Molecular Human Reproduction, Vol. 9, No. 11, 639-643,
November 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
Antioxidant enzymatic defences in human follicular fluid: characterization and age-dependent changes
Submitted on May 28, 2003; resubmitted on July 14, 2003. accepted on July 28, 2003
1 Department of Basic and Applied Biology, 2 Department of Biomedical Sciences and Technologies, 3 Department of Experimental Medicine, University of L’Aquila, via Vetoio, 67100 L’Aquila and 4 Centre for Assisted Reproduction, University of L’Aquila, Italy
5 To whom correspondence should be addressed. e-mail fernanda.amicarelli{at}univaq.it
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Abstract |
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The aim of this work was to study the antioxidant enzymatic defences in human follicular fluid and investigate their possible changes during reproductive ageing. To this end, we tested the specific activities and protein expression of enzymes involved in reactive oxygen species (ROS) scavenging and in detoxification of ROS byproducts in follicular fluid from young (range 27–32 years, n = 12) and older (range 39–45 years, n = 12) women participating in an IVF programme. Results show that all the tested enzymes [superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione transferase, glutathione reductase] were significantly expressed in human follicular fluid. However, when the two age groups were compared, we found that follicular fluid from older women exhibited a reduced level of glutathione transferase and catalase activities and a higher level of SOD activity. Immunoblot analysis revealed that ageing was associated with decreased protein expression of GST Pi isoform and did not affect SOD and catalase protein expression. Taken together, these findings indicate that reproductive ageing is accompanied by a change in the antioxidant enzymatic pattern that could impair ROS scavenging efficiency in the follicular environment.
Key words: ageing/antioxidant enzymes/follicular fluid/oxidative stress/reproductive potential
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Oxidative metabolism is essential for gamete and embryo energy production and is unavoidably associated with the generation of reactive oxygen species (ROS). Although at very low concentrations ROS are important second messengers capable of modulating the expression of genes that govern physiological processes of gametes and embryos (de Lamirande et al., 1998; Hensley et al., 2000; Droge, 2002), after an increase in their concentration oxidative stress can occur. Under these conditions, ROS are responsible for damage to molecules and structures, with deleterious effects on cellular functions.
It is known that an increase in peroxidative damage is associated with the process of ageing. The ‘free radical theory of ageing’ assumes that oxidative stress is one of the major causes of age-related cellular and molecular damage (Harman, 1984).
In this respect, oxidative stress might be responsible, at least in part, for the reduced reproductive potential associated with ageing. In fact, it is proven, in the mouse, that oral antioxidant administration counteracts the negative effects of female ageing on oocyte quantity and quality (Tarín et al., 2002). Moreover, these effects resemble those caused by exposure of oocytes and embryos to reactive oxygen species (Liu et al., 2000). Nevertheless, to our knowledge, no data exist on the age-related adaptive response of antioxidant defences in the oocyte and its surroundings.
All organisms have enzymatic and non-enzymatic mechanisms to scavenge oxidants, or to repair damage caused by ROS. Among the enzymatic defences, the removal of damaging oxygen products is catalysed by superoxide dismutase (SOD; EC 1.15.1.1.), catalase (EC 1.11.1.6) and selenium-dependent and independent glutathione peroxidases (GSH-Px; EC 1.11.1.9). SOD removes the superoxide anion in a dismutation reaction, producing hydrogen peroxide and molecular oxygen. The removal of hydrogen peroxide is catalysed by either catalase or GSH-Px. In the peroxidase reaction, reduced glutathione (GSH) is oxidized to GSSG (oxidized glutathione). The regeneration of GSH is, consequently, of fundamental importance for the ability of cells to challenge exposure to oxidizing metabolites. In the cell, GSSG is reduced by NADPH, through the action of glutathione reductase (GSSG-Rx; EC 1.6.4.27). Glutathione transferases (GST; EC 2.1.5.18) comprise a family of multifunctional enzymes that catalyse the conjugation to GSH of a large variety of electrophilic alkylating compounds, some of which are the products of the oxidative damage of biological membranes and macromolecules (Hayes and McLellan, 1999).
