Nuclear transfer leads to aberrant cell cycle regulation and translation, as determined by gene expression, and selection of mitochondrial DNA in porcine blastocysts

Abstract

Nuclear transfer technologies, such as metaphase II spindle transfer (MII-ST), are being developed to overcome poor oocyte quality and prevent children from acquiring severe mitochondrial DNA (mtDNA) disease. However, it is unclear whether these technologies perturb gene expression; and mtDNA carryover can be reduced to minimal levels. Using our pig model, we isolated individual karyoplasts with limited mtDNA carryover (306 ± 49 copies) from mature oocytes. Firstly, we reintroduced the spindle into its original cytoplast (autologous), fertilised, and cultured the resultant embryos to the hatching blastocyst stage of development. Following RNA-seq, 64 genes were differentially expressed at FDR < 0.05; and 1327 genes at P < 0.05. Pathways associated with cell cycle and epigenetic regulation and genes associated with nuclear-mitochondrial interactions were upregulated whilst protein translation pathways were downregulated. We then transferred karyoplasts to third party cytoplasts (heterologous) and undertook mtDNA next generation sequencing of 2-cell to hatching blastocyst stage preimplantation embryos. Nine of 20 embryos possessed two mitochondrial genomes. Our outcomes show that autologous nuclear transfer alone significantly changes hatching blastocyst gene expression; very low levels of mtDNA carryover can be preferentially replicated during early development; and nuclear and mitochondrial synchrony has not been reestablished.