Molecular basis of reproductive senescence: insights from model organisms

Abstract

Purpose

Reproductive decline due to parental age has become a major barrier to fertility as couples have delayed having offspring into their thirties and forties. Advanced parental age is also associated with increased incidence of neurological and cardiovascular disease in offspring. Thus, elucidating the etiology of reproductive decline is of clinical importance.

Methods

Deciphering the underlying processes that drive reproductive decline is particularly challenging in women in whom a discrete oocyte pool is established during embryogenesis and may remain dormant for tens of years. Instead, our understanding of the processes that drive reproductive senescence has emerged from studies in model organisms, both vertebrate and invertebrate, that are the focus of this literature review.

Conclusions

Studies of reproductive aging in model organisms not only have revealed the detrimental cellular changes that occur with age but also are helping identify major regulator proteins controlling them. Here, we discuss what we have learned from model organisms with respect to the molecular mechanisms that maintain both genome integrity and oocyte quality.

Introduction

Over the last 30 years, we have seen the average age at first conception increase by > 3.5 years as women delay childbearing. More and more couples suffer from infertility associated with maternal and paternal age. While assisted reproductive technologies (ART) offer increasing alternatives for men and women to bank reproductive cells and tissues, these technologies do not come without risks, including increased likelihood of imprinting disorders and failure to implant. ART also depends on the ability to procure healthy egg and sperm, and in cases of premature ovarian failure (POF) and advanced maternal age, this is often not feasible. POF is itself associated with increased risk for metabolic and cardiovascular disorders [1] that have led to the question of whether reproductive decline is itself driving deterioration of somatic tissues. Furthermore, reproductive decline is the earliest manifestations of aging experienced by most organisms and thus insight into its genesis and consequences will likely have profound implications for healthspan and aging interventions.

While the physiological and hormonal basis of reproductive senescence is well established, it is only in the last decade that we have developed a greater understanding of the molecular and cellular hallmarks of age-related fertility decline and have begun to obtain insights into the underlying mechanisms. Firstly, this explosion of knowledge is owed to the dramatic advances in high throughput, genomics technologies that have made it possible to document age-related changes in gene expression with unprecedented spatiotemporal resolution. Secondly, remarkable strides have been made in unraveling the fundamental biology of aging and correspondingly our knowledge of reproductive aging has advanced as well. These discoveries have often emerged from studies in model organisms, both vertebrate and invertebrate, and many have been subsequently substantiated in humans. The major cellular processes that have been implicated in loss of oocyte quality with advancing age have been genome instability, mitochondrial dysfunction, loss of proteostasis, and disturbances in meiotic chromosome segregation due to compromised function of the spindle assembly checkpoint (SAC) surveillance system. Here, we review the current state of knowledge on the molecular basis of reproductive aging and describe the contribution of model organism studies to our understanding of this subject.

Human perspectives

The pool of human oocytes is set in utero and diminishes continuously throughout a woman’s life. Age irreversibly alters eggs, both in terms of quantity as well as quality, leading to an increase in chromosomal segregation defects, reduced fertilizability of eggs, and defects in early embryogenesis. When the oocyte pool reaches fewer than one thousand, menopause ensues. This is driven primarily by altered hormonal signaling between the brain and the ovarian follicles and supporting granulosa cells. However, well before reproductive senescence is observed, egg quality diminishes and fertility rates decline leading to an increase in miscarriages and fetal chromosomal abnormalities. This phenomenon is observed in different model organisms as well, from mammals to invertebrate species that reproduce for about one-third to one-half of their lives and then experience reproductive senescence.

The cause of reproductive aging is multi-factorial with input from both germ cell intrinsic and extrinsic factors. Many of the factors that are thought to influence somatic aging also act during reproductive aging, but manifest differently in the male and female germlines. Because sperm are produced continuously and are not stored, the fidelity of DNA replication and repair of DNA lesions has a more profound effect on sperm than on eggs. Conversely, because oocytes persist for tens of years in dictyate arrest, they are subject to proteostasis breakdown and mitochondrial dysfunction.

Model organisms for studying reproductive aging

Worms, flies, and mice have been the primary model organisms used for the study of reproductive aging. The particularities of each system have facilitated dissection of individual aspects of this complex program. An overview of the reproductive organs and capacity of each is provided in this section.

Caenorhabditis elegans

The nematode C. elegans is a hermaphrodite that produces and stores sperm during larval development and switches to produce oocytes in the fourth larval stage and through adulthood. Each hermaphrodite has anterior- and posterior-positioned germline arms that are organized spatiotemporally from the distal proliferative zone, into meiosis, and to the most proximal, diakinesis-arrested oocytes awaiting ovulatory signals from stored sperm in the adjacent spermatheca. Peak fertility is seen between days 1.5 and 3 of adulthood and declines dramatically over the next several days with total broods of 250–300. Reproductive capacity is limited in part by sperm production. Males (which arise from random X chromosome nondisjunction) can mate with hermaphrodites, increasing reproductive capacity to 500–700 offspring. However, even mated worms with excessive sperm suffer reproductive decline by around days 9–10 of adulthood revealing an intrinsic program for reproductive senescence [2]. Feminized mutants of C. elegans do not produce self-sperm and thus hold their eggs until mating is achieved. The stored eggs in the feminized mutants are arrested at the diakinesis stage of meiosis, like human oocytes, and thus allow researchers to study impacts of genes and environment on aged, arrested oocytes. Indeed, a decline in reproductive fitness of oocytes, seen as changes in ploidy, DNA repair processes, proteostasis, mitochondrial function, is seen in these held oocytes of days 8–10 adults [3].

