Nuclear-Cytoplasmic Incompatibility in Inter-Species Cytoplasm Hybrid Embryos: Implications for Inter-Species Nuclear Transfer Hybrid Embryo.

Abstract

Interspecies somatic cell nuclear transfer (iSCNT) embryos reconstructed between other than closely related species rarely develop to the blastocyst stage and beyond. One explanation for this lack of development is an inability to reprogram the somatic genome. To explore this possibility we evaluated whether using a karyoplast that would require less extensive reprogramming such as an embryonic blastomere or the meiotic spindle from metaphase II oocytes would provide additional insight into the development of iSCNT embryos. Our results showed that karyoplasts of embryonic or oocyte origin are no different from somatic cells; all interspecies nuclear transfer embryos independent of karyoplast origin failed to develop to the blastocyst stage. We speculated that apart from faulty epigenetic reprogramming, nuclear-cytoplasmic incompatibility could be another reason for failure of embryonic development from interspecies nuclear transfer. To address nuclear-cytoplasmic incompatibility we used pig-mouse interspecies cytoplasmic hybrids as a model. Fertilized murine zygotes were reconstructed by fusing with porcine cytoplasts of varying cytoplasmic volumes (1/5 (small) and 1/3 (large) total volume of mouse zygote) produced from activated oocytes. The presence of pig cytoplasm significantly reduced in vitro development of mouse zygotes to the blastocyst stage as compared to control embryos at 120 h Post hCG (41% vs 6% vs 94%, P<0.05), (1/5, 1/3, control respectively). While copy numbers of the mitochondrial genome (mtDNA) remained relatively unchanged in cytoplasmic hybrids and control mouse embryos, genes responsible for mitochondrial replication (Tfam, PlogG, and PlogG2) mitochondrial fusion (mfn2) glucose transport (Glut 3 and Glut1) anti apopotosis (Bcl2 and hspb1) and pluripotency and lineage segregation (Oct4, Nanog, cdx2, Gata3, Tcfap2c) were significantly down regulated in cytoplasmic hybrid embryos. Methylation at promoter regions of Oct4 in cytoplasmic hybrids was slightly increased, however, methylation at LINE1 was not different from control embryos. We speculate that abnormal gene expression in cytoplasmic hybrids may be a consequence of the presence of foreign cytoplasmic components. RNA binding proteins, microRNAS and maternal transcripts of pig cytoplasmic origin may have adversely interfered with precise regulation of maternal murine transcripts and post-transcriptional and/or translation regulation of nascent mRNA synthesized from the mouse embryonic genome. These results demonstrate that the presence of even a small amount of porcine cytoplasm is detrimental to murine embryo development and suggest that a range of factors are likely to contribute to the failure of interspecies nuclear transfer embryos. (poster)