Redox Status of the Oviduct: DISCUSSION
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 and has long been recognized as a major obstacle to improving animal production in the tropics and subtropics. In particular, high-producing, lactating dairy cows are less tolerant of heat stress because of their enhanced heat production during milk synthesis. 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, GSH for antioxidant defense, and caspase for normal apoptotic function. 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. 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. 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 or high metabolic rate originating from acute exercise enhances the production of reactive oxygen species (ROS), which react with lipids, proteins, or nucleic acids in cells, resulting in cellular injury. 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]) and catalase, and antioxidant macromolecules, such as hypotaurine, taurine, and GSH, 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. Therefore, acute maternal heat stress for 12 h may have little effect on the antioxidative capacity of the oviduct. Bedaiwy et al. 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. Eukaryote cell division, including embryo division, is regulated by the activity of MPF kinase, which is a complex of cyclin B1 and Cdc2. 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. 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. 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. 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. The activation of Cdc25 phosphatase requires a reducing agent, and a recent study indicated that oxidative stress induces the degradation of Cdc25. 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.