Drosophila melanogaster

A fruit fly becomes fertile within hours of achieving adulthood. Peak reproduction occurs 5–10 days later with reproductive capacity lasting 30–50 days. Each female fly contains two ovaries that are composed of 12–16 strings of developing oocytes known as ovarioles. At the tip of the ovariole is a region known as the germarium which harbors both somatic and germline stem cells (GSCs). Complex signaling between the germline stem cell niche and the GSCs maintains reproduction [4, 5]. Fly eggs develop, as in humans, encapsulated by support cells, initially escort cells, and, later, follicle cells As a fly ages, hatching rates decrease and morphological defects increase, revealing decline in both egg production and egg quality with age [6–8].

Mus musculus

A mouse becomes sexually mature at 5 to 8 weeks after birth. Within mammalian species, the short reproductive cycle length, called estrous cycle, as well as the similarity of the germline development with humans, makes mice a very useful model organism to study reproductive features, especially reproductive aging. As in humans, mouse oogonia enter prophase I and differentiate into oocytes while developing in the fetal ovary. Female mice are born with a finite pool of oocytes, all arrested shortly after birth in the dictyate stage of late prophase in the developing follicles [9]. The number of eggs that resumes meiosis and is ovulated in each estrous cycle ranges between 6 and 16, depending on the strain. The gestation period is about 18.5 to 21 days, with a litter size ranging from 2 to 12 pups [10]. Mice have a productive breeding life of approximately 7 to 8 months although they can live to several years [10].

The balance between proliferation, meiotic entry, and apoptosis determines reproductive capacity

In flies, worms, and mammals, germ cell number is determined by rates of proliferation versus apoptosis. Although the timing of these events differs (oocyte proliferation and cell death occur before birth in mice and humans, but throughout much of adulthood in flies and worms), the molecular pathways that control proliferation and cell death are shared between these organism although often employed differently in the somatic and germline cells of different species or between the sexes of a given species.

During aging in worms, the number of proliferative germ cells decreases [2, 3, 11–16]. While this decline does not limit reproductive capacity, it does influence the rate of progression of oocytes through the germline and it does provide an important model for stem cell aging. In the proliferative zone, the balance between cell cycle length and meiotic entry ultimately controls proliferation in the worm germline. This balance is modulated to a great extent by interaction between the distal tip cell (DTC) that serves as the somatic germ cell niche in both hermaphrodites and males, and the GSCs. During development, the DTC directs the migration of the growing gonadal arms. In adulthood, the DTC encapsulates the anterior mitotic tip of the germline, extends projections into the proliferative zone that release GLP-1 Notch ligands to promote stem cell fate, and inhibits meiotic entry (reviewed in [17]. GLP-1 signaling is facilitated by direct association of the DTC with the GSC through innexin-based gap junctions, E-cadherin, and L1CAM [18, 19]. As cells are passively pushed down the syncytium, they lose contact with the DTC, get covered by gonadal sheath cells, and enter meiosis. Expression of the Notch effectors, LST-1 and SYGL-1, decreases in older animals resulting in a longer cell cycle and a decrease in the number of cells that enter meiosis [13]. The sheath cells extend distal projections, intercalating into the DTC plexus. Whereas the DTC plexus length was seen to be stable until day 4 of adulthood [20], it is unclear whether the attachments between the DTC and GSC continue to remain stable or whether they are lost with reproductive senescence.

In female flies, the tip of the germarium contains the terminal filament cells and cap cells that signal via the BMP ligand, Dpp [21], to promote self-renewal of the 2–3 GSCs that reside in the niche. By day 25 of adulthood, proliferation rates are decreased 2.67-fold and after day 25, it continue to decline, although less rapidly. Reduced dpp signaling in older Drosophila is a major contributor to the loss of proliferative capacity while overexpression of Dpp increases proliferation in older animals [6]. Nevertheless, reproductive capacity does not increase in these animals because of a concomitant increase in germline apoptosis (discussed below). BMP signaling is tightly regulated in both the GSCs and niche to ensure the proper maintenance of this tissue (reviewed in [22]), so the aging process can be seen as a deterioration of tissue homeostasis mechanisms.

In male flies, the niche is composed of hub cells that contact both the 6–12 GSC and somatic cyst stem cells (CySCs) through microtubule (MT)-based nanotubules. The GSCs divide to self-renew and form a gonialblast that in turn divides four times and differentiates into sperm. GSCs are lost at a rate of one every 2 weeks; though transient amplifying daughters can dedifferentiate to refill the niche, GSC numbers still decrease with paternal age [23]. Unpaired (Upd), the key mitogenic factor released from the hub, activates the JAK-STAT signaling pathway in GSCs to regulate the stem cell proliferation [24–26]. Upd concentrations decrease with paternal age and constitutive expression of Upd suppresses GSC loss supporting its pivotal role in maintaining the GSC pool [27]. upd mRNA levels are regulated by IGF-II messenger RNA-binding protein (Imp) which itself is targeted by the miRNA let-7. Increased let-7 leads to a reduction in Imp levels and increased degradation of upd RNAs resulting in a loss of proliferative capacity [28]. In aging hub cells, another miRNA, miR-9a, is also increased, leading to a downregulation of neural cadherin (N-cad) and a loss of hub cells’ contact with GSCs [29].

