Cells in the mammalian blastocyst segregate into three distinct lineages, namely, trophoblast, hypoblast, and epiblast. During development, these will form extraembryonic and embryonic tissues, respectively. In mouse, only epiblast cells can be directly converted into cultured pluripotent embryonic stem cells, capable of forming all adult cell types. This conversion is promoted by the double inhibition (i.e., 2i) of mitogen-activated protein kinase kinase (Map2k), antagonizing Fgf signaling, and of glycogen synthase kinase 3 (Gsk3), stimulating the Wnt pathway. We investigated the effect of 2i treatment on lineage segregation and pluripotency-related gene expression in bovine blastocysts. In vitro fertilized (IVF) embryos were cultured in the presence of dimethyl sulfoxide or inhibitors of MAP2K (0.4 μM PD0325901) and GSK3 (3 μM CHIR99021) from the zygote (Day 1) stage. Compared to vehicle controls, 2i conditions increased the abundance of cumulus cells in bovine IVF cultures, which compromised blastocyst formation. Following cumulus removal, 2i accelerated blastocyst development and increased inner cell mass (ICM) and trophoblast cell numbers by 30% and 27%, respectively. These developmental and morphological changes were accompanied by alterations in gene expression. Signal inhibition increased transcription of putative epiblast markers NANOG and SOX2 while repressing putative hypoblast marker GATA4. Using microsurgical blastocyst dissection, we found that the increase in NANOG and SOX2 levels was specific to the ICM and not due to ectopic expression in the trophoblast. Expression of other pluripotency-related (POU5F1, KLF4, DPPA3) or trophoblast-enriched (CDX2) genes was not affected. In summary, 2i conditions reprogrammed the transcriptional profile of bovine ICM but not trophoblast cells. By shifting the balance from hypoblast- to epiblast-associated gene expression, 2i culture may prime bovine epiblast for subsequent derivation of pluripotent stem cell cultures.
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Vol. 88 • No. 3