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Home , Generation time, Germline, Immortality, Somatic (biology)

Research Reviews 4 (2005) 67–82 www.elsevier.com/locate/arr

Viewpoints Achieving in the C. elegans Chris Smelickb, Shawn Ahmeda,b,*

aDepartment of , Coker Hall, University of North Carolina, Chapel Hill, NC 27599-3280, USA bDepartment of , Coker Hall, University of North Carolina, Chapel Hill, NC 27599-3280, USA

Received 12 August 2004; accepted 21 September 2004

Abstract

Germline immortality is a topic that has intrigued theoretical interested in aging for over a century. The germ lineage can be passed from one generation to the next, indefinitely. In contrast, cells are typically only needed for a single generation and are then discarded. Germ cells may, therefore, harbor mechanisms that enable them to proliferate for eons. Such processes are thought to be either absent from or down-regulated in somatic cells, although cell non- autonomous forms of rejuvenation are formally possible. A thorough description of mechanisms that foster in germ cells is lacking. The mysteries of germline immortality are being addressed in the nematode by studying mutants that reproduce normally for several generations but eventually become sterile. The mortal germline mutants probably become sterile as a consequence of accumulating various forms of heritable cellular damage. Such mutants are abundant, indicating that several different biochemical pathways are required to rejuvenate the germline. Thus, forward genetics should help to define mechanisms that enable the germline to achieve immortality. # 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Immortality; Germline; Disposable soma; Aging; ; C. elegans

1. Introduction

As humans have become aware of , of the nature of our transient existence on this planet, and of the biology of reproduction, suspicions have been raised that we are ‘vessels of biology’, somatic slaves to our physiological drives and the genes behind them (Dawkins, 1990; E. Henderson, personal communication). These suspicions arose from the concept that multicellular , in the most general sense, possess two classes of

* Corresponding author. Tel.: +1 919 843 4780; fax: +1 919 962 8472. E-mail address: [email protected] (S. Ahmed).

1568-1637/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.arr.2004.09.002 68 C. Smelick, S. Ahmed / Ageing Research Reviews 4 (2005) 67–82 cells, germline and somatic. Germline cells are defined by their ability to be passed down continuously from generation to generation. Somatic cells, on the other hand, exist for a single generation. The presence of these two distinct cell types alludes to a division of labor that evolved such that reproduction became the exclusive task of a subset of cells (germ cells), whereas somatic cells became specialized for the maintenance and propagation of the germ cells and thereby contribute to posterity vicariously. Germ cells have a vested interest in the creation of progeny that are vibrant, near-perfect versions of their parents, generation after generation, for . Given that the soma does not typically carry the burden of proliferative immortality, theoretical biologists have postulated that germline immortality is ensured by mechanisms that may be specifictoor enriched in germ cells (Kirkwood and Holliday, 1979; Medvedev, 1981). first made such arguments over a century ago (Weismann, 1882), but the premise that somatic cells are mortal simply because they are unable to create a new is not always viewed as compelling. Were somatic cells blessed with this ability, might we observe that they have retained the capacity for immortality after all? This conundrum is difficult to address, although transfer of somatic nuclei to enucleated may help to define nuclear boundaries drawn between the two lineages, as discussed later. Another blemish to the ‘immortal’ character of germ cells is that, contrary to their advertised intransigence to the perils of , germ cells do , often performing poorly or failing altogether in older individuals. Clear examples of this anomaly are apparent in aging women, where a 35-fold increase in the rate of non-disjunction occurs in oocytes, and where hormone-induced menopausal senescence of the germline occurs at 50–60 of age (Kirkwood, 1998). In older men, higher frequencies of spontaneous in result in 5- to 30-fold increases in heritable genetic disorders such as Apert’s syndrome and Achondroplasia (Crow, 2000). Similar fates befall older germ cells in organisms such as worms, flies and mice, indicating that the germline cannot withstand some effects of aging (Kaufmann, 1947; Byers and Muller, 1952; Purdom et al., 1968; Goldstein and Curis, 1987; Walter et al., 1998; Garigan et al., 2002). Instead, the lineage may achieve immortality with the aid of a rejuvenation process that somehow erases the effects of age that accumulate during one’s lifetime (Medvedev, 1981). While the germline is typically the only proliferatively immortal cell lineage in multicellular eukaryotes, many unicellular eukaryotes and prokaryotes divide by fission and are thought of as immortal cell lineages. However, stationary Escherichia coli cultures contain a population of oxidatively damaged cells, whose capacity to proliferate is very limited (Nystrom, 2002). Thus, high rates of proliferation in bacterial cultures may provide a fac¸ade of uniform vigor, when in fact it is the culture that is immortal. A clear example of this dichotomy in a unicellular culture is provided by the baker’s yeast Saccharomyces cerevisiae, where larger mother cells bud asymmetrically to produce small daughters. Mother cells represent a disposable cell type that can only divide about 25 and then perishes. The mother bud inherits more damage than its daughter in the form of bud scars, extrachromosomal rDNA circles, and oxidatively damaged proteins (Smeal et al., 1996; Sinclair and Guarente, 1997; Aguilaniu et al., 2003), suggesting that a mother cell may serve as a sink for some forms of damage that would otherwise build up to a harmful degree in its progeny. Could similar disposal processes operate actively in the germline thereby helping to cleanse it of macromolecular damage? C. Smelick, S. Ahmed / Ageing Research Reviews 4 (2005) 67–82 69

