ORCID Profile
0000-0002-3993-1449
Current Organisation
University of Adelaide
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Publisher: Oxford University Press (OUP)
Date: 08-2002
Abstract: The use of cytoplasmic transfer as an assisted reproductive technique has generated much attention. This arises as donor mitochondria are introduced into the cytoplasm of the recipient oocyte. The consequences are the possible transmission of two mitochondrial (mt)DNA populations to the offspring. This pattern of inheritance is in contrast to the strictly maternal manner in which mtDNA is transmitted following natural fertilization and ICSI. This paper discusses the advantages of using such a technique to enhance embryonic development from poor quality oocytes with respect to the low copy number of mtDNA found in some oocytes following superovulation protocols. However, it also cautions against using such a technique before a clearer understanding of the patterns of inheritance and transmission of mtDNA has been established and suggests that animal models be utilised to do so.
Publisher: Wiley
Date: 09-2015
DOI: 10.1111/NYAS.12873
Abstract: Mitochondrial DNA (mtDNA) copy number is strictly regulated during development and tumorigenesis. Pluripotent stem cells and cancer stem-like cells use glycolysis for energy metabolism, as they possess low mtDNA copy number, which promotes cell proliferation. As pluripotent stem cells can differentiate into all cell types of the body, they establish the mtDNA set point during early development, maintaining mtDNA copy number at low levels but enabling differentiating cells to acquire the appropriate numbers of mtDNA copy to meet their specific demands for OXPHOS-derived ATP, as they become specialized cells. This process is mediated by changes to DNA methylation at exon 2 of the catalytic subunit of the mitochondrial-specific polymerase, POLGA. Cancer stem-like cells, however, are hypermethylated and maintain low mtDNA copy number, resulting in their dependence on aerobic glycolysis. Their hypermethylation at exon 2 of POLGA also promotes their multipotent state. As a result, cancer cells are unable to increase their mtDNA content and differentiate into specific lineages unless they are treated with DNA demethylation agents or partially depleted of their mtDNA. This review describes these processes in depth and argues that DNA methylation of POLGA is instrumental in the fate of pluripotent stem cells and cancer cells.
Publisher: Mary Ann Liebert Inc
Date: 15-10-2017
Abstract: Mitochondrial deoxyribonucleic acid (mtDNA) copy number is tightly regulated during pluripotency and differentiation. There is increased demand of cellular adenosine triphosphate (ATP) during differentiation for energy-intensive cell types such as hepatocytes and neurons to meet the cell's functional requirements. During hepatocyte differentiation, mtDNA copy number should be synchronously increased to generate sufficient ATP through oxidative phosphorylation. Unlike bone marrow mesenchymal cells, mtDNA copy number failed to increase by 28 days of differentiation of human amniotic epithelial cells (hAEC) into hepatocyte-like cells (HLC) despite their expression of some end-stage hepatic markers. This was due to higher levels of DNA methylation at exon 2 of POLGA, the mtDNA-specific replication factor. Treatment with a DNA demethylation agent, 5-azacytidine, resulted in increased mtDNA copy number, reduced DNA methylation at exon 2 of POLGA, and reduced hepatic gene expression. Depletion of mtDNA followed by subsequent differentiation did not increase mtDNA copy number, but reduced DNA methylation at exon 2 of POLGA and increased expression of hepatic and pluripotency genes. We encapsulated hAEC in barium alginate microcapsules and subsequently differentiated them into HLC. Encapsulation resulted in no net increase of mtDNA copy number but a significant reduction in DNA methylation of POLGA. RNAseq analysis showed that differentiated HLC express hepatocyte-specific genes but also increased expression of inflammatory interferon genes. Differentiation in encapsulated cells showed suppression of inflammatory genes as well as increased expression of genes associated with hepatocyte function pathways and networks. This study demonstrates that an increase in classical hepatic gene expression can be achieved in HLC through encapsulation, although they fail to effectively regulate mtDNA copy number.
Publisher: Oxford University Press (OUP)
Date: 02-2006
DOI: 10.1095/BIOLREPROD.106.054536
Abstract: Cellular ATP is mainly generated through mitochondrial oxidative phosphorylation, which is dependent on mitochondrial DNA (mtDNA). We have previously demonstrated the importance of oocyte mtDNA for porcine and human fertilization. However, the role of nuclear-encoded mitochondrial replication factors during oocyte and embryo development is not yet understood. We have analyzed two key factors, mitochondrial transcription factor A (TFAM) and polymerase gamma (POLG), to determine their role in oocyte and early embryo development. Competent and incompetent oocytes, as determined by brilliant cresyl blue (BCB) dye, were assessed intermittently during the maturation process for TFAM and POLG mRNA using real-time RT-PCR, for TFAM and POLG protein using immunocytochemistry, and for mtDNA copy number using real-time PCR. Analysis was also carried out following treatment of maturing oocytes with the mtDNA replication inhibitor, 2',3'-dideoxycytidine (ddC). Following in vitro fertilization, preimplantation embryos were also analyzed. Despite increased levels of TFAM and POLG mRNA and protein at the four-cell stage, no increase in mtDNA copy number was observed in early preimplantation development. To compensate for this, mtDNA appeared to be replicated during oocyte maturation. However, significant differences in nuclear-encoded regulatory protein expression were observed between BCB(+) and BCB(-) oocytes and between untreated oocytes and those treated with ddC. These changes resulted in delayed mtDNA replication, which correlated to reduced fertilization and embryonic development. We therefore conclude that adherence to the regulation of the timing of mtDNA replication during oocyte maturation is essential for successful embryonic development.
Publisher: Springer Science and Business Media LLC
Date: 04-09-2005
DOI: 10.1038/NCHEMBIO730
Abstract: Gamete-gamete interactions are critically modulated by carbohydrate-protein interactions that rely on the carbohydrate-selective recognition of polyvalent carbohydrate structures. A galactose-binding protein has been identified in mammalian spermatozoa that has similarity to the well-characterized hepatic asialoglycoprotein receptor. With the aim of exploiting the ability of this class of proteins to bind and internalize macromolecules displaying galactose, we designed hybrid carbohydrate-antioxidant polymers to deliver antioxidant vitamin E (alpha-tocopherol) to porcine spermatozoa. Treatment of sperm cells with one hybrid polymer in particular produced large increases in intracellular sperm levels of alpha-tocopherol and greatly reduced endogenous fatty acid degradation under oxidative stress. The polymer-treated spermatozoa had enhanced physiological properties and longer half-lives, which resulted in enhanced fertilization rates. Our results indicate that hybrid polymer delivery systems can prolong the functional viability of mammalian spermatozoa and improve fertility rates, and that our functionally guided optimization strategy can be applied to the discovery of active glycoconjugate ligands.
Publisher: Oxford University Press (OUP)
Date: 07-2007
DOI: 10.1534/GENETICS.106.070177
Abstract: Mitochondrial DNA (mtDNA) is normally only inherited through the oocyte. However, nuclear transfer (NT), the fusion of a donor cell with an enucleated oocyte, can transmit both donor cell and recipient oocyte mtDNA. mtDNA replication is under the control of nuclear-encoded replication factors, such as polymerase gamma (POLG) and mitochondrial transcription factor A (TFAM). These are first expressed during late preimplantation embryo development. To account for the persistence of donor cell mtDNA, even when introduced at residual levels (mtDNAR), we hypothesized that POLG and TFAM would be upregulated in intra- and interspecific (ovine–ovine) and intergeneric (caprine–ovine) NT embryos when compared to in vitro fertilized (IVF) embryos. For the intra- and interspecific crosses, PolGA (catalytic subunit), PolGB (accessory subunit), and TFAM mRNA were expressed at the 2-cell stage in both nondepleted (mtDNA+) and mtDNAR embryos with protein being expressed up to the 16-cell stage for POLGA and TFAM. However, at the 16-cell stage, there was significantly more PolGA expression in the mtDNAR embryos compared to their mtDNA+ counterparts. Expression for all three genes first matched IVF embryos at the blastocyst stage. In the intergeneric model, POLG was upregulated during preimplantation development. Although these embryos did not persist further than the 16+-cell stage, significantly more mtDNAR embryos reached this stage. However, the vast majority of these embryos were homoplasmic for recipient oocyte mtDNA. The upreglation in mtDNA replication factors was most likely due to the donor cells still expressing these factors prior to NT.
Publisher: Springer New York
Date: 2016
Abstract: The mitochondrial genome resides in the mitochondrion of nearly all mammalian cells. It is important for energy production as it encodes 13 of the key subunits of the electron transfer chain, which generates the vast majority of cellular ATP through the process of oxidative phosphorylation. As cells establish pluripotency, they regulate their mtDNA copy number so that they possess few copies but sufficient that they can be replicated to match the differentiated cell-specific requirements for ATP derived through oxidative phosphorylation. However, the failure to strictly regulate this process prevents pluripotent cells from differentiating. We describe a series of protocols that analyze mtDNA copy number, DNA methylation within the nuclear-encoded mtDNA-specific polymerase, and gene expression of the other factors that drive replication of the mitochondrial genome. We demonstrate how to measure ATP-generating capacity through oxygen respiratory capacity and total cellular ATP and lactate levels. Finally, we also describe how to detect mtDNA variants in pluripotent and differentiating cells using next-generation sequencing protocols and how the variants can be confirmed by high-resolution melt analysis.
Publisher: Springer Science and Business Media LLC
Date: 07-01-2010
DOI: 10.1038/GT.2009.164
Publisher: Elsevier BV
Date: 07-2004
Publisher: Springer Science and Business Media LLC
Date: 30-08-2013
DOI: 10.1038/CDD.2013.115
Publisher: Cold Spring Harbor Laboratory
Date: 08-10-2020
DOI: 10.1101/2020.10.07.327478
Abstract: Increasing evidence in animal species ranging from mammals to insects has revealed phenotypes that are caused by ancestral life experiences including stress and diet. The descendent phenotypes themselves are wide ranging, and include changes to behaviour, disease risk, metabolism, and growth. Ancestral dietary macronutrient composition, and quantity (over- and under-nutrition) have been shown to alter descendent growth, metabolism and behaviour. Several studies have identified inherited molecules in gametes which are altered by ancestral diet and are required for the transgenerational effect. However, there is less understanding of the developmental pathways in the period between fertilisation and adulthood that are altered by the inherited molecules. Here we identify a key role of the MAPK signalling pathway in mediating changes to Drosophila larval developmental timing due to variation in ancestral diet. We exposed grand-parental and great grand-parental generations to defined protein to carbohydrate (P:C) dietary ratios and measured developmental timing. Descendent developmental timing was consistently faster in the period between the embryonic and pupal stages when the ancestor had a higher P:C ratio diet. Transcriptional analysis of embryos, larvae and adults revealed extensive and long-lasting changes to the MAPK signalling pathway which controlled growth rate through regulation of ribosomal RNA transcription. The importance of these processes was supported by pharmacological inhibition of MAPK and rRNA proteins which reproduced the ancestral diet-induced developmental changes. This work provides insight into the role of developmental growth signalling networks in mediating non-genetic inheritance in the period between fertilisation and adult. Ancestral, diet-induced descendent developmental timing changes are caused by alteration of MAPK signalling pathways in the period between the embryo and pupal stages in Drosophila .
