Rapid inhibition of mRNA synthesis during preimplantation embryo development: vital permeabilization by lysolecithin potentiates the action of alpha-amanitin.
Lysolecithin was used to permeabilize embryonic cells to impermeant compounds without compromising embryo viability. Within 2 min of exposure to 0.05% lysolecithin, mouse morulae became permeable to inositol 1,4,5-trisphosphate, leading to the release of calcium from intracellular stores. Although these morulae remained permeable for at least 2 h, they developed normally to the blastocyst stage during subsequent culture in vitro. Lysolecithin permeabilization was then used to potentiate the internalization of alpha-amanitin. Pretreatment with lysolecithin for 2 min markedly accelerated the onset of transcriptional inhibition from 3 h to 10 min after alpha-amanitin addition. The rapid inhibition by alpha-amanitin of mRNA synthesis in lysolecithin-treated embryos provided a precise method for delineating developmentally important transcriptional periods. Using this protocol, we found that mRNAs required for embryonic cavitation were synthesized between 87 and 91 h post-hCG, shortly before blastocoel formation commenced.
Khidir
M A
MA
C. S. Mott Center for Human Growth and Development, Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Stachecki
J J
JJ
Krawetz
S A
SA
Armant
D R
DR
D000546:Amanitins / Q000494:pharmacology*; D000818:Animals; D001755:Blastocyst / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects; D005314:Embryonic and Fetal Development; D008244:Lysophosphatidylcholines / Q000494:pharmacology*; D051379:Mice; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000096:biosynthesis*
D000546:Amanitins; D008244:Lysophosphatidylcholines; D012333:RNA, Messenger