Mitochondrial DNA transmission, replication and inheritance: a journey from the gamete through the embryo and into offspring and embryonic stem cells
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.
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ABSTRACT: Next generation sequencing (NGS) is now being used for detecting chromosomal abnormalities in blastocyst trophectoderm (TE) cells from in vitro fertilized embryos. However, few data are available regarding the clinical outcome, which provides vital reference for further application of the methodology. Here, we present a clinical evaluation of NGS-based preimplantation genetic diagnosis/screening (PGD/PGS) compared with single nucleotide polymorphism (SNP) array-based PGD/PGS as a control. A total of 395 couples participated. They were carriers of either translocation or inversion mutations, or were patients with recurrent miscarriage and/or advanced maternal age. A total of 1,512 blastocysts were biopsied on D5 after fertilization, with 1,058 blastocysts set aside for SNP array testing and 454 blastocysts for NGS testing. In the NGS cycles group, the implantation, clinical pregnancy and miscarriage rates were 52.6% (60/114), 61.3% (49/80) and 14.3% (7/49), respectively. In the SNP array cycles group, the implantation, clinical pregnancy and miscarriage rates were 47.6% (139/292), 56.7% (115/203) and 14.8% (17/115), respectively. The outcome measures of both the NGS and SNP array cycles were the same with insignificant differences. There were 150 blastocysts that underwent both NGS and SNP array analysis, of which seven blastocysts were found with inconsistent signals. All other signals obtained from NGS analysis were confirmed to be accurate by validation with qPCR. The relative copy number of mitochondrial DNA (mtDNA) for each blastocyst that underwent NGS testing was evaluated, and a significant difference was found between the copy number of mtDNA for the euploid and the chromosomally abnormal blastocysts. So far, out of 42 ongoing pregnancies, 24 babies were born in NGS cycles; all of these babies are healthy and free of any developmental problems. This study provides the first evaluation of the clinical outcomes of NGS-based pre-implantation genetic diagnosis/screening, and shows the reliability of this method in a clinical and array-based laboratory setting. NGS provides an accurate approach to detect embryonic imbalanced segmental rearrangements, to avoid the potential risks of false signals from SNP array in this study.
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ABSTRACT: We examined six types of cells that form the ovary of the earthworm Dendrobena veneta ogonia, prooocytes, vitellogenic oocytes, trophocytes, fully grown postvitellogenic oocytes and somatic cells of the gonad. The quantitative stereological method revealed a much higher "volume density" of mitochondria in all of the types of germ-line cells except for the somatic cells. Fluorescent vital stain JC-1, however, showed a much higher oxidative activity of mitochondria in the somatic cells than in the germ-line cells. The distribution of active and inactive mitochondria within the studied cells was assessed using the computer program ImageJ. The analysis showed a higher luminosity of inactive mitochondria in all of the types of germ-line cells and a higher luminosity of active mitochondria in somatic cells. The OXPHOS activity was found in somatic cells mitochondria and in the peripheral mitochondria of the vitellogenic oocytes. The detection of reactive oxygen species (ROS) revealed a differentiated distribution of ROS in the different cell types. The amount of ROS substances was lower in somatic cells than in younger germ-line cells. The ROS level was also low in the cytoplasm of fully grown postwitellogenic oocytes. The distribution of the MnSOD enzyme that protects mitochondria against destructive role of ROS substances was high in the oogonia and in prooocytes and it was very high in vitellogenic and postvitellogenic oocytes. However, a much lower level of this protective enzyme was observed in the trophocytes and the lowest level was found in the cytoplasm of somatic cells. The lower mitochondrial activity and higher level of MnSOD activity in germ-line cells when compared to somatic cells testifies to the necessity of the organisms to protect the mitochondria of oocytes against the destructive role of the ROS that are produced during oxidative phosphorylation. The protection of the mitochondria in oocytes is essential for the transfer of healthy organelles to the next generation.PLoS ONE 01/2015; 10(2):e0117187. DOI:10.1371/journal.pone.0117187 · 3.53 Impact Factor
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ABSTRACT: S Background: The aim of the current study was to assess the mRNA levels of two mitochondria-related genes, including nuclear-encoded NRF1 (nuclear respiratory factor 1), mitochondrial transcription factor A (TFAM), and mitochondrial-encoded cytochrome c oxidase subunit 1 (MT-CO1) genes in various stages of the human oocyte maturation. Methods: Oocytes were obtained from nine infertile women with male factor undergoing in vitro fertilization (IVF)/intra-cytoplasmic sperm injection protocol. Mitochondrial-related mRNA levels were performed by single-cell TaqMan real-time PCR. Results: the expression level of the target genes was low at the germinal vesicle stage (P>0.05). Although the mRNA level of NRF1gene remained stable in metaphase I, the mRNA level of TFAM and MT-CO1 increased significantly (P<0.05).In metaphase II, the expression level of all genes increased compared to metaphase I (P<0.05). Conclusion: The overexpression levels of NRF1, TFAM, and MT-CO1 genes are related to the oocyte maturation. Therefore, the current study could be used clinically to improve the success rate of IVF. Iran. Biomed. J. 19 (1): 23-28, 2015 INTRODUCTION any factors influence the fertilizability of an oocyte. Relatively, unexplained infertility is considerably seen in infertile women. In some cases, infertility occurs because oocyte fails to mature when reaches the metaphase II (MII) stage. Oocyte maturation is a well-regulated event, including nuclear and cytoplasmic maturation; both necessary for the fertilization and embryo development . Various factors affect oocyte maturation, including maturation promoting factor, gonadotropin hormones (e.g. luteinizing hormone and follicle-stimulating hormone), insulin-like growth factor, transforming growth factor β family such as bone morphogenetic, growth differentiation factor-9, anti-mullerian hormone activins and inhibins. Furthermore, an extensive production and reorganization of organelles occur during the oocyte maturation. However, the mitochondrial genome must be replicated with great accuracy because mitochondria are inherited by the zygote exclusively from the oocyte . Mitochondria, as the most predominant organelle in oocytes, play a critical role in this event by producing the primary supply of adenosine triphosphate through the oxidative-phosphorylation process . Although altering the mitochondrial distribution (as well as their metabolic activity) known to be vital for the oocyte cytoplasmic maturation and developmental competence, little is known about the occurrence of cytoplasmic maturation during the oocyte maturation . Decrease in mitochondrial activity has been reported to impair the oocyte maturation . In fact, mitochondria function is associated with the mitochondrial DNA (mtDNA) . A single copy of this genome exists in the mitochondria of human oocyte . mtDNA number is expanded to signify-MIranian biomedical journal 02/2015; 19(1):23-28. DOI:10.6091/ibj.1400.2015