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Reproduction, Fertility and Development, 2009, 21, 10–14 www.publish.csiro.au/journals/rfd

Telomeres and reproductive aging

David L. KeefeA,C and Lin LiuA,B

ADepartment of Ob/Gyn, University of South Florida, Tampa, FL 33606, USA.BCollege of Life Sciences, Nankai University, Tianjin 300071, China.CCorresponding author. Email: dkeefe@hsc.usf.edu

Abstract. Infertility, miscarriage and aneuploid offspring increase with age in women, and meiotic dysfunction underliesreproductive aging. How aging disrupts meiotic function in women remains unclear, but as women increasingly delayhaving children, solving this problem becomes an urgent priority. Telomeres consist of a (TTAGGG)n repeated sequenceand associated proteins at chromosome ends, mediate aging in mitotic cells and may also mediate aging during meiosis.Telomeres shorten both during DNA replication and from the response to oxidative DNA damage. Oocytes do not divide inadult mammals, but their precursors do replicate during fetal oogenesis; eggs ovulated from older females have traversedmore mitotic cell cycles before entering meiosis during fetal oogenesis than eggs ovulated from younger females.Telomeresalso would be expected to shorten from inefficient DNA repair of oxidative damage, because the interval between fetaloogenesis and ovulation is exceptionally prolonged in women. We have tested the hypothesis that telomere shorteningdisrupts meiosis by shortening telomeres experimentally in mice, which normally do not exhibit age-related meioticdysfunction. Interestingly, mouse telomeres are much longer than human telomeres, but genetic or pharmacologicalshortening of mouse telomeres recapitulates in mice the human reproductive aging phenotype as the mouse telomeresreach the length of telomeres from older women. These observations led us to propose a telomere theory of reproductiveaging. Moreover, chronological oxidative stress increases with reproductive aging, leading to DNA damage preferentiallyat (TTAGGG)n repeats. Finally, if telomeres shorten with aging, how do they reset across generations? Telomerase couldnot play a significant role in telomere elongation during early development, because this enzyme is not active until theblastocyst stage, well after the stage when telomere elongation takes place. Rather, telomeres lengthen during the earlycell cycles of development by a novel mechanism involving recombination and sister chromatid exchange. Telomeredysfunction resulting from oxidative stress, a DNA damage response or aberrant telomere recombination may contributeto reproductive aging-associated meiotic defects, miscarriage and infertility.

Additional keywords: aneuploidy, infertility, meiosis, reproduction.

Meiotic dysfunction increases with age in women

Women experience a marked increase in meiotic dysfunction asthey age, beginning over a decade before depletion of primaryfollicles at menopause. Fertility in women begins to decline bytheir mid-30s and any conceptions that do occur result in highrates of miscarriage and/or aneuploid offspring (Navot et al.1991; Hassold and Hunt 2001; Verlinsky et al. 2004; Munneet al. 2005). Indeed, meiotic non-disjunction in the oocyte isthe single most common genetic cause of mental retardationin children (Hassold and Hunt 2001). The effects of reproduc-tive aging on women have become more important as womenincreasingly delay attempts at child bearing. Moreover, the emo-tional, medical and social costs of aneuploid offspring representa major burden to society, so elucidation of the mechanism ofreproductive aging should be an urgent research priority.

Pathobiology of reproductive aging in women

Most aneuploid oocytes and embryos typically undergo apop-tosis before implantation, producing infertility (Navot et al.

1991; Hassold and Hunt 2001; Liu et al. 2002b; Verlinsky et al.2004; Munne et al. 2005), but some escape only to miscarrylater during development or give rise to offspring with a higherincidence of congenital problems. Several processes predisposeoocytes from older women to meiotic non-disjunction, includ-ing reduced chiasmata and recombination (Abruzzo and Hassold1995; Hassold and Hunt 2001), structural abnormalities of themeiotic spindle–chromosome complex (Battaglia et al. 1996),mitochondrial (mt) DNA mutations (Keefe et al. 1995; Barrittet al. 1999, 2000) and late exit from a production line duringfetal oogenesis (Henderson and Edwards 1968; Edwards 1970;Polani and Crolla 1991). How aging affects such diverse cellularprocesses in such an orderly fashion in virtually every woman,even while the rest of her body continues to function normally,remains an enigma.We have proposed a telomere theory of repro-ductive aging because experimental shortening of telomeres inmice, which normally have long telomeres and exhibit none ofthe above abnormalities, recapitulates virtually the entire repro-ductive aging phenotype seen in women (see below). Geneticor pharmacological shortening of telomeres produces oocytes