Publisher: MDPI AG
Date: 14-02-2023
DOI: 10.3390/IJMS24043783
Abstract: Mitochondrial DNA (mtDNA) deficiency correlates with poor oocyte quality and fertilisation failure. However, the supplementation of mtDNA deficient oocytes with extra copies of mtDNA improves fertilisation rates and embryo development. The molecular mechanisms associated with oocyte developmental incompetence, and the effects of mtDNA supplementation on embryo development are largely unknown. We investigated the association between the developmental competence of Sus scrofa oocytes, assessed with Brilliant Cresyl Blue, and transcriptome profiles. We also analysed the effects of mtDNA supplementation on the developmental transition from the oocyte to the blastocyst by longitudinal transcriptome analysis. mtDNA deficient oocytes revealed downregulation of genes associated with RNA metabolism and oxidative phosphorylation, including 56 small nucleolar RNA genes and 13 mtDNA protein coding genes. We also identified the downregulation of a large subset of genes for meiotic and mitotic cell cycle process, suggesting that developmental competence affects the completion of meiosis II and first embryonic cell ision. The supplementation of oocytes with mtDNA in combination with fertilisation improves the maintenance of the expression of several key developmental genes and the patterns of parental allele-specific imprinting gene expression in blastocysts. These results suggest associations between mtDNA deficiency and meiotic cell cycle and the developmental effects of mtDNA supplementation on Sus scrofa blastocysts.
Publisher: MDPI AG
Date: 08-08-2019
DOI: 10.3390/CELLS8080852
Abstract: Mitochondria and mitochondrial DNA have important roles to play in development. In primordial germ cells, they progress from small numbers to populate the maturing oocyte with high numbers to support post-fertilization events. These processes take place under the control of significant changes in DNA methylation and other epigenetic modifiers, as well as changes to the DNA methylation status of the nuclear-encoded mitochondrial DNA replication factors. Consequently, the differentiating germ cell requires significant synchrony between the two genomes in order to ensure that they are fit for purpose. In this review, I examine these processes in the context of female germline stem cells that are isolated from the ovary and those derived from embryonic stem cells and reprogrammed somatic cells. Although our knowledge is limited in this respect, I provide predictions based on other cellular systems of what is expected and provide insight into how these cells could be used in clinical medicine.
Publisher: Springer Science and Business Media LLC
Date: 31-03-2016
Publisher: Bioscientifica
Date: 06-2004
DOI: 10.1530/REP.1.00138
Abstract: The introduction of nuclear transfer (NT) and other technologies that involve embryo reconstruction require us to reinvestigate patterns of mitochondrial DNA (mtDNA) transmission, transcription and replication. MtDNA is a 16.6 kb genome located within each mitochondrion. The number of mitochondria and mtDNA copies per organelle is specific to each cell type. MtDNA is normally transmitted through the oocyte to the offspring. However, reconstructed oocytes often transmit both recipient oocyte mtDNA and mtDNA associated with the donor nucleus. We argue that the transmission of two populations of mtDNA may have implications for offspring survival as only one allele might be actively transcribed. This could result in the offspring phenotypically exhibiting mtDNA depletion-type syndromes. A similar occurrence could arise when nucleo–cytoplasmic interactions fail to regulate mtDNA transcription and replication, especially as the initiation of mtDNA replication post-implantation is a key developmental event. Furthermore, failure of the donor somatic nucleus to be reprogrammed could result in the early initiation of replication and the loss of cellular mtDNA specificity. We suggest investigations should be conducted to enhance our understanding of nucleo–cytoplasmic interactions in order to improve NT efficiency.
Publisher: Springer Science and Business Media LLC
Date: 09-2007
DOI: 10.1038/NCB436
Abstract: Scientists are seeking permission to generate human embryonic stem cells to study disease by introducing human genetic material into an animal oocyte. This has raised ethical questions that centre on whether the entities being generated are actually human. The answer to these questions will determine how this area of research will be regulated and whether such work will be legal. The function of the extra-nuclear mitochondrial genome lies at the heart of these issues and forms the focus of this commentary.
Publisher: Oxford University Press (OUP)
Date: 31-08-2012
DOI: 10.1093/NAR/GKS770
Publisher: Wiley
Date: 08-12-2013
Publisher: Springer Science and Business Media LLC
Date: 14-06-2012
DOI: 10.1007/S00441-012-1444-2
Abstract: The mitochondrial genome contributes key proteins to the electron-transfer chain, which through oxidative phosphorylation, generates the vast majority of cellular ATP. This maternally inherited genome is transmitted to subsequent generations through the oocyte. Its transmission, inheritance and replication are strictly regulated so that fully mature cells can be appropriately populated with mitochondrial DNA once they mature into adult cells. However, gametes do not always acquire the appropriate numbers of mitochondrial DNA copy this often renders them inappropriate for successful fertilisation outcome. Furthermore, the number of assisted reproductive technologies that can overcome problems associated with infertility and that can provide enhanced genetic outcomes for the offspring is increasing. However, such techniques could also have a detrimental effect on offspring survival. If we are to introduce these technologies into in vitro fertilisation clinics and animal production, then we first need to validate their use carefully.
Publisher: Wiley
Date: 03-05-2018
DOI: 10.1002/MRD.22985
Abstract: 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.
Publisher: CSIRO Publishing
Date: 2018
DOI: 10.1071/RD17408
Abstract: In mammalian species, the mitochondrial genome is between 16.2 and 16.7 kb 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.
Publisher: Springer Science and Business Media LLC
Date: 18-05-2016
Publisher: Publiverse Online S.R.L
Date: 2011
Publisher: Springer International Publishing
Date: 2019
DOI: 10.1007/102_2018_3
Abstract: Mitochondrial DNA (mtDNA) encodes proteins for the electron transport chain which produces the vast majority of cellular energy. MtDNA has its own replication and transcription machinery that relies on nuclear-encoded transcription and replication factors. MtDNA is inherited in a non-Mendelian fashion as maternal-only mtDNA is passed onto the next generation. Mutation to mtDNA can cause mitochondrial dysfunction, which affects energy production and tissue and organ function. In somatic cell nuclear transfer (SCNT), there is an issue with the mixing of two populations of mtDNA, namely from the donor cell and recipient oocyte. This review focuses on the transmission of mtDNA in SCNT embryos and offspring. The transmission of donor cell mtDNA can be prevented by depleting the donor cell of its mtDNA using mtDNA depletion agents prior to SCNT. As a result, SCNT embryos harbour oocyte-only mtDNA. Moreover, culturing SCNT embryos derived from mtDNA depleted cells in media supplemented with a nuclear reprograming agent can increase the levels of expression of genes related to embryo development when compared with non-depleted cell-derived embryos. Furthermore, we have reviewed how mitochondrial supplementation in oocytes can have beneficial effects for SCNT embryos by increasing mtDNA copy number and the levels of expression of genes involved in energy production and decreasing the levels of expression of genes involved in embryonic cell death. Notably, there are beneficial effects of mtDNA supplementation over the use of nuclear reprograming agents in terms of regulating gene expression in embryos. Taken together, manipulating mtDNA in donor cells and/or oocytes prior to SCNT could enhance embryo production efficiency.
Publisher: Elsevier BV
Date: 04-2016
DOI: 10.1016/J.SEMCDB.2016.01.028
Abstract: Until recently, it was thought that the role of the mitochondrial genome was confined to encoding key proteins that generate ATP through the process of oxidative phosphorylation in the electron transfer chain. However, with increasing new evidence, it is apparent that the mitochondrial genome has a major role to play in a number of diseases and phenotypes. For ex le, mitochondrial variants and copy number have been implicated in the processes of fertilisation outcome and development and the onset of tumorigenesis. On the other hand, mitochondrial DNA (mtDNA) haplotypes have been implicated in a variety of diseases and most likely account for the adaptation that our ancestors achieved in order that they were fit for their environments. The mechanisms, which enable the mitochondrial genome to either protect or promote the disease phenotype, require further elucidation. However, there appears to be significant 'crosstalk' between the chromosomal and mitochondrial genomes that enable this to take place. One such mechanism is the regulation of DNA methylation by mitochondrial DNA, which is often perturbed in reprogrammed cells that have undergone dedifferentiation and affects mitochondrial DNA copy number. Furthermore, it appears that the mitochondrial genome interacts with the chromosomal genome to regulate the transcription of key genes at certain stages during development. Additionally, the mitochondrial genome can accumulate a series of mtDNA variants, which can lead to diseases such as cancer. It is likely that a combination of certain mitochondrial variants and aberrant patterns of mtDNA copy number could indeed account for many diseases that have previously been unaccounted for. This review focuses on the role that the mitochondrial genome plays especially during early stages of development and in cancer.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 06-2016
Publisher: Wiley
Date: 20-07-2020
DOI: 10.1111/IMCB.12363
Publisher: Elsevier BV
Date: 2008
DOI: 10.1111/J.1432-0436.2007.00247.X
Abstract: Gametes of both sexes (sperm and oocyte) are highly specialized and differentiated but within a very short time period post-fertilization the embryonic genome, produced by the combination of the two highly specialized parental genomes, is completely converted into a totipotent state. As a result, the one-cell-stage embryo can give rise to all cell types of all three embryonic layers, including the gametes. Thus, it is evident that extensive and efficient reprogramming steps occur soon after fertilization and also probably during early embryogenesis to reverse completely the differentiated state of the gamete and to achieve toti- or later on pluripotency of embryonic cells. However, after the embryo reaches the blastocyst stage, the first two distinct cell lineages can be clearly distinguished--the trophectoderm and the inner cells mass. The de-differentiation of gametes after fertilization, as well as the differentiation that is associated with the formation of blastocysts, are accompanied by changes in the state and properties of chromatin in in idual embryonic nuclei at both the whole genome level as well as at the level of in idual genes. In this contribution, we focus mainly on those events that take place soon after fertilization and during early embryogenesis in mammals. We will discuss the changes in DNA methylation and covalent histone modifications that were shown to be highly dynamic during this period moreover, it has also been documented that abnormalities in these processes have a devastating impact on the developmental ability of embryos. Special attention will be paid to somatic cell nuclear transfer as it has been shown that the aberrant and inefficient reprogramming may be responsible for compromised development of cloned embryos.
Publisher: Elsevier BV
Date: 09-1997
Publisher: Springer Science and Business Media LLC
Date: 11-09-2017
DOI: 10.1038/CDDISCOVERY.2017.62
Abstract: 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.
