Effect Of In Vitro Culture Conditions On Early Development And Epiblast Stem Cell Isolation Of Mouse Embryos

Distinct difference exists between pre-implantation and implanted mouse embryos, which are regulated by maternal materials of oocytes during early development. Simulation of female mouse oviduct gives possibility to culture embryos in vitro. Since the activation of zygotic genome, mouse embryos develop and differentiate into inner cell mas (ICM), trophoblast, primitive endoderm,epiblast. The early development of mouse embryos are regulated by intra- and inter-cell signaling pathways, including transcription factors Oct4, Nanog and Sox2. Pluripotent Embryonic stem cells (mESCs) and epiblast stem cells (EpiSCs) maintaining self-renewal were isolated from the ICM and epiblast in vitro, respectively. The present study was aim at analyzing signaling pathways through which exogenous factors in the culture conditions affect mouse early embryo development.At first,8-cell stage mouse embryos were cultured in KSOM with small molecules as designed as follow:KSOM (as control), KSOM/PD0325901 (KPD), KSOM/PD0325901/CHIR99021 (K2i), KSOM/2i/LIF (K2L). The results showed that blastocyst rate between each group are similar, while hatching rate of KPD, K2i and K2L were significantly higher than control, and cells of each embryo were fewer than control. At the same time, Oct4 positive cells became more and Gata4 positive cells became fewer during the in vitro culture days.Then, blastocysts were cultured in the same conditions as described. The same results as 8-cell embryos were showed:fewer cells of single embryo, more Oct4 positive cells, and fewer Gata4 positive cells. However, exogenous small molecules did not affect hatch rate of blastocysts during in vitro culture.Finally, in vitro cultured embryos and in vivo embryos were plated into basal medium supplemented with N2, B27, Activin A and bFGF at parallel embryo developing days to establish EpiSCs. All the in vitro cultured embryos and in vivo blastocysts could not give rise to EpiSCs, while in vivo embryos at 5.5 days and 6.5 days could.Taken together, addition of exogenous small molecules, which inhibit WNT, MEK/ERK and LIF/STAT pathway, could maintain the pluripotency of cultured E2.5 and E3.5 embryos, as well as epiblast. Gene expression and transcription regulation were different between pre-implantation and implanted embryos, and embryos developed in vivo and in vitro. Although pluripotency of epiblast was maintained in vitro, exogenous small molecules could not support the develop conditions that embryos relying on in vivo. This could be the reason why EpiSCs did not isolated from in vitro cultured embryos.The embryos culturing system has the advantage that affects of exogenous factors on early embryo development could be directly analyzed. This study provided basic insights about the mechanism of early development of mouse embryos.