Mitochondrial DNA is an extranuclear genome normally maternally inherited through the oocyte. However, the use of nuclear transfer can result in both donor cell and recipient oocyte mitochondrial DNA persisting through to blastocyst and being transmitted to the offspring. The degree of donor mitochondrial DNA transmission appears to be random and currently no evidence exists to explain this phenomenon. To determine whether this is a dilution factor or directly related to the transcriptional status of the donor cell in respect of mitochondrial DNA transcription factors, we have generated sheep nuclear transfer embryos using donor cells: (1) possessing their full mitochondrial DNA complement, (2) those partially depleted, and (3) those depleted but containing residual levels. For each donor type, donor mitochondrial DNA persisted in some blastocysts. It is evident from the donor cells used that nuclearencoded mitochondrial DNA transcription and replication factors persist even after mitochondrial DNA depletion, as do transcripts for some of the mitochondrial-encoded genes. These cells are therefore still programmed to drive mitochondrial DNA replication and transcription. In nuclear transfer-derived embryos, we have observed the persistence of these nuclear-encoded mitochondrial DNA transcription and replication factors but not in those embryos generated through in vitro fertilization. Consequently, nucleo-mitochondrial interaction following nuclear transfer is out of sequence as the onset of mitochondrial replication is a postimplantation event.

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 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 1 ) 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 1 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 161-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.

As human embryonic stem cells (hESCs) undergo differentiation, they express genes characteristic of the lineage for which they are destined. However, fully differentiated individual cell types can be characterized by the number of mitochondria they possess and the copies of the mitochondrial genome per mitochondrion. These characteristics are indicative of a specific cell's requirement for adenosine triphosphate (ATP) and therefore cellular viability and function. Consequently, failure for an ESC to possess the full complement of mitochondria and mitochondrial DNA (mtDNA) could limit its final commitment to a particular fate. We describe a series of protocols that analyze the process of cellular mitochondrial and mtDNA differentiation during hESC differentiation. In addition, mtDNA transcription and replication are key events in cellular differentiation that require interaction between the nucleus and the mitochondrion. To this extent, we describe a series of protocols that analyze the initiation of these key events as hESCs progress from their undifferentiated state to the fully committed cell. Last, we describe real-time polymerase chain reaction protocols that allow both the identification of mtDNA copy number and determine whether mtDNA copy is uniform (homoplasmy) in its transmission or heterogeneous (heteroplasmy).

The introduction of nuclear transfer (NT) and other technologies that involve embryo reconstruction require us to reinvestigate patterns of mitochondrial DNA (mtDNA) transmission, transcription and replication. MtDNA is a 16.6 kb genome located within each mitochondrion. The number of mitochondria and mtDNA copies per organelle is specific to each cell type. MtDNA is normally transmitted through the oocyte to the offspring. However, reconstructed oocytes often transmit both recipient oocyte mtDNA and mtDNA associated with the donor nucleus. We argue that the transmission of two populations of mtDNA may have implications for offspring survival as only one allele might be actively transcribed. This could result in the offspring phenotypically exhibiting mtDNA depletion-type syndromes. A similar occurrence could arise when nucleo -cytoplasmic interactions fail to regulate mtDNA transcription and replication, especially as the initiation of mtDNA replication post-implantation is a key developmental event. Furthermore, failure of the donor somatic nucleus to be reprogrammed could result in the early initiation of replication and the loss of cellular mtDNA specificity. We suggest investigations should be conducted to enhance our understanding of nucleo -cytoplasmic interactions in order to improve NT efficiency.

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