| Oocytes contain a large stock of mitochondria. These are essential because, following fertilization, the embryo does not resume mitochondrial DNA (mtDNA) replication until near the time of implantation; therefore, a large stock is essential to ensure that each blastomere inherits a sufficient number of mitochondria. However, the timing and control of mtDNA accumulation in oocytes are poorly understood. We have developed a PCR-based assay that enables the mtDNA content of individual mouse oocytes to be measured. We report that the quantity of mtDNA increases progressively during oocyte growth, reaching ∼175,000 copies per cell. When oocytes reach full-size, however, mtDNA ceases to accumulate suggesting that accumulation of mtDNA is closely correlated with oocyte growth. To investigate the mechanism of accumulation, we analyzed mtDNA content in mouse oocytes grown in vitro. As in vivo, we found that mtDNA content increased during oocyte growth in vitro. Unexpectedly, although oocytes did not grow to the same size in vitro as in vivo, the mtDNA accumulated to the same extent under both conditions. This suggests that mtDNA accumulation is mechanistically uncoupled from oocyte growth. To test this, we incubated growing oocytes in the presence of LY294002, an inhibitor of phosphoinosotide-3 kinase, or in the absence of the surrounding granulosa cells. As expected, oocyte growth was inhibited under both conditions. In contrast, mtDNA accumulated in the oocytes despite the growth inhibition. These results indicate that the accumulation of mtDNA is independent of increase in size during oocyte growth. We then examined the expression of nuclear-encoded genes required for mtDNA replication. The amount of Tfam, Polga, and Polgb increased co-coordinately with oocyte growth. However, their quantity subsequently declined in fully grown oocytes. We propose that mtDNA ceases to accumulate in fully grown oocytes owing to the loss of the mRNAs encoding these essential components of the replication machinery. Thus, these mRNAs may be limiting factors that determine the rate and extent of mtDNA accumulation during oogenesis. We also evaluated the ATP content during oogenesis as a function of mtDNA activity. Growing oocytes showed a higher ATP content than fully grown oocytes and as oocytes developed toward meiotic maturation ATP level significantly decreased. These results suggest that growing oocytes may have increased energy needs than the subsequent developmental stages. |
