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

Abstract

The best results of inter-species somatic cell nuclear transfer (iSCNT) in mammals were obtained using closely related species that can hybridise naturally. However, in the last years, many reports describing blastocyst development following iSCNT between species with distant taxonomical relations (inter-classes, inter-order and inter-family) have been published. This indicates that embryonic genome activation (EGA) in xeno-cytoplasm is possible, albeit very rarely. Using a bovine-pig (inter-family) iSCNT model, we studied the basic characteristics of EGA: expression and activity of RNA polymerase II (RNA Pol II), formation of nucleoli (as an indicator of RNA polymerase I (RNA Pol I) activity), expression of the key pluripotency gene NANOG and alteration of mitochondrial mass. In control embryos (obtained by IVF or iSCNT), EGA was characterised by RNA Pol II accumulation and massive production of poly-adenylated transcripts (detected with oligo dT probes) in blastomere nuclei, and formation of nucleoli as a result of RNA Pol I activity. Conversely, iSCNT embryos were characterised by the absence of accumulation and low activity of RNA Pol II and inability to form active mature nucleoli. Moreover, in iSCNT embryos, NANOG was not expressed, and mitochondria mass was significantly lower than in intra-species embryos. Finally, the complete developmental block at the 16-25-cell stage for pig-bovine iSCNT embryos and at the four-cell stage for bovine-pig iSCNT embryos strongly suggests that EGA is not taking place in iSCNT embryos. Thus, our experiments clearly demonstrate poor nucleus-cytoplasm compatibility between these animal species.

Nuclear–cytoplasmic incompatibility and inefficient development of pig–mouse cytoplasmic hybrid embryos

“…Control SPNT cow and sheep embryos as expected developed to the blastocyst stage; however, none of the cow-sheep or sheep-cow iSPNT embryos developed beyond the 8-to 16-cell stage, again the stage at which EGA occurs in both species. Taken together, our results support the argument that development of inter-species nuclear transfer embryos is highly inefficient with developmental arrest occurring at the stage of EGA specific to recipient cytoplast species (Chung et al 2009, Lagutina et al 2010. These recent reports have clearly demonstrated that the donor nucleus in iSCNT is only partially remodeled by the heterologous cytoplasm, and iSCNT embryos need to overcome nuclear-cytoplasmic compatibility issues before any further embryonic development to the blastocyst or later stages can be expected (Chung et al 2009, Lagutina et al 2010.…”

“…However, all intra-species SCNT embryos produced under the same conditions used for iSCNT developed to blastocyst stage. Our results were not different from several other recent reports (Chung et al 2009, Lagutina et al 2010, who had also reported that iSCNT embryos failed to reactivate the embryonic genome and arrested at the stage of EGA specific to the recipient oocyte. Conversely, inter-genus (Oh et al 2006, Yin et al 2006, inter-family (Dominko et al 1999, Zhao et al 2007, inter-order (Yang et al 2003, Wen et al 2005, Illmensee et al 2006, and even inter-class iSCNT (Chen et al 2002, Liu et al 2004 nuclear transfer embryos were reported to have Pig-mouse cytoplasmic hybrid embryos developed to the blastocyst stage.…”

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

“…These embryos were characterized by prevailing nucleoplasmic nucleolin localization with occasional concentration around NPBs in some bovine and porcine nuclei. This pattern of nucleolin staining suggests the inability of these embryos to activate RNA polymerase I transcription and nucleoli formation and was identical to the pattern of nucleolin labeling in bovine and porcine SCNT embryos after inhibition of RNA polymerase I activity or EGA (Lagutina et al 2010). A similar pattern of nucleolin staining was found in all studied iSCNT embryos that did not form nucleoli in our experiments and was also shown for porcine-ovine (Hamilton et al 2004) and rhesus monkey-bovine (Song et al 2009) iSCNT embryos.…”

“…A similar pattern of nucleolin staining was found in all studied iSCNT embryos that did not form nucleoli in our experiments and was also shown for porcine-ovine (Hamilton et al 2004) and rhesus monkey-bovine (Song et al 2009) iSCNT embryos. The block of EGA in bovineporcine or porcine-bovine iSCNT embryos was confirmed by the absence of RNA polymerase II accumulation and its low activity, the absence of Nanog gene expression, a significantly lower level of mitochondrial mass in comparison with intra-species embryos, and a complete block of development at the 16-to 25-cell stage and 4-cell stage of iSCNT embryos, produced with bovine and porcine oocytes respectively (Lagutina et al 2010). Our data contradict previous reports (Dominko et al 1999, Uhm et al 2007) of 4-8% BL rates, implying at least partial xeno-genome activation in a wide set of iSCNT embryos.…”

“…We also failed to reproduce the development of porcine-bovine iSCNT BLs in CR1 (Dominko et al 1999, Uhm et al 2007. SOF (synthetic oviduct fluid) in vitro embryo culture medium that is equally efficient for bovine and porcine IVF/NT preimplantation development not only failed to support the development but also was not able to initiate EGA in porcine-bovine iSCNT embryos (Lagutina et al 2010). We were not able to obtain ovine-bovine iSCNT BLs in SOF (Hua et al 2008) despite nucleoli formation as a sign of EGA.…”