Caenorhabditis elegans Reproductive Aging:
Regulation and Underlying Mechanisms
Shijing Luo and Coleen T. Murphy*
Department of Molecular Biology, Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton,
Received 20 October 2010; Revised 16 November 2010; Accepted 17 November 2010
Summary: Female reproductive decline is one of the
first aging phenotypes in humans, manifested in
increasing rates of infertility, miscarriage, and birth
defects in children of mothers over 35. Recently, Cae-
norhabditis elegans (C. elegans) has been developed as
a model to study reproductive aging, and several stud-
ies have advanced our knowledge of reproductive aging
regulation in this organism. In this review, we describe
our current understanding of reproductive cessation in
C. elegans, including the relationship between oocyte
quality, ovulation rate, progeny number, and reproduc-
tive span. We then discuss possible mechanisms of
oocyte quality control, and provide an overview of the
signaling pathways currently identified to be involved in
reproductive span regulation in C. elegans. Finally, we
extend the relevance of C. elegans reproductive aging
studies to the issue of human female reproductive
decline, and we discuss ideas concerning the relation-
ship between reproductive aging and somatic longevity.
genesis 49:53–65, 2011.
C 2010 Wiley-Liss, Inc.
Key words: reproduction;
aging;germ line; oocyte;
C. elegans as a Model for Longevity Studies
Over the last two decades, our understanding of lon-
gevity mechanisms has been dramatically expanded with
breakthrough genetic studies using model organisms, in
particular the nematode Caenorhabditis elegans. Many
useful genetic manipulation tools and resources have
been developed for C. elegans research, including whole-
genome RNA interference libraries, chemical, UV, and
transposon mutagenesis, and repositories for large num-
bers of mutant and transgenic strains. These tools and the
fact that C. elegans has a short life span (2–3 weeks) have
allowed rapid analysis of survival and aging phenotypes.
There is a strong conservation of longevity pathways
from C. elegans to humans (Kenyon, 2005; Suh et al.,
2008). Moreover, C. elegans is a multicellular eukaryotic
organism with multiple tissues and complicated behav-
iors, making it a superb model organism to study the
senescence of many biological functions and processes,
including loss of mobility (Herndon et al., 2002; Huang
et al., 2004; Iwasa et al., 2010), decline in muscle
integrity (Herndon et al., 2002), increased cancer
susceptibility (Pinkston et al., 2006; Pinkston-Gosse and
Kenyon, 2007), and declines in chemotaxis, learning,
and memory (Kauffman et al., 2010).
The C. elegans Reproductive System
The transparency of C. elegans’ tissues and the spatio–
temporal layout of its reproductive system (Fig. 1) have
made it a powerful model to study the germ line and
reproduction, including germ line stem cell biology,
germ cell apoptosis, the transition from mitosis to meio-
sis, oocyte maturation, and fertilization (Hubbard and
Greenstein, 2000; Kimble and Crittenden, 2007). The
*Correspondence to: Coleen T. Murphy, Department of Molecular Bio-
logy, Lewis-Sigler Institute for Integrative Genomics, Princeton University,
Princeton, NJ 08544, USA. E-mail: firstname.lastname@example.org
Contract grant sponsor: NIH New Innovator, Contract grant number:
1DP2OD004402-01; Contract grant sponsor: March of Dimes (Basil
O’Connor Starting Scholar awards)
Published online 23 November 2010 in
Wiley Online Library (wileyonlinelibrary.com).
Abbreviations: ARD, adult reproductive diapause; DTC, distal tip cell;
IIS, insulin/IGF-1 signaling; MSP, major sperm protein; TGF-b, transforming
' 2010 Wiley-Liss, Inc.genesis 49:53–65 (2011)
C. elegans hermaphrodite germ line is a U-shaped tube
structure containing two symmetric arms (Fig. 1). In
each arm, the distal germ line receives the proprolifera-
tive LIN-12/Notch signal from the somatic distal tip cell
(DTC) located at the tip of the gonad, whereas the first
few rows of the syncytial distal germ line contain the
stem cell nuclei that give rise to the rest of the prolifer-
ating germ cell nuclei in the mitotic region (Cinquin
et al., 2010; Crittenden et al., 2006). In the transition
zone, the germ cell nuclei begin meiosis and enter into
the pachytene stage of meiotic prophase I. At the end
of the pachytene stage, which occurs in the bend
region, the syncytial germ cell nuclei begin to cellular-
ize and grow larger. The bend is also the region where
apoptosis takes place. The surviving germ cells then
complete the diplotene stage and arrest at diakinesis. In
the presence of sperm (either made prior to oogenesis
or provided by males), the most proximal oocyte is acti-
vated by major sperm protein (MSP), completes meiotic
maturation, and is ovulated into the spermatheca,
where sperm are stored. The oocyte is fertilized in the
spermatheca, and the fertilized embryo is pushed into
the uterus and finally laid through the vulva.
In C. elegans, interactions between the soma and
germ line occur at many levels to control reproduction.
The somatic DTC maintains proliferation of stem cell
nuclei in the distal germ line (Kimble and White, 1981).
