A somatic checkpoint through NHR-49/HNF4α governs reproductive investment and longevity in C. elegans

A somatic checkpoint through NHR-49/HNF4α governs reproductive investment and longevity in C. elegans | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A somatic checkpoint through NHR-49/HNF4α governs reproductive investment and longevity in C. elegans Siu Sylvia Lee, Sharada Gopal, Amaresh Chaturbedi, Tej Ramachandrula, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7217416/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The nuclear hormone receptor NHR-49, a homolog of mammalian PPARα and HNF4α, is a key transcriptional regulator of nutrition sensing and fatty acid metabolism in Caenorhabditis elegans . Here we uncovered a new function of NHR-49 in reproduction - controlling oocyte activation and ovulation. Loss of NHR-49 causes inappropriate oocyte activation and laying of unfertilized oocytes in the absence of sperm, resulting in rapid loss of yolk and stored fat, and drastically shortening of lifespan. We further demonstrated that prevention of yolk transfer into the oocytes largely restore fat storage and partially rescue lifespan in the nhr-49 mutants. Additionally, NHR-49 appears to couple germline proliferation to nutritional status, as evidenced by its requirement for starvation-induced reduction in germline proliferation. Mechanistically, we showed that NHR-49 primarily acts in somatic cells, rather than the germline itself, to regulate oocyte activation and ovulation. We further demonstrated that NHR-49 binds to the promoter of GSA-1 and may stimulate its expression. GSA-1 encodes a G-protein coupled receptor known to act in the gonadal sheath cells to couple sperm sensing and oocyte activation. Our findings therefore suggest a model whereby NHR-49 regulates the expression of GSA-1, which in turn regulates oocyte activation in response to sperm signal. Overall, our findings suggest a mechanistic link between nutrition sensing and fertility and point to regulated retention of reproductive resources to be critical for maintaining longevity. Biological sciences/Cell biology/Cell signalling/Nuclear receptors Biological sciences/Genetics/Gene regulation Biological sciences/Developmental biology/Ageing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Reproduction is one of the most energetically demanding biological processes 1 , requiring organisms to tightly coordinate reproductive activity with metabolic status 2 , 3 . In nutrient-poor conditions, animals often suppress germline proliferation, delay ovulation, or halt reproduction entirely to conserve resources 4 . These adjustments are especially crucial for optimizing reproductive success and ensuring the survival of both parent and offspring – 8 . However, the molecular mechanisms that link nutritional cues to reproductive timing remain incompletely understood. C. elegans are primarily hermaphrodites and possess both oocytes and sperm, making them capable of self-fertilization 9 . During the larval stage 4 (L4), the germline stem cells in the distal region actively proliferate, and the cells further away from the distal tip cell undergo meiosis, where they take up the sperm fate and the developed sperm are stored in the spermatheca 10 . From the young adult stage onwards, germ cells switch fate to an oogenic fate and the developing oocytes are arrested in meiosis I and stored in the oviduct 11 . A highly regulated process enables the oocytes most proximal to the spermatheca to sense the presence of sperm, become activated, i.e. begin to progress pass meiosis I, and simultaneously be ovulated into the spermatheca where they become fertilized; the fertilized embryos continue to develop in the uterus until they are laid outside of the parent at around 300 cells stage 12 . The FEM (feminization) and FOG (feminization of germline) genes are crucial regulators of sex determination and spermatogenesis in C. elegans 1 . Loss-of-function mutations of fem-3 , fog-2 or fog-3 result in defective spermatogenesis and feminization of worms that are incapable of self-fertilization 13 ( Fig. 1 A ) . Similar to hermaphrodites, the germline in these mutants contain mitotic and meiotic cells; However, the germ cells do not take up the spermatogenic fate during development. This results in an organism that has no sperm in their spermatheca. In the absence of males, these mutants accumulate ~ 20 oocytes in their oviduct and the process of germline stem cell proliferation and differentiation appear paused. Upon mating with males, or when sperm are provided 14 , the feminized mutants undergo normal stem cell proliferation and differentiation into oocytes, which are fertilized by the sperm. Mated feminized mutants produce similar number of healthy progenies as for mated hermaphrodites. Thus, feminized mutants in C. elegans resemble females of non-hermaphroditic species 14 . Feminized C. elegans mutants have been crucial in revealing the molecular pathways that regulate how oocytes sense sperm and become activated and ovulated 15 – 17 . Interestingly, we recently reported that unmated feminized mutants store elevated levels of fat and exhibit extended lifespan, likely due to a lack of progeny production and consequent reduced energy expenditure 18 . In C. elegans , the nuclear hormone receptor NHR-49—a putative functional homolog of mammalian PPARα and structural homolog of HNF4—is a central regulator of lipid metabolism and energy homeostasis 19 . Like all nuclear hormone receptors, NHR-49 is a DNA binding transcription factor that is regulated through ligand binding 20 , 21 . NHR-49 is known to regulate the expression of both lipid synthesis and lipid hydrolysis genes 22 . For example, NHR-49 transcriptionally regulates stearoyl-CoA desaturation genes fat-6 and fat-7 , which maintain membrane fluidity and polyunsaturated fatty acid synthesis 23 . It also activates acs-2 , a gene involved in mitochondrial β-oxidation 19 , 23 . In addition to fat metabolism, NHR-49 is known to also regulate oxidative stress response 24 , hypoxia adaptation 25 , peroxisome proliferation 26 , pathogen avoidance 27 , innate immunity 28 , and longevity 29 . Interestingly, NHR-49 has been suggested to participate in regulating adult reproductive diapause, a state in which severely starved L4 worms undergo extensive germline attrition and the animal can survive for a period of several weeks 30 , 31 , 32 , and the germline can regenerate when food become available again. Using feminized mutant C. elegans as a model, we uncovered a previously unrecognized role for NHR-49 in regulating oocyte activation and ovulation in a cell non-autonomous manner. We observed that loss of nhr-49 results in inappropriate oocyte activation and ovulation, and laying of unfertilized oocytes even in the absence of sperm signal. This inappropriate laying of unfertilized oocytes leads to excessive yolk and fat loss and drastically shortening of lifespan. Notably, when yolk transport to oocytes is blocked, thereby retaining yolk within the parent, fat storage is largely restored in nhr-49 mutant and their shortened lifespan is also partially rescued. Furthermore, nhr-49 mutants failed to halt oocyte proliferation under nutrient-restricted conditions, suggesting a disruption in the organism’s ability to couple reproductive output to nutritional status. Together, our findings suggest that NHR-49 plays a broader role in coordinating energy metabolism and reproductive restraint, and in doing so, orchestrate the balance between parental resource management and reproductive success, with a major impact on longevity. Results NHR-49 is required for the longer lifespan and elevated fat storage of feminized C. elegans Our lab recently reported that when not mated, feminized mutants exhibit significant lifespan extension and elevated fat storage compared to wild-type N2 (Wild-type) hermaphroditesat 25°C (Fig. 1B) 2 . While investigating the molecular factors required for the lifespan extension and fat metabolism of feminized worms, we identified NHR-49, a key metabolic regulator 28,28,29,33,34 , as essential for the enhanced lifespan of feminized worms (Fig. 1B) . Loss of nhr-49 drastically reduced the lifespan of feminized worms, where the double mutant nhr-49(gk405);fem-3(e1996) had lifespan similar to the short lifespan of the nhr-49(gk405) single mutant 33 . While characterizing the nhr-49(-);fem-3(-) double mutant, we noticed a surprising and interesting phenotype, where these feminized mutants laid unfertilized oocytes during early adulthood ( Fig. 1C ). In C. elegans , oocyte maturation and ovulation are strictly dependent on the presence of sperm, and consequently, feminized mutants lacking sperm retain their oocytes within the oviduct 15 . Accordingly, we observed no oocytes laid on the plate for fem-3(-) mutants (Fig. 1C and 1D) 3 . When examining nhr-49(-);fem-3(-) double mutants, we observed that these worms laid their unfertilized oocytes even though they lack sperm (Fig. 1 C and D) . Similar observation was made with a different feminized mutant strain 35 fog-2(-) and nhr-49(-);fog-2(-) (Fig. 1D) . NHR-49, and its homologs in other species, have not been implicated in regulating oocyte ovulation. To test whether NHR-49 uniquely regulates oocytes retention only in feminized mutants or whether it represents a more general role of NHR-49, we monitored nhr-49(-) mutant hermaphrodites. C. elegans hermaphrodites reach the end of their reproductive period around day 4 of adulthood at 20°C, when their sperm supply is depleted and the remaining oocytes are retained in the oviduct, mirroring the reproductive state of feminized mutants 36 . We first examined nhr-49 single mutant hermaphrodites at this post-reproductive stage and observed that nhr-49(-) mutants also laid unfertilized oocytes when examined on day 6 of adulthood (Fig. S1B) , a phenotype similar that observed for nhr-49(-);fem-3(-) double mutants (Fig. 1C) . We additionally monitored egg laying of nhr-49(-) single mutant compared to wild-type hermaphrodites and observed that nhr-49(-) laid reduced number of fertilized embryos, but significantly higher number of unfertilized oocytes, throughout the reproductive period, as well as during the post-reproductive period (i.e. >120 hrs after reaching adulthood) (Fig. S1B, S1D) . This unexpected finding likely explains the previously reported lower brood size phenotype associated with the nhr-49(-) mutant 26 ( Fig. S1A, C) . Together, our results uncovered a new and fundamental role of NHR-49 in retention of unfertilized oocytes, with this function becoming particularly evident in feminized mutants where sperm is absent throughout development. Different types of self-sterile C. elegans mutants, including feminized fem-3(-) mutants, accumulate excess fat 18 . Here, we found that the increased fat accumulation in fem-3(-) worms is dependent on NHR-49 . We used Oil red O (ORO) staining 37 to visualize storage lipid, and observed that both hermaphrodites and feminized mutant worms lacking nhr-49 showed significantly lower ORO staining on day 6 adulthood (Fig. 2 A and B) . Interestingly, when comparing the ORO levels between day 1 and day 6 of adulthood in the different strains, wild type showed significant increase in stored fat as they progressed towards post-reproductive period, as previously published 38 - 39 . fem-3(-) mutants showed no significant change during this timeframe. Hermaphrodites and feminized mutants lacking nhr-49 showed drastic reduction in ORO from day 1 to day 6 (Fig. 2 A and B) , suggesting a depletion of stored fat in worms lacking nhr-49 , which appears coincidental to the time-period where mutants lacking nhr-4 9 are expelling their unfertilized oocytes (Fig. S1) . Adult C. elegans hermaphrodites are known to transfer lipid-bound yolk to their oocytes to support embryonic development 40 . Feminized worms have increased yolk accumulation due to a lack of fertilization and embryo formation, which can be visualized using VIT-2::GFP 41 , a reporter strain for one of the vitellogenin proteins. We observed that nhr-49(-);fem-3(-) double mutants, which lay their unfertilized oocytes, display reduced VIT-2::GFP signal by day 2 of adulthood, which parallel the reduced ORO in day 6 fem-3(-); nhr-49(-) double mutants (Fig. S2A) . Since both hermaphrodites 33 and feminized mutants lacking nhr-49(-) are short-lived (Fig. 1B) and we found that they rapidly lose yolk by day 2 of adulthood (Fig. S2) , we hypothesized that attenuating yolk loss would help to preserve stored fat and lifespan. In C. elegans , yolk produced in the intestine is transported to oocytes via the endocytic receptor RME-2 42 . As expected, RNAi knockdown of rme-2 resulted in yolk retention in nhr-49(-);fem-3(-) double mutants, observed as increased VIT-2::GFP reporter signal (Fig. S2A and B) . Yolk retention also largely restored fat storage, as indicated by ORO staining (Fig. 2 C and D) . Interestingly, knockdown of rme-2 partially restored the lifespan of both nhr-49(-);fem-3(-) double mutants and nhr-49(-) single mutants (Fig. 2E) . Taken together, these findings indicated that laying of unfertilized oocytes is accompanied by shunting of yolk, which largely accounts for the rapid fat loss and partially accounts for the shortened lifespan of both hermaphrodites and feminized worms lacking nhr-49(-). NHR-49 prevents oocyte activation and ovulation in the absence of sperm We uncovered a new role of NHR-49 in preventing the laying of unfertilized oocytes, which appears to be coupled to the fat loss and shortened lifespan of nhr-49(-) mutants. To understand the molecular basis of inappropriate oocyte lay upon loss of nhr-49 , we first characterized the activation status of the oocytes. In C. elegans , oocytes are naturally arrested in meiotic prophase I and require sperm-derived signals to resume meiotic progression, as part of oocyte maturation, ovulation, and subsequent fertilization. Doubly phosphorylated MPK-1 (dpMPK-1), a homolog of the ERK MAP kinase, is a well-established marker of oocyte activation 16 (Fig. 3A) . We performed dpMPK-1 immunostaining of day-1 adults to examine the status of oocyte activation in hermaphrodites and feminized mutants with or without nhr-49 . While fertile wild-type worms showed the expected dpMPK-1 signal in the oocytes proximal to the spermatheca, feminized mutants, both fem-3(-) and fog-3(-) 17 , did not (Fig. 3A, B and S3A) , indicative of a loss of oocyte activation in the absence of sperm. Loss of nhr-49 did not substantially impact dpMPK-1 signal in reproductive hermaphrodites on day 1 of adulthood (Fig. 3A and B) . Interestingly, loss of nhr-49 resulted in prominent dpMPK-1 signal in the proximal oocytes of feminized worms (Fig. 3A, B, S3A, B) , which was comparable to that in hermaphrodites, indicating inappropriate activation of the oocyte maturation pathway despite the absence of sperm. Similar inappropriate dpMPK-1 activation was also observed in post reproductive nhr-49(-) mutant hermaphrodites (Fig. S3C, D) . Oocytes arrested in meiotic prophase I maintain minimal protein quality control activity, resulting in the accumulation of protein aggregates throughout the cytoplasm 5 . During oocyte maturation, proximal oocytes eliminate these protein aggregates to prepare for fertilization 43 (Fig. 3A) . To further assess the activation status of the oocytes in mutants lacking nhr-49 , we examined protein aggregate clearance in feminized strains with or without nhr-49 , using the HCP-1::GFP reporter strain that has been previously used to detect protein aggregates in C. elegans oocytes 43 . In day-1 adults, as expected, fem-3(-) single mutants had significantly higher HCP-1::GFP signal (Fig. 3B) in their proximal oocytes compared to reproductive wild-type hermaphrodites, consistent with that the oocytes are not activated and accumulate protein aggregates. RNAi knockdown of nhr-49 in feminized worms resulted in aberrant loss of HCP-1::GFP signal (Fig. 3B) in the proximal oocytes, further supporting inappropriate activation of the oocytes when nhr-49 is lost. In summary, in both post-reproductive hermaphrodites and feminized mutants, loss of nhr-49 leads to misactivation of oocytes, as well as laying of unfertilized oocytes. We therefore conclude that the indiscriminate laying of oocytes in the absence of sperm upon loss of NHR-49 occurs through misactivation of oocytes and their subsequent ovulation. In C. elegans , successful reproduction requires continuous oocyte production to replace those that are fertilized and laid. This process depends on two key processes: active proliferation of the mitotic stem cells in the distal region of the germline, and subsequent oogenesis in the meiotic region. Since unfertilized oocytes were laid in worms lacking nhr-49 , we predicted that germline stem cell proliferation and/or oogenesis would be enhanced. We assessed mitotic proliferation by EdU labelling 44 , which marks nuclei undergoing DNA synthesis, and oogenesis by immunostaining for dpMPK-1. Feminized mutants normally produce only a fixed number of oocytes due to them being retained in the oviduct, and as expected, they showed reduced number of EdU+ nuclei in the mitotic region (Fig. 3C) and lower