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Originally published as Genetics Published Articles Ahead of Print on May 16, 2007.

Genetics, Vol. 176, 1511-1526, July 2007, Copyright © 2007
doi:10.1534/genetics.106.070177

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Contrasting Effects of in Vitro Fertilization and Nuclear Transfer on the Expression of mtDNA Replication Factors

Emma J. Bowles*, Joon-Hee Lee{dagger},1, Ramiro Alberio{dagger}, Rhiannon E. I. Lloyd*,2, Dov Stekel{ddagger}, Keith H. S. Campbell{dagger} and Justin C. St. John*,3

* The Mitochondrial and Reproductive Genetics Group, The Medical School, University of Birmingham, Birmingham B15 2TT, United Kingdom, {dagger} Animal Development and Biotechnology Group, University of Nottingham, School of Biosciences, Leicestershire LE12 5RD, United Kingdom and {ddagger} School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom

3 Corresponding author: Mitochondrial and Reproductive Genetics Group, Room EF20A, The Medical School, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
E-mail: j.stjohn.1{at}bham.ac.uk

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 (mtDNAR), we hypothesized that POLG and TFAM would be upregulated in intra- and interspecific (ovine–ovine) and intergeneric (caprine–ovine) NT embryos when compared to in vitro fertilized (IVF) embryos. For the intra- and interspecific crosses, PolGA (catalytic subunit), PolGB (accessory subunit), and TFAM mRNA were expressed at the 2-cell stage in both nondepleted (mtDNA+) and mtDNAR 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 mtDNAR embryos compared to their mtDNA+ counterparts. Expression for all three genes first matched IVF embryos at the blastocyst stage. In the intergeneric model, POLG was upregulated during preimplantation development. Although these embryos did not persist further than the 16+-cell stage, significantly more mtDNAR 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.




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