Ouabain-sensitive Rb+ uptake in mouse eggs and preimplantation conceptuses.
The results of histochemical and immunocytochemical studies have been used elsewhere to support the hypothesis that Na+/K(+)-ATPase expression is initiated or increases dramatically in preimplantation mouse conceptuses just before they begin to cavitate. Moreover, localization of the enzyme in the inner membrane of the mural trophoblast is thought to be involved directly in formation and maintenance of the blastocyst cavity. Presumably, Na+/K(+)-ATPase extrudes the cation, Na+, and therefore water into the cavity. The cation transporting activity of the enzyme can be determined by measuring ouabain-sensitive Rb+ uptake by cells. Therefore, we measured Rb+ uptake in mouse eggs and preimplantation conceptuses at various stages of development. 86Rb+ uptake by conceptuses increased linearly with time for at least 60 min in medium containing 0.7 mM total Rb+ plus K+ in the absence or presence of 1.0 mM ouabain, and ouabain inhibited more than 70% of 86Rb+ uptake. The ouabain concentration at 1/2 of maximum inhibition of the ouabain-sensitive component of 86Rb+ uptake was about 10-20 microM in eggs and conceptuses at all stages of preimplantation development. Moreover, ouabain-sensitive Rb+ uptake had a twofold higher Vmax value in blastocysts than in eggs or conceptuses at earlier stages of development (i.e., approximately 173 vs 70-100 fmole.conceptus-1.min-1), although the total cell surface area also was probably about two times greater in blastocysts than in eggs or other conceptuses. Ouabain-sensitive Rb+ transport in eggs and conceptuses may have occurred via a single ouabain-sensitive Rb+ transporter with a Hill coefficient of 1.5-1.8 (Hill plots). When it was assumed that the Hill coefficient had a value of 2.0, however, eggs and conceptuses appeared to contain at least two forms of Na+/K(+)-ATPase activity. These studies are the first to show that the cation transporting activity of Na+/K(+)-ATPase can be measured quantitatively in mammalian eggs and preimplantation conceptuses. Inclusion of this assay in experiments designed to determine how Na+/K(+)-ATPase activity is controlled in oocytes and conceptuses should yield further insight into the role of this enzyme in oogenesis and preimplantation development.
Van Winkle
L J
LJ
Department of Biochemistry, Chicago College of Osteopathic Medicine, Downers Grove, Illinois 60515.
Campione
A L
AL
D000818:Animals; D001755:Blastocyst / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D047108:Embryonic Development / Q000187:drug effects*; D005260:Female; D007700:Kinetics; D051379:Mice; D008813:Mice, Inbred ICR; D010042:Ouabain / Q000494:pharmacology*; D010063:Ovum / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D011247:Pregnancy; D012414:Rubidium Radioisotopes; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
D012414:Rubidium Radioisotopes; D010042:Ouabain; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase