoxia induces S-phase cell-cycle arrest and p21(Cip1/Waf1)-independent Cdk2 inhibition in human carcinoma T47D-H3 cells.

Little is known about cell-cycle checkpoint activation by oxidative stress in mammalian cells. The effects of hyperoxia on cell-cycle progression were investigated in asynchronous human T47D-H3 cells, which contain mutated p53 and fail to arrest at G1/S in response to DNA damage. Hyperoxic exposure (95% O(2), 40-64 h) induced an S-phase arrest associated with acute inhibition of Cdk2 activity and DNA synthesis. In contrast, exit from G2/M was not inhibited in these cells. After 40 h of hyperoxia, these effects were partially reversible during recovery under normoxic conditions. The inhibition of Cdk2 activity was not due to degradation of Cdk2, cyclin E or A, nor impairment of Cdk2 complex formation with cyclin A or E and p21(Cip1). The loss of Cdk2 activity occurred in the absence of induction and recruitment of cdk inhibitor p21(Cip1) or p27(Kip1) in cyclin A/Cdk2 or cyclin E/Cdk2 complexes. In contrast, Cdk2 inhibition was associated with increased Cdk2-Tyr15 phosphorylation, increased E2F-1 recruitment, and decreased PCNA contents in Cdk2 complexes. The latter results indicate a p21(Cip1)/p27(Kip1)-independent mechanism of S-phase checkpoint activation in the hyperoxic T47D cell model investigated.