Ouabain sensitivity and expression of Na/K-ATPase alpha- and beta-subunit isoform genes during bovine early development.
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.
Betts
D H
DH
Molecular Genetics Unit, University of Western Ontario, London, Canada.
MacPhee
D J
DJ
Kidder
G M
GM
Watson
A J
AJ
D000818:Animals; D001483:Base Sequence; D001757:Blastomeres / Q000187:drug effects* / Q000378:metabolism; D002417:Cattle; D046508:Culture Techniques; D018076:DNA, Complementary; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D010042:Ouabain / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D012333:RNA, Messenger; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase