Modulation of WNT and ERK signalling blocks SOX17 expression without increasing NANOG in the bovine ICM

YNSURRALDE; GUBERMAN A; ALBERIO R; VAZQUEZ ECHEGARAY C; ALLER ATUCHA J; ALBERIO RH; CANIZO J; SUVA M; SALAMONE D

Congreso; IX Latin American Society for Developmental Biology Meeting 2017; 2017

Blocking primitive endoderm (PE) specification during early embryogenesis by modulation of signalling pathways has been used for the derivation of naïve embryonic stem cells in mouse and human. WNT and ERK inhibition (IWP2 and PD325901 respectively) prevents PE differentiation in marmoset embryos, and the WNT inhibitor was also successfully used in primates for establishment of pluripotent stem cells (PSCs) in the presence of FGF. Here we investigated the effect of IWP2 and PD32 during early bovine embryogenesis. We also tested different concentrations of PD32 in a dose response analysis. Bovine embryos were produced in vitro and cultured in SOF supplemented with 2.5% FBS until day 5 and then in N2B27 medium containing: 2.5 µM IWP2 + 10 µM PD32, each inhibitor alone or DMSO (control). Next, in an additional experiment, bovine embryos produced by IVF were cultured in vitro in N2B27 with 2.5; 5 and 10 µM of PD32 from day 5 to 8. Embryos fixed at day 8 were analysed by immunofluorescence for NANOG and SOX17, epiblast and hypoblast markers, respectively. In the first experiment, blastocyst development was highest in PD32 group. After immunostaining we observed a complete ablation of SOX17 in PD32 treated groups compared to control and IWP2 only group (57 ± 5 for control and 59 ± 6, respectively). The reduction in SOX17 was not compensated by an increase in NANOG expression in the ICM (42 ± 3 for control, 33 ± 3 for PD32, 23 ± 3 for IWP2 + PD32 and 52 ± 4 for IWP2 alone). Treatments with different PD32 concentrations reduced the total number of ICM cells at all concentration, as a result of SOX17 inhibition. The effect on NANOG expression was variable, with higher expression at 5 µM (43 ± 3) compared with 2.5 µM (29 ± 3) and 10 µM (33 ± 3) of PD32 (p value < 0.05). In conclusion, the results show that low PD32 (2.5 µM) can block PE specification, however combined inhibition of WNT did not result in increased proportion of NANOG expressing cells in bovine blastocysts.