1. Introduction
Besides the nuclear genome, a typical animal cell also has from 100 to 1000 copies of mitochondrial DNA (mtDNA) that encode core subunits of electron transport chain complexes [
1]. While converting energy to ATP and carrying out biosynthesis, mitochondria also generate free radicals that can damage DNA, proteins and lipids nearby [
2]. Mitochondrial genome has no histone protection and lacks homologues recombination or other efficient repair mechanisms. As a result, mtDNA is particularly prone to accumulating mutations [
3]. To make matter worse, inefficient electron transport chain (ETC) complexes caused by mtDNA mutations generate more free radicals and exacerbate the mitochondrial damage in a feed-forward cycle [
2]. Accumulation of mtDNA mutations during lifetime has been postulated to cause age-related decline of energy metabolism and impairment of tissue homeostasis [
4,
5]. Mitochondrial “mutator” mice with an elevated rate of mtDNA mutagenesis display premature aging, which, in principle, substantiates the correlation between mtDNA mutations and aging [
6,
7]. However, mtDNA mutations from various tissues of normally aged human or experimental animals are found to be too low to possibly elicit any pathological consequences [
8], which argues against a causative role of mtDNA mutations in physiological aging, particularly in the post mitotic tissues [
9].
DNA replication is the source of mutations [
10]. In adulthood, most tissues consist of post mitotic cells that have a slow turnover rate of mitochondria and mtDNA [
11], which might explain the low mtDNA mutations frequency in post mitotic tissues. Therefore, the quest for connection between mtDNA mutations and aging might have focused on the wrong target from the very beginning. On the other hand, one would expect that mtDNA mutations in actively dividing cells, such as cancer cells and stem cells, could reach a high level during the aging process [
12]. In fact, there is increasing evidence demonstrating the accumulation of mtDNA mutations in aged stem cells [
13,
14,
15,
16]. Stem cells are essential for tissue homeostasis and wound repair. Age dependent deterioration of stem cells contributes to several hallmarks of aging such as impaired capability of tissue repair and increased susceptibility to cancers and infectious diseases, and thereby has been proposed to play an important role in the natural aging process [
17]. Stem cells in a quiescent state often emphasize glycolysis, presumably to maintain the genomic integrity by minimizing the production of damaging free radicals [
18]. During active proliferation, stem cells undergo a metabolic reprograming to activate mitochondrial respiration, which is critical for their progenitors differentiation [
18,
19]. In principle, accumulation of mtDNA mutations in stem cells could cause age dependent deterioration of stem cells’ activity and competence. Nonetheless, the interplay between mtDNA mutations and stem cell aging remains to be explored. Besides the chronological aging, stem cells are also subject to proliferative aging, the accumulation of division cycles [
20]. The continuous proliferation of stem cells would lead to not only the accumulation of damaged mutations, but also potentially the shortening of telomeres [
21]. Telomere shortening has been observed in various types of aged stem cells in mammals, and hence ascribed as a factor contributing to the age associated decline of proliferation competency of stem cells [
20]. Despite the progress in the field, our knowledge on how aging impacts stem cell functions remains limited. The cellular deficiencies caused by accumulation of proliferative cycles that contribute to the decline of stem cells’ activities remain to be defined.
Drosophila oogenesis represents one of the best-characterized systems to study the behaviors of stem cells and the development of their progenitors. Germline stem cell (GSC) maintenance and division are regulated by bone morphogenic protein (BMP) signals from the surrounding niche [
22]. The asymmetric division of GSC renews itself and generates a cystoblast, which continues to divide an additional four rounds with incomplete cytokinesis, generating 16 interconnected germ cells. One of them will develop into the eventual oocyte, while the remaining 15 become nurse cells. Over the adult life span, the decline of BMP signals in aged ovaries causes reduced proliferation and a gradual loss of the stem cell population [
23]. About 70% of GSCs remain in two-month-old ovaries, and 40% of these are actively proliferating [
23]. However, most female flies cease to lay eggs 40 days after eclosion, despite having viable GSCs [
24]. As female flies age, there is a gradual reduction of hatching rate and increased incidences of developmental abnormalities of their eggs [
25]. These observations suggest that some undefined deficiencies must have occurred in germ cells of aged female flies and impede the oogenesis and the development of their progeny.
In current study, we utilized a physiological approach to manipulate GSC division cycle independent of their chronological age, and examine its impact on GSC aging and female reproductive physiology. We demonstrated that the accumulation of division cycles played a major role in maternal age dependent decline of eggs’ fitness and contributed to the age dependent decline of female fecundity. We also found that accumulation of division cycles led to a dramatic decline of cytochrome C oxidase activity, but did not cause telomere shortening in aged ovaries. Additionally, we detected increased mutations on mtDNA and observed impaired mtDNA replication in aged ovaries. The strong correlation between the decline of stem cell activity and mitochondrial dysfunction in aged ovaries, suggests that mtDNA mutations caused by proliferative cycles may contribute to stem cell aging.
