Small interfering RNAs bound to Argonautes ALG-3 and ALG-4 (ALG-3/4) in spermatocytes regulate fertility and aging in C. elegans. ALG-3/4 cell non-autonomously repress DAF-18/PTEN in the oocyte, thereby limiting the activity of DAF-16/FOXO and longevity. We find that the DAF-16 and ALG-3/4 target gene lea-1 is downregulated in precursor germ cells and oocytes of alg-3/4 mutants while being upregulated in age-1(hx546) PI3K mutants, where DAF-16 is activated. The downregulation of lea-1 in alg-3/4 mutants is inherited in progeny, unlike the elevation in DAF-18 levels. We therefore propose that LEA-1 and DAF-18 are regulated by ALG-3/4 through two distinct non-autonomous mechanisms.
","acknowledgements":"","authors":[{"affiliations":["Department of Biochemistry & Cell Biology, Chobanian & Avedisian School of Medicine, Boston University, 72 East Concord Street, Boston, MA, 02118, USA","Graduate Program in Genetics and Genomics, Chobanian & Avedisian School of Medicine, Boston University, Boston, MA, 02118, USA"],"departments":["",""],"credit":["conceptualization","formalAnalysis","investigation","visualization","writing_originalDraft","writing_reviewEditing"],"email":"tliontis@bu.edu","firstName":"Thomas","lastName":"Liontis","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0003-3376-1511"},{"affiliations":["Department of Biochemistry & Cell Biology, Chobanian & Avedisian School of Medicine, Boston University, 72 East Concord Street, Boston, MA, 02118, USA","Genome Science Institute, Boston University, Boston, MA, 02118, USA"],"departments":["",""],"credit":["conceptualization","fundingAcquisition","project","supervision","writing_originalDraft","writing_reviewEditing"],"email":"agrishok@bu.edu","firstName":"Alla","lastName":"Grishok","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-8922-7575"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was supported by National Institutes of Health grants [R01 GM135199] to AG and [P40 OD010440] to the Caenorhabditis Genetics Center, and a Hevolution Foundation grant [HF-AGE-23-1268260-52] to AG.
","image":{"url":"https://portal.micropublication.org/uploads/11a5b5c7b486d5ccdc15c6ac16861323.png"},"imageCaption":"(A) Representative images of endogenously-tagged mNeonGreen::3xFLAG::AID::LEA-1 (mNG::LEA-1) and differential interference contrast (DIC) microscopy of L4 male WT and alg‑3(tm1155); alg‑4(ok1041) mutant worms. Yellow arrow indicates spermatocytes. (B) Quantification of mean mNG::LEA-1 in spermatocytes from panel A. (C) Representative mNG::LEA-1 and DIC images of WT, alg-3/4 mutants, age-1(hx546), and triple mutant age-1; alg-3/4 young adult hermaphrodites. Red arrows indicate pachytene-stage germ cells, blue arrows indicate the −1 oocyte. (D – E) Quantification of mNG::LEA-1 in pachytene-stage germ cells from panel C. (G) Quantification of mNG::LEA-1 in −1 oocytes from panel C. (H) Mean mNG::LEA-1 fluorescence in the −1 oocyte of young adult F1 progeny arising from the indicated crosses. alg-3/4(-): alg-3; alg-4 double mutants. (I) Model of the regulation of LEA-1 by IIS and ALG-3/4. ALG-3/4 bind to siRNAs which repress certain msp genes (Liontis et al., 2026) and promote lea‑1 at the mRNA level in spermatocytes. Mutating alg-3/4 causes a decrease in lea-1 mRNA and protein levels in spermatocytes, resulting in decreased LEA-1 protein in the oocyte through an unknown mechanism (arrow with \"?\"), likely separate from the derepressed MSP activity that results in disrupted EphR (VAB-1) localization and increased DAF-18 levels (Liontis et al., 2026). DAF-18 is a phosphatase that dephosphorylates PIP3. In contrast, DAF-2 promotes the activation of AGE-1, which phosphorylates PIP2 to PIP3, resulting in repressed DAF-16 activity. In daf-2 or age-1 mutants, DAF-16 is activated and upregulates lea-1 mRNA and protein levels. Scale bars: 50 μm.
","imageTitle":"ALG-3/4 promote lea-1 expression cell non-autonomously in pachytene germ cells and oocytes
","methods":"C. elegans strains and maintenance
Worms were cultured and assayed at 20 °C on solid nematode growth media (NGM) seeded with E. coli OP50. The mNG::LEA-1 strain LP858 was outcrossed once to eliminate an unexpected dumpy (dpy)-like phenotype. The resulting strain was then crossed to the age-1 and alg-3; alg-4 mutant strains, which have already been outcrossed six times.
