title: Connexin trafficking and the control of gap junction assembly in mouse preimplantation embryos.
abstract: Gap junction assembly in the preimplantation mouse embryo is a temporally regulated event, beginning a few hours after the third cleavage during the morphogenetic event known as compaction. Recently, we demonstrated that both mRNA and protein corresponding to connexin43, a gap junction protein, accumulate through preimplantation development beginning at least as early as the 4-cell stage. Using an antibody raised against a synthetic C-terminal peptide of connexin43, this protein was shown 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. Kidder (1991). Molec. Reprod. Dev. 30, 18-26). The purpose of the present study was to follow the fate of nascent connexin43 during preimplantation development, from synthesis to plaque insertion, and to learn more about the control of gap junction assembly during compaction. Cell fractionation and reverse transcription-polymerase chain reaction were employed to show that connexin43 mRNA is in polyribosomes at the 4-cell stage, suggesting that synthesis of connexin43 begins at least one cell cycle in advance of when gap junctions first form. The fate of nascent connexin43 was then followed throughout preimplantation development 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 immunoreactivity could be seen as well. Later, connexin43 becomes differentially distributed in the apposed plasma membranes of morulae and blastocysts: a zonular distribution predominates between outside blastomeres and trophectoderm cells whereas plaque-like localizations predominate between inside blastomeres and cells of the inner cell mass. The cytoplasmic immunoreactivity in morulae was deemed to be nascent connexin en route to the plasma membrane since it could be abolished by treatment with cycloheximide, and redistributed by treatment with monensin or brefeldin-A, known inhibitors of protein trafficking. Treatment of uncompacted 8-cell embryos with either monensin or brefeldin-A inhibited the appearance of gap junction-like structures and the onset of gap junctional coupling in a reversible manner. These data demonstrate that the regulated 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.
authors.affiliation: Department of Zoology, University of Western Ontario, London, Canada.
authors.forename: P A
authors.initials: PA
authors.lastname: De Sousa
authors.forename: G
authors.initials: G
authors.lastname: Valdimarsson
authors.forename: B J
authors.initials: BJ
authors.lastname: Nicholson
authors.forename: G M
authors.initials: GM
authors.lastname: Kidder
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000502:physiology*; D015139:Blotting, Southern; D015153:Blotting, Western; D020126:Brefeldin A; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D003517:Cyclopentanes / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D007365:Intercellular Junctions / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008815:Mice, Inbred Strains; D008985:Monensin / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011132:Polyribosomes / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism
chemical_list: D018031:Connexin 43; D003517:Cyclopentanes; D012333:RNA, Messenger; D020126:Brefeldin A; D008985:Monensin