Genome reprogramming during the first cell cycle in in vitro produced porcine embryos

Abstract

ABSTRACT:Conflicting data still exist regarding the extent of paternal pronuclear DNA demethylation in one cell-stage mammalian embryos. Demethylation of paternal pronuclear DNA was observed in in vivo produced porcine zygotes, whereas in vitro produced embryos do not show any or only weak paternal genome demethylation. In our experiments, we have used and compared two in vitro techniques commonly used for in vitro embryo production (in vitro fertilization and intracytoplasmic sperm injection) and then we evaluated the extent of labelling in both these groups after 5-methylcytosine (5-MeC) or dimethyl H3/K9 labelling. We have found no differences in the methylation pattern between both those techniques used for the production of embryos. Moreover, we did not detect any demethylation of paternal DNA after 5-MeC labelling at all. Contrary to this, labelling with dimethyl H3/K9 antibodies showed differences in labelling intensity between maternal and paternal genomes in 42% of zygotes after in vitro fertilization and in 44% of zygotes after intracytoplasmic sperm injection. Our results indicate that in vitro matured pig oocytes exhibit rather inconsistent methylation patterns. This inconsistency probably resulted from insufficient cytoplasmic maturation of oocytes and to a lesser extent from the in vitro technique for embryo production.

Dynamics of epigenetic remodeling in interspecies porcine zygotes

“…There was no difference in the epigenetic labeling between interspecies zygotes produced from in vitro matured and ovulated oocytes. The labeling of interspecies zygotes was similar to patterns typical of intraspecies zygotes H3/K9-me2 = dimethylation on lysine 9 of histone 3; H3/K4-me3 = trimethylation of lysine 4 on histone 3 + = positive labeling; -= weak/asymmetrical labeling *labeling in 7 h after injection (intermediate labeling) is different than in 22 h after injection (positive labeling) a variable antibody labeling, the pronucleus is sometimes labeled and sometimes not labeled (Barnetova and Okada, 2010;Sega et al, 2007) b Jeong et al (2007b) positively labeled (pronuclei apposition, Figure 2). Quantitative analysis of fluorescence intensities in interspecies zygotes confirmed our observation ( Table 2).…”

“…In contrast, in intraspecies zygotes the difference in activation ability between in vitro matured and ovulated oocytes is not so evident. According to our previous results observed in intraspecies zygotes produced from in vitro matured oocytes (Barnetova and Okada, 2010) and the last results observed in control zygotes produced from ovulated oocytes, the difference in activation ability of oocytes in intraspecies zygotes was not confirmed significantly as well (Table 1). It must be noted that all the ICSI zygotes formed pronuclei without any additional activation.…”

“…Here, in naturally fertilized embryos, an extensive demethylation of paternal DNA has been reported (Dean et al, 2001;. In contrast, the paternal genome was not demethylated in zygotes from in vitro matured oocytes (Jeong et al, 2007b;Barnetova and Okada, 2010). As shown additionally, the oocyte demethylation activity depends on the quality of oocyte maturation, namely on the period of germinal vesicle breakdown (Gioia et al, 2005).…”

“…As controls we have injected porcine spermatozoa into ovulated oocytes. Porcine in vitro matured oocytes were used for boar spermatozoa injection in our previous experiments (Barnetova and Okada, 2010). Data obtained in this study were thus compared with data from the previous work.…”

Epigenetic Characteristics of Paternal Chromatin in Interspecies Zygotes

“…Our results clearly show that the male porcine genome undergoes active demethylation in the mouse ovulated oocyte, although it is not demethylated in the porcine in vitro matured oocyte [14,22]. This indicates that the absence of DNA demethylation in the porcine zygote is rather influenced by the quality of the oocyte.…”