The gamma-subunit of the Na-K-ATPase as a potential regulator of apical and basolateral Na+-pump isozymes during development of bovine pre-attachment embryos

Abstract

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.

Na,K-ATPase from Mice Lacking the γ Subunit (FXYD2) Exhibits Altered Na+ Affinity and Decreased Thermal Stability

“…Characteristics of ␥-Deficient Mice-The ␥ subunit has been detected in preimplantation mouse and cow embryos where the blastocyst trophectoderm, with basolaterally localized Na,KATPase, acts as a transporting epithelium similar in some ways to the nephron (11,45). The absence of ␥ did not disrupt blastocyst development or implantation, however, even in crosses involving null mutant females.…”

FXYD Proteins: New Tissue-Specific Regulators of the Ubiquitous Na,K-ATPase

“…Because antisense disruption of FXYD2 expression delays blastocoel formation, the authors concluded that FXYD2 has a role in transepithelial Na + reabsorption, and hence in blastocoel formation, that is independent of its association with the Na,K-ATPase (42). However, a recent study (43) reports that apical FXYD2 localizes with an apically expressed α3 isoform (43), suggesting that FXYD2 may function in the modulation of Na,K-ATPase activity in both the apical and basolateral membranes of the trophectoderm.…”

Deletion of the Na/K-ATPase α1-subunit gene (Atp1a1) does not prevent cavitation of the preimplantation mouse embryo

“…Increases in Na/K-ATPase activity occur concurrently with the onset of cavitation Tupper, 1975, 1977;Dumoulin et al, 1993;Betts et al, 1998) and are associated with increases in Na þ -pump subunit mRNA (Gardiner et al, 1990;Watson et al, 1990b;Betts et al, 1997) and protein expression (Benos, 1981;Watson and Kidder, 1988;Overstrom et al, 1989;Gardiner et al, 1990). Multiple isoforms of a-and b-subunits (as many as six isozymes) are expressed by murine (Watson and Kidder, 1988;Betts et al, 1997Betts et al, , 1998Jones et al, 1997;Waelchli et al, 1997;MacPhee et al, 2000;Barcroft et al, 2002;Jones et al, 2001 reviewed by Watson and Barcroft, 2001;Kidder, 2002) and bovine (Betts et al, 1997(Betts et al, , 1998 preimplantation embryos. The a1-subunit, however, appears to be the predominant isoform expressed within the basolateral membranes of the trophectoderm in all species examined (Watson and Kidder, 1988;Betts et al, 1998;MacPhee et al, 2000;Waelchli et al, 1997).…”