Ageing Research Reviews
4 (2005) 67–82
www.elsevier.com/locate/arr
Viewpoints
Achieving immortality in the C. elegans germline
Chris Smelickb, Shawn Ahmeda,b,*
a
Department of Genetics, Coker Hall, University of North Carolina, Chapel Hill, NC 27599-3280, USA
Department of Biology, Coker Hall, University of North Carolina, Chapel Hill, NC 27599-3280, USA
b
Received 12 August 2004; accepted 21 September 2004
Abstract
Germline immortality is a topic that has intrigued theoretical biologists interested in aging for
over a century. The germ cell lineage can be passed from one generation to the next, indefinitely. In
contrast, somatic cells are typically only needed for a single generation and are then discarded. Germ
cells may, therefore, harbor rejuvenation 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 nonautonomous forms of rejuvenation are formally possible. A thorough description of mechanisms that
foster eternal youth in germ cells is lacking. The mysteries of germline immortality are being
addressed in the nematode Caenorhabditis elegans 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; Senescence; C. elegans
1. Introduction
As humans have become aware of evolution, 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 organisms, 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
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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 eternity. 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 specific to or
enriched in germ cells (Kirkwood and Holliday, 1979; Medvedev, 1981). August
Weismann 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
organism 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 oocytes
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 time, germ cells do age, 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 chromosome non-disjunction occurs
in oocytes, and where hormone-induced menopausal senescence of the germline occurs at
50–60 years of age (Kirkwood, 1998). In older men, higher frequencies of spontaneous
mutation in sperm 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 germ cell
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 façade 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 times 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?
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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 ciliates, unicellular eukaryotes that contain two distinct nuclei: a diploid germline
micronucleus that is used for meiosis, and a polyploid somatic macronucleus that is
discarded during sexual reproduction. 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 ciliate Paramecium 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 autogamy (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 (Fagerström 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 cancer 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 telomeres,
the ends of linear chromosomes (Bodnar et al., 1998; Counter et al., 1998). Telomere length
in most organisms is maintained by telomerase, 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.
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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 death (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 mutation rate of various mouse tissues was estimated via analysis of
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mutations in an array of 50–70 copies of lacI or lacZ reporter transgenes 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,
transversions, 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 transgene 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 second 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 homologous recombination 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 species 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 differentiation. 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).
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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 genome 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 apoptosis during development and maturation (Matsui, 1998). Although
its functional significance is unclear, apoptosis is a conserved feature of metazoan germline
development 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 gamete development, the
frequency of spontaneous mutation has been observed to decrease in successive stages of
spermatogenesis 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 gametes. 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 technology that may provide insight into the relative importance of each of the three
classes of molecular mechanisms with regards to germline immortality is reproductive
cloning by somatic cell nuclear transfer. Reproductive cloning is achieved by
microinjecting the nucleus of a somatic cell into an enucleated oocyte, which is then
activated to undergo parthenogenesis, 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 term 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).
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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
germlines 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 multicellular organism whose germline and
soma are distinct from the first cell division 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
bacteria are seeded with six L1 larvae and scored a week 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).
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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 generation time of roughly 3.5 days, allowing
for 20 or 30 generations of growth to occur in only a few months’ 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 80˚ 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 1000fold 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 lossof-function mutation in a particular gene occurs, on average, once in every 4000 EMSmutagenized 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
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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 vertebrates 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 200fold 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
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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 metabolism 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 vertebrate 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
Aguilaniu, H., Gustafsson, L., Rigoulet, M., Nystrom, T., 2003. Asymmetric inheritance of oxidatively damaged
proteins during cytokinesis. Science 299, 1751–1753.
Ahmed, S., Hodgkin, J., 2000. MRT-2 checkpoint protein is required for germline immortality and telomere
replication in C. elegans. Nature 403, 159–164.
Albertson, D.G., Thomson, J.N., 1993. Segregation of holocentric chromosomes at meiosis in the nematode.
Caenorhabditis elegans Chromosome Res. 1, 15–26.
Anderson, P., 1995. Mutagen. Methods Cell Biol. 48, 31–58.
Arantes-Oliveira, N., Apfeld, J., Dillin, A., Kenyon, C., 2002. Regulation of life-span by germ-line stem cells in
Caenorhabditis elegans. Science 295, 502–505.
