Mouse models are widely employed to study mitochondrial inheritance, which have implications to several human diseases caused by mutations in the mitochondrial genome (mtDNA). These mouse models take advantage of polymorphisms between the mtDNA of the NZB/BINJ and the mtDNA of common inbred laboratory (i.e., C57BL/6) strains to generate mice with two mtDNA haplotypes (heteroplasmy). Based on PCR followed by restriction fragment length polymorphism (PCR-RFLP), these studies determine the level of heteroplasmy across generations and in different cell types aiming to understand the mechanisms underlying mitochondrial inheritance. However, PCR-RFLP is a time-consuming method of low sensitivity and accuracy that dependents on the use of restriction enzyme digestions. A more robust method to measure heteroplasmy has been provided by the use of real-time quantitative PCR (qPCR) based on allelic refractory mutation detection system (ARMS-qPCR). Herein, we report an ARMS-qPCR assay for quantification of heteroplasmy using heteroplasmic mice with mtDNA of NZB/BINJ and C57BL/6 origin. Heteroplasmy and mtDNA copy number were estimated in germline and somatic tissues, providing evidence of the reliability of the approach. Furthermore, it enabled single-step quantification of heteroplasmy, with sensitivity to detect as low as 0.1% of either NZB/BINJ or C57BL/6 mtDNA. These findings are relevant as the ARMS-qPCR assay reported here is fully compatible with similar heteroplasmic mouse models used to study mitochondrial inheritance in mammals.

Mitochondria play a fundamental role during development of the female germline. They are fragmented, round, and small. Despite these characteristics suggesting that they are inactive, there is accumulating evidence that mitochondrial dysfunctions are a major cause of infertility and generation of aneuploidies in humans. In addition, mitochondria and their own genomes (mitochondrial DNA-mtDNA) may become damaged with time, which might be one reason why aging leads to infertility. As a result, mitochondria have been proposed as an important target for evaluating oocyte and embryo quality, and developing treatments for female infertility. On the other hand, mutations in mtDNA may cause mitochondrial dysfunctions, leading to severe diseases that affect 1 in 4,300 people. Moreover, very low levels of mutated mtDNA seem to be present in every person worldwide. These may increase with time and associate with late-onset degenerative diseases such as Parkinson disease, Alzheimer disease, and common cancers. Mutations in mtDNA are transmitted down the maternal lineage, following a poorly understood pattern of inheritance. Recent findings have indicated existence in the female germline of a purifying filter against deleterious mtDNA variants. Although the underlying mechanism of this filter is largely unknown, it has been suggested to rely on autophagic degradation of dysfunctional mitochondria or selective replication/transmission of non-deleterious variants. Thus, understanding the mechanisms regulating mitochondrial inheritance is important both to improve diagnosis and develop therapeutic tools for preventing transmission of mtDNA-encoded diseases.

Oocyte mitochondria are increased in number, smaller, and rounder in appearance than mitochondria in somatic cells. Moreover, mitochondrial numbers and activity are narrowly tied with oocyte quality because of the key role of mitochondria to oocyte maturation. During oocyte maturation, mitochondria display great mobility and cluster at specific sites to meet the high energy demand. Conversely, oocyte mitochondria are not required during early oogenesis as coupling with granulosa cells is sufficient to support gamete's needs. In part, this might be explained by the importance of protecting mitochondria from oxidative damage that result in mutations in mitochondrial DNA (mtDNA). Considering mitochondria are transmitted exclusively by the mother, oocytes with mtDNA mutations may lead to diseases in offspring. Thus, to counterbalance mutation expansion, the oocyte has developed specific mechanisms to filter out deleterious mtDNA molecules. Herein, we discuss the role of mitochondria on oocyte developmental potential and recent evidence supporting a purifying filter against deleterious mtDNA mutations in oocytes.