A quantitative PCR method for measuring absolute telomere length

We describe a simple and reproducible method to measure absolute telomere length (aTL) using quantitative real-time polymerase chain reaction (qPCR). This method is based on the Cawthon method for relative measurement of telomere length (TL) but modified by introducing an oligomer standard to measure aTL. The method describes the oligomer standards, the generation of the standard curve and the calculations required to calculate aTL from the qPCR data. The necessary controls and performance characteristics of the assay are described in detail and compared relative to other methods for measuring TL. Typical results for this assay for a variety of human tissue samples are provided as well as a troubleshooting schedule. This method allows high throughput measurement of aTL using small amounts of DNA making it amenable for molecular epidemiological studies. Compared to the traditional relative TL qPCR assays, the aTL method described in this protocol enables a more direct comparison of results between experiments within and between laboratories.     

TeloTool: a new tool for telomere length measurement from terminal restriction fragment analysis with improved probe intensity correction

Telomeres comprise the protective caps of natural chromosome ends and function in the suppression of DNA damage signaling and cellular senescence. Therefore, techniques used to determine telomere length are important in a number of studies, ranging from those investigating telomeric structure to effects on human disease. Terminal restriction fragment (TRF) analysis has for a long time shown to be one of the most accurate methods for quantification of absolute telomere length and range from a number of species. As this technique centers on standard Southern blotting, telomeric DNA is observed on resulting autoradiograms as a heterogeneous smear. Methods to accurately determine telomere length from telomeric smears have proven problematic, and no reliable technique has been suggested to obtain mean telomere length values. Here, we present TeloTool, a new program allowing thorough statistical analysis of TRF data. Using this new method, a number of methodical biases are removed from previously stated techniques, including assumptions based on probe intensity corrections. This program provides a standardized mean for quick and reliable extraction of quantitative data from TRF autoradiograms; its wide application will allow accurate comparison between datasets generated in different laboratories.     

Absolute standards as a useful addition to the avian quantitative PCR telomere assay

Bird populations provide excellent systems to investigate variation in longevity in the wild since individuals can often be monitored over their lifetime. A number of recent studies suggest that the dynamics of protective telomere chromosome caps (telomere length and rate of loss) are indicative of biological state and potentially useful as indicators of future longevity. Currently, Terminal Restriction Fragment (TRF) analysis and relative quantitative PCR (qPCR) are used to measure telomeres in birds, but with limitations. TRF analysis is time consuming, while relative qPCR gives a within‐study relative value making it difficult to compare across experiments. Utilising an approach first developed in humans of using synthetic oligomer telomeric (TTAGGG)_n and normaliser gene standards of known length to calibrate qPCR values, we describe a methodological adaptation to the avian qPCR telomere assay to make results comparable within, and potentially between, bird species. We evaluate this absolute qPCR method in the Seychelles warbler Acrocephalus sechellensis against relative qPCR measurements on the same samples. Telomere estimates from both methods showed an age‐related decline in telomere length, and were highly correlated (r = 0.99). Absolute qPCR avian telomere analysis may prove a useful means of estimating telomere lengths in a calibrated, sensitive, and efficient way using small amounts of archived bird sample.     

Nestling telomere shortening,but not telomere length,reflects developmental stress and predicts survival in wild birds

Developmental stressors often have long-term fitness consequences, but linking offspring traits to fitness prospects has remained a challenge. Telomere length predicts mortality in adult birds, and may provide a link between developmental conditions and fitness prospects. Here, we examine the effects of manipulated brood size on growth, telomere dynamics and post-fledging survival in free-living jackdaws. Nestlings in enlarged broods achieved lower mass and lost 21% more telomere repeats relative to nestlings in reduced broods, showing that developmental stress accelerates telomere shortening. Adult telomere length was positively correlated with their telomere length as nestling (r = 0.83). Thus, an advantage of long telomeres in nestlings is carried through to adulthood. Nestling telomere shortening predicted post-fledging survival and recruitment independent of manipulation and fledgling mass. This effect was strong, with a threefold difference in recruitment probability over the telomere shortening range. By contrast, absolute telomere length was neither affected by brood size manipulation nor related to survival. We conclude that telomere loss, but not absolute telomere length, links developmental conditions to subsequent survival and suggest that telomere shortening may provide a key to unravelling the physiological causes of developmental effects on fitness.     

