A DOSE-DEPENDENT RESPONSE TO MEK INHIBITION DETERMINES HYPOBLAST FATE IN BOVINE EMBRYOS

CANIZO, JESICA; YNSAURRALDE RIVOLTA, AMADA E.; SUVÁ, MARIANA; ALBERIO, VIRGILIA; GUBERMAN, ALEJANDRA; SALAMONE, DANIEL F.; VAZQUEZ ECHEGARAY, CAMILA; ALBERIO, RAMIRO; ALLER, JUAN F.; ALBERIO, RICARDO H.

Chascomús

Taller; IV Taller de Biología Celular y del Desarrollo; 2018

IIB-INTECH

The segregation of the hypoblast and the emergence of the pluripotent epiblast mark the final stages of blastocyst formation in mammalian embryos. In bovine embryos the hypoblast has been partially studied, and evidence shows that MEK signaling plays a limited role during the formation of this lineage. To gain understanding of hypoblast segregation in the bovine embryo, we explored the dose response of MEK inhibition (0.1 to 10 μM of PD0325901) in the segregation of this lineage during in vitro development. We used a combination of immunofluorescence and RT-qPCR analysis of NANOG and SOX17 expression as readouts of epiblast and hypoblast, respectively. Our results show that NANOG is first detected from 8-cell stage; whereasSOX17 starts to be expressed in 16-32-cell stage embryos. SOX17 is first co-expressed with NANOG, but these markers become mutually exclusive by the late blastocyst stage. By assessing the expression kinetics of NANOG/SOX17 we show that low inhibition of MEK signalling (1μM) can eliminate SOX17 in bovine blastocysts, without altering NANOGexpression. In conclusion, SOX17 is a reliable early marker of hypoblast fate in the bovine embryo, which we determined to be fully segregated in day 7 blastocysts. SOX17 expression is abolished with 1μM of PD0523901, without affecting NANOG expression in the epiblast. These results suggest that for the long term in vitro culture of pluripotent stem cells derived from bovine embryos, it will be necessary, not only to avoid differentiation, but also to promote NANOG expression independently.