New Evidence Confirms That the Mitochondrial Bottleneck Is Generated without Reduction of Mitochondrial DNA Content in Early Primordial Germ Cells of Mice

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Abstract
In mammals, observations of rapid shifts in mitochondrial DNA (mtDNA) variants between generations have led to the creation of the bottleneck theory for the transmission of mtDNA. The bottleneck could be attributed to a marked decline of mtDNA content in germ cells giving rise to the next generation, to a small effective number of mtDNA segregation units resulting from homoplasmic nucleoids rather than the single mtDNA molecule serving as the units of segregation, or to the selective transmission of a subgroup of the mtDNA population to the progeny. We have previously determined mtDNA copy number in single germ cells and shown that the bottleneck occurs without the reduction in germline mtDNA content. Recently one study suggested that the bottleneck is driven by a remarkable decline of mtDNA copies in early primordial germ cells (PGCs), while another study reported that the mtDNA genetic bottleneck results from replication of a subpopulation of the mtDNA genome during postnatal oocyte maturation and not during embryonic oogenesis, despite a detected a reduction in mtDNA content in early PGCs. To clarify these contradictory results, we examined the mtDNA copy number in PGCs isolated from transgenic mice expressing fluorescent proteins specifically in PGCs as in the aforementioned two other studies. We provide clear evidence to confirm that no remarkable reduction in mtDNA content occurs in PGCs and reinforce that the bottleneck is generated without reduction of mtDNA content in germ cells. Mutations of mtDNA are responsible for many types of mitochondrial diseases in humans, including myopathy and neurological disorders. Females carrying a mixture of mutant and wild-type mtDNA variants transmit a variable amount of mutant mtDNA to each offspring. The proportion of mutated mtDNA inherited from the mother determines the onset and severity of diseases. Studies have suggested that the mtDNA genome is transmitted through a bottleneck, but the underlying mechanism remains controversial. By detecting mtDNA copy number in single cells, we previously showed that the bottleneck occurs without reduction of mtDNA content in germline cells. However, recently a study reported a marked decline of mtDNA copies in embryonic germ cells and attributed this reduction to the creation of the bottleneck. Yet another study concluded that the bottleneck occurs during postnatal oocyte maturation and not during embryonic oogenesis. To resolve these controversies, we examined mtDNA copies in embryonic germ cells identified using the same methodology as in the other two studies. We show solid evidence to confirm our previous findings. This confirmation is important because the understanding of mtDNA content in female germ cells will facilitate the development of therapeutic strategies preventing the transmission of mitochondrial diseases from mother to offspring.