Intercellular junctional coupling in preimplantation mouse embryos: effect of blocking transcription or translation

Gap junction formation in cleavage stage mouse embryos was examined by testing for ionic coupling or by observing the intercellular movement of the fluorescent dye, Lucifer yellow CH. Our results confirm that embryo-wide cell coupling, mediated by cell-to-cell membrane channels (gap junctions), is acquired in the 8-cell stage after compaction has begun. However, not all partially compacted embryos were found to be ionically or dye coupled, suggesting that the initiation of gap junction assembly is not necessarily triggered by the onset of cell flattening. The rate of fluorescent dye movement throughout the embryo was found to increase as embryos proceed through compaction and beyond, indicating that the number of gap junctional channels between blastomeres increases as development progresses. The inhibitors alpha-amanitin and cycloheximide were used to assess the requirement of new transcription and protein synthesis, respectively, for the onset of intercellular coupling and its progressive increase during compaction. Treatment conditions were chosen to bring about suppression of mRNA and protein synthesis within 2 hr. Ionic coupling was detected in almost all compacted 8-cell embryos treated with either inhibitor from the 4-cell stage. On the other hand, dye coupling was weak or undetectable in such embryos. We propose that a limited supply of junctional components is present by the 4-cell stage to serve as a pool of precursors for the first gap junctions to be assembled in the 8-cell stage. However, it is apparent that continued embryonic gene expression is required for the full extent of junctional coupling to be established.