Blastocyst formation is dependent on the differentiation of a transporting epithelium, the trophectoderm, which is coordinated by the embryonic expression and cell adhesive properties of E-cadherin. The trophectoderm shares differentiative characteristics with all epithelial tissues, including E-cadherin-mediated cell adhesion, tight junction formation, and polarized distribution of intramembrane proteins, including the Na\p=n-\K ATPase. The present study was conducted to characterize the mRNA expression and distribution of polypeptides encoding E-cadherin, (Boiler et al, 1985; Gumbiner and Simons, 1987; Gumbiner et al, 1988), resulting in the establishment of distinct apical and basolateral plasma membrane domains (Vestweber et al, 1987; D'Angelo Siliciano and Goodenough, 1988; Fleming and Johnson, 1988; Rodrigez-Boulan and Nelson, 1989;Wiley et al, 1990;Watson, 1992;Watson et al, 1992a; Collins and Fleming, 1995 1991 Citi, 1993; Kidder 1993 (Nagafuchi and Takeichi, 1988; Kemler and Ozawa, 1989; Gumbiner and McCrea, 1993;McNeill et al, 1993;Ranscht, 1994 (Stevenson et al, 1986Anderson et al., 1988; Citi et al, 1988 Citi et al, , 1993 Furuse et al, 1993 al., 1988;Stevenson et al, 1988;Watson, 1992; Citi, 1993 (Biggers et al, 1988;Watson, 1992; Kidder, 1993 (Stevenson et al, 1986;Takeichi, 1988; Li and Poznansky, 1990;Ozawa et al, 1990; Behrens et al, 1993;Su et al, 1993). Routine (Davis, 1993). The

Expression and activity of the Na-K-ATPase within the basolateral membrane domains of the trophectoderm epithelium provide the driving force for accumulation of Na(+) and Cl(-) across the nascent epithelium, mediating fluid movement into the forming blastocoel. Within the trophectoderm of the bovine blastocyst, multiple isozymes of the Na-K-ATPase are expressed. Immunolocalization has demonstrated that the alpha1-isozyme localizes within the basolateral membrane, whereas the alpha 3-isozyme localizes to the apical cell margins. Gene-specific RT-PCR and wholemount indirect immunofluorescence confocal laser scanning microscopy were used to examine expression of the Na-K-ATPase gamma-subunit (a regulatory subunit of the Na-K-ATPase) throughout development of bovine preattachment embryos in vitro. Expression of mRNA transcripts for the gamma-subunit was detected throughout bovine pre-attachment development from the fertilized one-cell embryo to the blastocyst stage. A similar pattern of expression was also observed for gamma-subunit protein, and immunofluorescence was detected within the membranes of embryonic blastomeres at all stages of development. In contrast to the expression patterns observed for the alpha-subunits, gamma-subunit proteins were detected in both the basolateral and apical cell margins of the trophectoderm, and surrounding all cells of the inner cell mass. Co-localization studies demonstrated that gamma-subunit peptides are co-expressed with the alpha1-subunit in the basolateral domains of the trophectoderm. These results indicate a role for the gamma-subunit of the Na-K-ATPase in modulating Na(+)-pump activity in both apical and basolateral margins of the trophectoderm during formation and expansion of the bovine blastocyst, and adds a further level of complexity to Na(+)-pump regulation of cavitation.

No abstract