The high reactivity of ROS requires (i) that activation of defence mechanisms occurs promptly after ROS generation and (ii) that defence systems are present at the site of ROS production.
ROS may originate either directly from gametes and embryos or from their surroundings. Therefore, the prevention against ROS formation must be linked both to internal and external protection. Although enzymatic antioxidant defences are present in mammalian oocytes and embryos (Mouatassim et al., 1999; Tarín et al., 2000; Cetica et al., 2001; Guérin et al., 2001), and follicular and tubal fluids have been reported to be endowed with non-enzymatic antioxidants (Gardiner et al., 1998; Guyader-Joly et al., 1998; Guérin et al., 2001), reports describing antioxidant enzymes in these environments are very exiguous (Paszkowsky et al., 1995; Bisseling et al., 1997).
On this basis, the aim of this work was to study the enzymatic antioxidant defences in the human follicular fluid and investigate their behaviour with ageing. In particular, we studied the levels of antioxidant enzymes such as SOD, catalase and GSH-Px as well as the levels of enzymes such as GST and GSSG-Rx not directly involved in ROS scavenging, but nonetheless having a pivotal role against oxidative stress injury.
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Materials and methods |
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Antibodies and chemical reagents
Anti-Cu-Zn SOD (mouse monoclonal SD-G6) was from Sigma Chemical Co. (St Louis, USA), anti-catalase (rabbit polyclonal) was from Rockland (Gilbertsville, USA). Anti-Pi GST raised against human GST Pi from placenta, anti-Mu GST raised against human recombinant GST Mu 1-1 and anti-Alpha GST raised against human recombinant GST Alpha 1-1 were produced in our laboratory according to standard procedure (Di Ilio et al., 1991).
Reagents for enzymatic assays were purchased from Sigma and were of the purest grade, unless stated otherwise.
Patient selection and sample collection
Samples were obtained from patients undergoing IVF treatment at the Center for Assisted Reproduction of University of L’Aquila after informed consent was obtained using a protocol approved by our internal ethics committee.
A total of 24 patients aged 27–45 years participated in this study, categorized on the basis of age: young women (range 27–32 years, n = 12) and older women (range 39–45 years, n = 12).
All patients were stimulated with a standard long protocol. GnRHa (Enantone Depot 3.75 mg; Takeda, Italy) was administered from day 23 of the cycle. From day 3 of the next cycle, all patients were treated with s.c. injection of recombinant (r)FSH, 200 IU per day (Puregon; Organon, Italy). The regimen started with 75 IU of FSH-HP a day. hCG (Profasi; Serono, Italy) 10 000 IU, was administered i.m. 24 h after the last rFSH injection to induce ovulation when three or more leading follicles had reached a diameter of 16–17 mm. Oocyte retrieval was performed by transvaginal aspiration 36 h after the hCG administration. The only primary indication for IVF treatment was male factor infertility and ICSI was performed.
In each patient, the volume of fluid was recorded and the follicular fluids from three mature follicles (18–20 mm diameter) were individually aspirated. After removal of the oocytes, samples were pooled and centrifuged at 3000 g for 10 min to remove debris, blood and granulosa cells as previously described (Tao et al., 1997). Then follicular fluid supernatant was transferred to sterile polypropylene tubes and stored at –80°C until assay.
Follicular fluids that were contaminated with significant quantities of blood cells were not used for analysis.
Enzymatic assays
Catalase
Catalase activity was measured spectrophotometrically, as previously described (Amicarelli et al., 1999, 2000). The reaction mixture contained 100 mmol/l potassium phosphate buffer pH 6.8, 10 mmol/l H2O2 and appropriate amounts of follicular fluid samples. The decomposition of H2O2 was monitored continuously at 240 nm (
240 nm = 0.040 mmol/l–1cm –1) and at 25°C with a Perkin–Elmer spectrophotometer and only the initial linear rate was used to estimate the catalase activity. The blank did not contain H2O2 and the activity was calculated after subtraction of the blank value. Reaction rate proportionality was assessed by using different sample aliquots for the activity measurements. The amount of enzyme activity that decomposed 1 mmol of H2O2 per min was defined as one unit of activity. Specific activities were expressed as mIU/mg protein. The assay detection level was 0.25 mIU/ml.
In each patient, catalase activity was calculated at least four times. The intra-assay coefficient of variation (CV) was always <3%.
Superoxide dismutase
SOD activity was assayed by its ability to inhibit the auto-oxidation of adrenaline, determined by the increase in the absorbance at 480 nm and at 30°C, as previously described (Amicarelli et al., 1999, 2000). The reaction was carried out in 50 mmol/l sodium carbonate buffer, pH 10.2, and was initiated by the addition of 0.1 mmol/l adrenaline. In each patient, an appropriate volume (30–80 µl) of sample was used to obtain 
50% inhibition of the adrenaline autoxidation. A standard curve, with a purified Cu-Zn bovine SOD, was obtained by plotting the inverse values of the amount of enzyme used and the percentage inhibition observed. This standard curve was used to determine the amount of follicular fluid necessary for a 50% inhibition. One unit of SOD was defined as the amount of the enzyme required to halve the rate of substrate auto-oxidation. Specific activities were expressed as mIU/mg protein. In each patient, SOD activity was calculated at least four times. The intra-assay CV was always <2%.
Glutathione peroxidase
GSH-Px was measured as previously described (Amicarelli et al., 1999, 2000). The reaction mixture contained 50 mmol/l potassium phosphate pH 7.0, 1 mmol/l EDTA, 1.5 mmol/l sodium azide, 0.15 IU of purified glutathione reductase from bakers’ yeast, 0.45 mmol/l GSH, 0.2 mmol/l NADPH, 1.2 mmol/l cumene hydroperoxide or 0.25 mmol/l as substrate and appropriate amounts of follicular fluid sample. The blank did not contain follicular fluid and the activity was calculated after subtraction of the blank value. Reaction rate proportionality was assessed by using different sample aliquots for the activity measurements. The activity of the Se-independent GSH-Px was determined by measuring total GSH-Px with cumene hydroperoxide as substrate, and then subtracting from this activity the Se-dependent GSH-Px activity measured with H2O2 as substrate. The rate of NADPH oxidation was monitored at 25°C with a Perkin–Elmer spectrophotometer on the basis of the decrease of absorbance at 340 nm (340 nm = 6.22 mmol/l–1 cm–1). One unit of enzyme activity was defined as 1 µmol of GSH oxidized/min at 25°C. Specific activities were expressed as mIU/mg protein. The assay detection level was 1.6 mIU/ml. In each patient, the selenium-dependent and independent GSH-Px activity was calculated at least four times. The intra-assay CV was always <3%.
Glutathione transferase
GST activity was recorded at 340 nm and at 25°C, as previously described (Amicarelli et al., 1999, 2000). The conjugation reaction of 1-chloro-2,4-dinitro-benzene to GSH was monitored with a Perkin–Elmer spectrophotometer by the increase of absorbance at 340 (340 nm = 9.6 mmol/l–1 cm–1). The reaction mixture contained 0.1 mol/l potassium phosphate buffer pH 6.5, 1 mmol/l EDTA, 2 mmol/l GSH and 1 mmol/l 1-chloro-2,4-dinitrobenzene and appropriate amounts of follicular fluid samples. The blank did not contained follicular fluids and the activity was calculated after subtraction of the blank value. Reaction rate proportionality was assessed by using different sample aliquots for the activity measurements. One unit of enzyme activity was defined as 1 µmol of GSH conjugated/min at 25°C. Specific activities were expressed as mIU/mg protein. The assay detection level was 1.0 mIU/ml. In each patient, the transferase activity was calculated at least four times. The intra-assay CV was always <3%.
Glutathione reductase
GSSG-Rx activity was measured as previously described (Amicarelli et al., 1999, 2000). The reaction mixture contained 0.1 mol/l potassium phosphate buffer pH 7.4, 1 mmol/l GSSG, and 0.16 mmol/l NADPH and appropriate amounts of follicular samples. The rate of NADPH oxidation was monitored at 25°C with a Perkin–Elmer spectrophotometer, after the decrease of absorbance at 340 nm (340 nm = 6.22 mmol/l–1 cm–1). The blank did not contain GSSG and the activity was calculated after subtraction of the blank value. Reaction rate proportionality was assessed by using different sample aliquots for the activity measurements. One unit of enzyme activity was defined as 1 µmol of NADPH oxidized/min at 25°C. Specific activities were expressed as mIU/mg protein. The assay detection level was 1.6 mIU/ml. In each patient, the GSH reductase activity was calculated at least four times. The intra-assay CV was always <4%.
Protein assay
The protein concentration was determined by the biuret method, using bovine serum albumin as the standard (Sigma).
Western blot analysis
Follicular fluids from three young women and three older women used to determine enzymatic activity were also processed for Western blot analysis, according to a method previously described (Amicarelli et al., 2001). Briefly, samples containing the same amount of protein (15 µg) were run on 12% polyacrylamide gels. Protein bands were transferred onto polyvinylidene difluoride sheets by wet electrophoretic transfer. Non-specific binding sites were blocked overnight at 4°C with 5% no-fat dry milk in Tris-buffered saline (TBS) containing 0.05% Tween 20/TBS. Membranes were incubated with the primary antibody at the appropriate dilution, according to the manufacturer’s instructions for 2 h at room temperature, followed by incubation with goat anti-rabbit or anti-mouse peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech, USA) (1:2000 in blocking solution) for 1 h at room temperature. After rinsing, the specific immune complexes were detected by ECL kit (Amersham Pharmacia Biotech). Semiquantification of immunoreactive bands was performed using Adobe Photoshop version 4.0 (Adobe Systems, USA). The mean pixel intensity of a preselected area, set to include the largest band, was obtained and adjusted for background intensity of our gel. The mean intensity of the bands in young women was used as the baseline value to which it was arbitrarily assigned the value of 1. The intensity of the bands in older women was calculated relative to this value. The experiment was performed in quadruplicate.
Statistical analysis
Enzymatic activity results were analysed by using Sigma Stat software (Jandel Scientific Software Corporation, USA). When the data passed normality and equal variance tests, Student’s t-test was applied. Otherwise we used the Mann–Whitney rank sum test. The null hypothesis was rejected at P < 0.05. All numerical data reported are expressed as the mean ± SEM. The SEM never exceeded 5% of the mean value.
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Results |
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The volume of aspirated follicular fluid obtained from single mature follicles were not significantly different in the patients from the two age groups (mean volume ± SEM: 3.80 ± 0.13 and 3.60 ± 0.13 ml in young and older women respectively). Protein assay performed on samples obtained by pooling follicular fluids from three follicles per patient revealed the presence of a similar amount of protein (40.0 ± 2.0 mg/ml).
Biochemical analyses revealed that human follicular fluids from both young and older women display detectable amounts of all the enzymes investigated.
Effect of ageing on catalase, SOD and glutathione-dependent enzymatic activities
We observed significant variations between the two groups in the activities of enzymes directly involved in ROS scavenging. In particular, in older women the specific activity of catalase, the enzyme that catalyses the removal of hydrogen peroxide, is significantly lower, 60% (P < 0.0005), than young women (Figure 1A). By contrast, the activity of SOD, that removes superoxide anions, reveals a significant increase to 25% in women of advanced reproductive age (P < 0.01) (Figure 1A). Neither glutathione peroxidases nor glutathione reductase specific activities were affected by age, although a trend to increased activity was observed in the older group (Figure 1B). The changes in SOD and catalase activities cause a reduction in the catalase/SOD ratio and a slight decrease in GSH-Px/SOD ratio in older women. On the contrary, the GSSG-Rx/GSH-Px ratio was similar in the two groups (Table I).
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Glutathione transferase activity showed statistically significant variation with age as it decreased
20% (P < 0.05) in older women compared with young women (Figure 1B).
Effect of ageing on SOD, catalase and GST isoenzyme expression
To determine whether the difference in SOD, catalase and GST specific activities found between young and older women were due to different protein expression, we investigated whether these enzymes are expressed differently in the two groups of patients.