dpMPK-1 signal intensity in the meiotic region (Fig. S4A) compared to wild-type control. Examination of nhr-49(-);fem-3(-) double mutant worms revealed aberrant proliferation of the distal germline stem cells as evident by higher levels of EdU+ nuclei in the mitotic region and inappropriate oogenesis as indicated by increased dpMPK-1 staining in the meiotic region (Fig. 3C and S4A) . Consistently, post-reproductive nhr-49 mutant hermaphrodites also showed a significant elevation of dpMPK-1 signal in the meiotic region compared to wild-type worms at the same age, suggestive of aberrant oogenesis (Fig. S4B) . Collectively, these data revealed that both proliferation of the mitotic stem cells and oogenesis are aberrantly activated upon loss of NHR-49, despite the absence of sperm. However, it is not clear whether aberrant activation of oocytes and subsequent laying of unfertilized oocytes triggered distal germline proliferation and oogenesis or vice versa in nhr-49 mutants. NHR-49 is required to couple nutritional status to germline proliferation NHR-49 is a well-established metabolic regulator, with particular importance in maintaining lipid homeostasis upon starvation 23,45 . NHR-49 has also been suggested to participate in starvation-induced germline attrition, a process known as adult reproductive diapause 46 . We therefore wondered whether the role of NHR-49 in the germline, as detailed above, is also sensitive to nutritional status. We monitored germline proliferation in reproductive hermaphrodites using EdU 44 labeling and, as expected, observed that germline proliferation was significantly reduced in response to starvation (24 hours) in wild-type worms. We found that nhr-49(-) single mutant showed reduced germline proliferation as evident by lower EdU+ nuclei in the distal region. Notably, we did not observe further reduction of EdU+ nuclei upon starvation in the nhr-49(-) mutants (Fig. 4A), suggesting that NHR-49 is required for coupling nutritional status to germline proliferation. NHR-49 may regulate oocyte activation through the somatic gonadal sheath cells To dissect the pathway through which NHR-49 regulates oocyte activation and ovulation, we examined its genetic interactions with known regulators of ovulation that act in distinct tissues. MSPs (Major Sperm Proteins) are sperm-derived cytoskeletal proteins that are also secreted as signaling molecules to promote oocyte maturation and sheath cell contraction 47 . vab-1 , encoding an ephrin receptor expressed on oocytes, and ceh-18 , a homeodomain transcription factor essential for sheath cell function, are both required for MSP-dependent oocyte activation. Given that sheath cells act as key MSP sensors, analogous to mammalian cumulus granulosa cells, these factors provide an entry point to determine the tissue of NHR-49 action. In feminized animals, RNAi knockdown of vab-1 or ceh-18 induces oocyte laying, where the effect of vab-1 RNAi is modest, consistent with previous findings 17 . Compared to vab-1 RNAi, knockdown of nhr-49 led to a significantly higher number of oocytes laid—nearly 6–10 times more than vab-1 knockdown alone. Knockdown of nhr-49 in vab-1 mutants resulted in an oocyte-laying phenotype that was comparable to nhr-49 RNAi alone (Fig. 5A) , suggesting a shared or converging pathway; However, given the mild effect of vab-1 loss, it is also possible that an additive effect would be difficult to detect. Combining RNAi knockdown of nhr-49 and ceh-18 mutation also did not show additive effect, again suggesting that nhr-49 functions in the same pathway as ceh-18 (Fig. 5B) . These results suggested that NHR-49 might act in both the oocytes and the sheath cells to regulate oocyte ovulation. To further test whether NHR-49 acts cell-autonomously in oocytes or from somatic tissues to regulate oocyte ovulation, we used a germline-specific RNAi approach, where rde-1 null mutant is complemented with functional RDE-1 only in the germline ( rde-1(-) ; Psun-1 ::RDE-1) 48 . Germline-specific knockdown of nhr-49 in feminized worms failed to recapitulate the oocyte-laying phenotype and dpMPK-1 misregulation observed with whole-body nhr-49 loss (Fig. 5C–E) . These results indicate that NHR-49 is largely dispensable in the germline and instead acts through somatic tissues—likely the sheath cells—to control oocyte activation. Genome-wide analyses identify metabolic and stress-related transcriptional targets of NHR-49 To further investigate the transcriptional outputs of NHR-49, we performed CUT&RUN, a chromatin profiling method that maps protein-DNA interactions, in day 1 adult wild-type animals using the CRISPR-engineered nhr-49(hj293) strain, which harbors a GFP::TEV::3xHA tag at the C - terminus of the endogenous nhr-49 locus 26 . This profiling revealed 2,227 high-confidence NHR-49 binding sites, with the majority of peaks localized to gene promoters and transcription start sites (Fig. S8A–B, Table 3) consistent with a role of NHR-49 in transcriptional regulation. Gene set enrichment analysis 49 of NHR-49-bound genes highlighted key categories related to lipid metabolism, mitochondrial fatty acid oxidation, and detoxification pathways (Fig. S8C–D) . To assess the functional impact of NHR-49 binding, we performed RNA-seq on nhr-49(-) mutants and wild-type controls at the day 1 of adulthood. Differential expression analysis revealed significant up- and downregulation of genes associated with lipid metabolism, innate immunity, and extracellular matrix remodeling (Fig. S7A–B, Table 4) . As an additional quality control, we compared our nhr-49(-) RNA-seq data to previously published transcriptomic profiles of nhr-49 mutants. We found that the three data sets showed significant overlap, confirming a shared core set of NHR-49–dependent gene expression changes across studies (Fig. S7E–F) . However, each dataset also displays distinct subsets of regulated genes, suggesting that chronic loss of nhr-49 function can lead to compensatory or context-specific transcriptional adaptations. Integrating CUT&RUN and RNA-seq data revealed 27 genes that were both bound by NHR-49 and differentially expressed in nhr-49 mutants (Fig. S7C, Table 5) , suggesting they represent direct transcriptional targets. Gene set enrichment analysis indicated that the upregulated genes are enriched for lipid biosynthesis and pathogen stress responses, and the downregulated genes are enriched for mitochondrial function. NHR-49 represses gsa-1 expression to prevent inappropriate oocyte activation Among the NHR-49-bound targets, we identified gsa-1 , which encodes a Gαs subunit known to act in the gonadal sheath cells to mediate MSP signaling and regulate oocyte ovulation 50 . NHR-49 binding peaks were detected at the promoter and second intron of gsa-1 (Fig. 6C) . Interestingly, RNA-seq indicated that gsa-1 RNA expression was modestly elevated in nhr-49 mutant (Fig. 6A) , albeit not reaching significance. Importantly, functional experiments showed that RNAi knockdown of gsa-1 suppressed the excessive oocyte laying phenotype of nhr-49(-);fem-3(-) animals (Fig. 6B) , indicating that elevated gsa-1 expression likely contributes to the aberrant laying of unfertilized oocytes. Taken together, the data suggested that NHR-49 acts, at least in part, through repressing gsa-1 expression to constrain inappropriate oocyte activation. Discussion Our findings uncover a previously unrecognized role for the nuclear hormone receptor NHR-49 in regulating oocyte activation and ovulation in C. elegans . This reproductive role appears to be mediated through the transcriptional repression of gsa-1 , a Gαs protein that promotes oocyte activation, and likely occurs via the gonadal sheath cells rather than the germline itself. We find that inappropriate oocyte activation and laying of unfertilized oocytes lead to premature yolk loss and depletion of parental resources. Suppressing yolk loss can partially restore lifespan, linking the reproductive phenotype to diminished somatic maintenance, and offers a partial explanation of the previously described lifespan shortening phenotype of the nhr-49 mutant. While NHR-49 has been extensively characterized as a regulator of lipid metabolism and lifespan, our data add a new dimension to the understanding of how metabolic regulators like NHR-49 influence longevity—not only by altering fat metabolism, but by controlling the timing and scale of reproductive investment. Our data suggest NHR-49 is required to suppress germline proliferation in adult worms subjected to acute nutrient deprivation. We found that nhr-49 mutants fail to reduce mitotic activity 4 in the distal germline following short-term starvation, suggesting that NHR-49 mediates nutrient-responsive germline restraint. NHR-49 is also known to be required for adult reproductive diapause (ARD) 30 —a distinct starvation-induced state in L3/L4 animals characterized by drastic germline shrinkage. While the mechanisms underlying these two responses may differ, the shared requirement for NHR-49 raises the possibility that it acts as a conserved metabolic regulator coordinating reproductive quiescence in response to energetic stress. Given that specific dietary and microbiota-derived metabolites can activate NHR-49 40 , these findings support a broader model in which NHR-49 functions as a metabolic sensor linking environmental inputs to germline decisions. Our CUT&RUN profiling revealed that NHR-49 binds near the loci of over 100 other nuclear hormone receptors, suggesting that it may serve as a broader transcriptional regulator of the NHR network itself. This opens the possibility that NHR-49 tunes hormonal signaling pathways across tissues in response to nutrient state. Future CUT&RUN studies in fed versus starved animals could determine whether NHR-49 differentially engages or recruits other NHRs depending on metabolic conditions. Additionally, future studies employing acute depletion system will likely reveal the immediate, direct transcriptional responses to NHR-49 loss from secondary effects that accumulate in constitutive mutants. NHR-49 regulates gsa-1 , a Gαs protein that functions in the gonadal sheath cells to promote oocyte activation and ovulation. Germline-specific RNAi suggests that NHR-49 does not act through the germline to regulate laying of unfertilized oocytes. Based on the known site of action of GSA-1 in the gonadal sheath cells, the observation that gsa-1 expression appears elevated in nhr-49 mutants, and the binding of NHR-49 to the promoter and intron regions of gsa-1 , we suggest that NHR-49 constrain oocyte activation and ovulation in the absence of sperm by repressing gsa-1 transcription in the sheath cells. Future tissue-specific studies will be important to confirm this proposed site of action. Together, these findings position NHR-49 as a metabolic gatekeeper that couples environmental and internal nutrient status to reproductive restraint. By limiting oocyte activation and germline proliferation when resources or sperm are lacking, NHR-49 helps align reproductive effort with metabolic capacity, promoting long-term survival and maternal resource preservation. Material and Methods C. elegans culture methods and strains C. elegans strains were maintained using standard protocols. Unless otherwise stated, all strains were grown on nematode growth medium (NGM) plates seeded with E. coli OP50-1 as a food source. The following strains were obtained from the Caenorhabditis Genetics Center (CGC): N2, DG2189 [ fog-3(q443) I/hT2 [bli-4(e937) let-?(q782) qIs48] (I;III); tnIs13 [pie-1p::vab-1::GFP + unc-119(+)] ltIs44 [pie-1p::mCherry::PH(PLC1delta1) + unc-119(+) ]; DG2190 [ fog-3(q443) I/hT2 [bli-4(e937) let-?(q782) qIs48] (I;III); tnIs13 [pie-1p::vab-1::GFP + unc-119(+)]V ]; VC470 [ nhr-49 (gk405) ]; DCL569 [ mkcSi13[sun-1p::rde-1::sun-1 3'UTR + unc-119(+)] II; rde-1(mkc36)V ]. These strains were previously generated in our laboratory, combining strains obtained from the CGC: IU613 [ fem-3(e1996)/nT1 [qIs51] (IV;V)]; IU614 [fem-3(e1996)/nT1 [qIs51] (IV;V) ] Lifespan Analysis Lifespan assays were performed as previously described 18 .10-15 gravid worms were placed on plates containing the desired bacterial food to obtain approximately 150-200 embryos. Upon reaching day one of adulthood, 30 worms were transferred to each of three RNAi plates and monitored every other day. Worms were gently transferred to fresh plates every other day to prevent starvation and mixing of generations. Care was taken to avoid transferring larvae. Lack of movement in response to gentle touch at the nose was used to confirm death. Worms that died due to rupturing, bursting, or leaving the plate were considered censored. The lifespan data was analyzed using the OASIS online survival analysis tool 78 available at https://sbi.postech.ac.kr/oasis2/, and log-rank tests were used to determine statistical significance between groups. All lifespan assays were performed at least twice. RNAi Treatment The RNAi procedure was carried out as previously described 18 . RNAi was conducted by feeding the worms with HT115 bacteria that expressed double-stranded RNA of the target gene. The RNAi clones were obtained from RNAi feeding libraries created by Julie Ahringer and Marc Vidal's laboratories 76,77 . pMPK-1 Immunostaining Approximately 100 worms were grown on OP50-1 seeded NGM plates, then washed off using PBST (PBS + 0.1% Tween 20) and collected in a 1.5 ml Eppendorf tube. The worms were spun for 15 seconds at 2000 rpm, the supernatant was removed, and the pellet was washed with 1X PBS twice, using 1 ml each time. Approximately 50 worms in 100 µl of PBS buffer were then transferred using a cut 200 µl micropipette tip onto a border-painted glass slide. To anesthetize the worms, 5 µl of 5 mM levamisole was added. Using 25- or 26-gauge needles, a cut was made near the pharyngeal bulb of each worm to release gonads, and all animals were dissected within 5–6 minutes. Dissected gonads were collected into a 1.5 ml tube using PBS-Tween–coated, cut-end 200 µl tips. Excess liquid was removed, and 200 µl of 4% formaldehyde fixative (prepared by mixing 100 µl 10X phosphate buffer, 100 µl 37% formaldehyde, and 800 µl ddH₂O to make a total volume of 1 ml fixative) was added. Samples were incubated at room temperature for 20-30 minutes. Fixative was then removed, and worms were incubated in 1 ml of chilled methanol (–20 °C) for 5 minutes. After removing methanol, worms were washed three times with PBST (5 minutes each). Excess liquid was removed, and worms were resuspended in 200 µl blocking buffer (0.1% Triton X-100, 0.1% Tween-20 and 2% BSA (Bovine Serum Albumin) in 1X PBS) and incubated for 1–2 hours. Primary antibody diluted 1:400 in Blocking buffer (50 µl per sample) was added and incubated overnight at 4 °C with rocking. Samples were then washed three times with blocking buffer for 10 minutes each. Secondary antibody (1:200 dilution in blocking buffer, 50 µl per sample) along with DAPI (final concentration 5 µg/ml) was added, and samples were incubated for 2 hours at room temperature in the dark with rocking. Following three washes with blocking buffer, worms were resuspended in 10 µl of Vectashield mounting medium . Worms were mounted on a 2% agarose pad prepared on a glass slide and covered with a coverslip for microscopy. EdU (5-ethynyl-2'-deoxyuridine) Staining EdU staining was performed as previously described, with modifications 82 , using the Click-iT® EdU Imaging Kit (ThermoFisher Cat. No. C10337). For EdU incorporation, 50-100 worms were washed and collected from plates using M9 buffer containing 0.1% Triton X-100 (M9T) and washed twice with 1 mL M9T. 50 µL of M9T containing the worms were transferred into a fresh 1.5 mL microcentrifuge tube using a cut tip, followed by the addition of 50 µL of 10 mM EdU. The worms were gently mixed and incubated on a shaker for 40 minutes at room temperature (22–25°C). Worms were washed twice with 1 mL M9T. Next, 30 µL of EBT (prepared by mixing 110 µL 10X egg buffer, 10 µL 10% Tween-20, 6.5 µL 10% tetramisole, and 850 µL distilled water to a final volume of 976.5 µL) was pipetted onto a slide, and approximately 50 worms were transferred into the EBT drop. The worms were anesthetized for 10-15 seconds, and their heads were cut to extrude the gonads. The gonads were collected into a 1.5 mL microcentrifuge tube, fixed with 4% formaldehyde in egg buffer for 20-30 minutes, and washed twice with 1 mL of 3% BSA in PBS. For permeabilization, the gonads were incubated in 1 mL of 1% Triton X-100 in PBS at room temperature for 20 minutes. After permeabilization, the buffer was removed, and the samples were washed twice with 1 mL of 3% BSA in PBS. Subsequently, 0.5 mL of the Click-iT® reaction cocktail (as per the kit instructions) was added, and the worms were incubated at room temperature for 30 minutes on a shaker and covered to protect from light. The reaction cocktail was removed, and the samples were washed twice with 1 mL of 3% BSA in PBS. For the EdU chase experiment, after the 10 mM EdU incorporation step, worms were transferred to the feeding plates for 24 hours, and the subsequent steps were performed as described above. Finally, 10 µL of Vectashield was added, and the worms were mounted on a 2% agarose pad