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Measuring vertebrate telomeres: applications and limitations

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  • Box 1. Telomeres, factors regulating telomere length, and aging

    Telomeres comprise many copies of an evolutionarily conserved DNA repeat, found at the natural ends of all linear eukaryotic chromosomes. The repeats consist of a short G-rich sequence; in vertebrates, the telomeric repeat (TTAGGG)n is conserved (Meyne et al. 1989) (Fig. 1). This natural end, along with associated proteins, provides chromosome stability, preventing degradation and chromosome fusion (while also anchoring chromosomes in the nuclear matrix); chromosome ends created by breakage are prone to fuse with other chromosomes (Blackburn 1991; Greider 1996). Telomere-associated proteins such as TRF1 and TRF2 are also involved in the regulation of telomere length (Fig. 1) (Broccoli et al. 1997; van Steensel & de Lange 1997; Karlseder et al. 2002). Telomeres play an essential part in DNA replication. At each cell division, a small number of telomeric repeats is lost because DNA replication is incomplete at the 3′ end of the double strands (i.e. the end-replication problem) (Watson 1972), leaving G-rich strand overhangs [whose length is a determinant in the rate of telomere shortening (Huffman et al. 2000)].

    Telomerase is a ribonucleoprotein reverse transcriptase that restores telomere repeats (Lingner et al. 1997). Telomerase activity is found in continually proliferating germ cells and stem cells, but for other types of somatic cell, various or zero activity levels are observed in different species (Prowse & Greider 1995; Venkatesan & Price 1998). The shortening of telomeres has been suggested to be one of the main mechanisms underlying ageing and age-related diseases, because loss of telomere function can lead to genome instability and cell replicative senescence (Harley et al. 1992; Campisi 1996). Mechanistic explanations of ageing involve the accumulation of mutations in genes, the shortening of telomeres and damage in mitochondrial DNA, in all of which oxidative damage plays a crucial role (Goyns 2002) [for free radical theories of ageing, see Finkel & Holbrook (2000)]. Oxidative stress incurred by reactive oxygen species (ROS) increases the rate of telomere shortening per cell division and also the rate of cell turnover (Fig. 1).

    Interestingly, in humans, telomere shortening (Benetos et al. 2001) occurs at different rates in males and females, presumably as a response to different levels of oestrogen between the sexes, which can stimulate telomerase activity and attenuate ROS (Aviv 2002b). Sex differences in telomere shortening have not been observed in any other species, but the possibility needs to be considered if telomere length is to become a tool useful for molecular ecological studies.

  • Box 2. Telomere length, telomere length rate of change and lifespan in birds and mammals

    Maximum telomere length and the telomere length rate of change (TROC) differ among species, as does maximum lifespan among species (Table 2 and Fig. 2). Haussmann and colleagues (Haussmann et al. 2003b; Vleck et al. 2003) found that telomere length at a given life stage did not correlate with lifespan but TROC correlated with lifespan in birds and mammals (Fig. 2c). Telomere length and TROC vary among individuals of the same species and among tissues from an individual (Table 1). Inter-species, interindividual and intertissue differences are accounted for mainly by factors such as different rates of cell replication, levels of telomerase activity and levels of oxidative stress (Fig. 1) (Aviv 2002b). It is particularly interesting that the telomeres of Leach's storm-petrels (Oceanodroma leucorhoa), an unusually long-lived species, lengthen with age (Fig. 2b), because lengthening of telomeres (i.e. telomerase activity) is associated usually with cancer [cancer cells are immortalized through telomerase activity or alternative processes that lengthen telomeres (Henson et al. 2002; Blasco 2003)]. It is proposed that the low or zero telomerase activity found in somatic cells may have been selected for to reduce the frequency of cancer (Harley et al. 1994). How Leach's storm-petrels avoid the tumour-susceptibility imposed by telomerase activity is of considerable potential interest in medical research (Haussmann et al. 2003b).

    Table 2.  Lifespan, telomere length, and telomere length rate of change (TROC) in birds and mammals from selected published literaturea
    SpeciesMaximum lifespan (years)bMaximum observed telomere length (bp)cTROC (bp/year)Tissue sampledRefs
    • a

      Modified from Haussmann et al. (2003b).

    • b

      From Haussmann et al. (2003b) and references therein.

    • c

      c These are approximate values observed in the study and the values come from different age stages (mostly at the age of zero).

    • d

      d Telomere length increased with age in these cells.