In mice, BMP4 and Nanog signaling pathways repress the activity of the transcriptional repressor Otx2 which represses the primordial germ cell (PGC) determinants Blimp1, Prdm14, and AP2gamma (reviewed in [30]. GSCs migrate into the gonadal ridge between days E8.5 and E12.5, increasing in number from ~ 45 to > 4000 at E12.5 and reaching > 25,000 cells by E13.5. Reducing the initial pool of PGCs by altering proliferation rate, e.g., by reduced Blimp1 expression [31, 32] or loss of the conserved germline determinant Vasa (Mvh in mice) [33], can have a profound impact on overall fertility, leading to reduced litter sizes and fewer broods.

In addition to proliferation, apoptosis also influences the size of the germ cell pool. In aged flies, cell death eliminates nearly 50% of developing oocytes [6]. Furthermore, on a poor diet, inhibition of TOR signaling by GATOR1 is required to protect previtellogenic follicles against apoptosis [34], suggesting that apoptosis serves as a quality control mechanism for the developing oocytes. In worms, nearly half of all oocytes are apoptosed and serve as nurse cells for the remaining oocytes [35] and, as in other organisms, germ cell apoptosis is also activated in response to DNA damage. Yet in older worms, activation of apoptosis is attenuated compared with younger animals [3] and inhibiting physiologic apoptosis leads to a substantial increase in defective oocytes [36]. Thus, while physiologic apoptosis in early life creates micronutrients to support oocyte growth, DNA damage–induced apoptosis maintains oocyte quality control by eliminating defective oocytes.

Inhibiting apoptosis in mice, as in worms, can increase the size of the oocyte pool, but it does so with a trade-off in oocyte quality. In female mice, germ cell proliferation occurs during embryogenesis, producing a huge excess of primordial germ cells. Atresia then reduces this germ cell pool almost 10-fold. Ongoing atresia during follicle development further reduces the oocyte pool postnatally. Inhibition of cell death (e.g., via mutation in the apoptosis gene, Bax) leads to an increased oocyte pool at advanced ages, but these are still unable to support pregnancy [37]. In both mice and worms mutant for the protein synthesis regulator, eEF2K/EFK-1 that phosphorylates the eukaryotic elongation factor 2 (eEF2), the size of the ovarian follicle pool (mice), or total egg counts (worms) is increased, but viability of the offspring is decreased [38]. Recent evidence supports the idea that a subset of these apoptosed oocytes has activated DNA damage checkpoint and has evidence of active transposons or meiotic damage [39, 40], supporting the idea that atresia functions to eliminate (at least some) defective oocytes.

Nutrition is a major determinant of reproductive capacity

Stress and environmental cues, including nutritional status, can influence reproductive capacity and outcomes across a range of species. For example, in female flies, GSCs are highly responsive to nutritional availability [41] and flies reared on poor-quality food show a higher rate of GSC decline [42]. Similarly, in the fly testis, GSC proliferation rate and numbers decrease under nutrient poor conditions [43, 44] although dietary restriction (DR, lifespan extension induced by sustained reduction in calorie intake) was shown to preserve GSCs in testis [45]. These data show that germ cell number is malleable and suggest that reproductive aging may be exacerbated by poor nutrition and environmental stress. Accordingly, nutrient-sensing signaling pathways have emerged as central players in the control of reproductive aging. Insulin signaling (IIS), AMPK signaling, and the TOR pathway have significant roles in both establishing the size of the initial progenitor pools and in GSC maintenance in worms [14, 46–51] and flies [34, 42, 52–58]. Apoptosis, while important in wild-type worms for oocyte quality control, does not appear to be the mechanism by which animals extend reproduction under nutrient stress [3].

In worms, TBFß pathway mutations prolong lifespan and reproduction [59]. Interestingly, this function is cell non-autonomous and executed by TGFß activity within the skin-like tissue of worms, the hypodermis [3]. TGFß inhibits the transcription factor, cAMP response-element binding protein, CREB, within hypodermal cells, which in turn helps produce a secreted protein, WRT-10, likely to function in the Hedgehog (Hh) signaling pathway. WRT-10 in turn activates Hh receptor proteins, PTC-1 and PTC-2, expressed on germ cells [60]. This elaborate inter-tissue dialogue allows the animal to coordinate reproductive output with physiological features such as nutrition. Both the TGFß and insulin signaling pathway mutations decrease the rates of aneuploidy [3], revealing a direct impact of these nutrient sensing pathways on chromosome behaviors. These studies point to the critical interplay between somatic and germline tissues in the regulation of gonadal health.

While IGF, AMPK, TOR, and TGFß pathways are continuously active in worms and flies to control reproductive (and somatic) lifespan, there are hints that they may also function to influence the size of the oocyte pool in humans and/or the integrity of the human gonadal environment with age (for reviews see [61–63]). Thus, understanding these networks, their function, and the myriad downstream pathways they regulate may have important implications for both male and female fertility preservation with age. What ultimately drives the changes in gene expression and decreased signaling between the stem cells and their niches is still unclear, although morphological changes in the aging gonad suggest that decreased cell-to-cell contacts and alterations in the extracellular matrix (ECM) stroma may contribute to the loss in signaling capabilities.