定量荧光原位杂交测定端粒长度

目的:应用定量荧光原位杂交(Q-FISH)方法测定端粒长度。方法:选取4种端粒长度均一的标准细胞株采用Q-FISH的方法做出荧光亮度与端粒长度的标准曲线,从而得出实验细胞株的端粒长度,与DNA印迹法测定末端限制性片段(TRF)长度进行二者之间的相关性分析。结果:检测荧光强度的最佳线性曝光时间为400ms,相对于DNA印迹法,定量荧光原位杂交(Q-FISH)法所需标本量少,实验周期短,端粒长度结果与Southern杂交法具有很好的相关性。结论:采用定量荧光原位杂交方法测端粒长度具有重复性好、精确可靠的特点,适用于对珍贵标本的端粒改变进行分析。     

Telomere dynamics in human mesenchymal stem cells after exposure to acute oxidative stress

A gradual shortening of telomeres due to replication can be measured using the standard telomere restriction fragments (TRF) assay and other methods by measuring the mean length of all the telomeres in a cell. In contrast, stress-induced telomere shortening, which is believed to be just as important for causing cellular senescence, cannot be measured properly using these methods. Stress-induced telomere shortening caused by, e.g. oxidative damage happens in a stochastic manner leaving just a few single telomeres critically short. It is now possible to visualize these few ultra-short telomeres due to the advantages of the newly developed Universal single telomere length assay (STELA), and we therefore believe that this method should be considered the method of choice when measuring the length of telomeres after exposure to oxidative stress. In order to test our hypothesis, cultured human mesenchymal stem cells, either primary or hTERT immortalized, were exposed to sub-lethal doses of hydrogen peroxide, and the short term effect on telomere dynamics was monitored by Universal STELA and TRF measurements. Both telomere measures were then correlated with the percentage of senescent cells estimated by senescence-associated β-galactosidase staining. The exposure to acute oxidative stress resulted in an increased number of ultra-short telomeres, which correlated strongly with the percentage of senescent cells, whereas a correlation between mean telomere length and the percentage of senescent cells was absent. Based on the findings in the present study, it seems reasonable to conclude that Universal STELA is superior to TRF in detecting telomere damage caused by exposure to oxidative stress. The choice of method should therefore be considered carefully in studies examining stress-related telomere shortening as well as in the emerging field of lifestyle studies involving telomere length measurements.     

Strain-specific telomere length revealed by single telomere length analysis in Caenorhabditis elegans

Terminal restriction fragment analysis is the only method currently available for measuring telomere length in Caenorhabditis elegans. Its limitations include low sensitivity and interference by the presence of interstitial telomeric sequences in the C.elegans genome. Here we report the adaptation of single telomere length analysis (STELA) to measure the length of telomeric repeats on the left arm of chromosome V in C.elegans. This highly sensitive PCR-based method allows telomere length measurement from as few as a single worm. The application of STELA to eight wild-type C.elegans strains revealed considerable strain-specific differences in telomere length. Within individual strains, short outlying telomeres were observed that were clearly distinct from the bulk telomere length distributions, suggesting that processes other than end-replication losses and telomerase-mediated lengthening may generate telomere length heterogeneity in C.elegans. The utility of this method was further demonstrated by the characterization of telomere shortening in mrt-2 mutants. We conclude that STELA appears to be a valuable tool for studying telomere biology in C.elegans.     

Telomere analysis by fluorescence in situ hybridization and flow cytometry.

Determination of telomere length is traditionally performed by Southern blotting and densitometry, giving a mean telomere restriction fragment (TRF) value for the total cell population studied. Fluorescence in situ hybridization (FISH) of telomere repeats has been used to calculate telomere length, a method called quantitative (Q)-FISH. We here present a quantitative flow cytometric approach, Q-FISHFCM, for evaluation of telomere length distribution in individual cells based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA)3probe and DNA staining with propidium iodide. A simple and rapid protocol with results within 30 h was developed giving high reproducibility. One important feature of the protocol was the use of an internal cell line control, giving an automatic compensation for potential differences in the hybridization steps. This protocol was tested successfully on cell lines and clinical samples from bone marrow, blood, lymph nodes and tonsils. A significant correlation was found between Southern blotting and Q-FISHFCMtelomere length values ( P = 0.002). The mean sub-telomeric DNA length of the tested cell lines and clinical samples was estimated to be 3.2 kbp. With the Q-FISHFCMmethod the fluorescence signal could be determined in different cell cycle phases, indicating that in human cells the vast majority of telomeric DNA is replicated early in S phase.     