Somatic gonadal sheath cells and spermathecal lineage
cells also play critical roles in germ cell nuclei mitotic
proliferation, meiotic differentiation, oocyte growth,
meiotic maturation, and ovulation (Govindan et al.,
2009; Hall et al., 1999; McCarter et al., 1997; Miller
et al., 2003). A recent study found that the C. elegans
early growth response factor family member egrh-1
functions in both the intestine and somatic gonad to
regulate oocyte development (Clary and Okkema,
C. elegans as a Model of Reproductive Aging
While many studies have addressed phenotypes of
late aging, fewer have focused on early aging phenom-
ena. In humans, female reproductive capacity declines
dramatically after the mid-30s, and is marked by
an increased risk of infertility, birth defects, and
miscarriage, making it perhaps the earliest age-related
decline that humans experience. These reproductive
aging problems are thought to be due to declining
oocyte quality rather than lack of oocytes because the
problems arise a decade prior to menopause. As more
and more women opt to have children later in life,
addressing the issue of female reproductive aging has
become increasingly important. As in humans, repro-
duction declines early in C. elegans, lasting only one-
third of its life span. Recently, C. elegans has been devel-
oped as a model to study reproductive capacity decline
with age (Andux and Ellis, 2008; Hughes et al., 2007;
Luo et al., 2009, 2010). These studies established that
(1) C. elegans reproductive aging is a genetically-regu-
lated process; (2) C. elegans reproductive aging is lim-
ited by oocyte quality decline, as in humans; and (3)
reproductive aging is normally coupled to, but also
distinct from somatic aging.
MECHANISMS OF C. ELEGANS
REPRODUCTIVE AGING REGULATION
Reproductive Aging Is Independent
of Progeny Number
Four possible models could explain reproductive
cessation in C. elegans: (1) C. elegans hermaphrodites
produce only ?300 sperm, therefore exhaustion of
self sperm could cause termination of reproduction;
(2) C. elegans may only generate a limited number of
oocytes; (3) C. elegans can generate an unlimited num-
ber of oocytes, but there is a limited number of high-
quality oocytes; or (4) C. elegans oocyte quality declines
with and is determined by maternal age, regardless of
oocyte number. To rule out the first possibility, that of
limitation by self-sperm, hermaphrodites (or spermless
mutant hermaphrodites) were provided with sufficient
sperm by mating with young wild-type males (hereafter
referred to as ‘‘mated reproductive span’’). When pro-
vided with excess sperm, reproduction still ceases early
in adulthood (Hughes et al., 2007; Luo et al., 2009),
suggesting that sperm number is not a limiting factor in
C. elegans mated hermaphrodite reproductive aging.
Among the three remaining possibilities, the second
and the third would suggest a usage-dependent mecha-
Notch signals to the distal germ line, maintaining the proliferative state of mitotic cell nuclei. Mitotic germ cell nuclei enter meiosis in the tran-
sition zone (TZ), marked by crescent-shaped nuclei. Germ cell nuclei exit the transition zone and enter the pachytene stage, complete the
diplotene stage, and become arrested at diakinesis. Mature oocytes are fertilized by sperm in the spermatheca, and the fertilized embryo is
stored in the uterus before finally being laid through the vulva.
The C. elegans gonad. The C. elegans hermaphrodite germ line contains two symmetric arms. Distal tip cells (DTC) send LIN-12/
LUO AND MURPHY
nism: in these two particular scenarios, the cessation of
reproduction would be a consequence of exhaustion of
good oocytes. If this were true, an extension of the
reproductive period could be achieved through slower
usage of oocytes in the early phase of reproduction.
Totest this hypothesis,
manipulated early reproduction by mating wild-type or
spermless mutants (spe-8 and fog-2 mutants) with
young wild-type males at different ages, and character-
ized their progeny production (Hughes et al., 2007).
They found that the decline in late progeny production
is independent of the number of progeny produced in
the early phase of reproduction, showing that reproduc-
tive cessation in C. elegans is not due to a usage-depend-
ent mechanism. Also, Andux and Ellis (2008) reported
that mated older fog-2 (spermless) mutant hermaphro-
dites produce more unhatched embryos and fewer
fertilized embryos than younger fog-2 mutant mothers.
This suggests that (1) reproductive cessation is due to a
decline in progeny quality, and (2) such decline is deter-
mined by maternal age rather than by the number of
progeny produced. Furthermore, Luo et al. (2009)
showed that neither slower usage of oocytes due to
slowed ovulation rate nor a smaller brood size, as in the
case of many small-body mutants, extends reproductive
span (i.e., the reproductive period) (Luo et al., 2009),
which again suggests that a usage-dependent mecha-
nism does not underlie reproductive cessation. Addi-
tionally, ovulation rate and progeny number are not
correlated with reproductive span (Luo et al., 2010).
Together, these studies show that reproductive cessa-
tion in C. elegans is usage-independent, and that simply
reducing the number of oocytes used or delaying
progeny production does not extend reproductive
span-that is, C. elegans, like humans, have a ‘‘use it or
lose it’’ limitation of reproduction.
