EVIDENCE FOR THE PRESENCE OF SODIUM-DEPENDENT AND POTASSIUM-DEPENDENTADENOSINE-TRIPHOSPHATASE ALPHA-1 AND BETA-1 SUBUNIT ISOFORMS AND THEIR PROBABLE ROLE IN BLASTOCYST EXPANSION IN THE PREATTACHMENT HORSE CONCEPTUS

The unusual hypotonicity of equine blastocyst fluid has prompted us to investigate the role of sodium-and potassium-dependent adenosine trip hosphatase (Na+,K+-ATPase) in the process of fluid accumulation in the horse conceptus. Nine mares were used for the experiments. Reverse tr anscriptase polymerase chain reaction was conducted on two sets of fiv e conceptuses recovered between 12 and 28 days (+/-1 day) after ovulat ion. Messenger RNAs encoding the alpha 1 and beta 1 subunit isoforms o f Na+,K+-ATPase were detected in all embryonic tissues examined, Weste rn blot analysis showed that alpha 1 and beta 1 subunits are both pres ent in Day 15 conceptuses, Trophoblast tissues from 19 conceptuses bet ween 8 and 31 days after ovulation were stained immunohistochemically using primary antibodies against the alpha 1 and beta 1 subunit isofor ms of the Na+,K+-ATPase. Both isoforms were detected in all sections. Trophoblastic vesicles, prepared from 6 conceptuses between 12 and 14 days after ovulation, were used to investigate the inhibition of blast ocyst expansion with ouabain after collapse induced with cytochalasin D. In normal medium there was a mean 3-fold increase, and in ouabain ( 10(-6) M) a mean 3-fold decrease, in the volume of vesicles that had b een partially collapsed with cytochalasin D. We therefore conclude tha t, despite the hypotonicity of the blastocyst fluid in the early horse conceptus, the Na+,K+-ATPase plays a role in its accumulation, as in other species.