Results show that the bands corresponding to catalase and SOD from young women are similar to those in older women (Figure 2A and B respectively), contary to what was observed for enzymatic activities. As for SOD protein, it is important to note that immunoblot analysis reveals the presence of extracellular Cu-Zn SOD which is known to be a secretory tetrameric glycoprotein with a molecular weight of 135 kDa (Marklund, 1984).
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We found detectable amounts of both GST Alpha and GST Pi isoforms in human follicular fluid. Moreover, the signal corresponding to GST Alpha isoenzyme from young women was not different from that in older women (Figure 2C) while the signal relative to GST Pi isoenzyme from young women is higher than that from older women (Figure 2D). The expression of GST Mu was not detectable in both young or older women.
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Discussion |
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In this study we demonstrate, for the first time, the presence of the major antioxidant and detoxifying enzymes in human follicular fluid. In particular, the activity of SOD, catalase and GSH-Px ensure an efficient scavenging action against reactive oxygen intermediates, thus preventing them from rapidly diffusing into oocyte membranes during the period of resumed meiotic activity. It is well known that germ cell membranes are particularly vulnerable to attack by ROS, being very rich in polyunsaturated fatty acids (Zeron et al., 2001; Lenzi et al., 2002). The high level of GST activity in this compartment can further contribute to an efficient detoxification from ROS byproducts. The presence of the Alpha and Pi GST isoenzymes in human follicular fluid has already been described (Bisseling et al., 1997). Our findings confirm this expression pattern and, in addition, show significant amounts of Se-independent GSH-Px activity, therefore emphasizing the antioxidant function of GST in follicular fluid. Finally, the significant levels of GSSG-Rx in this compartment may efficiently supply GSH, which, besides being a cofactor essential for both GSH-Px and GST activity, is also one of the most efficient non-enzymatic antioxidants. As a final result, the coordinate action of antioxidant enzymes, possibly working in concert with non-enzymatic antioxidants, may provide maximum protection against ROS and their byproducts. Data from the literature describing the absence of oxidative stress markers in human follicular fluid (Jozwik et al., 1999) are consistent with this conclusion.
Moreover, our data indicate that ageing can significantly affect the follicular fluid antioxidant enzymatic pattern, suggesting a change in ROS generation in the follicle. In particular, we found a significant increase in SOD specific activity, as well as a decrease in catalase specific activity. On the basis of Western blot analysis, neither of these changes is associated with variations in the protein level, suggesting that they are due to post-translational processes.
As shown in Table I, the age-dependent changes in SOD and catalase activities cause a reduction in the catalase/SOD ratio and a slight decrease in GSH-Px/SOD ratio, thus suggesting a lowering in ROS scavenging efficiency with ageing. On the contrary, ageing does not affect GSH recycling efficiency, since the GSSG-Rx/GSH-Px ratio is similar in the two groups (Somani et al., 1996). Although in this case we cannot state that the observed decrease in catalase/SOD ratio results in reduced scavenging efficiency, it is important to note that similar age-related ratio decreases found in some mammalian organs strongly affected their response to oxidative stress challenge (Amicarelli et al., 1997, 1999).
Ageing also seems to cause a significant reduction in GST specific activity, which on the basis of Western blot investigation, may be due to a reduction in the amount of the Pi isoform, recently recognized as one of the most efficient in detoxifying toxic oxidative stress byproducts (Yin et al., 2000).
Since follicular fluid analysed in this study in both age groups was from follicles of similar diameter, it can be suggested that the reported changes in antioxidant activities may reflect an age-dependent weakening of antioxidant defences in the follicular environment that surrounds the oocyte during maturation and ovulation. At the moment, however, it cannot be established whether, under these conditions, ROS are maintained at those ‘controlled levels’ at which they may play a positive role for gamete function (de Lamirande and Gagnon, 1995; Blondin et al., 1997).
In conclusion, although we are aware of the limited population size of the present study, the two major findings of this work are that significant amounts of antioxidant enzymes are present in human follicular fluid and that follicular fluid from older women display a different pattern of antioxidant enzymatic defences. This condition might be due to an age-linked altered metabolism in granulosa cells, perhaps as a result of hypoxic episodes caused by defective microcirculation within the follicles (Friedman et al., 1997), which, in turn, could affect antioxidant enzymatic expression (Schreck and Baeuerle, 1991). Work is in progress to investigate the possible different adaptive responses to oxidative stress injury in granulosa cells from women of advanced reproductive age.