Oocyte Quality Decline Limits Reproductive Span
In humans, reproductive cessation occurs a decade
before menopause, suggesting that declining oocyte
quality, rather than quantity, is the major cause of mater-
nal age-associated infertility and birth defects (ESHRE
Capri Workshop Group, 2005). Mammalian oocytes
exhibit increased abnormalities in fertilization, chromo-
response with age (Blondin et al., 1997; ESHRE Capri
Workshop Group, 2005; Goud et al., 1999; Hiroshi
Tamura et al., 2008; Jones, 2008; Tarin, 1996; te Velde
and Pearson, 2002). Similarly, several C. elegans studies
show that oocyte quality is the limiting factor for repro-
ductive capacity decline (Andux and Ellis, 2008;
Luo et al., 2010) (Fig. 2a). In mated hermaphrodites
provided with sufficient sperm, every oocyte that is
capable of being fertilized will acquire an egg shell and
become a fertilized embryo that is easily distinguished
from an unfertilized oocyte (Luo et al., 2010). Mated
embryos in early reproduction, but start producing
unfertilized oocytes as they age (Luo et al., 2010).
Likewise, older worms are more likely to have a cluster
of unfertilized oocytes in the uterus, where fertilized
embryos are normally stored before being laid (Luo
et al., 2010). These data indicate that the fertilizability
of oocytes becomes compromised with age.
The developmental competence of oocytes also
declines with age. Older hermaphrodites produce more
unhatched embryos, a phenotype caused by develop-
mental defects, such as chromosomal segregation errors
(Andux and Ellis, 2008; Luo et al., 2010). While
increased embryonic lethality can result from autosomal
nondisjunction, increased male production indicates
an increase in X chromosome segregation errors. In
C. elegans, XO (male) progeny are produced from XX
hermaphrodite mothers through meiotic X chromo-
some nondisjunction (Hodgkin et al., 1979). Nondis-
junction occurs at a low frequency in young hermaph-
rodites and males are relatively rare in the population,
but more male progeny are produced with increasing
maternal age (Luo et al., 2010; Rose and Baillie, 1979;
Tang et al., 2010). Together, the data suggest that
chromosomal abnormalities increase with age. This is
confirmed by the fact that the oocytes of older
worms are more likely to contain an abnormal number
of DAPI-stained bodies in their nuclei (Luo et al., 2010).
Older embryos are also more susceptible to hypo-
chlorite treatment (i.e., bleaching) and ionizing irradia-
tion treatment, suggesting that their stress resistance
declines with age (Luo et al., 2010).
In addition to the declines in their functional charac-
teristics, oocyte morphology is also degraded in older
mothers (Fig. 2b). Andux and Ellis reported that older
virgin hermaphrodites have stacked oocytes in their
proximal gonads, and embryos developed from stacked
oocytes are less likely to hatch, suggesting that stacking
at diakinesis of meiotic prophase I is correlated with
defective oocytes (Andux and Ellis, 2008). Older wild-
type animals produce more small embryos and tiny,
embryo-like objects. This phenotype is elevated in
apoptosis-defective mutants, so the increased frequency
of smaller embryos is more likely to be the result of
fertilization of smaller oocytes rather than due to the
breakdown of larger oocytes. Consistent with these
observations, Luo and colleagues found a small-oocyte
phenotype in day-8 mated wild-type gonads, and
showed that this morphology marker can be used as
one of the predictors of the reproductive capacity of a
worm (Luo et al., 2010). Small oocytes may lack suffi-
cient resources required for proper embryogenesis.
Since oocyte stacking is more frequently observed
prior to small oocytes in the gonad (Luo and Murphy,
unpublished data), it is possible that stacking is an
REPRODUCTIVE AGING REGULATION IN C. elegans
intermediate step in producing small oocytes. However,
it is not yet known whether stacked oocytes and small
oocytes reduce quality through the same mechanism or
independently. Small oocyte size cannot be solely re-
sponsible for oocyte quality, since not all unhatched
embryos are small (Andux and Ellis, 2008; Luo et al.,
2010); so normal-sized old oocytes may also be insuffi-
ciently equipped or be defective in maturation and/or
Oocyte size is just one of the morphological markers
of oocyte quality, however. In young worms, oocytes
are packed closely together and make extensive contact
with somatic tissues, while there are more cavities
between neighboring oocytes and between oocytes and
somatic gonad tissues in aging mothers (Luo et al.,
2010). Thus, cellular contact may be crucial for normal
signal transduction and uptake of important molecules,
and contact failure may partially explain defective
oocytes, regardless of their size.
Apoptosis and Germ Line Stem Cells
in Oocyte Quality Control
At least half of the oogenic germ cell nuclei
are removed by apoptosis in C. elegans (Gumienny
et al., 1999). In insects, such as the cockroach, ovarian
apoptosis increases under starvation and reduces
‘‘female reproductive life span’’ (Edvardsson et al.,
2009; Terashima and Bownes, 2004). In human oocytes,
(Wu et al., 2000). Thus, apoptosis may be a conserved
mechanism involved in reproductive maintenance.
