The transgenerational effects of oocyte mitochondrial supplementation.

Many women suffer from either failed fertilisation or their embryos arrest early during development. Autologous mitochondrial supplementation has been proposed as an assisted reproductive technology to overcome these problems. However, its safety remains to be tested in an animal model to determine if there are transgenerational effects. We have supplemented oocytes with autologous populations of mitochondria to generate founders. We mated the female founders and their offspring to produce three generations. We assessed litter size, the ovarian reserve, and weight gain and conducted a full histopathological analysis from each of the three generations. Across the generations, we observed significant increases in litter size and in the number of primordial follicles in the ovary matched by changes in global gene expression patterns for these early-stage oocytes. However, full histopathological analysis revealed that cardiac structure was compromised in first and second generation offspring, which could seriously affect the health of the offspring. Furthermore, the offspring were prone to increased weight gain during early life. Mitochondrial supplementation appears to perturb the regulation of the chromosomal genome resulting in transgenerational phenotypic gains and losses. These data highlight the need for caution when using autologous mitochondrial supplementation to treat female factor infertility.

The association of mitochondrial DNA haplotypes and phenotypic traits in pigs.

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.

The effects of mitochondrial DNA supplementation at the time of fertilization on the gene expression profiles of porcine preimplantation embryos.

Mitochondrial DNA (mtDNA) deficient metaphase II porcine oocytes are less likely to fertilize and more likely to arrest during preimplantation development. However, they can be supplemented with autologous populations of mitochondria at the time of fertilization, which significantly increases mtDNA copy number by the 2-cell stage due to the modulation of DNA methylation at a CpG island of the gene encoding the mtDNA-specific polymerase, POLG, and promotes preimplantation development. Although mitochondrial supplementation does not increase development rates or mtDNA copy number in oocytes with normal levels of mtDNA copy number, we tested whether this approach would also impact on chromosomal gene expression patterns in these oocytes at each stage of preimplantation development. Here, we have compared the gene expression profiles of embryos produced by mitochondrial supplementation at the time of fertilization with embryos produced by in vitro fertilization (IVF) using a panel of genes associated with different stages of preimplantation development. When compared to IVF-derived embryos, 27 (34%) genes were differentially expressed in supplemented embryos but this was restricted to one or two developmental stages. However, 53 (66%) genes were comparably expressed across all six stages and by the blastocyst stage 4 (5%) genes were differentially expressed. We conclude that additional copies of mtDNA can induce changes in gene expression at various stages of preimplantation development with the first changes occurring prior to maternal-to-zygotic transition (MZT). However, these changes appear to be transitory suggesting that some genomic resetting is taking place.

The molecular characterisation of mitochondrial DNA deficient oocytes using a pig model.

STUDY QUESTION: What are the molecular differences between mitochondrial DNA (mtDNA)-deficient and mtDNA-normal oocytes and how does mitochondrial supplementation alter these? SUMMARY ANSWER: Changes to DNA methylation in a 5' cytosine-phosphate-guanine 3' (CpG) island in the mtDNA-specific replication factor (DNA polymerase gamma (POLG)) of mtDNA-deficient oocytes mediates an increase in mtDNA copy number by the 2-cell stage that positively modulates the expression of nuclear genes, which affect cellular and metabolic processes, following autologous mitochondrial supplementation. WHAT IS KNOWN ALREADY: Too few copies of mtDNA in mature oocytes can lead to fertilisation failure or preimplantation embryo arrest. mtDNA-deficient oocytes that progress to blastocyst express genes associated with poor cellular and metabolic processes, transcriptional activation and mitochondrial biogenesis. STUDY DESIGN, SIZE, DURATION: Using a pig oocyte model, we assessed mtDNA-deficient and mtDNA-normal oocytes during in vitro maturation for mtDNA variants and levels of DNA methylation in POLG. We supplemented mtDNA-deficient oocytes with autologous populations of mitochondria to determine if there were changes to DNA methylation in POLG that coincided with increases in mtDNA copy number. We assessed metaphase II mtDNA-deficient and mtDNA-normal oocytes by RNA sequencing to identify differentially expressed genes and compared their profiles to blastocysts derived from mtDNA-normal, mtDNA-deficient and supplemented mtDNA-deficient oocytes. PARTICIPANTS/MATERIALS, SETTING, METHODS: mtDNA variant analysis (n = 24), mtDNA copy number (n = 60), POLG gene expression (n = 24), and RNA sequencing (n = 32 single; and 12 pooled cohorts of n = 5) were performed on oocytes and embryos. DNA methylation of a CpG island in POLG was determined quantitatively by pyrosequencing on oocytes to 2-cell embryos (n = 408). Bioinformatics tools were used to assess differences between mtDNA-normal and mtDNA-deficient oocytes and between mtDNA-normal and mtDNA-deficient oocytes and supplemented oocytes and their blastocyst stage equivalents. MAIN RESULTS AND THE ROLE OF CHANCE: Whilst mtDNA-deficient oocytes regulated variants less stringently during maturation (P < 0.05), there were no differences in the ratio of variants in mature-stage oocytes. However, mtDNA-normal mature oocytes had significantly more molecules affected due to their higher copy number (P < 0.0001). Normal mature oocytes differently DNA methylated a CpG island in POLG compared with mtDNA-deficient oocytes (P < 0.01). Supplementation of mtDNA-deficient oocytes modulated DNA methylation at this CpG island leading to a mtDNA replication event prior to embryonic genome activation inducing significant increases in mtDNA copy number. RNA-Seq identified 57 differentially expressed genes (false discovery rate (FDR) < 0.05) between the two cohorts of oocytes with blastocyst stage gene expression altered by supplementation of mtDNA-deficient oocytes (P < 0.05) including genes associated with metabolic disorders. One key factor was branched chain amino acid transaminase 2 (BCAT2), a regulator of amino acid metabolism and associated with diabetes. LARGE SCALE DATA: Sequence data are available on the NCBI Sequence Read Archive under the project number PRJNA422295. RNA sequencing data were deposited into NCBI Gene Expression Omnibus, under the accession number GSE108900. LIMITATIONS, REASONS FOR CAUTION: Whilst this work was conducted in a species that is highly relevant to human reproduction, the outcomes need to be tested in human oocytes and blastocysts prior to clinical application. WIDER IMPLICATIONS OF THE FINDINGS: The outcomes demonstrate a mechanism of action following mtDNA supplementation of mtDNA-deficient oocytes that results in improved gene expression at the blastocyst stage of development. STUDY FUNDING/COMPETING INTERESTS: This work was funded by OvaScience Inc. OvaScience did not influence the study design, analysis of results or interpretation of the data.

