Heat Shock-Induced Apoptosis in Preimplantation Bovine Embryos: RESULTS(1)

Heat-Induced Apoptosis in Embryos >16 Cells

Figure 1, A-C, displays representative confocal digital images of embryos that were collected on Day 5 after insemination (if cell number was >16 cells), cultured at either 38.5°C for 24 h or at 41°C for 9 h and 38.5°C for 15 h, and then subjected to the TUNEL reaction. An embryo exposed to 38.5°C is shown in Figure 1A, whereas embryos exposed to 41 °C are shown in Figure 1, B and C. An increase in the proportion of nuclei labeling positive for TU-NEL (yellow in color) is apparent in the embryos exposed to 41°C. The percentage of cells undergoing apoptosis following heat shock was determined in several experiments. In the first experiment, exposure of embryos >16 cells to 41 or 42°C for 9 h increased (P < 0.05) the percentage of cells undergoing apoptosis 24 h after initiation of heat shock compared with embryos cultured at 38.5°C for 24 h (Fig. 2A). Heat shock also tended to reduce (P = 0.07) total number of cells per embryo 24 h after initiation of treatment (Fig. 2B). The effect of heat shock was similar for 41 °C or 42°C.

In a second experiment, embryos were exposed to temperatures more characteristic of rectal temperatures experienced by cows during heat stress. In addition, the effect of different durations of heat shock were examined. Exposure to either 40°C (P < 0.05) or 41°C (P < 0.001) increased the percentage of cells that were apoptotic compared those at 38.5°C (Fig. 3A). When embryos were subjected to 40°C, there was a quadratic (P < 0.01) effect of time on the percentage of cells that were apoptotic. The percentage of apoptosis was higher for embryos at 40°C than for embryos at 38.5°C, but the percentage of apoptosis at 40°C was similar for durations of 3, 6, and 9 h. The effect of time on the induction of apoptosis was even more pronounced at 41 °C. Again, there was a quadratic (P < 0.001) increase in the percentage of apoptotic cells as time of exposure increased. Neither 40°C nor 41 °C affected the total number of cells per embryo (Fig. 3B).

Lack of Heat-Induced Apoptosis in Bovine Embryos at the 2- or 4-Cell Stage

Representative confocal digital images of embryos assessed by TUNEL for apoptosis 24 h after collection at the 2-cell stage are shown in Figure 1D (embryo at 38.5°C continuously) and Figure 1, E and F (embryos exposed to 41°C for 9 h and 38.5°C for 15 h, respectively, before TU-NEL labeling). Note the absence of TUNEL labeling in all embryos and the increased incidence of nuclear fragmentation and reduced cell number in heat-shocked embryos.

When data were analyzed, exposure of 2- or 4-cell embryos to 41 °C for 9 h had no effect on the percentage of cells undergoing apoptosis 24 h after initiation of heat shock compared with embryos cultured continuously at 38.5°C. Indeed, the proportion of apoptotic cells at these stages of embryonic development was very low and ranged from 0.04%-0.66% (Fig. 4A). This compares to 7.8%-8.0% apoptosis in > 16-cell embryos collected at Day 5 and cultured at 38.5°C (Figs. 2A and 3A). Although heat shock did not affect apoptosis, at the 2-cell (38.5 vs. 41°C, P < 0.001) and 4-cell stages (38.5 vs. 41°C, P < 0.001), heat shock reduced total embryo cell number 24 h after initiation of treatment (Fig. 4B).
fIG1Heat Shock-Induced Apoptosis
FIG. 1. Representative confocal images illustrating the frequency of apoptotic nuclei in bovine embryos subjected to TUNEL analysis 24 h after initiation of temperature treatment. Embryos were labeled with fluorescein isothiocyanate-conjugated dUTP (green channel) and propidium iodide (red channel). Each panel is a display of a single embryo. A-C) Embryos treated at the > 16-cell stage on Day 5 after insemination and D-F) embryos treated at the 2-cell stage. Analysis by TUNEL was performed after embryos were cultured at 38.5°C for 24 h (A and D) or after embryos were exposed to a heat shock of 41 °C for 9 h followed by 38.5°C for 15 h (B-C and E-F). Arrows point to apoptotic nuclei. Nuclei labeled with TUNEL were frequently fragmented. Note that Day 5 embryos exposed to 41°C for 9 h (C) displayed a higher frequency of TUNEL-positive cells compared with embryos exposed to 38.5°C (A). In contrast, 2-cell embryos exposed to 41 °C for 9 h did not exhibit apoptosis (E and F) Heat-shocked 2-cell embryos exhibited nuclear fragmentation and had a lower cell number compared with embryos cultured at 38.5°C. (Note that embryos in D and F were greater than 2cell in number at the time of TUNEL analysis because of cleavage following initiation of treatment).

Fig2Heat Shock-Induced Apoptosis
FIG. 2. Heat-induced apoptosis in bovine embryos (>16 cells) treated on Day 5 after insemination. The experiment was replicated four times using 50-85 embryos/group. Results are least-squares means ± SEM. Compared with embryos cultured at 38.5°C continuously for 24 h, heat shock of 41 or 42°C for 9 h increased (P < 0.05) the percentage of cells undergoing apoptosis at 24 h after initiation of heat shock and tended to reduce (P = 0.07) the total number of cells per embryo at this time. There was no significant difference between 41 or 42°C.

Fig3Heat Shock-Induced Apoptosis
FIG. 3. Time dependence of heat-induced apoptosis in bovine embryos (>16 cells) treated on Day 5 after insemination and assessed by TUNEL 24 h after initiation of heat shock. The experiment was replicated four times using 30-70 embryos/group. Results are least-squares means ± SEM. Zero hours represents embryos cultured at 38.5°C for 24 h. Heat shock of 40°C (P < 0.05) (open circles) and 41 °C (P < 0.001) (closed circles) increased the percentage of cells undergoing apoptosis at 24 h after initiation of heat shock. There was a quadratic effect of time at 40°C (P < 0.01) and 41 °C (P < 0.001). Heat shock had no effect on total cell number per embryo.

Fig4Heat Shock-Induced Apoptosis
FIG. 4. Lack of heat-induced apoptosis in bovine embryos treated at the 2- or 4-cell stage and assessed by TUNEL 24 h after initiation of heat shock. The experiment was replicated seven times using 94-146 embryos/ group. Results are least-squares means ± SEM. Open bars are control embryos and hatched bars are embryos exposed to heat shock. Heat shock of 41 °C for 9 h at the 2- or 4-cell stage had no effect on the percentage of cells undergoing apoptosis at 24 h after initiation of heat shock. However total number of cells per embryo at 24 h after initiation of treatment was reduced by heat shock at the 2 (38.5 vs. 41 °C, P < 0.001)- and 4 (38.5 vs. 41 °C, P < 0.001)-cell stage.

Category: Embryo

Tags: Apoptosis, Early development, Embryo, Stress

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