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BOR - Papers in Press, published online ahead of print March 17, 2004.
Biol Reprod 2004, 10.1095/biolreprod.103.022152
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BIOLOGY OF REPRODUCTION 71, 291–296 (2004)
DOI: 10.1095/biolreprod.103.022152
© 2004 by the Society for the Study of Reproduction, Inc.

Embryo

Redox Status of the Oviduct and Cdc2 Activity in 2-Cell Stage Embryos in Heat-Stressed Mice1

Manabu Ozawa, Takaya Matsuzuka, Miho Hirabayashi, and Yukio Kanai2

Institute of Agriculture and Forestry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian preimplantation embryos are vulnerable to heat stress. However, the mechanisms by which maternal heat stress compromises embryonic development are unclear. We hypothesized that the loss of developmental competence in maternally heat-stressed embryos results from enhanced oxidative stress in the oviducts. In experiment 1, oviducts and zygotes were collected from mice that were heat-stressed at 35°C and 60% relative humidity for 12 h on the day of pregnancy as well as from control mice. The zygotes were cultured for 84 h to assess their development, and the H2O2 level, glutathione concentration, and free radical scavenging activity (FRSA) were measured in the oviduct. In experiment 2, zygotes were cultured for 22 h to reach the late G2 phase in the 2-cell stage, and Cdc2 activity was assessed using immunoblotting. A high percentage (87.6%) of control embryos developed to morulae or blastocysts, whereas the majority (67.4%) of the heat-stressed group arrested at the 2-cell stage. Although heat stress did not alter the FRSA or glutathione concentration in the oviducts, the H2O2 level (P < 0.01) and its ratio to the FRSA (P < 0.05) significantly increased in the heat-stressed group. The Cdc2 activation at the 2-cell stage, as shown by the ratio of the dephosphorylated form to the phosphorylated form, was evident in control embryos but absent in heat-stressed embryos, and the level was similar to that in embryos blocked at the 2-cell stage (positive control). These results indicate that maternal heat stress enhances oxidative stress in the oviducts and that loss of developmental competence in maternally heat-stressed embryos correlates with a defect in Cdc2 activity at the 2-cell stage.

early development, embryo, female reproductive tract, oviduct, stress


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Because embryos are sensitive to heat stress during early development, an increase in maternal body temperature often leads to embryonic death. Such heat stress-induced early embryonic loss has been reported in a wide range of mammals and is prominent in domestic animals with enhanced metabolic rates and high productivity [1–3]. In lactating dairy cows with high milk yields, hyperthermia caused by heat stress becomes evident at temperatures as low as 27°C [4], resulting in reduced fertility [1–3, 5, 6].

Previous studies in cattle and mice have shown that the susceptibility of preimplantation embryos to heat stress is stage-dependent (i.e., the zygote is more sensitive than the morula or blastocyst to high temperatures) [6–8]. Based on results using in vitro culture systems, this phenomenon has been ascribed to the stage-dependent acquisition of thermotolerance [9] and the ability to synthesize glutathione (GSH) [10]. In cultured embryos, thermotolerance can be induced by short-term, mild heat shock at the 8-cell [9] or morula stage [10] but not at the zygote or 2-cell stage. However, the thermotolerance theory cannot fully explain the susceptibility of early embryos to maternal heat stress, because subsequent studies have revealed that bovine 2-cell embryos can synthesize increased heat shock protein 70 (HSP70) in response to heat shock but cannot develop thermotolerance [11]. In addition, exposure of bovine zygotes to fluctuating high temperatures carefully mimicking the rectal temperature of heat-stressed cows that experienced early embryonic death did not compromise normal development to the blastocyst stage [12]. Recently, we observed a similar phenomenon in mice: The deleterious effects of maternal heat stress on zygotes were not related to high body temperature alone but were mediated via physiological changes in the maternal environment that increased intracellular oxidative stress, as shown by the increased H2O2 concentrations and reduced GSH content within the embryo [13].