Several different types of apoptosis take place in the
C. elegans germ line. ‘‘Physiological germ cell apopto-
sis’’ refers to the reduction of nuclei in the apoptotic
zone prior to theircellularization
(Gumienny et al., 1999). Other forms of apoptosis are
triggered by stress: ‘‘DNA-damage checkpoint-induced
apoptosis’’ is caused by ionizing irradiation (Gartner
undergo declining reproductive ability with age, demonstrated through reduced embryo hatching and stress resistance; reduced oocyte
fertilizability, chromosome segregation fidelity, and degraded oocyte morphology; and degraded distal germ line morphology and germ cell
nuclei proliferation (Luo et al., 2010). DNA damage-induced apoptosis decreases significantly with age, independently of IIS and TGF-b
regulation, while physiological apoptosis may also decrease in aged wild-type animals (Andux and Ellis, 2008). (b) Oocytes of young (day 1)
and old (day 8) wild-type worms exhibit differences in morphology (Luo et al., 2010).
Summary of cellular processes that are better maintained in mutants with extended reproductive spans. (a) Wild-type worms
LUO AND MURPHY
et al., 2000), while meiotic recombination and pairing
checkpoints (Bhalla and Dernburg, 2005; Gartner et al.,
2000), and environmental stresses, especially pathogen
infections (Aballay and Ausubel, 2001; Salinas et al.,
2006), can also induce apoptosis.
Two models could explain the role of apoptosis
in oogenesis and oocyte quality maintenance with age:
(1) physiological apoptosis may provide nurse cells for
the remaining oocytes to ensure their proper growth
and maturation (the ‘‘nurse cell model’’) and (2) DNA
damage or other stress-induced apoptosis might elimi-
nate defective germ cell nuclei, thereby maintaining
genomic integrity of the germ line (the ‘‘elimination
model’’) (Andux and Ellis, 2008; Gartner et al., 2008;
Gumienny et al., 1999).
Andux and Ellis found that the apoptosis-defective
ced-3 and ced-4 mutants produce more unhatched
embryos, and that this effect on embryonic lethality is
maternal and therefore reflects a decrease in oocyte qual-
ity. However, two other mutants that are defective only
in DNA-damage-induced apoptosis but not physiological
germ cell apoptosis, egl-1 and ced-9 mutants, do not
seem to increase embryonic lethality, at least not to the
same degree that ced-3 and ced-4 mutants do (Andux
and Ellis, 2008). Wild-type animals produce more small
embryos with age (Andux and Ellis, 2008; Luo et al.,
2010), and this phenotype becomes more dramatic in
ced-3 and ced-4 mutants (Andux and Ellis, 2008).
Together, the data suggest the model that physiological
apoptosis controls oocyte quality by reducing the number
of germ cell nuclei to ensure proper resource allocation.
The levels of apoptosis after DNA damaging ionizing
irradiation dramatically decrease in wild-type animals
from day 2 to day 6 of adulthood (Luo et al., 2010), but
the authors observed only a slight decrease in the level
of physiological germ cell apoptosis during this interval
(Luo et al., 2010) (Fig. 2a). Taken together, these data
suggest that both the nurse cell model and the elimina-
tion model may play a role in regulating oocyte mainte-
nance. It is possible that the DNA damage checkpoint
may play a larger role when genotoxic stress accumu-
lates and cells are more susceptible to damage, e.g., in
late reproduction. Such a role would be magnified
when there is excessive DNA damage triggered by ioniz-
ing irradiation, but may be unnoticed in untreated
conditions early in reproduction because of lower
amounts of DNA damage. In support of this notion,
ced-9 mutants exhibit increased embryonic lethality
compared with wild type, but at a much later time than
observed in ced-3 and ced-4 mutants, perhaps reflecting
its increasing importance in late reproduction (Andux
and Ellis, 2008). Physiological apoptosis, however, may
play an important role early in reproduction, and a
slight decrease in physiological apoptosis may be suffi-
cient to make a difference in oocyte quality. While ovar-
ian apoptosis may underlie fertility decline in insects
(Edvardsson et al., 2009; Terashima and Bownes,
2004), it is required for oocyte quality maintenance in
C. elegans (Andux and Ellis, 2008). Perhaps the correct
level of apoptosis is required for optimal reproduction
in each organism.
Another possible explanation for oocyte quality
decline is a decrease in the quality of the germ cell
nuclei prior to apoptosis (Fig. 2a). Garigan and
colleagues reported that the distal germ lines of aged
C. elegans are degraded (Garigan et al., 2002). The
syncytial nuclei are often disrupted by cavities and
grainy material in older animals, and frequently the
nuclei become prematurely cellularized, resulting in
shriveled gonads (Garigan et al., 2002). Luo et al.
(2010) confirmed these aging signs in the distal germ
line of older animals, and also found a positive correla-
tion between these signs of distal germ line aging and
the markers of oocyte morphology degradation in the
same population of worms. Since the distal germ line is
upstream of oocyte maturation, distal germ line integ-
rity may influence oocyte quality. However, more direct
evidence is needed to test whether there is a causal
role, or whether the two tissues degrade simultaneously
but mechanistically independently. The distal germ cell
nuclei must be maintained at high quality so that these
precursors can eventually develop into good oocytes. If
they are degraded, since there is still an excess of pre-
cursor nuclei, the apoptosis machinery may then try to
select the better nuclei through the elimination mecha-
nism. Alternatively, the nurse cell model suggests
that the reduced resources would be allocated to fewer
surviving germ cell nuclei to ensure that they are
still loaded with sufficient components (Fig. 2a).
