Connexin Gene Expression And The Control Of Gap Junction Assembly In Preimplantation Development

Gap junctional communication in preimplantation mouse embryos was explored by investigating: (1) the expression pattern of the connexin (Cx: gap junction protein) multigene family in maturing mouse oocytes and preimplantation embryos; (2) the relative role of Cx43 in gap junctional communication in embryos; and (3) the control of the acquisition of gap junctional coupling.;Both Cx43 and Cx32 transcripts were detected in maturing oocytes but these transcripts declined to undetectable levels shortly after ovulation. Cx43 transcripts were detected again at the 4-cell stage and accumulated steadily thereafter to reach a maximum in blastocysts. Cx26 and Cx46 transcripts could not be detected in embryos. By Western blotting Cx43 was detected in 1-cell embryos, the levels decreased by the 2-cell stage, and then increased gradually from the 4-cell stage to the blastocyst stage. Oogenetic expression of Cx32 and 43 is therefore replaced by exclusive Cx43 expression in the embryos and it is probably Cx43 that is the main contributor to the assembly of gap junctions at the 8-cell stage. To attempt to inhibit Cx43 gene expression, embryos were either microinjected with Cx43 antisense deoxyoligonucleotides, or cultured in their presence. Dye coupling and Cx43 mRNA levels were not noticeably affected by the presence of the antisense reagents.;Treatment of 4-cell embryos with an activator of protein kinase C triggered premature compaction without an accompanying establishment of gap junctional communication. On the other hand, when coupled, 8-cell embryos were treated with the activator, intercellular coupling was diminished. Western blot analysis revealed that Cx43 becomes phosphorylated between the late 4-cell stage and the late morula stage. Aphidicolin experiments showed that the third and fourth rounds of DNA synthesis are not required for the normal acquisition of gap junctional communication. However, embryos in which the second round of DNA synthesis was delayed by 10 hours failed to initiate gap junctional coupling. Immunofluorescence localization of Cx43 in these communication-deficient embryos revealed extensive cytoplasmic Cx43 staining but punctate interblastomeric staining, indicative of gap junction plaques, was absent.;These results are consistent with a model in which the regulated step in the onset of gap junction assembly involves the phosphorylation of cytoplasmic Cx43