Avise, J.C., 1993. The evolutionary biology of aging, sexual reproduction, and DNA repair. Evolution 47, 1293–
1301.
Bell, G., 1988. Sex and Death in Protozoa: The History of an Obsession. Cambridge University Press, Cambridge.
Bermudez, V.P., Lindsey-Boltz, L.A., Cesare, A.J., Maniwa, Y., Griffith, J.D., Hurwitz, J., Sancar, A., 2003.
Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17replication factor C complex in vitro. Proc. Natl. Acad. Sci. U.S.A. 100, 1633–1638.
Bestor, T.H., 2003. Cytosine methylation mediates sexual conflict. Trends Genet. 19, 185–190.
Byers, H., Muller, H.J., 1952. Influence of aging at two different temperatures on the spontaneous mutation rate of
mature spermatozoa of Drosophila melanogaster. Genetics 37, 570–571.
Blasco, M.A., Lee, H.W., Hande, M.P., Samper, E., Lansdorp, P.M., DePinho, R.A., Greider, C.W., 1997.
Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 91, 25–34.
Bodnar, A.G., Ouellette, M., Frolkis, M., Holt, S.E., Chiu, C.P., Morin, G.B., Harley, C.B., Shay, J.W., et al., 1998.
Extension of life-span by introduction of telomerase into normal human cells. Science 279, 349–352.
Boulton, S.J., Gartner, A., Reboul, J., Vaglio, P., Dyson, N., Hill, D.E., Vidal, M., 2002. Combined functional
genomic maps of the C. elegans DNA damage response. Science 295, 127–131.
Buszczak, M., Cooley, L., 2000. Eggs to die for: cell death during Drosophila oogenesis. Cell Death Differ. 7,
1071–1074.
Chin, G.M., Villeneuve, A.M., 2001. C. elegans mre-11 is required for meiotic recombination and DNA repair but
is dispensable for the meiotic G(2) DNA damage checkpoint. Genes Dev. 15, 522–534.
Collins, J., Saari, B., Anderson, P., 1987. Activation of a transposable element in the germ line but not the soma of
Caenorhabditis elegans. Nature 328, 726–728.
Counter, C.M., Hahn, W.C., Wei, W., Caddle, S.D., Beijersbergen, R.L., Lansdorp, P.M., Sedivy, J.M., Weinberg,
R.A., 1998. Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization. Proc. Natl. Acad. Sci. U.S.A. 95, 14723–14728.
Crow, J., 2000. The origins, patterns and implications of human spontaneous mutation. Nat. Rev. Genet. 1, 40–47.
D’Mello, N.P., Jazwinski, S.M., 1991. Telomere length constancy during aging of Saccharomyces cerevisiae. J.
Bacteriol. 173, 6709–6713.
Dawkins, R., 1990. The Selfish Gene. Oxford University Press, Oxford.
80
C. Smelick, S. Ahmed / Ageing Research Reviews 4 (2005) 67–82
Degtyareva, N.P., Greenwell, P., Hofmann, E.R., Hengartner, M.O., Zhang, L., Culotti, J.G., Petes, T.D., 2002.
Caenorhabditis elegans DNA mismatch repair gene msh-2 is required for microsatellite stability and
maintenance of genome integrity. Proc. Natl. Acad. Sci. U.S.A. 99, 2158–2163.
Estes, S., Phillips, P.C., Denver, D.R., Thomas, W.K., Lynch, M., 2004. Mutation accumulation in populations of
varying size: the distribution of mutational effects for fitness correlates in Caenorhabditis elegans. Genetics
166, 1269–1279.
Fagerström, T., Briscoe, D.A., Sunnucks, P., 1998. Evolution of mitotic cell lineages in multicellular organisms.
Tree 13, 117–120.
Filion, A.M., Laughrea, M., 1985. Translation fidelity in the aging mammal: studies with an accurate in vitro
system on aged rats. Mech. Ageing Dev. 29, 125–142.
Garigan, D., Hsu, A.L., Fraser, A.G., Kamath, R.S., Ahringer, J., Kenyon, C., 2002. Genetic analysis of tissue aging
in Caenorhabditis elegans. a role for heat-shock factor and bacterial proliferation. Genetics 161, 1101–1112.
Gems, D., Partridge, L., 2001. Insulin/IGF signalling and ageing: seeing the bigger picture. Curr. Opin. Genet.
Dev. 11, 287–292.
