Dynamics of DNA‐demethylation in early mouse and rat embryos developed in vivo and in vitro

Abstract

Virtually all mammalian species including mouse, rat, pig, cow, and human, but not sheep and rabbit, undergo genome-wide epigenetic reprogramming by demethylation of the male pronucleus in early preimplantation development. In this study, we have investigated and compared the dynamics of DNA demethylation in preimplantation mouse and rat embryos by immunofluorescence staining with an antibody against 5-methylcytosine. We performed for the first time a detailed analysis of demethylation kinetics of early rat preimplantation embryos and have shown that active demethylation of the male pronucleus in rat zygotes proceeds with a slower kinetic than that in mouse embryos. Using dated mating we found that equally methylated male and female pronuclei were observed at 3 hr after copulation for mouse and 6 hr for rat embryos. However, a difference in methylation levels between male and female pronuclei could be observed already at 8 hr after copulation in mouse and 10 hr in rat. At 10 hr after copulation, mouse male pronuclei were completely demethylated, whereas rat zygotes at 16 hr after copulation still exhibited detectable methylation of the male pronucleus. In addition in both species, a higher DNA methylation level was found in embryos developed in vitro compared to in vivo, which may be one of the possible reasons for the described aberrations in embryonic gene expression after in vitro embryo manipulation and culture.

Epigenetic reprogramming of embryos derived from sperm frozen at −20°C

“…After fertilization a rapid paternal-specific active demethylation is observed. Thereafter, there is a step-wise decline in methylation until the morula stage and the de novo methylation occurs at blastocyst stage [35,36]. In our experiments, the dynamic process of active demethylation of the paternal genome and de novo methylation at the blastocyst stage was detected in the control group and in the frozen sperm group ( Figure 2A1-F1 and a1-f1).…”

“…First, in the pronuclear stage, the paternal genome undergoes active DNA demethylation, then the embryo maintains low DNA methylation status until the blastocyst stage when de novo methylation begins [32][33][34][35]. In the present study, we used a well-documented 5-methylcytidine antibody to visualize the methylation patterns during the different stages [32,36]. We found that the dynamic DNA methylation patterns in embryos from the fresh and frozen sperm groups were similar (Figure 2).…”

DNA methylation reprogramming and DNA repair in the mouse zygote

“…Though sometimes being variable in the degree of progression, the zygotic paternal demethylation seems to be common among mammals and is so far reported for mouse (Oswald et al, 2000;Rougier et al, 1998), rat (Dean et al, 2001;Zaitseva et al, 2007), human (Fulka et al, 2004), pig (Dean et al, 2001;, cattle (Dean et al, 2001), sheep (Hou et al, 2008) and rabbit (Lepikhov et al, 2008). Mouse zygotes serve as typical and relatively well characterized model to study epigenetic reprogramming in mammalian preimplantation embryos, therefore most of research data described and discussed here relate to the experiments on mouse embryos and gametes.…”

Differential Susceptibility of Rat Embryos to Methyl Methanesulfonate During the Pregastrulation Period

“…Active demethylation of the male pronucleus in rat zygotes proceeds with a slower kinetic than that in mouse embryo, and the rat zygotes at 16 hr after copulation still exhibited detectable methylation on the male pronucleus (Zaitseva et al, 2007). Fertilization of oocytes by spermatozoa with DNA damage induced by exposure to cyclophosphamide, an alkylating agent, 8.4 ± 0.6 4.0 ± 0.0 3.6 ± 0.4 5.6 ± 0.6 1/90 1/6 Control 7.2 ± 1.3 3.9 ± 0.2 3.5 ± 0.5 4.7 ± 1.0 3/62 2/7 GD0 8.6 ± 0.9 4.0 ± 0.0 3.8 ± 0.3 5.7 ± 0.2 0/53 0/5 GD1 8.6 ± 1.0 4.0 ± 0.0 3.7 ± 0.2 5.7 ± 0.3 1/44 1/5 GD2 7.8 ± 0.9 4.0 ± 0.1 3.6 ± 0.5 5.2 ± 0.9 1/40 1/4 GD3 8.2 ± 0.7 3.9 ± 0.1 3.6 ± 0.4 4.6 ± 1.4 3/50 2/5 GD4 7.4 ± 0.8* 3.8 ± 0. resulted in aberrant epigenetic reprogramming in the preimplantation rat embryo (Barton et al, 2005).…”