Early stage embryos are also sensitive to oxidative stress. The exposure of early embryos to oxidative stress under culture conditions disrupts normal development [14, 15]. When mouse zygotes are exposed to oxidative stress, development is consistently arrested at the 2-cell stage [16– 18]. Although the cytological mechanisms of the 2-cell block induced by oxidative stress are not completely understood, an embryo blocked at the 2-cell stage is arrested at the G2/M phase of the cell cycle [19]. This cell block is caused by the inactivation of maturation-promoting factor (MPF) or a defect in the dephosphorylation of Cdc2 [20, 21], an active component of MPF and a key molecule in the cell cycle [22]. These findings led us to speculate on the possible involvement of oxidative stress-mediated cell block in heat stress-induced early embryonic death. Therefore, the present series of experiments was designed to determine whether maternal heat stress enhances oxidative stress in the oviduct or oviduct fluid, which determines the embryo microenvironment, and whether a deficiency in developmental competence in maternally heat-stressed zygotes is correlated with a defect in Cdc2 activity at the 2-cell stage.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Postpubertal female ICR mice (age, 8–12 wk) and male BDF1 mice (age, >8 wk) were purchased from Charles River Japan, Inc. (Yokohama, Japan). The mice were housed at 25°C and 50% relative humidity under a 12L:12D photoperiod (lights-on, 0600 h) until use. All the experimental protocols and animal handling procedures were reviewed and approved by the Animal Care and Use Committee of the University of Tsukuba.

Materials

Mineral oil, GSH, glutathione disulfide reductase, ß-nicotinamide adenine dinucleotide phosphate (reduced form; NADPH), 5,5'-dithio-bis-2-nitrobenzoic acid (DTNB), Na2-EDTA, and 2',7'-dichlorodihydro-fluorescein diacetate (DCH-FDA) were purchased from Sigma Chemical Co. (St. Louis, MO). The eCG (Serotropin) and hCG (Gonatropin) were purchased from Teikokuzouki Pharmaceutical Co. (Tokyo, Japan). All the chemical components in the embryo culture medium (KSOM) [23]; the buffers for the immunoblotting, GSH, and H2O2 measurements; and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Heat Treatment of Pregnant Mice and Oviduct Collection

Female mice were superovulated by i.p. injections of 5 IU of eCG and 5 IU of hCG given 48 h apart. Then, each female was housed with a male during the dark period, and vaginal plug formation was confirmed at 0600 h the next day (Day 1 of pregnancy). The mated females were exposed to a temperature of 35°C with 60% relative humidity for 12 h during the light period on Day 1 (heat stress) or were not treated (control). Mice in the heat-stress and control groups were killed by cervical dislocation at 1800 h on Day 1, and oviducts containing zygotes were recovered and used in the experiments described below.

Monitoring Rectal Temperature

The rectal temperature of each mouse was measured inside an environmental chamber at 1800 h on Day 1 with a thermistor instrument (D611; Takara Thermistor Co., Tokyo, Japan) by inserting a probe (length, 1 cm) into the rectum for 20 sec as described in our previous study [13].

Assessing Embryo Developmental Competence

Embryos were recovered by flushing the oviducts with KSOM. Then, the collected embryos were washed three times in fresh KSOM and cultured as a litter in 50 µl of KSOM under mineral oil at 37.5°C in 5% CO2 in humid air for 84 h to determine their developmental ability.

GSH Concentration of the Oviduct Flush and Oviduct Tissue

Recovered oviducts were flushed with 50 µl of sodium phosphate buffer (0.2 M) with 10 mM Na2-EDTA. The oviduct flush was recovered, transferred to a 1.5-ml microfuge tube, and centrifuged at 10 000 x g for 5 min at 4°C. Then, the supernatant was recovered and used for the GSH concentration assay. The flushed oviducts were washed three times in fresh phosphate buffer and transferred to a 1.5-ml microfuge tube with 200 µl of buffer. Then, the oviducts were homogenized using a sonicator (Ultrasonic Disruptor; Tomy Seiko Co., Tokyo, Japan). The homogenate was centrifuged at 10 000 x g for 15 min at 4°C, and the supernatant was recovered and used for the GSH concentration assay. The GSH concentration in each sample was measured using the DTNB-glutathione disulfide reductase recycling assay as described by Anderson [24] (n = 10 each in heat-stress and control groups).