The links between nutritional status and reproduction have led to extensive study of the impact of various diets on reproductive fitness. For example, recent studies in flies have shown lower reproductive decline in male flies grown on polyunsaturated fatty acids (PUFAs )[64]. Alternatively, worm studies have shown the detrimental effects of lipids and sugars on spawning and reproductive capacity [65]. Similarly, mice undergoing DR also exhibited a slight increase in their follicle number; a decrease in chromosome alignment defects, spindle defects, and mitochondrial aggregation; and a decline of ATP production, compared with age-matched ad-libitum fed controls [66]. The ability to link specific micronutrients or microbiota to fertility outcomes is a burgeoning research topic and can provide potential therapies to extend reproduction.

Sister chromatid cohesion is the major contributor to chromosome segregation errors seen in aging oocytes

Maternal age was first shown to impact recombination rates and nondisjunction in Drosophila [67, 68] and has since been well documented in nematodes, mice, and humans [69–71]. In humans, defects in the formation of crossovers between parental chromosomes are the most prevalent cause of miscarriage and a significant contributor to age-related decline in oocytes.

In both males and females of any species, the two divisions that take place during meiosis are crucial for the production of haploid gametes. At the onset of meiosis I, chromosomes replicate to produce identical sister chromatids that become attached along their length by cohesin complexes, tetrameric complexes that form rings enveloping the replicated chromosomes [72]. As prophase I proceeds, DNA strand exchanges between homologous chromosomes are established and mature into chiasmata which, together with cohesins, hold the homologs together on the meiosis I spindle. At anaphase I, homologs separate when chiasmata are resolved into crossovers by resolvase enzymes and sister chromatid cohesion is released from the chromosome arms. In C. elegans, where chromosomes are holocentric, cohesion is removed from the crossover site to the nearest end of the chromosome. Centromeric cohesion permits sister chromatids to remain tethered until anaphase II, when cleavage of cohesin complexes facilitates their release and segregation to opposite poles [73]. Aneuploidy can result either from defects in crossover formation or from inappropriate release of cohesion at MI or MII or both.

In humans and mice, crossovers are established during embryogenesis in females and the oocytes are arrested at the diakinesis stage prior to maturation. By preventing mating in flies [74, 75] or using feminized mutants in worms [2], oocytes can also experience aging similar to humans. With age, certain crossovers configurations, particularly those near telomeres and centromeric heterochromatin, as well as uncharacterized, chromosome-specific features [76] make a chromosome more prone to missegregation. The Bickel and Hunt labs [75, 77, 78] discovered, in flies and mice, respectively, that defects in proteins required for meiotic chromosome cohesion substantially increased nondisjunction, providing the first hints that maintenance of cohesion deteriorated with maternal age. In flies, the loss of solo or ord gene function also led to decreased maintenance of the cohesin complex with maternal age [79–81]. In mice, the loss of the Smc1β and the cohesin subunit, Rec8, altered crossover distribution leading to an increase in distal chiasmata [77, 82] that are also more sensitive to cohesion loss. Both mutations also decreased the amount of the chromosome-associated protein, Shugoshin 2 (Sgo 2), that plays a critical role in protecting centromeric cohesion in meiosis I [83]. Ultimately, centromeric cohesion is destroyed by Separase, an enzyme that cleaves the cohesin ring. Separase activity is kept in check by two independent mechanisms, inhibition by Securin and inhibitory phosphorylation by CDK1 [84]. Mutations that allow for premature activation of Separase in oocytes led to premature sister separation in young oocytes that mimic age-related defects, suggesting that the dual mechanisms of Separase control may be critically important in the face of altered cohesion with age [85]. In fact, loss of Securin function has recently been implicated in maternal age-dependent nondisjunction [86].

In theory, cohesion subunits could be replaced by new pools of proteins during aging. However, recent studies in mice using tagged proteins induced after the completion of DNA replication show that the meiosis-specific kleisin subunit of cohesin, Rec8, cannot confer cohesion [87]. While studies in yeast and worms have suggested that cohesion can be induced by double-strand breaks [88, 89], the results from mouse suggest that endogenous DNA damage cannot reach sufficient levels to confer cohesion during mouse meiosis. Whether the much greater time frames that the human oocyte experiences in meiotic arrest expose an oocyte to increased damage that can induce cohesion is not known.

Other factors that could contribute to aged-related loss of cohesion in oocytes are oxidative damage and alteration of intracellular pH (discussed below). Recent findings have shown that genetically enhancing oxidative stress increases cohesion loss and segregation errors in Drosophila oocytes [90, 91]. In addition, extrinsic factors such as obesity can exacerbate reproductive aging by accelerating the loss of cohesion during meiosis [92]. Obese mice showed accelerated weakening of centromeric cohesion with advancing age and increase of chiasma terminalization and premature separation of sister centromere [92], possibly due to an increase of oxidative stress. Aged oocytes in mice have elevated intracellular pH that could cause an increase in aneuploidy associated with the loss of cohesion [93]. The increase in pH might damage the cohesion complex ring structure due to a reduction of the chromosome-associated cohesin protein SMC3, increasing sister inter-kinetochore distance and finally leading to chromosome misalignment and impaired segregation [93].