There is usually no distinction between germline and soma for single-celled eukaryotes, despite the fact that they can reproduce sexually. One interesting exception to this rule is found in , unicellular eukaryotes that contain two distinct nuclei: a diploid germline micronucleus that is used for , and a polyploid somatic macronucleus that is discarded during . Ciliates therefore provide an example of one of the most fundamental separations between germline and soma within a single cell. The first experiments on germline immortality were conducted by Loren Woodruff, who propagated a vegetative culture of a single mating type of the for almost 40 years in the early 1900’s and concluded that sexual reproduction was not required for cellular immortality (Bell, 1988). This persistent investigator did not appreciate the possibility that the ciliates in his ‘non-mating’ culture almost certainly underwent (meiosis and fertilization within a single cell) from time to time. It is now understood that in the absence of sexual reproduction, many ciliates will succumb to clonal senescence, a phenomenon that describes a population of cells with a common ancestor that ceases to proliferate (Smith-Sonneborn, 1987). Studies of senescence in ciliates suggest that germline immortality may be intertwined with aspects of sexual reproduction such as meiosis (Kirkwood and Holliday, 1979). While most multicellular eukaryotes propagate sexually, there are exceptions to this rule (Fagerstro¨m et al., 1998). Organisms such as grasses can reproduce clonally using somatic cells, while flatworms of the taxonomic families Dugesiidae and Planariidae can literally divide themselves in half and replace the missing portion using a population of pluripotent somatic stem cells. Thus, the trait of proliferative immortality can be bestowed upon the soma, although it is typically the exclusive domain of germ cells (Avise, 1993). Germline immortality may therefore have little or nothing to do with sexual reproduction itself. One reason that somatic cells are typically thought of as ‘mortal’ is that primary human fibroblasts have a limited replicative lifespan if grown in vitro (Hayflick and Moorhead, 1961), a phenomenon known as replicative senescence. In contrast, many cell lines can proliferate indefinitely under identical conditions, suggesting that replicative senescence may help to protect against cancer in humans (Shay and Roninson, 2004). The cause of replicative senescence in human fibroblasts is sequence erosion at , the ends of linear (Bodnar et al., 1998; Counter et al., 1998). length in most organisms is maintained by , a ribonucleoprotein that uses its RNA component as a template for telomere replication (Harrington, 2003). The catalytic subunit of telomerase is repressed in primary human fibroblasts that senesce, but not in most immortal cell lines (Kim et al., 1994; Meyerson et al., 1997; Nakamura et al., 1997). In addition, expression of this subunit of telomerase is critical for immortalization of human primary cells (Bodnar et al., 1998; Counter et al., 1998; Jiang et al., 1999; Morales et al., 1999). Mice that are deficient for the RNA subunit of telomerase are initially viable and healthy, but become sterile after six generations, thereby clearly defining a biological process required for germline immortality (Blasco et al., 1997). Work in S. cerevisiae has shown that clonal senescence occurs for mutants with telomere replication defects after roughly 85 cell divisions (Lundblad and Szostak, 1989). Thus, telomere replication is required for multigenerational proliferation of the germline in multicellular organisms and for mitotic proliferation in unicellular eukaryotes. 70 C. Smelick, S. Ahmed / Ageing Research Reviews 4 (2005) 67–82