Publisher: Oxford University Press (OUP)
Date: 30-09-2014
Abstract: What are the effects on fertility of cigarette smoke-induced toxicity on male offspring exposed during the gestational/weaning period? Maternal cigarette smoke exposure during the gestational/weaning period causes long-term defects in male offspring fertility. Cigarette smoke is a well-known reproductive toxicant which is particularly harmful to both fetal and neonatal germ cells. However, recent studies suggest a significant portion of young mothers in the developed world still smoke during pregnancy. In the context of male reproductive health, our understanding of the effects of in utero exposure on offspring fertility is limited. In this study, 27 C57BL/6 5-week-old female mice were exposed via the nose-only to cigarette smoke (treatment) or 27 were exposed to room air (control) for 6 weeks before being housed with stud males to produce litters. In the treatment group, smoke exposure continued throughout mating, pregnancy and lactation until weaning of pups at 21 days post birth. Male offspring were examined at post-natal days 3, 6, 12, 21 and 98 (adult). Approximately 108 maternal smoke-exposed C57BL/6 offspring and controls were examined. Spermatogenesis was examined using testicular histology and apoptosis/DNA damage was assessed using caspase immunohistochemistry and TUNEL. Sertoli cell morphology and fluctuations in the spermatogonial stem cell population were also examined using immunohistochemistry. Microarray and QPCR analysis were performed on adult testes to examine specific long-term transcriptomic alteration as a consequence of maternal smoke exposure. Sperm counts and motility, zona/oolemma binding assays, COMET analysis and mitochondrial genomic sequencing were also performed on spermatozoa obtained from adult treated and control mice. Fertility trials using exposed adult male offspring were also performed. Maternal cigarette smoke exposure caused increased gonocyte and meiotic spermatocyte apoptosis (P < 0.01) as well as germ cell depletion in the seminiferous tubules of neonatal and juvenile offspring. Aberrant testicular development characterized by abnormal Sertoli and germ cell organization, a depleted spermatogonial stem cell population (P < 0.01), atrophic seminiferous tubules and increased germ cell DNA damage (P < 0.01) persisted in adult offspring 11 weeks after exposure. Microarray analysis of adult offspring testes associated these defects with meiotic germ cell development, sex hormone metabolism, oxidative stress and Sertoli cell signalling. Next generation sequencing also revealed a high mitochondrial DNA mutational load in the testes of adult offspring (P < 0.01). Adult maternal smoke-exposed offspring also had reduced sperm counts with spermatozoa exhibiting morphological abnormalities (P < 0.01), affecting motility and fertilization potential. Odf2, a spermatozoa flagellum component required for coordinated ciliary beating, was also significantly down-regulated (P < 0.01) in maternal smoke-exposed adult offspring, with aberrant localization along the spermatozoa flagellum. Adult maternal smoke-exposed offspring took significantly longer to impregnate control females and had a slight but significant (P < 0.01) reduction in litter size. This study examined only one species (mouse) using a smoking model which only simulates human cigarette smoke exposure. This study represents the first comprehensive animal model of maternal smoking on male offspring reproductive function, suggesting that exposure during the gestational/weaning period causes long-term defects in male offspring fertility. This is due to a compromised spermatogonial stem cell population resulting from gonocyte apoptosis and impaired spermatogenic development. This results in significant germ cell damage and Sertoli cell dysfunction, impacting germ cell number, tubule organization, DNA damage and spermatozoa in adult offspring. This study strengthens the current literature suggesting that maternal exposure impairs male offspring fertility, which is currently debated due to conflicting studies. This study was funded by the Australian Research Council, Hunter Medical Research Institute, National Health and Medical Research Council of Australia and the Newcastle Permanent Building Society Charitable Trust. The authors declare no conflict of interest.
Publisher: Elsevier BV
Date: 2002
DOI: 10.1016/S0093-691X(01)00661-6
Abstract: Mitochondria, among other functions, generate energy in the form of ATP. The chondrial genome, located within each mitochondrion, encodes some of the polypeptides associated with the electron transfer chain (ETC) and ATP production. Transcription and replication of mitochondrial DNA (mtDNA) is dependent upon the import of transcription and replication factors encoded by the nucleus. Certain point mutations and large-scale deletions to mtDNA can be either severely debilitating or lethal. The transmission and inheritance of mtDNA [not readable: see to offspring is strictly regulated and specific to each species. In many mammalian systems, paternal mtDNA is eliminated very early during embryonic development. However, it is possible that the paternal molecule could be extruded to those cells destined to become trophoblasts and may act as a regulator of embryonic cell fate. Furthermore, the increasing use of more sophisticated assisted reproductive techniques has led to the incorporation of extraneous mtDNA in both the reconstructed oocyte and embryo with transmission to the offspring at varying degrees.
Publisher: Oxford University Press (OUP)
Date: 24-11-2014
Publisher: Elsevier BV
Date: 2004
DOI: 10.1016/S1472-6483(10)60496-8
Abstract: The recent introduction of more invasive assisted reproductive techniques offers the possibility to provide a wider treatment profile to patients. However, some of these technologies are of considerable concern as they are fraught with the possible transmission of genetic abnormalities to the offspring they create. To date, much analysis of these technologies has been conducted at the chromosomal DNA level. While some analysis has been conducted on the extranuclear, mitochondrial genome (mtDNA), this has been mainly descriptive. In the vast majority of cases, it appears that mtDNA is maternally inherited. The impact that leakage of sperm mtDNA transmission might have for the offspring is discussed in the light of the recent identification of sperm mtDNA presence in a patient with mtDNA disease. The implications of introducing donor mtDNA into a recipient oocyte through both cytoplasmic and nuclear transfer are also discussed. Again, the implications for offspring survival are discussed and suggestions made as to why the techniques might provide valuable insights into mtDNA transmission, replication and transcription. In order to be confident that patients and their offspring are being offered safe treatment, it is argued that potentially some of these treatments may be of considerable benefit in the future but significant scientific research is required before these treatments can be effectively employed in the clinic.
Publisher: Oxford University Press (OUP)
Date: 10-01-2008
DOI: 10.1634/STEMCELLS.2007-0747
Abstract: Following fertilization, mitochondrial DNA is inherited from the oocyte and transmitted homoplasmically. However, following nuclear transfer, mitochondrial DNA can be transmitted from both the donor cell and recipient oocyte, resulting in a state of heteroplasmy. To determine whether the genetic ersity between donor cell and recipient cytoplast mitochondrial DNA influences development, we generated bovine embryos by fusing a donor cell to one or more enucleated cytoplasts. Analysis of mitochondrial DNA from embryos, fetal tissues, and blood s les from offspring revealed that early preimplantation embryos from two or three cytoplasts had significantly more mitochondrial DNA variants than fetal tissues. Phylogenic analysis of embryos generated using single cytoplasts ided the mitochondrial DNA sequence variants into three separate groups with various amounts of genetic ergence from the donor cell line. In heteroplasmic tissue and blood s les, the predominant mitochondrial DNA population was significantly more ergent from the donor cell than the less frequent allele. Furthermore, analysis of the mitochondrially encoded cytochrome B gene showed that two heteroplasmic alleles encoded for different amino acids, and the ratios of mitochondrial DNA/mRNA for each allele differed significantly between tissues. The degree of evolutionary distance between the donor cell and the cytoplast and the variability in heteroplasmy between tissues may have an impact on more ergent intergeneric nuclear transfer and the use of this approach for the generation of embryonic stem cells. Disclosure of potential conflicts of interest is found at the end of this article.
Publisher: Mary Ann Liebert Inc
Date: 02-2016
Abstract: The vast majority of cellular ATP is produced by the oxidative phosphorylation (OXPHOS) system, which comprises the four complexes of the electron transfer chain plus the ATP synthase. Complex I is the largest of the OXPHOS complexes, and mutation of the genes encoding either the subunits or assembly factors of Complex I can result in Complex I deficiency, which is the most common OXPHOS disorder. Mutations in the Complex I gene NDUFS4 lead to Leigh syndrome, which is the most frequent presentation of Complex I deficiency in children presenting with progressive encephalopathy shortly after birth. Symptoms include motor and intellectual retardation, often accompanied by dystonia, ataxia, and growth retardation, and most patients die by 3 years of age. To understand the origins of this disease, we have generated a series of mouse embryonic stem cell lines from blastocysts that were wild type, heterozygous, and homozygous for the deletion of the Ndufs4 gene. We have demonstrated their pluripotency and potential to differentiate into all cell types of the body. Although the loss of Ndufs4 did not affect the stability of the mitochondrial and nuclear genomes, there were significant differences in patterns of chromosomal gene expression following both spontaneous differentiation and directed neural differentiation into astrocytes. The defect also affected the potential of the cells to generate beating embryoid bodies. These outcomes demonstrate that defects associated with Complex I deficiency affect early gene expression patterns, which escalate during early and later stages of differentiation and are mediated by the defect and not other chromosomal or mitochondrial DNA defects.
Publisher: Elsevier BV
Date: 09-1999
Publisher: Oxford University Press (OUP)
Date: 27-10-2009
DOI: 10.1002/STEM.244
Abstract: During development, cell differentiation is accompanied by the progressive loss of pluripotent gene expression and developmental potential, although de-differentiation in specialized cells can be induced by reprogramming strategies, indicating that transdifferentiation potential is retained in adult cells. The stromal niche provides differentiating cues to epithelial stem cells (SCs), but current evidence is restricted to tissue types within the same developmental germ layer lineage. Anticipating the use of adult SCs for tissue regeneration, we examined if stroma can enforce lineage commitment across germ layer boundaries and promote transdifferentiation of adult epithelial SCs. Here, we report tissue-specific mesenchyme instructing epithelial cells from a different germ layer origin to express dual phenotypes. Prostatic stroma induced mammary epithelia (or enriched Lin−CD29HICD24+/MOD mammary SCs) to generate glandular epithelia expressing both prostatic and mammary markers such as steroid hormone receptors and transcription factors including Foxa1, Nkx3.1, and GATA-3. Array data implicated Hh and Wnt pathways in mediating stromal-epithelial interactions (validated by increased Cyclin D1 expression). Other recombinants of prostatic mesenchyme and skin epithelia, or preputial gland mesenchyme and bladder or esophageal epithelia, showed foci expressing new markers adjacent to the original epithelial differentiation (e.g., sebaceous cells within bladder urothelium), confirming altered lineage specification induced by stroma and evidence of cross-germ layer transdifferentiation. Thus, stromal cell niche is critical in maintaining (or redirecting) differentiation in adult epithelia. In order to use adult epithelial SCs in regenerative medicine, we must additionally regulate their intrinsic properties to prevent (or enable) transdifferentiation in specified SC niches. Disclosure of potential conflicts of interest is found at the end of this article.
