Antisense c-myc effects on preimplantation mouse embryo development.
Antisense DNA inhibition of gene expression was explored as an approach toward elucidating mechanisms regulating development of preimplantation mammalian embryos. Specifically, a role for the c-myc protooncogene was examined. Detection of c-myc mRNA and immunoreactive nuclear c-myc protein in preimplantation mouse embryos at the eight-cell/morula and blastocyst stages suggested that this DNA-binding protein could be important during early embryo-genesis. The effects of c-myc oligodeoxyribonucleotides (oligos) on the in vitro development of two-cell mouse embryos were examined. Embryos cultured in medium containing an unmodified (phosphodiester) antisense c-myc oligo complementary to the translation initiation codon and spanning the first seven codons exhibited a dose-dependent arrest at the eight-cell/morula stage. At lower concentrations (7.5 microM) this inhibitory effect was specific to the antisense oligo and did not occur with the sense-strand complement or with duplexes of the antisense and sense oligos. However, at 4-fold higher concentrations of DNA (30 microM), all unmodified c-myc oligos were embryotoxic, causing embryos to arrest at the two-cell to four-cell stages. In contrast, almost all (98%) two-cell embryos cultured with a modified (chimeric phosphorothioate/phosphodiester) antisense c-myc oligo (7.5 microM) exhibited developmental arrest at the eight-cell/morula stage, whereas no developmental arrest occurred following incubation with high concentrations of the modified sense complement (30 microM). Culture of freshly recovered eight-cell embryos with antisense c-myc led to the absence of c-myc protein but no change in epidermal growth factor receptor in those embryos that developed a blastocoel. These effects on c-myc were specific for the antisense oligo. These results suggest that c-myc function becomes particularly critical for preimplantation mouse embryos at the eight-cell/morula stage of development and establish that antisense DNA can be successfully applied as an approach toward elucidating the roles of specific genes in preimplantation mammalian embryo development.
D000818:Animals; D001483:Base Sequence; D001755:Blastocyst / Q000187:drug effects / Q000502:physiology*; D002455:Cell Division; D005314:Embryonic and Fetal Development / Q000187:drug effects*; D005260:Female; D016259:Genes, myc; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D009028:Morula / Q000187:drug effects / Q000502:physiology*; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D016133:Polymerase Chain Reaction / Q000379:methods; D011247:Pregnancy; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase
D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase