Dynamics of epigenetic remodeling in interspecies porcine zygotes

Abstract

The process of active paternal chromatin demethylation after fertilization in the pig is not fully understood and very inconsistent data have been published by different research groups. We have applied the interspecies intracytoplasmic sperm injection (iICSI) to evaluate remodeling capabilities of porcine oocytes in more details. We injected mouse frozen-thawed sperm heads into porcine in vitro matured or ovulated oocytes, respectively. Embryos produced by intracytoplasmic sperm injection (ICSI) of boar spermatozoa into porcine ovulated oocytes (intraspecies) served as controls. Zygotes with 2-pronuclei were labeled with antibodies against certain epigenetic modifications (5-methylcytosine, 5-MeC; heterochromatin protein 1, HP1; trimethylation of H3/K4, H3/K4-me3; and dimethylation of H3/K9, H3/K9-me2). The labeling patterns were not different between zygotes produced from in vitro matured and ovulated oocytes. Both pronuclei were symmetrically labeled with 5-MeC, HP1, and H3/K4-me3 antibodies. Asymmetrical labeling was observed only with H3/K9-me2 antibody. The labeling of interspecies zygotes was similar to that of intraspecies zygotes. Moreover, the DNA demethylation was observed neither in control zygotes (intraspecies). The only difference observed between zygotes produced from in vitro matured and ovulated oocytes was in their ability to be activated. Intraspecies zygotes produced from ovulated oocytes were able to form the paternal pronucleus without additional activation; the zygotes produced from in vitro matured oocytes formed the paternal pronucleus only after additional activation with electric pulses. Our results show that the remodeling abilities of in vitro matured and ovulated oocytes are essentially similar. Moreover, it seems that reasons of inconsistent data reporting the active demethylation in the pig are more complicated and they are not associated exclusively with the oocyte quality.