Meiotic spindle attachments and a relaxed spindle assembly checkpoint allow for chromosome missegregation in older oocytes

In order to correctly segregate during meiosis, chromosomes must reach a stable bioriented configuration before anaphase. During meiosis I, sister chromatids are attached to the same pole and co-segregate, while homologous chromosomes connected by chiasmata segregate to opposite poles. During meiosis II, sister chromatids’ kinetochores become attached to MTs from opposite poles enabling an accurate segregation which result in haploid cells. Due to the important role that REC8 plays in the mono-orientation of sister chromatids [94], loss of Rec8 could contribute to an increase of bi-directional kinetochore orientation and ultimately to chromosome missegregation. One of the main signs of centromeric cohesion weakening in aged oocytes is the increase of inter-kinetochore distances, both during meioses I and II [82, 92]. The underlying mechanism responsible for this separation of associated kinetochores has been described recently, showing that experimental depletion of Smc3 in young mouse oocytes produces decompaction of centromeric chromatin and kinetochores fragmentation, reproducing the age-related changes observed in mammalian kinetochore and centromere architecture due to loss of cohesion [95].

The kinetochore serves as the docking site for spindle MTs and proteins at the kinetochore-MT interface monitor chromosome attachment through tension. During assembly of the spindle, erroneous attachments are corrected by activation of spindle assembly checkpoint (SAC)[96, 97]. Numerous studies performed in mice have shown that kinetochore MT establishment is compromised in aged oocytes due to weakened centromeric cohesion, increasing the frequency of merotelic attachments that could lead to chromosomes missegregation [73, 98, 99]. Merotelic kinetochore attachment is characterize by sister kinetochore attachment to both spindle poles simultaneously causing opposite pulling forces in anaphase. This phenomenon might help age-related aneuploid eggs in MII to elude SAC mechanism and enter into anaphase II, resulting in chromosome lagging and trailing, and lastly leading to the delay of anaphase II completion [100]. Moreover, this particular kind of aberrant attachments could enhance kinetochore fragmentation observed in aged oocytes [95]. Decreased efficacy of the SAC itself also appears to contribute to age-associated defects [101, 102] and is driven in part by decreased expression of SAC subunits [103]. The discovery that the SAC promotes metaphase I arrest in response to DNA damage [104–106] suggests that the age-related impairment in SAC function may both decrease genome integrity and increase chromosome segregation errors.

Chromosome segregation may falter with age to increase nondisjunction

Given the high frequency of non-recombinant (achiasmate) chromosomes seen in human miscarriages and birth defects (REF), one might wonder whether a system exists to help segregate these chromosomes to opposite poles. Indeed, such an achiasmate system has been described in Drosophila [107]. In Drosophila sperm, crossing over does not occur and homologous chromosomes rely on distributive pairing for segregation. In Drosophila females, the small, mostly heterochromatic, 4th chromosomes also do not experience crossing over, and their proper segregation is mediated by pericentric heterochromatic attachments [108, 109] that persist after the SC has disassembled [110, 111]. The importance of heterochromatin has recently been reinforced by the finding that heterochromatin-associated proteins HP1a, a histone H3 lysine 9 (H3K9) methyltransferase, and its binding partner, Piwi, are required for accurate segregation of achiasmate chromosomes [112]. In addition, heterozygosity of the ord locus, which functions in sister chromatid cohesion, predisposes achiasmate chromosomes to missegregate in older oocytes, underscoring the importance of sister-chromatid cohesion for segregation of both exchange (discussed above) and non-exchange chromosomes in meiosis I. Mutations in Axs and nod, both of which have roles in spindle assembly, also impair achiasmate segregation, suggesting additional layers of complexity for this system (reviewed in [113]).

Strong evidence that an achiasmate system exists in humans comes from pedigree analysis of 576 gametes in which non-exchange chromosomes segregated correctly in up to 7% of meioses, well above the rate expected by random chance [114, 115]. Meiotic centromere associations are well characterized in mice [116, 117] and the centromeric cohesion protein, Shugoshin, has recently been implicated in centromere pairing in mice and in promoting achiasmate segregation in yeast [118]. Determining whether these centromere attachments prevent age-related nondisjunction remains to be tested.

Elegant studies over the past 5 years in both human and worm oocytes have suggested that there also exist mechanisms of “meiotic correction.” Examination of polar bodies in human oocytes and early embryos revealed a surfeit of monosomic and trisomic chromosomes suggesting that oocytes contain an intrinsic mechanism to maintain normal chromosome number [119, 120]. Using mutants that increase meiotic chromosome errors, specifically on the X chromosome, the McNally group has shown that in C. elegans oocytes, meiotic spindle orients to segregate extra chromosomes into the polar body [121]. While it is not known whether this system is exacerbated by maternal age, the changes in kinetochore attachments and tension described above would suggest that erosion of this process could profoundly increase nondisjunction rates.

Mitochondrial dysfunction is a conserved feature of aging oocytes

Fertilization and development are energy intensive processes heavily reliant upon mitochondria for their ATP supply. Mitochondria are also the platform, and source, for many of the signaling pathways that influence every aspect of reproduction from germ-cell proliferation to embryonic development. In particular, oocyte mitochondria have uniquely crucial roles because they provide the cellular energy for proper chromosome alignment and segregation during meiosis, and their level directly determines the survival and health of the zygote before embryonic transcription can take over. Additionally, the integrity of maternal mitochondrial genome is crucial as they are the sole providers of the mitochondrial pool after destruction of the paternal mitochondria. Mitochondria have central roles in determining organismal aging itself, and mitochondrial dysfunction has emerged as a leading hallmark of aging [122]. Hence, it is not surprising that mitochondrial dysfunction has been found to be closely associated with reproductive senescence as well.

