REGULATION OF NA-ATPASE ALPHA-SUBUNIT GENE-EXPRESSION DURING MOUSE PREIMPLANTATION DEVELOPMENT(,K+)

Several lines of evidence support the hypothesis that a sodium flux, d riven by Na+,K+-ATPase in the basolateral plasma membranes of mural tr ophectoderm, drives fluid transport during blastocoel formation in eut herians. In light of the importance of this enzyme for preimplantation development, attention has been focused on the regulation of expressi on of its alpha and beta subunits. Here we report on the spatial distr ibution and translation of the alpha subunit mRNA. Although this mRNA accumulates from the 2-cell stage onward the alpha subunit itself coul d not be detected by immunofluorescence prior to the late morula stage , after which it becomes concentrated in the mural trophectoderm. In t he present study we have used a wholemount, fluorescent in situ hybrid ization technique that takes advantage of the optical sectioning capab ility of the confocal microscope to show that alpha subunit mRNA, in c ontrast to the the alpha subunit itself, accumulates in all cells of t he early blastocyst. This finding demonstrates that the spatial distri bution of the alpha subunit is regulated post-transcriptionally. We ha ve 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 subu nit in the late morula stage as revealed by immunofluorescence must be determined by post-translational events. In the Discussion, we consid er the hypothesis that synthesis of the beta subunit of the enzyme is the rate limiting step in functional expression of the alpha subunit. (C) 1994 Academic Press, Inc.