Mitochondrial DNA haplotypes induce differential patterns of DNA methylation that result in differential chromosomal gene expression patterns

Mitochondrial DNA copy number is strictly regulated during development as naive cells differentiate into mature cells to ensure that specific cell types have sufficient copies of mitochondrial DNA to perform their specialised functions. Mitochondrial DNA haplotypes are defined as specific regions of mitochondrial DNA that cluster with other mitochondrial sequences to show the phylogenetic origins of maternal lineages. Mitochondrial DNA haplotypes are associated with a range of phenotypes and disease. To understand how mitochondrial DNA haplotypes induce these characteristics, we used four embryonic stem cell lines that have the same set of chromosomes but possess different mitochondrial DNA haplotypes. We show that mitochondrial DNA haplotypes influence changes in chromosomal gene expression and affinity for nuclear-encoded mitochondrial DNA replication factors to modulate mitochondrial DNA copy number, two events that act synchronously during differentiation. Global DNA methylation analysis showed that each haplotype induces distinct DNA methylation patterns, which, when modulated by DNA demethylation agents, resulted in skewed gene expression patterns that highlight the effectiveness of the new DNA methylation patterns established by each haplotype. The haplotypes differentially regulate α-ketoglutarate, a metabolite from the TCA cycle that modulates the TET family of proteins, which catalyse the transition from 5-methylcytosine, indicative of DNA methylation, to 5-hydroxymethylcytosine, indicative of DNA demethylation. Our outcomes show that mitochondrial DNA haplotypes differentially modulate chromosomal gene expression patterns of naive and differentiating cells by establishing mitochondrial DNA haplotype-specific DNA methylation patterns

THE EFFECTS OF MITOCHONDRIAL DNA ON PORCINE PREIMPLANTATION EMBRYO DEVELOPMENT

The overall aim of my thesis was to determine the role of mitochondrial DNA (mtDNA) during porcine oocyte maturation and preimplantation embryo development. We have found that mtDNA affects oocyte quality and litter sizes. Using the pig model, we have developed a method to identify oocytes that are deficient in mtDNA. To increase mtDNA, we supplemented those oocytes with a pure population of mitochondria at the time of in vitro fertilisation, to deliver good quality mtDNA, for the improvement of oocyte quality. The outcomes of my work are applicable to farm animal production and assisted reproduction in the clinic

The association of mitochondrial DNA haplotypes and phenotypic traits in pigs

Abstract Background The mitochondrial genome (mtDNA) is an emerging determiner of phenotypic traits and disease. mtDNA is inherited in a strict maternal fashion from the population of mitochondria present in the egg at fertilisation. Individuals are assigned to mtDNA haplotypes and those with sequences that cluster closely have common origins and their migration patterns can be mapped. Previously, we identified five mtDNA haplotypes in the commercial breeding lines of Australian pigs, which defined their common origins, and showed how these mtDNA haplotypes influenced litter size and reproductive function in terms of egg and embryo quality and fertilisation efficiency. Results We have determined whether mtDNA haplotypes influence other phenotypic traits. These include fat density; muscle depth; fat to leanness ratios; lifetime daily gain; teat quality; muscle score; front and rear leg assessments; percentage offspring weaned; weaning to oestrus intervals; gilt age at selection; and gestational length. In all, we assessed 5687 pigs of which 2762 were females and 2925 were males. We assessed all animals together and then by gender. We further assessed by gender based on whether a sire had joined with females from only one haplotype or from more than one haplotype. We determined that fat density, muscle depth, fat to leanness ratios, lifetime daily gain and teat quality were influenced by mtDNA haplotype and that there were gender specific effects on teat quality. Conclusions Our data illustrate that mtDNA haplotypes are associated with a number of important phenotypic traits indicative of economic breeding values in breeding pigs with gender-specific differences. Interestingly, there are ‘trade offs’ whereby some mtDNA haplotypes perform better for one selection criterion, such as muscle depth, but less so for another, for example teat quality, indicating that pig mtDNA haplotypes are afforded an advantage in one respect but a disadvantage in another

Additional file 1: of The association of mitochondrial DNA haplotypes and phenotypic traits in pigs

Table S1. Number of animals analysed per mtDNA haplotype. (DOCX 47 kb

The relationship between mitochondrial DNA haplotype and the reproductive capacity of domestic pigs (Sus scrofa domesticus)

Frequencies of mtDNA variants for all 33 samples sequenced by Next Generation Sequencing. (XLSX 140 kb

Additional file 4: of The association of mitochondrial DNA haplotypes and phenotypic traits in pigs

Table S3. Distribution of sires across haplotypes. (DOCX 29 kb

Additional file 2: of The association of mitochondrial DNA haplotypes and phenotypic traits in pigs

Table S2. Distribution of breeds covering each haplotype. (DOCX 91 kb

Additional file 3: of The association of mitochondrial DNA haplotypes and phenotypic traits in pigs

Figure S1. Molecular Phylogenetic analysis by Maximum Likelihood method. Time of divergence was estimated using the RelTime method and based on the Asian European split of 750,000 YBP. The estimated divergence time for mtDNA haplotypes A and B was 50,000 YBP; A and C 90,000 YBP; A and D 750,000 YBP; and D and E 50,000 YBP. (DOCX 52 kb

Additional file 5: of The relationship between mitochondrial DNA haplotype and the reproductive capacity of domestic pigs (Sus scrofa domesticus)

Breed distribution across the mtDNA haplotypes for the 216 commercial pigs. (DOCX 42 kb

Restoration of normal embryogenesis by mitochondrial supplementation in pig oocytes exhibiting mitochondrial DNA deficiency

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