44 LIMITATIONS OF PORCINE OOPLASM TO REPROGRAM BOVINE SOMATIC CELLS

Abstract

The oocyte cytoplasm (ooplasm) constitutes a unique mixture of factors that are critical for successful reprogramming of haploid maternal and paternal genomes at fertilization as well as the diploid somatic cell genome after somatic cell nuclear transfer (SCNT). The 1-cell-stage embryos (produced by transfer of bovine fibroblast into porcine enucleated ooplast; interspecies SCNT, iSCNT) were processed at different time points (2, 4, 8, and 12 h post-activation, hpa) for detailed nuclear and nucleolar analysis by transmission electron microscopy (TEM; 4 to 5 embryos per group), and immunofluorescence (4 and 12 hpa) for visualization of nucleolar proteins related to transcription (upstream binding factor, UBF) and processing (fibrillarin; 8 to 9 embryos per group). The parameters of interspecies embryos were compared with porcine parthenogenetic (PA) counterparts (4 to 5 embryos per group for TEM analysis; 8 to 9 embryos per group for immunofluorescence analysis). At all evaluated time points, embryos in both groups displayed pronucleus-like nuclei with abundant euchromatin and characteristic porcine nucleolus precursor body (NPB), indicating maternal origin of nucleolar components. Fibrillarin was in both groups localized into shell-like intranuclear entities surrounding NPB. On the contrary, UBF in PA embryos was at 12 hpa colocalized with fibrillarin, whereas in iSCNT embryos, UBF staining was absent at both time points. Despite the similar morphology and localization of processing factor fibrillarin, striking differences in localization of transcription factor UBF suggest limitation of porcine ooplasm to mediate initial phases of nucleolar remodelling.

This work was supported by VEGA 1/0077/11.