Defects in either of the two mechanisms would likely
be detrimental to oocyte quality.
Interestingly, in the recently discovered starvation-
induced ‘‘adult reproductive diapause’’ (ARD) state, the
germ line shrinks significantly, with only a small popula-
tion of germ line stem cell nuclei remaining (Angelo
and Van Gilst, 2009). Once the ARD state is released by
feeding, the surviving germ line stem cell nuclei regen-
erate a new germ line and fully recover its function.
This suggests that, at least in starvation-induced ARD,
maintenance of germ line stem cell nuclei proliferation
is essential to the maintenance of reproductive capacity.
This may be also true in germ line aging. Garigan et al.
(2002) observed that older gonads frequently contain
relatively few nuclei, and there are fewer mitotic region
cells in older animals (Killian and Hubbard, 2005). Simi-
larly, Luo et al. (2010) found that the number of mitotic
germ cell nuclei in this region decreases significantly
with age. Thus, a decrease in mitotic germ cell nuclei
proliferation may contribute to the aging of the germ
line (Fig. 2a). However, we should note that Crittenden
and colleagues reported that although the mitotic
region shortens with age, the number of mitotic germ
REPRODUCTIVE AGING REGULATION IN C. elegans
cell nuclei remains constant from day 1 to day 6 of
adulthood (Crittenden et al., 2006). Also, there is a vast
excess of germ cell nuclei (>5,000) compared with the
maximum number of oocytes made (Crittenden et al.,
2006). Thus, further analysis is required to more
precisely dissect the relationship between germ line
stem cell nuclei proliferation and germ line and oocyte
IN AGED OOCYTES
In addition to the gross morphological and functional
changes in aging oocytes, transcriptional comparisons
between young and old oocytes reveal striking changes
and indicate molecular processes that may be particu-
larly susceptible to maternal age (Luo et al., 2010). Old
oocytes exhibit declines in the expression of many
genes that are likely to maintain oocyte function, includ-
ing chromosome segregation, cell cycle, DNA damage
response and repair, and proteolytic pathway genes
(Luo et al., 2010). Functional analyses of these genes
reveal that some gene classes are likely generally
required for oocyte development (e.g., chromosome
segregation fidelity and cell cycle regulation genes),
while others may become increasingly important with
age (e.g., DNA damage response/repair genes) (Luo
et al., 2010). For example, proteasome activity is
required to eliminate oxidatively-damaged proteins at
the time of oocyte maturation (Goudeau and Aguilaniu,
2010), suggesting that proteolysis genes are important
in aged worms that have accumulated carbonylated pro-
teins in the germ line. Strikingly, many of the genes that
decline with age in C. elegans oocytes also decline in
aging mammalian oocytes, including the SMC conden-
sins, cyclins, DNA mismatch repair components, and
proteolytic pathway members, suggesting that molecu-
lar processes crucial for oocyte quality maintenance are
conserved between worms and mammals (Hamatani
et al., 2004; Luo et al., 2010; Steuerwald et al., 2007).
In addition, the C. elegans oocyte expression analysis
identified several unknown genes that are downregu-
lated in older oocytes and are also required for success-
ful reproduction, revealing possible functions for those
uncharacterized genes (Luo et al., 2010).
SIGNALING PATHWAYS THAT REGULATE
C. ELEGANS REPRODUCTIVE AGING
In C. elegans, the insulin/IGF-1 signaling (IIS) path-
way is best known for its role in longevity and dauer
state regulation (Kenyon et al., 1993; Kimura et al.,
1997; Lin et al., 1997; Ogg et al., 1997; Riddle et al.,
1981). There are about 40 insulin-like molecules in the
C. elegans genome (Gregoire et al., 1998; Hua et al.,
2003; Kawano et al., 2006; Li et al., 2003; Murphy
et al., 2007; Pierce et al., 2001), but only one insulin-
like receptor, DAF-2. Mutations in daf-2 can more than
double the worm’s life span (Kenyon et al., 1993)
(Fig. 3a). Insulin-like agonist binding activates DAF-2,
triggering a kinase cascade that ultimately phosphoryl-
ates the FoxO transcription factor DAF-16, excluding it
from the nucleus and preventing its transcriptional
activity (Hertweck et al., 2004; Kimura et al., 1997; Lin
et al., 1997; Morris et al., 1996; Ogg et al., 1997; Paradis
et al., 1999; Paradis and Ruvkun, 1998). In loss-of-func-
mutant daf-2(e1370) and the dietary restriction mutant eat-2(ad465) both significantly extend life span compared to wild type, while the
TGF-b Sma/Mab signaling mutant sma-2(e502) does not (Luo et al., 2009). (b) daf-2(e1370), eat-2(ad465), and sma-2(e502) mutants all
significantly extend mated reproductive span (Luo et al., 2009).
