IQUIBICEN   23947
INSTITUTO DE QUIMICA BIOLOGICA DE LA FACULTAD DE CIENCIAS EXACTAS Y NATURALES
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Modulation of in vitro bovine embryos by t2iGöLIF increases the number of Nanog expressing cells in the ICM but does not prevent the activation of Sox17
Autor/es:
FISHER P; ALLER ATUCHA J; ALBERIO R; YNSAURRALDE RIVOLTA AE; GUBERMAN A; ALBERIO RH; CANIZO J; SUVA M; SALAMONE D
Reunión:
Congreso; Proceedings of the 30th Annual Meeting of the Brazilian Embryo Technology Society (SBTE); 2016
Resumen:
The combination of inhibitors known as 2i (MAPK and GSK3b inhibitors) + LIF is used for culturing mouse naïveembryonic stem cells (ESC), the only type of cells able to colonize the germline efficiently. In human ESC 2i+LIF isnot sufficient to confer naïve properties, but with the addition of a PKC inhibitor (Gö6983) and a modifiedconcentration of GSK3B inhibitor (known as t2iGöLIF), it is possible to capture naïve pluripotency in newlyderived cell lines. In this study we analysed the effect of t2iGöLIF treatment during early bovine embryogenesis.Bovine embryos were produced by IVF and cultured in vitro in serum free medium BBH7 until day 5 and then inN2B27 medium with 20 ng/ml of h-LIF or t2iGö, t2iGöLIF and DMSO (control). Embryos fixed at day 8 wereanalysed by immunocytochemistry for Nanog and Sox17, epiblast and hypoblast markers, respectively. Comparisonof blastocyst development showed no significant difference between control medium with DMSO (0.24 ± 0.05) vs.LIF (0.22 ± 0.04) and t2iGöLIF (0.32 ± 0.03), however t2iGö resulted in decreased embryo production (0.15 ±0.03). After immunostaining we found that all treatments produced an increased inner cell mass (ICM) totrophectoderm (TE) ratio, ICM:TE were 0.25 ± 0.05 vs 0.39 ± 0.08, 0.50 ± 0.08 and 0.38 ± 0.08 (control vs t2iGö,LIF and t2iGöLIF, respectively). In all treatments we detected more Nanog positive cells than control (25 ± 4) vs.t2iGö (40 ± 7), LIF (44 ± 7 A) and t2iGöLIF (45 ± 8). The number of Sox17 positive cells was reduced in t2iGöLIF(19 ± 3) and was unchanged in t2iGö (38 ± 5) compared to control (34 ± 5). Surprisingly, more Sox17 positive cellswere found in LIF treatment (61 ± 8) indicating that not only Nanog, but Sox17 increased with LIF. Furthermore,the number of cells that co-expressed both markers was higher in LIF treated groups (13 ± 4 vs 4 ± 1 in control). Inconclusion, the results indicate that t2iGöLIF is the only treatment that reduces the number of primitive endodermcells (hypoblast). These results suggest that additional signalling pathways may need to be blocked to prevent thedifferentiation of the early epiblast in cattle.