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Acknowledgements |
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This research was supported by a grant from the ‘Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale.’ The authors thank Prof. M.Moscarini for his invaluable contribution to this work and helpful discussions.
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References |
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Amicarelli, F., Di Ilio, C., Masciocco, L., Bonfigli, A., Zarivi, O., D’Andrea, M.R., Di Giulio, C. and Miranda, M. (1997) Aging and detoxifying enzymes responses to hypoxic or hyperoxic treatment. Mech. Ageing Dev., 97, 15–226.[CrossRef][ISI][Medline]
Amicarelli, F., Ragnelli, A.M., Aimola, P., Bonfigli, A., Colafarina, S., Di Ilio, C. and Miranda, M. (1999) Age-dependent ultrastructural alterations and biochemical response of rat skeletal muscle after hypoxic or hyperoxic treatments. Biochim. Biophys. Acta, 1453, 105–114.[Medline]
Amicarelli, F., Tiboni, G.M., Colafarina, S., Bonfigli, A, Iammarrone, E., Miranda, M. and Di Ilio, C. (2000) Antioxidant and GSH-related enzyme response to a single teratogenic exposure to the anticonvulsant phenytoin: temporospatial evaluation. Teratology, 62, 100–107.[CrossRef][ISI][Medline]
Amicarelli, F., Ragnelli, A.M., Aimola, P., Cattani, F., Bonfigli, A., Zarivi, O., Miranda, M. and Di Ilio, C. (2001) Developmental expression and distribution of amphibian glutathione transferases. Biochim. Biophys. Acta, 1526, 77–85.[Medline]
Bisseling, J.G.A., Knapen, M.F.C.M., Goverde, H.J.M., Mulder, T.P.J., Peters, W.H.M., Willemsen, W.N.P., Thomas, C.M.G. and Steegers, E.A.P. (1997) Glutathione S-transferases in human ovarian follicular fluid. Fertil. Steril., 68, 907–911.[CrossRef][ISI][Medline]
Blondin, P., Coenen, K. and Sirard, M. (1997) The impact of reactive oxygen species on bovine sperm fertilizing ability and oocyte maturation. J. Androl., 18, 454–460.
Cetica, P.D., Pintos, L.N., Dalvit, G.C. and Beconi, M.T. (2001) Antioxidant enzyme activity and oxidative stress in bovine oocyte in vitro maturation. IUBMB Life, 51, 57–64.[CrossRef][ISI][Medline]
deLamirande, E. and Gagnon, C. (1995) Impact of reactive species on spermatozoa: a balancing act between beneficial and detrimental effects. Hum. Reprod., 10, 15–21.
deLamirande, E., Harakat, A. and Gagnon, C. (1998) Human sperm capacitation induced by biological fluids and progesterone, but not by NADH or NADPH, is associated with the production of superoxide anion. J. Androl., 19, 215–225.
DiIlio, C., Aceto, A., Bucciarelli, T., Angelucci, S., Felaco, M., Grilli, A., Zezza, A., Tenaglia, R. and Federici, G. (1991) Glutathione transferase isoenzymes in normal and neoplastic human kidney tissue. Carcinogenesis, 12, 1471–1475.
Droge, W. (2002) Free radicals in the physiological control of cell function. Physiol. Rev., 82, 47–95.
Friedman, C., Danforth, D.R., Herbosa-Encarnacion, C., Arbogast, L., Alak, B.M. and Seifer, D.B. (1997) Follicular fluid vascular endothelial growth factor concentrations are elevated in women of advanced reproductive age undergoing ovulation induction. Fertil. Steril., 68, 607–612.[CrossRef][ISI][Medline]
Gardiner, C.S., Salmen, J.J., Brandt, C.J. and Stover, S.K. (1998) Glutathione is present in reproductive tract secretions and improves development of mouse embryos after chemically induced glutathione depletion. Biol. Reprod., 59, 431–436.
Guérin, P., Moutassim, S.E.I. and Ménézo, Y. (2001) Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings. Hum. Reprod. Update, 7, 175–189.
