Inhibition of ERK and GSK3 signalling pathways promotes development and expression of pluripotent markers in bovine blastocysts
Harris, D. (2012). Inhibition of ERK and GSK3 signalling pathways promotes development and expression of pluripotent markers in bovine blastocysts (Thesis, Master of Science (MSc)). University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/6602
Permanent Research Commons link: https://hdl.handle.net/10289/6602
Embryo-derived pluripotent stem cells (ePSCs) naturally derive from different founder tissues, namely the early and late epiblast, that vary in their cellular characteristics. Recent evidence in mice suggests these sources of starting material correspond with two states of pluripotency referred to as ‘naïve’ and ‘primed’, respectively. Pluripotency is defined as the ability of a cell to give rise to any foetal or subsequent adult cell type. Naïve and primed ePSCs have different fundamental properties and only naïve cells can give rise to all cell types of an animal, including functional gametes (Nichols & Smith 2009). Until recently, naïve ePSCs (also known as embryonic stem cell or ESCs) were only available in mouse. After almost 30 years, the first non-mouse ESCs were derived in rat using a combination of specific inhibitors that block the FGF-ERK differentiation pathway and simultaneously maintain self-renewal through inhibition of GSK3 (Buehr et al. 2008; Ying et al. 2008). The three inhibitor combination, or 3i, included inhibitors of the FGF receptor (PD173074), downstream ERK activity (PD0325901) and the GSK3 pathway (CHIR99021). The dual inhibition of PD0325901 and CHIR99021, termed 2i, was found to be effective for ESC culture; therefore the requirement for PD173074 became dispensable. If pluripotent mechanisms are conserved between species, we hypothesise that these inhibitors would aid the derivation of naïve ESCs in livestock species i.e. cattle. The aim of this study was to assess the effects of these inhibitors on the developing bovine embryo. We hypothesised that the use of the inhibitors in culture medium would enhance the pluripotent characteristics of blastocysts for subsequent ESC culture. To test this idea we cultured in vitro fertilised bovine embryos in the presence of the inhibitors to initially assess the effect on embryo development. To assess embryo quality, blastocysts were analysed to evaluate the total number of cell nuclei. Additionally, mRNA and protein analyses were undertaken to characterise the expression of core pluripotent genes and assess if the inhibitors had an effect on their expression. We found that culturing bovine embryos in either 3i or 2i did not influence the total number of embryos that progressed to the blastocyst stage. A significant decrease was observed in the proportion of early vs. late stage blastocysts in 2i, which suggested acceleration in the rate of development. In 2i medium, the number of cell nuclei in D7 blastocysts was significantly increased in both the inner cell mass (ICM) and the outer trophectoderm (TE). Additionally, embryos cultured in 2i showed a >2-fold increase in NANOG, a transcription factor at the core of the pluripotency network. The ICM-specific pluripotency marker SOX2 was also up-regulated in the presence of 2i. Both these genes were found to be significantly enriched in the ICM of bovine blastocysts. Overall, we conclude that bovine blastocysts differ from mouse in their response to 2i signal inhibition and molecular control of pluripotency.
University of Waikato
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