Publisher: Springer Science and Business Media LLC
Date: 04-04-2016
DOI: 10.1038/CDDISCOVERY.2016.16
Abstract: Although mitochondrial DNA has been implicated in diseases such as cancer, its role remains to be defined. Using three models of tumorigenesis, namely glioblastoma multiforme, multiple myeloma and osteosarcoma, we show that mitochondrial DNA plays defining roles at early and late tumour progression. Specifically, tumour cells partially or completely depleted of mitochondrial DNA either restored their mitochondrial DNA content or actively recruited mitochondrial DNA, which affected the rate of tumorigenesis. Nevertheless, non-depleted tumour cells modulated mitochondrial DNA copy number at early and late progression in a mitochondrial DNA genotype-specific manner. In glioblastoma multiforme and osteosarcoma, this was coupled with loss and gain of mitochondrial DNA variants. Changes in mitochondrial DNA genotype affected tumour morphology and gene expression patterns at early and late progression. Importantly, this identified a subset of genes that are essential to early progression. Consequently, mitochondrial DNA and commonly expressed early tumour-specific genes provide novel targets against tumorigenesis.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 05-2001
Publisher: Elsevier
Date: 2007
Publisher: Annual Reviews
Date: 16-02-2021
DOI: 10.1146/ANNUREV-ANIMAL-080520-083353
Abstract: Our understanding of the interactions between the nuclear and mitochondrial genomes is becoming increasingly important as they are extensively involved in establishing early development and developmental progression. Evidence from various biological systems indicates the interdependency between the genomes, which requires a high degree of compatibility and synchrony to ensure effective cellular function throughout development and in the resultant offspring. During development, waves of DNA demethylation, de novo methylation, and maintenance methylation act on the nuclear genome and typify oogenesis and pre- and postimplantation development. At the same time, significant changes in mitochondrial DNA copy number influence the metabolic status of the developing organism in a typically cell-type-specific manner. Collectively, at any given stage in development, these actions establish genomic balance that ensures each developmental milestone is met and that the organism's program for life is established.
Publisher: Wiley
Date: 23-10-2003
DOI: 10.1002/CM.10151
Abstract: Mutations in the human LIS1 gene cause the devastating brain disorder lissencephaly. LIS1 also regulates microtubule dynamics it interacts with the molecular motor cytoplasmic dynein and its cofactor dynactin, and is necessary for neuronal migration. Recently, LIS1 has been suggested to mediate pronuclear migration during fertilization. Here we use rhesus monkey and bovine oocytes, as well as pronucleate-stage bovine zygotes, to determine: Lis1 RNA expression using reverse transcription-polymerase chain reaction LIS1 protein association with dynactin using immunoprecipitation, Western blot analysis, and immunocytochemistry and LIS1 function in mediating genomic union using antibody transfection. We find that Lis1 RNA expression increases during fertilization, that LIS1 and dynactin subunit p150/(Glued) co-immunoprecipitate and co-localize to pronuclear surfaces, and that anti-LIS1 antibodies transfected into zygotes dramatically inhibit pronuclear migration and apposition. LIS1 is, therefore, essential to mediate genomic union in a process that involves the dynein-dynactin complex. These results shed light on an additional role for LIS1 and raise implications for human reproduction.
Publisher: Elsevier BV
Date: 03-2006
DOI: 10.1016/J.FERTNSTERT.2005.09.017
Abstract: To determine the content of mitochondrial DNA (mtDNA) in oocytes from a range of patients with fertilization success and failure. Analysis of mtDNA content in fertilized and unfertilized oocytes and embryos by real-time polymerase chain reaction (PCR). University hospital infertility and research center. Fifty-four women seeking treatment for infertility. None. A total of 142 fertilized and unfertilized oocytes were classified into three main groups. Group I consisted of 35 fertilized oocytes from 21 patients group II, 65 unfertilized oocytes from 36 patients and group III, 42 degenerate oocytes from 23 patients. Mitochondrial DNA content was determined by SYBR Green real-time PCR-based assay. The mean mtDNA copy number for the fertilized oocytes was 250,454, whereas for the unfertilized group it was 163,698. There were significant differences for mtDNA copy number between the male factor and female factor infertility unfertilized oocytes and between the unexplained infertility and female factor infertility groups. The mean copy number for the degenerate oocyte group was 44,629, which was significantly different from the other sub isions in this group. Mitochondrial DNA content is critical to fertilization outcome and serves as an important marker of oocyte quality, explaining some cases of fertilization failure.
Publisher: Oxford University Press (OUP)
Date: 07-2017
Abstract: The mixing of mitochondrial DNA (mtDNA) from the donor cell and the recipient oocyte in embryos and offspring derived from somatic cell nuclear transfer (SCNT) compromises genetic integrity and affects embryo development. We set out to generate SCNT embryos that inherited their mtDNA from the recipient oocyte only, as is the case following natural conception. While SCNT blastocysts produced from Holstein (Bos taurus) fibroblasts were depleted of their mtDNA, and oocytes derived from Angus (Bos taurus) cattle possessed oocyte mtDNA only, the coexistence of donor cell and oocyte mtDNA resulted in blastocysts derived from nondepleted cells. Moreover, the use of the reprogramming agent, Trichostatin A (TSA), further improved the development of embryos derived from depleted cells. RNA-seq analysis highlighted 35 differentially expressed genes from the comparison between blastocysts generated from nondepleted cells and blastocysts from depleted cells, both in the presence of TSA. The only differences between these two sets of embryos were the presence of donor cell mtDNA, and a significantly higher mtDNA copy number for embryos derived from nondepleted cells. Furthermore, the use of TSA on embryos derived from depleted cells positively modulated the expression of CLDN8, TMEM38A, and FREM1, which affect embryonic development. In conclusion, SCNT embryos produced by mtDNA depleted donor cells have the same potential to develop to the blastocyst stage without the presumed damaging effect resulting from the mixture of donor and recipient mtDNA.
Publisher: Springer Science and Business Media LLC
Date: 06-07-2018
Publisher: Springer Science and Business Media LLC
Date: 03-04-2009
DOI: 10.1007/S12015-009-9058-0
Abstract: Pluripotent blastomeres of mammalian pre-implantation embryos and embryonic stem cells (ESCs) are characterized by limited oxidative capacity and great reliance on anaerobic respiration. Early pre-implantation embryos and undifferentiated ESCs possess small and immature mitochondria located around the nucleus, have low oxygen consumption and express high levels of glycolytic enzymes. However, as embryonic cells and ESCs lose pluripotency and commit to a specific cell fate, the expression of mtDNA transcription and replication factors is upregulated and the number of mitochondria and mtDNA copies/cell increases. Moreover, upon cellular differentiation, mitochondria acquire an elongated morphology with swollen cristae and dense matrices, migrate into wider cytoplasmic areas and increase the levels of oxygen consumption and ATP production as a result of the activation of the more efficient, aerobic metabolism. Since pluripotency seems to be associated with anaerobic metabolism and a poorly developed mitochondrial network and differentiation leads to activation of mitochondrial biogenesis according to the metabolic requirements of the specific cell type, it is hypothesized that reprogramming of somatic cells towards a pluripotent state, by somatic cell nuclear transfer (SCNT), transcription-induced pluripotency or creation of pluripotent cell hybrids, requires acquisition of mitochondrial properties characteristic of pluripotent blastomeres and ESCs.
Publisher: Springer Science and Business Media LLC
Date: 02-2008
DOI: 10.1038/451627A
Publisher: American Society for Microbiology
Date: 24-04-2012
DOI: 10.1128/JB.00377-12
Publisher: Elsevier BV
Date: 2000
DOI: 10.1016/S0140-6736(99)03842-8
Abstract: Paternal mitochondrial DNA is normally eliminated from mammalian embryos. We have shown the presence of paternal mtDNA at the blastocyst stage in some abnormal human embryos.
Publisher: Public Library of Science (PLoS)
Date: 27-04-2011
Publisher: Springer New York
Date: 2015
DOI: 10.1007/978-1-4939-2848-4_18
Abstract: The mitochondrial genome has a major role to play in establishing and maintaining pluripotency. Furthermore, mitochondrial DNA (mtDNA) copy is strictly regulated during differentiation. Undifferentiated, pluripotent cells possess fewer than 300 copies of mtDNA, which establishes the mtDNA set point and promotes cell proliferation and, as a result, these cells rely on glycolysis with some support from oxidative phosphorylation (OXPHOS) for the generation of ATP. The mtDNA set point provides the starting point from which cells increase their mtDNA copy number as they differentiate into mature functional cells. Dependent on cell types, mtDNA copy number ranges from ~10 copies in sperm to several thousand in cardiomyocytes. Consequently, differentiating cell types can acquire the appropriate numbers of mtDNA copy to meet their specific requirements for ATP generated through OXPHOS. However, as reprogrammed somatic cells do not always achieve this, it is essential to analyze them for their OXPHOS potential and ability to regulate mtDNA copy number. Here, we describe how to assess mtDNA copy number in pluripotent and differentiating cells using real-time PCR protocols assess expression of the mtDNA specific replication factors through real-time RT-PCR identify mtDNA variants in embryonic and induced pluripotent stem cells determine DNA methylation patterns of the mtDNA-specific replication factors and assess mitochondrial OXPHOS capacity.
Publisher: Wiley
Date: 21-03-2005
DOI: 10.1111/J.1365-2605.2005.00515.X
Abstract: Summary Human mitochondrial DNA (mtDNA) encodes 13 of the polypeptides associated with the process of oxidative phosphorylation (OXPHOS), the cells most important ATP generating pathway. Until recently, the effects of mtDNA rearrangements on male fertility have been largely ignored. However, it is becoming increasingly evident that both point mutations and large-scale deletions may have an impact on sperm motility and morphology. We discuss the implications of these rearrangements in the context of the clinical setting. We further discuss the possible consequences resulting from the transmission of sperm mtDNA deletions to the offspring. The role of nucleo-cytoplasmic interaction is investigated in the context of nuclear transcription and replication factors that regulate mtDNA transcription and replication.
Publisher: Springer Science and Business Media LLC
Date: 08-05-2018
DOI: 10.1038/S41598-018-25516-3
Abstract: We generated cattle embryos using mitochondrial supplementation and somatic cell nuclear transfer (SCNT), named miNT, to determine how additional mitochondrial DNA (mtDNA) modulates the nuclear genome. To eliminate any confounding effects from somatic cell mtDNA in intraspecies SCNT, donor cell mtDNA was depleted prior to embryo production. Additional oocyte mtDNA did not affect embryo development rates but increased mtDNA copy number in blastocyst stage embryos. Moreover, miNT-derived blastocysts had different gene expression profiles when compared with SCNT-derived blastocysts. Additional mtDNA increased expression levels of genes involved in oxidative phosphorylation, cell cycle and DNA repair. Supplementing the embryo culture media with a histone deacetylase inhibitor, Trichostatin A (TSA), had no beneficial effects on the development of miNT-derived embryos, unlike SCNT-derived embryos. When compared with SCNT-derived blastocysts cultured in the presence of TSA, additional mtDNA alone had beneficial effects as the activity of glycolysis may increase and embryonic cell death may decrease. However, these beneficial effects were not found with additional mtDNA and TSA together, suggesting that additional mtDNA alone enhances reprogramming. In conclusion, additional mtDNA increased mtDNA copy number and expression levels of genes involved in energy production and embryo development in blastocyst stage embryos emphasising the importance of nuclear-mitochondrial interactions.