Life spans and reproductive spans of wild-type, IIS, dietary restriction, and TGF-b mutant worms. (a) The insulin/IGF-1 signaling
LUO AND MURPHY
tion daf-2 mutants, however, DAF-16 is constitutively
translocated into the nucleus and its transcriptional pro-
gram is activated. In addition to its effect on life span,
mutations in daf-2 significantly extend mated reproduc-
tive span (Hughes et al., 2007; Luo et al., 2009) (Fig. 3b).
daf-2 mutations extend reproductive span by improving
oocyte and germ line maintenance (Luo et al., 2010) (Ta-
ble 1). Old daf-2 mutants produce fewer unfertilized
oocytes, unhatched embryos, and males than wild-type
animals of the same age. In addition, the oocytes and dis-
tal germ lines of daf-2 mutants degrade more slowly than
those of wild type. Although daf-2 mutants have fewer
proliferating germ cell nuclei, both in larvae and adults
(Luo et al., 2010; Michaelson et al., 2010), there is a
smaller decrease in mitotic cell number with age in daf-2
mutants compared with wild type (Luo et al., 2010).
The reproductive span extension of daf-2 mutants is
genetically dependent upon daf-16 (Hughes et al.,
2007; Luo et al., 2009). Intestinal DAF-16 activity plays
a major role, and neuronal DAF-16 makes a small contri-
bution in daf-2’s regulation of longevity, while muscular
DAF-16 is not required (Libina et al., 2003). By contrast,
both intestinal and muscle DAF-16 are required for
daf-2’s reproductive span extension, while neuronal
DAF-16 has no effect (Luo, et al. 2010). IIS regulates
both life span and reproductive span in adulthood, but
IIS activity late in development also affects progeny pro-
duction and reproductive span (Dillin et al., 2002; Luo
et al., 2010). Thus, while IIS regulates both reproduc-
tive span and life span, the sites and timing of IIS activ-
ity are slightly different for the two processes.
Another manipulation known to extend both life
span and reproductive span is dietary restriction (DR).
In fact, life span extension and slowing of reproductive
activity are the hallmarks of dietary restriction. DR
reduces progeny number and extends and delays the
reproductive period of C. elegans hermaphrodites
(Huang et al., 2004; Hughes et al., 2007), female Dro-
sophila (Chapman and Partridge, 1996), and female
rodents (Holehan and Merry, 1985; McShane and Wise,
1996; Selesniemi et al., 2008). eat-2, which encodes a
mutation in the acetylcholine receptor, is a genetic
model of dietary restriction in C. elegans due to its
reduced ability to ingest food (Lakowski and Hekimi,
1998) (Fig. 3b). Like daf-2, the role of eat-2 in reproduc-
tive span regulation was originally identified through its
longevity phenotype (Huang et al., 2004; Hughes et al.,
2007; Luo et al., 2009) (Fig. 3a,b), and both its life span
and reproductive span are genetically dependent on the
FoxA transcription factor PHA-4 (Luo et al., 2009; Pan-
owski et al., 2007).
In the extreme case of starvation, adult worms can
also undergo a long period of reproductive diapause
until food becomes available again (Angelo and Van
Gilst, 2009). Furthermore, food availability also affects
C. elegans’s reproductive strategy, since worms modify
their production of males and outcrossing frequency
after starvation to increase genetic variation (Morran
et al., 2009). Therefore, it is possible that modifying the
reproductive schedule and preserving reproductive
capacity, awaiting an improved environment, is an
evolved strategy that worms and other species use to
adapt to suboptimal conditions.
TGF-b Sma/Mab Signaling
The Sma/Mab pathway is one of the two canonical
TGF-b signaling pathways in C. elegans. Mutants of the
TGF-b Sma/Mab pathway are small and have defective
male tails (Savage et al., 1996). DBL-1 is the TGF-b-
related ligand (Morita et al., 1999; Suzuki et al., 1999),
and SMA-6 and DAF-4 are type I and type II receptors.
The signal transducers include the R-Smads SMA-2 and
SMA-3 and the Co-Smad SMA-4, and SMA-9 is a transcrip-
tion cofactor (Liang et al., 2003; Savage et al., 1996;
Savage-Dunn, 2005; Savage-Dunn et al., 2003). Upon
binding of the DBL-1 ligand, the type I and type II recep-
tors assemble and phosphorylate the Smad signal trans-
ducers, which then enter the nucleus, bind to DNA, and
recruit transcription cofactors and factors to activate
and suppress target gene expression (Massague ´, 2000).
The ligand DBL-1 is mainly expressed in neurons, while
the receptor and Smad proteins are expressed in multi-
ple tissues, including intestine, pharynx, hypodermis,
muscle, and the somatic reproductive system.
Summary of Age-Affected Cellular Processes in daf-2 and sma-2 Mutants
EmbryoOocyteProximal germ line, apoptotic zoneDistal germ line
aExample genes upregulated in sma-2 mutant oocytes affecting the processes (Luo et al., 2010).
bCellular processes that are maintained with age in daf-2 and sma-2 mutants. More 1 indicates better maintenance.
cCellular processes that are not maintained with age in daf-2 and sma-2 mutants.
REPRODUCTIVE AGING REGULATION IN C. elegans
In addition to its other roles, the TGF-b Sma/Mab
pathway also regulates reproductive aging: mutants of
the pathway extend reproduction, in some cases dou-
bling the reproductive span (Luo et al., 2009) (Fig. 3b).
