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Updated: Jun 27, 2026
Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction
Published on: July 12, 2022
Variations in mouse mitochondrial DNA copy number from fertilization to birth are associated with oxidative stress
Catherine Em Aiken1, Tereza Cindrova-Davies, Martin H Johnson
1Department of Physiology, Development and Neuroscience, and Centre for Trophoblast Research, The Anatomy School, Downing Street, Cambridge CB2 3DY, UK.
Abstract:
Mitochondria are inherited maternally via the oocyte, which in the mouse contains 150-250 x 10(3) copies of mitochondrial DNA (mtDNA). The number of mtDNA copies/embryo is thought to be stable during cleavage, being progressively diluted/cell with each round of cell division, until replication begins at an undefined time post-implantation. Post-natally, tissues differ in copy number of mtDNA/cell, but when and how these differences arise is unclear. A ratiometric quantitative real-time polymerase chain reaction assay of the levels of a single mitochondrial gene against a single copy nuclear gene was used to estimate the average copy value of mtDNA/per cell from zygote to birth. A novel Bayesian statistical model was used to identify day 5.15-6.15 as the time at which replication recommences, consistent with the viability patterns of embryos carrying mitochondrial mutations. Mitochondrial DNA copy number/cell in a range of post-day 9.5 fetal and placental tissues showed tissue-specific temporal expression patterns. Western blotting was used to quantify post-day 9.5 tissue markers for oxidative stress and manganese superoxide dismutase, and revealed correlations with the changes in mtDNA copy number. These findings have potential implications for fetal programming, in-vitro embryo culture, and the mechanism underlying the mitochondrial bottleneck.
Insights
Mitochondrial DNA copy number is stable until replication restarts around days 5-6 post-implantation. Post-natal tissue differences in mitochondrial DNA levels correlate with oxidative stress markers.
Area of Science:
- Developmental Biology
- Cellular Biology
- Genetics
Background:
- Mitochondria and their DNA (mtDNA) are maternally inherited.
- The number of mtDNA copies per cell changes throughout embryonic development, but the exact timing of replication and factors influencing tissue-specific differences remain unclear.
Purpose of the Study:
- To determine when mtDNA replication recommences during mouse embryonic development.
- To investigate the establishment of tissue-specific mtDNA copy numbers post-implantation.
- To explore correlations between mtDNA copy number and oxidative stress markers.
Main Methods:
- Quantitative real-time PCR was used to measure mtDNA copy number from zygote to birth.
- A Bayesian statistical model identified the timing of mtDNA replication.
- Western blotting assessed oxidative stress markers and manganese superoxide dismutase levels.
Main Results:
- mtDNA replication was identified to recommence between days 5.15 and 6.15 post-implantation.
- Post-day 9.5, fetal and placental tissues exhibited distinct temporal patterns of mtDNA copy number.
- Changes in mtDNA copy number correlated with levels of oxidative stress and manganese superoxide dismutase.
Conclusions:
- The study pinpoints the critical window for mtDNA replication restart during mouse embryogenesis.
- Tissue-specific mtDNA copy numbers are established post-implantation and linked to metabolic and oxidative stress factors.
- Findings offer insights into fetal programming, in-vitro embryo culture, and the mitochondrial bottleneck mechanism.
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