Mitochondria in Ovarian Aging and Reproductive Longevity

Mitochondrial dysfunction is one of the hallmarks of aging. Consistently mitochondrial DNA (mtDNA) copy number and function decline with age in various tissues. There is increasing evidence to support that mitochondrial dysfunction drives ovarian aging. A decreased mtDNA copy number is also reported during ovarian aging. However, the mitochondrial mechanisms contributing to ovarian aging and infertility are not fully understood. Additionally, investigations into mitochondrial therapies to rejuvenate oocyte quality, select viable embryos and improve mitochondrial function may help enhance fertility or extend reproductive longevity in the future. These therapies include the use of mitochondrial replacement techniques, quantification of mtDNA copy number, and various pharmacologic and lifestyle measures. This review aims to describe the key evidence and current knowledge of the role of mitochondria in ovarian aging and identify the emerging potential options for therapy to extend reproductive longevity and improve fertility.     

Mitochondrial DNA segregation in the developing embryo

Mitochondrial (mt)DNA is strictly maternally inherited in mammals; new mutations thus segregate along maternal lineages without the benefit of homologous recombination with mtDNA of paternal origin. Despite the high mtDNA copy number (approximately 100000 or more) in mature oocytes, and despite the relatively small number of cell divisions during oogenesis, mtDNA sequence variants segregate rapidly between generations. This paradoxical behaviour has been ascribed to the presence of a mtDNA 'bottleneck' in oogenesis or early embryogenesis. The nature and size of this bottleneck have been the subject of much controversy. This review argues that segregation of mtDNA sequence variants in the female germline occurs primarily during mitosis in the oocyte precursor population. Segregation is rapid because the precursor cells (primordial germ cells and oogonia) contain a relatively small number of mtDNA templates (the bottleneck) and because the replication of mtDNA is under relaxed control. For the most part, the process appears similar in mice segregating polymorphic sequence variants and in human pedigrees segregating pathogenic point mutations. In particular, there is no evidence for selection against high levels of pathogenic mtDNA point mutations in oogenesis, in early embryonic development, or in fetal development, thus suggesting that efficient respiratory chain function is not critical until post-natal life. These results have important practical implications for clinical genetics.     

Transfer of paternal mitochondrial DNA during fertilization of honeybee (Apis mellifera L.) eggs

Strict maternal inheritance of mitochondrial (mt) DNA is believed to be the rule in most eukaryotic organisms because of exclusion of paternal mitochondria from the egg cytoplasm during fertilization. In honeybees, polyspermic fertilization occurs, and many spermatozoa, including their mitochondria-rich flagellum, can completely penetrate the egg, thus allowing for a possibly high paternal leakage. In order to identify paternal mtDNA in honeybee eggs, restriction fragment length polymorphisms (RFLP) of different subspecies were used. Total DNA extracts of different developmental stages of an Apis mellifera carnica x Apis mellifera capensis hybrid brood were tested with a radioactively-labelled diagnostic mtDNA probe. Densitograms of autoradiographs indicated that the male contribution represents up to 27% of the total mitochondrial DNA in the fertilized eggs 12 h after oviposition. In subsequent developmental stages the portion of paternal mtDNA slowly decreased until hatching of the larvae when only traces were found. Although rapid disintegration of paternal mtDNA does not occur, the initially high paternal mitochondrial contribution is not maintained in the adult animal.     

No evidence for paternal mtDNA transmission to offspring or extra-embryonic tissues after ICSI

There is a risk that ICSI may increase the transmission of mtDNA diseases to children born after this technique. Knowledge of the fate and transmission of paternal mitochondrial DNA is important since mutations in mitochondrial DNA have been described in oligozoospermic males. We have used an adaptation of solid phase mini-sequencing to exclude the presence of levels of paternal mtDNA >0.001% in ICSI families. This method is more sensitive than those used in previous studies and is sufficient to detect the likely paternal contribution (approximately 0.1-0.5% from simple calculations of expected dilution during fertilization). Using this method, we were able to detect concentrations as low as 0.001% paternal mtDNA in a maternal mtDNA background. No paternal mtDNA was detected in the embryonic (blood or buccal swabs) tissue of children born after ICSI nor in extra-embryonic tissue (placenta or umbilical cord). In conclusion, we did not detect paternal mtDNA in blood, buccal swabs, placenta or umbilical cord of children born after ICSI. We have found no evidence that ICSI increases the risk of paternal transmission of mtDNA and hence of mtDNA disorders.     

