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Regular Article |
-Glucoside During In Vitro Maturation1
a School of Bioresources, Hiroshima Prefectural University, Shobara, Hiroshima 727-0023, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-glucoside (AA-2G), a stable ascorbate derivative, on the sustenance of cytoplasmic maturation responsible for subsequent developmental competence after in vitro fertilization of porcine oocytes. Cumulus-oocyte complexes were cultured for 44 h in North Carolina State University 37 medium supplemented with cysteine, gonadotropins, 10% (v:v) porcine follicular fluid, and 0–750 µM AA-2G. When oocytes were matured in the presence of 250 µM AA-2G, their ability to promote transformation of the sperm nucleus into the male pronucleus (MPN) was strongly enhanced after in vitro fertilization. Similarly, the presence of 25 µM ß-mercaptoethanol (ME) enhanced the degree of progression to MPN of penetrated sperm by associating with the increase in intracellular glutathione (GSH) content. Although the AA-2G treatment during oocyte maturation showed no influence on the GSH concentration, significantly higher levels of ascorbic acid (AsA) were detected in these oocytes than in those oocytes cultured without AA-2G (P < 0.05). The length of DNA migration encompassed by reactive oxygen species (ROS), generated by the hypoxanthine-xanthine oxidase system, was not increased in the oocytes treated with AA-2G, whereas ME treatment could not block the DNA damage by ROS. These findings indicate that AA-2G in maturation medium can potentiate the cellular protection of oocytes against oxidative stress by continuously supplying AsA. The proportion of development to the blastocyst stage after in vitro insemination was significantly increased in oocytes matured with AA-2G (P < 0.05), and this proportion showed no difference in comparison with that of oocytes treated with ME. These findings suggest that a critical concentration of intracellular AsA, supplied by AA-2G during in vitro maturation, plays an important role in supporting the cytoplasmic maturation responsible for developmental competence after fertilization by prevention of oxidative stress against porcine oocytes.
early development, fertilization, ovum, stress
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In our previous study [18], cumulus cells during IVM efficiently protected porcine oocytes against cell damage caused by oxidative stress resulting in significantly increased concentrations of intracellular GSH in cumulus-oocyte complexes (COCs). However, the GSH content in cumulus-denuded oocytes was markedly decreased, and exposure of denuded oocytes to reactive oxygen species (ROS) resulted in an increased frequency of apoptotic cell death. With these findings, we suggested that cumulus cells play a critical role in protecting oocytes against oxidative stress-induced apoptosis through the enhancement of GSH content in oocytes. It is thus suggested that mediation of antioxidants or free-radical scavengers to prevent damage to porcine oocytes caused by oxidative stress plays an important role in acquiring developmental competence, but it remains unknown whether antioxidants contribute to functional roles by supporting cytoplasmic maturation in mammalian oocytes.
A large amount of L-ascorbic acid (AsA) was contained in the preovulatory follicles in rat ovaries [19]. In general, AsA functions in many biological processes, such as biosynthesis of collagen and other components of the extracellular matrix, and it has been considered to be the most important antioxidant in extracellular fluids [20–22]. In fact, AsA added to media could prevent follicular apoptosis in cultured rat [23] and mouse [24] follicles. However, AsA is unstable under various oxidative conditions, such as exposure to neutral pH, heat, light, and heavy metals, which results in rapid degradation [25, 26]. Thus, few studies have focused on the functional roles of AsA in oocyte maturation during IVM culture. In contrast to AsA, ascorbic acid 2-O--glucoside (AA-2G) is characterized by its high stability toward thermal and oxidative degradation in aqueous solutions and its nonreducibility [26, 27]. Treatment with AA-2G is capable of enhancing both antibody production in cultured murine splenocytes [28] and collagen synthesis in confluent cultures of human skin fibroblasts [29]. Because the intact form of AA-2G is not detected in these cells [28, 29], AA-2G is hydrolyzed by membrane-bound -glucosidase-like enzyme(s), and the resultant AsA is transported into the cells. Therefore, the present study was conducted to clarify whether addition of AA-2G to maturation medium supported the cytoplasmic maturation responsible for subsequent developmental competence in porcine oocytes. Meiotic maturation, in vitro fertilization (IVF) parameters, and subsequent development to the blastocyst stage were investigated in oocytes treated with AA-2G, and the improving effects of AA-2G were compared with the functional roles of ME leading to increased intracellular GSH levels.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Collection of Oocytes
Ovaries were collected from maturing gilts at a local slaughterhouse and transported to the laboratory in 0.9% (w:v) NaCl containing 100 mg/L kanamycin sulfate (Meiji Seika, Tokyo, Japan) at 30°C. Within 2 h after slaughter, the follicular contents were recovered by excising the visible small antral follicles (diameter, 2–6 mm) on the ovarian surface using a razor and by scraping the inner surface of the follicle walls with a disposable surgical blade. Only COCs with a uniform ooplasm and a compact cumulus cell mass were collected and washed three times with HEPES-buffered Tyrode medium containing 0.01% (w:v) polyvinyl alcohol (H-TL-PVA) [1].