Gensler, H.L., Bernstein, H., 1981. DNA damage as the primary cause of aging. Q. Rev. Biol. 56, 279–303.
Gilley, D., Blackburn, E.H., 1994. Lack of telomere shortening during senescence in Paramecium. Proc. Natl.
Acad. Sci. U.S.A. 91, 1955–1958.
Goldstein, P., Curis, M., 1987. Age-related changes in the meiotic chromosomes of the nematode Caenorhabditis
elegans. Mech. Ageing Dev. 40, 115–130.
Goldstein, S., Wojtyk, R.I., Harley, C.B., Pollard, J.W., Chamberlain, J.W., Stanners, C.P., 1985. Protein synthetic
fidelity in aging human fibroblasts. Adv. Exp. Med. Biol. 190, 495–508.
Haber, J.E., 2000. Partners and pathways repairing a double-strand break. Trends Genet. 16, 259–264.
Hajkova, P., Erhardt, S., Lane, N., Haaf, T., El-Maarri, O., Reik, W., Walter, J., Surani, M.A., 2002. Epigenetic
reprogramming in mouse primordial germ cells. Mech. Dev. 117, 15–23.
Harrington, L., 2003. Biochemical aspects of telomerase function. Cancer Lett. 194, 139–154.
Hayflick, L., Moorhead, P.S., 1961. The serial cultivation of human diploid cell strains. Exp. Cell Res. 25, 585–
621.
Hill, K.A., Buettner, V.L., Halangoda, A., Kunishige, M., Moore, S.R., Longmate, J., Scaringe, W.A., Sommer,
S.S., 2004. Spontaneous mutation in Big Blue mice from fetus to old age: tissue-specific time courses of
mutation frequency but similar mutation types. Environ. Mol. Mutagen. 43, 110–120.
Hofmann, E.R., Milstein, S., Boulton, S.J., Ye, M., Hofmann, J.J., Stergiou, L., Gartner, A., Vidal, M., et al., 2002.
Caenorhabditis elegans HUS-1 is a DNA damage checkpoint protein required for genome stability and EGL1-mediated apoptosis. Curr. Biol. 12, 1908–1918.
Holliday, R., 1984. The biological significance of meiosis. Symp. Soc. Exp. Biol. 38, 381–394.
Hsin, H., Kenyon, C., 1999. Signals from the reproductive system regulate the lifespan of C. elegans. Nature 399,
362–666.
Intano, G.W., McMahan, C.A., Walter, R.B., McCarrey, J.R., Walter, C.A., 2001. Mixed spermatogenic germ cell
nuclear extracts exhibit high base excision repair activity. Nucleic Acids Res. 29, 1366–1372.
Jiang, X.R., Jimenez, G., Chang, E., Frolkis, M., Kusler, B., Sage, M., Beeche, M., Bodnar, A.G., et al., 1999.
Telomerase expression in human somatic cells does not induce changes associated with a transformed
phenotype. Nat. Genet. 21, 111–114.
Kaufmann, B.P., 1947. Spontaneous mutation rate in Drosophila. Am. Nat. 81, 77–80.
Kelly, K.O., Dernburg, A.F., Stanfield, G.M., Villeneuve, A.M., 2000. Caenorhabditis elegans msh-5 is required
for both normal and radiation-induced meiotic crossing over but not for completion of meiosis. Genetics 156,
617–630.
Kim, N.W., Piatyszek, M.A., Prowse, K.R., Harley, C.B., West, M.D., Ho, P.L., Coviello, G.M., Wright, W.E.,
et al., 1994. Specific association of human telomerase activity with immortal cells and cancer. Science 266,
2011–2015.
Kirkland, J.L., 2002. The biology of senescence: potential for prevention of disease. Clin. Geriatr. Med. 18, 383–
405.
Kirkwood, T.B., 1998. Ovarian ageing and the general biology of senescence. Maturitas 30, 105–111.
Kirkwood, T.B., Holliday, R., 1979. The evolution of ageing and longevity. Proc. R. Soc. Lond. B Biol. Sci. 205,
531–546.
C. Smelick, S. Ahmed / Ageing Research Reviews 4 (2005) 67–82
81
Kirkwood, T.B.L., 1987. Immortality of the germ-line versus disposability of the soma. In: Woodhead, A.D.,
Thompson, K.H. (Eds.), Evolution of Longevity in Animals: a Comparative Approach. Plenum Press, New
York, pp. 209–218.
