Influence of glucose and oxygen tension on the trophectoderm and the inner cell mass of in vitro produced bovine embryos

The first cell differentiation event that occurs in the embryo determines the inner cell mass (ICM) and the trophectoderm (TE). In the mouse, glucose (GLC) is essential for this process, while oxygen tension (O2) also interferes with TE formation. The roles of GLC and O2 in this event in bovine embryos are not completely elucidated. We hypothesized that the absence of glucose and a higher O2 tension negatively impact ICM and TE cell allocation in the bovine embryo. The objective of this study was to evaluate the effect of GLC within different O2 levels on the formation of the TE. In vitro-produced embryos were cultured in serum-free KSOM medium and randomly submitted to treatments on the day of IVC, according to a 2x2 factorial model, in which GLC (present [+GLC] or absent [-GLC]) and O2 (low [5%O2] or high [20%O2]) were the independent variables. Cleavage and blastocyst rates were obtained at D4 and D8, respectively. Embryos at D8 were subjected to autofluorescence analysis to quantitate NADH and FAD+ or fixed for GATA3 and YAP1 immunostaining using a laser scanning confocal microscope. Total, TE, and ICM cell counts were obtained. Embryos were also harvested for gene expression quantification of GATA3, YAP1, SOX2, CDX2, TFAP2C and OCT4. Results indicate that there was an effect of O2 (p=0.018) on cleavage rates, although no differences were observed in blastocyst rates. NADH was higher in -GLC compared to +GLC (p=0.014) and no differences in FAD+ were observed. Total cell count data were not different between variables. There was an increase in the ICM cell count in the +GLC 5%O2 condition compared to the other three conditions. No effects of GLC, O2, or their interactions were observed on TE cell count or the TE/total cell ratio. CDX2 (p=0.007) and TFAP2C (p=0.038) were increased in -GLC 20%O2 compared to +GLC 20%O2. SOX2 was decreased in +GLC 20%O2 compared to +GLC 5%O2 (p=0.027) or compared to -GLC 20%O2 (p=0.005). GATA3, YAP1, and OCT4 genes did not present differences among conditions. In conclusion, both GLC and high oxygen tension did not impair TE formation and TE cell number, although a +GLC-low oxygen environment led to a higher number of ICM cells. Interestingly, the expression of TE-related gene CDX2 was increased in the absence of glucose within higher O2 tension. Our results implicate that according to the oxygen tension used in IVC, glucose can exert different effects on blastocyst cell allocation or gene expression.