TGF-ß Sma/Mab Signaling Mutations Uncouple Reproductive Aging from Somatic Aging

Female reproductive cessation is one of the earliest age-related declines humans experience, occurring in mid-adulthood. Similarly, Caenorhabditis elegans' reproductive span is short relative to its total life span, with reproduction ceasing about a third into its 15–20 day adulthood. All of the known mutations and treatments that extend C. elegans' reproductive period also regulate longevity, suggesting that reproductive span is normally linked to life span. C. elegans has two canonical TGF-ß signaling pathways. We recently found that the TGF-ß Dauer pathway regulates longevity through the Insulin/IGF-1 Signaling (IIS) pathway; here we show that this pathway has a moderate effect on reproductive span. By contrast, TGF-ß Sma/Mab signaling mutants exhibit a substantially extended reproductive period, more than doubling reproductive span in some cases. Sma/Mab mutations extend reproductive span disproportionately to life span and act independently of known regulators of somatic aging, such as Insulin/IGF-1 Signaling and Dietary Restriction. This is the first discovery of a pathway that regulates reproductive span independently of longevity and the first identification of the TGF-ß Sma/Mab pathway as a regulator of reproductive aging. Our results suggest that longevity and reproductive span regulation can be uncoupled, although they appear to normally be linked through regulatory pathways.


Introduction
Among human age-related declines, female reproductive cessation is one of the earliest to occur, with infertility and maternal age-related birth defects arising during the fourth decade of life [1].While artificial reproductive technologies have improved late conception success [2][3][4][5], the underlying molecular regulators of reproductive cessation remain largely unknown.Like longevity, the ability to produce progeny with advanced age is likely to be genetically regulated.Thus, understanding the processes that regulate reproductive aging may allow us to address the problems of maternal age-related infertility and birth defects.
Although C. elegans produces large broods of progeny and does not care for its young, its reproductive schedule is similar to human females' in that its fertility and reproduction sharply decline in early/mid-adulthood, followed by a long postreproductive period [6,7].The similarities between C. elegans and human reproductive schedules suggest the intriguing possibility that studies in this model organism may reveal mechanisms regulating reproductive cessation across species.While many C. elegans studies have focused on reproductive fitness, measuring total numbers of offspring produced and average generation time, we are instead interested in identifying regulators of late-life reproduction (i.e., the ability of individual mothers to continue to reproduce viable progeny as they age).The standard assessments of fitness and fertility do not ascertain the length of time that an individual is capable of successful reproduction.However, we and others [6][7][8] have recently become interested in the latter aspect of reproduction, because of the obvious possible parallels with human reproductive aging, in particular, Advanced Maternal Age (AMA) and its related clinical problems.In other words, these model organism studies of reproductive aging are focused on determining the capacity to reproduce successfully late in life, rather than on total progeny production or evolutionary fitness.Hughes, et al. recently showed that worms undergo reproductive aging, a process that is dependent neither on tissue wear (as manipulation of early progeny number had no influence) nor on sperm availability [7].Thus, the reproductive system of C. elegans ages significantly during the first week of adulthood, which is also reflected in the degree of germ line degeneration and oocyte quality decline [8,9].This germ line aging results in reproductive cessation days to weeks prior to death and a relatively long post-reproductive life span, similar to human females' long post-reproductive life span.
The C. elegans mutants currently known to delay reproductive aging were originally identified through their longevity phenotypes [7].These longevity mutants include the insulin/IGF-1 receptor mutant daf-2 [7,10,11], and a model of Dietary Restriction (DR), eat-2 [6,12].Insulin/IGF-1 signaling (IIS) and FOXO transcription factor activity have been implicated in the regulation of reproduction in several other organisms, including Drosophila [13], mice [14,15], and humans [16].Life span extension and slowing of reproductive activity are also hallmarks of Dietary Restriction.Dietary Restriction reduces progeny number and lengthens the reproductive period of C. elegans hermaphrodites [7], female Drosophila [16], and female rodents [18,19].C. elegans shifts its reproductive strategy after starvation, modifying its production of males and its outcrossing frequency [17], and many animals adjust their reproductive life span in response to predation levels [18].Together, these data indicate that the reproductive schedule is flexible, poised to respond to environmental and molecular perturbations, and that the mechanisms regulating reproductive aging, like longevity, are likely to be evolutionarily conserved.
Here we show that the Dauer pathway has a moderate effect on reproductive span, mediated at least in part by insulin/FOXO activity.More importantly, we have found that its shared member with the TGF-ß Sma/Mab pathway, daf-4, and the entire TGF-ß Sma/Mab pathway, strongly influence reproductive aging.Reduced TGF-ß Sma/Mab signaling extends reproductive span disproportionately to life span, and is genetically independent of known longevity regulators.The TGF-ß Sma/Mab pathway is a novel regulator of reproductive aging, and the first regulator of reproductive aging to be identified independently of somatic aging regulation.Our results demonstrate that somatic aging and reproductive aging can be uncoupled, suggesting that different molecular mechanisms underlie the two processes, but may normally be linked.

