The mitochondrion is explicitly involved in cytoplasmic regulation and is the cell's major generator of ATP. Our aim was to determine whether mitochondria alone could influence fertilisation outcome. In vitro, oocyte competence can be assessed through the presence of glucose-6-phosphate dehydrogenase (G6PD) as indicated by the dye, brilliant cresyl blue (BCB). Using porcine in vitro fertilisation (IVF), we have assessed oocyte maturation, cytoplasmic volume, fertilisation outcome, mitochondrial number as determined by mtDNA copy number, and whether mitochondria are uniformly distributed between blastomeres of each embryo. After staining with BCB, we observed a significant difference in cytoplasmic volume between BCB positive (BCB 1 ) and BCB negative (BCB 2 ) oocytes. There was also a significant difference in mtDNA copy number between fertilised and unfertilised oocytes and unequal mitochondrial segregation between blastomeres during early cleavage stages. Furthermore, we have supplemented BCB 2 oocytes with mitochondria from maternal relatives and observed a significant difference in fertilisation outcomes following both IVF and intracytoplasmic sperm injection (ICSI) between supplemented, sham-injected and non-treated BCB 2 oocytes. We have therefore demonstrated a relationship between oocyte maturity, cytoplasmic volume, and fertilisation outcome and mitochondrial content. These data suggest that mitochondrial number is important for fertilisation outcome and embryonic development. Furthermore, a mitochondrial pre-fertilisation threshold may ensure that, as mitochondria are diluted out during post-fertilisation cleavage, there are sufficient copies of mtDNA per blastomere to allow transmission of mtDNA to each cell of the post-implantation embryo after the initiation of mtDNA replication during the early postimplantation stages.Reproduction (

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.