Abstract
BackgroundInterspecies somatic cell nuclear transfer (iSCNT) has been proposed as a tool to address basic developmental questions and to improve the feasibility of cell therapy. However, the low efficiency of iSCNT embryonic development is a crucial problem when compared to in vitro fertilization (IVF) and intraspecies SCNT. Thus, we examined the effect of donor cell species on the early development of SCNT embryos after reconstruction with bovine ooplasm.ResultsNo apparent difference in cleavage rate was found among IVF, monkey-bovine (MB)-iSCNT, and bovine-bovine (BB)-SCNT embryos. However, MB-iSCNT embryos failed to develop beyond the 8- or 16-cell stages and lacked expression of the genes involved in embryonic genome activation (EGA) at the 8-cell stage. From ultrastructural observations made during the peri-EGA period using transmission electron microscopy (TEM), we found that the nucleoli of MB-iSCNT embryos were morphologically abnormal or arrested at the primary stage of nucleologenesis. Consistent with the TEM analysis, nucleolar component proteins, such as upstream binding transcription factor, fibrillarin, nucleolin, and nucleophosmin, showed decreased expression and were structurally disorganized in MB-iSCNT embryos compared to IVF and BB-SCNT embryos, as revealed by real-time PCR and immunofluorescence confocal laser scanning microscopy, respectively.ConclusionThe down-regulation of housekeeping and imprinting genes, abnormal nucleolar morphology, and aberrant patterns of nucleolar proteins during EGA resulted in developmental failure in MB-iSCNT embryos. These results provide insight into the unresolved problems of early embryonic development in iSCNT embryos.
Highlights
Interspecies somatic cell nuclear transfer has been proposed as a tool to address basic developmental questions and to improve the feasibility of cell therapy
Monkey and bovine mitochondrial DNA (mtDNA) were detected in 8-cell Interspecies somatic cell nuclear transfer (iSCNT) (Figure 1C)
MB-iSCNT embryos derived from donor monkey fibroblasts and bovine recipient oocytes did not develop to the blastocyst stage
Summary
Interspecies somatic cell nuclear transfer (iSCNT) has been proposed as a tool to address basic developmental questions and to improve the feasibility of cell therapy. We examined the effect of donor cell species on the early development of SCNT embryos after reconstruction with bovine ooplasm. The derivation of human embryonic stem cells (hESCs) from somatic cell nuclear transfer (SCNT) blastocysts represents an innovative strategy for overcoming immune rejection during transplantation. Interspecies SCNT (iSCNT) shows promise as a technique for examining nucleocytoplasmic interactions [1], stem cells [2], and the cloning of endangered animals whose oocytes are difficult to obtain [3,4]. The most important application of iSCNT lies in its potential to facilitate the reprogramming of human somatic cells into embryonic stem cells, avoiding ethical issues associated with using human oocytes. ISCNT may increase the feasibility of human therapeutic cloning by providing comprehensive information about a variety of developmental events
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