However, the pathway has an insignificant effect on life
span (Fig. 3a); thus, it is the first reported pathway that
regulates reproductive span independently of somatic
longevity (Luo et al., 2009). TGF-b Sma/Mab mutants
extend reproductive span independently of the two
major somatic aging regulators, daf-16 (IIS pathway
transcription factor) and pha-4 (dietary restriction path-
way transcription factor) (Luo et al., 2009). Similar to
reduced IIS, reduced TGF-b Sma/Mab signaling delays
reproductive aging by better maintaining fertilizability,
chromosome segregation fidelity, stress resistance,
oocyte morphology, distal germ line morphology,
and distal germ line proliferation (Luo et al., 2010)
(Table 1). Mosaic analysis and tissue-specificity studies
reveal that signals from the soma regulate aging of the
reproductive system: TGF-b Sma/Mab signaling acts in
the hypodermis to promote aging of the germ line and
oocytes, while IIS acts in the intestine and muscle (Luo
et al., 2010). The fact that TGF-b Sma/Mab signaling
acts in hypodermis to regulate reproductive aging
suggests that a signal is sensed in the soma and is trans-
mitted to the germ line; this signal has not yet been
identified (Luo et al., 2010).
TGF-b Sma/Mab signaling in the hypodermis is neces-
sary and sufficient for body size regulation (Wang et al.,
2002; Yoshida et al., 2001). Interestingly, hypodermal
SMA-3 activity is also necessary and sufficient to restore
the long reproductive span of sma-3 mutants to normal
(short) reproductive span (Luo et al., 2010). Therefore,
the pathway regulates two different biological pro-
cesses, growth and reproduction, through its activity in
the same tissue. However, the fact that reproductive
span regulation is independent of body size is sup-
ported by several lines of evidence. First, none of the
non-TGF-b small mutants that have been tested extend
reproductive span (Luo et al., 2009). Secondly, while
the activity of the SMA-9 transcription cofactor is
required during early larval development to regulate
body size growth (Liang et al., 2003), it acts in adult-
hood to regulate reproductive span (Luo et al., 2010).
Last but not least, the transcriptional targets regulated
by TGF-b Sma/Mab signaling in oocytes are distinct
from those involved in body size regulation (Liang
et al., 2007; Luo et al., 2010).
COORDINATION OF SOMATIC AGING AND
While daf-2 and eat-2 mutants have both long life spans
and long reproductive spans, TGF-b Sma/Mab mutants
slow the rate of reproductive aging without concomi-
tantly slowing somatic aging (Luo et al., 2009). Reduced
TGF-b Sma/Mab signaling has very little effect on lon-
gevity, and matricide (mortality induced by progeny
hatching within the mother) increases at the same rate
as in wild-type animals. While matricide stops in wild
type around day 7–8, in concert with reproductive
cessation, it continues in TGF-b Sma/Mab mutants due
to their extended reproduction. Matricide is caused by
defects in egg-laying, likely due to declining muscle
integrity. Thus increasing matricide with age suggests
that the rate of somatic decline is similar in wild type
and TGF-b Sma/Mab mutants. The daf-2 and eat-2 lon-
gevity mutants experience less age-related matricide
than TGF-b Sma/Mab mutants at the same age, likely
due to theirimproved somatic (muscle) health.
Thus, aging of the reproductive system is normally
coupled with aging of the soma in wild-type animals as
well as daf-2 and eat-2 mutants. TGF-b Sma/Mab signal-
ing may normally mediate soma-to-germ line communi-
cation to adjust the reproductive rate, but such cou-
pling is broken in the TGF-b Sma/Mab mutants (Luo
et al., 2009).
The increased rate of matricide with age indicates
that reproduction itself requires the soma to be in peak
physical condition, otherwise reproduction may be det-
rimental to the mother and to unproduced progeny, as
in the case of old TGF-b Sma/Mab mutants. The soma
can merely survive postreproductively well below the
peak level of function. In fact, in both worms and
humans, many biological functions, including motility,
pathogen resistance, learning, and memory, etc., all
peak during the reproductive period, but begin to
decline soon afterwards, offering one model to explain
the long post-reproductive life span of both worms and
GERM LINE REGULATION OF SOMATIC AGING
In addition to the somatic regulation of reproductive
aging, signals from the germ line regulate somatic aging.
Removal of the germ line extends longevity, an effect
that depends on DAF-16 nuclear localization in the
intestine and that requires the presence of the somatic
gonad and an ankyrin-repeat protein, KRI-1 (Berman
and Kenyon, 2006; Hsin and Kenyon, 1999; Libina
et al., 2003; Lin et al., 2001). The daf-12 dafachronic
acid pathway is required both for DAF-16 nuclear local-
ization and the somatic gonad effect on longevity
(Berman and Kenyon, 2006; Gerisch et al., 2007; Yama-
waki et al., 2010). Recently, a histone H3K4 methyl-
transferase/demethylase complex was found to act in
the germ line to regulate life span (Greer et al., 2010).
Thus, a bidirectional flow of information between
somatic and reproductive tissues may coordinate
their rates of aging, possibly to optimize reproductive
success and adaptation to adverse conditions.
