Academic journal article Genetics

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

Academic journal article Genetics

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

Article excerpt

ABSTRACT

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^sup R^), we hypothesized that POLG and TFAM would be upregulated in intra- and interspecific (ovine-ovine) and intergeneric (caprine-ovine) NTembryos 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^sup +^) and mtDNA^sup 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^sup R^ embryos compared to their mtDNA^sup +^ 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 mtDNA^sup 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.

NUCLEAR transfer (NT) involves the transfer of a donor cell, or a donor cell nucleus, into an enucleated oocyte. Subsequent fusion and activation of the reconstructed oocyte results in an embryo that can give rise to offspring and embryonic stem cells possessing nuclear genetic material identical to that of the donor cell (Campbell et al. 1996;Wakayama et al. 2001). Poor developmental success rates and the abnormalities observed in NT embryos, fetuses, and offspring have been associated with a number of factors. These include epigenetic abnormalities and irregular patterns of gene expression (Ohgane et al. 2001; Humpherys et al. 2002; Li et al. 2005, 2006). Failure of the recipient oocyte to completely reprogram the donor nucleus also results in incomplete reactivation of genes associated with pluripotency, such as Oct4 (Bortvin et al. 2003).

The donor cell also contributes mitochondria to the reconstructed oocyte. Mitochondria, which reside in the cytoplasm of all eukaryotic cells, contain one or more copies of the double-stranded circular mitochondrial genome (mtDNA). mtDNA encodes 13 of the >70 subunits of the electron transfer chain (ETC), the intracellular apparatus that generates the vast majority of a cell's ATP through the process of oxidative phosphorylation (OXPHOS). The remainder of the subunits are encoded by chromosomal DNA (Anderson et al. 1981). During normal sexual reproduction, mtDNA is almost exclusively transmitted from the mtDNA present in the unfertilized oocyte and is therefore maternally inherited (Giles et al. 1980; Birky 1995). These mtDNA genomes originate from a few mitochondria segregated to each primordial germ cell shortly after the onset of gastrulation (Jansen and de Boer 1998). The mtDNA in these precursor germ cells are then clonally amplified during the later stages of oogenesis (Hauswirth and Laipis 1982; Poulton et al. 1998). In the vast majority of cases, this process leads to the transmission of only a single type of mtDNA, a state known as homoplasmy. However, due to the donor cell contributing mtDNA to the reconstructed oocyte, mtDNA can be inherited either from the recipient oocyte only or from both the donor cell and recipient oocyte (heteroplasmy). Such transmission has been observed in both NTembryos (Steinborn et al. …

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