While telomere shortening can be detrimental to cell proliferation, replicative senescence of human cells in vitro can also occur as a consequence of stress or aberrant signaling-induced senescence, a form of senescence that is initiated by stressful cell culture conditions such as treatment with chemotheraputic agents (Shay and Roninson, 2004). Given that telomere erosion and stress-induced culture shock both produce senescent cells, it is possible these two forms of replicative senescence in human primary cells occur as a result of a common molecular switch, such as DNA damage foci at telomeres or elsewhere. In agreement with the possibility of multiple triggers for replicative senescence, mouse fibroblasts, Paramecium cultures and wildtype S. cerevisiae mother cells all senesce, but not as a consequence of telomere shortening (D’Mello and Jazwinski, 1991; Gilley and Blackburn, 1994; Smeal et al., 1996; Wright and Shay, 2000). Thus, observations from the field of cellular senescence suggest that the germline may need to ward off multiple types of damages in order to achieve immortality.

2. Theory and putative mechanisms for perpetual germline proliferation

Germline immortality has been previously characterized as the avoidance of a progressive accumulation of defects across generations. Kirkwood has grouped putative immortality mechanisms into three general categories: (1) higher levels of maintenance and repair in germ cells versus somatic cells, (2) repair mechanisms that act to specifically rejuvenate germ cells, and (3) selective processes that allow only robust germ cells to propagate (Kirkwood, 1987). For humans and mice, telomerase-mediated telomere elongation would fall under the ‘higher levels of maintenance and repair’ mechanism of germline immortality, because telomerase is active in the germline but repressed in a proportion of somatic cells in both organisms (Kim et al., 1994; Prowse and Greider, 1995; Meyerson et al., 1997; Nakamura et al., 1997). In contrast, telomerase is required for continuous proliferation of unicellular eukaryotic organisms such as yeast and protozoa (Lundblad and Szostak, 1989), as might be expected for organisms that are clonally immortal. An initial hypothesis regarding maintenance and repair suggested that the germline might synthesize proteins more accurately than somatic cells. Orgel’s ‘‘error catastrophe’’ hypothesis of aging postulated that age-related errors in protein synthesis might lead to progressive accumulation of such errors in the translational machinery, thereby resulting in an exponential increase in the synthesis of altered proteins and eventual cellular senescence and (Orgel, 1963). However, translational fidelity does not change with age (Filion and Laughrea, 1985; Goldstein et al., 1985), leaving the error catastrophe hypothesis untenable. A more likely hypothesis is the ‘‘disposable soma’’ theory, a contemporary extension of the Weismann germline/soma distinction, which suggests that natural selection might favor allocating greater energy resources for maintenance and repair of the germline rather than the soma (Kirkwood and Holliday, 1979). This theory suggests that higher levels of DNA repair in the germline might result in a lower rate of spontaneous mutation in germ versus somatic cells, a possibility that has received experimental support. The spontaneous of various mouse tissues was estimated via analysis of C. Smelick, S. Ahmed / Ageing Research Reviews 4 (2005) 67–82 71 in an array of 50–70 copies of lacI or lacZ reporter integrated at a chromosomal locus (Hill et al., 2004). A lower mutation frequency was observed over all ages for germline cells versus all other tissues examined (cerebellum, forebrain, thymus, liver, and adipose tissue). The types of mutations observed included base transitions, , deletions and insertions, suggesting that the germline may be more resistant than somatic cells to a broad range of DNA lesions. These results have been corroborated by other studies in mouse and fish using similar reporters (Kohler et al., 1991; Winn et al., 2000). To the extent that mutation rates measured using repetitive transgenes reflect those of native genes, these data support the prediction of higher levels of maintenance and repair occurring in the germline. Note that non-dividing somatic tissues and proliferating germline tissues may possess inherently different abilities to repair DNA damage, as high fidelity recombinational repair occurs primarily during S-phase (Takata et al., 1998). An alternative to studying the frequency of spontaneous mutation in the germline is to measure levels of DNA repair directly. When the activity of uracil-DNA glycosylase-initated base excision repair was examined for a variety of mouse tissues, it was found to be highest in germ cells, a result that was consistent across three different strains (two inbred, one outbred) (Intano et al., 2001). Taken together, these in vitro and in vivo studies support the possibility that a dichotomy exists for the levels of DNA repair in germ and somatic cells, although additional studies would lend greater validity to this hypothesis. The category of immortality mechanisms is thought to specifically rejuvenate germ cells by cleansing them of accumulated damage. Although many authors have speculated that the process of recombination during meiosis could be a means to repair DNA damage that accumulates with aging (Gensler and Bernstein, 1981; Medvedev, 1981; Holliday, 1984; Strehler, 1986), the types of damage (i.e., double-strand breaks) envisioned to be healed by are typically lethal if unrepaired (Haber, 2000), and are therefore unlikely to be passed through several mitotic germ cell divisions and then repaired during meiosis. Nevertheless, meiotic recombination could help to resolve specific kinds of damage to DNA or chromatin. From the evolutionary perspective, the products of meiotic recombination can be used for cross-fertilization, which may help to reduce the negative influence of deleterious mutations that occur in the germline. Note, however, that the number of successful self-fertilizing on the planet would suggest that processes that occur concomittant with sexual reproduction, rather than outcrossing itself, may be critical for rejuvenation of the germline in multicellular organisms. Another meiotic rejuvenation theory suggests that polar bodies generated by oocytes of multicellular organisms may represent potential sinks for macromolecular damage (Medvedev, 1981), a process that might resemble the sequestration of oxidatively damaged proteins by yeast mother cells (Aguilaniu et al., 2003). Germline-specific mechanisms of rejuvenation could also be accomplished by epigenetic modifications such as histone acetylation and DNA methylation, which regulates transcriptional activity and chromatin structure. These processes play a significant role in establishing tissue-specific gene expression profiles during differentia- tion. Distinct epigenetic processes are active exclusively in the germline, including erasure and re-establishment of sex-specific imprinting of a number of genes in primordial germ cells (Surani, 1998; Hajkova et al., 2002; Reik et al., 2003; Santos and Dean, 2004). 72 C. Smelick, S. Ahmed / Ageing Research Reviews 4 (2005) 67–82