Real-time quantitative PCR assay for measurement of avian telomeres

We present the application of a real-time quantitative PCR assay, previously developed to measure relative telomere length in humans and mice, to two bird species, the zebra finch Taeniopygia guttata and the Alpine swift Apus melba . This technique is based on the PCR amplification of telomeric (TTAGGG)_n sequences using specific oligonucleotide primers. Relative telomere length is expressed as the ratio (T/S) of telomere repeat copy number (T) to control single gene copy number (S). This method is particularly useful for comparisons of individuals within species, or where the same individuals are followed longitudinally. We used glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a single control gene. In both species, we validated our PCR measurements of relative telomere length against absolute measurements of telomere length determined by the conventional method of quantifying telomere terminal restriction fragment (TRF) lengths using both the traditional Southern blot analysis (Alpine swifts) and in gel hybridization (zebra finches). As found in humans and mice, telomere lengths in the same sample measured by TRF and PCR were well correlated in both the Alpine swift and the zebra finch.. Hence, this PCR assay for measurement of bird telomeres, which is fast and requires only small amounts of genomic DNA, should open new avenues in the study of environmental factors influencing variation in telomere length, and how this variation translates into variation in cellular and whole organism senescence.     

Work-related exhaustion and telomere length: a population-based study

Background

Psychological stress is suggested to accelerate the rate of biological aging. We investigated whether work-related exhaustion, an indicator of prolonged work stress, is associated with accelerated biological aging, as indicated by shorter leukocyte telomeres, that is, the DNA-protein complexes that cap chromosomal ends in cells.

Methods

We used data from a representative sample of the Finnish working-age population, the Health 2000 Study. Our sample consisted of 2911 men and women aged 30–64. Work-related exhaustion was assessed using the Maslach Burnout Inventory - General Survey. We determined relative leukocyte telomere length using a quantitative real-time polymerase chain reaction (PCR) -based method.

Results

After adjustment for age and sex, individuals with severe exhaustion had leukocyte telomeres on average 0.043 relative units shorter (standard error of the mean 0.016) than those with no exhaustion (p = 0.009). The association between exhaustion and relative telomere length remained significant after additional adjustment for marital and socioeconomic status, smoking, body mass index, and morbidities (adjusted difference 0.044 relative units, standard error of the mean 0.017, p = 0.008).

Conclusions

These data suggest that work-related exhaustion is related to the acceleration of the rate of biological aging. This hypothesis awaits confirmation in a prospective study measuring changes in relative telomere length over time.     

Is telomere length a molecular marker of past thermal stress in wild fish?

Telomeres protect eukaryotic chromosomes; variation in telomere length has been linked (primarily in homoeothermic animals) to variation in stress, cellular ageing and disease risk. Moreover, telomeres have been suggested to function as biomarker for quantifying past environmental stress, but studies in wild animals remain rare. Environmental stress, such as extreme environmental temperatures in poikilothermic animals, may result in oxidative stress that accelerates telomere attrition. However, growth, which may depend on temperature, can also contribute to telomere attrition. To test for associations between multitissue telomere length and past water temperature while accounting for the previous individual growth, we used quantitative PCR to analyse samples from 112 young‐of‐the‐year brown trout from 10 natural rivers with average water temperature differences of up to 6°C (and an absolute maximum of 23°C). We found negative associations between relative telomere length (RTL) and both average river temperature and individual body size. We found no indication of RTL–temperature association differences among six tissues, but we did find indications for differences among the tissues for associations between RTL and body size; size trends, albeit nonsignificant in their differences, were strongest in muscle and weakest in fin. Although causal relationships among temperature, growth, oxidative stress, and cross‐sectional telomere length remain largely unknown, our results indicate that telomere‐length variation in a poikilothermic wild animal is associated with both past temperature and growth.     

