Regulation of Na+,K(+)-ATPase alpha subunit gene expression during mouse preimplantation development

Several lines of evidence support the hypothesis that a sodium flux, driven by Na+,K(+)-ATPase in the basolateral plasma membranes of mural trophectoderm, drives fluid transport during blastocoel formation in eutherians. In light of the importance of this enzyme for preimplantation development, attention has been focused on the regulation of expression of its alpha and beta subunits. Here we report on the spatial distribution and translation of the alpha subunit mRNA. Although this mRNA accumulates from the 2-cell stage onward the alpha subunit itself could not be detected by immunofluorescence prior to the late morula stage, after which it becomes concentrated in the mural trophectoderm. In the present study we have used a wholemount, fluorescent in situ hybridization technique that takes advantage of the optical sectioning capability of the confocal microscope to show that alpha subunit mRNA, in contrast to the alpha subunit itself, accumulates in all cells of the early blastocyst. This finding demonstrates that the spatial distribution of the alpha subunit is regulated post-transcriptionally. We have also examined the translational regulation of alpha subunit mRNA by preparing polyribosomal and subribosomal ribonucleoprotein fractions for mRNA assay by reverse transcription-polymerase chain reaction. We found that alpha subunit mRNA is in polyribosomes continuously from at least the 4-cell stage. Thus, the abrupt appearance of the alpha subunit in the late morula stage as revealed by immunofluorescence must be determined by post-translational events. In the Discussion, we consider the hypothesis that synthesis of the beta subunit of the enzyme is the rate limiting step in functional expression of the alpha subunit.