Aberrant epigenetic reprogramming in the germline can result in epimutations that are equivalent to deleterious DNA mutations and may be passed on from one generation to the next (Rakyan et al., 2001; Suter et al., 2004). Maintenance of epigenetic silencing of repetitive elements such as transposons is another critical feature of the reprogramming process that is essential for stability (Hajkova et al., 2002; Bestor, 2003). Carefully coordinated epigenetic silencing and reprogramming could, therefore, constitute a chromatin maintenance mechanism that contributes to germline immortality. The third class of mechanisms that might help the germline to achieve immortality is selection, which can operate at all stages of the reproductive cycle, from primordial germ cell selection to phenotypic selection of the progeny. Mammalian germ cells undergo several waves of during development and maturation (Matsui, 1998). Although its functional significance is unclear, apoptosis is a conserved feature of metazoan that may help to dispose of damaged germ cells (Buszczak and Cooley, 2000; Walter et al., 2003). With regards to selection at early stages of development, the frequency of spontaneous mutation has been observed to decrease in successive stages of at a time when apoptosis is occurring (Walter et al., 1998). Thus, a surprising form of meiotic quality control may be able to assess the mutational load of developing . Another method of selection could occur for haploid gametes, where aneuploidy or deleterious mutations that affect basic cellular functions could impair the ability of a gamete to achieve syngamy (Medvedev, 1981). Thus, haploid selection may help to suppress the frequency of spontaneous mutation for the subset of genes responsible for essential cellular functions, thereby accomplishing a task similar to that of DNA repair. A that may provide insight into the relative importance of each of the three classes of molecular mechanisms with regards to germline immortality is reproductive by nuclear transfer. Reproductive cloning is achieved by microinjecting the nucleus of a somatic cell into an enucleated , which is then activated to undergo , thereby producing an animal or ‘‘clone’’ that is genetically identical to the donor somatic cell. If a female clone is fertile, then the cloning process could potentially be repeated for any number of generations, an experimental routine termed ‘serial cloning’. If serial cloning were successful, then neither meiotic recombination nor haploid selection could be factors that influence germline immortality. Instead, either the oocyte cytoplasm or an inductive signal from the somatic gonad would be sufficient to confer immortality to somatic nuclei. The success rates of reproductive cloning research are still extremely low, with current efficiency levels at 2–3% for full mouse development (Tamashiro et al., 2000). Although such births are commonly accompanied by somatic developmental defects, adult clones are typically fertile (Kubota et al., 2004). These results demonstrate that despite an evolved lower investment in the upkeep of the soma, adult somatic nuclei can occasionally create new, fertile beings, perhaps encouraging cautious interpretation of the disposable soma theory. At the same time, the common developmental defects of cloned animals might be due to a decreased investment in somatic nuclei, although this may simply reflect a defect in the reprogramming of imprinted genes, which occurs for prospective germline nuclei during early embryogenesis (Surani, 1998; Hajkova et al., 2002; Reik et al., 2003; Santos and Dean, 2004). C. Smelick, S. Ahmed / Ageing Research Reviews 4 (2005) 67–82 73