H2O2 Concentration and Free Radical Scavenging Ability in Oviduct Tissue

The oviducts from each mouse were recovered. The right oviduct was used to measure the free radical scavenging ability (FRSA), and the left was used to measure the H2O2 concentration (n = 9 each in heat-stress and control groups). Each oviduct was flushed with PBS (136 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, 1.47 mM KH2PO4), and the embryos were collected. The FRSA of the oviduct was measured as described by Kojima et al. [25] with some modification. Briefly, the oviduct was washed twice in assay buffer (0.01 µM Tris-HCl, 320 mM sucrose, 1 mM Na2-EDTA; pH 7.4) and homogenized in 500 µl of assay buffer using a sonicator. The homogenate was centrifuged at 13 000 x g for 30 min at 4°C, and 250 µl of supernatant were mixed with 200 µl of 200 µM DPPH in absolute ethanol and 50 µl of assay buffer. The absorbance at 517 nm was recorded using a spectrophotometer (V-550; JASCO International Co., Tokyo, Japan) at 1-min intervals for 20 min.

The left oviduct was used for the H2O2 assay according to the method described by Bejma and Ji [26]. The oviduct was washed twice in H2O2 assay buffer (130 mM KCl, 5 mM MgCl2, 20 mM NaH2PO4, 20 mM Tris-HCl, 30 mM glucose) and homogenized in 500 µl of assay buffer using a sonicator. The homogenate was centrifuged at 10 000 x g for 15 min at 4°C. Then, the supernatant, which was equivalent to 40 µg of protein, was transferred to a 1.5-ml microfuge tube, and assay buffer (total volume, 396 µl) and 4 µl of DCH-FDA (1 mM in dimethyl sulfoxide) were added. Subsequently, the samples were incubated at 37°C for 15 min. The fluorescence intensity, as a relative indicator of the intracellular H2O2 level, was monitored for 30 min after excitation at 488 nm and emission at 515 nm using a fluorescence spectrophotometer (RF-5300PC; Shimadzu Co., Kyoto, Japan). The protein concentration in each sample was also measured using Advanced Protein Assay Reagent (Cytoskeleton, Inc., Denver, CO) according to the manufacturer's instructions.

Immunoblotting Analysis of Cdc2 Activity in Embryos

Zygotes were collected from each treated mouse at 1800 h on Day 1 by flushing the oviduct with KSOM. Recovered zygotes were cultured in KSOM at 37.5°C in 5% CO2 in humid air for 18 h (48 h post-hCG injection; mid G2 phase) or 22 h (52 h post-hCG injection; late G2 phase) [20]. As a positive control (2-cell block embryos), zygotes from nonheat-stressed mice were cultured at a low CO2 concentration (1%) to induce 2-cell block. Subsequently, each group of 40–80 embryos, pooled from two to six animals per group, was lysed in SDS sample buffer, boiled for 5 min at 100°C to denature the proteins, and stored at –80°C until use. Samples were electrophoresed on an SDS/10% (w/v) polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore Co., Billerica, MA). The membrane was blocked with 3% (w/v) skimmed milk in PBS containing 0.1% (v/v) Tween-20 (PBST) for 1 h at room temperature. Subsequently, the blocked membrane was incubated with primary antibody against Cdc2 (Sc-54; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in PBST containing 0.1% (w/v) skimmed milk overnight at 4°C (1:250 dilution). Then, the membrane was incubated with secondary antibody (NA931; Amersham Pharmacia Biotech Ltd., Buckinghamshire, U.K.) in PBST containing 5% (w/v) skimmed milk at 37°C for 45 min (1:1000 dilution). The blots were detected using Western Blotting Detection Reagent (RPN2109; Amersham Pharmacia Biotech Ltd., Piscataway, NJ) according to the manufacturer's instructions. As a positive Cdc2 control, HeLa cells were used. The intensity of the upper (inactive form) and lower (active form) Cdc2 bands was determined using NIH Image software (National Institutes of Health, Bethesda, MD), and activities of Cdc2 kinase were assessed by the ratio of the lower band to the upper band (n = 5 measurements) according to the method described previously [20, 21, 27].