Fluorescence microscopy
Compound microscopy was performed using the Zeiss AxioImager Z1. Worms were mounted on 2% agarose pads, paralyzed with 10–20 mM levamisole in M9 buffer, placed under a thin glass coverslip, and immediately imaged. A constant exposure time was always used for images shown in the same figure panel. In addition, controls were always present on the exact same agarose pad as experimental animals (e.g., mutants). The ImageJ (Fiji) software was used for quantification of mNeonGreen::LEA-1 fluorescence and this analysis was performed while blinded to the strains' genotype. Mean fluorescence was normalized to controls in each independent experimental replicate, and results were pooled to produce the graphs shown in the figure. Random counterbalancing of the order in which strains were imaged was done between each independent replicate. Regions of interest were delimited manually: spermatocytes for L4 males; pachytene-stage germ cells in the proximal Rachis region and the proximal −1 oocyte's cytoplasmic region for young Day 1 adult hermaphrodites. At this stage, oocytes were large and mature but no more than 1 embryo was present.
Statistical analyses and graphs
Statistics were analyzed as was done previously (Liontis et al., 2026). Briefly, biological replicates (n) represent the number of animals, whereas independent experimental replicates (N) represent the number of independent experiments started on a different day with independent populations of animals. When comparing means, the F-test for variance and Anderson-Darling, D'Agostino-Pearson, Shapiro-Wilk, or Kolmogorov-Smirnov tests for normality were computed first. The resulting appropriate unpaired Student's t-test (equal vs. unequal variance) or Mann-Whitney test (nonparametric) was then used to assess a two-tailed significant difference between two groups. Tests were computed and graphs were generated using GraphPad Prism 10.
Error bars represent the standard error of the mean.
Parental effects on LEA-1
All strains were homozygous for mNeonGreen::LEA-1. Three males and one hermaphrodite at the L3 – L4 stage were transferred to each plate, with several plates for each cross. Hermaphrodites were considered to have mated when a significant number of progeny (F1) were males (up to 50%). F1 late L4 hermaphrodites that were identified as cross-progeny, i.e. not older than their sibling F1 males, were transferred to a new plate to prevent new crossing as a confounding factor. These F1 animals were imaged 7 hours after being transferred, at which point they reached young adulthood.
","reagents":"Strain name | Genotype | Source |
|---|---|---|
C. elegans wild type | Caenorhabditis Genetics Center | |
Caenorhabditis Genetics Center | ||
Caenorhabditis Genetics Center | ||
Caenorhabditis Genetics Center | ||
AGK lab | ||
AGK lab | ||
age-1(hx546) II; alg-4(ok1041) III; alg-3(tm1155) IV; lea-1(cp431) V | AGK lab | |
E. coli OP50 | Caenorhabditis Genetics Center |
In C. elegans, exogenous and endogenous double-stranded RNAs are recognized and bound by RDE-4, which directs them to Dicer for cleavage (Parker et al., 2006; Tabara et al., 2002; Thivierge et al., 2011). Subsequent processing of these RNAs results in double-stranded primary small interfering RNAs (siRNAs) (Duchaine et al., 2006; Lee et al., 2006; Yigit et al., 2006), which bind to Argonaute proteins. The Argonautes ALG-3 and ALG-4 (ALG-3/4) are redundant and expressed in spermatocytes during spermatogenesis (Conine et al., 2010; Han et al., 2009; Vasale et al., 2010). Their disruption causes defects in spermatocyte maturation and sperm function (Conine et al., 2010). ALG-3/4 are able to repress or promote the expression of their targets, with the latter process being enhanced at 25 °C (Conine et al., 2010, 2013). One gene with decreased levels of corresponding endo-siRNA, mRNA, and protein in alg-3/4 mutant males, i.e. a direct target of ALG-3/4, is lea-1 (Conine et al., 2013).
The lea-1 gene is robustly upregulated in long-lived mutants of the insulin/IGF-1 (IIS) pathway such as the insulin/IGF-1 receptor daf-2 mutant (Chen et al., 2015) and PI3K catalytic subunit age-1 mutant (Liontis et al., 2026). As is the case for the lifespan extension of IIS mutants (Kenyon et al., 1993; Klass, 1983; Murakami & Johnson, 1996), the upregulation of lea-1 in daf-2 mutants requires the downstream activation of the forkhead box O (FOXO) transcription factor DAF-16 (Chen et al., 2015). However, lea-1 does not appear to play a role in aging and is dispensable for the longevity of daf-2 mutants (Hibshman & Goldstein, 2021; Zečić et al., 2022).
We previously found that alg-3/4 mutations cause the disruption of Eph receptor (EphR) perimembrane localization and the elevation of PTEN protein levels in the neighboring mature oocyte (Liontis et al., 2026). These effects caused by alg-3/4 mutations were associated with the enhancement of the lifespan and healthspan of age-1(hx546) mutant hermaphrodites, but not males (Liontis et al., 2026). In addition, paternal sperm carrying the alg-3/4 mutations was not sufficient to elevate oocyte PTEN levels in progeny, and paternal sperm carrying wild-type alg-3/4 was also not sufficient to rescue the particularly low brood size of age-1; alg-3/4 mutants (Liontis et al., 2026). Taken together, our previous findings are consistent with spermatogenically-expressed ALG-3/4 regulating the oocyte in the same generation in a cell non-autonomous manner.