LUO AND MURPHY
CONSERVATION WITH HUMAN OOCYTE
AGING AND IMPLICATIONS FOR
Despite their vastly different life histories, chronological
time frames, and reproductive strategies, the cellular
and molecular regulation of C. elegans and human
reproductive aging are strikingly similar. Although
oocytes are continually produced in worms, while
humans’ total oocyte supply is produced at birth, both
human and C. elegans females have long post-reproduc-
tive life spans, and undergo significant reproductive
aging on proportional time scales (Cant and Johnstone,
2008; Luo et al., 2009). Both human and C. elegans
oocytes are cell-cycle arrested at meiotic prophase I,
and their release from arrest by hormone is coordinated
with oocyte maturation (Greenstein, 2005; Mehlmann,
2005). The mechanisms underlying oocyte maturation
are highly conserved between the two organisms. Most
importantly, in both humans and worms, oocyte quality,
rather than quantity, is the major cause of reproductive
cessation. In particular, fertilizability, chromosome
segregation fidelity, stress resistance, and morphology
are compromised with age in both organisms (Blondin
et al., 1997; ESHRE Capri Workshop Group, 2005;
Goud et al., 1999; Hiroshi Tamura et al., 2008; Jones,
2008; Luo et al., 2010; Magli et al., 2007; Rose and
Baillie, 1979; Rubio et al., 2003; Tang et al., 2010; Tarin,
1996; te Velde and Pearson, 2002). C. elegans reproduc-
tive aging is not only limited by oocyte quality, as it is in
humans, but similar underlying molecular factors also
contribute to oocyte quality maintenance in the two
IIS and dietary restriction regulate longevity from
worms to mammals (Kenyon et al., 1993; Suh et al.,
2008; Tu et al., 2002; Willcox et al., 2006, 2008). These
pathways have been implicated in the regulation of
mammalian reproductive aging (Castrillon et al., 2003;
Holehan and Merry, 1985; Klein et al., 2000; McShane
and Wise, 1996; Selesniemi et al., 2008) and C. elegans
reproductive aging (Huang et al., 2004; Hughes et al.,
2007; Luo et al., 2009, 2010). A recent genome-wide
association study also identified ARHGEF7, a gene that
interacts with FOXO3a, the human homolog of DAF-16/
FOXO, as a candidate gene associated with age at meno-
pause (Ong et al., 2009). Additionally, Foxo3a knockout
mice exhibit a defect in follicular activation (Castrillon
et al., 2003), again linking IIS and FoxO to fertility and
TGF-b superfamily ligands influence mammalian
reproduction through the regulation of follicle develop-
ment (Knight and Glister, 2006; Trombly et al., 2009).
Additionally, TGF-b signaling has been implicated in
reproductive aging, as TGF-b members are upregulated
in aged mouse oocytes (Hamatani et al., 2004). Our C.
elegans transcriptional analyses show that many genes
upregulated in TGF-b Sma/Mab mutant oocytes are simi-
lar to human and mouse oocyte genes that decline with
age, including the condensin genes required for proper
chromosome segregation, cell cycle genes, DNA mis-
match repair genes, proteolytic pathway genes, and
many others (Hamatani et al., 2004; Luo et al., 2010;
Steuerwald et al., 2007). lin-28, which is upregulated in
TGF-b Sma/Mab mutant oocytes, is also associated with
(He et al., 2009; Ong et al., 2009; Perry et al., 2009;
Sulem et al., 2009). Transcriptional studies suggest that
many of the molecular mechanisms underlying oocyte
quality maintenance are likely shared between C. ele-
gans and humans through TGF-b signaling. Therefore,
while mammals have a more complex and diverse TGF-
b family that carries out many different functions, it is
likely that some branch of TGF-b signaling may be
involved in the regulation of reproductive cessation. If
so, modulation of TGF-b signaling may offer new ave-
nues to delay human reproductive aging, and our stud-
ies in C. elegans may provide insights into potential
therapies for maternal age-associated infertility and
CONCLUSIONS AND FUTURE DIRECTIONS
While human postreproductive life span has been
greatly extended through improvements in medicine,
nutrition, hygiene, and environment (Centers for Dis-
ease Control and Prevention, 1999; Finch and Crim-
mins, 2004), these factors have not influenced female
reproductive span. Here, we have described recent
studies on reproductive aging mechanisms in C. elegans
and our current understanding of its regulation. These
studies were the first efforts to establish C. elegans as a
model to study reproductive capacity decline, and have
laid the foundation for future research in the field.
While several reproductive aging signaling pathways
have been identified, many questions still remain. One
immediate challenge is to identify soma-to-germ line sig-
nals that mediate the regulation of germ line and oocyte
quality by TGF-b Sma/Mab and IIS. Direct experimental
evidence is also required to address the relationship
between germ cell nuclei proliferation, germ line integ-
rity, and oocyte quality. In addition, the mechanism of
reproductive span extension by dietary restriction must
also be dissected. Finally, forward genetic screens will
facilitate the discovery of additional novel regulators of
reproductive span. Such studies will help us better
understand how aging of the reproductive system is
regulated in C. elegans, shed light on the relationship
between reproductive and somatic aging, and further
elucidate the implications for human reproductive
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