prepared on a glass slide for imaging. Images were acquired using a Keyence BZ-X810 microscope. Fluorescence quantification and imaging For imaging, 10-20 worms were immobilized on a 2% agarose pad prepared on a glass slide in 10 µL of 5 mM levamisole in PBS. Images were acquired using the appropriate fluorescence channel on Leica DM5000B compound microscope or a Zeiss LSM 710 or LSM 880 inverted confocal microscope. Images were acquired as Z-stacks, and image tiling was used to scan at 63X magnification under oil immersion. For data processing, ZEN software was used for image stitching and FIJI and was used for image analysis and processing. To generate representative images, Adobe Photoshop was used. RNA isolation and library preparation for RNA-seq RNA isolation and RNA-seq library preparation were performed as previously described 18 . For the collection of whole worm samples, approximately 200 worms were collected in TRI reagent (MRC, Cat. No. TR 118). For tissue-specific analysis, germlines were surgically isolated from 100-150 animals using fine-gauge needle dissection, with 80-100 intact germlines collected per sample in TRI reagent. Two to three biological replicates were collected for each genotype. RNA-seq libraries were prepared using the QuantSeq 3‘ mRNA-Seq Library Prep Kit for Illumina (FWD) (Lexogen), and sequencing was performed by the Biotechnology Resource Center (BRC) of the Cornell Institute of Biotechnology. RNA-seq data analysis RNA-seq data analysis was performed as previously described 18 with minor modifications. To obtain the total read count of RNA-seq data were processed using Linux on the BioHPC server at Cornell University. Raw sequencing data underwent quality control and preprocessing using Trim Galore (v0.6.5), which incorporates Cutadapt 51 (v3.4) and FastQC (v0.11.8). Parameters were set to retain reads with quality scores ≥20 (-q 20) while performing FastQC quality assessment. Preprocessed reads were mapped to the C. elegans reference genome (ce11/WBcel235) using STAR aligner (v2.7.9a) 52 , with parameters configured for strict alignment: two computing threads (--runThreadN 2), gene count quantification (--quantMode GeneCounts), unique mapping only (--outFilterMultiMapNmax 1), a maximum of two mismatches (--outFilterMismatchNmax 2), and coordinate-sorted BAM files (--outSAMtype BAM SortedByCoordinate). For 3' RNA-seq data analysis, the middle column of counts from the STAR-generated tab-delimited files was used to construct expression matrices for comparative analysis. Matrices were organized either by genotype (comparing different mutant strains under identical conditions) or by treatment (comparing identical genotypes under different RNAi conditions). Differential expression analysis was performed in R (RStudio) using DESeq2 (v1.34.0) 53 . To improve statistical power, genes with fewer than 10 reads across fewer than 2 samples were excluded. Principal component analysis was conducted on variance-stabilizing transformed data (using the vst function), visualized using the plotPCA function with default parameters, and further refined using ggplot2 (v3.3.6). Statistically significant differentially expressed genes were identified using the standard DESeq2 workflow with an adjusted p-value threshold of 0.05. For genes of interest, normalized counts were visualized using DESeq2's plotCounts function. Gene set comparisons between genotypes or between RNA-seq and CUT&RUN datasets were performed using DeepVenn (https://www.deepvenn.com/) 54 online software, with statistical significance determined by Fisher's exact test. Gene set enrichment analysis was conducted using WormCat 2.0 (www.wormcat.com) 49 , which also employs Fisher's test with FDR correction. For heatmap generation, log 2 fold-change values were extracted from DESeq2 results tables without filtering out low-expression genes. Heatmaps were created using the ComplexHeatmap package (v2.10.0) with row clustering enabled (cluster_rows = TRUE), column clustering disabled (cluster_columns = FALSE), and row names hidden (show_row_names = FALSE). For clustered analyses, heatmaps were partitioned (split = 3), and genes from each cluster were extracted using the row_order function. CUT&RUN for NHR-49 binding sites in the genome CUT&RUN experiments were performed using our previously published protocol 55,56 . A sufficient number of embryos were collected from gravid adult worms grown on 10-cm NGM plates seeded with OP50-1. For each CUT&RUN reaction, 3000 embryos were plated onto 15-cm NGM plates supplemented with 1 mL of 25× concentrated OP50-1 culture (grown overnight). Sample preparation was scaled to ensure sufficient worm numbers (3000 animals per reaction) for each experimental condition. Plates were incubated at 25°C for approximately 48-52 hours until populations reached the predominantly young adult stage. At this developmental stage, worms were harvested by washing plates with PBS buffers, and CUT&RUN experiments were performed according to a detailed protocol previously published 55 which follows standard CUT&RUN methodology with minor modifications specific to C. elegans samples. Sequencing libraries were constructed using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, catalog numbers E7645S and E7335S). Library amplification was performed with 14 PCR cycles according to the manufacturer's guidelines, with slight procedural adjustments as described in the published protocol 57 .The resulting libraries underwent paired-end sequencing (2 × 32 bp) on an Illumina NextSeq 500 platform, performed by the Biotechnology Resource Center (BRC) of the Cornell Institute of Biotechnology. All CUT&RUN experiments were conducted with at least two independent biological replicates. CUT&RUN Analysis CUT&RUN analysis was performed as previously described 55,57–60 . Declarations Acknowledgments We would like to thank personnel at Cornell Genomics Center, Microscopy core and statistical consulting, and the Caenorhabditis Genetics Center (CGC) for all the worm strains used in this study. We thank B. Sanketi, A. Ren, N. Kurpios, R. Sardana, K. Liu, D. Lin, G. Hollopeter members of the Cornell worm club, P. Cohen and members of the Cornell Reproductive Sciences Center, and the members of the Lee lab for their valuable discussion and feedback. Funding National Institute of Aging R01 AG024425 (SSL); NIH 1S10RR025502 for Cornell Institute of Biotechnology. Author contributions Conceptualization, S.G., A.C., S.S.L.; Methodology, S.G., A.C., S.S.L.; Investigation, S.G., A.C.; Investigation: assistance with selected experiments, T.C., J.O., R.R., Data analysis, S.G., A.C.; Data curation, S.G., A.C.; visualization, S.G., A.C., S.S.L.; funding acquisition, S.S.L.; supervision, S.S.L.; writing – original draft, S.G.; and writing – review and editing, A.C., S.S.L. Materials and correspondence Requests for materials may be directed to [email protected] (SSL). Declaration of interests The authors declare no competing interests. References Stearns, S. C. Trade-Offs in Life-History Evolution. Funct. Ecol. 3 , 259–268 (1989). Della Torre, S., Benedusi, V., Fontana, R. & Maggi, A. Energy metabolism and fertility: a balance preserved for female health. Nat. Rev. Endocrinol. 10 , 13–23 (2014). Mircea, C. N., Lujan, M. E. & Pierson, R. A. Metabolic fuel and clinical implications for female reproduction. J. Obstet. Gynaecol. Can. JOGC J. Obstet. Gynecol. Can. JOGC 29 , 887–902 (2007). Lopez, A. L. et al. DAF-2 and ERK Couple Nutrient Availability to Meiotic Progression during Caenorhabditis elegans Oogenesis. Dev. Cell 27 , 227–240 (2013). Thondamal, M., Witting, M., Schmitt-Kopplin, P. & Aguilaniu, H. Steroid hormone signalling links reproduction to lifespan in dietary-restricted Caenorhabditis elegans. Nat. Commun. 5 , 4879 (2014). Templeman, N. M. & Murphy, C. T. Regulation of reproduction and longevity by nutrient-sensing pathways. J. Cell Biol. 217 , 93–106 (2018). Cao, W. & Pocock, R. Mechanisms of germ cell survival and plasticity in Caenorhabditis elegans. Biochem. Soc. Trans. 50 , 1517–1526 (2022). Sahu, S. & Armstrong, A. R. Translation components in adult Drosophila melanogaster adipocytes regulate the ovarian germline stem cell lineage. 2024.08.31.610632 Preprint at https://doi.org/10.1101/2024.08.31.610632 (2024). Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77 , 71–94 (1974). Pazdernik, N. & Schedl, T. Introduction to Germ Cell Development in C. elegans. Adv. Exp. Med. Biol. 757 , 1–16 (2013). Kimble, J. & Crittenden, S. L. Germline proliferation and its control. WormBook Online Rev. C Elegans Biol. 1–14 (2005) doi:10.1895/wormbook.1.13.1. Davis, G. M., Hipwell, H. & Boag, P. R. Oogenesis in Caenorhabditis elegans. Sex. Dev. 17 , 73–83 (2023). Hodgkin, J. Sex Determination in the Nematode C. ELEGANS: Analysis of tra-3 Suppressors and Characterization of fem Genes. Genetics 114 , 15–52 (1986). Schedl, T. & Kimble, J. Fog-2, a Germ-Line-Specific Sex Determination Gene Required for Hermaphrodite Spermatogenesis in Caenorhabditis Elegans. Genetics 119 , 43–61 (1988). Cheng, H., Govindan, J. A. & Greenstein, D. Regulated trafficking of the MSP/Eph receptor during oocyte meiotic maturation in C. elegans. Curr. Biol. CB 18 , 705–714 (2008). Miller, M. A. et al. A Sperm Cytoskeletal Protein That Signals Oocyte Meiotic Maturation and Ovulation. Science 291 , 2144–2147 (2001). Miller, M. A., Ruest, P. J., Kosinski, M., Hanks, S. K. & Greenstein, D. An Eph receptor sperm-sensing control mechanism for oocyte meiotic maturation in Caenorhabditis elegans. Genes Dev. 17 , 187–200 (2003). Chaturbedi, A. & Lee, S. S. Different gametogenesis states uniquely impact longevity in Caenorhabditis elegans. 2023.06.13.544885 Preprint at https://doi.org/10.1101/2023.06.13.544885 (2023). Doering, K. R. S., Ermakova, G. & Taubert, S. Nuclear hormone receptor NHR-49 is an essential regulator of stress resilience and healthy aging in Caenorhabditis elegans. Front. Physiol. 14 , 1241591 (2023). Scholtes, C. & Giguère, V. Transcriptional control of energy metabolism by nuclear receptors. Nat. Rev. Mol. Cell Biol. 23 , 750–770 (2022). Sever, R. & Glass, C. K. Signaling by Nuclear Receptors. Cold Spring Harb. Perspect. Biol. 5 , a016709 (2013). Taubert, S., Van Gilst, M. R., Hansen, M. & Yamamoto, K. R. A Mediator subunit, MDT-15, integrates regulation of fatty acid metabolism by NHR-49-dependent and -independent pathways in C. elegans. Genes Dev. 20 , 1137–1149 (2006). Van Gilst, M. R., Hadjivassiliou, H., Jolly, A. & Yamamoto, K. R. Nuclear hormone receptor NHR-49 controls fat consumption and fatty acid composition in C. elegans. PLoS Biol. 3 , e53 (2005). Goh, G. Y. S. et al. NHR-49/HNF4 integrates regulation of fatty acid metabolism with a protective transcriptional response to oxidative stress and fasting. Aging Cell 17 , e12743 (2018). Doering, K. R. et al. Nuclear hormone receptor NHR-49 acts in parallel with HIF-1 to promote hypoxia adaptation in Caenorhabditis elegans. eLife 11 , e67911 (2022). Zeng, L. et al. Nuclear receptors NHR-49 and NHR-79 promote peroxisome proliferation to compensate for aldehyde dehydrogenase deficiency in C. elegans. PLOS Genet. 17 , e1009635 (2021). Kwon, S., Park, K.-S. & Yoon, K. Regulator of Lipid Metabolism NHR-49 Mediates Pathogen Avoidance through Precise Control of Neuronal Activity. Cells 13 , 978 (2024). Dasgupta, M. et al. NHR-49 Transcription Factor Regulates Immunometabolic Response and Survival of Caenorhabditis elegans during Enterococcus faecalis Infection. Infect. Immun. 88 , (2020). Naim, N. et al. Cell nonautonomous roles of NHR-49 in promoting longevity and innate immunity. Aging Cell 20 , e13413 (2021). Eustice, M. et al. Nutrient sensing pathways regulating adult reproductive diapause in C. elegans. PloS One 17 , e0274076 (2022). Seidel, H. S. & Kimble, J. The oogenic germline starvation response in C. elegans. PloS One 6 , e28074 (2011). Heestand, B., Simon, M., Frenk, S., Titov, D. & Ahmed, S. Transgenerational Sterility of Piwi Mutants Represents a Dynamic Form of Adult Reproductive Diapause. Cell Rep. 23 , 156–171 (2018). Ratnappan, R. et al. Germline signals deploy NHR-49 to modulate fatty-acid β-oxidation and desaturation in somatic tissues of C. elegans. PLoS Genet. 10 , e1004829 (2014). Nuclear hormone receptor NHR-49 acts in parallel with HIF-1 to promote hypoxia adaptation in Caenorhabditis elegans | eLife. https://elifesciences.org/articles/67911. Schedl, T. & Kimble, J. fog-2, a germ-line-specific sex determination gene required for hermaphrodite spermatogenesis in Caenorhabditis elegans. Genetics 119 , 43–61 (1988). Scharf, A., Pohl, F., Egan, B. M., Kocsisova, Z. & Kornfeld, K. Reproductive Aging in Caenorhabditis elegans: From Molecules to Ecology. Front. Cell Dev. Biol. 9 , 718522 (2021). Rapid Lipid Quantification in Caenorhabditis elegans by Oil Red O and Nile Red Staining. https://en.bio-protocol.org/en/bpdetail?id=4340&type=0. Hou, N. S. & Taubert, S. Function and Regulation of Lipid Biology in Caenorhabditis elegans Aging. Front. Physiol. 3 , (2012). Mullaney, B. C. & Ashrafi, K. C. elegans Fat Storage and Metabolic Regulation. Biochim. Biophys. Acta 1791 , 474–478 (2009). Evolutionarily related host and microbial pathways regulate fat desaturation in C. elegans | Nature Communications. https://www.nature.com/articles/s41467-024-45782-2. Balklava, Z., Pant, S., Fares, H. & Grant, B. D. Genome-wide analysis identifies a general requirement for polarity proteins in endocytic traffic. Nat. Cell Biol. 9 , 1066–1073 (2007). Grant, B. & Hirsh, D. Receptor-mediated Endocytosis in the Caenorhabditis elegans Oocyte. Mol. Biol. Cell 10 , 4311–4326 (1999). Bohnert, K. A. & Kenyon, C. A lysosomal switch triggers proteostasis renewal in the immortal C. elegans germ lineage. Nature 551 , 629–633 (2017). Kocsisova, Z., Mohammad, A., Kornfeld, K. & Schedl, T. Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU. J. Vis. Exp. JoVE 58339 (2018) doi:10.3791/58339. Watterson, A. et al. Intracellular lipid surveillance by small G protein geranylgeranylation. Nature 605 , 736–740 (2022). Angelo, G. & Van Gilst, M. R. Starvation Protects Germline Stem Cells and Extends Reproductive Longevity in C. elegans . Science 326 , 954–958 (2009). Yang, Y., Han, S. M. & Miller, M. A. MSP Hormonal Control of the Oocyte MAP Kinase Cascade and Reactive Oxygen Species Signaling. Dev. Biol. 342 , 96–107 (2010). Construction of a germline-specific RNAi tool in C. elegans. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6382888/. Holdorf, A. D. et al. WormCat: An Online Tool for Annotation and Visualization of Caenorhabditis elegans Genome-Scale Data. Genetics 214 , 279–294 (2020). Govindan, J. A., Cheng, H., Harris, J. E. & Greenstein, D. Gαo/i and Gαs Signaling Function in Parallel with the MSP/Eph Receptor to Control Meiotic Diapause in C. elegans. Curr. Biol. 16 , 1257–1268 (2006). Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17 , 10–12 (2011). Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. BIOINFORMATICS 29 , 15–21 (2013). Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15 , 550 (2014). Hulsen, T., de Vlieg, J. & Alkema, W. BioVenn – a web application for the comparison and visualization of biological lists using area-proportional Venn diagrams. BMC Genomics 9 , 488 (2008). Emerson, F. J. & Lee, S. S. CUT&RUN for Chromatin Profiling in Caenorhabditis elegans. Curr. Protoc. 2 , e445 (2022). Skene, P. J., Henikoff, J. G. & Henikoff, S. Targeted in situ genome-wide profiling with high efficiency for low cell numbers. Nat. Protoc. 13 , 1006–1019 (2018). Skene, P. J. & Henikoff, S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. eLife 6 , e21856 (2017). Li, C.-L. et al. Region-specific H3K9me3 gain in aged somatic tissues in Caenorhabditis elegans. PLOS Genet. 17 , e1009432 (2021). Emerson, F. J. et al. The chromatin factors SET-26 and HCF-1 oppose the histone deacetylase HDA-1 in longevity and gene regulation in C. elegans. Nat. Commun. 