Kohler, S.W., Provost, G.S., Fieck, A., Kretz, P.L., Bullock, W.O., Sorge, J.A., Putman, D.L., Short, J.M., 1991.
Spectra of spontaneous and mutagen-induced mutations in the lacI gene in transgenic mice. Proc. Natl. Acad.
Sci. U.S.A. 88, 7958–7962.
Kubota, C., Tian, X.C., Yang, X., 2004. Serial bull cloning by somatic cell nuclear transfer. Nat. Biotechnol. 22,
693–694.
Lim, D.S., Hasty, P., 1996. A mutation in mouse RAD51 results in an early embryonic lethal that is suppressed by a
mutation in p53. Mol. Cell. Biol. 16, 7133–7143.
Lundblad, V., Szostak, J.W., 1989. A mutant with a defect in telomere elongation leads to senescence in yeast. Cell
57, 633–643.
Matsui, Y., 1998. Developmental fates of the mouse germ cell line. Int. J. Dev. Biol. 42, 1037–1042.
Medvedev, Z.A., 1981. On the immortality of the germ line: genetic and biochemical mechanism: A review. Mech.
Ageing Dev. 17, 331–359.
Meyerson, M., Counter, C.M., Eaton, E.N., Ellisen, L.W., Steiner, P., Caddle, S.D., Ziaugra, L., Beijersbergen,
R.L., et al., 1997. hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells
and during immortalization. Cell 90, 785–795.
Morales, C.P., Holt, S.E., Ouellette, M., Kaur, K.J., Yan, Y., Wilson, K.S., White, M.A., Wright, W.E., et al., 1999.
Absence of cancer-associated changes in human fibroblasts immortalized with telomerase. Nat. Genet. 21,
115–118.
Naito, T., Matsuura, A., Ishikawa, F., 1998. Circular chromosome formation in a fission yeast mutant defective in
two ATM homologues. Nat. Genet. 20, 203–206.
Nakamura, T.M., Cooper, J.P., Cech, T.R., 1998. Two modes of survival of fission yeast without telomerase.
Science 282, 493–496.
Nakamura, T.M., Morin, G.B., Chapman, K.B., Weinrich, S.L., Andrews, W.H., Lingner, J., Harley, C.B., Cech,
T.R., 1997. Telomerase catalytic subunit homologs from fission yeast and human. Science 277, 955–959.
Nystrom, T., 2002. Aging in bacteria. Curr. Opin. Microbiol. 5, 596–601.
Orgel, L.E., 1963. The maintenance of the accuracy of protein synthesis and its relevance to ageing. Proc. Natl.
Acad. Sci. U.S.A. 49, 517–521.
Partridge, L., Gems, D., 2002. Mechanisms of ageing: public or private? Nat. Rev. Genet. 3, 165–175.
Plasterk, R.H.A., van Leunen, H.G.A.M., 1997. Transposons. In: Riddle, D.L., Blumenthal, T., Meyer, B.J.,
Priess, J.R. (Eds.), C. elegans II. Cold Spring Harbor Laboratory Press, Plainview, pp. 97–116.
Prowse, K.R., Greider, C.W., 1995. Developmental and tissue-specific regulation of mouse telomerase and
telomere length. Proc. Natl. Acad. Sci. U.S.A. 92, 4818–4822.
Purdom, C.E., Dyer, K.F., Papworth, D.G., 1968. Spontaneous mutation in Drosophila: studies on the rate of
mutation in mature and immature male germ cells. Mutat. Res. 5, 133–146.
Rakyan, V.K., Preis, J., Morgan, H.D., Whitelaw, E., 2001. The marks, mechanisms and memory of epigenetic
states in mammals. Biochem. J. 356, 1–10.
Reik, W., Santos, F., Mitsuya, K., Morgan, H., Dean, W., 2003. Epigenetic asymmetry in the mammalian zygote and
early embryo: relationship to lineage commitment? Philos. Trans. R. Soc. Lond. B Biol. Sci. 358, 1403–1409.
Santos, F., Dean, W., 2004. Epigenetic reprogramming during early development in mammals. Reproduction 127,
643–651.
Seydoux, G., Schedl, T., 2001. The germline in C. elegans: origins, proliferation, and silencing. Int. Rev. Cytol.
