SUMMARYAt the blastocyst stage of mammalian pre-implantation development, three distinct cell lineages have formed: trophectoderm, hypoblast (primitive endoderm) and epiblast. The inability to derive embryonic stem (ES) cell lines in a variety of species suggests divergence between species in the cell signaling pathways involved in early lineage specification. In mouse, segregation of the primitive endoderm lineage from the pluripotent epiblast lineage depends on FGF/MAP kinase signaling, but it is unknown whether this is conserved between species. Here we examined segregation of the hypoblast and epiblast lineages in bovine and human embryos through modulation of FGF/MAP kinase signaling pathways in cultured embryos. Bovine embryos stimulated with FGF4 and heparin form inner cell masses (ICMs) composed entirely of hypoblast cells and no epiblast cells. Inhibition of MEK in bovine embryos results in ICMs with increased epiblast precursors and decreased hypoblast precursors. The hypoblast precursor population was not fully ablated upon MEK inhibition, indicating that other factors are involved in hypoblast differentiation. Surprisingly, inhibition of FGF signaling upstream of MEK had no effects on epiblast and hypoblast precursor numbers in bovine development, suggesting that GATA6 expression is not dependent on FGF signaling. By contrast, in human embryos, inhibition of MEK did not significantly alter epiblast or hypoblast precursor numbers despite the ability of the MEK inhibitor to potently inhibit ERK phosphorylation in human ES cells. These findings demonstrate intrinsic differences in early mammalian development in the role of the FGF/MAP kinase signaling pathways in governing hypoblast versus epiblast lineage choices.

A mammalian oocyte within an ovarian follicle is surrounded by cumulus cells, together this structure is known as the cumulus-oocyte complex (COC). Cumulus cells are important for the development of the oocyte, they support the maturation process of the oocyte within the ovary and aid in sperm recognition. Because it is known that a Dicer knockout leads to infertility, microRNAs (miRNA) are focused to have an important role in oocyte development. MiRNAs are small noncoding RNA sequences that act as transcriptional regulators. Little is known about the expression of miRNA in cumulus cells or how cumulus-derived miRNA may regulate or be used to indicate the developmental competence of the maturing oocyte. Our aim was to investigate miRNA expression in oocytes and to identify and establish how specific miRNA influence the acquisition of developmental competence by bovine oocytes. Normalization of qPCR data requires stable reference genes. To this end, we tested the expression of various miRNA with respect to their ability to be used as reference miRNA for bovine cumulus cells; these included miR-103, miR-93, miR-26, let-7a, miR-191, and the small noncoding nuclear RNA U6. Cumulus-oocyte complexes were recovered from the ovaries of slaughtered cows and matured in vitro. Small samples of cumulus cells were collected from these COC before and after maturation. From the cumulus cell groups recovered at different stages, small RNA were extracted and cDNA was synthesised, followed by qRT-PCR. To identify the optimal combination of reference genes, the geNorm algorithm was used. MiR-26a and let-7a were identified as the most stably expressed miRNAs, whereas U6 showed the most variable expression levels. Future investigations are planned to identify miRNA in cumulus cells that can be used as markers for oocyte developmental competence. Using a single oocyte-embryo culture system will enable us to retrospectively relate cumulus miRNA expression to the developmental capacity of the oocyte.This work was supported by EU FP7 EpiHealthNet (N°317146).