15 , 2320 (2024). Zhang, Y. et al. Model-based Analysis of ChIP-Seq (MACS). Genome Biol. 9 , R137 (2008). Tables Tables 1 to 5 are available in the Supplementary Files section. Additional Declarations There is NO Competing Interest. Supplementary Files Table1.xlsx Lifespan Source Data Table2.xlsx Lifespan Source Data Table3.xlsx NHR-49 binding targets Table4.xlsx RNAseq Analysis Table5.xlsx CUT&RUN and RNAseq nhr-49 overlapping targets suppFigure.docx Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7217416","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":493989074,"identity":"b46f936c-8706-4e61-9f20-0e7f721c81a0","order_by":0,"name":"Siu Sylvia Lee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYBACPgYGxgOMDWA244EHFUCKmbkBrxY2IIZpYTiQcAakBcYlSktiG9g2AlrYmw8c+LnDTs58NpCROK82mr8dqOVHxTbcWniOJRzsPZNsLHPnWMKBxG3Hc2ccZmxg7DlzG7cWiRyDA7xtBxJngBiJ247lNgC1MDO24dEi//7Dwb9gLfkfDiTOOZY7n6AWCR6Gw1BbgN5vqMndQFALT5rBYVmgXyQk0gwOJBw7kLsRqOUgPr/wsx9++PAtMMQkJJIfPvhQU5c77/zhgw9+VODWgg4Og8kDRKsHgjpSFI+CUTAKRsEIAQChRGNKJHDEygAAAABJRU5ErkJggg==","orcid":"","institution":"Cornell University","correspondingAuthor":true,"prefix":"","firstName":"Siu","middleName":"Sylvia","lastName":"Lee","suffix":""},{"id":493989075,"identity":"154156a5-e2fe-4e4a-a857-ec5a2f443626","order_by":1,"name":"Sharada Gopal","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"Sharada","middleName":"","lastName":"Gopal","suffix":""},{"id":493989076,"identity":"419d8bcf-a7ae-4260-93a4-57e20a413eef","order_by":2,"name":"Amaresh Chaturbedi","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"Amaresh","middleName":"","lastName":"Chaturbedi","suffix":""},{"id":493989077,"identity":"3b8f80c6-8994-44ed-bd0e-942d85c67ea6","order_by":3,"name":"Tej Ramachandrula","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"Tej","middleName":"","lastName":"Ramachandrula","suffix":""},{"id":493989078,"identity":"39b57ce8-f051-41c9-9b38-87859b2f7008","order_by":4,"name":"John OuYang","email":"","orcid":"","institution":"Cornell University","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"","lastName":"OuYang","suffix":""},{"id":493989079,"identity":"3723e263-d9cb-4f85-a5e7-41c9a90c5051","order_by":5,"name":"Rebecca Rodell","email":"","orcid":"","institution":"Stanford University","correspondingAuthor":false,"prefix":"","firstName":"Rebecca","middleName":"","lastName":"Rodell","suffix":""}],"badges":[],"createdAt":"2025-07-25 22:10:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7217416/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7217416/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90506333,"identity":"8ca73534-4a38-4b56-a617-385b6f409a91","added_by":"auto","created_at":"2025-09-03 12:50:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":451809,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of NHR-49 shortens lifespan and induces oocyte ovulation in feminized worms.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Schematic representation of feminization of \u003cem\u003eC. elegans \u003c/em\u003egermline. \u003cstrong\u003e(B)\u003c/strong\u003e Survival curves for N2\u003cem\u003e \u003c/em\u003e(wild-type hermaphrodite), \u003cem\u003enhr-49(gk405) \u003c/em\u003e(hermaphrodite)\u003cem\u003e, fem-3(e1996) (\u003c/em\u003efeminized\u003cem\u003e) and fem-3(e1996);nhr-49(gk405)\u003c/em\u003e. Quantitative data and Kaplan-Meier statistics are provided in Table 1. \u003cstrong\u003e(C)\u003c/strong\u003eRepresentative images showing an absence of laid oocytes on plates with \u003cem\u003efem-3(e1996)\u003c/em\u003emutants, and numerous oocytes (marked by arrowheads) were laid on plates with \u003cem\u003efem-3(e1996);nhr-49(gk405) \u003c/em\u003edouble mutants\u003cem\u003e \u003c/em\u003eon day 1 of adulthood. \u003cstrong\u003e(D)\u003c/strong\u003e Bar graphs showing the number of oocytes laid by \u003cem\u003efem-3(e1996) \u003c/em\u003eand \u003cem\u003efem-3(e1996);nhr-49(gk405) \u003c/em\u003edouble mutants, and \u003cem\u003efog-2\u003c/em\u003e(\u003cem\u003eq71\u003c/em\u003e) and \u003cem\u003efog-2\u003c/em\u003e(\u003cem\u003eq71\u003c/em\u003e);\u003cem\u003enhr-49(gk405) \u003c/em\u003eon day 1 of adulthood. The number of oocytes laid were counted over a period of 24-hour period starting from the larval stage L4 (see Methods for details), (N \u0026gt;30) for each genotype, collected from two independent biological replicates. Statistical comparisons were performed using an unpaired t-test and are presented as mean ± SEM.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7217416/v1/e09017482e442a8d6d82abc3.png"},{"id":90507557,"identity":"89b9fed6-acc8-4dc5-b88c-609d8654af95","added_by":"auto","created_at":"2025-09-03 12:58:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":235344,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLifespan shortening and fat loss in NHR-49 mutants are partially dependent on RME-2 mediated yolk transport into oocytes.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003eRepresentative images of Oil Red O staining in N2\u003cem\u003e \u003c/em\u003ewild-type (hermaphrodite), \u003cem\u003efem-3(e1996) \u003c/em\u003e(feminized)\u003cem\u003e, nhr-49(gk405) \u003c/em\u003e(hermaphrodite)\u003cem\u003e, \u003c/em\u003eand \u003cem\u003efem-3(e1996);nhr-49(gk405) \u003c/em\u003e(feminized) worms on day 1 (1 DA) and day 6 (6 DA) of adulthood. \u003cstrong\u003e(B)\u003c/strong\u003e Oil red O (ORO) intensity in the indicated strains at day 1 and day 6 of adulthood. ORO intensity is shown as the mean intensity per-worm (N \u0026gt;30) for each genotype, collected from two independent biological replicates. Statistical comparisons were performed using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons test. \u003cstrong\u003e(C)\u003c/strong\u003e Loss of RME-2 restored fat accumulation, as shown by ORO staining. Representative images of ORO staining in \u003cem\u003efem-3(-);nhr-49(-) \u003c/em\u003ewith and without \u003cem\u003erme-2\u003c/em\u003e RNAi on day 2 of adulthood. \u003cstrong\u003e(D)\u003c/strong\u003e Bar graphs showing ORO intensity in the indicated strains (day 1 adults). ORO intensity is shown as the mean intensity per-worm (N \u0026gt;30) for each genotype, collected from two independent biological replicates. Two-tailed p-values were computed using unpaired t-test. \u003cstrong\u003e(E)\u003c/strong\u003e Survival curves for \u003cem\u003efem-3(e1996)\u003c/em\u003e, and \u003cem\u003efem-3(e1996);nhr-49(gk405) \u003c/em\u003edouble mutants on empty vector control and \u003cem\u003erme-2\u003c/em\u003e RNAi from one representative experiment. Quantitative data and Kaplan-Meier statistics are provided in Table 2.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7217416/v1/acd2c7450ba779825882b22f.png"},{"id":90506338,"identity":"ac7563d2-a430-4215-babe-570fe069fec0","added_by":"auto","created_at":"2025-09-03 12:50:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":382689,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNHR-49 negatively regulates oocyte activation in feminized mutants. (A)\u003c/strong\u003eSchematic diagram representing dpMPK-1 activation and protein aggregation states in the proximal region of the germline. \u003cstrong\u003e\u0026nbsp;(B)\u003c/strong\u003e Representative images of dissected day-1 adult gonads immunostained with anti-dpMPK-1 antibody, which recognizes the di-phosphorylated, active form of MPK-1 (red). The position of the oocyte most proximal to the spermatheca in each image is marked by a yellow arrowhead. DAPI staining (blue) was used to indicate germ cell nuclei. \u003cstrong\u003e(C)\u003c/strong\u003e Bar graph showing dpMPK-1 signal intensity in the proximal oocytes of the indicated strains. Statistical comparisons were performed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. More than 45 worms from 2 independent replicates per genotype were analyzed. \u003cstrong\u003e(D)\u003c/strong\u003eRepresentative images of dissected day-1 adult gonads from wild-typeor \u003cem\u003efem-3(-)\u003c/em\u003e worms expressing HCP-1::GFP fusion protein, treated with control RNAi (EV) or \u003cem\u003enhr-49\u003c/em\u003eRNAi. The position of the oocyte most proximal to the spermatheca in each image is marked by a yellow arrowhead. ‘p’ and ‘d’ indicate distal or proximal region of the gonad respectively. \u003cstrong\u003e(E)\u003c/strong\u003eQuantification of GFP fluorescence intensity in wild-type and \u003cem\u003efem-3(-)\u003c/em\u003eworms expressing HCP-1::GFP fusion protein, treated with control RNAi (EV) and \u003cem\u003enhr-49\u003c/em\u003eRNAi. Statistical comparisons were performed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. More than 45 worms from 2 independent replicates per genotype were analyzed. \u003cstrong\u003e(F\u003c/strong\u003e) Representative images of dissected day-1 adult gonads stained with EdU (green), marking proliferating nuclei, and counterstained with DAPI (purple) to visualize germ cell nuclei. Shown are gonads from N2, \u003cem\u003enhr-49(gk405)\u003c/em\u003e, \u003cem\u003efem-3(e1996),\u003c/em\u003eand \u003cem\u003efem-3(e1996);nhr-49(gk405)\u003c/em\u003e worms. (G) Bar graph showing the number of EdU positive nuclei in the indicated strains. Statistical comparisons were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. More than 45 worms from two independent replicates per genotype were analyzed. All scale bars = 50μm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7217416/v1/b44644a303ca11b886b3e8ca.png"},{"id":90507553,"identity":"00e9404e-c5b6-46f1-b003-4eb3eda3fdfb","added_by":"auto","created_at":"2025-09-03 12:58:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":138606,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNHR-49 may mediate starvation response of the germline. (A)\u003c/strong\u003e Representative images of EdU-stained dissected gonads from \u003cem\u003eN2 \u003c/em\u003eand \u003cem\u003enhr-49(gk405) \u003c/em\u003eworms under fed condition or after 24 hours of starvation started as late L4 stage and scored at day 1 of adulthood. Wildtype N2 germlines showed a significant decline in mitotic proliferation as indicated by EdU+ nuclei. \u003cem\u003enhr-49(gk405) \u003c/em\u003emutantshowed reduced EdU+ nuclei under fed condition but did not show further decrease in response to starvation. ‘d’ indicates distal region of the gonad. All scale bars = 50μm.\u003cstrong\u003e (B)\u003c/strong\u003e Bar graph showing the number of EdU+ nuclei in the indicated strains. Statistical comparisons were performed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test, based on more than 20 worms from two independent replicates per genotype.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7217416/v1/c22d4ca052182993f2afaa39.png"},{"id":90506334,"identity":"d8595874-f195-4286-be2e-68b4e07ea1db","added_by":"auto","created_at":"2025-09-03 12:50:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":217454,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNHR-49 primarily regulates oocyte activation through somatic tissues. (A)\u003c/strong\u003eBar graph showing the number of unfertilized oocytes laid by \u003cem\u003efog-2(q71),\u003c/em\u003e \u003cem\u003evab-1(dx31);fog-2(q71) \u003c/em\u003emutants with and without \u003cem\u003enhr-49 \u003c/em\u003eRNAi. \u003cstrong\u003e(B) \u003c/strong\u003eBar graph showing the number of unfertilized oocytes laid by \u003cem\u003efog-2(q71),\u003c/em\u003e \u003cem\u003efog-2(q71);ceh-18(mg57) \u003c/em\u003ewith and without \u003cem\u003enhr-49 \u003c/em\u003eRNAi. Comparisons were statistically tested by one-way analysis of variance (ANOVA) followed by multiple comparisons test with Tukey's correction for \u0026gt;45 worms from 2 replicates for each genotype. \u003cstrong\u003e(C)\u003c/strong\u003eRepresentative images of dpMPK-1 immunostained dissected gonads from day-1 feminized mutants with either whole-body RNAi knockdown of \u003cem\u003enhr-49\u003c/em\u003e (right column) or germline-specific RNAi knockdown (second column) using \u003cem\u003ePsun-1::RDE-1 \u003c/em\u003estrain\u003cem\u003e \u003c/em\u003e(see Methods for details of the germline-specific RNAi strain). dpMPK-1 signal (red) was observed in feminized mutants upon whole-body knockdown of \u003cem\u003enhr-49,\u003c/em\u003e but not when \u003cem\u003enhr-49\u003c/em\u003e was specifically knocked down in the germline. \u003cstrong\u003e(D)\u003c/strong\u003e Bar graph showing dpMPK-1 signal intensity in the proximal oocytes of the indicated strains. \u003cem\u003erde-1\u003c/em\u003e ‘+’ indicates a strain background that can undergo whole-body RNAi; \u003cem\u003ePsun-1::RDE-1\u003c/em\u003e‘+’ indicates a strain background that can undergo germline-specific RNAi. Comparisons were statistically tested by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. More than 25 worms from two independent replicates per genotype were analyzed. \u003cstrong\u003e(E)\u003c/strong\u003e Bar graph showing the number of unfertilized oocytes laid in strains undergoing whole-body ( \u003cem\u003erde-1\u003c/em\u003e‘+’) or germline specific (\u003cem\u003ePsun-1::RDE-1\u003c/em\u003e ‘+’) RNAi knockdown of \u003cem\u003enhr-49\u003c/em\u003e. Comparisons were statistically tested by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. More than 25 worms from two independent replicates per genotype were analyzed. All scale bars = 50μm.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7217416/v1/258dc4f64ac475abd472c0dc.png"},{"id":90507882,"identity":"f853073c-7bba-4fcc-b597-572b87aee6d7","added_by":"auto","created_at":"2025-09-03 13:06:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":136030,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNHR-49 may regulate oocyte activation by repressing sheath cell-mediated sperm sensing. (A)\u003c/strong\u003eBar graph showing a trend of elevated \u003cem\u003egsa-1 \u003c/em\u003emRNA expression in \u003cem\u003enhr-49(gk405)\u003c/em\u003eworms compared to \u003cem\u003eN2 \u003c/em\u003e(wild-type) based on RNAseq results. Two-tailed p-values were computed using an unpaired t-test. N \u0026gt;30 2 independent biological replicates \u003cstrong\u003e(B)\u003c/strong\u003e Bar graph showing the number of unfertilized oocytes laid by \u003cem\u003efem-3(e1996);nhr-49(gk405) \u003c/em\u003edouble mutants treated with control (EV) and \u003cem\u003egsa-1\u003c/em\u003eRNAi. Two-tailed p-values were computed using an unpaired t-test. N \u0026gt;30 2 independent biological replicates \u003cstrong\u003e(C) \u003c/strong\u003eScreenshot from Integrative Genomics Viewer (IGV) showing NHR-49-HA binding at the promoter and intragenic regions of \u003cem\u003egsa-1\u003c/em\u003e, as detected by CUT\u0026amp;RUN chromatin profiling. Darker blue at promoter region indicates stronger signal with signalValue: 2.26408 pValue: 82.6164 qValue: 79.556 and lighter blue signal at the intron region suggests comparatively weaker signal with a signalValue: 1.49912 pValue: 5.58139 qValue: 4.01701. Fluorescent blue signal in the middle of the peak indicates peak summit.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7217416/v1/18bd0739d4a5bf060ab6b66c.png"},{"id":90507558,"identity":"2132c319-0cbb-462f-bad6-83d6d2ffece6","added_by":"auto","created_at":"2025-09-03 12:58:12","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":230409,"visible":true,"origin":"","legend":"\u003cp\u003eNHR-49 represses GSA-1 in sheath cells to regulate oocyte activation: We propose that NHR-49 responds to nutritional cues and represses the expression of GSA-1 in sheath cells, which results in aberrant oocyte activation and ovulation. This leads to yolk/fat loss and shortening of parental lifespan.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7217416/v1/3ea697f95a902da2fc177ff5.png"},{"id":90509151,"identity":"5eaf5fb8-a885-4bbf-ab3d-05cbec866398","added_by":"auto","created_at":"2025-09-03 13:14:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3014415,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7217416/v1/c6851f87-8555-4f9e-aff9-3f93cd158169.pdf"},{"id":90506341,"identity":"00668973-2b5c-46d5-82f6-5488062eba75","added_by":"auto","created_at":"2025-09-03 12:50:12","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11066,"visible":true,"origin":"","legend":"Lifespan Source Data","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7217416/v1/623c9b9ddadaf6b5e2ae38f7.xlsx"},{"id":90506336,"identity":"8115184c-02bb-44a3-a7a4-3133b1979d54","added_by":"auto","created_at":"2025-09-03 12:50:12","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13282,"visible":true,"origin":"","legend":"Lifespan Source Data","description":"","filename":"Table2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7217416/v1/f559ef895d25557c9668b82c.xlsx"},{"id":90506340,"identity":"a15eb206-00bd-4ec6-8e03-2a61a15a14c9","added_by":"auto","created_at":"2025-09-03 12:50:12","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":91708,"visible":true,"origin":"","legend":"NHR-49 binding targets","description":"","filename":"Table3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7217416/v1/5b76928deeb821b183b21357.xlsx"},{"id":90506344,"identity":"a72a6411-54eb-4896-b9ee-426b39607b12","added_by":"auto","created_at":"2025-09-03 12:50:12","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1256094,"visible":true,"origin":"","legend":"RNAseq Analysis","description":"","filename":"Table4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7217416/v1/a93621310e9baeefcd9901b7.xlsx"},{"id":90507555,"identity":"7865b279-cb5c-41c3-a8da-87bf71d9fdf1","added_by":"auto","created_at":"2025-09-03 12:58:12","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":10538,"visible":true,"origin":"","legend":"CUT\u0026RUN and RNAseq nhr-49 overlapping targets","description":"","filename":"Table5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7217416/v1/32635efd7b71e443f0d81c7b.xlsx"},{"id":90507556,"identity":"7a11bba4-3841-41ab-a3af-76c29d76dacf","added_by":"auto","created_at":"2025-09-03 12:58:12","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":2912353,"visible":true,"origin":"","legend":"","description":"","filename":"suppFigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-7217416/v1/b89e35b54b7901a91a178b3d.