Telomere Formation by Rap1p Binding Site Arrays Reveals End-Specific Length Regulation Requirements and Active Telomeric Recombination

Rap1p, the major telomere repeat binding protein in yeast, has been implicated in both de novo telomere formation and telomere length regulation. To characterize the role of Rap1p in these processes in more detail, we studied the generation of telomeres in vivo from linear DNA substrates containing defined arrays of Rap1p binding sites. Consistent with previous work, our results indicate that synthetic Rap1p binding sites within the internal half of a telomeric array are recognized as an integral part of the telomere complex in an orientation-independent manner that is largely insensitive to the precise spacing between adjacent sites. By extending the lengths of these constructs, we found that several different Rap1p site arrays could never be found at the very distal end of a telomere, even when correctly oriented. Instead, these synthetic arrays were always followed by a short ( approximately 100-bp) "cap" of genuine TG repeat sequence, indicating a remarkably strict sequence requirement for an end-specific function(s) of the telomere. Despite this fact, even misoriented Rap1p site arrays promote telomere formation when they are placed at the distal end of a telomere-healing substrate, provided that at least a single correctly oriented site is present within the array. Surprisingly, these heterogeneous arrays of Rap1p binding sites generate telomeres through a RAD52-dependent fusion resolution reaction that results in an inversion of the original array. Our results provide new insights into the nature of telomere end capping and reveal one way by which recombination can resolve a defect in this process.     

Early-Life Telomere Dynamics Differ between the Sexes and Predict Growth in the Barn Swallow (Hirundo rustica)

Telomeres are conserved DNA-protein structures at the termini of eukaryotic chromosomes which contribute to maintenance of genome integrity, and their shortening leads to cell senescence, with negative consequences for organismal functions. Because telomere erosion is influenced by extrinsic and endogenous factors, telomere dynamics may provide a mechanistic basis for evolutionary and physiological trade-offs. Yet, knowledge of fundamental aspects of telomere biology under natural selection regimes, including sex- and context-dependent variation in early-life, and the covariation between telomere dynamics and growth, is scant. In this study of barn swallows (Hirundo rustica) we investigated the sex-dependent telomere erosion during nestling period, and the covariation between relative telomere length and body and plumage growth. Finally, we tested whether any covariation between growth traits and relative telomere length depends on the social environment, as influenced by sibling sex ratio. Relative telomere length declined on average over the period of nestling maximal growth rate (between 7 and 16 days of age) and differently covaried with initial relative telomere length in either sex. The frequency distribution of changes in relative telomere length was bimodal, with most nestlings decreasing and some increasing relative telomere length, but none of the offspring traits predicted the a posteriori identified group to which individual nestlings belonged. Tail and wing length increased with relative telomere length, but more steeply in males than females, and this relationship held both at the within- and among-broods levels. Moreover, the increase in plumage phenotypic values was steeper when the sex ratio of an individual’s siblings was female-biased. Our study provides evidence for telomere shortening during early life according to subtly different dynamics in either sex. Furthermore, it shows that the positive covariation between growth and relative telomere length depends on sex as well as social environment, in terms of sibling sex ratio.     

Childhood Physical and Sexual Abuse History and Leukocyte Telomere Length among Women in Middle Adulthood

Objective

Abuse victimization in childhood is associated with a variety of age-related cardiometabolic diseases, but the mechanisms remain unknown. Telomeres, which form the protective caps at the ends of chromosomes, have been proposed as measures of biological age, and a growing body of research suggests that telomere attrition may help to explain relationships between stress and cardiometabolic degradation. We examined the association between childhood abuse victimization and leukocyte telomere length among 1,135 participants in the Nurses’ Health Study II (NHSII).

Methods

The NHSII ascertained physical and sexual child abuse histories in 2001. Telomere length was measured in genomic DNA extracted from peripheral blood leukocytes collected between 1996 and 1999. The ratio of telomere repeat copy number to a single gene copy number (T/S) was determined by a modified version of the quantitative real-time PCR telomere assay. Telomere length was log-transformed and corrected for assay variation across batch. We regressed telomere length on childhood abuse exposure variables and covariates using linear regression.

Results

We observed a reduction in telomere length associated with moderate physical abuse versus no physical abuse, but there was no evidence of a dose-response relationship for increased severity of physical abuse. No associations were noted for sexual abuse.

Conclusions

We found no evidence of an association between severity of childhood physical or sexual abuse and leukocyte telomere length in the NHSII.     

Medaka fish exhibits longevity gender gap,a natural drop in estrogen and telomere shortening during aging: a unique model for studying sex-dependent longevity

Introduction

Females having a longer telomere and lifespan than males have been documented in many animals. Such linkage however has never been reported in fish. Progressive shortening of telomere length is an important aging mechanism. Mounting in vitro evidence has shown that telomere shortening beyond a critical length triggered replicative senescence or cell death. Estrogen has been postulated as a key factor contributing to maintenance of telomere and sex-dependent longevity in animals. This postulation remains unproven due to the lack of a suitable animal system for testing. Here, we introduce a teleost model, the Japanese medaka Oryzias latipes, which shows promise for research into the molecular mechanism(s) controlling sex difference in aging.

