89 INHIBITION OF MAPKK AND GSK3 SIGNALLING PROMOTES DEVELOPMENT AND EPIBLAST-SPECIFIC EXPRESSION OF PLURIPOTENCY MARKERS IN BOVINE BLASTOCYSTS

During blastocyst development, the inner cell mass segregates into the epiblast and the hypoblast. These 2 tissues form morphologically and molecularly distinct cell populations that subsequently develop into the embryo proper and some extraembryonic components, respectively. In mouse, isolated epiblast cells can be directly converted into pluripotent embryonic stem cells, capable of differentiating into all cell types of an adult animal. Epiblast pluripotency is promoted by pharmacological inhibition of mitogen-activated protein kinase kinase (Mapkk). This shields epiblast cells from secreted fibroblast growth factor (Fgf), which would otherwise instruct them to exit pluripotency and differentiate into extraembryonic lineages. Indirect stimulation of the Wnt pathway by inhibiting glycogen synthase kinase 3 (GSK3) further antagonises inductive Fgf/Mapkk signalling. Thus the double inhibition (2i) of Mapkk and Gsk3 effectively promotes pluripotency (Q. L. Ying et al. 2008 Nature 453, 519–523; J. Nichols et al. 2009 Development 136, 3215–3222). We investigated the effect of 2i culture on bovine blastocysts. The IVF embryos were cultured in the presence of dimethyl sulfoxide or inhibitors of MAPKK (0.4 µM PD0325901) and GSK3 (3 µM CHIR99021) from the zygote (Day 1) stage onward. Compared to vehicle controls, 2i increased the abundance of cumulus cells in bovine IVF cultures, compromising blastocyst formation in cumulus-intact (248/823 = 30% v. 211/824 = 26%, respectively, n = 10; P < 0.05) but not cumulus-free cultures (546/1653 = 33% v. 572/1674 = 34%, respectively, n = 15; P = 0.51). In all subsequent experiments, we therefore cultured cumulus-free zygotes in 2i v. dimethyl sulfoxide until the blastocyst stage. This treatment increased the proportion of hatching (19/433 = 4% v. 7/416 = 2%, respectively, n = 10; P < 0.05) at the expense of early blastocysts (70/433 = 16% v. 93/416 = 22%, respectively, n = 11; P < 0.05). Differential staining of expanded IETS grade 1 and 2 blastocysts showed that 2i culture increased putative inner cell mass, trophectoderm, and total cell nuclei numbers by about 30% compared with controls (57 v. 43, 89 v. 69, and 146 v. 112, respectively; P < 0.01). Accelerated development and increased cell numbers were accompanied by gene expression changes in grade 1 and 2 blastocysts. Under 2i conditions, mRNA abundance of putative epiblast markers NANOG and SOX2 was >3-fold increased (P < 0.0001 and P < 0.01, respectively), and the putative hypoblast marker GATA4 was 2-fold reduced (P < 0.05). Other lineage-related markers (POU5F1, KLF4, DPPA3, and CDX2) showed no significant changes. Using microsurgical blastocyst dissection, we found that the increase in NANOG and SOX2 levels was specific to the inner cell mass-containing portion (7-fold for NANOG and 3-fold for SOX2; P < 0.00005 and P < 0.05, respectively) and not due to ectopic expression in the trophoblast-containing part, which showed similarly low expression levels for both genes. In summary, 2i treatment primed bovine blastocysts for pluripotency in the epiblast. Supported by MSI C10X1002.