Maturation Culture of Oocytes
The basic medium used for oocyte maturation was BSA-free North Carolina State University (NCSU) 37 medium [7] supplemented with 0.6 mM cysteine, 0.04 U/ml of ovine FSH, 0.02 U/ml of ovine LH, 10% (v:v) porcine follicular fluid, and various concentrations of agents. Porcine follicular fluid was aspirated from follicles (diameter, 2–6 mm) and stored at -30°C until use. After washing in basic medium, groups of 20 COCs were transferred into 100-µl droplets of the basic medium, which had been previously equilibrated in a CO2 incubator. After 20 h of maturation culture, the oocytes were washed and transferred to 100-µl droplets of the basic medium without hormonal supplementation for an additional 24 h of culture. All media containing COCs were covered with mineral oil and cultured at 39°C in an atmosphere of 5% (v/v) CO2 in air. After total maturation culture of 44 h, COCs were sucked through a narrow-bore pipette to remove their cumulus cells in H-TL-PVA containing 0.1% (w:v) hyaluronidase.
In Vitro Fertilization
Denuded oocytes were washed three times with modified Tris-buffered medium (mTBM) [4], designated as IVF medium, supplemented with 2 mM caffeine sodium benzoate and 0.1% (w:v) BSA. After washing, 25–30 oocytes were transferred to 50-µl droplets of IVF medium that had been covered with warm mineral oil. The droplets containing oocytes were kept in the incubator for 30–45 min until spermatozoa were added for fertilization. For sperm preparation, frozen-ejaculated boar spermatozoa were thawed (39°C) and washed two times by centrifugation at 400 x g for 4 min in Dulbecco PBS (Gibco BRL, Grand Island, NY) supplemented with 0.1% (w:v) PVA at pH 7.2. At the end of the washing procedure, the sperm pellet was resuspended at 4 x 108 cells/ml in mTBM supplemented with 4 mM caffeine sodium benzoate and 0.4% (w:v) BSA and then incubated for 90 min at 39°C. After sperm preincubation, 50 µl of diluted sperm suspension in IVF medium was added to a droplet containing oocytes for a final sperm concentration of 1 x 106 cells/ml. Oocytes were coincubated with spermatozoa for 7 h at 39°C in an atmosphere of 5% CO2 in air.
Embryo Culture
After insemination, oocytes were removed from fertilization drops, washed three times with NCSU37 medium, and cultured in 50 µl of NCSU37 medium at 39°C in an atmosphere of 5% CO2 in air. At 48 and 168 h after IVF, cleavage rate and blastocyst formation, respectively, were evaluated under a stereomicroscope.
Experimental Design
In experiment 1, IVM of porcine oocytes was carried out in the presence of 0, 100, 250, 500, or 750 µM AA-2G (Hayashibara Biochem. Lab., Okayama, Japan) to evaluate the effect of AA-2G on oocyte maturation and fertilization parameters after IVF. After IVM culture, a portion of oocytes was fixed to examine for nuclear maturation or prepared for assay of the intracellular AsA concentration, and the remaining oocytes were inseminated to examine for fertilization parameters.