Publisher: Oxford University Press (OUP)
Date: 31-03-2006
Abstract: Mitochondria are the organelles responsible for producing the majority of a cell's ATP and also play an essential role in gamete maturation and embryo development. ATP production within the mitochondria is dependent on proteins encoded by both the nuclear and the mitochondrial genomes, therefore co-ordination between the two genomes is vital for cell survival. To assist with this co-ordination, cells normally contain only one type of mitochondrial DNA (mtDNA) termed homoplasmy. Occasionally, however, two or more types of mtDNA are present termed heteroplasmy. This can result from a combination of mutant and wild-type mtDNA molecules or from a combination of wild-type mtDNA variants. As heteroplasmy can result in mitochondrial disease, various mechanisms exist in the natural fertilization process to ensure the maternal-only transmission of mtDNA and the maintenance of homoplasmy in future generations. However, there is now an increasing use of invasive oocyte reconstruction protocols, which tend to bypass mechanisms for the maintenance of homoplasmy, potentially resulting in the transmission of either form of mtDNA heteroplasmy. Indeed, heteroplasmy caused by combinations of wild-type variants has been reported following cytoplasmic transfer (CT) in the human and following nuclear transfer (NT) in various animal species. Other techniques, such as germinal vesicle transfer and pronuclei transfer, have been proposed as methods of preventing transmission of mitochondrial diseases to future generations. However, resulting embryos and offspring may contain mtDNA heteroplasmy, which itself could result in mitochondrial disease. It is therefore essential that uniparental transmission of mtDNA is ensured before these techniques are used therapeutically.
Publisher: Springer Science and Business Media LLC
Date: 02-1997
DOI: 10.1038/NM0297-124C
Publisher: Oxford University Press (OUP)
Date: 13-03-2018
Abstract: What are the molecular differences between mitochondrial DNA (mtDNA)-deficient and mtDNA-normal oocytes and how does mitochondrial supplementation alter these? 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. 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. 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. 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. 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. 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. 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. 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. This work was funded by OvaScience Inc. OvaScience did not influence the study design, analysis of results or interpretation of the data.
Publisher: Frontiers Media SA
Date: 07-02-2022
DOI: 10.3389/FGENE.2021.762177
Abstract: Polycystic Ovary Syndrome (PCOS) is a multifactorial syndrome with reproductive, endocrine, and metabolic symptoms, affecting about 10% women of reproductive age. Pathogenesis of the syndrome is poorly understood with genetic and fetal origins being the focus of the conundrum. Genetic predisposition of PCOS has been confirmed by candidate gene studies and Genome-Wide Association Studies (GWAS). Recently, the expression of PCOS candidate genes across gestation has been studied in human and bovine fetal ovaries. The current study sought to identify potential upstream regulators and mechanisms associated with PCOS candidate genes. Using RNA sequencing data of bovine fetal ovaries (62–276 days, n = 19), expression of PCOS candidate genes across gestation was analysed using Partek Flow. A supervised heatmap of the expression data of all 24,889 genes across gestation was generated. Most of the PCOS genes fell into one of four clusters according to their expression patterns. Some genes correlated negatively (early genes C8H9orf3 , TOX3 , FBN3 , GATA4 , HMGA2 , and DENND1A ) and others positively (late genes FDFT1 , LHCGR , AMH , FSHR , ZBTB16 , and PLGRKT ) with gestational age. Pathways associated with PCOS candidate genes and genes co-expressed with them were determined using Ingenuity pathway analysis (IPA) software as well as DAVID Bioinformatics Resources for KEGG pathway analysis and Gene Ontology databases. Genes expressed in the early cluster were mainly involved in mitochondrial function and oxidative phosphorylation and their upstream regulators included PTEN , ESRRG/A and MYC . Genes in the late cluster were involved in stromal expansion, cholesterol biosynthesis and steroidogenesis and their upstream regulators included TGFB1/2/3 , TNF, ERBB2/3 , VEGF , INSIG1 , POR , and IL25 . These findings provide insight into ovarian development of relevance to the origins of PCOS, and suggest that multiple aetiological pathways might exist for the development of PCOS.
Publisher: Mary Ann Liebert Inc
Date: 06-2010
Abstract: Generally in mammals, in idual animals contain only maternally inherited mitochondrial DNA (mtDNA), as paternal (sperm)-derived mitochondria are usually eliminated during early development. Somatic cell nuclear transfer (SCNT) bypasses the normal routes of mtDNA inheritance and introduces not only a different nuclear genome into the recipient cytoplast (in general an enucleated oocyte) but also somatic mitochondria. Differences in mtDNA genotype between recipient oocytes and potential mtDNA heteroplasmy due to persistence and replication of somatic mtDNA means that offspring generated by SCNT are not true clones. However, more importantly, the consequences of the presence of somatic mtDNA, mtDNA heteroplasmy, or possible incompatibility between nuclear and mtDNA genotypes on subsequent development and function of the embryo, fetus and offspring are unknown. Following sexual reproduction, mitochondrial function requires the biparental control of maternally inherited mtDNA, whereas following SCNT incompatibility between the recipient cell mitochondrial and transplanted nuclear genomes, or mtDNA heteroplasmy, may result in energy imbalance and initiate the onset of mtDNA-type disease, or disruption of normal developmental events. To remove the potentially adverse effects of somatic mtDNA following SCNT we have previously produced embryos using donor cells depleted to residual levels of mtDNA (mtDNA). We now report that these cells support development to term and produced live lambs in which no donor somatic mtDNA was detected, the lambs being homoplasmic for recipient oocyte DNA.
Publisher: Elsevier BV
Date: 09-2006
Publisher: Elsevier BV
Date: 09-2008
DOI: 10.1016/J.TIBTECH.2008.06.001
Abstract: The recent vote in the British Parliament allows scientists in principle to create hybrid embryos by transferring human somatic cell nuclei into animal oocytes. This vote opens a fascinating new area of research with the central aim of generating interspecific lines of embryonic stem cells (ESCs) that could potentially be used to understand development, differentiation, gene expression and genomic compatibility. It will also promote human cell therapies, as well as the pharmaceutical industry's search for new drug targets. If this approach is to be successful, many biological questions need to be answered and, in addition, some moral and ethical aspects must be taken into account.
Publisher: Impact Journals, LLC
Date: 28-06-2017
Publisher: Oxford University Press (OUP)
Date: 05-2015
Abstract: Body weight and adiposity are determined by the balance between energy intake, energy expenditure, and nutrient deposition. We have identified differences in appetite-regulating peptides in sheep selectively bred to be either lean or fat, wherein gene expression for orexin and melanin-concentrating hormone are elevated in the lean group. Despite this, the underlying mechanisms leading to differences in body composition in the lean and fat lines remains unknown. We measured postprandial temperature in adipose tissue and muscle to ascertain whether a difference in thermogenesis is associated with the difference in body composition in genetically lean (n = 8) and fat (n = 12) ewes. Body weight was higher (P < 0.01) but percent fat mass was lower (P < 0.001) in the lean group. The percent lean mass was similar in lean and fat groups. Animals received intracerebroventricular cannulae and temperature probes implanted into the retroperitoneal fat and the hind-limb skeletal muscle (vastus lateralis). Animals were meal fed (1100-1600 h) to entrain postprandial thermogenesis. Food intake was similar between lean and fat animals. Postprandial thermogenesis was greater (P < 0.05) in the retroperitoneal adipose tissue of lean animals but not in skeletal muscle. Intracerebroventricular infusion of leptin reduced (P< 0.05) food intake by an equal extent in both groups. Postprandial expression of UCP1 mRNA was greater (P < 0.05) in retroperitoneal fat of lean animals, with similar UCP3 expression in skeletal muscle. Mitochondrial genome sequencing indicated haplotypic clustering in lean and fat animals within both the encoding and nonencoding regions. This demonstrates that differences in body composition may be underpinned by differences in thermogenesis, specifically within adipose tissue. Furthermore, thermogenic differences may be associated with specific mitochondrial DNA haplotypes, suggesting a strong genetic component inherited through the maternal lineage.
Publisher: Wiley
Date: 12-2005
DOI: 10.1002/MRD.20370
Abstract: Double nuclear transfer begins with the transfer of nuclear DNA from a donor cell into an enucleated recipient oocyte. This reconstructed oocyte is allowed to develop to the pronuclear stage, where the pronuclei are transferred into an enucleated zygote. This reconstructed zygote is then transferred to a surrogate sow. The genetic integrity of cloned offspring can be compromised by the transmission of mitochondrial DNA from the donor cell, the recipient oocyte and the recipient zygote. We have verified through the use of sequence analysis, restriction fragment length polymorphism analysis, allele specific PCR and primer extension polymorphism analysis that following double nuclear transfer the donor cell mtDNA is eliminated. However, it is likely that the recipient oocyte and zygote mitochondrial DNA are transmitted to the offspring, indicating bimaternal mitochondrial DNA transmission. This pattern of mtDNA inheritance is similar to that observed following cytoplasmic transfer and violates the strict unimaternal inheritance of mitochondrial DNA to offspring. This form of transmission raises concerns regarding the genetic integrity of cloned offspring and their uses in studies that require metabolic analysis or a stable genetic environment where only one variable is under analysis, such as in knockout technology.
Publisher: Oxford University Press (OUP)
Date: 06-2004
DOI: 10.1534/GENETICS.103.025049
Abstract: Offspring produced by nuclear transfer (NT) have identical nuclear DNA (nDNA). However, mitochondrial DNA (mtDNA) inheritance could vary considerably. In sheep, homoplasmy is maintained since mtDNA is transmitted from the oocyte (recipient) only. In contrast, cattle are heteroplasmic, harboring a predominance of recipient mtDNA along with varying levels of donor mtDNA. We show that the two nonhuman primate Macaca mulatta offspring born by NT have mtDNA from three sources: (1) maternal mtDNA from the recipient egg, (2) maternal mtDNA from the egg contributing to the donor blastomere, and (3) paternal mtDNA from the sperm that fertilized the egg from which the donor blastomere was isolated. The introduction of foreign mtDNA into reconstructed recipient eggs has also been demonstrated in mice through pronuclear injection and in humans through cytoplasmic transfer. The mitochondrial triplasmy following M. mulatta NT reported here forces concerns regarding the parental origins of mtDNA in clinically reconstructed eggs. In addition, mtDNA heteroplasmy might result in the embryonic stem cell lines generated for experimental and therapeutic purposes (“therapeutic cloning”).
Publisher: Wiley
Date: 20-08-2022
DOI: 10.1002/MRD.23640
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. In idual 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 ex le, 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.
Publisher: Oxford University Press (OUP)
Date: 06-12-2012
Publisher: Oxford University Press (OUP)
Date: 25-01-2016
DOI: 10.1534/GENETICS.115.181321
Abstract: 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 s les 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.
