No abstract

The mitochondrial genome resides in the mitochondria present in nearly all cell types. The porcine (Sus scrofa) mitochondrial genome is circa 16.7 kb in size and exists in the multimeric format in cells. Individual cell types have different numbers of mitochondrial DNA (mtDNA) copy number based on their requirements for ATP produced by oxidative phosphorylation. The oocyte has the largest number of mtDNA of any cell type. During oogenesis, the oocyte sets mtDNA copy number in order that sufficient copies are available to support subsequent developmental events. It also initiates a program of epigenetic patterning that regulates, for example, DNA methylation levels of the nuclear genome. Once fertilized, the nuclear and mitochondrial genomes establish synchrony to ensure that the embryo and fetus can complete each developmental milestone. However, altering the oocyte's mtDNA copy number by mitochondrial supplementation can affect the programming and gene expression profiles of the developing embryo and, in oocytes deficient of mtDNA, it appears to have a positive impact on the embryo development rates and gene expression profiles. Furthermore, mtDNA haplotypes, which define common maternal origins, appear to affect developmental outcomes and certain reproductive traits. Nevertheless, the manipulation of the mitochondrial content of an oocyte might have a developmental advantage.

Maternal obesity can impair offspring metabolic health, however the precise mechanism underpinning programming is unknown. Ten-Eleven translocase (TET) enzymes demethylate DNA using the TCA cycle intermediary α-ketoglutarate and may be involved in programming offspring health. Whether TETs are disrupted by maternal obesity is unknown. 5-6 week old C57Bl/6 female mice were fed a control diet (CD; 6% fat, n=175) or a high-fat diet (HFD; 21% fat, n=158) for six weeks. After superovulation oocytes were collected for metabolic assessment, or females were mated and zygotes cultured for embryo development, foetal growth, and assessment of global DNA methylation (5mC, 5hmC, 5fC and 5caC) in the 2-cell embryo. Zygotes collected from superovulated CBAF1 females were cultured in media containing α-ketoglutarate (0mM, 1.4mM, 3.5mM, or 14.0mM) or with 2-hydroxyglutarate (2HG) (0mM or 20mM), a competitive inhibitor of α-ketoglutarate, with methylation and blastocyst differentiation assessed. After HFD, oocytes showed increased pyruvate oxidation and intracellular ROS, with no changes in Tet3 expression, while 2-cell embryo global 5hmC DNA methylation was reduced and 5fC increased. Embryos cultured with 1.4 mM α-ketoglutarate had decreased 2-cell 5mC, whilst 14.0mM α-ketoglutarate increased the 5hmC:5mC ratio. In contrast supplementation with 20 mM 2HG increased 5mC and decreased the 5fC:5mC and 5caC:5mC ratios. α-ketoglutarate up to 3.5mM did not alter embryo development, whilst culturing in 14.0mM α-ketoglutarate blocked development at the 2-cell. Culture with 2HG delayed embryo development past the 4-cell and decreased blastocyst total cell number. In conclusion, disruptions in metabolic intermediates in the preimplantation embryo may provide a link between maternal obesity and programming offspring for ill health.

Oocytes can be supplemented with extra copies of mitochondrial DNA (mtDNA) to enhance developmental outcome. Pigs generated through supplementation with mtDNA derived from either sister (autologous) or third-party (heterologous) oocytes have been shown to exhibit only minor differences in growth, physiological and biochemical assessments, and health and well-being do not appear affected. However, it remains to be determined whether changes in gene expression identified during preimplantation development persisted and affected the gene expression of adult tissues indicative of high mtDNA copy number. It is also unknown if autologous and heterologous mtDNA supplementation resulted in different patterns of gene expression. Our transcriptome analyses revealed that genes involved in immune response and glyoxylate metabolism were commonly affected in brain, heart and liver tissues by mtDNA supplementation. The source of mtDNA influenced the expression of genes associated with oxidative phosphorylation (OXPHOS), suggesting a link between the use of third-party mtDNA and OXPHOS. We observed a significant difference in parental allele-specific imprinted gene expression in mtDNA-supplemented-derived pigs, with shifts to biallelic expression with no effect on expression levels. Overall, mtDNA supplementation influences the expression of genes in important biological processes in adult tissues. Consequently, it is important to determine the effect of these changes on animal development and health.