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.
The temperatures that induced apoptosis included those characteristic of heat-stressed cows (40 and 41 °C), suggesting that apoptosis could be induced in preimplantation embryos exposed to maternal hyperthermia. Moreover, the degree of apoptosis experienced by heat-shocked embryos generally reflected the severity of heat shock. The percent of cells that were positive for apoptosis increased with increasing heat shock temperatures and, at least at 41 °C, increased with duration of heat shock. At 40°C, induction of apoptosis was also time dependent, with no increase in apoptosis seen after an exposure of 80 min (Fig. 6), but an increase in the percentage of apoptotic cells after 3, 6, or 9 h of exposure (Fig. 3). The observation that the degree of apoptosis following exposure to 40°C for 3, 6, and 9 h was generally similar could possibly reflect time-dependent cellular adaptation to prevent apoptosis following prolonged exposure to a mild heat shock of 40°C that was not possible for a more severe heat shock of 41 °C.