Publisher: Springer Science and Business Media LLC
Date: 26-06-2017
Publisher: Oxford University Press (OUP)
Date: 24-03-2013
DOI: 10.1002/STEM.1313
Abstract: Mitochondrial DNA haplotypes are associated with various phenotypes, such as altered susceptibility to disease, environmental adaptations, and aging. Accumulating evidence suggests that mitochondrial DNA is essential for cell differentiation and the cell phenotype. However, the effects of different mitochondrial DNA haplotypes on differentiation and development remain to be determined. Using embryonic stem cell lines possessing the same Mus musculus chromosomes but harboring one of Mus musculus, Mus spretus, or Mus terricolor mitochondrial DNA haplotypes, we have determined the effects of different mitochondrial DNA haplotypes on chromosomal gene expression, differentiation, and mitochondrial metabolism. In undifferentiated and differentiating embryonic stem cells, we observed mitochondrial DNA haplotype-specific expression of genes involved in pluripotency, differentiation, mitochondrial energy metabolism, and DNA methylation. These mitochondrial DNA haplotypes also influenced the potential of embryonic stem cells to produce spontaneously beating cardiomyocytes. The differences in gene expression patterns and cardiomyocyte production were independent of ATP content, oxygen consumption, and respiratory capacity, which until now have been considered to be the primary roles of mitochondrial DNA. Differentiation of embryonic stem cells harboring the different mitochondrial DNA haplotypes in a 3D environment significantly increased chromosomal gene expression for all haplotypes during differentiation. However, haplotype-specific differences in gene expression patterns were maintained in this environment. Taken together, these results provide significant insight into the phenotypic consequences of mitochondrial DNA haplotypes and demonstrate their influence on differentiation and development. We propose that mitochondrial DNA haplotypes play a pivotal role in the process of differentiation and mediate the fate of the cell.
Publisher: The Company of Biologists
Date: 15-11-2007
DOI: 10.1242/JCS.016972
Abstract: Oxidative phosphorylation (OXPHOS), the intracellular process that generates the majority of the ATP of a cell through the electron-transfer chain, is highly dependent on proteins encoded by the mitochondrial genome (mtDNA). MtDNA replication is regulated by the nuclear-encoded mitochondrial transcription factor A (TFAM) and the mitochondrial-specific DNA polymerase gamma, which consists of a catalytic (POLG) and an accessory (POLG2) subunit. Differentiation of pluripotent embryonic stem cells (ESCs) into specific cell types requires expansion of discrete populations of mitochondria and mtDNA replication to meet the specific metabolic requirements of the cell. We determined by real-time PCR that expression of pluripotent markers is reduced before the upregulation of Polg, Polg2 and Tfam in spontaneously differentiating R1 murine (m)ESCs, along with transient increases in mtDNA copy number. In D3 mESCs, the initial transient increase did not take place. However, precursors of neuronal and cardiomyocyte differentiation were positive for both POLG and TFAM. Similar-stage ESCs also showed active mtDNA replication, identified by 5-bromo-2′-deoxy-uridine labelling, as mtDNA copy number increased. Retinoic-acid-induced differentiation resulted in more consistent patterns of replication and upregulation of Polg, Polg2 and Tfam, whereas siRNA knockdown demonstrated that steady-state expression of POLG is essential for maintaining pluripotency.
Publisher: Public Library of Science (PLoS)
Date: 27-06-2011
Publisher: Mary Ann Liebert Inc
Date: 03-2009
Publisher: Oxford University Press (OUP)
Date: 04-2006
DOI: 10.1534/GENETICS.105.055145
Abstract: Mitochondrial DNA is an extranuclear genome normally maternally inherited through the oocyte. However, the use of nuclear transfer can result in both donor cell and recipient oocyte mitochondrial DNA persisting through to blastocyst and being transmitted to the offspring. The degree of donor mitochondrial DNA transmission appears to be random and currently no evidence exists to explain this phenomenon. To determine whether this is a dilution factor or directly related to the transcriptional status of the donor cell in respect of mitochondrial DNA transcription factors, we have generated sheep nuclear transfer embryos using donor cells: (1) possessing their full mitochondrial DNA complement, (2) those partially depleted, and (3) those depleted but containing residual levels. For each donor type, donor mitochondrial DNA persisted in some blastocysts. It is evident from the donor cells used that nuclear-encoded mitochondrial DNA transcription and replication factors persist even after mitochondrial DNA depletion, as do transcripts for some of the mitochondrial-encoded genes. These cells are therefore still programmed to drive mitochondrial DNA replication and transcription. In nuclear transfer-derived embryos, we have observed the persistence of these nuclear-encoded mitochondrial DNA transcription and replication factors but not in those embryos generated through in vitro fertilization. Consequently, nucleo-mitochondrial interaction following nuclear transfer is out of sequence as the onset of mitochondrial replication is a postimplantation event.
Publisher: Elsevier BV
Date: 09-2019
Publisher: Elsevier BV
Date: 04-2014
Publisher: Springer Science and Business Media LLC
Date: 12-2002
DOI: 10.1007/BF03402090
Publisher: Mary Ann Liebert Inc
Date: 09-2005
Abstract: Mitochondrial biogenesis and activation of both oxidative phosphorylation, as well as transcription and replication of the mitochondrial genome, are key regulatory events in cell differentiation. Mitochondrial DNA transcription and replication are highly dependent on the interaction with nuclear-encoded transcription factors translocated from the nucleus. Using a human embryonic stem cell line, HSF 6, we analyzed the proliferation of mitochondria and the expression of mtDNA-specific transcription factors in undifferentiated, migratory embryonic stem cells and spontaneously derived cardiomyocytes. Mitochondrial proliferation and mtDNA transcription are initiated in human embryonic stem cells as they undergo spontaneous differentiation in culture into beating cardiomyocytes. Undifferentiated, pluripotent human embryonic stem cells have few mitochondria, and, as they differentiate, they polarize to one extremity of the cell and then bipolarize the differentiating cell. The differentiated cell then adopts the cytoplasmic configuration of a somatic cell as evidenced in differentiating cardiomyocytes. Transcription and replication of the extranuclear mitochondrial genome is dependent on nuclear encoded factors exported to the mitochondrion. However, the differentiating cardiomyocytes have reduced or absent levels of these transcription and replication factors, namely mitochondrial transcription factors A, B1, B2, and nuclear respiratory factor 1 and polymerase gamma. Therefore, final embryonic stem cell commitment may be influenced by mitochondrial proliferation and mtDNA transcription. However, it is likely that differentiating cardiomyocytes are in mitochondrial arrest, awaiting commitment to a final cell fate.
Publisher: MDPI AG
Date: 23-10-2019
DOI: 10.3390/CELLS8111306
Abstract: It is becoming increasingly apparent that cells require cooperation between the nuclear and mitochondrial genomes to promote effective function. However, it was long thought that the mitochondrial genome was under the strict control of the nuclear genome and the mitochondrial genome had little influence on cell fate unless it was extensively mutated, as in the case of the mitochondrial DNA diseases. However, as our understanding of the roles that epigenetic regulators, including DNA methylation, and metabolism play in cell fate and function, the role of the mitochondrial genome appears to have a greater influence than previously thought. In this review, I draw on ex les from tumorigenesis, stem cells, and oocyte pre- and post-fertilisation events to discuss how modulating one genome affects the other and that this results in a compromise to produce functional mature cells. I propose that, during development, both of the genomes interact with each other through intermediaries to establish genomic balance and that establishing genomic balance is a key facet in determining cell fate and viability.
Publisher: Oxford University Press (OUP)
Date: 2001
Abstract: It is believed that one cause of sperm dysfunction might arise through multiple mitochondrial DNA deletions (Delta mtDNA) resulting in the formation of an incomplete electron transport chain. This study investigates the incidence of multiple Delta mtDNA in human spermatozoa prepared on Percoll gradients. Firstly, we investigated for the presence of two frequently analysed Delta mtDNA, the 4977 and 7.4 kb deletions, using conventional polymerase chain reaction (PCR). These two deletions are characteristically flanked by direct repeats. We further analysed the incidence of one other deletion, the 15 bp deletion in the cytochrome c oxidase subunit III (COX III) of complex IV to determine whether other deletions flanked by direct repeats could be equally predictive. The incidence of these three deletions was not clearly associated with the diagnostic categorization of male infertility. However, the use of long PCR showed that s les harbouring high numbers of Delta mtDNA were associated with the diagnostic categorization of male infertility. We propose that these deletions could arise through a free radical-driven event occurring at the spermatogonial cell stage resulting in the replication of Delta mtDNA molecules at the expense of wild-type molecules. These anomalies in ejaculated sperm mtDNA could account for reproductive failure in some men.
Publisher: Wiley
Date: 10-09-2013
DOI: 10.1096/FJ.13-238345
Abstract: Subjects characterized as cortisol high responders (HRs) consume more calories after stress, but it is unknown whether cortisol responsiveness predicts a propensity for obesity. Female sheep with either high or low cortisol responses to adrenocorticotropin (ACTH) were identified. Body composition was similar in HRs and cortisol low responders (LRs), but the HRs had greater (P<0.01) adiposity than did the LRs (40.5±0.7 vs. 35.8±1.4%) after high-energy feeding, despite comparable food intake. Postprandial thermogenesis in muscle temperature was 0.8 ± 0.08°C higher in the LRs than in the HRs (P<0.01), whereas feeding-induced changes in fat temperature were similar. Leptin and insulin sensitivity were similar in the HRs and LRs. Feeding lowered (P<0.001) the respiratory control ratio in muscle (HRs 9.2±0.8-5.2±1.2 LRs 8.4±0.5-5.2±0.7), indicative of increased uncoupled respiration. Also in muscle, the feeding-induced increases in uncoupling protein (UCP)-3 (fold increase: HRs, 2.4 LRs, 2.0), ryanodine 1 receptor (RyR1 fold increase: HRs 3.1 LRs 2.1), and sarcoendoplasmic reticulum Ca(2+)-dependent ATPase (fold increase: HRs 1.5 LRs 1.6) were equivalent in the HRs and LRs. Sequencing of mitochondrial DNA revealed no haplotypic differences between the 2 groups. We conclude that predisposition to obesity can be predicted by cortisol responsiveness to an ACTH challenge and that the response is due to innate differences in muscle thermogenesis.
Publisher: Oxford University Press (OUP)
Date: 14-01-2022
Abstract: Mitochondrial DNA (mtDNA) methylation in vertebrates has been hotly debated for over 40 years. Most contrasting results have been reported following bisulfite sequencing (BS-seq) analyses. We addressed whether BS-seq experimental and analysis conditions influenced the estimation of the levels of methylation in specific mtDNA sequences. We found false positive non-CpG methylation in the CHH context (fpCHH) using unmethylated Sus scrofa mtDNA BS-seq data. fpCHH methylation was detected on the top lus strand of mtDNA within low guanine content regions. These top lus strand sequences of fpCHH regions would become extremely AT-rich sequences after BS-conversion, whilst bottom/minus strand sequences remained almost unchanged. These unique sequences caused BS-seq aligners to falsely assign the origin of each strand in fpCHH regions, resulting in false methylation calls. fpCHH methylation detection was enhanced by short sequence reads, short library inserts, skewed top/bottom read ratios and non-directional read mapping modes. We confirmed no detectable CHH methylation in fpCHH regions by BS- licon sequencing. The fpCHH peaks were located in the D-loop, ATP6, ND2, ND4L, ND5 and ND6 regions and identified in our S. scrofa ovary and oocyte data and human BS-seq data sets. We conclude that non-CpG methylation could potentially be overestimated in specific sequence regions by BS-seq analysis.
