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.
Absence of heat-induced apoptosis in embryos heat shocked at the 2- and 4-cell stage is associated with a lack of activation of group II caspases. Failure of activation of these execution caspases is probably not a reflection of the absence of these enzymes or the absence of much of the signaling pathway for caspase activation. Experiments using the protein kinase C inhibitor staurosporine indicates that the cell death machinery is constitutively present in early cleavage embryos. Staurosporine could induce apo-ptosis in mouse embryos at the 1- to 4-cell stage and bovine embryos at the 1- to 16-cell stage. Also, mRNA for caspase-2, -3, -6, and -12 are present throughout preimplantation embryonic development in the mouse, as are transcripts for the antiapoptotic Bcl-2, Bcl-xl, and Bcl-w and the proapoptotic Bax. However, certain aspects of cell death are altered in early embryos because the time required for staurosporine to induce apoptosis in zygotes and 1- to 4-cell-stage embryos was longer than for blastocysts or other cell types. One possibility is that antia-poptotic proteins are very high in early embryos and that heat-induced apoptosis becomes possible when amounts of these proteins decline during development.
It is also possible that the particular mechanisms for heat-induced apoptosis are dysfunctional in early cleavage-stage embryos. For heat shock and oxidative stress, induction of apoptosis is initiated upon activation of the enzyme sphingomyelinase, which hydrolyzes the membrane phospholipid sphingomyelin to generate the second-messenger ceramide. Perhaps amounts of this enzyme or its substrate early in development make the early embryo unable to generate sufficient ceramide in response to heat shock for apoptosis to occur. Finally, that heat-induced apoptosis first occurs at a stage of development that is coincident with embryonic genome activation in the cow means that it is possible that heat-induced apoptosis is dependent on transcriptionally controlled events in the embryo.