Date of Award
1996
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Abstract
The Na,K-ATPase or sodium pump is an enzyme that is found in most animal cells. The enzyme pumps sodium and potassium ions against their concentration gradients and is hypothesized to be a mediator of fluid accumulation during mouse preimplantation development. The purpose of the present study was to determine, using reverse transcriptase-polymerase chain reaction (RT-PCR), whether all {dollar}\alpha\ (\alpha1,\ \alpha2,\ \alpha3,\ \alpha4{dollar}) and {dollar}\beta\ (\beta1{dollar} and {dollar}\beta{dollar}2) isoforms of the sodium pump were expressed during preimplantation development. Previous work by Watson and Kidder (1988) had shown that the {dollar}\alpha{dollar} subunit was localized to the basolateral domains of mural trophectoderm surrounding the blastocoel. The spatial localization of {dollar}\alpha1{dollar} transcripts in mouse morulae and blastocysts was examined with the development of a non-radioactive wholemount in situ hybridization procedure and the use of a laser scanning confocal microscope.;{dollar}\alpha3{dollar} and {dollar}\beta2{dollar} isoform transcripts were detected throughout mouse preimplantation stages. {dollar}\alpha2{dollar} transcripts were only detected in oocytes while {dollar}\alpha4{dollar} transcripts were not detected in any embryo stages. None of the {dollar}\alpha3,\ \alpha2{dollar}, or {dollar}\beta2{dollar} isoform subunits were detected, by immunocytochemistry, in membranes of preimplantation stages. {dollar}\alpha3{dollar} subunits were detected in morulae/blastocyst total protein on a western blot.;{dollar}\alpha1{dollar} subunits were found in membranes of all preimplantation stages while {dollar}\beta{dollar}1 subunits were only detectable from the morulae stage onwards. {dollar}\alpha{dollar}1 transcripts were detected throughout the cytoplasm of mouse morulae and blastocysts correlating with the presence of {dollar}\alpha{dollar}1 subunits in membranes. These results provide evidence that {dollar}\alpha1{dollar} and {dollar}\beta1{dollar} subunits are the most probable subunit isoforms of the sodium pump, to mediate fluid accumulation in mouse morulae and blastocysts.;Using antisense oligodeoxynucleotides (ODNs), {dollar}\beta1{dollar} subunit mRNA was targeted in mouse preimplantation embryo to help determine the role of the {dollar}\beta1{dollar} subunit during cavitation. The number of cavitating embryos were significantly decreased (p {dollar}<{dollar} 0.10) compared to controls, but future experiments will require a better understanding of ODN uptake characteristics in mouse embryos in order to increase the observed effects. The results do suggest that the {dollar}\beta{dollar}1 subunit could be a trigger for cavitation to occur.;Sodium pump gene expression was also examined in another mammalian species, the horse, to provide further evidence that the enzyme is a mediator of mammalian blastocyst development. {dollar}\alpha1{dollar} and {dollar}\beta1{dollar} transcripts were detected between day 12 and 28 of horse development, a period of maximal blastocoel expansion. The two corresponding subunits were also detected, by immunocytochemistry, in trophoblast of day 16 conceptuses. The presence of the two subunits corresponds to the enzyme's potential role of pumping sodium ions into the developing blastocoel, creating osmotic pressure, resulting in the movement of fluid into the blastocoel.
Recommended Citation
Macphee, Daniel James, "Expression Of Sodium Pump Subunit Genes During Mouse Preimplantation Development" (1996). Digitized Theses. 2651.
https://ir.lib.uwo.ca/digitizedtheses/2651