Na/K-ATPase-mediated 86Rb+ uptake and asymmetrical trophectoderm localization of α1 and α3 Na/K-ATPase isoforms during bovine preattachment development

This study evaluated Na/K-ATPase alpha 1- and alpha 3-subunit isoform polypeptide expression and localization during bovine preattachment development. Na/K-ATPase cation transport activity from the one-cell to blastocyst stage was also determined by measuring ouabain-sensitive Rb-86(+) uptake. Both alpha 1- and alpha 3-subunit polypeptides were detected by immunofluorescence to encircle the entire cell margins of each blastomere of inseminated zygotes, cleavage stage embryos, and morulae. immunofluorescent localization of alpha 1-subunit polypeptide in bovine blastocysts revealed an alpha 1 immunofluorescence signal confined to the basolateral membrane margins of the trophectoderm and encircling the cell periphery of each inner cell mass (ICM) cell. In contrast, alpha 3-subunit polypeptide immunofluorescence was localized primarily to the apical cell surfaces of the trophectoderm with a reduced signal present in basolateral trophectoderm regions. There was no apparent alpha 3-subunit signal in the ICM. Analysis of Rb-86(+) transport in vitro demonstrated ouabain-sensitive activity throughout development from the one-cell to the six-to eight-cell stage of bovine development. Rb-86(+) uptake by morulae (day 6 postinsemination) did not vary significantly from uptake detected in cleavage stage embryos; however, a significant increase was measured at the blastocyst stage (P < 0.05). Treatment of embryos with cytochalasin D (5 mu g/ml) did not influence Rb-86(+) uptake in cleavage stage embryos. Cytochalasin D treatment however was associated with a significant rise in ion transport in morulae and blastocysts (13.49 and 61.57 fmol/embryo/min, respectively) compared to untreated controls (2.65 and 22.83 fmol/embryo/min, respectively). Our results, for the first time, demonstrate that multiple Na/K-ATPase Lu-subunit isoforms are distributed throughout the first week of mammalian development and raise the possibility that multiple isozymes of the Na/K-ATPase contribute to blastocyst formation. (C) 1998 Academic Press.