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"A somatic checkpoint through NHR-49/HNF4α governs reproductive investment and longevity in C. elegans","fulltext":[{"header":"Introduction","content":"\u003cp\u003eReproduction is one of the most energetically demanding biological processes\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, requiring organisms to tightly coordinate reproductive activity with metabolic status\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In nutrient-poor conditions, animals often suppress germline proliferation, delay ovulation, or halt reproduction entirely to conserve resources\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. These adjustments are especially crucial for optimizing reproductive success and ensuring the survival of both parent and offspring\u003csup\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. However, the molecular mechanisms that link nutritional cues to reproductive timing remain incompletely understood.\u003c/p\u003e\u003cp\u003e\u003cem\u003eC. elegans\u003c/em\u003e are primarily hermaphrodites and possess both oocytes and sperm, making them capable of self-fertilization\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. During the larval stage 4 (L4), the germline stem cells in the distal region actively proliferate, and the cells further away from the distal tip cell undergo meiosis, where they take up the sperm fate and the developed sperm are stored in the spermatheca\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. From the young adult stage onwards, germ cells switch fate to an oogenic fate and the developing oocytes are arrested in meiosis I and stored in the oviduct\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. A highly regulated process enables the oocytes most proximal to the spermatheca to sense the presence of sperm, become activated, i.e. begin to progress pass meiosis I, and simultaneously be ovulated into the spermatheca where they become fertilized; the fertilized embryos continue to develop in the uterus until they are laid outside of the parent at around 300 cells stage\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. The FEM (feminization) and FOG (feminization of germline) genes are crucial regulators of sex determination and spermatogenesis in \u003cem\u003eC. elegans\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Loss-of-function mutations of \u003cem\u003efem-3\u003c/em\u003e, \u003cem\u003efog-2\u003c/em\u003e or \u003cem\u003efog-3\u003c/em\u003e result in defective spermatogenesis and feminization of worms that are incapable of self-fertilization \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e\u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. Similar to hermaphrodites, the germline in these mutants contain mitotic and meiotic cells; However, the germ cells do not take up the spermatogenic fate during development. This results in an organism that has no sperm in their spermatheca. In the absence of males, these mutants accumulate\u0026thinsp;~\u0026thinsp;20 oocytes in their oviduct and the process of germline stem cell proliferation and differentiation appear paused. Upon mating with males, or when sperm are provided \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, the feminized mutants undergo normal stem cell proliferation and differentiation into oocytes, which are fertilized by the sperm. Mated feminized mutants produce similar number of healthy progenies as for mated hermaphrodites. Thus, feminized mutants in \u003cem\u003eC. elegans\u003c/em\u003e resemble females of non-hermaphroditic species\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Feminized \u003cem\u003eC. elegans\u003c/em\u003e mutants have been crucial in revealing the molecular pathways that regulate how oocytes sense sperm and become activated and ovulated\u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Interestingly, we recently reported that unmated feminized mutants store elevated levels of fat and exhibit extended lifespan, likely due to a lack of progeny production and consequent reduced energy expenditure\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn \u003cem\u003eC. elegans\u003c/em\u003e, the nuclear hormone receptor NHR-49\u0026mdash;a putative functional homolog of mammalian PPARα and structural homolog of HNF4\u0026mdash;is a central regulator of lipid metabolism and energy homeostasis\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Like all nuclear hormone receptors, NHR-49 is a DNA binding transcription factor that is regulated through ligand binding\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. NHR-49 is known to regulate the expression of both lipid synthesis and lipid hydrolysis genes\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. For example, NHR-49 transcriptionally regulates stearoyl-CoA desaturation genes \u003cem\u003efat-6\u003c/em\u003e and \u003cem\u003efat-7\u003c/em\u003e, which maintain membrane fluidity and polyunsaturated fatty acid synthesis\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. It also activates \u003cem\u003eacs-2\u003c/em\u003e, a gene involved in mitochondrial β-oxidation\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In addition to fat metabolism, NHR-49 is known to also regulate oxidative stress response\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, hypoxia adaptation\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, peroxisome proliferation\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, pathogen avoidance\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, innate immunity\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003c/sup\u003e and longevity\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Interestingly, NHR-49 has been suggested to participate in regulating adult reproductive diapause, a state in which severely starved L4 worms undergo extensive germline attrition and the animal can survive for a period of several weeks \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, and the germline can regenerate when food become available again.\u003c/p\u003e\u003cp\u003eUsing feminized mutant C. elegans as a model, we uncovered a previously unrecognized role for NHR-49 in regulating oocyte activation and ovulation in a cell non-autonomous manner. We observed that loss of \u003cem\u003enhr-49\u003c/em\u003e results in inappropriate oocyte activation and ovulation, and laying of unfertilized oocytes even in the absence of sperm signal. This inappropriate laying of unfertilized oocytes leads to excessive yolk and fat loss and drastically shortening of lifespan. Notably, when yolk transport to oocytes is blocked, thereby retaining yolk within the parent, fat storage is largely restored in nhr-49 mutant and their shortened lifespan is also partially rescued. Furthermore, \u003cem\u003enhr-49\u003c/em\u003e mutants failed to halt oocyte proliferation under nutrient-restricted conditions, suggesting a disruption in the organism\u0026rsquo;s ability to couple reproductive output to nutritional status. Together, our findings suggest that NHR-49 plays a broader role in coordinating energy metabolism and reproductive restraint, and in doing so, orchestrate the balance between parental resource management and reproductive success, with a major impact on longevity.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eNHR-49 is required for the longer lifespan and elevated fat storage of feminized \u003cem\u003eC. elegans\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003eOur lab recently reported that when not mated, feminized mutants exhibit significant lifespan extension and elevated fat storage compared to wild-type N2 (Wild-type) hermaphroditesat 25\u0026deg;C\u003cstrong\u003e (Fig. 1B)\u003c/strong\u003e\u003csup\u003e2\u003c/sup\u003e. While investigating the molecular factors required for the lifespan extension and fat metabolism of feminized worms, we identified NHR-49, a key metabolic regulator \u003csup\u003e28,28,29,33,34\u003c/sup\u003e, as essential for the enhanced lifespan of feminized worms \u003cstrong\u003e(Fig. 1B)\u003c/strong\u003e. Loss of \u003cem\u003enhr-49\u003c/em\u003e drastically reduced the lifespan of feminized worms, where the double mutant \u003cem\u003enhr-49(gk405);fem-3(e1996)\u003c/em\u003e had lifespan similar to the short lifespan of the \u003cem\u003enhr-49(gk405)\u003c/em\u003e single mutant\u003csup\u003e33\u003c/sup\u003e. \u003c/p\u003e\n\n\u003cp\u003eWhile characterizing the \u003cem\u003enhr-49(-);fem-3(-) \u003c/em\u003edouble mutant, we noticed a surprising and interesting phenotype, where these feminized mutants laid unfertilized oocytes during early adulthood (\u003cstrong\u003eFig. 1C\u003c/strong\u003e). In \u003cem\u003eC. elegans\u003c/em\u003e, oocyte maturation and ovulation are strictly dependent on the presence of sperm, and consequently, feminized mutants lacking sperm retain their oocytes within the oviduct\u003csup\u003e15\u003c/sup\u003e. Accordingly, we observed no oocytes laid on the plate for \u003cem\u003efem-3(-)\u003c/em\u003e mutants \u003cstrong\u003e(Fig. 1C and 1D)\u003c/strong\u003e\u003csup\u003e3\u003c/sup\u003e. When examining \u003cem\u003enhr-49(-);fem-3(-)\u003c/em\u003e\u003cem\u003e \u003c/em\u003edouble mutants, we observed that these worms laid their unfertilized oocytes even though they lack sperm \u003cstrong\u003e(Fig. 1 C and D)\u003c/strong\u003e\u003cem\u003e.\u003c/em\u003e Similar observation was made with a different feminized mutant strain\u003csup\u003e35\u003c/sup\u003e \u003cem\u003efog-2(-)\u003c/em\u003e and \u003cem\u003enhr-49(-);fog-2(-)\u003c/em\u003e \u003cstrong\u003e(Fig. 1D)\u003c/strong\u003e.\u003cem\u003e \u003c/em\u003eNHR-49, and its homologs in other species, have not been implicated in regulating oocyte ovulation. To test whether NHR-49 uniquely regulates oocytes retention only in feminized mutants or whether it represents a more general role of NHR-49, we monitored \u003cem\u003enhr-49(-) \u003c/em\u003emutant\u003cem\u003e \u003c/em\u003ehermaphrodites. \u003cem\u003eC. elegans\u003c/em\u003e hermaphrodites reach the end of their reproductive period around day 4 of adulthood at 20\u0026deg;C, when their sperm supply is depleted and the remaining oocytes are retained in the oviduct, mirroring the reproductive state of feminized mutants\u003csup\u003e36\u003c/sup\u003e. We first examined \u003cem\u003enhr-49\u003c/em\u003e single mutant hermaphrodites at this post-reproductive stage and observed that \u003cem\u003enhr-49(-)\u003c/em\u003e mutants also laid unfertilized oocytes when examined on day 6 of adulthood \u003cstrong\u003e(Fig. S1B)\u003c/strong\u003e, a phenotype similar that observed for \u003cem\u003enhr-49(-);fem-3(-) \u003c/em\u003edouble mutants \u003cstrong\u003e(Fig. 1C)\u003c/strong\u003e. We additionally monitored egg laying of \u003cem\u003enhr-49(-)\u003c/em\u003e single mutant compared to wild-type hermaphrodites and observed that \u003cem\u003enhr-49(-)\u003c/em\u003e laid reduced number of fertilized embryos, but significantly higher number of unfertilized oocytes, throughout the reproductive period, as well as during the post-reproductive period (i.e. \u0026gt;120 hrs after reaching adulthood) \u003cstrong\u003e(Fig. S1B, S1D)\u003c/strong\u003e. This unexpected finding likely explains the previously reported lower brood size phenotype associated with the \u003cem\u003enhr-49(-)\u003c/em\u003e mutant\u003csup\u003e26\u003c/sup\u003e (\u003cstrong\u003eFig. S1A, C)\u003c/strong\u003e. Together, our results uncovered a new and fundamental role of NHR-49 in retention of unfertilized oocytes, with this function becoming particularly evident in feminized mutants where sperm is absent throughout development.\u003c/p\u003e\n\n\u003cp\u003eDifferent types of self-sterile \u003cem\u003eC. elegans\u003c/em\u003e mutants, including feminized \u003cem\u003efem-3(-)\u003c/em\u003e mutants, accumulate excess fat\u003csup\u003e18\u003c/sup\u003e. Here, we found that the increased fat accumulation in \u003cem\u003efem-3(-)\u003c/em\u003e worms is dependent on NHR-49\u003cem\u003e. \u003c/em\u003eWe used Oil red O (ORO) staining\u003csup\u003e37\u003c/sup\u003e to visualize storage lipid, and observed that both hermaphrodites and feminized mutant worms lacking \u003cem\u003enhr-49\u003c/em\u003e showed significantly lower ORO staining on day 6 adulthood \u003cstrong\u003e(Fig. 2 A and B)\u003c/strong\u003e. Interestingly, when comparing the ORO levels between day 1 and day 6 of adulthood in the different strains, wild type showed significant increase in stored fat as they progressed towards post-reproductive period, as previously published \u003csup\u003e38\u003c/sup\u003e\u003csup\u003e-\u003c/sup\u003e\u003csup\u003e39\u003c/sup\u003e. \u003cem\u003efem-3(-)\u003c/em\u003e mutants showed no significant change during this timeframe. Hermaphrodites and feminized mutants lacking \u003cem\u003enhr-49\u003c/em\u003e showed drastic reduction in ORO from day 1 to day 6 \u003cstrong\u003e(Fig. 2 A and B)\u003c/strong\u003e, suggesting a depletion of stored fat in worms lacking \u003cem\u003enhr-49\u003c/em\u003e, which appears coincidental to the time-period where mutants lacking \u003cem\u003enhr-4\u003c/em\u003e9 are expelling their unfertilized oocytes \u003cstrong\u003e(Fig. S1)\u003c/strong\u003e\u003cem\u003e. \u003c/em\u003eAdult \u003cem\u003eC. elegans \u003c/em\u003ehermaphrodites are known to transfer lipid-bound yolk to their oocytes to support embryonic development\u003csup\u003e40\u003c/sup\u003e. Feminized worms have increased yolk accumulation due to a lack of fertilization and embryo formation, which can be visualized using VIT-2::GFP\u003csup\u003e41\u003c/sup\u003e, a reporter strain for one of the vitellogenin proteins. We observed that \u003cem\u003enhr-49(-);fem-3(-)\u003c/em\u003e double mutants, which lay their unfertilized oocytes, display reduced VIT-2::GFP signal by day 2 of adulthood, which parallel the reduced ORO in day 6 fem-3(-); \u003cem\u003enhr-49(-)\u003c/em\u003e double mutants \u003cstrong\u003e(Fig. S2A)\u003c/strong\u003e. \u003c/p\u003e\n\n\u003cp\u003eSince both hermaphrodites\u003csup\u003e33\u003c/sup\u003e and feminized mutants lacking \u003cem\u003enhr-49(-) \u003c/em\u003eare short-lived \u003cstrong\u003e(Fig. 1B)\u003c/strong\u003e and we found that they rapidly lose yolk by day 2 of adulthood \u003cstrong\u003e(Fig. S2)\u003c/strong\u003e, we hypothesized that attenuating yolk loss would help to preserve stored fat and lifespan. In \u003cem\u003eC. elegans\u003c/em\u003e, yolk produced in the intestine is transported to oocytes via the endocytic receptor RME-2\u003csup\u003e42\u003c/sup\u003e. As expected, RNAi knockdown of \u003cem\u003erme-2 \u003c/em\u003eresulted in yolk retention in \u003cem\u003enhr-49(-);fem-3(-)\u003c/em\u003e\u003cem\u003e \u003c/em\u003edouble mutants, observed as increased VIT-2::GFP reporter signal \u003cstrong\u003e(Fig. S2A and B)\u003c/strong\u003e. Yolk retention also largely restored fat storage, as indicated by ORO staining \u003cstrong\u003e(Fig. 2 C and D)\u003c/strong\u003e. Interestingly, knockdown of \u003cem\u003erme-2 \u003c/em\u003epartially restored the lifespan of both \u003cem\u003enhr-49(-);fem-3(-)\u003c/em\u003e\u003cem\u003e \u003c/em\u003edouble mutants and \u003cem\u003enhr-49(-) \u003c/em\u003esingle mutants \u003cstrong\u003e(Fig. 2E)\u003c/strong\u003e. Taken together, these findings indicated that laying of unfertilized oocytes is accompanied by shunting of yolk, which largely accounts for the rapid fat loss and partially accounts for the shortened lifespan of both hermaphrodites and feminized worms lacking \u003cem\u003enhr-49(-). \u003c/em\u003e\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eNHR-49 prevents oocyte activation and ovulation in the absence of sperm\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003eWe uncovered a new role of NHR-49 in preventing the laying of unfertilized oocytes, which appears to be coupled to the fat loss and shortened lifespan of \u003cem\u003enhr-49(-)\u003c/em\u003e mutants. To understand the molecular basis of inappropriate oocyte lay upon loss of \u003cem\u003enhr-49\u003c/em\u003e, we first characterized the activation status of the oocytes. In \u003cem\u003eC. elegans\u003c/em\u003e, oocytes are naturally arrested in meiotic prophase I and require sperm-derived signals to resume meiotic progression, as part of oocyte maturation, ovulation, and subsequent fertilization. Doubly phosphorylated MPK-1 (dpMPK-1), a homolog of the ERK MAP kinase, is a well-established marker of oocyte activation\u003csup\u003e16\u003c/sup\u003e \u003cstrong\u003e(Fig. 3A)\u003c/strong\u003e. We performed dpMPK-1 immunostaining of day-1 adults to examine the status of oocyte activation in hermaphrodites and feminized mutants with or without \u003cem\u003enhr-49\u003c/em\u003e. While fertile wild-type worms showed the expected dpMPK-1 signal in the oocytes proximal to the spermatheca, feminized mutants, both \u003cem\u003efem-3(-)\u003c/em\u003e and \u003cem\u003efog-3(-)\u003c/em\u003e\u003csup\u003e17\u003c/sup\u003e, did not \u003cstrong\u003e(Fig. 3A, B and S3A)\u003c/strong\u003e, indicative of a loss of oocyte activation in the absence of sperm. Loss of \u003cem\u003enhr-49\u003c/em\u003e did not substantially impact dpMPK-1 signal in reproductive hermaphrodites on day 1 of adulthood \u003cstrong\u003e(Fig. 3A and B)\u003c/strong\u003e. Interestingly, loss of \u003cem\u003enhr-49 \u003c/em\u003eresulted in prominent dpMPK-1 signal in the proximal oocytes of feminized worms \u003cstrong\u003e(Fig. 3A, B, S3A, B)\u003c/strong\u003e, which was comparable to that in hermaphrodites, indicating inappropriate activation of the oocyte maturation pathway despite the absence of sperm. Similar inappropriate dpMPK-1 activation was also observed in post reproductive \u003cem\u003enhr-49(-)\u003c/em\u003e mutant hermaphrodites \u003cstrong\u003e(Fig. S3C, D)\u003c/strong\u003e.