Results

Using the medaka, we demonstrate for the first time in teleost that (i) sex differences (female?>?male) in telomere length and longevity also exist in fish, and (ii) a natural, ‘menopause’-like decline of plasma estrogen was evident in females during aging. Estrogen levels significantly correlated with telomerase activity as well as telomere length in female organs (not in males), suggesting estrogen could modulate telomere length via telomerase activation in a sex -specific manner. A hypothetical in vivo ‘critical’ terminal restriction fragment (TRF, representing telomere) length of approximately 4 kb was deduced in medaka liver for prediction of organismal mortality, which is highly comparable with that for human cells. An age conversion model was also established to enable age translation between medaka (in months) and human (in years). These novel tools are useful for future research on comparative biology of aging using medaka.

Conclusion

The striking similarity in estrogen profile between aging female O. latipes and women enables studying the influence of “postmenopausal” decline of estrogen on telomere and longevity without the need of invasive ovariectomy. Medaka fish is advantageous for studying the direct effect of increased estrogen on telomere length and longevity without the breast cancer complications reported in rodents. The findings strongly support the notion that O. latipes is a unique non-mammalian model for validation of estrogenic influence on telomere and longevity in vertebrates. This laboratory model fish is of potential significance for deciphering the ostensibly conserved mechanism(s) of sex-associated longevity in vertebrates.

     

Reliability and short-term intra-individual variability of telomere length measurement using monochrome multiplexing quantitative PCR

Background

Studies examining the association between telomere length and cancer risk have often relied on measurement of telomere length from a single blood draw using a real-time PCR technique. We examined the reliability of telomere length measurement using sequential samples collected over a 9-month period.

Methods and Findings

Relative telomere length in peripheral blood was estimated using a single tube monochrome multiplex quantitative PCR assay in blood DNA samples from 27 non-pregnant adult women (aged 35 to 74 years) collected in 7 visits over a 9-month period. A linear mixed model was used to estimate the components of variance for telomere length measurements attributed to variation among women and variation between time points within women. Mean telomere length measurement at any single visit was not significantly different from the average of 7 visits. Plates had a significant systematic influence on telomere length measurements, although measurements between different plates were highly correlated. After controlling for plate effects, 64% of the remaining variance was estimated to be accounted for by variance due to subject. Variance explained by time of visit within a subject was minor, contributing 5% of the remaining variance.

Conclusion

Our data demonstrate good short-term reliability of telomere length measurement using blood from a single draw. However, the existence of technical variability, particularly plate effects, reinforces the need for technical replicates and balancing of case and control samples across plates.     

A ‘higher order’ of telomere regulation: telomere heterochromatin and telomeric RNAs

Protection of chromosome ends from DNA repair and degradation activities is mediated by specialized protein complexes bound to telomere repeats. Recently, it has become apparent that epigenetic regulation of the telomric chromatin template critically impacts on telomere function and telomere‐length homeostasis from yeast to man. Across all species, telomeric repeats as well as the adjacent subtelomeric regions carry features of repressive chromatin. Disruption of this silent chromatin environment results in loss of telomere‐length control and increased telomere recombination. In turn, progressive telomere loss reduces chromatin compaction at telomeric and subtelomeric domains. The recent discoveries of telomere chromatin regulation during early mammalian development, as well as during nuclear reprogramming, further highlights a central role of telomere chromatin changes in ontogenesis. In addition, telomeres were recently shown to generate long, non‐coding RNAs that remain associated to telomeric chromatin and will provide new insights into the regulation of telomere length and telomere chromatin. In this review, we will discuss the epigenetic regulation of telomeres across species, with special emphasis on mammalian telomeres. We will also discuss the links between epigenetic alterations at mammalian telomeres and telomere‐associated diseases.     