Statistical Analysis

The data are expressed as the mean ± SEM. The rectal temperatures of the mice and the Cdc2 kinase activities were analyzed using one-way ANOVA followed by the Fisher protected least-significant difference test. The percentages of embryos that underwent first cleavage or that developed into morulae or blastocysts were arc-sine transformed and then analyzed using the Student t-test. The GSH concentrations of oviduct tissues or oviduct fluids, H2O2 levels, and FRSA of oviduct tissues were compared using the Student t-test. Differences were considered to be significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Maternal Heat Stress on Rectal Temperature of Mice

The rectal temperatures of the heat-stressed groups were increased (39.6 ± 0.1°C for the GSH assay group, 39.5 ± 0.1°C for the H2O2 and FRSA group, and 39.7 ± 0.1°C for the Western blot analysis group) significantly (P < 0.001) compared with the control groups (37.6 ± 0.2°C for the GSH assay group, 37.7 ± 0.2°C for the H2O2 and FRSA group, and 37.6 ± 0.2°C for the Western blot analysis group). No significant differences were detected among the control groups or among the heat-stressed groups.

Effects of Maternal Heat Stress on Embryonic Developmental Competence In Vitro

The in vitro developmental competence of the embryos is shown in Table 1. The percentage of embryos that underwent first cleavage was not affected by maternal heat stress, whereas the percentage of embryos that reached the morula or blastocyst stage from the 2-cell stage significantly (P < 0.001) decreased in maternally heat-stressed embryos as compared with control embryos. In addition, the majority of heat-stressed embryos were arrested at the 2-cell stage, and the difference was significant (P < 0.001) compared with the controls.


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  		TABLE 1. In vitro development competence of embryos after maternal heat exposure during the zygote stage.a

Effect of Maternal Heat Stress on Redox Status in the Oviducts

No significant differences were found in the GSH concentrations of oviduct tissues (35.8 ± 1.2 vs. 37.4 ± 2.3 pmol GSH per µg protein for control vs. heat, respectively) or oviduct fluids (44.7 ± 7.9 vs. 37.5 ± 4.9 pmol GSH per µg protein for control vs. heat, respectively), as shown in Figure 1. The intracellular H2O2 levels in oviduct tissues, as expressed in fluorescence intensity units (FIU) of 2', 7'-dichlorofluorescein (DCF), were significantly (P < 0.01) greater in the heat group than in the control group (0.15 ± 0.01 vs. 0.12 ± 0.01 FIU per mg protein, respectively) (Fig. 2A). Maternal heat stress tended to decrease FRSA, as determined by the DPPH scavenging activity, in oviduct tissues (0.078 ± 0.003 vs. 0.070 ± 0.003 {Delta} absorbance per mg protein per min in control vs. heat, respectively; P = 0.09) (Fig. 2B). The ratios of the redox status, as determined by the H2O2 levels per FRSA, significantly (P < 0.05) leaned toward oxidation in maternally heat-stressed oviducts (2.08 ± 0.12) as compared to controls (1.62 ± 0.11) (Fig. 2C).



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  		FIG. 1. Effect of maternal heat stress on the glutathione concentrations in oviduct fluid and oviduct tissue. Pregnant females were exposed to heat stress for 12 h on Day 1 (heat) or were not treated (control). Oviducts were recovered at 1800 h on Day 1. Then, the oviducts were flushed, and glutathione was measured in the oviduct homogenate and oviduct flush using the DTNB-glutathione reductase recycling assay. Data are expressed as the mean ± SEM of 10 replicates. No significant differences were found between treatments in either oviduct fluid or oviduct tissue



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  		FIG. 2. Effect of maternal heat stress on (A) H2O2 level, (B) FRSA, and (C) redox status in oviduct tissue. Pregnant females were exposed to heat stress for 12 h on Day 1 (heat) or were not treated (control). Oviducts were recovered at 1800 h on Day 1. Then, each oviduct was homogenized in buffer. The homogenate of the left oviduct was used for H2O2 measurement using DCF fluorescence, and the right oviduct was used for FRSA measurement using the DPPH scavenging activity. The redox status in the oviduct was expressed as the H2O2 level/FRSA. Data are expressed as the mean ± SEM of nine replicates. 1 = fluorescence intensity unit; 2 = FRSA. *P < 0.05 vs. control

