Objective: To determine the content of mitochondrial DNA (mtDNA) in oocytes from a range of patients with fertilization success and failure. Design: Analysis of mtDNA content in fertilized and unfertilized oocytes and embryos by real-time polymerase chain reaction (PCR). Setting: University hospital infertility and research center. Patient(s): Fifty-four women seeking treatment for infertility. Intervention(s): None. Main Outcome Measure(s): A total of 142 fertilized and unfertilized oocytes were classified into three main groups. Group I consisted of 35 fertilized oocytes from 21 patients; group II, 65 unfertilized oocytes from 36 patients; and group III, 42 degenerate oocytes from 23 patients. Mitochondrial DNA content was determined by SYBR Green real-time PCR-based assay. Result(s):The mean mtDNA copy number for the fertilized oocytes was 250,454, whereas for the unfertilized group it was 163,698. There were significant differences for mtDNA copy number between the male factor and female factor infertility unfertilized oocytes and between the unexplained infertility and female factor infertility groups. The mean copy number for the degenerate oocyte group was 44,629, which was significantly different from the other subdivisions in this group. Conclusion(s):Mitochondrial DNA content is critical to fertilization outcome and serves as an important marker of oocyte quality, explaining some cases of fertilization failure. (Fertil Steril 2006;85:584 -91.

Oxidative phosphorylation (OXPHOS), the intracellular process that generates the majority of the ATP of a cell through the electron-transfer chain, is highly dependent on proteins encoded by the mitochondrial genome (mtDNA). MtDNA replication is regulated by the nuclear-encoded mitochondrial transcription factor A (TFAM) and the mitochondrial-specific DNA polymerase gamma, which consists of a catalytic (POLG) and an accessory (POLG2) subunit. Differentiation of pluripotent embryonic stem cells (ESCs) into specific cell types requires expansion of discrete populations of mitochondria and mtDNA replication to meet the specific metabolic requirements of the cell. We determined by real-time PCR that expression of pluripotent markers is reduced before the upregulation of Polg, Polg2 and Tfam in spontaneously differentiating R1 murine (m)ESCs, along with transient increases in mtDNA copy number. In D3 mESCs, the initial transient increase did not take place. However, precursors of neuronal and cardiomyocyte differentiation were positive for both POLG and TFAM. Similar-stage ESCs also showed active mtDNA replication, identified by 5-bromo-2′-deoxy-uridine labelling, as mtDNA copy number increased. Retinoic-acid-induced differentiation resulted in more consistent patterns of replication and upregulation of Polg, Polg2 and Tfam, whereas siRNA knockdown demonstrated that steady-state expression of POLG is essential for maintaining pluripotency.

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 (

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Cellular ATP is mainly generated through mitochondrial oxidative phosphorylation, which is dependent on mitochondrial DNA (mtDNA). We have previously demonstrated the importance of oocyte mtDNA for porcine and human fertilization. However, the role of nuclear-encoded mitochondrial replication factors during oocyte and embryo development is not yet understood. We have analyzed two key factors, mitochondrial transcription factor A (TFAM) and polymerase gamma (POLG), to determine their role in oocyte and early embryo development. Competent and incompetent oocytes, as determined by brilliant cresyl blue (BCB) dye, were assessed intermittently during the maturation process for TFAM and POLG mRNA using real-time RT-PCR, for TFAM and POLG protein using immunocytochemistry, and for mtDNA copy number using real-time PCR. Analysis was also carried out following treatment of maturing oocytes with the mtDNA replication inhibitor, 2',3'-dideoxycytidine (ddC). Following in vitro fertilization, preimplantation embryos were also analyzed. Despite increased levels of TFAM and POLG mRNA and protein at the four-cell stage, no increase in mtDNA copy number was observed in early preimplantation development. To compensate for this, mtDNA appeared to be replicated during oocyte maturation. However, significant differences in nuclear-encoded regulatory protein expression were observed between BCB(+) and BCB(-) oocytes and between untreated oocytes and those treated with ddC. These changes resulted in delayed mtDNA replication, which correlated to reduced fertilization and embryonic development. We therefore conclude that adherence to the regulation of the timing of mtDNA replication during oocyte maturation is essential for successful embryonic development.

Prevention of the transmission of mtDNA disease from one generation to the next is still restricted by our lack of understanding as to how to ensure that a donor karyoplast transferred to an enucleated oocyte is free of accompanying mutant mtDNA. Techniques still need to be developed if stem cells are to be used to treat mtDNA disease in those patients already suffering from the phenotype.

Mitochondrial biogenesis and activation of both oxidative phosphorylation, as well as transcription and replication of the mitochondrial genome, are key regulatory events in cell differentiation. Mitochondrial DNA transcription and replication are highly dependent on the interaction with nuclear-encoded transcription factors translocated from the nucleus. Using a human embryonic stem cell line, HSF 6, we analyzed the proliferation of mitochondria and the expression of mtDNA-specific transcription factors in undifferentiated, migratory embryonic stem cells and spontaneously derived cardiomyocytes. Mitochondrial proliferation and mtDNA transcription are initiated in human embryonic stem cells as they undergo spontaneous differentiation in culture into beating cardiomyocytes. Undifferentiated, pluripotent human embryonic stem cells have few mitochondria, and, as they differentiate, they polarize to one extremity of the cell and then bipolarize the differentiating cell. The differentiated cell then adopts the cytoplasmic configuration of a somatic cell as evidenced in differentiating cardiomyocytes. Transcription and replication of the extranuclear mitochondrial genome is dependent on nuclear encoded factors exported to the mitochondrion. However, the differentiating cardiomyocytes have reduced or absent levels of these transcription and replication factors, namely mitochondrial transcription factors A, B1, B2, and nuclear respiratory factor 1 and polymerase gamma. Therefore, final embryonic stem cell commitment may be influenced by mitochondrial proliferation and mtDNA transcription. However, it is likely that differentiating cardiomyocytes are in mitochondrial arrest, awaiting commitment to a final cell fate.

An increasing number of women fail to achieve pregnancy due to either failed fertilization or embryo arrest during preimplantation development. This often results from decreased oocyte quality. Indeed, reduced mitochondrial DNA copy number (mitochondrial DNA deficiency) may disrupt oocyte quality in some women. To overcome mitochondrial DNA deficiency, whilst maintaining genetic identity, we supplemented pig oocytes selected for mitochondrial DNA deficiency, reduced cytoplasmic maturation and lower developmental competence, with autologous populations of mitochondrial isolate at fertilization. Supplementation increased development to blastocyst, the final stage of preimplantation development, and promoted mitochondrial DNA replication prior to embryonic genome activation in mitochondrial DNA deficient oocytes but not in oocytes with normal levels of mitochondrial DNA. Blastocysts exhibited transcriptome profiles more closely resembling those of blastocysts from developmentally competent oocytes. Furthermore, mitochondrial supplementation reduced gene expression patterns associated with metabolic disorders that were identified in blastocysts from mitochondrial DNA deficient oocytes. These results demonstrate the importance of the oocyte’s mitochondrial DNA investment in fertilization outcome and subsequent embryo development to mitochondrial DNA deficient oocytes.