Morphology and/or hatching ability of in vitro produced bovine embryos is no reliable indicator of inner cell mass cell number

Abstract

This study documents the spatial and temporal distribution of Oct-4, Cdx-2 and acetylated H4K5 (H4K5ac) by immunocytochemistry staining using in-vivo-derived rabbit embryos at different stages: day-3 compact morulae, day-4 early blastocysts, day-4 expanded blastocysts, day-5 blastocysts, day-6 blastocysts and day-7 blastocysts. The Oct-4 signal was stronger in the inner cell mass (ICM)/epiblast cells than in the trophectoderm (TE) cells in all blastocyst stages except day-4 expanded blastocysts, where the signal was similarly weak in both the ICM and TE cells. The Cdx-2 signal was first detected in a small number of TE cells of day-4 early blastocysts, and became evident in the TE cells exclusively afterwards. A consistently strong H4K5ac signal was observed in the TE cells in all blastocyst stages examined. In particular, this signal was stronger in the TE than in the ICM cells in day-4 early blastocysts, day-4 expanded blastocysts and day-5 blastocysts. Double staining of H4K5ac with either Oct-4 or Cdx-2 on embryos at different blastocyst stages confirmed these findings. This work suggests that day 4 is a critical timing for lineage formation in rabbit embryos. A combination of Oct-4, Cdx-2 and H4K5ac can be used as biomarkers to identify different lineage cells in rabbit blastocysts.There are very limited studies on key transcription factors and epigenetic programming events in preimplantation-stage rabbit embryos. Previously we reported the spatial and temporal distribution of Oct-4 and acetylated H4K5 (H4K5ac) in-vitro-cultured rabbit embryos. In the present study, we report the similar distribution patterns of Oct-4 and H4K5ac using in-vivo-derived rabbit embryos at different stages: day-3 compact morulae, day-4 early blastocysts, day-4 expanded blastocysts, day-5 blastocysts, day-6 blastocysts and day-7 blastocysts. The Oct-4 signal was stronger in the inner cell mass (ICM)/epiblast cells than in the trophectoderm (TE) cells in all blastocyst stages except day-4 expanded blastocysts, where such signal was similarly weak in both the ICM and the TE cells. A consistent strong H4K5ac signal was observed in the TE cells in all stages examined. In particular, this signal was stronger in the TE than in the ICM cells in day-4 early blastocysts, day-4 expanded blastocysts and day-5 blastocysts. Importantly, we also report, as far as is known for the first time, the distribution pattern of Cdx-2 in rabbit embryos. The Cdx-2 signal was first detected in a small number of TE cells of day-4 early blastocysts. It became evident in the TE cells exclusively afterwards. The present study provides novel information on key transcription factors and epigenetic events during rabbit embryo development, and demonstrates that a combination of Oct-4, Cdx-2 and H4K5ac could be used as biomarkers to identify different lineage cells in rabbit blastocysts.