Date of Award
Doctor of Philosophy
This study determined the time of appearance and cellular distribution of Na/K-ATPase during preimplantation development, the direct involvement of this enzyme in the process of cavitation (blastocoel formation) and the pattern of expression of the Na/K-ATPase alpha (catalytic) subunit mRNA during preimplantation development. Na/K-ATPase was first detected by immunofluorescence in late morulae (84 hr post-hCG). During cavitation, the enzyme changed in its distribution to become restricted to the basolateral membrane domain of the mural trophectoderm and trophectodermal projections. Wheat germ agglutinin treatment of 2-, 4- and 8-cell embryos confirmed the timing of appearance of the enzyme and also showed that this appearance is independant of cytokinesis. Embryos treated with cycloheximide for 8 hr or ouabain for 4 hr following cytochalasin B-induced blastocoel collapse indicated that both new protein synthesis and an active Na/K-ATPase are required for re-expansion of the blastocoel cavity. Cytochalasin B treatment resulted in Na/K-ATPase becoming restricted to the apical membrane domain of the mural trophectoderm and also in the disruption of the apical junctional complex. Recovery from cytochalasin B treatment involved the return of the enzyme to the basolateral membrane domain of the trophectoderm and the reappearance of the apical junctional complex. Treatment of embryos with antiserum raised against uvomorulin resulted in the blockage of cavitation, the formation of multiple fluid-filled cavities within each blastomere and the disruption of the apical junctional complex. Na/K-ATPase was localized within the cytoplasm of all treated blastomeres and also as a ring encircling each fluid-filled cavity. The fluid-filled cavities likely developed from the coalescence of smaller cytoplasmic vesicles, containing nascent but active Na/K-ATPase. Northern blot analysis of three identical RNA blots only resulted in the hybridization of a Na/K-ATPase alpha 1 isoform cDNA. Analysis of a preimplantation developmental RNA series northern blot probed with the alpha 1 cDNA produced a hybridization profile with the weakest signal at the 2-cell stage, after which it approximately doubled in intensity for each subsequent cleavage division, attaining its strongest signal in blastocysts.
Watson, Andrew John, "Regulation Of Sodium,potassium-atpase Expression During Mouse Embryogenesis" (1989). Digitized Theses. 1857.