2022, Vol. 26 ›› Issue (31): 5047-5052

Effects of re-vitrification at blastocyst stage on histone epigenetic modification and pluripotency gene expression in mouse embryos

Wang Jianwu1, Zhao Yanhua2, Li Jingyu2, Wang Jiaqiang1   

Abstract: BACKGROUND: In assisted reproduction work, re-vitrification can effectively improve embryo utilization. Our previous research results show that the secondary vitrification at different developmental stages significantly affects embryo development, but the mechanism is still unclear.  
OBJECTIVE: To discuss the potential mechanism of secondary vitrification in different periods affecting embryo development potential.
METHODS:   These 2-cell embryos fertilized in vivo were collected and cultured in vitro, and randomly divided into four groups: control, vitrified at the 8-cell stage (8C), vitrified at the 8-cell stage, and re-vitrified at the 8-cell (8C-8C) or early blastocyst stage (8C-BL). Immunofluorescence was utilized to analyze changes in the expression levels of H3K4me3, H3K9me2, and H3K9AC at the blastocyst stage. Real-time fluorescent quantitative PCR was applied to analyze changes in the expression levels of pluripotency genes Cdx2, Oct4, and Sox2.  
RESULTS AND CONCLUSION: (1) Immunofluorescence results showed that first and second freezing led to a significant increase in the levels of H3K9me2 (P < 0.01) and H3K9AC (P < 0.000 1) at the blastocyst stage. (2)The first freezing did not affect the level of H3K4me3, but the level of H3K4me3 in the second freezing decreased significantly, and the decrease was more serious in the 8C-BL group (P < 0.000 1). (3) The results of real-time fluorescent quantitative PCR showed that the first freezing and the second freezing at the 8-cell stage did not affect the expression levels of embryo pluripotency genes Cdx2, Oct4 and Sox2, but the expression levels of embryo pluripotency genes in the 8C-BL group changed significantly. The level of Oct4 decreased significantly (P < 0.01), while the expression level of Cdx2 (P < 0.001) and Sox2 (P < 0.01) increased significantly. (4) The results show that the secondary vitrification has a certain negative impact on the epigenetics of mouse embryos, and the secondary freezing at the blastocyst stage significantly affects the H3K4me3 modification and pluripotency-related gene expression levels.
Key words: early embryos, re-vitrification, mouse, assisted reproductive, histone epigenetics, pluripotency gene