Heat Shock-Induced Apoptosis in Preimplantation Bovine Embryos: DISCUSSION(4)

DISCUSSION(4)

Heat shock also compromised embryonic viability as determined by the total number of cells per embryo at 24 h after the initiation of heat shock. The deleterious effect of heat shock on embryo cell number depended upon the magnitude of heat shock and stage of embryonic development. Thus, heat shock at the 2- to 4-cell stage caused a larger reduction in embryo cell number than heat shock at the morula stage. This finding is in agreement with earlier studies in which heat shock caused a greater reduction in development when applied at the 2-cell stage than the morula stage or at Day 3 after fertilization than at Day 4. Therefore, those embryos that were at stages of development that were capable of heat-induced apoptosis were more resistant to the deleterious effect of heat shock on development.

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Heat Shock-Induced Apoptosis in Preimplantation Bovine Embryos: DISCUSSION(3)

Previous exposure of Day 4 embryos at the 8- to 16-cell stage to a mild and transient heat shock of 40°C for 80 min blocked the induction of apoptosis induced by a subsequent severe heat shock of 41 °C for 9 h. Such a phenomenon has been observed for others cells also. The biochemical mechanisms by which mild heat shock prevents apo-ptosis induced by a more severe heat shock presumably involves HSP70. Besides protecting cells from heat shock, HSP70 can protect cells against several apoptotic stimuli, including DNA damage, UV irradiation, serum withdrawal, and chemotherapeutic agents.

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Heat Shock-Induced Apoptosis in Preimplantation Bovine Embryos: DISCUSSION(2)

DISCUSSION(2)

Acquisition of heat-induced apoptosis is developmentally regulated and does not occur until approximately Day 4 after insemination at the 8- to 16-cell stage. Spontaneous apoptosis in bovine embryos is also first observed at the 8-to 16-cell stage. The failure to observe apoptosis in 8-to 16-cell embryos collected at Day 3 after insemination may mean that either 1) development of apoptosis mechanisms are controlled by time as well as by the number of cleavage divisions, 2) that Day 4 embryos are more likely to have completed more cleavage divisions than Day 3 embryos, or 3) that Day 4 embryos include retarded embryos that are more susceptible to apoptosis.

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

One function of apoptosis is to eliminate cells that are damaged by stress. Heat shock, for example, induces apoptosis in many cell types. Although several recent studies have demonstrated that preimplantation embryos undergo apoptosis in a stage-specific manner, few studies have evaluated the role of stress in induction of apoptosis in embryos. The present study demonstrated that heat shock, which is a cellular stress associated with embryonic loss in vivo and in vitro, can induce apoptosis as determined by TUNEL reaction. Nuclei could conceivably be TUNEL-positive because of necrosis or because of DNA strand breaks that occur as artifacts of sample preparation and fixation. However, TUNEL labeling of apoptotic nuclei is confined to the nuclei, as seen in the current study, whereas labeling of necrotic cells can spread throughout the cytoplasm. Further evidence that the TUNEL-positive blastomeres in the present study represent apoptosis was the observation of nuclear fragmentation that is characteristic of apoptosis and increased activity of group II caspases.

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Heat Shock-Induced Apoptosis in Preimplantation Bovine Embryos: RESULTS(2)

RESULTS(2)

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).

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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.

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Heat Shock-Induced Apoptosis in Preimplantation Bovine Embryos: MATERIALS AND METHODS(6)

MATERIALS AND METHODS(6)

Heat-induced apoptosis in bovine embryos at the 2- or 4-cell stage. Embryos at the 2- or 4-cell stage were collected at 28-30 and 37-39 h after insemination, respectively. Embryos were transferred to a new drop of modified KSOM and cultured at 38.5°C for 24 h or were heat shocked at 41°C for 9 h followed by 38.5°C for 15 h. Embryos were fixed in 4% paraformaldehyde, transferred to a poly-L-lysine coated slide and saved at 4°C until analysis by TUNEL. The experiment was replicated seven times using 491 embryos (94-146 embryos/group).

Heat-induced apoptosis in bovine embryos at the 8- to 16-cell stage on Day 3 after insemination. Embryos at the 8- to 16-cell stage were collected on Day 3 after insemination and transferred to a new drop of modified KSOM. Embryos were then cultured at 38.5°C for 24 h or were heat shocked at 40 or 41°C for 9 h followed by 38.5°C for 15 h. At the end of culture, embryos were fixed and subjected to TUNEL analysis. The experiment was replicated three times using 188 embryos (52-64 embryos/group).

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