Publisher: Springer Science and Business Media LLC
Date: 18-03-2016
DOI: 10.1038/SREP23229
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.
Publisher: Hindawi Limited
Date: 24-11-2014
DOI: 10.1002/HUMU.22694
Abstract: Mitochondrial DNA (mtDNA) is replicated throughout life in postmitotic cells, resulting in higher levels of somatic mutation than in nuclear genes. However, controversy remains as to the importance of low-level mtDNA somatic mutants in cancerous and normal human tissues. To capture somatic mtDNA mutations for functional analysis, we generated synaptosome cybrids from synaptic endings isolated from fresh hippoc us and cortex brain biopsies. We analyzed the whole mtDNA genome from 120 cybrid clones derived from four in idual donors by chemical cleavage of mismatch and Sanger sequencing, scanning around two million base pairs. Seventeen different somatic point mutations were identified, including eight coding region mutations, four of which result in frameshifts. Examination of one cybrid clone with a novel m.2949_2953delCTATT mutation in MT-RNR2 (which encodes mitochondrial 16S rRNA) revealed a severe disruption of mtDNA-encoded protein translation. We also performed functional studies on a homoplasmic nonsense mutation in MT-ND1, previously reported in oncocytomas, and show that both ATP generation and the stability of oxidative phosphorylation complex I are disrupted. As the mtDNA remains locked against direct genetic manipulation, we demonstrate that the synaptosome cybrid approach can capture biologically relevant mtDNA mutants in vitro to study effects on mitochondrial respiratory chain function.
Publisher: Elsevier BV
Date: 02-2023
Publisher: Portland Press Ltd.
Date: 27-09-2016
DOI: 10.1042/BCJ20160008
Abstract: Mitochondrial DNA replication is critical for maintaining mtDNA copy number to generate sufficient cellular energy that is required for development and for functional cells. In early development, mtDNA copy number is strictly regulated at different stages, and, as a result, the establishment of the mtDNA set point is required for sequential cell lineage commitment. The failure to establish the mtDNA set point results in incomplete differentiation or embryonic arrest. The regulation of mtDNA copy number during differentiation is closely associated with cellular gene expression, especially with the pluripotency network, and DNA methylation profiles. The findings from cancer research highlight the relationship between mitochondrial function, mtDNA copy number and DNA methylation in regulating differentiation. DNA methylation at exon 2 of DNA polymerase gamma subunit A (POLGA) has been shown to be a key factor, which can be modulated to change the mtDNA copy number and cell fate of differentiating and tumour cells. The present review combines multi-disciplinary data from mitochondria, development, epigenetics and tumorigenesis, which could provide novel insights for further research, especially for developmental disorders and cancers.
Publisher: Springer Science and Business Media LLC
Date: 12-2018
Publisher: MDPI AG
Date: 19-04-2023
DOI: 10.3390/IJMS24087545
Abstract: 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.
Publisher: Springer Science and Business Media LLC
Date: 02-01-2014
Publisher: Springer Science and Business Media LLC
Date: 30-04-2019
DOI: 10.1038/S41598-019-43135-4
Abstract: 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.
Publisher: Springer Science and Business Media LLC
Date: 13-10-2013
DOI: 10.1007/S12015-011-9318-7
Abstract: Undifferentiated mouse embryonic stem cells (ESCs) possess low numbers of mitochondrial DNA (mtDNA), which encodes key subunits associated with the generation of ATP through oxidative phosphorylation (OXPHOS). As ESCs differentiate, mtDNA copy number is regulated by the nuclear-encoded mtDNA replication factors, which initiate a major replication event on Day 6 of differentiation. Here, we examined mtDNA replication events in somatic cells reprogrammed to pluripotency, namely somatic cell-ES (SC-ES), somatic cell nuclear transfer ES (NT-ES) and induced pluripotent stem (iPS) cells, all at low-passage. MtDNA copy number in undifferentiated iPS cells was similar to ESCs whilst SC-ES and NT-ES cells had significantly increased levels, which correlated positively and negatively with Nanog and Sox2 expression, respectively. During pluripotency and differentiation, the expression of the mtDNA-specific replication factors, PolgA and Peo1, were differentially expressed in iPS and SC-ES cells when compared to ESCs. Throughout differentiation, reprogrammed somatic cells were unable to accumulate mtDNA copy number, characteristic of ESCs, especially on Day 6. In addition, iPS and SC-ES cells were also unable to regulate ATP content in a manner similar to differentiating ESCs prior to Day 14. The treatment of reprogrammed somatic cells with an inhibitor of de novo DNA methylation, 5-Azacytidine, prior to differentiation enabled iPS cells, but not SC-ES and NT-ES cells, to accumulate mtDNA copies per cell in a manner similar to ESCs. These data demonstrate that the reprogramming process disrupts the regulation of mtDNA replication during pluripotency but this can be re-established through the use of epigenetic modifiers.
Publisher: Springer Science and Business Media LLC
Date: 26-02-2015
Abstract: Mitochondrial DNA (mtDNA) copy number is strictly regulated during differentiation so that cells with a high requirement for ATP generated through oxidative phosphorylation have high mtDNA copy number, whereas those with a low requirement have few copies. Using immunoprecipitation of DNA methylation on 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), which distinguish between de nov o DNA methylation and demethylation, respectively, we set out to determine whether DNA methylation at exon 2 of the human mtDNA-specific polymerase (DNA polymerase gamma A ( POLGA )) regulates cell-specific mtDNA copy number in highly proliferative and terminally differentiated cells. Highly proliferative cancer and pluripotent and multipotent cells possessed low mtDNA copy number and were highly methylated at exon 2 of POLGA in contrast to post-mitotic cells. Unlike neural stem cells, cancer cells were unable to differentiate and remained extensively DNA methylated at exon 2 of POLGA. However, mtDNA depletion of cancer cells reduced DNA methylation at exon 2 of POLGA as they replenished mtDNA to form tumours in mice. Glioblastoma cells treated with the DNA demethylation agent 5-azacytidine over 28 days of astrocyte-induced differentiation demethylated exon 2 of POLGA leading to increased mtDNA copy number and expression of the astrocyte endpoint marker glial fibrillary acidic protein ( GFAP ). However, the demethylation agent vitamin C (VitC) was unable to sustain increased mtDNA copy number and differentiation, as was the case when VitC was withdrawn after short-term treatment. These data demonstrate that DNA demethylation of POLGA is an essential regulator of mtDNA copy number and cellular fate and that cancer cells are only able to modulate DNA methylation of POLGA and mtDNA copy number in the presence of a DNA demethylation agent that inhibits de novo methyltransferase 1 activity.
Publisher: Oxford University Press (OUP)
Date: 15-03-2010
Abstract: Mitochondrial DNA (mtDNA) encodes key proteins associated with the process of oxidative phosphorylation. Defects to mtDNA cause severe disease phenotypes that can affect offspring survival. The aim of this review is to identify how mtDNA is replicated as it transits from the fertilized oocyte into the preimplantation embryo, the fetus and offspring. Approaches for deriving offspring and embryonic stem cells (ESCs) are analysed to determine their potential application for the prevention and treatment of mtDNA disease. The scientific literature was investigated to determine how mtDNA is transmitted, replicated and segregated during pluripotency, differentiation and development. It was also probed to understand how the mtDNA nucleoid is regulated in somatic cells. mtDNA replication is strictly down-regulated from the fertilized oocyte through the preimplantation embryo. At the blastocyst stage, the onset of mtDNA replication is specific to the trophectodermal cells. The inner cell mass cells restrict mtDNA replication until they receive the key signals to commit to specific cell types. However, it is necessary to determine whether somatic cells reprogrammed through somatic cell nuclear transfer, induced pluripotency or fusion to an ESC are able to regulate mtDNA replication so that they can be used for patient-specific cell therapies and to model disease. Prevention of the transmission of mtDNA disease from one generation to the next is still restricted by our lack of understanding as to how to ensure that a donor karyoplast transferred to an enucleated oocyte is free of accompanying mutant mtDNA. Techniques still need to be developed if stem cells are to be used to treat mtDNA disease in those patients already suffering from the phenotype.
Publisher: Springer Science and Business Media LLC
Date: 12-09-2018
Publisher: Springer Science and Business Media LLC
Date: 15-04-2022
DOI: 10.1186/S13072-022-00442-X
Abstract: Mitochondrial DNA (mtDNA) copy number in oocytes correlates with oocyte quality and fertilisation outcome. The introduction of additional copies of mtDNA through mitochondrial supplementation of mtDNA-deficient Sus scrofa oocytes resulted in: (1) improved rates of fertilisation (2) increased mtDNA copy number in the 2-cell stage embryo and (3) improved development of the embryo to the blastocyst stage. Furthermore, a subset of genes showed changes in gene expression. However, it is still unknown if mitochondrial supplementation alters global and local DNA methylation patterns during early development. We generated a series of embryos in a model animal, Sus scrofa , by intracytoplasmic sperm injection (ICSI) and mitochondrial supplementation in combination with ICSI (mICSI). The DNA methylation status of ICSI- and mICSI-derived blastocysts was analysed by whole genome bisulfite sequencing. At a global level, the additional copies of mtDNA did not affect nuclear DNA methylation profiles of blastocysts, though over 2000 local genomic regions exhibited differential levels of DNA methylation. In terms of the imprinted genes, DNA methylation patterns were conserved in putative imprint control regions and the gene expression profile of these genes and genes involved in embryonic genome activation were not affected by mitochondrial supplementation. However, 52 genes showed significant differences in expression as demonstrated by RNAseq analysis. The affected gene networks involved haematological system development and function, tissue morphology and cell cycle. Furthermore, seven mtDNA-encoded t-RNAs were downregulated in mICSI-derived blastocysts suggesting that extra copies of mtDNA affected tRNA processing and/or turnover, hence protein synthesis in blastocysts. We also showed a potential association between differentially methylated regions and changes in expression for 55 genes due to mitochondrial supplementation. The addition of just an extra ~ 800 copies of mtDNA into oocytes can have a significant impact on both gene expression and DNA methylation profiles in Sus scrofa blastocysts by altering the epigenetic programming established during oogenesis. Some of these changes may affect specific tissue-types later in life. Consequently, it is important to determine the longitudinal effect of these molecular changes on growth and development before considering human clinical practice.