Effect of Maternal Heat Stress on Cdc2 Activity in 2-Cell Stage Embryos

The Cdc2 band pattern in the Western blot analysis of each treated 2-cell stage embryo is shown in Figure 3. The upper band is the phosphorylated (nonactive) form of Cdc2, and the lower band is the dephosphorylated (active) form. At 48 h post-hCG injection, the ratios did not differ among the treatments. In contrast, the Cdc2 kinase activity at 52 h was significantly (P < 0.01) higher in the control group (3.3 ± 0.2) compared to the heat (2.2 ± 0.3) and 2-cell block (1.0 ± 0.1) groups.



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  		FIG. 3. Western blot analysis of Cdc2 in mouse 2-cell stage embryos. Pregnant mice were heat-stressed for 12 h on Day 1 (heat) or were untreated (control and artificial 2-cell block). Then, embryos were recovered from oviducts and cultured at 37.5°C and 5% CO2 (control and heat) or at 37.5°C and 1% CO2 (artificial 2-cell block). Cultured embryos were harvested (A) 48 h (mid G2 phase) or (B) 52 h (late G2 phase ) after hCG injection and used for Western blot analysis. The upper band is the phosphorylated (nonactive) form of Cdc2, and the lower band is the dephosphorylated (active) form. The intensity of the upper and lower Cdc2 band was determined using NIH Image software. Data are expressed as the mean ± SEM of five replicates. Values with different letters differ significantly (P < 0.01, ANOVA)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results indicate that the redox status of the oviduct in heat-stressed mice shifts toward oxidation with increased H2O2 production. Moreover, the developmental competence of the majority of maternally heat-stressed zygotes decreases in vitro, and they arrest at the 2-cell stage because of a lack of Cdc2 activation at the G2 stage of the second cell cycle. These results confirm the hypothesis that, in addition to the deleterious effect of high temperature alone, the heat stress-mediated generation of oxidative stress in the oviduct is important for understanding the mechanisms underlying early embryonic death in heat-stressed animals.

Heat stress-induced early embryonic loss or reduced fertility in heat-stressed females occurs in many mammalian species [1, 28–30] and has long been recognized as a major obstacle to improving animal production in the tropics and subtropics [3]. In particular, high-producing, lactating dairy cows are less tolerant of heat stress because of their enhanced heat production during milk synthesis [2, 3]. Using an in vitro culture system that exposed embryos to high temperatures directly, the vulnerability of early bovine embryos to heat shock at the 2- to 4-cell stage was attributed to their inability to produce certain proteins that protect cells from stress, such as HSP70 for induced thermotolerance [11], GSH for antioxidant defense [10], and caspase for normal apoptotic function [31]. These previous findings deepened our understanding of the cellular mechanism that results in the vulnerability of early embryos to heat stress, but it remained unclear why exposure of embryos to fluctuating temperatures similar to the rectal temperatures of cows experiencing heat stress does not decrease embryonic development in vitro unless the heat stress continues for several days [12]. Recently, we observed that the development of maternally heat-stressed mouse zygotes is compromised after the 2-cell stage because of reduced intracellular GSH and enhanced H2O2 concentrations and that these responses were not reproduced when the zygotes were heat-stressed in vitro [13]. These results confirmed our previous finding that maternal heat stress enhances the oxidative stress of embryos, and they further revealed that heat stress-induced oxidative stress occurs in the oviduct, which determines the microenvironment of preimplantation embryos.

