57 GENOME ACTIVATION IN MOUSE EMBRYOS OF DIFFERENT ORIGIN

Abstract

Major genome activation is a key event in early embryonic development occurring at the late 2-cell stage in the mouse. Concomitantly occurring molecular and ultrastructural changes in the nucleolus, where the ribosomal RNA genes are transcribed and their transcripts processed, enable the use of this organelle as a sensitive marker of genome activation in embryos produced by different techniques. The aim of this study was to evaluate and compare the genome activation in mouse embryos of different origin using the nucleolus as a marker. Early and late 2-cell- and late 4-cell-stage embryos, prepared by in vitro fertilization (IVF), parthenogenetic activation (PG), and somatic cell nuclear transfer of mouse embryonic fibroblast (MEF), and mouse HM1 embryonic stem cells (HM1) were processed for autoradiography following 3H-uridine incubation and transmission electron microscopy (5 embryos per group) and for immunofluorescence for detection of nucleolar proteins involved in rRNA synthesis (upstream binding factor; UBF) and processing (nucleophosmin; B23) (10–21 embryos per group). Early 2-cell embryos in all groups showed transcriptional activity in the nucleoplasm, but not over nucleolar precursor bodies (NPBs). UBF was localized diffusely in the cytoplasm. B23 was, likewise, localized in the cytoplasm and, in 30% of embryos, in the nucleoplasm. Late 2-cell IVF and PG embryos displayed transcriptional labelling over nucleoplasm and NPBs, which, ultrastructurally, were in the process of transformation into fibrillo-granular nucleoli presenting fibrillar centers, a dense fibrillar component, and a granular component. MEF and HM1 embryos displayed transcriptional labelling over nucleoplasm, but not over NPBs, and the transformation into functional nucleoli was never observed at this stage of development. UBF and B23 were in all groups localized in the nucleoplasm and, in 40–50% of cases, distinctly in the developing nucleoli. At the late 4-cell stage, all embryos presented transcriptional labelling over nucleoplasm and NPBs, which were at different levels of transformation into fibrillo-granular nucleoli. UBF and B23 were distinctly localized in these developing nucleoli. However, whereas fully transformed reticulated fibrillo-granular nucleoli without remnants of NPBs were found in IVF and PG embryos, despite the distinct localization of nucleolar proteins, the nucleoli in MEF and HM1 embryos were not reticulated and still displayed remnants of NPBs. Conclusively, embryos reconstructed by nuclear transfer, independent of cell origin, displayed well-timed extranucleolar genomic activation, but delayed transformation of NPBs into reticulated fibrillo-granular nucleoli. Moreover, the proper nucleolar activation noted in PG embryos activated in the same manner as MEF and HM1 embryos demonstrate that somatic and embryonic stem cell factors exert an influence on nucleolar activation and may cause reduced embryo viability.

This work was supported by the Specific Targeted Project (MED-RAT; contract LSHG-CT-2006-518240) and Marie Curie ResearchTraining Networks (CLONET; contract 035468-2).