Replication Errors Made During Oogenesis Lead to Detectable De Novo mtDNA Mutations in Zebrafish Oocytes with a Low mtDNA Copy Number

Abstract

Of all pathogenic mitochondrial DNA (mtDNA) mutations in humans, 25% is de novo, although the occurrence in oocytes has never been directly assessed. We used next-generation sequencing to detect point mutations directly in the mtDNA of 3-15 individual mature oocytes and three somatic tissues from eight zebrafish females. Various statistical and biological filters allowed reliable detection of de novo variants with heteroplasmy $1.5%. In total, we detected 38 de novo base substitutions, but no insertions or deletions. These 38 de novo mutations were present in 19 of 103 mature oocytes, indicating that 20% of the mature oocytes carry at least one de novo mutation with heteroplasmy $1.5%. This frequency of de novo mutations is close to that deducted from the reported error rate of polymerase gamma, the mitochondrial replication enzyme, implying that mtDNA replication errors made during oogenesis are a likely explanation. Substantial variation in the mutation prevalence among mature oocytes can be explained by the highly variable mtDNA copy number, since we previously reported that 20% of the primordial germ cells have a mtDNA copy number of #73 and would lead to detectable mutation loads. In conclusion, replication errors made during oogenesis are an important source of de novo mtDNA base substitutions and their location and heteroplasmy level determine their significance.

Deep sequencing shows that oocytes are not prone to accumulate mtDNA heteroplasmic mutations during ovarian ageing

“…More probably, they correspond to mutations acquired during germ line quiescence or development. This result is in accordance with previous studies showing mtDNA mutations in 27% of human oocytes (Jacobs et al, 2007), and de novo variants in 20% of zebra fish oocytes (Otten et al, 2016).…”

Mitochondrial DNA Heteroplasmy and Purifying Selection in the Mammalian Female Germ Line

“…They observed a marked decrease from 2.0 × 10 7 copies per cell in the fertilized oocyte to 170 copies per cell in the PGC (Otten et al . ), suggesting a strong bottleneck effect, similar to that observed in mice. Measurements of mtDNA copy number have also been performed at later stages during oogenesis in mammalian species.…”

Mutations of mitochondrial DNA are not major contributors to aging of fruit flies

“…Interestingly, transmission of mtDNA mutations from either young or old females had no effect on the lifespan of the progeny. This somewhat surprising finding may be explained by the fact that most of the replication errors of mtDNA are made in early development of fruit flies ( 34 ), zebrafish ( 64 ), mice ( 65 ), and humans ( 66 ). In somatic tissues, the levels of mtDNA mutations fluctuate because of random genetic drift ( 58 , 66 ), and in mammals this leads to focal OXPHOS dysfunction in a subset of cells in aging tissues ( 10 ).…”