High body temperature [32] or high metabolic rate originating from acute exercise [33, 34] enhances the production of reactive oxygen species (ROS), which react with lipids, proteins, or nucleic acids in cells, resulting in cellular injury [35]. In the present study, we found an apparent increase in the H2O2 concentration in the oviducts, whereas the reducing status of the oviduct, as determined by the FRSA and GSH concentration, was not suppressed by maternal heat stress. In mammals, many antioxidant enzymes, such as glutathione peroxidase (both Cu, Zn-type, and Mn-type superoxide dismutase [SOD]) [36] and catalase [37], and antioxidant macromolecules, such as hypotaurine, taurine [38], and GSH [39], are expressed in oviduct epithelial cells. These substances are secreted into the oviduct fluid and protect preimplantation embryos from oxidative stress. These defense systems against oxidative stress have very complex, multiple, and complementary actions [35, 36]. Therefore, acute maternal heat stress for 12 h may have little effect on the antioxidative capacity of the oviduct. Bedaiwy et al. [40] reported that oviduct fluid from patients with hydrosalpinges (hydrosalpingeal fluid [HSF]), which is a disease of embryo toxicity that reduces embryo viability, contains significantly elevated amounts of ROS compared to normal oviduct fluid. When HSF is added to mouse embryo culture medium, it causes a dose-dependent decrease in the number of embryos that develop to the blastocyst stage, and the proportion of blastocysts is positively correlated with the ROS concentration in the HSF. Interestingly, Bedaiwy et al. also reported that the total antioxidant capacity of HSF does not differ from that of normal oviduct fluid, which is similar to our present observation, indicating that an increment of ROS concentration is not necessarily correlated with a noticeable reduction of antioxidant capacity. Taken together, these findings suggest that maternal heat stress could shift the redox status of the oviduct toward oxidation, thus enhancing the oxidative stress on the embryos. Further studies are required to analyze the mechanisms of ROS production in the oviduct attributed to maternal heat stress.

The 2-cell block in vitro occurs when cultured mouse zygotes are subjected to oxidative stress [16–18]. Eukaryote cell division, including embryo division, is regulated by the activity of MPF kinase, which is a complex of cyclin B1 and Cdc2 [41]. During the G1, S, and early G2 phases, Cdc2 is phosphorylated, and kinase activity is low. At the late G2 phase, Cdc2 is dephosphorylated and binds to cyclin B1, and the cell cycle continues to the M phase [42]. Previous studies have clarified that the Cdc2 activity in 2-cell block embryos remains low during the second cell cycle, arresting cleavage at the G2 phase in 2-cell embryos [19, 20]. Moreover, block-released embryos, which are cultured in media with added SOD or thioredoxin, show a similar Cdc2 activity pattern throughout the second cell cycle [19]. These previous findings demonstrate that the inactivation of Cdc2 in the G2 phase during the second cell cycle is the direct cause of the 2-cell block. The present study indicates that the development of maternally heat-stressed embryos arrests mainly at the 2-cell stage, and Cdc2 activation was less evident. Therefore, the developmental arrest of embryos because of maternal heat stress likely is caused by mechanisms similar to those causing in vitro 2-cell block.

Dephosphorylation of Cdc2 is regulated by Cdc25 phosphatase at the G2/M transition [22]. The activation of Cdc25 phosphatase requires a reducing agent [43], and a recent study indicated that oxidative stress induces the degradation of Cdc25 [44]. Therefore, it is possible that heat stress-induced oxidative stress in the oviduct results in a failure to activate Cdc25 phosphatase, which results in the developmental arrest of maternally heat-stressed embryos. Further analysis of Cdc25 activity in maternally heat-stressed embryos is required.

In conclusion, maternal heat stress shifts the redox status of the oviduct to oxidation, and the development of maternally heat-stressed zygotes is compromised after the 2-cell stage because of defective Cdc2 activity in the second cell cycle. Our finding that oxidative stress is involved in the physiological cues leading to heat stress-induced early embryonic death provides new insight regarding the development of practical countermeasures against reduced fertility in heat-stressed animals.


   ACKNOWLEDGMENTS
 
The authors thank I. Ohshima and A. Ushitani for their kind assistance, M. Kajihara for her generous guidance with the embryo culture, Dr. N. Minami for technical advice concerning the GSH assays and Western blot analysis, and Dr. K. Sasaki for technical advice on measuring FRSA using DPPH.


   FOOTNOTES
 
1 Supported by a Grant-in-Aid for Exploratory Research (14656098) from the Japan Society for the Promotion of Science to Y.K. and in part by a grant from Morinaga Hoshikai to M.H. Back

2 Correspondence: Yukio Kanai, Institute of Agriculture and Forestry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan. FAX: 81 29 853 6617; kanaiy{at}sakura.cc.tsukuba.ac.jp Back

Received: 11 August 2003.

First decision: 8 September 2003.

Accepted: 9 March 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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