Publisher: Bioscientifica
Date: 02-2006
DOI: 10.1530/REP.1.00551
Abstract: The mitochondrion is explicitly involved in cytoplasmic regulation and is the cell’s major generator of ATP. Our aim was to determine whether mitochondria alone could influence fertilisation outcome. In vitro , oocyte competence can be assessed through the presence of glucose-6-phosphate dehydrogenase (G6PD) as indicated by the dye, brilliant cresyl blue (BCB). Using porcine in vitro fertilisation (IVF), we have assessed oocyte maturation, cytoplasmic volume, fertilisation outcome, mitochondrial number as determined by mtDNA copy number, and whether mitochondria are uniformly distributed between blastomeres of each embryo. After staining with BCB, we observed a significant difference in cytoplasmic volume between BCB positive (BCB + ) and BCB negative (BCB − ) oocytes. There was also a significant difference in mtDNA copy number between fertilised and unfertilised oocytes and unequal mitochondrial segregation between blastomeres during early cleavage stages. Furthermore, we have supplemented BCB − oocytes with mitochondria from maternal relatives and observed a significant difference in fertilisation outcomes following both IVF and intracytoplasmic sperm injection (ICSI) between supplemented, sham-injected and non-treated BCB − oocytes. We have therefore demonstrated a relationship between oocyte maturity, cytoplasmic volume, and fertilisation outcome and mitochondrial content. These data suggest that mitochondrial number is important for fertilisation outcome and embryonic development. Furthermore, a mitochondrial pre-fertilisation threshold may ensure that, as mitochondria are diluted out during post-fertilisation cleavage, there are sufficient copies of mtDNA per blastomere to allow transmission of mtDNA to each cell of the post-implantation embryo after the initiation of mtDNA replication during the early postimplantation stages.
Publisher: Springer Science and Business Media LLC
Date: 05-2000
DOI: 10.1007/BF03401783
Publisher: Elsevier BV
Date: 07-2016
DOI: 10.1016/J.DOMANIEND.2016.03.005
Abstract: The vast majority of cellular energy is generated through the process of oxidative phosphorylation, which takes place in the electron transport chain in the mitochondria. The electron transport chain is encoded by 2 genomes, the chromosomal and the mitochondrial genomes. Mitochondrial DNA is associated with a number of traits, which include tolerance to heat, growth and physical performance, meat and milk quality, and fertility. Mitochondrial genomes can be clustered into groups known as mtDNA haplotypes. Mitochondrial DNA haplotypes are a potential genetic source for manipulating phenotypes in farm animals. The use of assisted reproductive technologies, such as nuclear transfer, allows favorable chromosomal genetic traits to be mixed and matched with sought after mtDNA haplotype traits. As a result super breeds can be generated.
Publisher: Humana Press
Date: 2006
DOI: 10.1385/1-59745-046-4:347
Abstract: As human embryonic stem cells (hESCs) undergo differentiation, they express genes characteristic of the lineage for which they are destined. However, fully differentiated in idual cell types can be characterized by the number of mitochondria they possess and the copies of the mitochondrial genome per mitochondrion. These characteristics are indicative of a specific cell's requirement for adenosine triphosphate (ATP) and therefore cellular viability and function. Consequently, failure for an ESC to possess the full complement of mitochondria and mitochondrial DNA (mtDNA) could limit its final commitment to a particular fate. We describe a series of protocols that analyze the process of cellular mitochondrial and mtDNA differentiation during hESC differentiation. In addition, mtDNA transcription and replication are key events in cellular differentiation that require interaction between the nucleus and the mitochondrion. To this extent, we describe a series of protocols that analyze the initiation of these key events as hESCs progress from their undifferentiated state to the fully committed cell. Last, we describe real-time polymerase chain reaction protocols that allow both the identification of mtDNA copy number and determine whether mtDNA copy is uniform (homoplasmy) in its transmission or heterogeneous (heteroplasmy).
Publisher: Oxford University Press (OUP)
Date: 02-05-2018
DOI: 10.1093/NAR/GKY339
Publisher: UPV/EHU Press
Date: 2010
Abstract: Mitochondrial DNA (mtDNA) is a 16.6 kb genome that encodes for 13 of the 100+ subunits of the electron transfer chain (ETC), whilst the other subunits are encoded by chromosomal DNA. The ETC is responsible for the generation of the majority of cellular ATP through the process of oxidative phosphorylation (OXPHOS). mtDNA is normally inherited from the population present in the mature oocyte just prior to fertilisation. However, following somatic cell nuclear transfer (SCNT), mtDNA can be transmitted from both the donor cell and the recipient oocyte. This heteroplasmic transmission of mtDNA is a random event and does not appear to be related to the amount of mtDNA contributed by the donor cell. The distribution of mtDNA is randomly segregated between blastomeres and differentiating tissues, and therefore the mtDNA complement transmitted to offspring tissue cannot be predicted. mtDNA ergence between the cytoplast and the donor cell in intra- and inter-specific crosses favours a slightly more erse mtDNA haplotype. However, this is limited as interspecies SCNT (iSCNT) genetic ergence contributes to developmental failure. SCNT embryos demonstrate a plethora of aberrantly reprogrammed characteristics including the uncoordinated regulation of the mtDNA replication factors. This results in increased mtDNA copy number during preimplantation development and propagates the replication of donor cell mtDNA. These failures are likely to be a consequence of incompatible nuclear- and mtDNA -encoded proteins interacting within the ETC thus reducing ATP production. The outcomes would be similar to the severely debilitating or even fatal mtDNA diseases associated with genetic rearrangements to mtDNA or mtDNA depletion type syndromes and have serious implications for any form of karyoplast transfer approach. The only method to overcome the problems of heteroplasmy in SCNT embryos is to completely deplete the donor cell of its mtDNA prior to SCNT.
Publisher: Springer New York
Date: 2016
DOI: 10.1007/978-1-4939-3040-1_10
Abstract: Replication of mitochondrial DNA (mtDNA) is important for ensuring that cells have sufficient mtDNA copy number to meet their specific requirements for the generation of cellular energy through oxidative phosphorylation. A number of transcription and replication factors are required for this process, with a key factor being the nuclear-encoded mtDNA-specific DNA polymerase γ. DNA polymerase γ has a catalytic subunit (POLGA), whose gene has been shown to be DNA methylated at exon 2. This methylation is considered to be one of the key mechanisms that regulate mtDNA copy number. These findings have made it of great importance to establish optimal methods for investigating the effects of DNA methylation on mtDNA replication. Here, we provide methods to determine the extent of DNA methylation at exon 2 of POLGA as well as other gene targets of interest. We also show how mtDNA copy number is assessed and, from these two outputs, define the efficiency of mtDNA replication by calculating the mtDNA-replicative efficiency index.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 04-2004
Publisher: Oxford University Press (OUP)
Date: 05-03-2007
Abstract: Human mitochondrial DNA (mtDNA) encodes 13 polypeptides of the electron transfer chain. Its replication is dependent on the nuclear-encoded polymerase gamma (POLG) and mitochondrial transcription factor A (TFAM). For POLG, only the polyglutamine tract, characterized by a series of CAG repeats, has been investigated in human sperm. However, TFAM is associated with the reduction in mtDNA content of testicular sperm. We have determined whether POLG and TFAM have functional roles in post-ejaculatory sperm mtDNA. Sperm s les were categorized as: normals, s les with one or two abnormal sperm parameters and oligoasthenoteratozoospermics (OATs). These were analysed by fluorescent PCR to determine the number of CAG repeats, real-time PCR for mtDNA copy number and immunocytochemistry and western blotting for patterns of expression for POLG, TFAM and the mtDNA-encoded COXI. Only the OAT group presented with a significantly higher incidence of heterozygosity for CAG repeats, higher mtDNA content and a lower percentage of sperm expressing POLG and TFAM. Paradoxically, good-quality sperm had fewer mtDNA copies but significantly more sperm expressed POLG, TFAM and COXI. Our data support the original findings that an association between sperm quality and POLG CAG repeats does exist. However, the biological significance of these variants in male infertility remains unclear, as these do not seem to affect mtDNA maintenance. The reduction in mtDNA content in normal s les likely reflects normal spermiogenesis, whereas increases in POLG and TFAM expression possibly compensate for the low mtDNA content, maintaining mitochondrial homeostasis.
Publisher: Elsevier BV
Date: 04-2013
DOI: 10.1016/J.BBAGEN.2013.10.036
Abstract: Mitochondrial DNA (mtDNA) is important for energy production as it encodes some of the key genes of electron transfer chain, where the majority of cellular energy is generated through oxidative phosphorylation (OXPHOS). MtDNA replication is mediated by nuclear DNA-encoded proteins or enzymes, which translocate to the mitochondria, and is strictly regulated throughout development. It starts with approximately 200 copies in each primordial germ cell and these copies undergo expansion and restriction events at various stages of development. I describe the patterns of mtDNA replication at key stages of development. I explain that it is essential to regulate mtDNA copy number and to establish the mtDNA set point in order that the mature, specialised cell acquires the appropriate numbers of mtDNA copy to generate sufficient adenosine triphosphate (ATP) through OXPHOS to undertake its specialised function. I discuss how these processes are dependent on the controlled expression of the nuclear-encoded mtDNA-specific replication factors and that this can be modulated by mtDNA haplotypes. I discuss how these events are altered by certain assisted reproductive technologies, some of which have been proposed to prevent the transmission of mutant mtDNA and others to overcome infertility. Furthermore, some of these technologies are predisposed to transmitting two or more populations of mtDNA, which can be extremely harmful. The failure to regulate mtDNA replication and mtDNA transmission during development is disadvantageous. Manipulation of oocytes and embryos can lead to significant implications for the maternal-only transmission of mtDNA. This article is part of a Special Issue entitled Frontiers of mitochondrial research.
Publisher: Elsevier BV
Date: 12-2005
Publisher: Oxford University Press (OUP)
Date: 04-1998
DOI: 10.1093/HUMREP/13.SUPPL_1.51
Abstract: This review critically analyses the diagnostic value of conventional semen analysis and sperm function testing. It is clear from the data available that a high quality comprehensive semen assessment is a basic requirement in the diagnosis of the infertile couple. Sperm function testing has given disappointing results and a new generation of sperm function tests is required, which are cost effective, reliable and provide clinically significant information. In the future there will be an increasing use of molecular techniques to diagnose male infertility. Specific attention is given to the role of polymerase chain reaction in the diagnosis of genital infection and Y chromosomal and mitochondrial DNA deletions as ex les.
Publisher: Informa UK Limited
Date: 29-06-2022
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
Start Date: 2007
End Date: 2010
Funder: Medical Research Council
View Funded ActivityStart Date: 2021
End Date: 2025
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2018
End Date: 2020
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2012
End Date: 2015
Funder: Medical Research Council
View Funded ActivityStart Date: 2016
End Date: 2020
Funder: Australian Research Council
View Funded ActivityStart Date: 2013
End Date: 2015
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2012
End Date: 2014
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2019
End Date: 2020
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2015
End Date: 2018
Funder: National Health and Medical Research Council
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