Low oocyte mitochondrial DNA content in ovarian insufficiency

BACKGROUND: Mitochondrial biogenesis and bioenergetics play an important role in oocyte maturation and embryo development. We have investigated the relationship between defective mitochondrial biogenesis and the lack of oocyte maturity observed during IVF procedures with patients suffering from ovarian dystrophy and ovarian insufficiency. METHODS: We used real-time quantitative PCR to quantify mitochondrial DNA (mtDNA) in 116 oocytes obtained from 47 women undergoing the ICSI procedure. We compared the mtDNA content of oocytes from women with a normal ovarian profile with that of oocytes from women with ovarian dystrophy and ovarian insufficiency. RESULTS: We found an average of 256,000 +/- 213,000 mitochondrial genomes per cell. The mean mtDNA copy number was not significantly different in ovarian dystrophy compared with controls, but it was significantly lower in oocytes from women with ovarian insufficiency (100,000 +/- 99,000, P < 0.0001). CONCLUSIONS: Our results suggest that low mtDNA content is associated with the impaired oocyte quality observed in ovarian insufficiency.     

Twinkle helicase is essential for mtDNA maintenance and regulates mtDNA copy number

Mechanisms of mitochondrial DNA (mtDNA) maintenance have recently gained wide interest owing to their role in inherited diseases as well as in aging. Twinkle is a new mitochondrial 5'-3' DNA helicase, defects of which we have previously shown to underlie a mitochondrial disease, progressive external ophthalmoplegia with multiple mtDNA deletions. Mouse Twinkle is highly similar to the human counterpart, suggesting conserved function. Here, we have characterized the mouse Twinkle gene and expression profile and report that the expression patterns are not conserved between human and mouse, but are synchronized with the adjacent gene MrpL43, suggesting a shared promoter. To elucidate the in vivo role of Twinkle in mtDNA maintenance, we generated two transgenic mouse lines overexpressing wild-type Twinkle. We could demonstrate for the first time that increased expression of Twinkle in muscle and heart increases mtDNA copy number up to 3-fold higher than controls, more than any other factor reported to date. Additionally, we utilized cultured human cells and observed that reduced expression of Twinkle by RNA interference mediated a rapid drop in mtDNA copy number, further supporting the in vivo results. These data demonstrate that Twinkle helicase is essential for mtDNA maintenance, and that it may be a key regulator of mtDNA copy number in mammals.     

Transmission of mitochondrial DNA following assisted reproduction and nuclear transfer

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.     

Ooplasm donation in humans: the need to investigate the transmission of mitochondrial DNA following cytoplasmic transfer

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.     

Interference of nuclear mitochondrial DNA segments in mitochondrial DNA testing resembles biparental transmission of mitochondrial DNA in humans

PurposeReports have questioned the dogma of exclusive maternal transmission of human mitochondrial DNA (mtDNA), including the recent report of an admixture of two mtDNA haplogroups in individuals from three multigeneration families. This was interpreted as being consistent with biparental transmission of mtDNA in an autosomal dominant–like mode. The authenticity and frequency of these findings are debated.MethodsWe retrospectively analyzed individuals with two mtDNA haplogroups from 2017 to 2019 and selected four families for further study.ResultsWe identified this phenomenon in 104/27,388 (approximately 1/263) unrelated individuals. Further study revealed (1) a male with two mitochondrial haplogroups transmits only one haplogroup to some of his offspring, consistent with nuclear transmission; (2) the heteroplasmy level of paternally transmitted variants is highest in blood, lower in buccal, and absent in muscle or urine of the same individual, indicating it is inversely correlated with mtDNA content; and (3) paternally transmitted apparent large-scale mtDNA deletions/duplications are not associated with a disease phenotype.ConclusionThese findings strongly suggest that the observed mitochondrial haplogroup of paternal origin resulted from coamplification of rare, concatenated nuclear mtDNA segments with genuine mtDNA during testing. Evaluation of additional specimen types can help clarify the clinical significance of the observed results.     

