Redox Status of the Oviduct: RESULTS
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.
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 |xg protein for control vs. heat, respectively) or oviduct fluids (44.7 ± 7.9 vs. 37.5 ± 4.9 pmol GSH per |xg 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 A 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).
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.
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.
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.
TABLE 1. In vitro development competence of embryos after maternal heat exposure during the zygote stage.a
|% Embryos that|
|Treatment||Underwent first cleavage||Reached a morula or blastocyst from the 2-cell stage||Arrested at the 2-cell stage|
|ControlHeat||92.5 ± 3.0 85.4 ± 4.4||87.6 ± 6.5 8.0 ± 3.5b||2.5 ± 1.5 67.4 ± 7.3b|
a Data are expressed as the mean ± SEM of 10 replicates. b Significantly different from control (P < 0.001, percentages were arcsine transformed and then analyzed using the Student t-test).
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 phos-phorylated (nonactive) form of Cdc2, and the lower band is the dephos-phorylated (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).