The effects of embryo splitting on Cdx2, Sox2, Oct4, and Nanog gene expression in mouse blastocysts

Abstract

Background: Embryo splitting is utilized in reproduction biotechnology. The blastomeres resulting from the splitting of an embryo in two-, four- or eight-cell stages can develop into separate embryos that are genetically similar to the other blastomeres.

Aims: The present work studied the effects of splitting on embryo pluripotent gene expression (Cdx2, Sox2, Oct4, and Nanog) in mice.

Methods: Two-cell embryos were isolated from stimulated mice. The embryos were grouped into "split" and "non-split" groups. The zona pellucida was removed from the split group and the blastomeres were distributed before being co-cultured with mouse embryo fibroblasts to the blastocyst stage. Normal (non-split) blastocysts were co-cultured in the same way. The 3.5-day-old blastomeres were collected as the control group. For molecular evaluation, real-time PCR was conducted to analyze changes in Cdx2, Sox2, Oct4, and Nanog gene expression. Moreover, the blastocyst formation rate, overall blastocyst rate, and the number of newborns were statistically analyzed.

Results: The findings showed that embryo splitting increased blastocyst formation, overall blastocysts, developmental potential embryos, and the number of infants. Furthermore, the split and non-split (control) groups showed equal expression of pluripotent genes (Cdx2, Sox2, Oct4, and Nanog) in the molecular analysis.

Conclusion: It can be concluded that the growth and developmental potency of sister blastocysts derived from split two-cell stage mouse embryos are the same as those of normal blastocysts. So, there are no significant differences in gene expression between the split and non-split groups.

Keywords: Embryo splitting; Gene expression; Mouse blastocyst; Two-cell embryo.

Fig. 1

Embryo splitting and in vitro culturing. (A) Two-cell mouse donor embryos (×10), (B) Two-cell naked mouse embryos (removed ZP) (×10), and (C) Blastomere from in vitro culture of a naked embryo (×40)

Fig. 2

Relative expression of Oct4, Nanog, Sox2, and Cdx2 genes in groups. No significant difference was observed between groups. P-values shown on top of each column. S-B: Comparison between splitting and blastocyst in vivo, N: Splitting and non-splitting, and B: Blastocyst in vivo and non-splitting

Fig. 3

Blastocysts. (A) Split group (×40), and (B) Non-split group (×10)

Fig. 4

Newborn offspring from split and free zona embryos