Attempts at serial cloning have failed within three to six generations for bulls and mice, respectively (Wakayama et al., 2000; Kubota et al., 2004). Progressive telomere shortening was not observed in either of these cases, indicating that adult somatic nuclei may be defective for another biochemical pathway that is required for germline immortality. However, the process of cloning is clearly imperfect, and the failure of serial cloning could result from cumulative deleterious effects unrelated to germline immortality. If the inherent problems with the cloning process can be resolved and serial cloning still fails, then this would indicate that adult somatic nuclei contain at least one form of damage that is detrimental to immortality and is normally prevented in the germline. Such damage might resemble that experienced by senescent ciliates, where a rejuvenating bout of sexual reproduction is required from time to time (Bell, 1988).

3. Genetic analysis of germline immortality in C. elegans

An alternative to testing specific hypotheses regarding how the germline might achieve immortality, such as the DNA repair and serial cloning experiments discussed above, is to identify genes required for germline immortality by mutation. This concept is being developed in Caenorhabditis elegans by isolating mortal germline mutants, mutants whose can reproduce for several generations but eventually become sterile (Fig. 1a). The nematode C. elegans has a number of advantages that make it attractive for studies of germline immortality. First, it is a transparent whose germline and soma are distinct from the first of embryogenesis, providing a simple and precise context for studies of germline development (Strome and Wood, 1982). Second, the

Fig. 1. (a) Brood size drops progressively in C. elegans mortal germline mutants. Petri dishes with lawns of E. coli are seeded with six L1 larvae and scored a later for progeny. Fluorescence microscopy of oocyte nuclei reveals (b) six clearly separated bivalents in wildtype worms, and (c) chromosome fusions in C. elegans telomere replication mutants that are close to sterility (Photos courtesy of J. Boerckel). 74 C. Smelick, S. Ahmed / Ageing Research Reviews 4 (2005) 67–82

C. elegans germline is one of the largest tissues in the adult, and its development has been studied extensively (Seydoux and Schedl, 2001). Third, C. elegans worms are typically self-fertilizing hermaphrodites, so mutant lines can be propagated for multiple generations with ease (whereas for Drosophila, crosses of males and females would have to be set up every generation). Fourth, C. elegans has a of roughly 3.5 days, allowing for 20 or 30 generations of growth to occur in only a few ’ time (whereas generation time is 11 days for Drosophila and 2 months for mouse). Fifth, although C. elegans mutants with defects in germline immortality are expected to become sterile, fertile founders that initiate such mutant lineages can be kept as starved stocks or frozen at À80˚C, thereby allowing a mutation to be recovered after sterile progeny are identified (in contrast, balanced heterozygous stocks would have to be maintained for every mutagenized Drosophila line examined). These traits make C. elegans an ideal organism for defining most or all processes required for germline immortality by mutation, using a forward genetic approach. The first C. elegans strain reported to have a defect in germline immortality was actually a wild strain called Bergerac. This strain displayed high levels of transposition in its germline and had accumulated over 300 copies of the Tc1 transposon (Anderson, 1995). In contrast, transposon activation in the genetic background of Bristol N2, the standard wildtype C. elegans laboratory strain that has a low transposon copy number, does not compromise germline immortality (Plasterk and van Leunen, 1997). Transposons are normally silenced in the C. elegans germline, whereas somatic cells display about a 1000- fold increase in transposon activity (Collins et al., 1987). Thus, the Bergerac strain of C. elegans identified transposon silencing as germline-specific mechanism for maintaining genome stability and germline immortality. Ironically, studies of the Bergerac strain indicate that loss of some pathways required for germline immortality may result in useful properties in the wild, perhaps helping to increase genetic diversity in a species that is primarily composed of hermaphrodites that self-fertilize. Given that transposon-silencing defects can compromise germline immortality in strains with high transposon copy numbers, it was of interest to determine if other pathways might be required for germline immortality in strains with low numbers of transposons. Initially, a small pilot screen for mortal germline mutants was conducted to determine if such mutants could be identified by forward genetics. Bristol N2 wildtype worms were mutagenized with ethylmethanesulphonate (EMS), a standard mutagen, and 400 F2 lines were propagated clonally for 16 generations at 25˚C. Each of these mutant lines ought to have been homozygous for a number of EMS-induced loss-of-function mutations (Anderson, 1995), and 16 mortal germline mutants were identified (Ahmed and Hodgkin, 2000)(Fig. 1a). Each mutant originated from a different mutagenized grandparent, and therefore, became sterile as a result of an independent EMS mutation. A homozygous loss- of-function mutation in a particular gene occurs, on average, once in every 4000 EMS- mutagenized F2, suggesting that roughly 160 genes may be required for germline immortality in C. elegans. Complementation tests were not conducted with these mutants, as complementation is difficult to perform with two unbalanced delayed sterility mutations segregating in the progeny of a trans-heterozygote. Although some mortal germline mutants could represent multiple alleles of highly mutable genes, even a conservative estimate of 50 genes suggests that germ cells use several different biochemical pathways to C. Smelick, S. Ahmed / Ageing Research Reviews 4 (2005) 67–82 75 achieve immortality. The initial observation of a high frequency of mortal germline mutants has been borne out in large-scale screens (Liu, Boerckel and Ahmed, unpublished). The first mortal germline mutant to be cloned, mrt-2, became sterile as a result of defects in telomere replication. The mrt-2 mutant experiences progressive telomere shortening and end-to-end chromosome fusions, phenotypes that are hallmarks of yeast and mouse mutants that are defective for telomerase (Blasco et al., 1997; Naito et al., 1998; Nakamura et al., 1998; Ahmed and Hodgkin, 2000). End-to-end chromosome fusions in mammalian cells are frequently pulled to opposite sides of the mitotic spindle by their centromeres, resulting in broken chromosomes and genetic catastrophe (Blasco et al., 1997). However, C. elegans has holocentric centromeres that attach along the length of a chromosome, which allows for end-to-end fusions to be stably propagated during mitosis (Albertson and Thomson, 1993). As mrt-2 approaches sterility, chromosome number drops precipitously as judged by fluorescence microscopy (Fig. 1b and c), and mrt-2 mutants probably become sterile as a consequence of chromosome circularization, which results in meiotic failure in S. pombe (Nakamura et al., 1998). MRT-2 is a DNA damage checkpoint protein that physically interacts with HUS-1 and RAD-9 (Boulton et al., 2002). Orthologues of all three proteins heterotrimerize to form a PCNA-like ring that can be recruited to sites of DNA damage (Bermudez et al., 2003), where they may act as a clamp and processing factor for polymerases and possibly for telomerase. Consistent with the physical interaction between the MRT-2 and HUS-1 proteins, a null mutation in the C. elegans hus-1 gene eliminates telomere replication, resulting in a Mortal Germline phenotype (Hofmann et al., 2002). These telomere replication defects are identical to those observed in strains defective for the C. elegans catalytic subunit of telomerase ( Meier, Hodgkin and Ahmed, unpublished data). Together, these results are consistent with studies in mouse and yeast, where telomere replication defects cause either sterility after six generations or clonal senescence, respectively (Lundblad and Szostak, 1989; Blasco et al., 1997). In addition to its effects on telomere replication, the hus-1 DNA damage checkpoint mutant displays a 10-fold increase in the rate of spontaneous mutation in its germline (Hofmann et al., 2002). Given that in vivo and in vitro studies from suggest that germ cells are endowed with high levels of DNA repair, it is possible that a germline mutator phenotype could compromise the viability of C. elegans over multiple generations. However, the sole reason for the Mortal Germline phenotype observed in DNA damage checkpoint mutants such as hus-1 or mrt-2 is telomere erosion, as judged by the uniform presence of end-to-end chromosome fusions at sterility (Fig. 1b and c) (Ahmed and Hodgkin, 2000; Hofmann et al., 2002). Thus, a modest increase in the rate of spontaneous mutagenesis is of little consequence to the C. elegans germline, because sterility is not observed in the absence of chromosome fusions in hus-1 or mrt-2 mutants. Studies of C. elegans mutator strains with activated transposons indicate that a high transposon copy number can compromise germline immortality (Anderson, 1995). These results suggest that a large increase in the rate of spontaneous mutation may be generally detrimental to the C. elegans germline. Two independent studies recently reported a 200- fold increase in the rate of spontaneous mutation in the C. elegans mismatch repair mutants msh-2 and msh-6 (Degtyareva et al., 2002; Tijsterman et al., 2002). Further, more than half 76 C. Smelick, S. Ahmed / Ageing Research Reviews 4 (2005) 67–82 of msh-2 and msh-6 mutant lines became sterile when propagated between 10 and 20 generations, suggesting that mismatch repair might be required for germline immortality (Degtyareva et al., 2002; Tijsterman et al., 2002). However, these investigators passaged mutant lines by moving single worms between petri dishes, and might, therefore, have been selecting for homozygous sterile or maternal effect lethal mutations that arose as a result of mismatch repair defects. Note that when 100 lines of the wildtype strain of C. elegans, Bristol N2, were passaged by transferring single worms each generation, five lines became sterile by generation 50 (Vassilieva and Lynch, 1999), presumably as a consequence of spontaneous mutations that occasionally arise and become homozygous. When isolating mortal germline mutants in genetic screens, six worms are used for transfers between petri dishes, thereby ensuring that a line will only perish as a consequence of inherited damage that uniformly affects a small population of worms (Ahmed and Hodgkin, 2000)(Fig. 1a). Strikingly, when msh-2 mutant lines were passaged for 20 generations using 3 or more worms per transfer, no lines became sterile (Estes et al., 2004; S. Estes, personal communication). These results indicate that large increases in the rate of spontaneous mutation are tolerated by the C. elegans germline, even when bottlenecks of three individuals are repeatedly imposed. On the other hand, transposon activation in a background with large numbers of transposons is detrimental to germline immortality, perhaps because transposon silencing and mismatch repair protect different regions of the genome from mutation. Thus, a primary theory regarding the maintenance and repair category of mechanisms for germ cell immortality has received mixed experimental support in C. elegans. It is possible that levels of DNA repair may be more important for vertebrates, whose rate of may induce more oxidative damage and spontaneous DNA lesions than that of C. elegans. An interesting C. elegans gene that is required for germline immortality is mre-11, which is essential for double-strand break repair (Chin and Villeneuve, 2001). In mre-11 mutants, homologous chromosomes segregate as univalents because they fail to form crossovers during meiosis, which results in high levels of aneuploid gametes. However, C. elegans has only six chromosomes and a small fraction of the hundreds of embryos laid by a single mre-11 hermaphrodite will by chance contain the appropriate diploid complement of chromosomes and develop into normal F3 adults. Despite the survival of a small number of F3 progeny, mre-11 strains produce F4 only rarely and then become sterile (Chin and Villeneuve, 2001). Thus, either homologous recombination during meiosis or some aspect of the MRE-11-mediated double-stranded break repair is required for germline immortality. In order to distinguish between these two possibilities, C. elegans mutants defective for meiotic homologous recombination but not for double-strand break repair (Zalevsky et al., 1999; Kelly et al., 2000) were examined. In most cases, such mutants were able to proliferate continually, despite the fact that meiotic crossing over was severely reduced or eliminated (Chin and Villeneuve, 2001). Thus, germline immortality does not depend on high rates of meiotic recombination to cleanse chromosomes of DNA lesions (Gensler and Bernstein, 1981; Medvedev, 1981; Holliday, 1984; Strehler, 1986). When mre-11 mutants were examined near sterility, they displayed DNA aberrations that were indicative of damage induced by double-strand breaks (Chin and Villeneuve, 2001). Mouse strains that lack DNA repair genes such as mre-11 typically die as either as embryos or as embryonic stem cells as a consequence of endogenous DNA damage (Lim and Hasty, C. Smelick, S. Ahmed / Ageing Research Reviews 4 (2005) 67–82 77