\u003c/p\u003e\n\n\u003cp\u003eOocytes arrested in meiotic prophase I maintain minimal protein quality control activity, resulting in the accumulation of protein aggregates throughout the cytoplasm\u003csup\u003e5\u003c/sup\u003e. During oocyte maturation, proximal oocytes eliminate these protein aggregates to prepare for fertilization\u003csup\u003e43\u003c/sup\u003e \u003cstrong\u003e(Fig. 3A)\u003c/strong\u003e. To further assess the activation status of the oocytes in mutants lacking \u003cem\u003enhr-49\u003c/em\u003e, we examined protein aggregate clearance in feminized strains with or without \u003cem\u003enhr-49\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eusing the HCP-1::GFP reporter strain that has been previously used to detect protein aggregates in \u003cem\u003eC. elegans\u003c/em\u003e oocytes\u003csup\u003e43\u003c/sup\u003e. In day-1 adults, as expected, \u003cem\u003efem-3(-) \u003c/em\u003esingle mutants had significantly higher HCP-1::GFP signal \u003cstrong\u003e(Fig. 3B) \u003c/strong\u003ein their proximal oocytes compared to reproductive wild-type\u003cem\u003e \u003c/em\u003ehermaphrodites, consistent with that the oocytes are not activated and accumulate protein aggregates. RNAi knockdown of \u003cem\u003enhr-49 \u003c/em\u003ein feminized worms resulted in aberrant loss of HCP-1::GFP signal \u003cstrong\u003e(Fig. 3B)\u003c/strong\u003e in the proximal oocytes, further supporting inappropriate activation of the oocytes when \u003cem\u003enhr-49\u003c/em\u003e is lost. \u003c/p\u003e\n\u003cp\u003eIn summary, in both post-reproductive hermaphrodites and feminized mutants, loss of \u003cem\u003enhr-49\u003c/em\u003e leads to misactivation of oocytes, as well as laying of unfertilized oocytes. We therefore conclude that the indiscriminate laying of oocytes in the absence of sperm upon loss of NHR-49 occurs through misactivation of oocytes and their subsequent ovulation.\u003c/p\u003e\n\n\u003cp\u003eIn C. \u003cem\u003eelegans\u003c/em\u003e, successful reproduction requires continuous oocyte production to replace those that are fertilized and laid. This process depends on two key processes: active proliferation of the mitotic stem cells in the distal region of the germline, and subsequent oogenesis in the meiotic region. Since unfertilized oocytes were laid in worms lacking \u003cem\u003enhr-49\u003c/em\u003e, we predicted that germline stem cell proliferation and/or oogenesis would be enhanced. We assessed mitotic proliferation by EdU labelling\u003csup\u003e44\u003c/sup\u003e, which marks nuclei undergoing DNA synthesis, and oogenesis by immunostaining for dpMPK-1. Feminized mutants normally produce only a fixed number of oocytes due to them being retained in the oviduct, and as expected, they showed reduced number of EdU+ nuclei in the mitotic region \u003cstrong\u003e(Fig. 3C) \u003c/strong\u003eand lower dpMPK-1 signal intensity in the meiotic region \u003cstrong\u003e(Fig. S4A)\u003c/strong\u003e compared to wild-type control. Examination of \u003cem\u003enhr-49(-);fem-3(-)\u003c/em\u003e double mutant worms revealed aberrant proliferation of the distal germline stem cells as evident by higher levels of EdU+ nuclei in the mitotic region and inappropriate oogenesis as indicated by increased dpMPK-1 staining in the meiotic region \u003cstrong\u003e(Fig. 3C and S4A)\u003c/strong\u003e. Consistently, post-reproductive \u003cem\u003enhr-49\u003c/em\u003e mutant hermaphrodites also showed a significant elevation of dpMPK-1 signal in the meiotic region compared to wild-type\u003cem\u003e \u003c/em\u003eworms at the same age, suggestive of aberrant oogenesis \u003cstrong\u003e(Fig. S4B)\u003c/strong\u003e. Collectively, these data revealed that both proliferation of the mitotic stem cells and oogenesis are aberrantly activated upon loss of NHR-49, despite the absence of sperm. However, it is not clear whether aberrant activation of oocytes and subsequent laying of unfertilized oocytes triggered distal germline proliferation and oogenesis or \u003cem\u003evice versa\u003c/em\u003e in \u003cem\u003enhr-49\u003c/em\u003e mutants. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eNHR-49 is required to couple nutritional status to germline proliferation\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003eNHR-49 is a well-established metabolic regulator, with particular importance in maintaining lipid homeostasis upon starvation \u003csup\u003e23,45\u003c/sup\u003e. NHR-49 has also been suggested to participate in starvation-induced germline attrition, a process known as adult reproductive diapause\u003csup\u003e46\u003c/sup\u003e. We therefore wondered whether the role of NHR-49 in the germline, as detailed above, is also sensitive to nutritional status. We monitored germline proliferation in reproductive hermaphrodites using EdU\u003csup\u003e44\u003c/sup\u003e labeling and, as expected, observed that germline proliferation was significantly reduced in response to starvation (24 hours) in wild-type worms. We found that \u003cem\u003enhr-49(-)\u003c/em\u003e single mutant showed reduced germline proliferation as evident by lower EdU+ nuclei in the distal region. Notably, we did not observe further reduction of EdU+ nuclei upon starvation in the \u003cem\u003enhr-49(-)\u003c/em\u003e mutants \u003cstrong\u003e(Fig. 4A), \u003c/strong\u003esuggesting that NHR-49 is required for coupling nutritional status to germline proliferation. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eNHR-49 may regulate oocyte activation through the somatic gonadal sheath cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo dissect the pathway through which NHR-49 regulates oocyte activation and ovulation, we examined its genetic interactions with known regulators of ovulation that act in distinct tissues. MSPs (Major Sperm Proteins) are sperm-derived cytoskeletal proteins that are also secreted as signaling molecules to promote oocyte maturation and sheath cell contraction\u003csup\u003e47\u003c/sup\u003e. \u003cem\u003evab-1\u003c/em\u003e, encoding an ephrin receptor expressed on oocytes, and \u003cem\u003eceh-18\u003c/em\u003e, a homeodomain transcription factor essential for sheath cell function, are both required for MSP-dependent oocyte activation. Given that sheath cells act as key MSP sensors, analogous to mammalian cumulus granulosa cells, these factors provide an entry point to determine the tissue of NHR-49 action. In feminized animals, RNAi knockdown of \u003cem\u003evab-1\u003c/em\u003e or \u003cem\u003eceh-18\u003c/em\u003e induces oocyte laying, where the effect of \u003cem\u003evab-1\u003c/em\u003e RNAi is modest, consistent with previous findings \u003csup\u003e17\u003c/sup\u003e. Compared to \u003cem\u003evab-1\u003c/em\u003e RNAi, knockdown of \u003cem\u003enhr-49\u003c/em\u003e led to a significantly higher number of oocytes laid\u0026mdash;nearly 6\u0026ndash;10 times more than \u003cem\u003evab-1\u003c/em\u003e knockdown alone. Knockdown of \u003cem\u003enhr-49\u003c/em\u003e in \u003cem\u003evab-1\u003c/em\u003e mutants resulted in an oocyte-laying phenotype that was comparable to \u003cem\u003enhr-49\u003c/em\u003e RNAi alone \u003cstrong\u003e(Fig. 5A)\u003c/strong\u003e, suggesting a shared or converging pathway; However, given the mild effect of \u003cem\u003evab-1\u003c/em\u003e loss, it is also possible that an additive effect would be difficult to detect. Combining RNAi knockdown of \u003cem\u003enhr-49\u003c/em\u003e and \u003cem\u003eceh-18 \u003c/em\u003emutation also did not show additive effect, again suggesting that \u003cem\u003enhr-49\u003c/em\u003e functions in the same pathway as \u003cem\u003eceh-18\u003c/em\u003e \u003cstrong\u003e(Fig. 5B)\u003c/strong\u003e. These results suggested that NHR-49 might act in both the oocytes and the sheath cells to regulate oocyte ovulation. \u003c/p\u003e\n\u003cp\u003eTo further test whether NHR-49 acts cell-autonomously in oocytes or from somatic tissues to regulate oocyte ovulation, we used a germline-specific RNAi approach, where \u003cem\u003erde-1\u003c/em\u003e null mutant is complemented with functional RDE-1 only in the germline (\u003cem\u003erde-1(-)\u003c/em\u003e; \u003cem\u003ePsun-1\u003c/em\u003e::RDE-1)\u003csup\u003e48\u003c/sup\u003e. Germline-specific knockdown of \u003cem\u003enhr-49\u003c/em\u003e in feminized worms failed to recapitulate the oocyte-laying phenotype and dpMPK-1 misregulation observed with whole-body \u003cem\u003enhr-49\u003c/em\u003e loss \u003cstrong\u003e(Fig. 5C\u0026ndash;E)\u003c/strong\u003e. These results indicate that NHR-49 is largely dispensable in the germline and instead acts through somatic tissues\u0026mdash;likely the sheath cells\u0026mdash;to control oocyte activation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenome-wide analyses identify metabolic and stress-related transcriptional targets of NHR-49\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the transcriptional outputs of NHR-49, we performed CUT\u0026amp;RUN, a chromatin profiling method that maps protein-DNA interactions, in day 1 adult wild-type animals using the CRISPR-engineered \u003cem\u003enhr-49(hj293)\u003c/em\u003e strain, which harbors a GFP::TEV::3xHA tag at the C - terminus of the endogenous \u003cem\u003enhr-49\u003c/em\u003e locus \u003csup\u003e26\u003c/sup\u003e. This profiling revealed 2,227 high-confidence NHR-49 binding sites, with the majority of peaks localized to gene promoters and transcription start sites \u003cstrong\u003e(Fig. S8A\u0026ndash;B, Table 3)\u003c/strong\u003e consistent with a role of NHR-49 in transcriptional regulation. Gene set enrichment analysis \u003csup\u003e49\u003c/sup\u003e of NHR-49-bound genes highlighted key categories related to lipid metabolism, mitochondrial fatty acid oxidation, and detoxification pathways \u003cstrong\u003e(Fig. S8C\u0026ndash;D)\u003c/strong\u003e. \u003c/p\u003e\n\u003cp\u003eTo assess the functional impact of NHR-49 binding, we performed RNA-seq on \u003cem\u003enhr-49(-)\u003c/em\u003e mutants and wild-type controls at the day 1 of adulthood. Differential expression analysis revealed significant up- and downregulation of genes associated with lipid metabolism, innate immunity, and extracellular matrix remodeling \u003cstrong\u003e(Fig. S7A\u0026ndash;B, Table 4)\u003c/strong\u003e. As an additional quality control, we compared our \u003cem\u003enhr-49(-)\u003c/em\u003e RNA-seq data to previously published transcriptomic profiles of \u003cem\u003enhr-49\u003c/em\u003e mutants. We found that the three data sets showed significant overlap, confirming a shared core set of NHR-49\u0026ndash;dependent gene expression changes across studies \u003cstrong\u003e(Fig. S7E\u0026ndash;F)\u003c/strong\u003e. However, each dataset also displays distinct subsets of regulated genes, suggesting that chronic loss of \u003cem\u003enhr-49\u003c/em\u003e function can lead to compensatory or context-specific transcriptional adaptations. \u003c/p\u003e\n\u003cp\u003eIntegrating CUT\u0026amp;RUN and RNA-seq data revealed 27 genes that were both bound by NHR-49 and differentially expressed in \u003cem\u003enhr-49\u003c/em\u003e mutants \u003cstrong\u003e(Fig. S7C, Table 5)\u003c/strong\u003e, suggesting they represent direct transcriptional targets. Gene set enrichment analysis indicated that the upregulated genes are enriched for lipid biosynthesis and pathogen stress responses, and the downregulated genes are enriched for mitochondrial function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNHR-49 represses \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003egsa-1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e expression to prevent inappropriate oocyte activation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong the NHR-49-bound targets, we identified \u003cem\u003egsa-1\u003c/em\u003e, which encodes a G\u0026alpha;s subunit known to act in the gonadal sheath cells to mediate MSP signaling and regulate oocyte ovulation \u003csup\u003e50\u003c/sup\u003e. NHR-49 binding peaks were detected at the promoter and second intron of \u003cem\u003egsa-1\u003c/em\u003e \u003cstrong\u003e(Fig. 6C)\u003c/strong\u003e. Interestingly, RNA-seq indicated that \u003cem\u003egsa-1\u003c/em\u003e RNA expression was modestly elevated in \u003cem\u003enhr-49\u003c/em\u003e mutant \u003cstrong\u003e(Fig. 6A)\u003c/strong\u003e, albeit not reaching significance. Importantly, functional experiments showed that RNAi knockdown of \u003cem\u003egsa-1\u003c/em\u003e suppressed the excessive oocyte laying phenotype of \u003cem\u003enhr-49(-);fem-3(-)\u003c/em\u003e animals \u003cstrong\u003e(Fig. 6B)\u003c/strong\u003e, indicating that elevated gsa-1 expression likely contributes to the aberrant laying of unfertilized oocytes. Taken together, the data suggested that NHR-49 acts, at least in part, through repressing \u003cem\u003egsa-1\u003c/em\u003e expression to constrain inappropriate oocyte activation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur findings uncover a previously unrecognized role for the nuclear hormone receptor NHR-49 in regulating oocyte activation and ovulation in \u003cem\u003eC. elegans\u003c/em\u003e. This reproductive role appears to be mediated through the transcriptional repression of \u003cem\u003egsa-1\u003c/em\u003e, a Gαs protein that promotes oocyte activation, and likely occurs via the gonadal sheath cells rather than the germline itself. We find that inappropriate oocyte activation and laying of unfertilized oocytes lead to premature yolk loss and depletion of parental resources. Suppressing yolk loss can partially restore lifespan, linking the reproductive phenotype to diminished somatic maintenance, and offers a partial explanation of the previously described lifespan shortening phenotype of the \u003cem\u003enhr-49\u003c/em\u003e mutant. While NHR-49 has been extensively characterized as a regulator of lipid metabolism and lifespan, our data add a new dimension to the understanding of how metabolic regulators like NHR-49 influence longevity\u0026mdash;not only by altering fat metabolism, but by controlling the timing and scale of reproductive investment.\u003c/p\u003e\u003cp\u003eOur data suggest NHR-49 is required to suppress germline proliferation in adult worms subjected to acute nutrient deprivation. We found that \u003cem\u003enhr-49\u003c/em\u003e mutants fail to reduce mitotic activity\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e in the distal germline following short-term starvation, suggesting that NHR-49 mediates nutrient-responsive germline restraint. NHR-49 is also known to be required for adult reproductive diapause (ARD)\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e\u0026mdash;a distinct starvation-induced state in L3/L4 animals characterized by drastic germline shrinkage. While the mechanisms underlying these two responses may differ, the shared requirement for NHR-49 raises the possibility that it acts as a conserved metabolic regulator coordinating reproductive quiescence in response to energetic stress. Given that specific dietary and microbiota-derived metabolites can activate NHR-49\u003csup\u003e40\u003c/sup\u003e, these findings support a broader model in which NHR-49 functions as a metabolic sensor linking environmental inputs to germline decisions.\u003c/p\u003e\u003cp\u003eOur CUT\u0026amp;RUN profiling revealed that NHR-49 binds near the loci of over 100 other nuclear hormone receptors, suggesting that it may serve as a broader transcriptional regulator of the NHR network itself. This opens the possibility that NHR-49 tunes hormonal signaling pathways across tissues in response to nutrient state. Future CUT\u0026amp;RUN studies in fed versus starved animals could determine whether NHR-49 differentially engages or recruits other NHRs depending on metabolic conditions. Additionally, future studies employing acute depletion system will likely reveal the immediate, direct transcriptional responses to NHR-49 loss from secondary effects that accumulate in constitutive mutants.\u003c/p\u003e\u003cp\u003eNHR-49 regulates \u003cem\u003egsa-1\u003c/em\u003e, a Gαs protein that functions in the gonadal sheath cells to promote oocyte activation and ovulation. Germline-specific RNAi suggests that NHR-49 does not act through the germline to regulate laying of unfertilized oocytes. Based on the known site of action of GSA-1 in the gonadal sheath cells, the observation that \u003cem\u003egsa-1\u003c/em\u003e expression appears elevated in \u003cem\u003enhr-49\u003c/em\u003e mutants, and the binding of NHR-49 to the promoter and intron regions of \u003cem\u003egsa-1\u003c/em\u003e, we suggest that NHR-49 constrain oocyte activation and ovulation in the absence of sperm by repressing \u003cem\u003egsa-1\u003c/em\u003e transcription in the sheath cells. Future tissue-specific studies will be important to confirm this proposed site of action.