Mitophagy and the dynamics of mitochondrial DNA inheritance in early development and reproduction

Abstract

Inherited heteroplasmic mitochondrial DNA (mtDNA) mutations are a cause of severe disease of adults and children. Thus, methods have been developed to reduce inheritance of pathogenic mtDNA variants; preimplantation genetic diagnosis (PGD) and pronuclear transfer (PNT). However, it is unclear at which stage of development a biopsy better predicts embryo heteroplasmy in PGD. Meanwhile, a small amount of pathogenic variant-harbouring mtDNA is carried over during PNT. Recent work indicates that embryos undergo mitophagy during late preimplantion development. This coincides with a reported increase in intercellular variation of mtDNA heteroplasmy, raising the question of whether mitophagy contributes to segregation of mtDNA. In addition, manipulation of mitophagy may be applied to reduce carry-over of mtDNA variants during PNT. To investigate the mechanisms of mitophagy, using a large single cell RNA sequencing data from human embryos, I analysed the expression of key mitophagy genes. BNIP3 family genes were identified as probable mediators of mitophagy. Overexpression of a phosphomimetic BNIP3L protein upregulated mitophagy, which may be useful in preventing survival of carried over mtDNA in PNT. Furthermore, correlations emerged between mitophagy genes and genes driving the establishment of cell lineages in the blastocyst, suggesting lineage-specific regulation of mitophagy. Using the tRNAalanine (tRNAala) mouse model carrying a pathogenic mtDNA variant, I verified an increase in intercellular variation of heteroplasmy in the blastocyst. Analysis of the segregation of mtDNA suggested that the 8-cell embryo is the optimal stage for PGD biopsy. Upregulation of mitophagy had no detectable influence on the segregation of mtDNA, suggesting that mitophagy may not be a major source of intercellular variation in heteroplasmy. I also revealed an accumulation of mtDNA variants in the oocytes of aged tRNAala mice. Combined with the presence of a pathogenic mutation, mtDNA variants did not manifest a selection mechanism. This suggests acquired mtDNA variants only modestly impair the health of aged oocytes and age-related loss of fertility is primarily due to other causes. These findings elucidate mechanisms which may modulate the inheritance of mtDNA in early development. Furthermore, they provide insight into the impact of mitophagy in the preimplantation embryo, and identify an 8-cell embryo biopsy as preferable to that of a blastocyst in predicting embryo heteroplasmy during PGD. Identification of a method to upregulate mitophagy will help guide the process of preventing the inheritance of mtDNA mutations using PNT