    Zebra finch (Taeniopygia guttata)  5  9300–515ErythrocyteHaussmann et al. (2003b)
    Tree swallow (Tachycineta bicolor) 1117 300–391ErythrocyteHaussmann et al. (2003b)
    Adélie penguin (Pygoscelis adeliae) 20  9500–235ErythrocyteHaussmann et al. (2003b)
    Common tern (Sterna hirundo) 26  9800 –57ErythrocyteHaussmann et al. (2003b)
    Leach's storm-petrel (Ocenodroma leucorhoa) 3620 000  75dErythrocyteHaussmann et al. (2003b)
    Western wild mice (Mus spretus)  3.5  9500–600SpleenCoviello-McLaughlin & Prowse (1997)
    Cattle (Bos taurus) 3022 000–230LeucocytesMiyashita et al. (2002)
    Cynomolgus monkey (Macaca fascicularis) 3716 500 –63LeucocytesLee et al. (2002)
    Human (Homo sapiens)11010 000 –33LeucocytesHastie et al. (1990)
    Human (Homo sapiens)11010 500 –15FibroblastsAllsopp et al. (1992)
    Human (Homo sapiens)110  9000 –68Stem cellsVaziri et al. (1994)
    Human (Homo sapiens)11020 000  71dSpermAllsopp et al. (1992)

    Despite the apparently adverse conditions promoting ageing in birds, such as high oxygen consumption and high body temperature, avian species tend to live longer than mammals of comparable body sizes. For a given body size, birds produce less reactive oxygen species (ROS), which are one of the main sources of oxidative damage, and are more tolerant to oxidative damage than mammals (Ogburn et al. 2001). Also, in several mammalian species the production of ROS and levels of oxidative damage are higher in short-lived spices than long-lives ones (Barja & Herrero 2000). Clarification of the relationships between telomerase activity and both oxidative damage and lifespan in a range of avian species is currently under investigation (Vleck et al. 2003).

  • Box 3. Telomere length as a tool in vertebrate conservation management

    Telomere length assays may revolutionize the management of many endangered vertebrate species, where data on age may be lacking, but such knowledge might enhance dramatically predictive models and conservation recovery plans. This is especially true of long-lived k-selected vertebrate species that are grossly over-represented in the IUCN Red Book (IUCN 2003). The critically endangered kakapo (Strigops habroptilus) is an archetypical representative of this type of species. Kakapo are currently returning from near extinction after the discovery of remnant populations in Fiordland and Stewart Island in the 1970s (Elliott et al. 2001). In 2003 the population consisted of 86 individuals. However, none of the reproductively active individuals in the current population are of known ages (Elliott et al. 2001). With suggestions that some of the breeding birds may be 60+ and possibly older, and with demonstrably different levels of breeding success among this population (Miller et al. 2003), it would be useful to know what the age structure of this population is and how reproductive performance varies across individuals’ life spans in order to better predict the future trends of this species. Furthermore, knowledge of age, when used in conjunction with traditional molecular markers, such as microsatellites, would also enable the elucidation of family groups and pedigrees where these are unknown, greatly improving our understanding of the social organization and dynamics of species that have not been subject to long-term study. Given that blood samples of adequate size (> 50 µl) are available for every kakapo, it is possible to use both TRF and Q-PCR assays to provide the knowledge of age needed to help better manage kakapo recovery.

    The application of this technology to other species is no less impressive. For example, new molecular tools for ageing together with other established approaches might see an end to lethal sampling programmes such as the scientific whaling programme (Brownell et al. 2000; Aron 2001). In the past a desire to understand the age structure of natural populations might appear to be one of the few legitimate areas where lethal sampling might still be justifiable. However, if validated, telomere-based assays of age would make it possible to gain information on: (i) an individual's age via telomere length changes; (ii) species, sex and populational affinities using mtDNA, sex and microsatellite markers (Baker et al. 2000); (iii) physiological state via analysis of stress hormones (Wasser et al. 2000); (iv) diet through lipid analyses (Olsen & Grahl-Nielsen 2003); and (v) oceanic movement using trace element analyses (Kelly 2000), without lethal sampling.

Neil J. Gemmell. Fax: + 64 3364 2590; E-mail: neil.gemmell@canterbury.ac.nz

Abstract

Telomeres are short tandem repeated sequences of DNA found at the ends of eukaryotic chromosomes that function in stabilizing chromosomal end integrity. In vivo studies of somatic tissue of mammals and birds have shown a correlation between telomere length and organismal age within species, and correlations between telomere shortening rate and lifespan among species. This result presents the tantalizing possibility that telomere length could be used to provide much needed information on age, ageing and survival in natural populations where longitudinal studies are lacking. Here we review methods available for measuring telomere length and discuss the potential uses and limitations of telomeres as age and ageing estimators in the fields of vertebrate ecology, evolution and conservation.

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