© IETS 2009 10.1071/RD08229 1031-3613/09/010010

Telomeres and reproductive aging Reproduction, Fertility and Development 11

with reduced chiasmata and synapsis (Liu et al. 2004), abnor-mal spindles with abnormal chromosome alignment (Liu et al.2002a) and arrested embryonic development ending in apop-tosis (Liu et al. 2003; Keefe et al. 2005). Moreover, reactiveoxygen, such as would be expected to result from even low lev-els of mtDNA mutations, further shortens telomeres (Liu et al.2002c). Finally, many genetic or pharmacological interventionscan acutely induce one or another feature of reproductive agingin mice, but only telomere shortening provides a plausible tim-ing mechanism to explain the effects of aging itself on oocytefunction. We have proposed that telomere dysfunction in eggsfrom older women provides a unifying and parsimonious the-ory to explain how aging predisposes older women to manifoldaspects of reproductive dysfunction (Keefe et al. 2006).

Telomere biology

Telomeres are repetitive sequences of TTAGGG with severalassociated proteins that loop, and therefore cap, chromosomeends and prevent end-to-end fusions. In most cells, telomeresshorten with each cell division, as well as from exposure to reac-tive oxygen. Critically short telomeres trigger cell cycle arrestand apoptosis and promote genomic instability (Blasco et al.1999; Blackburn 2005a, 2005b). The tendency of telomeres toshorten with each round of DNA replication and from exposureto reactive oxygen (Liu et al. 2002c; Kurz et al. 2004; Passosand von Zglinicki 2005) makes them a promising candidate toexplain the effects of aging on oocytes.

Telomeres have an especially important role in meiosis,because during early meiosis telomeres tether chromosomesand facilitate their alignment, pairing, synapsis and crossingover (Scherthan et al. 1996; Bass et al. 1997; Roig et al. 2004;Scherthan 2006). During early meiosis, telomeres cluster to form‘bouquets’, which are stalk-like attachments to the nuclear enve-lope, to facilitate the homologue alignments required for theformation of synapsis and chiasmata. Elucidation of the molec-ular basis of telomere movement, and specifically the regulationof bouquet, synapsis and chiasmata formation, will advance ourunderstanding of reproductive aging.

Telomerase is a reverse transcriptase that prevents telomereshortening in some cells, including male germ, stem and cancercells. However, telomerase activity remains low in female germcells from the oocyte to the blastocyst stages of development(Wright et al. 1996). Thus, in mammals, oocyte telomeres areendowed with their appropriate length during early development.Interestingly, in the male, whose reproductive function does notdecline appreciably with age, spermatogonia express telomeraseactivity throughout the life of the animal. Because telomeres playimportant roles in chiasmata formation, spindle structure, celldivision and cell death, and become increasingly dysfunctionalwith age, they could play an important role in the age-relatedreproductive failure observed in women.

Telomeres shorten during DNA replicationand from a DNA damage response

Telomeres shorten by at least two mechanisms: (1) replica-tive senescence, which is the normal shortening resulting fromchromosomal replication (Blackburn 2005a, 2005b); and (2) as

a response to damage from reactive oxygen (Passos and vonZglinicki 2005). Once telomeres reach a critical length, the cellstops dividing and undergoes apoptosis and/or chromosomalfusions. A few cell types, including stem and cancer cells, canavoid replicative senescence by expressing telomerase, a reversetranscriptase, which adds telomeric sequence (Blasco et al. 1999;Blackburn 2005a, 2005b).

The DNA damage response to oxidation shortens telomeresby excising oxidised sequences (Passos and von Zglinicki 2005).Telomeres are especially susceptible to reactive oxygen species(ROS)-induced replication-independent telomere shorteningbecause their sequence is guanine rich (the nucleotide most sus-ceptible to oxidation), they sit in the nuclear membrane, wherethey are susceptible to lipid peroxidation, and they lack pro-tective proteins. Both replicative senescence and ROS-inducedtelomere shortening occur in mouse oocytes and early preim-plantation embryos (Liu et al. 2002c), the developmental stageswhen most non-disjunction occurs in older women (Hassold andHunt 2001;Verlinsky et al. 2004; Munne et al. 2005). Replicativesenescence seems implausible in oocytes, which do not dividein adults. Their precursors, however, did divide during fetal life,before entering meiosis. Indeed, DNA labelling studies haveshown that the last oocytes to ovulate in adults were the lastto exit the ‘production line’ during oogenesis (Henderson andEdwards 1968; Edwards 1970; Polani and Crolla 1991). There-fore, telomeres in eggs from older women started shorter becauseof late exit from the production line and shortened further fromexposure to reactive oxygen, produced by the unavoidable effectsof cellular metabolism during the prolonged interval between thebirth of the oocytes and their ovulation (up to 45 years in somewomen). Telomeres provide a cytogenetic mechanism to explainthe ‘two hits’ of aging on the female reproductive system, oneactive during fetal life and the other during adult life.

