Effect of Maternal Heat Stress on Viability of Early Preimplantation Mouse Embryos in vivo and in vitro

It is generally accepted that maternal heat stress during early pregnancy reduces fertility in mammalian species. Preimplantation embryos are sensitive to heat stress, and acute elevation of temperature causes a decrease of embryonic viability and subsequent development rate to the blastocyst stage in mouse (Arechiga et al., 1998). In dairy cows, high ambient temperatures around the time of insemination result in a significant decrease in the conception (Ingraham et al., 1976). The majority of embryonic mortality in cows exposed to heat stress occurred before Day7 of gestation (Monty et al., 1987). The objective of the present study was to characterise the effects of maternal heat stress soon after fertilisation on the development of early preimplantation mouse embryos in vivo and in vitro. Eight to twelve weeks old ICR mice were used. The day of detection of vaginal plugs in females was designated Day1. After detection of vaginal plugs, female mice were randomly allotted to control group and heat stress group. Animals were housed under 25°C and 50% relative humidity (RH) conditions with 12L-12D photoperiodic cycles lighting on at 6 am. Mice in heat stress group were exposed to 35 with 60% RH environment for 12 hours from 6 am on Day 1, while mice in control group remained untreated. In experiment 1, embryos were collected by flushing of the oviducts and uteri by modified Whitten medium on Days 2, 3 and 4 (n=10 for each group). Recovered embryos were observed for morphology and their developmental stages were recorded. In experiment 2, embryos were collected by flushing of the oviducts on Day1 at 6 pm, and cultured in modified Whitten medium+4mg/ml BSA at 37°C in 5%CO2 in humid air for 84h (equivalent to Day4 in vivo). Then, the developmental stages of cultured embryos were determined. In experiment 1, the number of collected embryos was not significantly different between control and heat stress groups. On Day 2, the majority of embryos was 2-cell stage in both groups (90.9% vs 76.3% for control and heat stress, respectively, and not significantly different). On Day 3, 4 to 8-cell embryos dominated in control group (77.5%), whereas a large number of embryos remained at 2-cell stage in heat stress group (53.4%). On Day 4, 97.6% of recovered embryos was the morula or blastocyst stage in control group, but the majority of embryos remained at 1 or 2-cell stage and only 2.4% (P<0.001) reached to the molura or blastocyst stage in heat stress group. In experiment 2, all embryos recovered were at 1-cell stage and there was no difference in their numbers between two groups. 91.1% of embryos in control group developed to the morula (42.7%) or the blastocyst stage (48.4%) after in vitro culture. However, none of embryo in heat stress group developed to the morula and blastocyst (0%, P<0.001). Instead, the number of embryos disrupted at 1-cell stage (12.8%) and 2-cell stage (42.7%) markedly increased in heat stress group. Furthermore, degenerated embryos, which were unable to define exactly the developmental stage, were observed at a higher rate in heat stress group than in control group (23.9%, vs 1.6%, P<0.001). These results demonstrate that maternal heat stress soon after fertilisation disrupts normal development of preimplantation embryos and leads to early embryonic death. Although the process of fertilisation is impaired as judged from a decreased percentage of 2-cell embryos on Day 2, the disruption of early embryonic development is presumably associates with a decrease in the viability of 2-cell embryos itself in part.