Differences in Strength and Timing of the mtDNA Bottleneck between Zebrafish Germline and Non-germline Cells

Abstract

We studied the mtDNA bottleneck in zebrafish to elucidate size, timing, and variation in germline and non-germline cells. Mature zebrafish oocytes contain, on average, 19.0 × 10(6) mtDNA molecules with high variation between oocytes. During embryogenesis, the mtDNA copy number decreases to ∼170 mtDNA molecules per primordial germ cell (PGC), a number similar to that in mammals, and to ∼50 per non-PGC. These occur at the same developmental stage, implying considerable variation in mtDNA copy number in (non-)PGCs of the same female, dictated by variation in the mature oocyte. The presence of oocytes with low mtDNA numbers, if similar in humans, could explain how (de novo) mutations can reach high mutation loads within a single generation. High mtDNA copy numbers in mature oocytes are established by mtDNA replication during oocyte development. Bottleneck differences between germline and non-germline cells, due to early differentiation of PGCs, may account for different distribution patterns of familial mutations.

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

“…Most likely in humans, these pathogenic mutations are filtered out by mitophagy (Song et al 2014) or are at high levels not compatible with embryonic survival and remain unnoticed at low levels. Given the high sequence homology (72%; NCBI blast performed) between the mtDNA genome of zebrafish and humans and the high evolutionary conservation of the mtDNA bottleneck in animal species (Wolff et al 2011;Guo et al 2013;Otten and Smeets 2015;Otten et al 2016), our results indicate that the de novo risk might be similar among zebrafish and humans. Indeed, a study in 26 human oocytes (Jacobs et al 2007), seven oocytes (26.9%) were found to harbor de novo variants.…”

“…Zebrafish oocytes (Otten et al 2016) possess a much higher absolute mtDNA content compared to human oocytes (Duran et al 2011;Murakoshi et al 2013) (factor 100), mostly due to different implantation patterns. In humans, implantation occurs rapidly (Wimsatt 1975), allowing a fast shift to the uterus for energy supply, while in zebrafish implantation is absent and energy must be supplied by the embryo itself.…”

“…The inheritance of the mtDNA through a bottleneck leads to low mtDNA levels in PGCs at the bottom of the bottleneck (Cree et al 2008), and a mutation originating at this point may lead to higher heteroplasmy levels. In a previous study (Otten et al 2016), we determined the mtDNA copy number in zebrafish PGCs isolated from several embryonic stages and found, on average, 171 mtDNA molecules at the bottom of the bottleneck, but with high variation in this number (SD = 111). Based on these parameters, we constructed a Gaussian distribution with mean 171 6 111 (SD).…”

Carotenoid glycosides from cyanobacteria are teratogenic in the zebrafish (Danio rerio) embryo model

“…Mature oocytes of the zebrafish contain remarkably high densities of maternally derived mitochondria. In a very recent study (Otten et al, 2016) the number of mitochondrial DNA (mtDNA) copies, for example, was found to be on the order of 20 × 10 6 copies per cell compared to five orders of magnitude fewer, i.e., 100–1000 copies, in somatic cells. In the proposed model, therefore, teratogenicity of the xanthophylls would result from sequential cleavage: during early stages of development, mitochondrial BCO2 would cleave xanthophylls to corresponding apocarotenoids, and subsequent CCE (i.e., ACO or BCO) activity, along with possible β-oxidation, would convert the resulting apocarotenoids to teratogenic retinoids.…”

Mitochondrial matters: Mitochondrial bottlenecks, self-assembling structures, and entrapment in the female germline

“…During this period of rapid di vision, no replication of mitochondrial DNA has been detected in several vertebrates examined [28, 35, 36]; thus, despite differences in onset between species each cleavage prior to zygotic genome activation and acquisition of pluripotency would generate cells with less mitochondrial DNA per cell. This period without mitochondrial replication has been identified as a genetic bottleneck in primates based on examination of heteroplasmic embryos [28].…”