Telomeres play a crucial role in synapsisand crossing over during early meiosis

In mammals, oocytes initiate meiosis during fetal development,which consists of pairing, synapsis and recombination. Chias-mata formed during fetal life influence chromosome disjunctionduring the preovulatory phase of the adult (Hassold and Hunt2001). During the first meiotic division (MI), chiasmata formedduring fetal oogenesis tether homologous chromosomes and pro-vide counter-traction to the meiotic spindle, which is critical forproper chromosomal segregation. Their deficiency predisposesto subsequent non-disjunction and aneuploidy (Lamb et al. 1997;Petronis 1999; Hassold and Hunt 2001), presumably because chi-asmata are needed to counter pole-ward pulling forces exertedby the meiotic spindle, at least until all chromosomes have con-gressed to the metaphase plate. Indeed, children with Down’ssyndrome, resulting from non-disjunction of chromosome 21,have decreased levels of genetic recombination compared withnormal children (Lamb et al. 1997; Petronis 1999).

Mice rarely exhibit age-related meiotic dysfunction and,intriguingly, their telomeres are almost an order of magni-tude longer than telomeres in humans. However, shorten-ing telomeres, with a null mutation of the telomerase gene,has markedly reduced chiasmata and synapsis compared with

12 Reproduction, Fertility and Development D. L. Keefe and L. Liu

wild-type animals with normal telomere length (Liu et al. 2004).A paucity of chiasmata, coupled with chiasmata-independenteffects of short telomeres, predisposes embryos to genomicinstability, cell cycle arrest and apoptosis, the hallmarks ofreproductive aging in women.

Telomere shortening disrupts metaphase chromosomealignment and spindle structure

Eggs from older women exhibit abnormalities in chromo-some congression and spindle dysmorphologies (Battaglia et al.1996). Structural abnormalities in the meiotic spindle, includ-ing asymmetry of the spindle poles and failure of chromosomesto align on the metaphase plate, appear in almost 80% of eggsaspirated from women over 40 years of age compared with only17% of eggs from young controls (Battaglia et al. 1996).

Telomeres shortened by several generations in thetelomerase-null state (Liu et al. 2002a), or exposure to pharma-cologically induced reactive oxygen in mice (Liu et al. 2003),produce similar abnormalities in meiotic spindles, with asymme-try and congression failure (Battaglia et al. 1996). These spindleand chromosome abnormalities should be especially disruptiveto meiosis, because check-points at the metaphase-to-anaphasetransition are less efficient in females than males (LeMaire-Adkins et al. 1997; Hassold and Hunt 2001). During femalemeiosis, progression from MI to MII occurs even in the presenceof chromosome misalignment because of this lack of efficientmetaphase check-point control (LeMaire-Adkins et al. 1997).

The mechanisms underlying chromosome misalignment anddisruption of meiotic spindles caused by telomere dysfunctionare not well understood, but could result from improper homol-ogous chromosome pairing and recombination during earlymeiosis. That female Mlh 1 mutant mice, which lack normalrecombination, also exhibit abnormal spindle assembly (Woodset al. 1999) fits this hypothesis. If meiotic spindles nucleate fromtelomeres, as well as from kinetechores, telomere shorteningwould disrupt spindle formation.

Mitochondrial dysfunction shortens telomeres

Mitochondrial dysfunction and associated oxidative stress havebeen implicated in cellular senescence, apoptosis and aging,including reproductive senescence (Sastre et al. 2002; Tarinet al. 2002). Uncoupling mitochondrial electron transport withpharmacologic agents administered to one-cell embryos dis-rupts mitochondrial membrane potential (��m), increases ROSproduction and compromises embryo development in a time-and dose-dependent manner (Liu et al. 2002c). Measurement oftelomere length in individual oocytes and embryos by quan-titative fluorescence in situ hybridisation (FISH; Lansdorp1996; Zijlmans et al. 1997) demonstrates that mitochondrialdysfunction causes telomere attrition and eventually chromo-some fusions and cellular apoptosis. Therefore, reactive oxygencan shorten telomeres and promote genomic instability. Theantioxidant N-acetylcysteine (NAC) quenches reactive oxygenand increases levels of glutathione, which also scavenges freeradicals. NAC prevents telomere shortening and cell deathinduced by the mitochondrial uncouplers carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) (Liu et al. 2002c)

or arsenite (Navarro et al. 2006). However, NAC does not res-cue embryos from telomere attrition, cell cycle arrest and celldeath when administered only after FCCP treatment, suggestingthat telomeres already damaged by ROS could not be rescued byanti-oxidants.