Telomere Length Dynamics in Pearl Millet (Pennisetum glaucum [L.] R. Br.) as Observed at Different Developmental Stages

Abstract: In plants, the developmental dynamics of telomere length have only been studied in a few species to date. Contrasting results have been reported. To search for the pattern(s) operating in plants, a study of the telomere length was made in pearl millet. Telomere length in cells representing different developmental stages: a) embryo, b) leaves of 1-week-old seedlings, 1-month-old plants and boot leaf and c) germ cells (pollen) were compared. The presence of the consensus plant telomere repeat sequence (5'-TTTAGGG-3') in pearl millet telomeres was first ascertained; the sensitivity of the sequences hybridizing with (TTTAGGG)₄ oligonucleotide to time course Bal 31 exonuclease digestions were studied on dot blots and gel blots. The exonuclease digestion kinetics revealed the presence of the consensus telomere repeat sequence in pearl millet telomeres. The average telomere length (ATL) was measured from autoradiograms of Hae III digested DNA, hybridized with labelled (5'-TTTAGGG-3')₄ oligonucleotide using "UVI band" software. No significant difference in the average telomere length was observed between the embryo, leaves of 1-week-old seedlings, boot leaf and pollen. The ATL in leaves of 1-month-old plants was slightly higher. The results of the present investigation and analysis of the reports in the other plants suggest that there is an occasional increase in telomere length in some telomeres but no significant decrease due to loss during DNA replication.     

Telomere length measurement by fluorescence in situ hybridization and flow cytometry: tips and pitfalls

BACKGROUND: Telomeres containing noncoding DNA repeats at the end of the chromosomes are essential for chromosomal stability and are implicated in regulating the replication and senescence of cells. The gradual loss of telomere repeats in cells has been linked to aging and tumor development and methods to measure telomere length are of increasing interest. At least three methods for measuring the length of telomere repeats have been described: Southern blot analysis and quantitative fluorescence in situ hybridization using either digital fluorescence microscopy (Q-FISH) or flow cytometry (flow-FISH). Both Southern blot analysis and Q-FISH have specific limitations and are time-consuming, whereas the flow-FISH technique requires relatively few cells (10(5)) and can be completed in a single day. A further advantage of the flow-FISH method is that data on the telomere length from individual cells and subsets of cells (lymphocytes and granulocytes) can be acquired from the same sample. In order to obtain accurate and reproducible results using the flow-FISH technique, we systematically explored the influence of various steps in the protocol on telomere length values and established an acceptable range for the most critical parameters. METHODS: Isolated leukocytes from whole blood are denatured by heat and 70%/75% formamide, then hybridized with or without a telomere-specific fluorescein isothiocyante (FITC)-conjugated peptide nucleic acid probe (PNA). Unbound telomere PNA is washed away, the DNA is counterstained, and telomere fluorescence is measured on a flow cytometer using an argon ion laser (488 nm) to excite FITC. For each sample, duplicates of telomere PNA-stained and unstained tubes are analyzed. RESULTS: Cell counts and flow-FISH telomere length measurements were performed on leukocytes and thymocytes of humans and other species. Leukocyte suspensions were prepared by two red blood cell lysis steps with ammonium chloride. Optimal denaturation of DNA was achieved by heating at 85-87 degrees C for 15 min in a solution containing 70%/75% formamide. Hybridization was performed at room temperature with a 0.3 microg/ml telomere-PNA probe for at least 60-90 min. Unbound telomere-PNA probe was diluted at least 4,000-40,000 times with wash steps containing 70%/75% formamide at room temperature. LDS 751 and DAPI were suitable as DNA counterstains as they did not show significant interference with telomere length measurement. CONCLUSIONS: The use of flow-FISH for telomere length measurements in nucleated blood cells requires tight adherence to an optimized protocol. The method described here can be used to determine rapidly the telomere length in subsets of nucleated blood cells.     

Age at maturation has sex- and temperature-specific effects on telomere length in a fish

Telomeres are highly conserved nucleoprotein structures which protect genome integrity. The length of telomeres is influenced by both genetic and environmental factors, but relatively little is known about how different hereditary and environmental factors interact in determining telomere length. We manipulated growth rates and timing of maturation by exposing full-sib nine-spined sticklebacks (Pungitius pungitius) to two different temperature treatments and quantified the effects of temperature treatments, sex, timing of maturation, growth rate and family (genetic influences) on telomere length. We did not find the overall effect of temperature treatment on the relative telomere length. However, we found that variation in telomere length was related to timing of maturation in a sex- and temperature-dependent manner. Telomere length was negatively related to age at maturation in elevated temperature and early maturing males and females differed in telomere length. Variation in growth rate did not explain any variation in telomere length. The broad sense heritability (h ²) of telomere length was estimated at h ² = 0.31 ? 0.47, suggesting predominance of environmental over genetic determinants of telomere length variability. This study provides the first evidence that age at maturation together with factors associated with it are influencing telomere length in an ectotherm. Future studies are encouraged to identify the extent to which these results can be replicated in other ectotherms.