Using extended growth of cattle embryos in culture to gain insights into bovine developmental events on embryonic days 8 to 10.

We have previously described an extended embryo culture system, based on uterine composition, growth factors and the cell culture additive B27, for growing cattle embryos in vitro beyond embryonic day 7. Here, extended in vitro embryos are compared to embryos developed in the uterus and are used to establish a developmental staging framework useful for understanding developmental events occurring until Day 10. Immunofluorescence or mRNA expression of the ICM/epiblast markers OCT4, SOX2 and NANOG, hypoblast markers GATA6, SOX17 and GATA4 and trophoblast genes CDX2, GATA3, ASCL2 and IFNT revealed the presence of four stages during this period that can be molecularly distinguished. These are expanded blastocyst, hatched blastocyst, hypoblast layering and early hypoblast migration. Interestingly NANOG and SOX17 show reciprocal expression at the expanded blastocyst stage, well before SOX2 and GATA6 expression refines to a similar so-called "salt and pepper" mutually exclusive expression in the ICM at the hatched blastocyst stage. GATA4 expression is only seen from stages when the hypoblast starts migrating around the blastocyst cavity. Intriguingly, trophoblast still expresses GATA6 and OCT4 in all cells during the expanded blastocyst phase, while SOX2 and SOX17 are seen in only some trophoblast cells. By the hypoblast-epiblast layering stage no trophoblast expression remains except for that of OCT4 protein, which starts waning in trophoblast once the hypoblast begins migrating. Lastly, it is shown that cultured embryos exhibit increased expression of the stress marker TP53 in the epiblast and hypoblast at late stages in comparison to embryos produced in the uterine environment.