Abstract
The well known and most important function of nucleoli is ribosome biogenesis. However, the nucleolus showed delayed development and malfunction in somatic cell nuclear transfer (NT) embryos. Previous studies indicated that nearly half rRNA genes (rDNA) in somatic cells were inactive and not transcribed. We compared the rDNA methylation level, active nucleolar organizer region (NORs) numbers, nucleolar proteins (upstream binding factor (UBF), nucleophosmin (B23)) distribution, and nucleolar-related gene expression in three different donor cells and NT embryos. The results showed embryonic stem cells (ESCs) had the most active NORs and lowest rDNA methylation level (7.66 and 6.76%), whereas mouse embryonic fibroblasts (MEFs) were the opposite (4.70 and 22.57%). After the donor cells were injected into enucleated MII oocytes, cumulus cells and MEFs nuclei lost B23 and UBF signals in 20 min, whereas in ESC-NT embryos, B23 and UBF signals could still be detected at 60 min post-NT. The embryos derived from ESCs, cumulus cells, and MEFs showed the same trend in active NORs numbers (7.19 versus 6.68 versus 5.77, p < 0.05) and rDNA methylation levels (6.36 versus 9.67% versus 15.52%) at the 4-cell stage as that in donor cells. However, the MEF-NT embryos displayed low rRNA synthesis/processing potential at morula stage and had an obvious decrease in blastocyst developmental rate. The results presented clear evidences that the rDNA reprogramming efficiency in NT embryos was determined by the rDNA activity in donor cells from which they derived.
Highlights
The nucleolus always shows delayed development and malfunction in somatic cell nuclear transfer (NT) embryos
Because the rDNA transcription machinery is still assembled during mitosis in active nucleolar organizer regions (NORs) and absent in inactive NORs [2], the spot pair numbers observed during metaphase plate could be directly considered as active NORs numbers
The results indicated that B23 and upstream binding factor (UBF) signals co-localized within donor cell nuclear, except for B23 in the ESCs nuclear transfer (ESNT) group (Fig. 5, E, F, G, and H)
Summary
The nucleolus always shows delayed development and malfunction in somatic cell nuclear transfer (NT) embryos. We compared the rDNA methylation level, active nucleolar organizer region (NORs) numbers, nucleolar proteins (upstream binding factor (UBF), nucleophosmin (B23)) distribution, and nucleolar-related gene expression in three different donor cells and NT embryos. Because the reprogramming competence of oocyte to somatic cell nuclear is limited, we wonder whether all those inactive rDNA/NORs could be fully activated at the 4-cell stage in mouse NT embryos when compared with normal ones. If not, will it impair ribosome synthesis and intracellular metabolism of early embryonic development? The rDNA methylation level, active NORs numbers, and nucleolarrelated gene expression were compared in donor cells and in corresponding NT embryos at different preimplantation development stages. The distribution of nucleolar protein (B23 and UBF) was compared before and after NT
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