In experiment 2, oocytes were cultured for 44 h in the absence or presence of 250 µM AA-2G, 250 µM AsA, and 25 µM ME. The concentration of AsA added to medium was reconciled with that of AA-2G, and the addition of AsA to IVM culture was performed to show the superior effects of AA-2G on protection against oxidative damage. In contrast, ME was used to compare the effects with those of AA-2G on the protection of oocytes from oxidative stress and the acquirement of developmental competence after IVF, because the presence of ME during IVM culture effectively increases the intracellular GSH synthesis, which is partly associated with cytoplasmic maturation of porcine oocytes [5, 16]. The concentration of ME was based on the results of a study reported by Abeydeera et al. [5]. After IVM culture, a portion of oocytes was prepared for assay of GSH contents or DNA damage, and another portion was inseminated to examine fertilization parameters and developmental competence to the blastocyst stage.
Assessment of Meiotic Maturation and Fertilization Parameters
After IVM culture or 10 h after IVF, groups of 30–40 oocytes were mounted, fixed in acetic acid : ethanol (1:3, v:v) for 72 h, stained with 1% (w:v) lacmoid in 45% (v:v) acetic acid, and examined for nuclear maturation or fertilization parameters, respectively, under a phase-contrast microscope at 400x magnification. Germinal vesicle breakdown (GVBD), maturation to metaphase II (M-II), sperm penetration, polyspermy, and MPN formation were assessed. Oocytes were considered to have been penetrated by spermatozoa at the M-II stage when they had two polar bodies, one or more swollen sperm heads, and/or MPN and their corresponding sperm tail.
Determination of AsA
Intracellular AsA levels of the oocytes were determined by high-performance liquid chromatography (HPLC) as described previously [30] but with several modifications. In this HPLC method, AsA was clearly distinguishable from AA-2G according to differences in their retention times [31]. Oocytes cultured in medium with 0, 100, 250, 500, and 750 µM AA-2G as well as oocytes freshly isolated from their follicles (freshly isolated oocytes) were vortexed in H-TL-PVA containing 0.1% (w:v) hyaluronidase at the maximum speed (3400 rpm) for 1 min to remove cumulus cells completely and then washed three times in cold-PBS supplemented with 0.01% (w:v) PVA. Immediately, groups of 100 oocytes were put into microtubes containing 10 µl of 1.5% (w:v) metaphosphoric acid solution (pH 3.5). The extract was centrifuged for 10 min at 12 000 x g and 4°C, and the supernatant was analyzed on a Shim-pack ODS column (6 x 150 mm; Shimadzu, Kyoto, Japan). The oocytes were extracted and chromatographed by HPLC on the same day. The HPLC system was equipped with a pulsation damper setting between the pump and injector, and a UV detector Model SPD-10A (Shimadzu). The mobile phase was 0.1 M KH2PO4-H3PO4 buffer (pH 2.0) containing 10 µg/ml of EDTA with a flow rate of 0.7 ml/min. The absorbance of AsA was monitored at 240 nm. The standard AsA solution was freshly prepared at a concentration of 1 mM in 1.5% (w:v) metaphosphoric acid solution and diluted immediately before use.
Assay of GSH
Oocytes treated with or without 250 µM AA-2G, 250 µM AsA, and 25 µM ME, in addition to freshly isolated oocytes, were assayed for GSH content. Denuded oocytes were washed three times in the stock buffer (0.2 M sodium phosphate buffer, pH 7.2, containing 10 mM EDTA). Groups of 40 oocytes in 5 µl of stock buffer were transferred to 1.5-ml microtubes, and 5 µl of 1.25 M H3PO4 were added. Samples were stored at -80°C until assay. The concentration of intracellular GSH in oocytes was determined using the 5,5-dithio-bis(2-nitro-benzoic acid)-glutathione disulfide (DTNB-GSSG) reductase recycling assay as described previously [18], which detects both GSH and GSSG in the oocyte.