203, 139–185.
Sgro, C.M., Partridge, L., 1999. A delayed wave of death from reproduction in Drosophila. Science 286, 2521–
2524.
Shay, J.W., Roninson, I.B., 2004. Hallmarks of senescence in carcinogenesis and cancer therapy. Oncogene 23,
2919–2933.
Sinclair, D.A., Guarente, L., 1997. Extrachromosomal rDNA circles—a cause of aging in yeast. Cell 91, 1033–
1042.
Smeal, T., Claus, J., Kennedy, B., Cole, F., Guarente, L., 1996. Loss of transcriptional silencing causes sterility in
old mother cells of S. cerevisiae. Cell 84, 633–642.
82
C. Smelick, S. Ahmed / Ageing Research Reviews 4 (2005) 67–82
Smith-Sonneborn, J., 1987. Longevity in the protozoa. Basic Life Sci. 42, 101–109.
Strehler, B.L., 1986. Genetic instability as the primary cause of human aging. Exp. Gerontol. 21, 283–319.
Strome, S., Wood, W.B., 1982. Immunofluorescence visualization of germ-line-specific cytoplasmic granules in
embryos, larvae, and adults of Caenorhabditis elegans. Proc. Natl. Acad. Sci. U.S.A. 79, 1558–1562.
Surani, M.A., 1998. Imprinting and the initiation of gene silencing in the germ line. Cell 93, 309–312.
Suter, C.M., Martin, D.I., Ward, R.L., 2004. Germline epimutation of MLH1 in individuals with multiple cancers.
Nat. Genet. 36, 497–501.
Takata, M., Sasaki, M.S., Sonoda, E., Morrison, C., Hashimoto, M., Utsumi, H., Yamaguchi-Iwai, Y., Shinohara,
A., et al., 1998. Homologous recombination and non-homologous end-joining pathways of DNA doublestrand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells.
EMBO J. 17, 5497–5508.
Tamashiro, K.L., Wakayama, T., Blanchard, R.J., Blanchard, D.C., Yanagimachi, R., 2000. Postnatal growth and
behavioral development of mice cloned from adult cumulus cells. Biol. Reprod. 63, 328–334.
Tijsterman, M., Pothof, J., Plasterk, R.H., 2002. Frequent germline mutations and somatic repeat instability in
DNA mismatch-repair-deficient Caenorhabditis elegans. Genetics 161, 651–660.
Vassilieva, L.L., Lynch, M., 1999. The rate of spontaneous mutation for life-history traits in Caenorhabditis
elegans. Genetics 151, 119–129.
Wakayama, T., Tateno, H., Mombaerts, P., Yanagimachi, R., 2000. Nuclear transfer into mouse zygotes. Nat.
Genet. 24, 108–109.
Walter, C.A., Intano, G.W., McCarrey, J.R., McMahan, C.A., Walter, R.B., 1998. Mutation frequency declines
during spermatogenesis in young mice but increases in old mice. Proc. Natl. Acad. Sci. U.S.A. 95, 10015–
10019.
Walter, C.A., Walter, R.B., McCarrey, J.R., 2003. Germline genomes–a biological fountain of youth? Sci. Aging
Knowl. Environ. PE4.
Weismann, A., 1882. Ueber die Dauer des Lebens (in German), G. Fischer, Jena.
Winn, R.N., Norris, M.B., Brayer, K.J., Torres, C., Muller, S.L., 2000. Detection of mutations in transgenic fish
carrying a bacteriophage lambda cII transgene target. Proc. Natl. Acad. Sci. U.S.A. 97, 12655–12660.
Wright, W.E., Shay, J.W., 2000. Telomere dynamics in cancer progression and prevention: fundamental
differences in human and mouse telomere biology. Nat. Med. 6, 849–851.
Xiao, Y., Weaver, D.T., 1997. Conditional gene targeted deletion by Cre recombinase demonstrates the
requirement for the double-strand break repair Mre11 protein in murine embryonic stem cells. Nucleic
Acids Res. 25, 2985–2991.
Zalevsky, J., MacQueen, A.J., Duffy, J.B., Kemphues, K.J., Villeneuve, A.M., 1999. Crossing over during
Caenorhabditis elegans meiosis requires a conserved MutS-based pathway that is partially dispensable in
budding yeast. Genetics 153, 1271–1283.