Age-related qualitative and quantitative alterations in mitochondria have been documented in numerous organisms from invertebrates to humans. In flies, worms, mice, hamsters, and non-human primates, oocyte mitochondrial number decreases with age [123–126]. mtDNA content is found to be lower in older oocytes compared with young ones in mouse and hamster models as well as in older women [124, 127]. Mitochondria from aged oocytes also appear more elongated as compared with younger ones in rat, mouse, and hamsters [123, 124, 128]. Older mouse oocytes exhibit more mitochondrial aggregates too while oocytes from older women appear larger and denser [129, 130].

The main functional changes observed in the mitochondria of aged oocytes include reduced membrane potential, activation of the mitochondrial permeability transition pore, and leakage of the mitochondrial matrix [131]. Importantly, ATP production in oocytes goes down significantly in aged mouse oocytes and this has been found to be a critical determinant of the oocyte’s fertilization potential as well as the embryo’s survivability [66]. Gene expression studies comparing the transcriptomes of oocytes from young vs. old mice have revealed a decrease in expression of numerous genes involved in mitochondrial functions [132]. In particular, enzymes involved in the synthesis of Coenzyme Q (CoQ), a key component of the electron transport chain, decrease significantly with age in murine models [133]. Mitochondrial ATP production is accompanied by the generation of reactive oxygen species (ROS), byproducts that damage cellular components. ROS are normally eliminated by the activity of antioxidant enzymes. However, with age, there is mitochondrial dysfunction that causes increased ROS, as well as reduced expression of ROS-scavenging enzymes [122, 134]. In Drosophila, there is evidence of higher ROS levels and lower antioxidant activity in oocytes, and this trend was also observed in ovarian follicles and interstitial cells of aging mice and non-human primates [135–137]. In women, there is a decline in levels of antioxidant enzymes with age, in the oocyte as well as the in the granulosa cells that surround the developing follicle, and cumulus cells which surround the oocyte [134, 138, 139]. Interestingly, circular RNAs (circRNAs) that potentially target and silence genes involved in oxidation-reduction reactions have been found to be enriched in ovaries of older females compared with their younger counterparts, providing a potential post-transcriptional mechanism for reduced mitochondrial gene expression [140].

The mitochondrial changes enumerated above have functional consequences. Studies in model organisms have not only helped discover the impact of aging on germ-cell mitochondria, but have also provided evidence for mitochondrial dysfunction causing age-related fertility diminution. Treatment with hydrogen peroxide (H₂O₂) that mimics the disruption of mitochondrial ATP production observed in older oocytes has been shown to cause meiotic spindle abnormalities in mice [141]. In older female oocytes, altered mitochondrial morphology has been found to be negatively correlated with embryo development based on the decline of mitochondria activity [142]. In addition to mitochondrial morphology, aneuploid human embryos manifest higher mtDNA content which corresponds with the abnormal levels of mtDNA in older oocytes [143].

Mitochondria are a major target in the search for anti-aging interventions. More recently, they have become the focus of molecular and therapeutic approaches to mitigate age-related fertility decline [131]. In part, this stems from observations that some lifespan-extending interventions have been found to retard reproductive decline as well. For instance, an exercise regimen that partially rescued the premature aging defects of a mtDNA mutator mouse carrying a γDNA polymerase deletion was reported to also mitigate the early reproduction decline seen in these mutants [144]. Treatment with antioxidants has been shown to maintain oocyte quality with age in mice further substantiating a role for ROS in aging oocytes [145]. Other interventions that improve mitochondrial bioenergetics have also been reported to benefit the quality of aging oocytes. Ben-Meir et al. found that by treating aged female mice with CoQ10, ovulation was increased, spindle defects were rescued, mitochondrial gene expression was restored, and more pups were born [133]. In addition to mitochondrial antioxidants, key regulators of metabolism with mitochondrial hubs are emerging as potent therapeutic targets. A recent report indicated that in mouse oocytes, the levels of nicotinamide adenine dinucleotide (NAD⁺) undergo an age-related decline [146]. NAD⁺ is a central metabolic coenzyme/substrate and redox factor involved in cellular energy metabolism and energy production. It plays a central role in oxidation-reduction reactions in cells and in mitochondrial function via participation in tricarboxylic acid cycle and oxidative phosphorylation. It also serves as a substrate for the histone deacetylase Sirtuin enzymes that catalyze posttranslational modifications in cytoplasmic and mitochondrial proteins. Excitingly, supplementation with the NAD⁺ precursor, nicotinamide mononucleotide (NMN), or overexpression of the NAD⁺-dependent Sirtuin, SIRT2, improved oocyte quality in old female animals, restored their fertility, and reversed the adverse impacts of maternal age on developing embryos [146].

A similar trend of dysfunctional mitochondria is observed in males as well, and impacts reproductive fitness with age. Function is also decreased in older rat testicular mitochondria [147]. Similar to oocytes, in a rat model, there was an increase in ROS and a decrease in superoxide dismutase enzymes in older sperm [148]. Using an aging mouse model, which has been injected with d-galactose to generate advanced glycation end products, there was decreased super dismutase activity in the testis, decreased sperm count, and more abnormal sperm [149].

Impaired DNA damage repair networks promote reproductive aging

A major target of ROS is the nuclear genome where it induces DNA damage and telomere shortening. The effects of ROS are further exacerbated during aging by compromised antioxidant defense systems [150, 151]. In C. elegans, gene expression profiling revealed that decrease in mismatch repair capacity may be mediated by age-related decrease in mitochondrial function and ATP production [152]. DNA damage is particularly a serious cause of concern for nondividing or slowly dividing cells (i.e., oocytes), where unrepaired damage will tend to accumulate over time. In somatic cells, it is well established that nuclear genome integrity is required for healthy aging [153, 154] and that repair rates decline in mammalian cells in culture [154, 155]. Due to the limited access to human oocytes, animal models are a powerful tool to study DNA damage accumulation and telomere attrition.