Analysis of DNA Damage
To clarify precisely the DNA damage caused by oxidative stress, the oocytes for this assay were cultured in the basic medium with 1 mM hypoxanthine and 1 mU/ml of xanthine oxidase during IVM, as reported previously [18]. Oocytes treated with or without 250 µM AA-2G, 250 µM AsA, and 25 µM ME, in addition to freshly isolated oocytes, were transferred into 0.1% (w:v) protease solution in H-TL-PVA at room temperature to remove the zona pellucida, then washed quickly in PBS containing 0.3% (w:v) BSA. The DNA damage in each oocyte was detected by single cell microgel electrophoresis (comet) assay according to the method reported by Singh et al. [32] and Tatemoto et al. [18], but with several modifications. Fifteen to twenty zona-free oocytes were mixed with 10-µl drops of 2% (w:v) low-melting agarose (SeaPlaque GTG agarose; FMC BioProducts, Rockland, ME) at 40°C on the glass slide, and the cell suspension was immediately covered with a coverslip. The space between the coverslip and the glass slide was filled with 2% low-melting agarose, and the slides were then kept at 4°C for 10 min to allow solidification of the agarose. After gently removing the coverslip, the slides stuck with the oocytes-embedded agarose were immersed in a lysing solution (1% N-lauroyl-sarcosine, 2.5 M NaCl, 20 mM EDTA, 10 mM Tris, and 1% Triton X-100, pH 10) for 1 h to lyse the cells and permit DNA unfolding. The slides were then placed on a horizontal gel electrophoresis unit and equilibrated for 20 min in an electrophoresis buffer (100 mM Tris, 90 mM boric acid, and 1 mM EDTA, pH 8.0). Electrophoresis was conducted for 20 min at 50 V. After electrophoresis, the slides were stained with 10 µg/ml of bis-benzimide Hoechst 33342 for 10 min, washed with distilled water, and then covered with a coverslip. The slides were sealed with clear nail polish and examined using a fluorescent microscope. The length of migrated DNA of 30–40 oocytes in each experimental group was individually measured using a micrometer.
Statistical Analysis
Statistical analyses of findings from four replicate trials for each treatment comparison were carried out by ANOVA and Fisher protected least significant difference test using the STATVIEW program (Abacus Concepts, Inc., Berkeley, CA). All percentage values were subjected to arc-sine transformation before statistical analysis. Values are expressed as the mean ± SEM. A probability of P < 0.05 was considered to be statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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When oocytes were cultured for 44 h in the absence or presence of increasing AA-2G concentrations (range, 100–750 µM), treatment with AA-2G at a concentration of 500 µM or greater showed a tendency to decrease the incidence of GVBD and oocyte maturation up to the M-II stage (Table 1). As shown in Table 2, no difference was found in the rates of penetration (85–89%), polyspermic fertilization (48–52%), and mean number of spermatozoa per oocyte (1.8–1.9) among the experimental groups. The oocytes matured in medium supplemented with 250 µM AA-2G showed a significant increase in the proportion of MPN formation after sperm penetration (78%) compared with that of oocytes treated without AA-2G (64%; P < 0.05). However, treatments with the higher concentrations (500 and 750 µM) of AA-2G during IVM culture showed a weak, but not significant, acceleration of MPN formation. From these findings, it was suggested that 250 µM of AA-2G added to maturation medium was optimal for supporting cytoplasmic maturation in porcine oocytes.
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As shown in Figure 1, the concentration of intracellular AsA in oocytes freshly isolated from their follicles (i.e., freshly isolated oocytes) was 820.2 ± 23.7 fmol/oocyte, whereas the oocytes matured without AA-2G showed a trace amount of AsA (6.0 ± 0.6 fmol/oocyte). However, the AsA content of oocytes increased progressively with the increase in the AA-2G concentration added to the maturation medium. The oocytes treated with 250 µM AA-2G during IVM possessed a significantly higher level of intracellular AsA (406.1 ± 12.3 fmol/oocyte) than untreated oocytes, and the addition of 500 or 750 µM AA-2G to IVM medium significantly increased in the intracellular concentration of AsA (802.6 ± 37.9 or 1253.3 ± 29.4 fmol/oocyte, respectively; P < 0.05).
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Effects of Treatment with AA-2G, AsA, or ME on GSH Content, DNA Damage, Fertilization Parameters, and Embryo Development
In freshly isolated oocytes, the average intracellular GSH was 6.5 ± 0.4 pmol/oocyte (Fig. 2A). The GSH content of oocytes cultured in medium with 250 µM AsA was not changed with the advance of oocyte maturation (6.8 ± 0.4 pmol/oocyte), and no significant difference was found in these values compared with those of oocytes without treatment (7.0 ± 0.2 pmol/oocyte). Treatment with 25 µM ME during IVM caused a significant increase in GSH content (12.3 ± 0.3 pmol/oocyte), whereas treatment with 250 µM AA-2G did not exert any eminent influence on GSH content.