4. Discussion
We demonstrated that the division cycle of GSCs, but not solely the chronological age, contributes to the age dependent decline of GSCs function, which is likely to be the major causative factor of the reduced hatching rate of embryos produced by old mothers. We also noticed that accumulation of proliferation cycles led to increased mtDNA mutation load in aged GSCs based on two indirect approaches: mtDNA mutations selection and RFLP analysis of AT-rich region. However, neither of these assays provided a quantitative measure for mutation frequency. Also, it is puzzling that we did not detect an obvious increase of mutation load in aged ovaries via mtDNA deep sequencing. It has been documented that more than 60% of mutation load, corresponding to a mutation frequency around 10
−4 per nucleotide is required to elicit any discernible phenotype in cultured cells [
8]. It is possible that the actual mutation rate might be below the systematic error rate at 1% [
33], but higher than 10
−4 in aged ovaries, which could disrupt ETC complexes or impede mtDNA replication as demonstrated in aged ovaries. Alternatively, the observed 10-fold increase of escapers carrying mtDNA mutations by old mothers might be simply due to the continual proliferation of few GSCs carrying high level of mtDNA mutations. Interestingly, a mosaic pattern of the ETC activity staining was observed in a few aged ovaries, in which some germ cells had normal mitochondrial activity, while others were clearly defective in complex IV (
Figure 4C, insert). This resembles the clonal expansions of mtDNA mutations that have been demonstrated in human tissues and cells [
13,
36]. Ideally, more elegant assays such as laser microdissection or single cell sequencing [
13,
36], shall be applied to quantitatively assess mtDNA mutation frequency, which would help to resolve this discrepancy. Additionally, other age-associated deficiencies such as mtDNA depletion [
37], and accumulation of oxidative damages [
2], could also contribute to the mitochondrial deterioration in aging stem cells. Therefore, the data presented in this study should be interpreted with caution.
The AT-rich region seems to be particularly susceptible to mutations based on our data, which is consistent with previous studies [
34,
35]. Although mammalian mitochondrial genomes lack AT-rich regions, the presence of single strand DNA structure in the D-loop could render them vulnerable to mutations accumulation [
38]. Thus, the impacts of the division cycle on stem cell function present a great challenge for long-lived mammals. Sophisticated mechanisms must be evolved to preserve the replication cycles of stem cells in order to maintain their fitness over a long lifespan. For example, in the process of mammalian hematopoiesis, the multipotent hematopoietic stem cell normally divides infrequently to produce committed progenitor cells, which divide multiple rounds before differentiating into mature blood cells [
39]. It has been proposed that the amplifying divisions of committed progenitors help to reduce the risk of replicative senescence caused by telomere shortening [
39]. However, rodents have active telomerase and rather long telomeres in somatic tissues [
40], yet still employ the same mechanism to reduce the replication cycles of stem cells. This argues against the idea that the risk of telomere shortening is the major evolution pressure underlying this conserved aspect of hematopoiesis in all mammals. In current study, we did not find significant telomere shortening in aged GSCs. Instead, continual division of GSCs led to the accumulation of mtDNA deficiencies, which could potentially disrupt mitochondrial respiration complexes and likely contributed to the age dependent decline of GSC activity as demonstrated by gradual decline of female fecundity.
In most long-lived post-mitotic tissues, mitochondria undergo continual proliferation, while the nuclear genome becomes quiescent [
11]. Age dependent accumulation of mtDNA mutations has been found in various tissues of experimental animals and humans, and thereby has been proposed to play a causative role in aging [
41]. However, mitochondrial mutations in most aged post-mitotic tissues are too low to cause any functional consequences [
8,
9], as mitochondria of aged tissues remain biochemically active [
8,
9]. It is confounding why the replication cycle of mtDNA only causes mutation accumulation in stem cells, but not in post-mitotic cells. It has been proposed that continual mitochondrial biogenesis and turnover in post-mitotic cells underlie a quality control mechanism to weed out defective mitochondria [
42]. Recent studies suggest that the autophagy pathway might be required to degrade defective mitochondria with low inner membrane potential, the electrochemical potential generated through active mitochondria respiration [
43]. Though the detailed mechanism of mitochondrial quality control remains to be fully elucidated, it is a reasonable assumption that active mitochondrial respiration is a prerequisite for a cell to distinguish healthy vs. defective mitochondria. Drosophila female GSCs have been suggested to emphasize glycolysis instead of oxidative phosphorylation to produce ATP [
44]. Mitochondrial enzymatic staining also confirmed that electron transport chain complexes in early-stage GSCs in the germarium were inactive (
Figure 4C), which may render GSCs incapable of recognizing mitochondria carrying mtDNA mutations. We propose that stem cells might be particularly prone to accumulation of mtDNA mutations due to two factors: i) continual replications that lead to mutation accumulation and ii) the lack of efficient quality control mechanism against defective mitochondria. It has been well documented that mitochondria are inactive in a few types of mammalian stem cells [
45,
46,
47,
48]. In addition, mtDNA mutations have been identified in mouse and human stem cells and have been suggested to play a causal role in stem cell aging [
13,
16]. Therefore, the replication cycle and the accumulation of mtDNA mutations might be a conserved mechanism underlying stem cell aging in metazoans.