Mitochondrial DNA content affects the fertilizability of human oocytes

Mitochondrial DNA content varies considerably in oocytes, even when collected from the same patient. In the present study, real-time quantitative polymerase chain reaction analysis of 113 unfertilized oocytes obtained from 43 patients revealed an average of 193,000 (range: 20,000 to 598,000) mitochondrial genomes per cell. We compared several groups of oocytes to investigate the relationship between mitochondrial DNA content and fertilizability. The average mitochondrial DNA copy number was significantly lower in cohorts suffering from fertilization failure compared to cohorts with a normal rate of fertilization. In addition, the mitochondrial copy number of oocytes from patients with fertilization failure due to unknown causes was significantly lower than that of oocytes from patients in which IVF failure was due mainly to a severe sperm defect. The lower mtDNA copy number could be due to defective cytoplasmic maturation of oocytes. We conclude that low mitochondrial DNA content, due to inadequate mitochondrial biogenesis or cytoplasmic maturation, may adversely affect oocyte fertilizability.     

Triple genetic identities for the complete hydatidiform mole, placenta and co-existing fetus after transfer of a single in vitro fertilized oocyte: case report and possible mechanisms

We found different genotypes for the complete hydatidiform mole (CHM), placenta and co-existing fetus derived from a single in vitro fertilized human oocyte by the analysis of short tandem repeat (STR) DNA markers. The molar tissue was found to be heterozygously androgenetic. The fetus and placenta contained identical maternal, but different paternal genomes. Two models were proposed to account for the identification of triple genetic identities in a single fertilized oocyte. In the first model, the oocyte was fertilized by a diploid sperm, resulting in diandric triploidy. Premature cytokinesis resulted in early splitting of a cytoplasmic fragment with one copy of the replicated sperm chromosome, which developed into a heterozygous CHM. The bipolar spindle in syngamy pulled the other copy of sperm chromosomes and replicated oocyte chromosomes to form two blastomeres, which develop into the fetus and placenta, respectively. In the second model, the oocyte was fertilized by two haploid sperms, followed by tripolar spindle formation. Whatever is the mechanism, this case provides direct evidence that CHM can be derived from an oocyte containing an intact maternal genome.     

线粒体能量代谢和DNA复制对小鼠卵子体外成熟、受精及发育的影响

目的 探讨胞质线粒体的氧化磷酸化（OXPHOS）活性或线粒体DNA复制在卵子成熟、受精和胚胎发育过程中的作用。方法 通过在小鼠体外成熟培养液中引入不同浓度的羰基氰4-(三氟甲氧基)苯腙 (FCCP, 10 nmol/L和100nmol/L)或2′,3′-双脱氧胞苷(ddC,10μmol/L和100μmol/L),抑制线粒体OXPHOS活性或线粒体DNA复制,统计分析各组卵子的体外生发泡破裂（GVBD）率、核成熟率、受精率及囊胚形成率,以分析线粒体功能抑制对卵子成熟、受精和胚胎发育的影响。 结果 线粒体OXPHOS活性和DNA复制功能在卵子和胚胎中所发挥的作用并不完全相同。FCCP抑制线粒体OXPHOS活性可显著降低卵子的核成熟率和囊胚形成率;但对卵子的GVBD的发生率和受精率无显著影响。而ddC抑制线粒体DNA复制不影响卵子的体外成熟和受精,但可显著抑制囊胚的形成。 结论 OXPHOS活性主要影响卵子成熟及胚胎发育;线粒体DNA复制则主要影响胚胎发育;而线粒体功能抑制不影响卵子的成熟启动和体外受精。     

Mitochondrial fusion increases the mitochondrial DNA copy number in budding yeast

Mitochondrial fusion plays an important role in mitochondrial DNA (mtDNA) maintenance, although the underlying mechanisms are unclear. In budding yeast, certain levels of reactive oxygen species (ROS) can promote recombination-mediated mtDNA replication, and mtDNA maintenance depends on the homologous DNA pairing protein Mhr1. Here, we show that the fusion of isolated yeast mitochondria, which can be monitored by the bimolecular fluorescence complementation-derived green fluorescent protein (GFP) fluorescence, increases the mtDNA copy number in a manner dependent on Mhr1. The fusion event, accompanied by the degradation of dissociated electron transport chain complex IV and transient reductions in the complex IV subunits by the inner membrane AAA proteases such as Yme1, increases ROS levels. Analysis of the initial stage of mitochondrial fusion in early log-phase cells produced similar results. Moreover, higher ROS levels in mitochondrial fusion-deficient mutant cells increased the amount of newly synthesized mtDNA, resulting in increases in the mtDNA copy number. In contrast, reducing ROS levels in yme1 null mutant cells significantly decreased the mtDNA copy number, leading to an increase in cells lacking mtDNA. Our results indicate that mitochondrial fusion induces mtDNA synthesis by facilitating ROS-triggered, recombination-mediated replication and thereby prevents the generation of mitochondria lacking DNA.