1996; Xiao and Weaver, 1997). Taken together, these results are consistent with the possibility that mre-11 plays an essential role in the repair of spontaneous DNA damage during cell proliferation, one whose effects are not fully felt for several generations in C. elegans.

4. Perspectives

The means by which the germline achieves immortality is largely a mystery composed of experimentally unverified theories. Three primary mechanisms have received attention: (1) higher levels of maintenance and repair in the germline, (2) rejuvenation mechanisms that transform germ cells to a revived resting state, and (3) removal of or selection against damaged cells (Kirkwood, 1987). A fourth mechanism might include cell non-autonomous contributions of the soma to germline immortality (Fig. 2). Evidence for higher levels of maintenance and repair has been provided by studies of both DNA repair and telomerase in mice and humans (Blasco et al., 1997; Walter et al., 2003). Curiously, one study observed a drop in the frequency of spontaneous mutation at successive stages of spermatogenesis, suggesting that damaged germline cells may be culled (Walter et al., 1998). Finally, attempts at serial cloning by transfer of adult somatic nuclei into enucleated oocytes have failed in various mammals (Wakayama et al., 2000; Kubota et al., 2004), possibly indicating that one or more of the above mechanisms is compromised in adult somatic nuclei. Work in C. elegans has addressed several issues related to germline immortality. Although biochemical and genetic evidence indicates that levels of DNA repair are higher in germlines, 10- to 200-fold increases in the rate of spontaneous mutation are in

