Repression of induced apoptosis in the 2-cell bovine embryo involves DNA methylation and histone deacetylation.
Apoptosis in the bovine embryo cannot be induced by activators of the extrinsic apoptosis pathway until the 8-16-cell stage. Depolarization of mitochondria with the decoupling agent carbonyl cyanide 3-chlorophenylhydrazone (CCCP) can activate caspase-3 in 2-cell embryos but DNA fragmentation does not occur. Here we hypothesized that the repression of apoptosis is caused by methylation of DNA and deacetylation of histones. To test this hypothesis, we evaluated whether reducing DNA methylation by 5-aza-2'-deoxycytidine (AZA) or inhibition of histone deacetylation by trichostatin-A (TSA) would make 2-cell embryos susceptible to DNA fragmentation caused by CCCP. The percent of blastomeres positive for TUNEL was affected by a treatment x CCCP interaction (P<0.0001). CCCP did not cause a large increase in the percent of cells positive for TUNEL in embryos treated with vehicle but did increase the percent of cells that were TUNEL positive if embryos were pretreated with AZA or TSA. Immunostaining using an antibody against 5-methyl-cytosine antibody revealed that AZA and TSA reduced DNA methylation. In conclusion, disruption of DNA methylation and histone deacetylation removes the block to apoptosis in bovine 2-cell embryos.
Carambula
Silvia F
SF
Department of Animal Sciences, University of Florida, Gainesville, FL 32611-0910, USA.
Oliveira
Lilian J
LJ
Hansen
Peter J
PJ
D044503:5-Methylcytosine / Q000032:analysis / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001374:Azacitidine / Q000031:analogs & derivatives; D001755:Blastocyst / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002417:Cattle; D003596:Cytosine / Q000378:metabolism*; D053938:DNA Fragmentation; D019175:DNA Methylation; D000077209:Decitabine; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D006877:Hydroxamic Acids / Q000494:pharmacology
D056572:Histone Deacetylase Inhibitors; D006657:Histones; D006877:Hydroxamic Acids; C012589:trichostatin A; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D044503:5-Methylcytosine; D000077209:Decitabine; D003596:Cytosine; D006655:Histone Deacetylases; D001374:Azacitidine