Mutations in mtDNA have been found in oocytes from olderwomen (Keefe et al. 1995; Barritt et al. 1999, 2000). How-ever, the impact of these mutations on energy metabolism inoocytes and early embryos must be minimal because mutantmtDNA represents only a tiny fraction of the 100 000–500 000mitochondria transmitted through the cytoplasm of each egg(Chen et al. 1995). Although energy dysfunction would beunlikely to result from such a low burden of mutant mtDNA,the mutant mtDNA could exert dominant negative effects ondevelopment by uncoupling oxidative phosphorylation and pro-ducing excess reactive oxygen, which preferentially attacks themore vulnerable, guanine-rich telomeres.

Telomere length of spare human eggs predicts pregnancyfollowing IVF

Experimentally induced telomere dysfunction can recapitulate inmice the phenotype of reproductive aging seen in women, so wewondered whether measurement of telomere length in humaneggs could predict reproductive potential (Keefe et al. 2007).Ethical constraints limit the production of human embryosstrictly for research, but a study of sister eggs, aspirated duringclinical IVF procedures, found that their telomere length pre-dicted the developmental potential of sibling oocytes. Maximal(19.3 ± 3.1 v. 13.9 ± 3.3 kb; P < 0.01) and mean (7.5 ± 1.2 v.6.2 ± 1.7 kb) telomere lengths were longer, whereas varia-tion, measured by standard deviation, was greater (4.4 ± 1.0 v.3.5 ± 1.1) in eggs from patients who become pregnant followingIVF embryo transfer compared with those who fail to becomepregnant. None of the patients whose mean telomere length wasless than 6.32 kb became pregnant. No other clinical parameters,including patient age, baseline FSH levels, egg number, bodymass index, ovarian stimulation protocol, number of prior IVFcycles, diagnosis or embryo morphology, distinguished the preg-nant and non-pregnant groups. Decreased egg telomere lengthportends poor reproductive outcome in infertile women undergo-ing IVF.Telomere length provides a better predictor of pregnancyoutcome following IVF than patient age itself or other clinicalparameters, even when telomere length was measured only inspare eggs. Telomere length in eggs correlates highly with that ofassociated first polar bodies (R2 = 0.98), suggesting that futurestudies could measure the telomere length of the actual embryostransferred, rather than estimation by measurement of telomeresin spare, sister eggs (Keefe et al. 2007).

Telomeres reset by recombination duringearly development

If telomeres shorten during oogenesis, as suggested by ourresearch, then how do they reset across generations? We foundthat telomeres do lengthen, in fact they lengthen dramati-cally, during the days following fertilisation (Liu et al. 2007).Telomerase is an unlikely contributor to this process becausetelomerase activity is low to non-existent from the oocyte until

Telomeres and reproductive aging Reproduction, Fertility and Development 13

the blastocyst stage of development. Moreover, the kinetics oftelomere restitution by telomerase does not fit the marked telo-mere elongation we found during early development. Indeed, weobserved telomere lengthening even in telomerase-null mice. Acontribution from sperm telomeres also cannot explain telom-ere elongation, because it occurred even in parthenogeneticallyactivated eggs. Telomere–telomere recombination is involvedin the so-called alternative lengthening of telomeres (ALT)observed in somatic cells lacking telomerase or those exposedto telomerase inhibitors. ALT involves DNA damage repairproteins, including Rad 50, MRE11, Bloom and WRN, anddepends on sister chromatid exchange. We identified these pro-teins by immunofluorescene until the morula stage (i.e. thesame time when telomeres elongate). We also found directevidence of telomere sister chromatid exchange, using chromo-some orientation fluorescence in situ hybridisation (CO-FISH).Telomere recombination during early preimplantation embryodevelopment, when embryos are racing to reach the blasto-cyst stage before the implantation window and when cell cyclecheck-point controls are lax, may predisposes embryos to non-disjunction from non-resolution of Holiday junctions involvedin recombination.

Summary

Telomeres play crucial roles in meiosis and likely also playan important role in reproductive aging. Clarification of themolecular mechanisms of telomere regulation of early meiosisprovides a promising avenue for research, with implications forunderstanding both normal development and human disease.

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