DAF-4, a shared TGF-ß co-receptor, regulates reproductive aging
In addition to its regulation of dauer formation [26], we recently found that the TGF-ß Dauer pathway (Figure 1A, Figure S1A) regulates longevity [19].However, whether this pathway also plays a role in the regulation of reproductive aging is unknown.To analyze the effect of TGF-ß Dauer mutants on reproduction, we determined the proportion of adults capable of progeny production as a function of age.The ''reproductive span'' calculated from such assays (see Materials and Methods) reflects the period of time animals produce viable progeny, as described previously [6,7].We found that members of the TGF-ß Dauer pathway moderately extended reproduction (Figure 1B; Figure S1B, S1C): while wild type's mean reproductive span was ,3.5 days, the means of daf-7, daf-1, daf-8, and daf-14 mutants were 4-5 days, extensions of 25-50% (Table S2; Figure S1D).In addition, their maximum reproductive spans were ,1 day longer than wild type's.At least part of the moderate reproductive span extension is likely a result of delayed onset of reproduction due to an egg-laying (Egl) defect (Figure 1D) [19,27,28]; by the end of daf-7's reproductive span, many progeny hatched into L1 larvae immediately upon being laid, as opposed to the typical 12-16 hour hatching time of wildtype eggs.daf-7 progeny do not develop into adults faster than wild type, so the advanced developmental stage of the progeny is likely due to egg retention in the mother.
Unlike the moderate reproductive span extensions of other TGF-ß Dauer pathway mutants, daf-4's ,8 day mean is more than double the reproductive span of wild-type animals (Figure 1C; Figure S1D; Table S2).daf-4 mutants continued to steadily produce progeny for several days after reproductive cessation in wild-type animals, and its maximum reproductive span was 4-5 days longer than wild type (Figure S1G).This dramatic difference cannot be explained by the egg-laying defect typical of the TGF-ß Dauer pathway mutants, which extends reproductive span a maximum of one day.daf-4 encodes C. elegans' sole ortholog of the type II TGF-ß co-receptor, and is utilized by both the Dauer pathway and a second TGF-ß pathway, the Sma/Mab pathway (Figure 1E, Figure S2A) [22][23][24][25].The large reproductive span extension that we observed in daf-4 animals, but not other TGF-ß Dauer mutants, suggested the possibility that the Sma/Mab pathway might be important in the regulation of reproductive aging.

TGF-ß Sma/Mab signaling regulates reproductive aging
We measured the reproductive spans of seven alleles of TGF-ß Sma/Mab pathway mutants (Figure 1E, Figure S2A), and found that decreased TGF-ß Sma/Mab signaling indeed increased reproductive span significantly: similar to daf-4, the mean reproductive spans of sma-2 and dbl-1 were over 7 days, compared to ,3.5 days in wild type; the rest of the mutants in this pathway (sma-3, sma-4, sma-6, and sma-9) also increased reproductive span substantially (Figure 1F and 1G; Table S1; Figure S2B, S2C).The hatching rates of Sma/Mab mutants were comparable to wild type (Figure 1D), the onset of progeny production was not delayed, and progeny were steadily produced beyond the age when wild-type reproduction ceased (Figure S3A, S3B, S3C).Similar to daf-2 and eat-2, mutants that also extend reproductive span [7], Sma/Mab mutants produce fewer total progeny over a longer period of time (Table S3; Figure S3).The reproductive span extensions and progeny production profiles of the Sma/Mab mutants contrast with the delayed onset and sharp decline in the number of progeny produced after peak reproduction by the TGF-ß Dauer mutants

