EMBRYONIC EXPRESSION OF THE PUTATIVE GAMMA-SUBUNIT OF THE SODIUM-PUMPIS REQUIRED FOR ACQUISITION OF FLUID TRANSPORT CAPACITY DURING MOUSE BLASTOCYST DEVELOPMENT

The sodium/potassium pump, Na+,K+-ATPase, is generally understood to f unction as a heterodimer of two subunits, a catalytic alpha subunit an d a noncatalytic, glycosylated beta subunit. Recently, a putative thir d subunit, the gamma subunit, was cloned. This small protein (6.5 kD) coimmunoprecipitates with the alpha and beta subunits and is closely a ssociated with the ouabain binding site on the holoenzyme, but its fun ction is unknown. We have investigated the expression of the gamma sub unit in preimplantation mouse development, where Na+,K+-ATPase plays a critical role as the driving force for blastocoel formation (cavitati on). Using reverse transcriptase-polymerase chain reaction, we demonst rated that the gamma subunit mRNA accumulates continuously from the ei ght-cell stage onward and that it cosediments with polyribosomes from its time of first appearance, Confocal immunofluorescence microscopy r evealed that the gamma subunit itself accumulates and is localized at the blastomere surfaces up to the blastocyst stage, In contrast with t he alpha and beta subunits, the gamma subunit is not concentrated in t he basolateral surface of the polarized trophectoderm layer, but is st rongly expressed al the apical surface as well. When embryos were trea ted with antisense oligodeoxynucleotide complementary to the gamma sub unit mRNA, ouabain-sensitive K+ transport (as indicated by Rb-86(+) up take) was reduced and cavitation delayed, However, Na+,K+-ATPase enzym atic activity was unaffected as determined by a direct phosphorylation assay (''back door'' phosphorylation) applied to plasma membrane prep arations, These results indicate that the gamma subunit, although not an integral component of Na+,K+-ATPase, is an important determinant of active cation transport and that, as such, its embryonic expression i s essential for blastocoel formation in the mouse.