Developmental features of DNA methylation in CpG islands of human gametes and preimplantation embryos

Abstract

The aim of current study was to apply the methylated DNA immunoprecipitation (MeDIP)-Chip method to investigate dynamic changes in CpG island methylation in human sperm, oocytes and various developmental stages of preimplantation embryos. Samples were divided into eight groups: 1, sperm (n=30); 2, MII oocyte (n=25); 3, two-pronuclear (2PN) period zygote (n=25); 4, 4-cell stage embryo (n=5); 5, 8-cell stage embryo (n=4); 6, morula embryo (n=6); 7, blastular inner cell mass (ICM) group (n=5); 8, blastular trophoblastic cells (TE) (n=5). DNA was extracted and hybridized to NimbleGen Human DNA microarray. Following this, chip methylation data were read and analyzed. The CpG island methylation level of sperm was highest (peak value=15604), followed by oocytes (peak value=6062). The methylation level of zygotes decreased from 2PN stage (peak value=3744) to 4-cell stage (peak value=2826). This methylation level began to rise from 8-cell stage (peak value=3073) to morula stage (peak value=5374), ICM stage (peak value=5706) and TE stage (peak value=8376). The proportion of sperm methylation signal that was in the promoter region was 73.7%, and that in the oocyte was 60.8%, 2PN stage was 57.9%, 4-cell stage was 52.2%, 8-cell stage was 50.3%, morula was 50.3%, ICM was 66.6% and TE was 66.8%. In conclusion, the current study indicated that CpG island methylation changes in human preimplantation embryos were divided into three stages. In the first stage from fertilization to 2PN, the level of CpG island methylation declined sharply. In the second stage from morula to blastular ICM, methylation rapidly increased. In the third stage, methylation was reestablished in the TE. Dynamic CpG island methylation changes were derived primarily from methylation in the promoter region.

Keywords: CpG island methylation; human oocyte; human sperm; preimplantation embryos.

Figure 1.

CpG island-associated peak M-value in sperm (n=15,604), oocyte (n=6,062), 2PN stage (n=3,744), 4-cell stage (n=2,826), 8-cell stage (n=3,073), morula stage (n=5,374), ICM (n=5,706) and TE (n=8,376). Horizontal axis: CpG-island associated peak M-value. The peak M-value of sperm was highest (n=15,604), followed by oocytes (n=6,062). The peak M-value of zygotes decreased from 2PN stage (n=3,744) and reached its minimum level at the 4-cell stage (n=2,826). This peak M-value began to rise from 8-cell stage (n=3,073) to morula stage (n=5,374) and to ICM stage (n=5,706) and TE stage (n=8,376). The peak M-value of the ICM stage was similar to that of oocytes. The peak M-value of TE fell between those of oocytes and sperm. ICM, inner cell mass; TE, trophoblast cells; 2PN, two pronuclei.

Figure 2.

Proportion of low, middle and high CpG island-associated peak M-values. CpG island methylation changes of human preimplantation embryos are divided into three stages. The proportion of high CpG island methylation regions (peak M-value ≥0.7) in 2PN stage was lowest. Following the 2PN stage, fluctuations in CpG island methylation tended to increase. The proportion of high CpG island methylation regions in the blastular ICM stage was similar to those in the TE stage. However, the results for the proportion of low CpG island methylation regions (peak M-value ≤0.4) were reversed. The proportion of low CpG island methylation regions (peak M-value ≤0.4) in 2PN stage was highest. Changes in the proportion of high CpG island methylation regions were not evident. ICM, inner cell mass; TE, trophoblast cells; 2PN, two pronuclei.

Figure 3.

CpG island methylation changes from gametes to various developmental stages of the preimplantation embryo. Dark blue represents the peak value of the changing methylation point, and light blue represents the peak value of the stable methylation point. In the first stage from fertilization to 2PN, the level of CpG island methylation declined sharply. In the second stage from morula to blastular ICM, methylation rapidly increased again. The third stage was the methylation reestablishment process of TE. ICM, inner cell mass; TE, trophoblast cells; 2PN, two pronuclei.

Figure 4.

CpG island-associated PeakScore in intragenic, intergenic and promoter regions. (A) Sperm, (B) oocyte, (C) 2PN stage, (D) 4-cell stage, (E) 8-cell stage, (F) morula stage, (G) ICM and (H) TE. The proportion of sperm methylation signal in the promoter region was 73.7%, and that in the oocyte was 60.8%, 2PN was 57.9%, 4-cell stage was 52.2%, 8-cell stage was 50.3%, morula stage was 68.8%, ICM was 66.6% and TE was 66.8%. Methylation in the intergenic region took second place. However, dynamic methylation changes in intragenic regions were not observed. ICM, inner cell mass; TE, trophoblast cells; 2PN, two pronuclei.

Figure 5.

Mean values of CpG island-associated peak M-value PeakScore in (A) intergenic, (B) intragenic and (C) promoter regions. Fluctuation at various stages of the preimplantation embryo was most evident in the promoter region. In promoter regions, the methylation level reached a minimum value in the 2PN stage, then it began to rise. The methylation level of ICM and TE in the promoter region fell between the levels observed in oocytes and sperm. The CpG methylation fluctuation pattern in intragenic regions was similar to those in intergenic regions. In intragenic and intergenic regions, the methylation level decreased from 2PN stage and reached a minimum value at the 4-cell stage, then it began to rise. The methylation level of ICM and TE in these regions was similar to that in oocytes. ICM, inner cell mass; TE, trophoblast cells; 2PN, two pronuclei.