Author Summary
Female reproductive cessation is the earliest aging phenotype humans experience, and its importance as a clinical issue is growing as more women opt to have children later in life.While much work has been done to understand the general aging process, little is currently known about the regulation of reproductive aging.Like longevity, the ability to produce progeny with advanced age is likely to be genetically regulated.Thus, understanding the processes that regulate reproductive aging may allow us to address the problems of maternal age-related infertility and birth defects.C. elegans and humans both have long post-reproductive life spans, leaving open the possibility that their reproductive spans might be extendable.C. elegans has been used previously to discover conserved regulators of aging, and here we use worms to identify a new regulator of reproductive aging, a highly conserved TGF-ß signaling pathway.We find that TGF-ß signaling regulates reproductive aging independently of somatic aging.This is the first identification of a pathway that breaks the coupling that normally links the two processes.Our work will provide new insights into the improvement of human fertility and prevention of agerelated birth defects, and it has implications for the evolutionary relationship between reproduction and longevity regulation.(Figure S1E, S1F), suggesting that the Dauer and Sma/Mab mutants are distinct in their reproductive aging phenotypes.
Sma/Mab mutants exhibited a highly penetrant late egg-laying defect and internal hatching (matricide, or ''bagging'') at the end of their reproductive period, in contrast to the Dauer mutants' very early egg-laying (Egl) and bagging phenotypes (Figure 1H, Figure S2E).In fact, several assays were terminated when a large fraction of the worms were still reproductive, due to the Sma/Mab mutants' late matricide phenotype (see asterisked sma-4 in Figure 1F and sma-3 in Figure 1G).It is likely that the full late reproductive capacity of the Sma/Mab mutants is masked by this late matricide defect.Thus, while the TGF-ß Dauer mutants' delayed onset of reproduction and Egl phenotypes may account for part of their moderate reproductive span increases (a maximum of one day), these factors are not likely the cause of TGF-ß Sma/Mab mutants' dramatic reproductive span extensions.

Sma/Mab reproductive span extension is independent of sperm contribution
C. elegans hermaphrodite sperm number limits wild-type selffertilized reproduction, but mating with young (day 1 adult) males, whose sperm are not limited and outcompete those of the hermaphrodite, increases and prolongs progeny production [29][30][31].To rule out the possibility that the reproductive span extensions we observed in TGF-ß Sma/Mab mutants are due to increased or extended sperm production or utilization, we mated Sma/Mab mutant hermaphrodites with young wild-type males.We found that Sma/ Mab mutants significantly and consistently increased mated reproductive span, from wild type's mean mated reproductive span of ,6.0 days to a mean of 10-11 days (Figure 2A-2D; Table S1).In fact, Sma/Mab mutants were usually still fertile through Day 12-13 of adulthood, compared to the complete cessation of reproduction by Day 8-9 in wild-type animals.(Interestingly, when mated with wild-type males at an older age (day 4), the Sma/Mab mutants still had significant reproductive span extensions (Figure S2D), further supporting the notion that sperm quality and number are not the limiting factor, as shown previously [7].) To further eliminate the possibility of sperm contribution, we also tested feminized (fem-1) mutant hermaphrodites, which fail to make sperm when raised at restrictive temperatures.fem-1 mutants mated with wild-type males have a mean reproductive span of 6.3 days, while more than 90% of the mated sma-2;fem-1 double mutants were still fertile at day 12 (Figure 2E).Notably, the self-fertilized reproductive spans of Sma/Mab mutants are even longer than wild type's mated reproductive span, highlighting the extreme extensions shown by Sma/Mab mutants (compare Figure 1F and Figure 2; Table S1).Additionally, neither the self-fertilized nor the mated Sma/Mab mutants delay the onset of progeny production, and both continue to produce progeny steadily beyond the age of wild-type reproductive cessation (Figure S3).
In self-fertilized worms, sperm is only made prior to oogenesis [24], and in mated worms sperm is in excess, thus the extended reproductive span we observed cannot be due to extended spermatogenesis.Our results, together with the Hughes, et al. data, suggest that significant reproductive aging already occurs prior to the cessation of sperm availability in self-fertilized animals, and that Sma/Mab mutants, like IIS (daf-2) and DR (eat-2) mutants [7], slow the rate of aging of the reproductive system.