DNA damage has a profound effect on GSCs, and more so in aged animals. Exposure of young flies to gamma irradiation (IR) leads to mitotic arrest, repair, and p53-dependent regeneration of the germline after a week [156, 157]. By contrast, a similar dose in 6-week-old flies leads to permanent cell cycle arrest of the GSCs [158], despite evidence of DNA repair. Moreover, the impairment in DNA repair capacity observed in oocytes with advanced maternal age could be due to molecular alterations and epigenetic modifications of genes involved in DNA double-strand break (DSB) repair. Studies in mouse and humans reported an accumulation of DNA DSBs in primordial follicles with age [159, 160]. Age-related decline in the expression of DNA DSB repair genes, including BRCA1 and RAD51, among others, was observed using quantitative PCR (qRT-PCR) on primordial follicles of female rats [161] and humans [159]. DNA damage and repair deficiency with age also affects the granulosa cells connected with oocytes in the ovary. These cells are crucial to maintain follicle reservoir, oocyte growth, and follicular development in mammals. Experiments in rhesus monkeys, a non-human primate model, showed an increase in DSBs in granulosa cells, accompanied with a decrease in DSB repair ability, measured by the number of BRCA1 foci: the number of BRCA1 foci declined both in granulosa cells and oocyte nuclei with maternal age [162]. Thus, oocytes and their support cells suffer from defects in DNA damage sensing, checkpoint activation, and repair itself.

A consequence of impaired DNA repair is reduced robustness of offspring. While mutations may or may not be deleterious in the immediate offspring, studies from model organisms have shown profound transgenerational effects including defects in DNA repair. For example, inability to repair DNA: protein crosslinks that arise in the germline, such as occurs by loss of the GCNA protein, results in transgenerational sterility in flies and worms [163], and impairment of mismatch repair by mutation of the worm msh-2 gene, lead to microsatellite repeat instability and reduced fecundity in later generations [164].

Telomere attrition also impact oocyte quality and have transgenerational consequences (reviewed in [165]). Telomeres transform the blunt end of chromosome DNA to a closed loop, preventing the chromosome end from being identified as DSBs, which would trigger a DNA damage response [166]. Telomere attrition occurs every round of replication in dividing cells, and in nondividing cells, such as the oocytes, results from the effects of ROS, and associated cellular responses. Telomeres are especially susceptible to ROS-induced DNA damage, because their sequence is rich in guanine and this nucleotide is most susceptible to oxidation [167]. Telomere attrition plays a central role in oocyte aging [168, 169] and shorter telomeres are associated with infertility (reviewed in [170]). Also, studies in mice showed a significant decline both in expression of TERT, the catalytic enzyme of telomerase, and in telomerase activity in oocytes of older females as compared with those from young ones [171]. In both mice and worms, telomerase mutations lead to progressive telomere shortening over generations resulting in female sterility due to abnormal spindles and misalignments of metaphase chromosomes in oocytes [167]. While alternative lengthening of telomeres (or ALT) pathways that use homologous recombination to lengthen telomeres can be induced in tissue culture and some cancers [172], it is unclear whether ALT can be activated during normal aging in oocytes. In worms, rfs-1, the paralog of the DNA repair protein RAD51D shows hallmarks of ALT including progressive telomere shortening and enhancement of telomerase-mediated mutations and transgenerational sterility that was exacerbated by maternal age [173]. While this points to a potential role for ALT-like mechanisms in germ cells, it is unclear whether ALT can be activated in dictyate arrested oocytes as a mechanism to retain telomere length.

Aging oocytes exhibit loss of multiple aspects of proteostasis

Proteostasis encompasses the production and maintenance of correctly folded proteins as well the efficient elimination of misfolded, damaged, or old proteins, mediated by the combined activities of the protein folding/chaperone systems as well as degradation executed by the proteasomal and lysosomal/autophagy pathways. Impediments to either protein synthesis or disposal can cause protein aggregation, a phenomenon linked to aging and disease [174]. Proteostasis in the immortal germline is especially sacrosanct to prevent transmission of accumulated damage across generations. Several mechanisms have been identified for maintenance of proteostasis in the germline and for ensuring that a newly fertilized embryo starts with a fresh proteostasis slate. In some species, this is achieved by maintaining high-fidelity protein quality control in the germline, as compared with somatic cells. In Drosophila for instance, the proteasome is more active in young eggs than in age-matched somatic cells, whereas, asymmetric segregation of damaged proteins during gametogenesis occurs in mammalian embryonic stem cells [175, 176]. In C. elegans, damage clearance seems to be linked to fertilization. Goudeau et al. first showed that carbonylation, a form of protein damage, and protein aggregation are widespread in immature oocytes, but carbonylated proteins are removed before fertilization via proteasomal degradation [177]. The signal(s) triggering this proteolysis were recently found to originate from sperm-secreted hormones which activate a lysosomal proton pump, the vacuolar H+ ATPase (V-ATPase), VHA-13 in the oocytes. The V-ATPase facilitates lysosomal acidification, and this is associated with mitochondrial changes and activation, including morphology, ROS levels, and the ATP/ADP ratio, which contribute towards degradation of aggregated proteins, through an unknown mechanism [126]. Interestingly, oocyte maturation in the frog, Xenopus laevis, is accompanied by a similar acidification suggesting this may be a conserved mechanism [126]. Interestingly, oocyte maturation in the frog, Xaenopus laevis, is accompanied by a similar acidification suggesting this may be a conserved mechanism [126]. In mice, early embryonic development is accompanied by an increase in 20S proteasome activity, which reduces the carbonylated and advanced glycation end product (AGE)–modified proteins [178]. In human oocytes, there is also an accumulation of AGE and their concentration is inversely correlated with positive IVF outcomes [179]. Besides generational integrity, protein homeostatic mechanisms also have unique reproductive roles that make them critical. For instance, degradation of paternal mitochondria upon fertilization is mediated by the autophagic machinery of the egg. Studies in worms, flies, and mice have shown that incapacitating maternal autophagy disrupts this with catastrophic consequences [180–182]. A single-cell transcriptomic analysis of ovaries from young and old cynomolgus monkeys identified an increase in apoptosis in older oocytes [135]. In aged men, there is a decline in apoptosis in the spermatogonia but an increase in apoptosis in spermatocytes [183].