\u003c/p\u003e\u003cp\u003eTogether, these findings position NHR-49 as a metabolic gatekeeper that couples environmental and internal nutrient status to reproductive restraint. By limiting oocyte activation and germline proliferation when resources or sperm are lacking, NHR-49 helps align reproductive effort with metabolic capacity, promoting long-term survival and maternal resource preservation.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eC. elegans\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e culture methods and strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eC. elegans\u003c/em\u003e strains were maintained using standard protocols. Unless otherwise stated, all strains were grown on nematode growth medium (NGM) plates seeded with E. coli OP50-1 as a food source. The following strains were obtained from the Caenorhabditis Genetics Center (CGC): N2, DG2189 [\u003cem\u003efog-3(q443) I/hT2 [bli-4(e937) let-?(q782) qIs48] (I;III); tnIs13 [pie-1p::vab-1::GFP + unc-119(+)] ltIs44 [pie-1p::mCherry::PH(PLC1delta1) + unc-119(+)\u003c/em\u003e]; DG2190 [\u003cem\u003efog-3(q443) I/hT2 [bli-4(e937) let-?(q782) qIs48] (I;III); tnIs13 [pie-1p::vab-1::GFP + unc-119(+)]V\u003c/em\u003e]; VC470 [\u003cem\u003enhr-49 (gk405)\u003c/em\u003e]; DCL569 [\u003cem\u003emkcSi13[sun-1p::rde-1::sun-1 3\u0026apos;UTR + unc-119(+)] II; rde-1(mkc36)V\u003c/em\u003e]. These strains were previously generated in our laboratory, combining strains obtained from the CGC: IU613 [\u003cem\u003efem-3(e1996)/nT1 [qIs51] (IV;V)]; IU614 [fem-3(e1996)/nT1 [qIs51] (IV;V)\u003c/em\u003e]\u003c/p\u003e\n\n\n\u003cp\u003e\u003cstrong\u003eLifespan Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLifespan assays were performed as previously described\u003csup\u003e18\u003c/sup\u003e .10-15 gravid worms were placed on plates containing the desired bacterial food to obtain approximately 150-200 embryos. Upon reaching day one of adulthood, 30 worms were transferred to each of three RNAi plates and monitored every other day. Worms were gently transferred to fresh plates every other day to prevent starvation and mixing of generations. Care was taken to avoid transferring larvae. Lack of movement in response to gentle touch at the nose was used to confirm death. Worms that died due to rupturing, bursting, or leaving the plate were considered censored. The lifespan data was analyzed using the OASIS online survival analysis tool\u003csup\u003e78\u003c/sup\u003e available at https://sbi.postech.ac.kr/oasis2/, and log-rank tests were used to determine statistical significance between groups. \u003cstrong\u003eAll lifespan assays were performed at least twice.\u003c/strong\u003e\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eRNAi Treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RNAi procedure was carried out as previously described\u003csup\u003e18\u003c/sup\u003e. RNAi was conducted by feeding the worms with HT115 bacteria that expressed double-stranded RNA of the target gene. The RNAi clones were obtained from RNAi feeding libraries created by Julie Ahringer and Marc Vidal\u0026apos;s laboratories\u003csup\u003e76,77\u003c/sup\u003e. \u003c/p\u003e\n\n\n\u003cp\u003e\u003cstrong\u003epMPK-1 Immunostaining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApproximately 100 worms were grown on OP50-1 seeded NGM plates, then washed off using PBST (PBS + 0.1% Tween 20) and collected in a 1.5 ml Eppendorf tube. The worms were spun for 15 seconds at 2000 rpm, the supernatant was removed, and the pellet was washed with 1X PBS twice, using 1 ml each time. Approximately 50 worms in 100 \u0026micro;l of PBS buffer were then transferred using a cut 200 \u0026micro;l micropipette tip onto a border-painted glass slide. To anesthetize the worms, 5 \u0026micro;l of 5 mM levamisole was added. Using 25- or 26-gauge needles, a cut was made near the pharyngeal bulb of each worm to release gonads, and all animals were dissected within 5\u0026ndash;6 minutes. Dissected gonads were collected into a 1.5 ml tube using PBS-Tween\u0026ndash;coated, cut-end 200 \u0026micro;l tips. Excess liquid was removed, and 200 \u0026micro;l of 4% formaldehyde fixative (prepared by mixing 100 \u0026micro;l 10X phosphate buffer, 100 \u0026micro;l 37% formaldehyde, and 800 \u0026micro;l ddH₂O to make a total volume of 1 ml fixative) was added. Samples were incubated at room temperature for 20-30 minutes. Fixative was then removed, and worms were incubated in 1 ml of chilled methanol (\u0026ndash;20 \u0026deg;C) for 5 minutes. After removing methanol, worms were washed three times with PBST (5 minutes each). Excess liquid was removed, and worms were resuspended in 200 \u0026micro;l blocking buffer (0.1% Triton X-100, 0.1% Tween-20 and 2% BSA (Bovine Serum Albumin) in 1X PBS) and incubated for 1\u0026ndash;2 hours. Primary antibody diluted 1:400 in Blocking buffer (50 \u0026micro;l per sample) was added and incubated overnight at 4 \u0026deg;C with rocking. Samples were then washed three times with blocking buffer for 10 minutes each. Secondary antibody (1:200 dilution in blocking buffer, 50 \u0026micro;l per sample) along with DAPI (final concentration 5 \u0026micro;g/ml) was added, and samples were incubated for 2 hours at room temperature in the dark with rocking. Following three washes with blocking buffer, worms were resuspended in 10 \u0026micro;l of Vectashield mounting medium . Worms were mounted on a 2% agarose pad prepared on a glass slide and covered with a coverslip for microscopy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003e \u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEdU (5-ethynyl-2\u0026apos;-deoxyuridine) Staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEdU staining was performed as previously described, with modifications\u003csup\u003e82\u003c/sup\u003e, using the Click-iT\u0026reg; EdU Imaging Kit (ThermoFisher Cat. No. C10337). For EdU incorporation, 50-100 worms were washed and collected from plates using M9 buffer containing 0.1% Triton X-100 (M9T) and washed twice with 1 mL M9T. 50 \u0026micro;L of M9T containing the worms were transferred into a fresh 1.5 mL microcentrifuge tube using a cut tip, followed by the addition of 50 \u0026micro;L of 10 mM EdU. The worms were gently mixed and incubated on a shaker for 40 minutes at room temperature (22\u0026ndash;25\u0026deg;C). Worms were washed twice with 1 mL M9T. Next, 30 \u0026micro;L of EBT (prepared by mixing 110 \u0026micro;L 10X egg buffer, 10 \u0026micro;L 10% Tween-20, 6.5 \u0026micro;L 10% tetramisole, and 850 \u0026micro;L distilled water to a final volume of 976.5 \u0026micro;L) was pipetted onto a slide, and approximately 50 worms were transferred into the EBT drop. The worms were anesthetized for 10-15 seconds, and their heads were cut to extrude the gonads. The gonads were collected into a 1.5 mL microcentrifuge tube, fixed with 4% formaldehyde in egg buffer for 20-30 minutes, and washed twice with 1 mL of 3% BSA in PBS. For permeabilization, the gonads were incubated in 1 mL of 1% Triton X-100 in PBS at room temperature for 20 minutes. After permeabilization, the buffer was removed, and the samples were washed twice with 1 mL of 3% BSA in PBS. Subsequently, 0.5 mL of the Click-iT\u0026reg; reaction cocktail (as per the kit instructions) was added, and the worms were incubated at room temperature for 30 minutes on a shaker and covered to protect from light. The reaction cocktail was removed, and the samples were washed twice with 1 mL of 3% BSA in PBS. For the EdU chase experiment, after the 10 mM EdU incorporation step, worms were transferred to the feeding plates for 24 hours, and the subsequent steps were performed as described above. Finally, 10 \u0026micro;L of Vectashield was added, and the worms were mounted on a 2% agarose pad prepared on a glass slide for imaging. Images were acquired using a Keyence BZ-X810 microscope.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFluorescence quantification and imaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor imaging, 10-20 worms were immobilized on a 2% agarose pad prepared on a glass slide in 10 \u0026micro;L of 5 mM levamisole in PBS. Images were acquired using the appropriate fluorescence channel on Leica DM5000B compound microscope or a Zeiss LSM 710 or LSM 880 inverted confocal microscope. Images were acquired as Z-stacks, and image tiling was used to scan at 63X magnification under oil immersion. For data processing, ZEN software was used for image stitching and FIJI and was used for image analysis and processing. To generate representative images, Adobe Photoshop was used.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eRNA isolation and library preparation for RNA-seq\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA isolation and RNA-seq library preparation were performed as previously described \u003csup\u003e18\u003c/sup\u003e. For the collection of whole worm samples, approximately 200 worms were collected in TRI reagent (MRC, Cat. No. TR 118). For tissue-specific analysis, germlines were surgically isolated from 100-150 animals using fine-gauge needle dissection, with 80-100 intact germlines collected per sample in TRI reagent. Two to three biological replicates were collected for each genotype. RNA-seq libraries were prepared using the QuantSeq 3\u0026lsquo; mRNA-Seq Library Prep Kit for Illumina (FWD) (Lexogen), and sequencing was performed by the Biotechnology Resource Center (BRC) of the Cornell Institute of Biotechnology.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eRNA-seq data analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA-seq data analysis was performed as previously described \u003csup\u003e18\u003c/sup\u003e with minor modifications. To obtain the total read count of RNA-seq data were processed using Linux on the BioHPC server at Cornell University. Raw sequencing data underwent quality control and preprocessing using Trim Galore (v0.6.5), which incorporates Cutadapt\u003csup\u003e51\u003c/sup\u003e (v3.4) and FastQC (v0.11.8). Parameters were set to retain reads with quality scores \u0026ge;20 (-q 20) while performing FastQC quality assessment. Preprocessed reads were mapped to the \u003cem\u003eC. elegans\u003c/em\u003e reference genome (ce11/WBcel235) using STAR aligner (v2.7.9a)\u003csup\u003e52\u003c/sup\u003e, with parameters configured for strict alignment: two computing threads (--runThreadN 2), gene count quantification (--quantMode GeneCounts), unique mapping only (--outFilterMultiMapNmax 1), a maximum of two mismatches (--outFilterMismatchNmax 2), and coordinate-sorted BAM files (--outSAMtype BAM SortedByCoordinate). For 3\u0026apos; RNA-seq data analysis, the middle column of counts from the STAR-generated tab-delimited files was used to construct expression matrices for comparative analysis. Matrices were organized either by genotype (comparing different mutant strains under identical conditions) or by treatment (comparing identical genotypes under different RNAi conditions). Differential expression analysis was performed in R (RStudio) using DESeq2 (v1.34.0)\u003csup\u003e53\u003c/sup\u003e. To improve statistical power, genes with fewer than 10 reads across fewer than 2 samples were excluded. Principal component analysis was conducted on variance-stabilizing transformed data (using the vst function), visualized using the plotPCA function with default parameters, and further refined using ggplot2 (v3.3.6). Statistically significant differentially expressed genes were identified using the standard DESeq2 workflow with an adjusted p-value threshold of 0.05. For genes of interest, normalized counts were visualized using DESeq2\u0026apos;s plotCounts function. Gene set comparisons between genotypes or between RNA-seq and CUT\u0026amp;RUN datasets were performed using DeepVenn (https://www.deepvenn.com/)\u003csup\u003e54\u003c/sup\u003e online software, with statistical significance determined by Fisher\u0026apos;s exact test. Gene set enrichment analysis was conducted using WormCat 2.0 (www.wormcat.com)\u003csup\u003e49\u003c/sup\u003e, which also employs Fisher\u0026apos;s test with FDR correction. For heatmap generation, log\u003csub\u003e2\u003c/sub\u003e fold-change values were extracted from DESeq2 results tables without filtering out low-expression genes. Heatmaps were created using the ComplexHeatmap package (v2.10.0) with row clustering enabled (cluster_rows = TRUE), column clustering disabled (cluster_columns = FALSE), and row names hidden (show_row_names = FALSE). For clustered analyses, heatmaps were partitioned (split = 3), and genes from each cluster were extracted using the row_order function.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCUT\u0026amp;RUN for NHR-49 binding sites in the genome\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCUT\u0026amp;RUN experiments were performed using our previously published protocol\u003csup\u003e55,56\u003c/sup\u003e. A sufficient number of embryos were collected from gravid adult worms grown on 10-cm NGM plates seeded with OP50-1. For each CUT\u0026amp;RUN reaction, 3000 embryos were plated onto 15-cm NGM plates supplemented with 1 mL of 25\u0026times; concentrated OP50-1 culture (grown overnight). Sample preparation was scaled to ensure sufficient worm numbers (3000 animals per reaction) for each experimental condition. Plates were incubated at 25\u0026deg;C for approximately 48-52 hours until populations reached the predominantly young adult stage. At this developmental stage, worms were harvested by washing plates with PBS buffers, and CUT\u0026amp;RUN experiments were performed according to a detailed protocol previously published\u003csup\u003e55\u003c/sup\u003e which follows standard CUT\u0026amp;RUN methodology with minor modifications specific to \u003cem\u003eC. elegans\u003c/em\u003e samples. Sequencing libraries were constructed using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, catalog numbers E7645S and E7335S). Library amplification was performed with 14 PCR cycles according to the manufacturer\u0026apos;s guidelines, with slight procedural adjustments as described in the published protocol\u003csup\u003e57\u003c/sup\u003e .The resulting libraries underwent paired-end sequencing (2 \u0026times; 32 bp) on an Illumina NextSeq 500 platform, performed by the Biotechnology Resource Center (BRC) of the Cornell Institute of Biotechnology. All CUT\u0026amp;RUN experiments were conducted with at least two independent biological replicates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCUT\u0026amp;RUN Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCUT\u0026amp;RUN analysis was performed as previously described\u003csup\u003e55,57\u0026ndash;60\u003c/sup\u003e. \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank personnel at Cornell Genomics Center, Microscopy core and statistical consulting, and the Caenorhabditis Genetics Center (CGC) for all the worm strains used in this study. We thank B. Sanketi, A. Ren, N. Kurpios, \u0026nbsp;R. Sardana, K. Liu, D. Lin, G. Hollopeter members of the Cornell worm club, P. Cohen and members of the Cornell Reproductive Sciences Center, and the members of the Lee lab for their valuable discussion and feedback. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNational Institute of Aging R01 AG024425 (SSL); NIH 1S10RR025502 for Cornell Institute of Biotechnology. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, S.G., A.C., S.S.L.; Methodology, S.G., A.C., S.S.L.; Investigation, S.G., A.C.; Investigation: assistance with selected experiments, T.C., J.O., R.R., Data analysis, S.G., A.C.; Data curation, S.G., A.C.; visualization, S.G., A.C., S.S.L.; funding acquisition, S.S.L.; supervision, S.S.L.; writing \u0026ndash; original draft, S.G.; and writing \u0026ndash; review and editing, A.C., S.S.L.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and correspondence\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRequests for materials may be directed to [email protected] (SSL). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eStearns, S. C. Trade-Offs in Life-History Evolution. \u003cem\u003eFunct. Ecol.\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 259\u0026ndash;268 (1989).\u003c/li\u003e\n\u003cli\u003eDella Torre, S., Benedusi, V., Fontana, R. \u0026amp; Maggi, A. Energy metabolism and fertility: a balance preserved for female health. \u003cem\u003eNat. Rev. Endocrinol.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 13\u0026ndash;23 (2014).\u003c/li\u003e\n\u003cli\u003eMircea, C. N., Lujan, M. E. \u0026amp; Pierson, R. A. Metabolic fuel and clinical implications for female reproduction. \u003cem\u003eJ. Obstet. Gynaecol. Can. JOGC J. Obstet. Gynecol. Can. JOGC\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 887\u0026ndash;902 (2007).\u003c/li\u003e\n\u003cli\u003eLopez, A. L. \u003cem\u003eet al.\u003c/em\u003e DAF-2 and ERK Couple Nutrient Availability to Meiotic Progression during \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e Oogenesis. \u003cem\u003eDev. Cell\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 227\u0026ndash;240 (2013).