Ouabain sensitivity and expression of Na/K-ATPase alpha- and beta-subunit isoform genes during bovine early development.

Abstract

The fluid movements that arise during blastocyst formation (cavitation) are, at least in part, driven by the Na/K-ATPase. In this study, the reverse transcriptase-polymerase chain reaction (RT-PCR) was used to survey bovine pre-attachment embryos for transcripts encoding known isoforms of the Na/K-ATPase alpha- and beta-subunits, including isoforms not previously detected during the first week of mammalian development. Transcripts encoding the Na-K-ATPase alpha 1, alpha 2, alpha 3 and beta 2 isoforms were detected throughout bovine preattachment development. This is the first indication that alpha 2, alpha 3 and beta 2 mRNAs are expressed during this early developmental interval. As in the mouse, beta 1-subunit transcripts were not detected until the morula stage and were also present in blastocysts. Thus, in two mammalian species an increase in abundance of beta 1 isoform transcripts in the morula stage is coincident with the onset of cavitation. Transcripts encoding the recently characterized alpha 4 isoform were not detected. The sensitivity of bovine blastocysts to ouabain (a potent inhibitor of Na/K-ATPase) was determined by assessing the ability of bovine blastocysts to recover in ouabain supplemental culture medium following cytochalasin-induced blastocyst collapse. Re-expansion of bovine blastocysts was inhibited in all ouabain concentrations down to 10(-9) M. Mouse blastocysts, in contrast, were sensitive to ouabain at or above 10(-3)M. These results have established that transcripts encoding multiple isoforms of both the alpha and beta subunits of the Na/K-ATPase are expressed throughout early bovine development and that bovine blastocysts display a greater sensitivity to ouabain than murine blastocysts. Future analysis will determine the possible individual and collective roles of these isoforms during blastocyst formation.