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As a prelude to investigations regarding DNA damage during IVM culture, our previous study [18] measured the lengths of DNA migration in oocytes under the normal culture condition and compared these lengths with those of freshly isolated oocytes. However, the lengths of DNA migration slightly increased (82.3 ± 3.7 µm) compared with those of freshly isolated oocytes (73.3 ± 3.0 µm), because cumulus cells coupled to oocytes had a critical role in protecting oocytes against oxidative stress. Therefore, COCs in this assay were cultured in the basic medium with 1 mM hypoxanthine and 1 mU/ml of xanthine oxidase to clarify more precisely the DNA damage caused by oxidative stress. The ROS produced by the hypoxanthine-xanthine oxidase system significantly increased the lengths of DNA migration of a single oocyte, despite treatment with or without AsA (136.0 ± 10.6 and 136.0 ± 12.1 µm, respectively), compared with those of freshly isolated oocytes (72.7 ± 3.0 µm; P <0.05). The same increase in the length of DNA migration was also observed in oocytes cultured with ME (136.5 ± 8.6 µm). In contrast, treatment with AA-2G could prevent DNA damage in oocytes from ROS during IVM by maintaining the length of DNA migration (85.5 ± 8.1 µm) similar to that of freshly isolated oocytes (Fig. 3).
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After in vitro insemination of oocytes matured in medium with or without AsA, ME, and AA-2G, no difference was detectable in the rates of penetration (87–95%), polyspermic fertilization (60–66%), and mean number of spermatozoa per oocyte (2.1–2.3) among the experimental groups (Table 3). However, the proportion of MPN formation in the oocytes treated with ME or AA-2G (90% and 77%, respectively) was significantly higher than that in oocytes treated with or without AsA (62% and 65%, respectively; P < 0.05), although treatment with AA-2G during IVM did not stimulate the degree of progression to MPN of penetrated sperm to a level comparable with that of ME treatment. As shown in Figure 4, the cleavage rate after 48 h of in vitro insemination showed no difference in oocytes matured with or without AsA, ME, and AA-2G (72–74%). However, compared with those of oocytes treated with or without AsA (6% or 8%, respectively), significantly higher proportions of putative IVM-IVF zygotes developed to the blastocyst stage only when oocytes were matured in medium supplemented with ME (18%) and AA-2G (16%), with no significant difference between them (P < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, however, treatment with AsA during IVM showed no effect on prevention of an increased length of DNA migration by ROS (Fig. 2B), promotion of MPN formation, or development to the blastocyst stage after in vitro insemination (Table 3 and Fig. 4). When bovine oocytes were matured in vitro in the presence of 10, 100, or 500 µM AsA, the developmental competence of the oocytes after IVF was not improved [41]. Similarly, the effectiveness of AsA treatment was not evident in the potentiation for developmental ability of bovine [42] and hamster [43] embryos cultured in vitro. Because AsA is easily oxidized in aqueous solutions [25, 26], it appeared likely that AsA added to the maturation medium of porcine oocytes was degraded rapidly in that medium, showing no effect of protecting oocytes from ROS and supporting cytoplasmic maturation. In fact, we found that porcine follicular fluid collected from small antral follicles within 2 h after slaughter contained 154.4 ± 0.5 µM AsA, although AsA in follicular fluid was degraded rapidly after preparation and its concentration was reduced to less than 50% within 8 h after incubation (unpublished data). In addition, when oocytes were cultured in maturation medium without AA-2G, their intracellular AsA was completely consumed to nearly zero (Fig. 1). This suggests that the oocytes were cultured under conditions distinct from those in vivo with respect to intra- and extracellular concentrations of AsA. Therefore, a stable and bioavailable ascorbate derivative should be added for supplementation of AsA throughout the IVM culture period of porcine oocytes.