\u003c/li\u003e\n\u003cli\u003eThondamal, M., Witting, M., Schmitt-Kopplin, P. \u0026amp; Aguilaniu, H. Steroid hormone signalling links reproduction to lifespan in dietary-restricted Caenorhabditis elegans. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 4879 (2014).\u003c/li\u003e\n\u003cli\u003eTempleman, N. M. \u0026amp; Murphy, C. T. Regulation of reproduction and longevity by nutrient-sensing pathways. \u003cem\u003eJ. Cell Biol.\u003c/em\u003e \u003cstrong\u003e217\u003c/strong\u003e, 93\u0026ndash;106 (2018).\u003c/li\u003e\n\u003cli\u003eCao, W. \u0026amp; Pocock, R. Mechanisms of germ cell survival and plasticity in Caenorhabditis elegans. \u003cem\u003eBiochem. Soc. Trans.\u003c/em\u003e \u003cstrong\u003e50\u003c/strong\u003e, 1517\u0026ndash;1526 (2022).\u003c/li\u003e\n\u003cli\u003eSahu, S. \u0026amp; Armstrong, A. R. Translation components in adult Drosophila melanogaster adipocytes regulate the ovarian germline stem cell lineage. 2024.08.31.610632 Preprint at https://doi.org/10.1101/2024.08.31.610632 (2024).\u003c/li\u003e\n\u003cli\u003eBrenner, S. The genetics of Caenorhabditis elegans. \u003cem\u003eGenetics\u003c/em\u003e \u003cstrong\u003e77\u003c/strong\u003e, 71\u0026ndash;94 (1974).\u003c/li\u003e\n\u003cli\u003ePazdernik, N. \u0026amp; Schedl, T. Introduction to Germ Cell Development in C. elegans. \u003cem\u003eAdv. Exp. Med. Biol.\u003c/em\u003e \u003cstrong\u003e757\u003c/strong\u003e, 1\u0026ndash;16 (2013).\u003c/li\u003e\n\u003cli\u003eKimble, J. \u0026amp; Crittenden, S. L. Germline proliferation and its control. \u003cem\u003eWormBook Online Rev. C Elegans Biol.\u003c/em\u003e 1\u0026ndash;14 (2005) doi:10.1895/wormbook.1.13.1.\u003c/li\u003e\n\u003cli\u003eDavis, G. M., Hipwell, H. \u0026amp; Boag, P. R. Oogenesis in Caenorhabditis elegans. \u003cem\u003eSex. Dev.\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 73\u0026ndash;83 (2023).\u003c/li\u003e\n\u003cli\u003eHodgkin, J. Sex Determination in the Nematode C. ELEGANS: Analysis of tra-3 Suppressors and Characterization of fem Genes. \u003cem\u003eGenetics\u003c/em\u003e \u003cstrong\u003e114\u003c/strong\u003e, 15\u0026ndash;52 (1986).\u003c/li\u003e\n\u003cli\u003eSchedl, T. \u0026amp; Kimble, J. Fog-2, a Germ-Line-Specific Sex Determination Gene Required for Hermaphrodite Spermatogenesis in Caenorhabditis Elegans. \u003cem\u003eGenetics\u003c/em\u003e \u003cstrong\u003e119\u003c/strong\u003e, 43\u0026ndash;61 (1988).\u003c/li\u003e\n\u003cli\u003eCheng, H., Govindan, J. A. \u0026amp; Greenstein, D. Regulated trafficking of the MSP/Eph receptor during oocyte meiotic maturation in C. elegans. \u003cem\u003eCurr. Biol. CB\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 705\u0026ndash;714 (2008).\u003c/li\u003e\n\u003cli\u003eMiller, M. A. \u003cem\u003eet al.\u003c/em\u003e A Sperm Cytoskeletal Protein That Signals Oocyte Meiotic Maturation and Ovulation. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e291\u003c/strong\u003e, 2144\u0026ndash;2147 (2001).\u003c/li\u003e\n\u003cli\u003eMiller, M. A., Ruest, P. J., Kosinski, M., Hanks, S. K. \u0026amp; Greenstein, D. An Eph receptor sperm-sensing control mechanism for oocyte meiotic maturation in Caenorhabditis elegans. \u003cem\u003eGenes Dev.\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 187\u0026ndash;200 (2003).\u003c/li\u003e\n\u003cli\u003eChaturbedi, A. \u0026amp; Lee, S. S. Different gametogenesis states uniquely impact longevity in Caenorhabditis elegans. 2023.06.13.544885 Preprint at https://doi.org/10.1101/2023.06.13.544885 (2023).\u003c/li\u003e\n\u003cli\u003eDoering, K. R. S., Ermakova, G. \u0026amp; Taubert, S. Nuclear hormone receptor NHR-49 is an essential regulator of stress resilience and healthy aging in Caenorhabditis elegans. \u003cem\u003eFront. Physiol.\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 1241591 (2023).\u003c/li\u003e\n\u003cli\u003eScholtes, C. \u0026amp; Gigu\u0026egrave;re, V. Transcriptional control of energy metabolism by nuclear receptors. \u003cem\u003eNat. Rev. Mol. Cell Biol.\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 750\u0026ndash;770 (2022).\u003c/li\u003e\n\u003cli\u003eSever, R. \u0026amp; Glass, C. K. Signaling by Nuclear Receptors. \u003cem\u003eCold Spring Harb. Perspect. Biol.\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, a016709 (2013).\u003c/li\u003e\n\u003cli\u003eTaubert, S., Van Gilst, M. R., Hansen, M. \u0026amp; Yamamoto, K. R. A Mediator subunit, MDT-15, integrates regulation of fatty acid metabolism by NHR-49-dependent and -independent pathways in C. elegans. \u003cem\u003eGenes Dev.\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 1137\u0026ndash;1149 (2006).\u003c/li\u003e\n\u003cli\u003eVan Gilst, M. R., Hadjivassiliou, H., Jolly, A. \u0026amp; Yamamoto, K. R. Nuclear hormone receptor NHR-49 controls fat consumption and fatty acid composition in C. elegans. \u003cem\u003ePLoS Biol.\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, e53 (2005).\u003c/li\u003e\n\u003cli\u003eGoh, G. Y. S. \u003cem\u003eet al.\u003c/em\u003e NHR-49/HNF4 integrates regulation of fatty acid metabolism with a protective transcriptional response to oxidative stress and fasting. \u003cem\u003eAging Cell\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, e12743 (2018).\u003c/li\u003e\n\u003cli\u003eDoering, K. R. \u003cem\u003eet al.\u003c/em\u003e Nuclear hormone receptor NHR-49 acts in parallel with HIF-1 to promote hypoxia adaptation in Caenorhabditis elegans. \u003cem\u003eeLife\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, e67911 (2022).\u003c/li\u003e\n\u003cli\u003eZeng, L. \u003cem\u003eet al.\u003c/em\u003e Nuclear receptors NHR-49 and NHR-79 promote peroxisome proliferation to compensate for aldehyde dehydrogenase deficiency in C. elegans. \u003cem\u003ePLOS Genet.\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, e1009635 (2021).\u003c/li\u003e\n\u003cli\u003eKwon, S., Park, K.-S. \u0026amp; Yoon, K. Regulator of Lipid Metabolism NHR-49 Mediates Pathogen Avoidance through Precise Control of Neuronal Activity. \u003cem\u003eCells\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 978 (2024).\u003c/li\u003e\n\u003cli\u003eDasgupta, M. \u003cem\u003eet al.\u003c/em\u003e NHR-49 Transcription Factor Regulates Immunometabolic Response and Survival of Caenorhabditis elegans during Enterococcus faecalis Infection. \u003cem\u003eInfect. Immun.\u003c/em\u003e \u003cstrong\u003e88\u003c/strong\u003e, (2020).\u003c/li\u003e\n\u003cli\u003eNaim, N. \u003cem\u003eet al.\u003c/em\u003e Cell nonautonomous roles of NHR-49 in promoting longevity and innate immunity. \u003cem\u003eAging Cell\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, e13413 (2021).\u003c/li\u003e\n\u003cli\u003eEustice, M. \u003cem\u003eet al.\u003c/em\u003e Nutrient sensing pathways regulating adult reproductive diapause in C. elegans. \u003cem\u003ePloS One\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, e0274076 (2022).\u003c/li\u003e\n\u003cli\u003eSeidel, H. S. \u0026amp; Kimble, J. The oogenic germline starvation response in C. elegans. \u003cem\u003ePloS One\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, e28074 (2011).\u003c/li\u003e\n\u003cli\u003eHeestand, B., Simon, M., Frenk, S., Titov, D. \u0026amp; Ahmed, S. Transgenerational Sterility of Piwi Mutants Represents a Dynamic Form of Adult Reproductive Diapause. \u003cem\u003eCell Rep.\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e, 156\u0026ndash;171 (2018).\u003c/li\u003e\n\u003cli\u003eRatnappan, R. \u003cem\u003eet al.\u003c/em\u003e Germline signals deploy NHR-49 to modulate fatty-acid \u0026beta;-oxidation and desaturation in somatic tissues of C. elegans. \u003cem\u003ePLoS Genet.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, e1004829 (2014).\u003c/li\u003e\n\u003cli\u003eNuclear hormone receptor NHR-49 acts in parallel with HIF-1 to promote hypoxia adaptation in Caenorhabditis elegans | eLife. https://elifesciences.org/articles/67911.\u003c/li\u003e\n\u003cli\u003eSchedl, T. \u0026amp; Kimble, J. fog-2, a germ-line-specific sex determination gene required for hermaphrodite spermatogenesis in Caenorhabditis elegans. \u003cem\u003eGenetics\u003c/em\u003e \u003cstrong\u003e119\u003c/strong\u003e, 43\u0026ndash;61 (1988).\u003c/li\u003e\n\u003cli\u003eScharf, A., Pohl, F., Egan, B. M., Kocsisova, Z. \u0026amp; Kornfeld, K. Reproductive Aging in Caenorhabditis elegans: From Molecules to Ecology. \u003cem\u003eFront. Cell Dev. Biol.\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 718522 (2021).\u003c/li\u003e\n\u003cli\u003eRapid Lipid Quantification in Caenorhabditis elegans by Oil Red O and Nile Red Staining. https://en.bio-protocol.org/en/bpdetail?id=4340\u0026amp;type=0.\u003c/li\u003e\n\u003cli\u003eHou, N. S. \u0026amp; Taubert, S. Function and Regulation of Lipid Biology in Caenorhabditis elegans Aging. \u003cem\u003eFront. Physiol.\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, (2012).\u003c/li\u003e\n\u003cli\u003eMullaney, B. C. \u0026amp; Ashrafi, K. C. elegans Fat Storage and Metabolic Regulation. \u003cem\u003eBiochim. Biophys. Acta\u003c/em\u003e \u003cstrong\u003e1791\u003c/strong\u003e, 474\u0026ndash;478 (2009).\u003c/li\u003e\n\u003cli\u003eEvolutionarily related host and microbial pathways regulate fat desaturation in C. elegans | Nature Communications. https://www.nature.com/articles/s41467-024-45782-2.\u003c/li\u003e\n\u003cli\u003eBalklava, Z., Pant, S., Fares, H. \u0026amp; Grant, B. D. Genome-wide analysis identifies a general requirement for polarity proteins in endocytic traffic. \u003cem\u003eNat. Cell Biol.\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 1066\u0026ndash;1073 (2007).\u003c/li\u003e\n\u003cli\u003eGrant, B. \u0026amp; Hirsh, D. Receptor-mediated Endocytosis in the Caenorhabditis elegans Oocyte. \u003cem\u003eMol. Biol. Cell\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 4311\u0026ndash;4326 (1999).\u003c/li\u003e\n\u003cli\u003eBohnert, K. A. \u0026amp; Kenyon, C. A lysosomal switch triggers proteostasis renewal in the immortal C. elegans germ lineage. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e551\u003c/strong\u003e, 629\u0026ndash;633 (2017).\u003c/li\u003e\n\u003cli\u003eKocsisova, Z., Mohammad, A., Kornfeld, K. \u0026amp; Schedl, T. Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU. \u003cem\u003eJ. Vis. Exp. JoVE\u003c/em\u003e 58339 (2018) doi:10.3791/58339.\u003c/li\u003e\n\u003cli\u003eWatterson, A. \u003cem\u003eet al.\u003c/em\u003e Intracellular lipid surveillance by small G protein geranylgeranylation. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e605\u003c/strong\u003e, 736\u0026ndash;740 (2022).\u003c/li\u003e\n\u003cli\u003eAngelo, G. \u0026amp; Van Gilst, M. R. Starvation Protects Germline Stem Cells and Extends Reproductive Longevity in \u003cem\u003eC. elegans\u003c/em\u003e. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e326\u003c/strong\u003e, 954\u0026ndash;958 (2009).\u003c/li\u003e\n\u003cli\u003eYang, Y., Han, S. M. \u0026amp; Miller, M. A. MSP Hormonal Control of the Oocyte MAP Kinase Cascade and Reactive Oxygen Species Signaling. \u003cem\u003eDev. Biol.\u003c/em\u003e \u003cstrong\u003e342\u003c/strong\u003e, 96\u0026ndash;107 (2010).\u003c/li\u003e\n\u003cli\u003eConstruction of a germline-specific RNAi tool in C. elegans. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6382888/.\u003c/li\u003e\n\u003cli\u003eHoldorf, A. D. \u003cem\u003eet al.\u003c/em\u003e WormCat: An Online Tool for Annotation and Visualization of Caenorhabditis elegans Genome-Scale Data. \u003cem\u003eGenetics\u003c/em\u003e \u003cstrong\u003e214\u003c/strong\u003e, 279\u0026ndash;294 (2020).\u003c/li\u003e\n\u003cli\u003eGovindan, J. A., Cheng, H., Harris, J. E. \u0026amp; Greenstein, D. G\u0026alpha;o/i and G\u0026alpha;s Signaling Function in Parallel with the MSP/Eph Receptor to Control Meiotic Diapause in C. elegans. \u003cem\u003eCurr. Biol.\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 1257\u0026ndash;1268 (2006).\u003c/li\u003e\n\u003cli\u003eMartin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. \u003cem\u003eEMBnet.journal\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 10\u0026ndash;12 (2011).\u003c/li\u003e\n\u003cli\u003eDobin, A. \u003cem\u003eet al.\u003c/em\u003e STAR: ultrafast universal RNA-seq aligner. \u003cem\u003eBIOINFORMATICS\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 15\u0026ndash;21 (2013).\u003c/li\u003e\n\u003cli\u003eLove, M. I., Huber, W. \u0026amp; Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. \u003cem\u003eGenome Biol.\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 550 (2014).\u003c/li\u003e\n\u003cli\u003eHulsen, T., de Vlieg, J. \u0026amp; Alkema, W. BioVenn \u0026ndash; a web application for the comparison and visualization of biological lists using area-proportional Venn diagrams. \u003cem\u003eBMC Genomics\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 488 (2008).\u003c/li\u003e\n\u003cli\u003eEmerson, F. J. \u0026amp; Lee, S. S. CUT\u0026amp;RUN for Chromatin Profiling in Caenorhabditis elegans. \u003cem\u003eCurr. Protoc.\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, e445 (2022).\u003c/li\u003e\n\u003cli\u003eSkene, P. J., Henikoff, J. G. \u0026amp; Henikoff, S. Targeted in situ genome-wide profiling with high efficiency for low cell numbers. \u003cem\u003eNat. Protoc.\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 1006\u0026ndash;1019 (2018).\u003c/li\u003e\n\u003cli\u003eSkene, P. J. \u0026amp; Henikoff, S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. \u003cem\u003eeLife\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, e21856 (2017).\u003c/li\u003e\n\u003cli\u003eLi, C.-L. \u003cem\u003eet al.\u003c/em\u003e Region-specific H3K9me3 gain in aged somatic tissues in Caenorhabditis elegans. \u003cem\u003ePLOS Genet.\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, e1009432 (2021).\u003c/li\u003e\n\u003cli\u003eEmerson, F. J. \u003cem\u003eet al.\u003c/em\u003e The chromatin factors SET-26 and HCF-1 oppose the histone deacetylase HDA-1 in longevity and gene regulation in C. elegans. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 2320 (2024).\u003c/li\u003e\n\u003cli\u003eZhang, Y. \u003cem\u003eet al.\u003c/em\u003e Model-based Analysis of ChIP-Seq (MACS). \u003cem\u003eGenome Biol.\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, R137 (2008).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 5 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7217416/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7217416/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe nuclear hormone receptor NHR-49, a homolog of mammalian PPARα and HNF4α, is a key transcriptional regulator of nutrition sensing and fatty acid metabolism in \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e. Here we uncovered a new function of NHR-49 in reproduction - controlling oocyte activation and ovulation. Loss of NHR-49 causes inappropriate oocyte activation and laying of unfertilized oocytes in the absence of sperm, resulting in rapid loss of yolk and stored fat, and drastically shortening of lifespan. We further demonstrated that prevention of yolk transfer into the oocytes largely restore fat storage and partially rescue lifespan in the \u003cem\u003enhr-49\u003c/em\u003e mutants. Additionally, NHR-49 appears to couple germline proliferation to nutritional status, as evidenced by its requirement for starvation-induced reduction in germline proliferation. Mechanistically, we showed that NHR-49 primarily acts in somatic cells, rather than the germline itself, to regulate oocyte activation and ovulation. We further demonstrated that NHR-49 binds to the promoter of GSA-1 and may stimulate its expression. GSA-1 encodes a G-protein coupled receptor known to act in the gonadal sheath cells to couple sperm sensing and oocyte activation. Our findings therefore suggest a model whereby NHR-49 regulates the expression of GSA-1, which in turn regulates oocyte activation in response to sperm signal. Overall, our findings suggest a mechanistic link between nutrition sensing and fertility and point to regulated retention of reproductive resources to be critical for maintaining longevity.\u003c/p\u003e","manuscriptTitle":"A somatic checkpoint through NHR-49/HNF4α governs reproductive investment and longevity in C. elegans","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-03 12:50:07","doi":"10.21203/rs.3.rs-7217416/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f92cfb11-29e6-45b5-a501-bb74c2bafde7","owner":[],"postedDate":"September 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":52463858,"name":"Biological sciences/Cell biology/Cell signalling/Nuclear receptors"},{"id":52463859,"name":"Biological sciences/Genetics/Gene regulation"},{"id":52463860,"name":"Biological sciences/Developmental biology/Ageing"}],"tags":[],"updatedAt":"2025-09-03T12:50:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-03 12:50:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7217416","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7217416","identity":"rs-7217416","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}