The investigation of the mechanisms controlling preimplantation development is necessary to optimize embryonic culture conditions, to improve our ability to assess embryonic developmental capacity prior to embryo transfer and to apply assisted reproductive technologies in the most efficient ways possible. The Na-K ATPase plays a pivotal role in preimplantation development, the period from oocyte-sperm union to uterine wall invasion. Activity of the Na-K ATPase pump, after assuming its basolateral position in the trophectoderm, establishes a trans-epithelial ionic gradient that facilitates the movement of water across the epithelium to create the fluid-filled blastocyst cavity. Reaching the blastocyst stage is crucial for implantation, and consequently, successful pregnancy. The Na-K ATPase is also implicated in regulating tight junction assembly and function and most recently in extracellular protein interactions. Signal transduction properties of the Na-K ATPase have been discovered upon binding of cardiotonic steroids (CTS), such as ouabain, to its α subunit leading to SRC activation. We hypothesize that pump activation induced by ouabain leads to SRC and subsequent MAPK activation, resulting in the regulation of trophectoderm tight junction formation and function during preimplantation development. To investigate this hypothesis, embryos were collected following application of superovulation methods applied to MF1 female mice. Morula stage embryos were subjected to treatment with 10-3M, 10-5M, 10-7M, 10-9M ouabain or bufalin to characterize affects of CTS treatment on blastocyst formation. Treatment with 10-5M, 10-7M, and 10-9M concentrations for up to 30 hr did not significantly affect the proportion of morulae that progressed to the blastocyst stage. However, embryos treated with 10-3M ouabain and bufalin did not progress to the blastocyst stage. Embryos treated with 10-5M, 10-7M, and 10-9M concentrations displayed a trend for increased blastocyst diameter and volume compared to untreated controls. Next experiments investigated the effects of treating blastocysts with ouabain at 10-3M, 10-3.5M, 10-4M, 10-4.5M and 10-5 M in KSOMaa medium for 1, 5, and 10 minutes to characterize influences of ouabain treatment on SRC phosphorylation by detecting variations in SRC phosphorylation using whole-mount immunofluorescence methods. The results from these experiments demonstrated an increase in phosphorylated SRC between KSOM controls and 10-3.5M ouabain-treated embryos. Those treated with 10-4M ouabain for 1 minute exhibited a marked increase in Src phosphorylation with a steady decrease at 10-4.5M and 10-5M concentrations. In addition RT-PCR methods were used to detect mRNAs encoding all SRC family members during preimplantation development. Preliminary results indicate that only mRNAs encoding SRC are detectable during preimplantation development while mRNAs encoding Lyn, Lck, Fyn and Yes are not. Our results demonstrate the likelihood of a CTS Na-K ATPase mediated mechanism for activating SRC during preimplantation development. Our future experiments will investigate the consequences of SRC blockade on preimplantation development and the role of SRC signaling in regulating tight junction assembly and function during preimplantation development. These studies contribute to our understanding of the mechanisms controlling preimplantation development. Research supported by operating grant from the Canadian Institutes of Health Research.