Dear Anumah,

The unusual hypotonicity of equine blastocyst fluid hasprompted us to investigate the role of sodium- and potassiumdependentadenosine triphosphatase (Na+,K+-ATPase) in theprocess of fluid accumulation in the horse conceptus. Ninemares were used for the experiments. Reverse transcriptasepolymerase chain reaction was conducted on two sets of fiveconceptuses recovered between 12 and 28 days (+ 1 day) afterovulation. Messenger RNAs encoding the ct1 and pi subunitisoforms of Na+,K+-ATPase were detected in all embryonic tissuesexamined. Western blot analysis showed that oal and 31subunits are both present in Day 15 conceptuses. Trophoblasttissues from 19 conceptuses between 8 and 31 days after ovulationwere stained immunohistochemically using primary antibodiesagainst the aol and 31 subunit isoforms of the Na+,K +-ATPase. Both isoforms were detected in all sections. Trophoblasticvesicles, prepared from 6 conceptuses between 12 and14 days after ovulation, were used to investigate the inhibitionof blastocyst expansion with ouabain after collapse induced withcytochalasin D. In normal medium there was a mean 3-fold increase,and in ouabain (10-6 M) a mean 3-fold decrease, in thevolume of vesicles that had been partially collapsed with cytochalasinD. We therefore conclude that, despite the hypotonicityof the blastocyst fluid in the early horse conceptus, the Na+,K +-ATPase plays a role in its accumulation, as in other species.

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