Reproductive span extension is independent of body size, ovulation rate, early progeny number, and brood size
We noticed that the Sma/Mab mutants produced fewer progeny than wild type each day in the early phase of reproduction (Figure S3; Figure S4D, S4E, S4F), and have smaller broods (Table S3, Table S4).This reduction in progeny number reflects slower ovulation rate of the mutants in early reproduction (Figure S4C), likely due to their small body size (Figure S4A, S4B).In fact, it has been suggested that reduction of C. elegans progeny number is linked to small body size via physical constraint of the maternal gonad and/or body size [32][33][34][35].The downstream transcription co-factor of the Sma/Mab pathway, sma-9, is required in early larval development for the regulation of body size before gametogenesis [25,36]; however, we find that reduction of sma-9 only in adulthood is sufficient to extend late reproduction (Figure S5), suggesting that the growth and reproductive aging functions of the Sma/Mab pathway are independent.
In mated assays, sperm number is not limiting; therefore, one possible explanation for extended reproductive span of the Sma/ Mab mutants is that oocyte number is limiting, and thus slower ovulation allows the mutants to use up their oocyte supply more slowly.To test this hypothesis as well as the body size effect on reproduction, we examined five small but non-TGF-ß mutants (dpy-6(e2762), sma-1(ru18), sma-1(e30), dpy-1(e1) and dpy-9(e12)) whose body sizes are similar to the TGF-ß Sma/Mab mutants (compare Figure S4A and S4B and Figure S6A and S6B).Importantly, none of these strains have been reported to have egglaying or embryonic developmental abnormalities or effects on longevity, and therefore serve as a fair set of samples for comparison.As expected, the five small mutants have slower ovulation rates (Figure 3A and 3B), and as a result produce fewer early progeny and fewer total progeny (Figure 3C and 3D).However, unlike the TGF-ß mutants, none of the small, non-TGFß mutants extended mated reproductive span (Figure 3E and 3F).In fact, all of the mutants had shorter reproductive spans.These data suggest that small body size and reductions in ovulation rate and progeny number do not increase reproductive span, but rather are usually associated with shorter reproductive spans.Therefore the TGF-ß Sma/Mab mutants are special in their extension of reproductive span.
We also addressed whether oocyte number becomes the limiting factor when sperm is no longer limiting by mating animals with young wild-type males, which is one basis for the assumption that slow ovulation extends reproductive span.On day 3, all wild-type animals (n = 12) produced only eggs that were able to hatch and develop to viable progeny (Figure 3G), but on day 7, 59% (n = 17) of the animals laid oocytes that failed to be fertilized (Figure 3H) and/or eggs that were unable to hatch (Figure 3H and 3I), resulting in cessation of viable progeny production.Therefore, the limiting factor is not number of oocytes, which are clearly still in excess, but the quality of the oocytes.By contrast, the majority of sma-2 mutants still produced exclusively viable eggs on day 7 (Figure 3J), with only 19% (n = 16, p = 0.03 compared with wild type) of the animals starting to lay unfertilized oocytes or unhatchable eggs.Our data, together with the observation that late reproduction is independent of early reproduction [7], suggest that Sma/Mab mutants extend reproductive span independently of body size, ovulation rate, early progeny number, and brood size, but instead by improving oocyte quality.

Sma/Mab signaling regulates reproductive span disproportionately to life span
While daf-2 and eat-2 regulate reproductive aging [6,7,11], they are known foremost for their roles in life span extension [10,12].Thus far, all of the known mutations and treatments that extend C. elegans reproductive period also regulate longevity [6,7].In addition, the link between longevity and reproduction has been suggested and/or reported in multiple organisms [37][38][39][40][41][42][43].These data suggest the possibility that the regulation of life span and reproductive span are coupled, or even regulated by the same mechanisms.Our TGF-ß Dauer pathway data further support this notion, as the mutants increase both life span and reproductive span (Figure 4E) [19].However, we found that Sma/Mab pathway mutants only mildly affect longevity, despite their dramatic effects on reproductive span.Some Sma/Mab mutants increased life span moderately (dbl-1, sma-6) or inconsistently (sma-2, sma-4), while others appeared to have no effect on longevity (sma-3, sma-9) (Figure 4A-4D; Table S6, S7, S8, S9, S10).Because these alleles are not nulls, the inconsistencies in longevity between mutants in the pathway could be due to varying hypomorphic effects.Therefore we compared each mutant allele's effect on reproductive span and life span (Figure 4E).While daf-4 mutants doubled both reproductive span and life span, and daf-7 mutations had a moderate effect on each, exclusive members of the Sma/Mab pathway disproportionately extended reproductive span compared to life span (Figure 4E).This effect was maintained when the mated reproductive spans were considered (compare sma-2 and daf-7 in Figures 4E, Figure 5G and  5H).daf-4's effect on the two processes is likely due to its dual roles in TGF-ß Dauer regulation of life span [19] and in TGF-ß Sma/ Mab regulation of reproductive span.
Matricide is a common event in the late reproductive period, but we noticed that the TGF-ß mutants are different from longevity mutants in this regard.In mated wild-type animals, the matricide frequency increased with age within the reproductive period (Figure 5A), perhaps reflecting aging of the musculature required for egg-laying.After day 9, reproduction stopped completely, therefore no matricide was observed.The matricide frequency of sma-2 mutants also increased with age at a similar rate as wild type (Figure 5A).Because sma-2 mutants continued to reproduce, however, the matricide rate continued to rise further, and therefore more mutant animals suffered from matricide than wild type.The matricide frequency of daf-2 and eat-2 mutants, however, increased at a much slower rate (Figure 5B).For example, on day 8 about 70% of worms died from matricide in both wild type and sma-2 animals, whereas only 40% of daf-2 and 30% of eat-2 animals died of matricide (Figure 5A and 5B).(daf-7 mutants exhibited high matricide rate from very early age (Figure 5B), due to their Egl defects (Figure 1D), therefore are different from the other strains.)The matricide frequency data suggest that late-reproduction matricide may be a somatically-controlled event, separate from reproductive aging.Together with the life span data, the matricide data suggest that sma-2's soma ages at a rate that is similar to wild type, unlike the daf-2 and eat-2 longevity mutants.
To further investigate sma-2's role in somatic aging, we compared the effects of mutations in sma-2, daf-2, eat-2, and daf-7 on life span and reproductive span.The other mutants have longer life spans than sma-2 (Figure 5C and 5D; Table S7 and Table S8), but their reproductive spans are either shorter or comparable to sma-2 (Figure 5E and 5F; Table S7, S8).In fact, daf-2 animals increase life span to a greater degree than reproductive span (Figure 5G and 5H), while sma-2 and eat-2 have greater effects on reproductive span than life span.sma-2's effect on reproductive span is the most disproportionate among all the mutants.For example, when comparing the increases in mated reproductive spans and life spans of all the mutants (Figure 5H), sma-2's increase in reproductive span is 10-fold its increase in lifespan, whereas eat-2's effect on reproductive span is only 4-fold, and the daf-7's and daf-2's are both less than one fold.Together, our data show that TGF-ß Sma/Mab signaling affects reproductive aging disproportionately to its effect on longevity compared to other reproductive span and life span mutants.

Sma/Mab signaling does not regulate reproductive aging through IIS or DR somatic aging pathways
The FOXO transcription factor DAF-16 is required for longevity of the IIS pathway mutant daf-2 [10] and is also required for daf-2's effects on reproduction [7] (W.Shaw & C.T. Murphy, unpublished data).Previously, we showed that TGF-ß Dauer signaling regulates longevity through its interactions with the IIS pathway, as the lifespan extension of daf-7 mutants is suppressed by loss of DAF-16/ FOXO transcription factor activity [19] (Figure 6A).To test the role of daf-16 in TGF-ß Dauer pathway regulation of reproduction, we compared the reproductive spans of daf-7(e1372), daf-16(mu86), and daf-16(mu86);daf-7(e1372) double mutants.We found that daf-7's reproductive span extension was significantly suppressed by loss of daf-16 activity (Figure 6B and 6C; Table S9), suggesting that DAF-16/FOXO activity is required for both the life span and reproductive span extensions of daf-7 mutants.Interestingly, loss of daf-16 activity also suppressed sma-2's small life span extension (Figure 6D, Figure S7A; Table S9); sma-2's occasional moderate effect on longevity may be due to cross-talk between the TGF-ß and IIS pathways rather than a primary output of TGF-ß Sma/Mab signaling, reminiscent of the cross-talk between the two TGF-ß pathways in dauer regulation [44,45], and TGF-ß Dauer/IIS crosstalk in longevity regulation [19].By contrast with its effect on sma-2 life span, loss of daf-16 activity failed to suppress sma-2 and dbl-1 selffertilized and mated reproductive span extensions (Figure 6E and 6F; Figure S7C and S7D; Table S9).However, the double mutant's peak matricide frequency shifted earlier (Figure S7B), consistent with daf-16;sma-2 and daf-16's shorter life spans.These results suggest that the TGF-ß Sma/Mab pathway's regulation of reproductive span is not mediated by DAF-16/FOXO activity.Together with its disproportionate effect on the two processes, the TGF-ß Sma/Mab pathway appears to have genetically uncoupled regulation of reproduction and longevity.
The FoxA transcription factor PHA-4 is required for the life span extension of the Dietary Restriction model eat-2 (Figure 7A) [46].We found that eat-2's reproductive span extension was also significantly suppressed by loss of PHA-4 activity (Figure 7C and 7D; Table S10) when treated from L4 onward.(To check the efficacy of pha-4 RNAi, we determined the fraction of arrested L1 progeny from L4-onward-fed mothers, and found that sma-2 and eat-2 animals are similarly sensitive to pha-4 RNAi (Figure 7B).)To test whether TGF-ß Sma/Mab signaling utilizes the Dietary Restriction pathway, we tested sma-2's requirement for PHA-4 activity.In contrast to eat-2's requirement for pha-4, we found that pha-4 RNAi treatment did not suppress the reproductive span extension of sma-2 mutants (Figure 7E and 7F; Table S10).Thus, while pha-4 is required for the reproductive span changes associated with Dietary Restriction, it is not required for sma-2's reproductive span extension.
If the reproductive span extension of Dietary Restriction animals required TGF-ß Sma/Mab signaling, we might expect the DBL-1 ligand to act downstream of the eat-2-induced DR effect, which is caused by eat-2 mutants' inability to pump their pharynx to properly ingest food.However, we observed that overexpression of the DBL-1 ligand is not sufficient to suppress eat-2's reproductive span extension (Figure 7G; Table S11), consistent with the hypothesis that Dietary Restriction and TGF-ß Sma/ Mab regulate reproductive span independently.(Interestingly, the eat-2;dbl-1 OE animals, like dbl-1 OE single mutants, are larger than wild type (Figure 7H) yet have a long reproductive span, further supporting the notion that there is no direct correlation between body size and reproductive span.) Our results show that in addition to its role in body size regulation, the TGF-ß Sma/Mab pathway is a novel regulator of reproductive aging.Sma/Mab signaling regulates reproductive aging independently of at least two known regulators of somatic aging, Dietary Restriction and IIS/FOXO signaling, and uncouples reproductive aging from somatic aging.S7 and Table S8.doi:10.1371/journal.pgen.1000789.g005S10.(G) dbl-1 overexpression does not suppress eat-2(ad465)'s reproductive span extension.Additional statistics are in Table S11.(H) Body size of wild type, eat-2(ad465), dbl-1 OE, and eat-2(ad465);dbl-1 OE. eat-2;dbl-1 OE animals are larger than wild type.sma-2;pha-4(RNAi) animals have a severe egg-laying defect, possibly masking even longer reproductive spans (Figure S8).doi:10.1371/journal.pgen.1000789.g007

TGF-ß Sma/Mab signaling is a novel regulator of reproductive aging
Here we have shown that loss of function of a canonical TGF-ß pathway significantly delays C. elegans reproductive aging.Intriguingly, TGF-ß Sma/Mab mutants extend reproductive span without a proportional extension of life span.We have also found that TGFß Sma/Mab reproductive span extension is genetically independent of regulation by Dietary Restriction and Insulin/IGF-1 Signaling.The uncoupling of reproductive and somatic aging in TGF-ß Sma/ Mab signaling mutants suggests that the molecular mechanisms underlying the maintenance of somatic [47,48] and reproductive tissues are distinct.This is the first identification of a pathway that regulates reproductive aging independently of somatic aging, and may lead to insights into mechanisms that specifically govern agerelated reproductive cessation.

Somatic aging and reproductive aging may be coupled
Signaling from the reproductive system to the soma to regulate aging has already been shown through germ line and somatic gonad ablation experiments in worms and flies [49,50].Because germ line and somatic gonad ablation both result in sterility, but have opposite effects on life span, direct resource allocation from the germ line to the soma cannot be the cause of this longevity.Instead, signals from the germ line and somatic gonad normally communicate with the rest of the soma to regulate life span, acting through the insulin/ FOXO and daf-12 nuclear hormone pathways [49][50][51].Our results suggest that the reproductive system may also normally receive signals through these regulatory pathways and the TGF-ß Sma/ Mab pathway to regulate its rate of aging, allowing the animal to adjust its reproductive rate to its environment.
The prevalence of matricide by the TGF-ß Sma/Mab mutants illustrates the importance of signaling that normally links reproductive and somatic aging.Many animals slow their reproductive rates in response to environmental factors, such as high predation and food shortages, in order to optimize their reproductive fitness [7,16,18,52].In some cases, such as under environmental stress, late reproduction can increase fitness, allowing increased genetic diversity through mating (''facultative outcrossing'') [17].Like daf-2 and eat-2 mutants, reduced TGF-ß Sma/Mab signaling slows the rate of reproductive aging and thus extends reproductive span.However, TGF-ß Sma/Mab mutants do not concomitantly slow somatic aging, and as a result they often suffer from high reproductive-age mortality induced by the physical stresses of reproduction, whereas same-aged longevity mutants daf-2 and eat-2 experience less age-related matricide (see Figures 2D and 2E and Figure 5A and 5B).From these results we infer that the slowing of somatic aging is required for successful delayed reproduction, which may be necessary under certain environmental conditions.Signals through the TGF-ß Sma/Mab pathway may allow the animal to adjust its reproductive rate to its environment.In turn, signals from the germ line and somatic gonad coordinate somatic aging rate with reproductive aging rate to allow successful reproduction.

Worms and humans utilize similar mechanisms to regulate post-reproductive life span
If reproductive and somatic aging are coupled, why do worms and humans live so long after reproduction has ceased?A popular but controversial theory specific to humans postulates that investment in grand-progeny by grandmothers increases fitness to a greater degree than would continuing reproduction, and thus downregulation of reproductive ability in mid-life is evolutionarily beneficial (the ''Grandmother Hypothesis'' [53,54]).However, as C. elegans does not care for its young, such an investment in its grand-progeny cannot explain its similarly early decline in reproduction and long post-reproductive life span.
Instead, we propose that reproduction itself requires the soma to function at its highest level, but the body can survive well below this threshold level of function.That is, if one were to plot every parameter of function (reproduction, motility, pathogen resistance, survival, etc.) in both worms and humans, these functions would all peak during the reproductive period, but begin to decline postreproductively, each at a different rate.Successful reproduction requires peak physical condition of the soma; the increased matricide rates in older, reproductive Sma/Mab mutants shows that increased oocyte quality in the absence of healthy somatic tissues can be catastrophic, at least to the mother and any unproduced late progeny.By contrast, ''survival'' is the lowest measurable function (assayed as live vs. dead) and thus persists much longer than reproductive activity.Humans survive well past the age of peak physical function, a period that overlaps with female reproduction.Improvements in medicine, nutrition, hygiene, and environment have extended human life span significantly [55,56], extending the post-reproductive life span but having little effect on maximum reproductive span.Analogously, the worm's life span in the low-predation and lowpathogen conditions of the laboratory is likely longer than in the wild, but likely largely affects post-reproductive life span.Therefore, the long post-reproductive life span of both worms and human females could be attributed to the high level of somatic function required for successful reproduction, essentially a side effect of the requirements for successful reproduction earlier in life.

Regulation of reproductive aging may be evolutionarily conserved
Longevity is regulated by insulin/IGF-1/FOXO signaling and by Dietary Restriction in worms through mammals [10,12,[57][58][59], despite large differences in chronological life spans of these organisms.Additionally, Insulin/IGF-1 Signaling and Dietary Restriction have been implicated in regulation of mammalian reproductive aging [14,52,60,61].Intriguingly, TGF-ß levels are upregulated in aged mouse oocytes [62], and TGF-ß activity regulates mammalian follicle cell activity [63].Thus, it is possible that regulation of reproductive aging, like the regulation of somatic aging by IIS and DR pathways, is evolutionarily conserved, and that TGF-ß signaling may regulate human reproductive cessation.
Despite the vast differences in their life histories and chronological time frames, our work suggests that the regulation of worms and humans' longevity and reproductive spans may be conserved.Future studies will determine whether Sma/Mab mutants use a conserved mechanism to slow reproductive aging in C. elegans.If so, modulation of TGF-ß signaling may offer new avenues to improve fertility and offspring health in mothers of advanced age.

Reproductive span analysis
Individual synchronized L4 hermaphrodites were moved to fresh plates daily until reproduction ceased for at least two days.The last day of viable progeny production was noted as the day of reproduction cessation for each individual.When matricide occurred, the animal was censored from the experiment on that day.All experiments were performed at 20uC, except that sma-2(e502);fem-1(hc17) and fem-1(hc17) worms were shifted to 25uC from L3 and back to 20uC after L4.All experiments were performed with at least 10 individuals per strain (most experiments included .25 individuals, as indicated in Supplementary Tables).The log-rank (Mantel-Cox) method was used to test the null hypothesis.In mating reproductive span assays, L4 hermaphrodites were mated to young wild-type males at a 1:3 ratio for 24 hours before being separated onto individual plates.Successful mating was ascertained by the fraction of male progeny each day.For the pha-4 RNAi reproductive span experiments, mothers were moved onto RNAi bacteria starting at L4.