The ability of oocytes to promote transformation of sperm nuclei into MPN was strongly enhanced by treatment with 250 µM AA-2G during IVM (Table 2), and significantly higher levels of AsA were detected in these oocytes than in those without AA-2G treatment (Fig. 1). Behrman et al. [38] reported that AsA uptake in rat granulosa cells is energy- and Na+-dependent, and that induction of AsA transporters occurred through multiple hormones, which ultimately influenced tyrosine-specific protein kinases. The mechanism of AsA uptake and the existence of AsA transporters in porcine COCs remains unclear, but to our knowledge, the present study is the first to show a stimulatory effect of AA-2G added to maturation medium on the progression to MPN of penetrated sperm through accumulation of AsA in their ooplasm. However, treatment with higher concentrations of AA-2G (500 or 750 µM) showed no effect on the induction of oocyte maturation and MPN formation, even in the presence of greatly higher levels of AsA uptake in these oocytes (Tables 1 and 2). As reported previously [44], AsA has two different actions: an antioxidant action at lower concentrations, and a pro-oxidant action at higher concentrations. Thus, it appears that accumulation of AsA beyond the optimum concentration ranges may have deleterious effects on maturation events occurring in both the nucleus and the cytoplasm.
The major nonproteinaceous sulfhydryl compound in mammalian cells is GSH, which plays an important role in protecting cells against oxidative stress [45]. In the present study, the GSH content of oocytes was significantly increased by treatment with ME (Fig. 2A), although the increase in the length of DNA migration caused by oxidative stress was not blocked in these oocytes (Fig. 2B). Even in the presence of ROS produced by the reaction of hypoxanthine with xanthine oxidase, the GSH content of porcine oocytes was significantly increased (to 9.8 ± 0.4 pmol/oocyte) by treatment with ME compared with that of untreated oocytes (5.8 ± 0.4 pmol/oocyte; P < 0.05), indicating that the increase in GSH content due to ME treatment was not inhibited by the produced ROS (data not shown). Thus, oocytes possessing high GSH content by treatment with ME subsequently stimulated the degree of progression to MPN of penetrated sperm, resulting in facilitation of development to the blastocyst stage. The intracellular GSH was associated with maintaining the redox state of matured oocytes, which might be responsible for enhancing MPN formation by reducing disulfide bonds in the penetrated sperm [46–48]. In contrast, the increasing length of DNA migration, encompassed by ROS, was not detected in oocytes treated with AA-2G, despite significantly lower levels of GSH than those in oocytes treated with ME (Figs. 2 and 3). These findings imply that the AA-2G treatment could potentiate the cellular protection of porcine oocytes against oxidative stress through the continuous release of AsA, which scavenges not only intracellular but also extracellular ROS. Afterward, the proportions of MPN formation and subsequent embryo development to the blastocyst stage could be increased by treatment with AA-2G during IVM culture, similar to the treatment with ME, irrespective of the lower values of GSH compared with those of oocytes treated with ME (Table 3 and Fig. 4). Very recently, we observed that when cumulus-denuded porcine oocytes were matured in the presence of 250 µM AA-2G, the intracellular GSH was significantly decreased with the advance of IVM culture, but the treatment with AA-2G caused a significant increase in the proportion of MPN formation following IVF, with a concomitant increase in the intracellular AsA levels (unpublished data). From these findings, the intracellular AsA clearly contributes to the acquirement of developmental competence after IVF by a mechanism, different from that of GSH, in which it functions to protect porcine oocytes against oxidative stress during IVM. However, further studies are necessary to improve the proportion of blastocyst development (e.g., the synergistic effect of AA-2G and ME on the cytoplasmic maturation must be examined).
In summary, the present study demonstrated that treatment with AA-2G, which is a stable form of AsA derivative, efficiently blocked DNA damage caused by oxidative stress in porcine oocytes, and that the oocytes treated with AA-2G at a certain concentration (250 µM) during IVM showed enhanced abilities to undergo MPN formation and to develop to the blastocyst stage after in vitro insemination. Therefore, it is suggested that AA-2G can support cytoplasmic maturation relating to developmental competence after fertilization by alleviating oxidative stress during oocyte maturation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants for scientific research from the Ito Memorial Foundation of Japan (to H.T.) and from the Ministry of Education, Science Sports and Culture of Japan (grant 11760196 to H.T.). 
2 Correspondence. FAX: 81 8247 4 0191; hidettmt{at}bio.hiroshima-pu.ac.jp
Accepted: August 2, 2001.
Received: May 22, 2001.
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