CONNEXIN TRAFFICKING AND THE CONTROL OF GAP JUNCTION ASSEMBLY IN MOUSE PREIMPLANTATION EMBRYOS

Gap junction assembly in the preimplantation mouse embryo is a tempora lly regulated event, beginning a few hours after the third cleavage du ring the morphogenetic event known as compaction. Recently, we demonst rated that both mRNA and protein corresponding to connexin43, a gap ju nction protein, accumulate through preimplantation development beginni ng at least as early as the 4-cell stage. Using an antibody raised aga inst a synthetic C-terminal peptide of connexin43, this protein was sh own to assemble into gap junction-like plaques beginning at compaction (G. Valdimarsson, P. A. De Sousa, E. C. Beyer, D. L. Paul and G. M. K idder (1991). Molec. Reprod. Dev. 30, 18-26). The purpose of the prese nt study was to follow the fate of nascent connexin43 during preimplan tation development, from synthesis to plaque insertion, and to learn m ore about the control of gap junction assembly during compaction. Cell fractionation and reverse transcription-polymerase chain reaction wer e employed to show that connexin43 mRNA is in polyribosomes at the 4-c ell stage, suggesting that synthesis of connexin43 begins at least one cell cycle in advance of when gap junctions first form. The fate of n ascent connexin43 was then followed throughout preimplantation develop ment by means of laser confocal microscopy, using two other peptide (C -terminal)specific antibodies. As was reported previously, connexin43 could first be detected in gap junction-like plaques beginning in the 8-cell stage, at which time considerable intracellular immunoreactivit y could be seen as well. Later, connexin43 becomes differentially dist ributed in the apposed plasma membranes of morulae and blastocysts: a zonular distribution predominates between outside blastomeres and trop hectoderm cells whereas plaque-like localizations predominate between inside blastomeres and cells of the inner cell mass. The cytoplasmic i mmunoreactivity in morulae was deemed to be nascent connexin en route to the plasma membrane since it could be abolished by treatment with c ycloheximide, and redistributed by treatment with monensin or brefeldi n-A, known inhibitors of protein trafficking. Treatment of uncompacted 8-cell embryos with either monensin or brefeldin-A inhibited the appe arance of gap junction-like structures and the onset of gap junctional coupling in a reversible manner. These data demonstrate that the regu lated step in the onset of gap junction assembly during compaction is downstream of transcription and translation and involves mobilization of connexin43 through trafficking organelles to plasma membranes.