Abstract
The objective of the present study was to develop an approach that could assess the chromosomal status and the mitochondrial DNA (mtDNA) content of oocytes and their corresponding polar bodies (PBs) with the goal of obtaining a comparative picture of the segregation process both for nuclear and mtDNA. After Whole Genome Amplification (WGA), sequencing of the whole mitochondrial genome was attempted to analyze the segregation of mutant and wild-type mtDNA during human meiosis. Three triads, composed of oocyte and corresponding PBs, were analyzed and their chromosome status was successfully assessed. The complete mitochondrial genome (mitogenome) was almost entirely sequenced in the oocytes (95.99% compared to 98.43% in blood), while the percentage of sequences obtained in the corresponding PB1 and PB2 was lower (69.70% and 69.04% respectively). The comparison with the mtDNA sequence in blood revealed no changes in the D-loop region for any of the cells of each triad. In the coding region of blood mtDNA and oocyte mtDNA sequences showed full correspondence, whereas all PBs had at least one change with respect to the blood-oocyte pairs. In all, 9 changes were found, either in PB1 or PB2: 4 in MT-ND5, 2 in MT-RNR2, and 1 each in MT-ATP8, MT-ND4, MT-CYTB. The full concordance between oocyte and blood in the 3 triads, and the relegation of changes to PBs, revealed the unexpected coexistence of different variants, giving a refined estimation of mitochondrial heteroplasmy. Should these findings be confirmed by additional data, an active mechanism could be postulated in the oocyte to preserve a condition of ‘normality’.
Highlights
As mitochondria are maternally inherited, the pool carried by the mature oocyte is the final complement for the conceptus throughout his life
The total amount of mitochondria in each daughter cell becomes reduced at each cell division: a few of them will be removed from the ooplasm by segregation into polar bodies (PBs), while in the preimplantation embryo, the number of mitochondria will be approximately halved at each mitosis
The standardization of methods implied the use of enough quantity of mitochondrial DNA (mtDNA) from PBs and oocytes for Sanger sequencing
Summary
As mitochondria are maternally inherited, the pool carried by the mature oocyte is the final complement for the conceptus throughout his life. Several lines of evidence indicate that dysfunctions in mitochondrial bioenergetic activities represent a major cause of chromosomal nondisjunction during meiotic and mitotic divisions, as well as of the reduced viability of oocytes and embryos. These defects seem to be especially relevant in advanced reproductive age as the damage by oxidative stress is more pronounced with negative effects on further development [2]
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