Contrasting effects of in vitro fertilisation and nuclear transfer on the expression of mtDNA replication factors

Mitochondrial DNA (mtDNA) is normally only inherited through the oocyte. However, nuclear transfer (NT), the fusion of a donor cell with an enucleated oocyte, can transmit both donor cell and recipient oocyte mtDNA. mtDNA replication is under the control of nuclear-encoded replication factors, such as Polymerase Gamma (POLG) and mitochondrial transcription factor A (TFAM). These are first expressed during late preimplantation embryo development. To account for the persistence of donor cell mtDNA, even when introduced at residual levels (mtDNA^R), we hypothesised that POLG and TFAM would be upregulated in intra- and inter-specific (ovine-ovine) and cross-species (caprine-ovine) NT embryos when compared to in vitro fertilised (IVF) embryos. For the intra- and inter-specific crosses, PolGA (catalytic subunit), PolGB (accessory subunit) and TFAM mRNA were expressed at the two-cell stage in both non-depleted (mtDNA⁺) and mtDNA^R-embryos with protein being expressed up to the 16-cell stage for POLGA and TFAM. However, at the 16-cell stage, there was significantly more PolGA expression in the mtDNA^R-embryos compared to their mtDNA⁺-counterparts. Expression for all three genes first matched IVF-embryos at the blastocyst stage. In the cross-species model, POLG was upregulated during preimplantation development. Although these embryos did not persist further than the 16+-cell stage, significantly more mtDNA^R-embryos reached this stage. However, the vast majority of these embryos were homoplasmic for recipient oocyte mtDNA. The upreglation in mtDNA replication factors was most likely due to the donor cells still expressing these factors prior to NT.

Key Words: Nuclear transfer, Polymerase Gamma, evolutionary distance, heteroplasmy, mitochondrial DNA

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