Heat-Induced Apoptosis and Induced Thermotolerance at the 8- to 16-Cell Stage
Two experiments were conducted to determine whether apoptosis occurs at the 8- to 16-cell stage. In the first study, exposure of Day 3 embryos at the 8- to 16-cell stage to 40°C or 41 °C for 9 h had no effect on the percentage of apoptotic cells (Fig. 5A) or on total embryo cell number (Fig. 5B) at 24 h after initiation of temperature treatments. In the second experiment (Fig. 6), 8- to 16-cell embryos were collected on Day 4 rather than on Day 3. In addition, we tested whether exposure to a mild heat shock of 40°C for 80 min would make embryos more resistant to apoptosis induced by a severe heat shock of 41 °C for 9 h. There was no difference between control embryos at 38.5°C and embryos subjected to 40°C for 80 min on the percentage of cells undergoing apoptosis or embryo cell number (Fig. 6A). In contrast, exposure to 41°C for 9 h increased (38.5 vs. 41 °C, P < 0.001) the proportion of cells that were ap-optotic 24 h after initiation of temperature treatments and reduced (38.5 vs. 41°C, P < 0.01) embryo cell number. Preincubation at 40°C for 80 min, however, blocked (40/ 41°C vs. 41°C, P <0.001) heat-induced apoptosis. Embryo cell number remained lower (38.5 vs. 40/41°C, P <0.001) for heat shocked embryos than controls, however, regardless of whether embryos were preincubated at 40°C (Fig. 6B).
Representative digital images illustrating caspase activity in 2-cell embryos and embryos a 16-cell stage are shown in Figure 2. Note the higher caspase activity for Day 5 embryos a 16-cell stage exposed to heat shock of 41 °C for 9 h (Fig. 7, D-F) compared with embryos exposed to 38.5°C (Fig. 7C). However, when 2-cell embryos were exposed to the same heat shock treatment there was no induction of caspase activity (Fig. 7, A and B).
The effect of heat shock (41 °C for 9 h) on caspase activity was dependent upon stage of development (stage x heat shock, P < 0.01). Heat shock increased (38.5 vs. 41°C, P < 0.001) caspase activity for Day 5 bovine embryos Fig. 8) compared with 38.5°C control. In contrast, caspase activity of 2-cell embryos was not affected by heat shock.
FIG. 5. Lack of heat-induced apoptosis in 8- to 16-cell bovine embryos treated on Day 3 after insemination and assessed by TUNEL 24 h after initiation of heat shock. The experiment was replicated three times using 52-64 embryos/group. Results are least-squares means ± SEM. Heat shock of 40°C or 41 °C for 9 h had no significant effect on subsequent percentage of cells undergoing apoptosis or total cell number per embryo.
FIG. 6. Induced thermotolerance in bovine embryos at the 8- to 16-cell stage treated on Day 4 after insemination and assessed by TUNEL 24 h after initiation of heat shock. The experiment was replicated four times using 111-120 embryos/group. Results are least-squares means ± SEM. Exposure of embryos to a mild heat shock of (40°C for 80 min) blocked apoptosis induced by exposure to a subsequent heat shock of 41 °C for 9 h (40/41 °C vs. 41 °C, P < 0.001). However, preexposure to 40°C did not prevent the subsequent reduction in total embryo cell number induced by 41 °C for 9 h (38.5°C vs. 40/41 °C, P < 0.05).
FIG. 7. Representative digital images illustrating group II caspase activity in embryos a16-cell stage and 2-cell embryos. A) Two-cell embryo and (C) a16-cell embryo cultured at 38.5°C. B) Two-cell embryo and D, E, and F) 16-cell embryos exposed to 41 °C for 9 h. Caspase activity was determined immediately after the end of the heat-shock period. Note that Day 5 embryos a16-cell stage exposed to heat shock of 41 °C for 9 h had higher caspase activity compared with embryos exposed to 38.5°C. However, exposure of 2-cell embryos to the same heat shock did not increase caspase activity.
FIG. 8. Group II caspase activity in bovine embryos. Results are least-squares means ± SEM of data from 135 embryos (22-42/group) in a total of two replicates. Open bars are control embryos and hatched bars are embryos exposed to heat shock. Exposure of embryos to 41 °C for 9 h did not increase caspase activity of 2-cell embryos but did increase caspase activity of embryos >16 cells on Day 5 after insemination (stage X temperature, P < 0.01).