The molecular characterization of porcine egg precursor cells.

Female-factor infertility can be caused by poor oocyte quality and depleted ovarian reserves. Egg precursor cells (EPCs), isolated from the ovarian cortex, have the potential to be used to overcome female infertility. We aimed to define the origins of EPCs by analyzing their gene expression profiles and mtDNA content using a mini-pig model. We characterized FAC-sorted DDX4+-derived porcine EPCs by performing RNA-sequencing and determined that they utilize pathways important for cell cycle and proliferation, which supports the existence of adult mitotically active oogonial cells. Expression of the pluripotent markers Sox2 and Oct4, and the primitive germ cell markers Blimp1 and Stella were not detected. However, Nanog and Ddx4 were expressed, as were the primitive germ cell markers Fragilis, c-Kit and Tert. Moreover, porcine EPCs expressed self-renewal and proliferation markers including Myc, Esrrb, Id2, Klf4, Klf5, Stat3, Fgfr1, Fgfr2 and Il6st. The presence of Zp1, Zp2, Zp3 and Nobox were not detected, indicating that porcine EPCs are not indicative of mature primordial oocytes. We performed mitochondrial DNA Next Generation Sequencing and determined that one mtDNA variant harbored by EPCs was present in oocytes, preimplantation embryos and somatic tissues over three generations in our mini-pig model indicating the potential germline origin of EPCs.

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 mitochondrial genome: how it drives fertility.

In mammalian species, the mitochondrial genome is between 16.2 and 16.7kb in size and encodes key proteins associated with the cell's major energy-generating apparatus, the electron transfer chain. The maternally inherited mitochondrial genome has, until recently, been thought to be only involved in the production of energy. In this review, we analyse how the mitochondrial genome influences the developing embryo and cellular differentiation, as well as fetal and offspring health and wellbeing. We make specific reference to two assisted reproductive technologies, namely mitochondrial supplementation and somatic cell nuclear transfer, and how modulating the mitochondrial content in the oocyte influences embryo viability and the potential to generate enhanced offspring for livestock production purposes. We also explain why it is important to ensure that the transmission of only one population of mitochondrial (mt) DNA is maintained through to the offspring and why two populations of genetically distinct mitochondrial genomes could be deleterious. Finally, we explain how mtDNA influences chromosomal gene expression patterns in developing embryos and cells primarily by modulating DNA methylation patterns through factors associated with the citric acid cycle. These factors can then modulate the ten-eleven translocation (TET) pathway, which, in turn, determines whether a cell is in a more or less DNA methylated state.

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

BACKGROUND: The maternally inherited mitochondrial genome encodes key proteins of the electron transfer chain, which produces the vast majority of cellular ATP. Mitochondrial DNA (mtDNA) present in the mature oocyte acts as a template for all mtDNA that is replicated during development to meet the specific energy requirements of each tissue. Individuals that share a maternal lineage cluster into groupings known as mtDNA haplotypes. MtDNA haplotypes confer advantages and disadvantages to an organism and this affects its phenotype. In livestock, certain mtDNA haplotypes are associated with improved milk and meat quality, whilst, other species, mtDNA haplotypes have shown increased longevity, growth and susceptibility to diseases. In this work, we have set out to determine whether mtDNA haplotypes influence reproductive capacity. This has been undertaken using a pig model. RESULTS: To determine the genetic diversity of domestic pigs in Australia, we have sequenced the D-loop region of 368 pigs, and identified five mtDNA haplotypes (A to E). To assess reproductive capacity, we compared oocyte maturation, fertilization and development to blastocyst, and found that there were significant differences for maturation and fertilization amongst the haplotypes. We then determined that haplotypes C, D and E produced significantly larger litters. When we assessed the conversion of developmentally competent oocytes and their subsequent developmental stages to offspring, we found that haplotypes A and B had the lowest reproductive efficiencies. Amongst the mtDNA haplotypes, the number of mtDNA variants harbored at >25 % correlated with oocyte quality. MtDNA copy number for developmentally competent oocytes positively correlated with the level of the 16383delC variant. This variant is located in the conserved sequence box II, which is a regulatory region for mtDNA transcription and replication. CONCLUSIONS: We have identified five mtDNA haplotypes in Australian domestic pigs indicating that genetic diversity is restricted. We have also shown that there are differences in reproductive capacity amongst the mtDNA haplotypes. We conclude that mtDNA haplotypes affect pig reproductive capacity and can be used as a marker to complement current selection methods to identify productive pigs.