Guyader-Joly, C., Guérin, P., Renard, J.P., Guillaud, J., Ponchon, S. and Ménézo, Y. (1998) Precursors of taurine in female genital tract: effects on developmental capacity of bovine embryo produced in vitro. Aminoacids, 15, 27–42.[CrossRef][ISI][Medline]
Harman, D. (1984) Free radical theory of aging. Free Radical Diseases. Age, 57, 111–131.[CrossRef]
HayesJ.D.andMcLellan, L.I. (1999) Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress. Free Rad. Res., 31, 273–300.[ISI][Medline]
Hensley, K., Robinson, K.A., Garitta, S.P., Salsman, S. and Floyd, R.A. (2000) Reactive Oxygen species, cell signalling and cell injury. Free Rad. Biol. Med., 28, 1456–1462.[CrossRef][ISI][Medline]
Jozwik, M., Wolczynski, S., Jozwik, M. and Szamatowicz, M. (1999) Oxidative stress markers in preovulatory follicular fluid in humans. Mol. Hum. Reprod., 5, 409–413.
Lenzi, A., Gandini, L., Lombardo, F., Picardo, M., Maresca, V., Panfili, E., Tramer, F., Boi, C. and Dondero, F. (2002) Polyunsaturated fatty acids of germ cell membranes, glutathione and blutathione-dependent enzyme-PHGPx: from basic to clinic. Contraception, 65, 301–304.[CrossRef][ISI][Medline]
Liu, L., Trimarchi, J.R. and Keefe, D.L. (2000) Involvement of mitochondria in oxidative stress-induced cell death in mouse zygotes. Biol. Reprod., 62, 1745–1753.
Marklund, S.L. (1984) Extracellular superoxide dismutase in human tissues and human cell lines. J. Clin. Invest., 74, 1398–1403.[ISI][Medline]
Mouatassim, S.EI., Guérin, P. and Ménézo, Y. (1999) Expression of genes encoding antioxidant enzymes in human and mouse oocytes during the final stage of maturation. Mol. Hum. Reprod., 5, 720–725.
Paszkowsky, T., Traub, A.I., Robinson, S.Y. and McMaster, D. (1995) Selenium dependent glutathione peroxidase activity in human follicular fluid. Clin. Chim. Acta, 236, 173–180.[CrossRef][ISI][Medline]
Schreck, R. and Baeuerle, P. (1991) A role for oxygen radicals as second messenger. Trends Cell. Biol., 1, 39–42.[CrossRef][Medline]
Somani, S.M., Husain, K. and Schlorff, E.C. (1996) Response of antioxidant system to physical and chemical stress. In Baskins, I.S. and Salem, H. (eds), Oxidant, Antioxidants and Free Radicals. Taylor & Francis, London, pp. 125–141.
Tao, M., Kodama, H., Kagabu, S., Fukuda, J., Murata, M., Shimizu, Y., Hirano, H. and Tanaka, T. (1997) Possible contribution of follicular interleukin-1ß to nitric oxide generation in human pre-ovulatory follicles. Hum. Reprod., 12, 2220–2225.
Tarín, J.J., Pérez-Albalá, S. and Cano, A. (2000) Consequences on offspring of abnormal function in ageing gametes. Hum. Reprod. Update, 6, 532–549.
Tarín, J.J., Pérez-Albalá, S. and Cano, A. (2002) Oral antioxidants counteract the negative effects of female aging on oocyte quantity and quality in the mouse. Mol. Reprod. Dev., 61, 385–397.[CrossRef][ISI][Medline]
Yin, Z., Ivanov, V.N., Habelsah, H. Tew, K. and Ronai, Z. (2000) Glutathione S-transferase p elicits protection against H2O2-induced cell induced death via coordinated regulation of stress kinases. Cancer Res., 60, 4053–4057.
Zeron, Y., Ocherentny, A., Kedar, O., Borochov, A., Sklan, D. and Arav, A. (2001) Seasonal changes in bovine fertility: relation to developmental competence of oocytes, membrane properties and fatty acid composition of follicles. Reproduction, 121, 447–54.[Abstract]
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