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Nucleologenesis and embryonic genome activation are defective in interspecies cloned embryos between bovine ooplasm and rhesus monkey somatic cells.

Song BS, Lee SH, Kim SU, Kim JS, Park JS, Kim CH, Chang KT, Han YM, Lee KK, Lee DS, Koo DB - BMC Dev. Biol. (2009)

Bottom Line: 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.These results provide insight into the unresolved problems of early embryonic development in iSCNT embryos.

View Article: PubMed Central - HTML - PubMed

Affiliation: Development and Differentiation Research Center, Korea Research Institute of Bioscience and Biotechnology, Gwahangno, Yuseong-gu, Daejeon, Republic of Korea. sbs6401@hanmail.net

ABSTRACT

Background: 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. 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.

Results: No 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.

Conclusion: The 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.

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Analysis of mitochondrial DNA (mtDNA) in monkey-bovine MB-iSCNT embryos on day 3. Rhesus monkey ear skin fibroblasts (A), image of MB-iSCNT embryos at day 3 (B), and expression of mtDNA in MB-iSCNT embryos. Bar, 50 μm. B, bovine; M, rhesus monkey.
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Figure 1: Analysis of mitochondrial DNA (mtDNA) in monkey-bovine MB-iSCNT embryos on day 3. Rhesus monkey ear skin fibroblasts (A), image of MB-iSCNT embryos at day 3 (B), and expression of mtDNA in MB-iSCNT embryos. Bar, 50 μm. B, bovine; M, rhesus monkey.

Mentions: Rhesus monkey ear fibroblast cells (Figure 1A) were fused with enucleated bovine oocytes (MB-iSCNT) using the same fusion parameters used for bovine fibroblasts (BB-SCNT). IVF bovine embryos were also cultured to control for the effect of the nuclear transfer (NT) procedure. Eight-cell stage IVF, BB-SCNT, and MB-iSCNT embryos were observed (Figure 1B) after 3 days of culture. PCR-based mitochondrial DNA (mtDNA) analysis was used to confirm that 8-cell-stage MB-iSCNT embryos had fused. PCR amplification of D-loop mtDNA was performed using species-specific primers. Monkey and bovine mtDNA were detected in 8-cell iSCNTs (Figure 1C). No difference in cleavage rate was observed among MB-iSCNT (89.3 ± 2.7%, 99/110), IVF (86.3 ± 1.3, 101/117), or BB-SCNT embryos (85.3 ± 2.5%, 83/99; Table 1). The developmental rates of IVF and BB-SCNT embryos were 33.5 ± 2.8% and 28.2 ± 2.7%, respectively. However, the MB-iSCNT embryos did not develop into blastocysts (Table 1).


Nucleologenesis and embryonic genome activation are defective in interspecies cloned embryos between bovine ooplasm and rhesus monkey somatic cells.

Song BS, Lee SH, Kim SU, Kim JS, Park JS, Kim CH, Chang KT, Han YM, Lee KK, Lee DS, Koo DB - BMC Dev. Biol. (2009)

Analysis of mitochondrial DNA (mtDNA) in monkey-bovine MB-iSCNT embryos on day 3. Rhesus monkey ear skin fibroblasts (A), image of MB-iSCNT embryos at day 3 (B), and expression of mtDNA in MB-iSCNT embryos. Bar, 50 μm. B, bovine; M, rhesus monkey.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2734572&req=5

Figure 1: Analysis of mitochondrial DNA (mtDNA) in monkey-bovine MB-iSCNT embryos on day 3. Rhesus monkey ear skin fibroblasts (A), image of MB-iSCNT embryos at day 3 (B), and expression of mtDNA in MB-iSCNT embryos. Bar, 50 μm. B, bovine; M, rhesus monkey.
Mentions: Rhesus monkey ear fibroblast cells (Figure 1A) were fused with enucleated bovine oocytes (MB-iSCNT) using the same fusion parameters used for bovine fibroblasts (BB-SCNT). IVF bovine embryos were also cultured to control for the effect of the nuclear transfer (NT) procedure. Eight-cell stage IVF, BB-SCNT, and MB-iSCNT embryos were observed (Figure 1B) after 3 days of culture. PCR-based mitochondrial DNA (mtDNA) analysis was used to confirm that 8-cell-stage MB-iSCNT embryos had fused. PCR amplification of D-loop mtDNA was performed using species-specific primers. Monkey and bovine mtDNA were detected in 8-cell iSCNTs (Figure 1C). No difference in cleavage rate was observed among MB-iSCNT (89.3 ± 2.7%, 99/110), IVF (86.3 ± 1.3, 101/117), or BB-SCNT embryos (85.3 ± 2.5%, 83/99; Table 1). The developmental rates of IVF and BB-SCNT embryos were 33.5 ± 2.8% and 28.2 ± 2.7%, respectively. However, the MB-iSCNT embryos did not develop into blastocysts (Table 1).

Bottom Line: 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.These results provide insight into the unresolved problems of early embryonic development in iSCNT embryos.

View Article: PubMed Central - HTML - PubMed

Affiliation: Development and Differentiation Research Center, Korea Research Institute of Bioscience and Biotechnology, Gwahangno, Yuseong-gu, Daejeon, Republic of Korea. sbs6401@hanmail.net

ABSTRACT

Background: 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. 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.

Results: No 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.

Conclusion: The 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.

Show MeSH