Mitochondrial DNA supplementation as an enhancer of female reproductive capacity.

PURPOSE OF REVIEW: There has been increasing interest in developing assisted reproductive technologies to overcome failure at fertilization and early embryonic arrest. Some of the affected patients harbor too few copies of mitochondrial DNA (mtDNA) in their eggs at the time of fertilization, which results in developmental failure. RECENT FINDINGS: In the last 3-5 years, there has been a drive to overcome mtDNA deficiency through mitochondrial supplementation protocols using autologous populations of mitochondrial DNA so as not to perturb the offspring's genetic identity or mediate a series of side-effects because of the mixing of two distinct populations of mitochondrial DNA. It is evident that there is strict regulation of mitochondrial DNA copy number from the primordial germ cell through to the time when tissues are specified during organogenesis. Supplementation of oocytes can give rise to better quality embryos, enhanced blastocyst rates, and ongoing pregnancies. It utilizes a key mitochondrial DNA replication event that takes place shortly after fertilization to stabilize the embryonic genome. SUMMARY: These findings provide a rationale for undertaking mitochondrial supplementation and propose a mechanism to explain why the process can enhance embryo development. They also take the approach a step closer to clinical practice.

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.

Segregation of Naturally Occurring Mitochondrial DNA Variants in a Mini-Pig Model.

The maternally inherited mitochondrial genome (mtDNA) is present in multimeric form within cells and harbors sequence variants (heteroplasmy). While a single mtDNA variant at high load can cause disease, naturally occurring variants likely persist at low levels across generations of healthy populations. To determine how naturally occurring variants are segregated and transmitted, we generated a mini-pig model, which originates from the same maternal ancestor. Following next-generation sequencing, we identified a series of low-level mtDNA variants in blood samples from the female founder and her daughters. Four variants, ranging from 3% to 20%, were selected for validation by high-resolution melting analysis in 12 tissues from 31 animals across three generations. All four variants were maintained in the offspring, but variant load fluctuated significantly across the generations in several tissues, with sex-specific differences in heart and liver. Moreover, variant load was persistently reduced in high-respiratory organs (heart, brain, diaphragm, and muscle), which correlated significantly with higher mtDNA copy number. However, oocytes showed increased heterogeneity in variant load, which correlated with increased mtDNA copy number during in vitro maturation. Altogether, these outcomes show that naturally occurring mtDNA variants segregate and are maintained in a tissue-specific manner across generations. This segregation likely involves the maintenance of selective mtDNA variants during organogenesis, which can be differentially regulated in oocytes and preimplantation embryos during maturation.

Mitochondrial dysfunction in oocytes of obese mothers: transmission to offspring and reversal by pharmacological endoplasmic reticulum stress inhibitors.

Over-nutrition in females causes altered fetal growth during pregnancy and permanently programs the metabolism of offspring; however, the temporal and mechanistic origins of these changes, and whether they are reversible, are unknown. We now show that, in obese female mice, cumulus-oocyte complexes exhibit endoplasmic reticulum (ER) stress, high levels of intracellular lipid, spindle abnormalities and reduced PTX3 extracellular matrix protein production. Ovulated oocytes from obese mice contain normal levels of mitochondrial (mt) DNA but have reduced mitochondrial membrane potential and high levels of autophagy compared with oocytes from lean mice. After in vitro fertilization, the oocytes of obese female mice demonstrate reduced developmental potential and form blastocysts with reduced levels of mtDNA. Blastocysts transferred to normal weight surrogates that were then analyzed at E14.5 showed that oocytes from obese mice gave rise to fetuses that were heavier than controls and had reduced liver and kidney mtDNA content per cell, indicating that maternal obesity before conception had altered the transmission of mitochondria to offspring. Treatment of the obese females with the ER stress inhibitor salubrinal or the chaperone inducer BGP-15 before ovulation increased the amount of the mitochondrial replication factors TFAM and DRP1, and mtDNA content in oocytes. Salubrinal and BGP-15 also completely restored oocyte quality, embryo development and the mtDNA content of fetal tissue to levels equivalent to those derived from lean mice. These results demonstrate that obesity before conception imparts a legacy of mitochondrial loss in offspring that is caused by ER stress and is reversible during the final stages of oocyte development and maturation.