49 EARLY ASPECTS OF NUCLEAR REPROGRAMMING FOLLOWING BOVINE SOMATIC CELL NUCLEAR TRANSFER

Abstract

This study was designed in order to evaluate the global transcription and morphology of reprogramming during somatic cell nuclear transfer (SCNT). Following 20–22 h of IVM, couplets of MII cytoplasts and starved bovine fibroblasts were, after 2 h co-culture, electrically fused, chemically activated by 5 µm ionomycin for 5 min, followed by a 3–4 h incubation in 2 mm 6-DMAP, and fixed at 0.5, 1, 1.5, 2, 3, 4, 8, 12, and 16 h post-activation (hpa). The reconstructed embryos were processed for lacmoid staining, autoradiography following [3H]uridine incubation, transmission electron microscopy (TEM), and immunofluorescence (4 and 12 hpa) in order to evaluate chromatin dynamics, transcriptional activity, nuclear and nucleolar ultrastructure, and localization of nucleolar proteins (upstream binding factor (UBF) and fibrillarin), respectively, during the first cell cycle. Likewise, starved fibroblasts were fixed and processed for autoradiography and TEM. The fibroblasts displayed strong transcriptional activity and active fibrillo-granular nucleoli. None of the reconstructed embryos, however, displayed transcriptional activity. During first 3 hpa, the majority of the embryos displayed a single block of condensed chromatin surrounded by a more or less complete nuclear envelope (NE) and an abundant population of elongated somatic cell mitochondria. This somatic cell complex was located peripherally in the ooplasm. At the subsequent time points, the embryos displayed pronuclear-like structures, which from 8 hpa were located centrally in the ooplasm. From about 4 hpa, the somatic cell complex had dispersed and the elongated mitochondria could no longer be tracked. The first nucleolus-related structures were observed at 1.5 hpa and only in nuclei with a complete NE. At 1.5 to 4 hpa, the nucleolus-related structures appeared either as bodies presenting a large fibrillar center and presumptive dense fibrillar component, but no granular component, or as electron-dense nucleolus precursor bodies (NPBs). From 4 hpa and onward, only compact NPBs were observed. At 4 and 12 hpa, UBF was localized into small discrete clusters of foci enclosed in a shell-like structure labeled by fibrillarin in the nucleus. In conclusion, at SCNT, the somatic cell cytoplasm remains structurally organized in a somatic cell complex over the initial 3–4 hpa. During the same period, the somatic cell chromatin undergoes condensation and the NE is partially dissolved. Subsequently, pronucleus-like euchromatic nuclei with typical NPBs are formed, although somatic cell nucleolar components may be temporarily seen. Throughout the process, transcription is repressed.

This work was supported by Marie Curie Intra-European Fellowships (MEIF-CT-2006-021629), and by grant VEGA 1/3255/06 and DFG.