Abstract
An increasing number of women fail to achieve pregnancy due to either failed fertilization or embryo arrest during preimplantation development. This often results from decreased oocyte quality. Indeed, reduced mitochondrial DNA copy number (mitochondrial DNA deficiency) may disrupt oocyte quality in some women. To overcome mitochondrial DNA deficiency, whilst maintaining genetic identity, we supplemented pig oocytes selected for mitochondrial DNA deficiency, reduced cytoplasmic maturation and lower developmental competence, with autologous populations of mitochondrial isolate at fertilization. Supplementation increased development to blastocyst, the final stage of preimplantation development, and promoted mitochondrial DNA replication prior to embryonic genome activation in mitochondrial DNA deficient oocytes but not in oocytes with normal levels of mitochondrial DNA. Blastocysts exhibited transcriptome profiles more closely resembling those of blastocysts from developmentally competent oocytes. Furthermore, mitochondrial supplementation reduced gene expression patterns associated with metabolic disorders that were identified in blastocysts from mitochondrial DNA deficient oocytes. These results demonstrate the importance of the oocyte’s mitochondrial DNA investment in fertilization outcome and subsequent embryo development to mitochondrial DNA deficient oocytes.
Highlights
There are a number of Mitochondrial DNA (mtDNA) disorders[6], which include mtDNA deficiency syndromes that manifest in somatic tissues and organs and primarily affect cells that are highly dependent on oxidative phosphorylation (OXPHOS) for the generation of ATP6
Oocytes can be selected by staining with Brilliant Cresyl Blue (BCB), a non-toxic dye that is reduced to a colorless compound by glucose-6-phosphate dehydrogenase (G6PD)[25]
To validate the use of BCB staining as a differential marker of oocyte maturation for oocytes that had not been synchronised to the S-phase of the cell cycle[10] (Supplementary Fig. 1), aspirated BCB+ and BCB− oocytes were assessed for Metaphase II (MII; polar body extrusion) after 44 hr of in vitro maturation (IVM)
Summary
There are a number of mtDNA disorders[6], which include mtDNA deficiency syndromes that manifest in somatic tissues and organs and primarily affect cells that are highly dependent on OXPHOS for the generation of ATP6. Human oocytes with low mtDNA copy number frequently fail to fertilise or arrest during preimplantation development[12,13,14,15,16,17] To this extent, the amount of mtDNA present in the oocyte at fertilisation is likely to be an investment in subsequent developmental events. During pig oocyte maturation, replication of mtDNA establishes a minimum investment of ~120 000 copies in oocytes that have the capacity to be fertilised[10,11,18,19,20] This investment ensures that sufficient mtDNA is available during organogenesis so that each mature cell type has sufficient mtDNA copy number to meet its required metabolic demands, in a cell specific manner. MtDNA replication and reduction events have been mapped in porcine oocytes and embryos[10,11], which are very similar to human oocytes and embryos[10,11,14]
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