Progeny production analysis
Individual synchronized L4 hermaphrodites were moved to fresh plates and the number of progeny produced by each individual counted daily until reproduction ceased for at least two days.When matricide occurred, the animal was censored from the experiment on that day.All experiments were performed at 20uC with at least 6 individuals per strain (most experiments included 20-40 individuals).

Matricide (bagging) rate
The assay was performed as described in Reproductive Span analysis, except the cumulative percentage of hermaphrodites that underwent matricide was calculated daily.The matricide frequency was determined as the frequency of reproductive worms that die of matricide; as matricide is caused by internal progeny hatching, non-reproductive worms by definition never die of matricide, and thus are not included in calculation.This number reflects the likelihood of matricide.

Hatching rate
Eggs were synchronized by hypochlorite treatment and allowed to develop at 20uC until day 3 of adulthood.,100 synchronized hermaphrodites were transferred to a new plate and allowed to lay progeny for 1 hour; eggs and L1 progeny were counted at 3-hour intervals.

Survival analysis
The first day of adulthood was defined as t = 0, and the log-rank (Mantel-Cox) method was used to test the null hypothesis in Kaplan-Meier survival analysis, as previously described [65].All experiments were carried out at 20uC with 50 mM FUdR starting at L4; n.60 in each experiment.

RNA interference
Bacterial feeding RNAi experiments were carried out as previously described [66] with IPTG at 1 mM.Each clone was verified by PCR and sequence analysis.

Figure 1 .
Figure 1.TGF-ß Sma/Mab pathway regulates reproductive span.(A, E) Schematic representation of the TGF-ß Dauer and TGF-ß Sma/Mab pathways in C. elegans.(B) Mutants of the TGF-ß Dauer pathway moderately extend reproductive span (two experiments are shown).(C) A mutant of the shared receptor between the two pathways, daf-4, doubles the reproductive span of wild type.(D) daf-7, but not sma-2, animals lay moredeveloped progeny than wild type: percentage of embryos hatched 0-1, 3-4, and 6-7 hours after laying from day 3 wild-type, daf-7 and sma-2 adults.(F, G) Mutants of the TGF-ß Sma/Mab pathway greatly extend reproductive span.(H) Cumulative percentage of matricide animals (caused by internal progeny hatching, same below).daf-7's matricide rate is high starting in early adulthood, while sma-2's increases with age, finally resulting in more matricide animals.All reproductive spans in this figure are measured in self-fertilized hermaphrodites.Asterisk indicates high matricide frequency.Additional statistics presented inTable S1 and Table S2.doi:10.1371/journal.pgen.1000789.g001

Figure 4 .
Figure 4. TGF-ß Sma/Mab mutants have larger effects on reproductive span than on life span.(A-D) Life span assay trials.dbl-1 and sma-6 mutants increase life span moderately (B, D), sma-2 and sma-4 mutants have inconsistent effects on life span (A-C), and sma-3 and sma-9 appear to have no effect on longevity (A, B). dbl-1 OE (over-expression) slightly shortens life span (B, D). (Additional statistics presented in Table S6.) (E) Average percent change from wild-type life span and reproductive span of TGF-ß Dauer and Sma/Mab mutants.Sma/Mab mutants all have larger effects on reproductive span than on life span.Asterisk indicates that the value is not available due to high matricide rate.doi:10.1371/journal.pgen.1000789.g004

Figure 5 .
Figure 5. TGF-ß Sma/Mab mutants extend reproductive span most disproportionately to longevity effects.(A, B) Matricide frequencies with age of wild-type, sma-2, daf-7, daf-2, and eat-2 animals (6SEP).Wild-type and sma-2 have similar rates of increasing matricide frequency with age, while daf-2 and eat-2 rates are lower, and daf-7's Egl defect causes very early matricide.(C, D) The life span of sma-2 is shorter than daf-7, daf-2, and eat-2.(E, F) The reproductive span of sma-2 is either longer or comparable to these mutants when self-fertilized (E) or mated (F).(G, H) Percent change from wild-type life span and self-fertilized reproductive span (G) or mated reproductive span (H) of sma-2, daf-7, daf-2, and eat-2.Numbers above reproductive span bars indicate the fold of reproductive span increase over life span increase.sma-2 has the largest effect on reproductive span, compared with its effect on life span.Note that (G) is calculated from (C, E), and (H) is calculated from (D, F).Additional statistics presented in TableS7and TableS8.doi:10.1371/journal.pgen.1000789.g005