The block to apoptosis in bovine two-cell embryos involves inhibition of caspase-9 activation and caspase-mediated DNA damage.
The capacity of the preimplantation embryo to undergo apoptosis in response to external stimuli is developmentally regulated. Acquisition of apoptosis does not occur in the cow embryo until between the 8- and 16-cell stages. The purpose of the present experiments was to determine the mechanism by which apoptosis is blocked in the bovine two-cell embryo. Heat shock (41 degrees C for 15 h) did not increase activity of caspase-9 or group II caspases (caspase-2, -3, and -7) in two-cell embryos but did in day 5 embryos. Exposure of embryos to carbonyl cyanide 3-chlorophenylhydrazone (CCCP) to depolarize mitochondria resulted in activation of caspase-9 and group II caspases at both stages of development. For day 5 embryos, CCCP also increased the proportion of blastomeres that underwent DNA fragmentation as determined by the TUNEL assay. In contrast, CCCP did not increase TUNEL labeling when applied at the two-cell stage. In conclusion, failure of heat shock to increase caspase-9 and group II caspase activity in the two-cell embryo indicates that the signaling pathway leading to mitochondrial depolarization and caspase activation is inhibited at this stage of development. The fact that CCCP treatment of two-cell embryos induced caspase-9 and group II-caspase activity indicates that caspase activation is possible following mitochondrial depolarization. However, since CCCP did not increase TUNEL labeling of two-cell embryos, actions of group II-caspases to activate DNases is inhibited.
Department of Animal Sciences, University of Florida, PO Box 110910, Gainesville, Florida 32611-0910, USA.
Amber M
AM
Brad
Katherine E M
KE
Hendricks
Peter J
PJ
Hansen
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001755:Blastocyst / Q000166:cytology* / Q000187:drug effects; D001757:Blastomeres / Q000166:cytology; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D053143:Caspase 2 / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism*; D002417:Cattle; D002970:Cleavage Stage, Ovum / Q000166:cytology* / Q000187:drug effects; D004249:DNA Damage; D053938:DNA Fragmentation; D003851:Deoxyribonucleases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods; D006358:Hot Temperature; D020287:In Situ Nick-End Labeling; D008297:Male; D008856:Microscopy, Fluorescence; D011247:Pregnancy; D015398:Signal Transduction / Q000187:drug effects; D014475:Uncoupling Agents / Q000494:pharmacology
D014475:Uncoupling Agents; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D003851:Deoxyribonucleases; D053143:Caspase 2; D053148:Caspase 3; D053179:Caspase 7; D053453:Caspase 9