Dynamics of epigenetic remodeling in interspecies porcine zygotes

Abstract

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.

The role of nitric oxide synthase isoforms in aged porcine oocytes

“…The nNOS localization in the cell nucleus is also known in somatic cells, and it is associated with transcription regulation (Yuan et al, 2004). For the genome reactivation of porcine embryos occurring in later embryonic development, nNOS localization may be either a form of parthenogenetic zygote preparation for embryonic genome reactivation or a reflection of early transcription of the same genes prior to complete genome reactivation (Latham and Schultz, 2001;Barnetová et al, 2012). The signal intensity decrease of NOS isoforms in aged porcine oocytes was observed.…”