06 Jan

Redox Status of the Oviduct: MATERIALS AND METHODS

Animals

Postpubertal female ICR mice (age, 8-12 wk) and male BDFj 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, p-nicotinamide adenine dinucleotide phosphate (reduced form; NADPH), 5,5′-dithio-Ms-2-nitrobenzoic acid (DTNB), Na2-EDTA, and 2′,7′-dichlorodihydro-fluores-cein 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); 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.

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 |xl 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 |xl 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 10000 X gfor 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 |xl 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 (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. with some modification. Briefly, the oviduct was washed twice in assay buffer (0.01 |xM Tris-HCl, 320 mM sucrose, 1 mM Na2-EDTA; pH 7.4) and homogenized in 500 |xl of assay buffer using a son-icator. The homogenate was centrifuged at 13 000 X g for 30 min at 4°C, and 250 |xl of supernatant were mixed with 200 |xl of 200 |xM DPPH in absolute ethanol and 50 |xl 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. 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 |xl 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 |xg of protein, was transferred to a 1.5-ml microfuge tube, and assay buffer (total volume, 396 |xl) and 4 |xl 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). As a positive control (2-cell block embryos), zygotes from nonheat-stressed mice were cultured at a low CO2 concentration (1%) to induce 2cell 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.

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 /-test. The GSH concentrations of oviduct tissues or oviduct fluids, H2O2 levels, and FRSA of oviduct tissues were compared using the Student /-test. Differences were considered to be significant at P < 0.05.

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