Effects of phlorizin and ouabain on the polarity of mouse 4-cell/16-cell stage blastomere heterokaryons

Abstract

Cell polarity is thought to be required for the efficient production of nascent blastocoele fluid, which begins at the 16-cell stage of mouse preimplantation development. In this study the 4-cell/16-cell blastomere heterokaryon was used to test the hypothesis that solute transport across the apical membrane domain induces the apical-basal axis of organelle distribution across polar 16-cell-stage blastomeres. Fusion of 4-cell/16-cell blastomere pairs resulted in a population of heterokaryons of which 65% were polar (contain an apical plasma membrane domain from a polar 16-cell-stage plasma membrane insert) and 30% were apolar (contain an apolar 16-cell-stage plasma membrane insert). Polar heterokaryons were distinguished from apolar ones by labeling their apical domains with fluorescent succinylated concanavalin A. In polar heterokaryons, both nuclei (labeled with Hoeschst 33242) were immediately subjacent to the apical plasma membrane domain, while in apolar heterokaryons both nuclei were located centrally. Two inhibitors of apical transmembrane solute transport—phlorizin, which inhibits brush border (apical) Na +/glucose symporters, and ouabain, which inhibits Na +/K +ATPase, thereby modifying the transmembrane Na + gradient—were examined for their effect on nuclear position in polar and apolar heterokaryons after a 4-hr incubation in either inhibitor. Both ouabain ( L. M. Wiley, 1984, Dev. Biol. 105, 330–342 ) and phlorizin (this study) had a biphasic effect on the rate of nascent blastocoele fluid accumulation such that at lower concentrations (ouabain, 10 −5 M; phlorizin, 10 −6 M) fluid accumulation was accelerated and at higher concentrations (both inhibitors, 10 −4 M) fluid accumulation was delayed. In polar heterokaryons, both concentrations of each inhibitor caused the nuclei to become displaced basally from their normal location against the apical plasma membrane domain. Both nuclei, however, remained on the axis of polarity passing through the apical domain. The magnitude of displacement was greater at higher concentrations of either inhibitor. Neither inhibitor affected nuclear position in apolar heterokaryons. These observations agree with the hypothesis that apical plasma membrane solute transport maintains the asymmetric organelle distribution across the apical-basal axis of polar 16-cell-stage blastomeres.