Connexin Trafficking And The Control Of Gap Junction Assembly In The Mouse Preimplantation Embryo

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De, Sousa Paul
Biology; Cell
thesis / dissertation description
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. The purpose of the present study was to determine which connexins contribute to gap junctions in the preimplantation mouse embryo and how their assembly into gap junctions at compaction is regulated. Using antibodies raised against different synthetic C-terminal peptides of connexin43 (Cx43), this protein was shown to assemble into gap junction-like plaques beginning with compaction. Prior to this time no Cx43 can be detected in the plasma membrane. Cell fractionation and reverse transcription-polymerase chain reaction (RT-PCR) were employed to show that Cx43 mRNA is in polyribosomes at the 4-cell stage, suggesting that synthesis of Cx43 begins at least one cell cycle in advance of when gap junctions first form. The fate of nascent Cx43 was followed throughout preimplantation development by means of immunofluorescence in conjunction with confocal laser scanning microscopy. In morulae and blastocysts Cx43 becomes differentially distributed in the apposed plasma membranes: 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. These latter two drugs were shown to cause specific alterations in the structure and distribution of a variety of organelles in morulae, especially those involved in protein trafficking and degradation.;No conclusive evidence for the contribution of connexin32 (Cx32) to gap junctions in morulae could be found. However, using immunogold electron microscopy, the contribution of Cx43 was confirmed. Cx43 was also found to contribute to gap junctions in cumulus-oocyte complexes (COCs), both between cumulus granulosa cells and these cells and the oocyte. Although 100% of gap junctions between cumulus cells were labeled with Cx43 specific antisera, over 30% of gap junctions in morulae were not. Thus other members of the connexin gene family may yet be found to contribute to gap junctions at this stage. Treatment of uncompacted 8-cell embryos with either monensin or brefeldin-A inhibited the appearance of gap junction-like structures containing Cx43 and the onset of gap junctional coupling in a reversible manner. These data demonstrate that the onset of gap junction assembly during compaction is regulated post-translationally and involves mobilization of connexin43 and possibly other members of the connexin gene family through trafficking organelles to plasma membranes.