Fig. 2. Theories and putative mechanisms for achieving germline immortality. Results of studies of putative mechanisms are described as: Yes, positive result; No, negative result; Mixed, positive and negative results; or Not tested. 78 C. Smelick, S. Ahmed / Ageing Research Reviews 4 (2005) 67–82 some cases permissible for germline immortality in C. elegans (Mechanism 1) (Degtyareva et al., 2002; Hofmann et al., 2002; Tijsterman et al., 2002; Estes et al., 2004). C. elegans mismatch repair mutants are not terribly robust after many generations of growth in the laboratory and might eventually perish. However, many C. elegans mutant strains become similarly sick after propogation for multiple generations, a phenotype that is distinct from the complete sterility observed in mortal germline mutants (Y. Liu, J. Boerckel and S. Ahmed, unpublished data). While the worm germline can withstand some insults to genome stability, both telomere replication and double-strand break repair are required for germline immortality in C. elegans (Ahmed and Hodgkin, 2000; Chin and Villeneuve, 2001; Hofmann et al., 2002). These DNA repair enzymes are likely to fall under the maintenance and repair umbrella of Mechanism 1 above. Although meiotic recombination has been proposed as a possible germ-line specific rejuvenation mechanism, C. elegans mutants with defects in this process have immortal germlines (Chin and Villeneuve, 2001). Among the advantages of using C. elegans to study germline immortality is that it is the foremost system for addressing the molecular genetics of aging, which may help to address theoretical arguments regarding the relationship between the aging process and germline immortality. For example, one might imagine some overlap between mechanisms that help to extend lifespan in C. elegans and those employed for maintenance and repair of germ cells (Mechanism 1). However, an initial test of one of the keystone genes required for lifespan extension in C. elegans has failed to reveal a role in germline immortality (S. Ahmed, unpublished results). Mortal germline mutants may become sterile as a consequence of accumulated forms of heritable damage, some of which might be passed on to somatic cells, possibly causing mutant adults to age more quickly in later generations. While this argument is logical with respect to molecular mechanisms that might compromise germline immortality, theoretical and experimental evidence suggests that evolutionary trade-offs occur between reproduction and aging, such that somatic lifespan is extended for animals that put less energy into reproduction (Partridge and Gems, 2002). For example, ablation of the germline extends lifespan in a number of organisms, including Drosophila and C. elegans, suggesting that the germline can antagonize the aging process (Hsin and Kenyon, 1999; Sgro and Partridge, 1999; Gems and Partridge, 2001; Arantes-Oliveira et al., 2002). Thus, it is formally possible that defects in germline immortality might provide beneficial resources to the soma, perhaps extending the lifespan of some mortal germline mutants. Much theoretical and experimental attention has been paid to nuclear functions that might affect germline immortality (Medvedev, 1981; Avise, 1993), and two failed attempts at serial cloning in mammals suggest that somatic nuclei may be unable to achieve germline immortality (Wakayama et al., 2000; Kubota et al., 2004). However, cytoplasmic processes such as mitochondrial maintenance or the disposal of misfolded proteins, oxidized lipids and lipofuscin, a pigment known to accumulate with age in many species, could be critically important for immortality of the germline. Although we currently only have a peripheral understanding of the essence of germ cell immortality (Fig. 2), the groundwork is being laid to define the pathways that regulate this fundamental biological process in C. elegans. A number of mortal germline mutants have already been identified, and their frequency suggests that multiple biochemical pathways act in concert to enable C. Smelick, S. Ahmed / Ageing Research Reviews 4 (2005) 67–82 79 the germline to achieve immortality. Once mechanisms that help to ensure the eternal youth of germ cells have been identified at the molecular level, it will be important to determine if ectopic, somatic activation of such processes can affect lifespan (Kirkland, 2002).

Acknowledgements

We thank members of the Ahmed lab for critical review of the manuscript and Jeff Sekelsky for discussion. S.A. is supported by an Ellison Medical Foundation New Scholar in Aging Award.

References

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