Loss of proteostasis has emerged as a central aspect of organismal aging and, accordingly, of reproductive senescence as well. This is an especially critical process in species such as humans, wherein a finite pool of oocytes is laid down and maintained for decades before being fertilized. Oocytes are therefore highly susceptible to age-related proteostasis dysfunction. Carbonylated proteins increase with age not only in C. elegans but also in Drosophila oocytes [176, 177]. In C. elegans, there is also increased protein aggregation seen in maturing oocytes, a phenomenon not observed in young mothers [184]. Aged mouse oocytes also have increased formation of large ubiquitinated aggregates, due to the reduction in oocyte proteasome activity [185]. Proteostasis defects occur in other reproductive tissues as well. In worms, extracellular proteins accumulate in the uterus [186]. In humans, dysregulation of autophagy (found due to mutations in in the autophagy gene, ATG4) was found to cause uterine fibroids [187]. These observations have been corroborated by gene expression studies and proteolytic activity assessments in many species. In Drosophila, both the 26S and 20S proteasome peptidase activities were found to decline in somatic tissue, but were maintained in aged gonads [136]. In C. elegans too, old oocytes exhibit decreased expression of proteolytic pathway genes [3]. In aged mouse oocytes, an RNA Seq study found differences between old and young transcriptomes in genes involved in multiple aspects of proteostasis including decreased expression of molecular chaperones in aged oocytes [132, 188]. In mature MII oocytes, and human embryonic stem cells, transcriptomics data suggested an increase in expression of genes encoding proteins involved in ubiquitination and the proteasome pathway although a corresponding increase in protein levels or enzymatic activities is not seen [189]. As with mitochondria, promising genetic and chemical interventions that improve proteostasis have also been found to improve age-related reproductive decline. For instance, long-term treatment of mice with melatonin, postulated to enhance autophagy as well as function as an antioxidant, was shown to result in a greater number of ovulated oocytes in older mice, compared with control older mice as well as elevated expression of autophagy markers [190].

Translation efficiency changes in older oocytes

In addition to the factors that directly affect the viability and health of the egg, aged oocytes themselves are also less able to produce healthy offspring. This is because the egg is responsible for providing the embryo with all of the proteins, RNAs, and organelles needed during the first several divisions prior to zygotic gene activation. It has long been appreciated that oocyte maturation and early embryonic divisions are exquisitely controlled by regulation of maternal RNA translation and turnover (e.g., see [191] for a recent review of Xenopus translational control). Recent studies in flies using a delayed mating strategy showed that ribosome-associated RNAs decrease with maternal age and can lead to spindle defects [192] and to cell-type specific impairments during embryogenesis [193]. However, little is understood about age-related changes in translation efficiency of oocytes and maternal RNA deposition. Revisiting whether the mRNA post-transcriptional modifications are impacted in older oocytes to drive the changes in translational efficiency will be particularly illuminating.

Paternal age is associated with epigenetic changes

While most of the focus on age-related fertility effects are associated with oocytes, advanced paternal age is a susceptibility factor for some human diseases, most notably autism spectrum disorder and schizophrenia (reviewed in [194]). Paternal age is not a major risk for nondisjunction [195] and a recent study suggests that mutation accumulation in the paternal genome may be explained by repair defects in zygotes derived from older mothers [196]. Instead, paternal age impacts the sperm epigenome [197, 198] with changes in methylation enriched at neurodevelopmental genes [199].

Conclusions

Whereas our initial understanding of reproductive aging placed meiotic chromosome segregation front and center, studies of model organisms in the last decade have revealed multiple cell autonomous and non-autonomous processes that impact oocyte quality. With the ability to perform single-cell “omics,” the next decade is likely to provide specific information on the genetic, epigenetic, and proteomic changes that drive reproductive aging. Model organisms offer the opportunity to screen for both the genetic and environmental determinants that impact aging and the ability to subject model organisms to large, compound libraries has the potential to identify therapeutic strategies that can delay or ameliorate reproductive decline.

Authors’ contributions

All authors contributed to the writing and editing of this manuscript.

Funding

This work was funded by grants from the National Institutes of Health (R01GM104007) to JLY and (R01AG051659) to AG, a grant from the Global Consortium for Reproductive Longevity and Equality to CQC, and a Children’s Hospital of Pittsburgh Research Advisory Committee (RAC) fellowship to JAL.

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The authors declare that they have no conflict of interest.

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