Bovine Ooplasm Partially Remodels Primate Somatic Nuclei following Somatic Cell Nuclear Transfer

Abstract

Interspecies somatic cell nuclear transfer (iSCNT) has the potential to become a useful tool to address basic questions about the nucleus-cytoplasm interactions between species. It has also been proposed as an alternative for the preservation of endangered species and to derive autologous embryonic stem cells. Using chimpanzee/ bovine iSCNT as our experimental model we studied the early epigenetic events that take place soon after cell fusion until embryonic genome activation (EGA). Our analysis suggested partial EGA in iSCNT embryos at the eight-cell stage, as indicated by Br-UTP incorporation and expression of chimpanzee embryonic genes. Oct4, Stella, Crabp1, CCNE2, CXCL6, PTGER4, H2AFZ, c-MYC, KLF4, and GAPDH transcripts were expressed, while Nanog, Glut1, DSC2, USF2, Adrbk1, and Lin28 failed to be activated. Although development of iSCNT embryos did not progress beyond the 8- to 16-cell stage, chromatin remodeling events, monitored by H3K27 methylation, H4K5 acetylation, and global DNA methylation, were similar in both intra- and interspecies SCNT embryos. However, bisulfite sequencing indicated incomplete demethylation of Oct4 and Nanog promoters in eight-cell iSCNT embryos. ATP production levels were significantly higher in bovine SCNT embryos than in iSCNT embryos, TUNEL assays did not reveal any difference in the apoptotic status of the nuclei from both types of embryos. Collectively, our results suggest that bovine ooplasm can partially remodel chimpanzee somatic nuclei, and provides insight into some of the current barriers iSCNT must overcome if further embryonic development is to be expected.

Development, embryonic genome activity and mitochondrial characteristics of bovine–pig inter-family nuclear transfer embryos

“…Porcine NANOG mRNA was not Bovine-pig iSCNT embryos at the time of EGA detected even in the best (R16-cell) pig-bovine iSCNT embryos at the time of porcine and bovine EGA as previously reported for chimpanzee-bovine iSCNT embryos (Wang et al 2009). Comparison of gene expression in 8-16-cell stage human-bovine or human-rabbit embryos and human IVF or NT embryos using single-embryo transcriptome profiling revealed general downregulation of human genes in the bovine and rabbit recipient cytoplasm (Chung et al 2009).…”

“…This permanent activity of RNA Pol II is enough to sustain GFP expression in MEF-bovine (Arat et al 2003) and panda-rabbit iSCNT embryos (Wen et al 2005), avian feather keratin expression in chicken-rabbit intra-classes iSCNT embryos , housekeeping gene expression in mouse-bovine iSCNT embryos (Arat et al 2003, Kim et al 2004. However, the abnormal pattern of housekeeping genes expression (Arat et al 2003, Kim et al 2004, Wang et al 2009 confirmed the inadequate embryonic genome activity in all tested iSCNT embryos.…”

“…Indeed, very few data are available on embryonic gene function before and at the time of EGA in iSCNT embryos (Arat et al 2003, Bjerregaard et al 2007, Chung et al 2009, Hamilton et al 2004, Kim et al 2004, Li et al 2008, Wang et al 2009, and all show abnormal genome activation and gene expression profiles in the iSCNT embryos studied. It is likely that the ineffectiveness of the iSCNT approach may be linked to abnormal (or absence of) EGA in such embryos due to aberrant nucleuscytoplasm cross-talk.…”

Formation of nucleoli in interspecies nuclear transfer embryos derived from bovine, porcine, and rabbit oocytes and nuclear donor cells of various species

“…The ability of oocyte to demethylate gene promoters has donor nucleus species-specific features (Wang et al 2009). Although the trend of global demethylation seems to be similar in bovine SCNT and chimpanzee-bovine iSCNT embryos, bovine ooplasm could not recapitulate in chimpanzee nuclei the DNA demethylation events observed in the bovine SCNT embryos.…”

Heterochromatin reprogramming in rabbit embryos after fertilization, intra-, and inter-species SCNT correlates with preimplantation development

“…After being introduced into recipient oocytes, differentiated somatic nuclei must be completely remodeled to initiate another round of embryonic development. Although the abnormal chromatin remodeling has been implicated as primary reasons for the low efficiency of the SCNT procedure (Arat et al 2003, Park et al 2004, Wang et al 2009), the associated mechanisms have not been completely elucidated. In order to obtain a deeper insight into these events, we focused on centromeric and pericentric heterochromatin, known to form higher-order chromatin structures within nuclei in the interphase.…”