To further understand the spatial dynamics of genes regulated by ALG-3/4, we aimed here to measure the non-autonomous effects of ALG-3/4 on the expression of their target gene LEA-1. In addition, we were interested in assessing the interaction between ALG-3/4 and IIS pathways in their regulation of a common gene target. Although LEA-1 does not appear to play a role in aging, it uniquely satisfies the conditions of being expressed in various tissues, including spermatocytes and oocytes, being regulated by both ALG-3/4 and IIS, and having a readily available reporter strain, LP858, where its coding region was endogenously tagged with a fluorescent protein using CRISPR (mNeonGreen::LEA-1) (Hibshman & Goldstein, 2021).
We confirmed that ALG-3/4 promote LEA-1 protein levels in males by observing a strong decrease in mNeonGreen::LEA-1 levels in the spermatocytes of alg-3(tm1155); alg-4(ok1041) mutant males (Figure 1A, B). We then assessed LEA-1 levels in hermaphrodites (Figure 1C–G). Intriguingly, we found a strong downregulation of mNeonGreen::LEA-1 in the pachytene stage germ cells and proximal (−1) oocyte of alg-3/4 mutant hermaphrodites (Figure 1C, D, G). Given that ALG-3 and ALG-4 are specifically expressed in spermatocytes (Charlesworth et al., 2021; Conine et al., 2010), this finding is consistent with non-autonomous effects of ALG-3/4. Consistent with lea-1 being the most statistically significantly upregulated gene in age-1(hx546) mutants according to our RNA-sequencing data (Liontis et al., 2026), mNeonGreen::LEA-1 levels were considerably increased throughout the germline of age-1 mutant hermaphrodites (Figure 1C, E, G). In these age-1 mutants, the alg-3/4 mutations caused a statistically significant but markedly attenuated reduction in mNeonGreen::LEA-1 levels (Figure 1C, F, G). In other words, the age-1(hx546) background largely (but not entirely) suppressed the effect of alg-3/4 mutations in reducing LEA-1 levels in pachytene germ cells and the proximal oocyte.
Finally, we crossed wild-type (WT) and alg-3/4 mutant worms to observe the effects of maternal or paternal loss of alg-3/4 on the LEA-1 levels of progeny. Cross-progeny of alg-3/4 mutant parents displayed significantly decreased mNeonGreen::LEA-1 levels in their proximal oocytes, compared to cross-progeny of WT worms, as expected from these control conditions (Figure 1H). Surprisingly, progeny of WT males crossed to alg-3/4 mutant hermaphrodites, like those of alg-3/4 mutant males crossed to WT hermaphrodites, exhibited a decrease of similar magnitude in mNeonGreen::LEA-1 levels in their proximal oocyte (Figure 1H). This suggests that the process resulting in the downregulation of lea-1 in alg-3/4 mutants can be inherited both maternally and paternally. Given that these progeny animals are heterozygous for the alg-3/4 mutations, an alternative explanation is that alg-3/4 are haploinsufficient, at least with respect to their regulation of lea-1. This interpretation is unlikely because alg-3/4 heterozygotes do not phenocopy the sterility at 25 °C of alg-3/4 homozygous mutants (Conine et al., 2010, 2013). In addition, it was shown that male alg-3/4 heterozygotes do not display a decrease in lea-1 pre-mRNA levels compared to WT, unless they descend directly from a homozygous alg-3/4 mutant parent (Conine et al., 2013). Overall, although it is unclear how ALG-3/4 promote LEA-1 expression outside of spermatocytes, ALG-3/4 must be functional in both the father and hermaphrodite mother to sustain LEA-1 expression in the oocyte of their progeny.
In this work, we found that ALG-3/4 not only promotes LEA-1 levels in male spermatocytes but also in the oocyte and precursor germ cells of hermaphrodites (Figure 1I). Given that LEA-1 is repressed by IIS, this is a system where ALG-3/4 and AGE-1 have opposite effects on gene expression. In contrast, ALG-3/4 and AGE-1 both inhibit DAF-16 activity in their regulation of lifespan (Liontis et al., 2026). The downregulation of LEA-1 despite the increased activation of DAF-16 in alg-3/4 mutants is paradoxical, suggesting that there is likely an additional signaling pathway regulated by ALG-3/4, separate from the MSP/EphR/PTEN system (Liontis et al., 2026), at play (Figure 1I). This is further supported by the fact that the increase in DAF-18 (PTEN) levels in the oocyte of alg-3/4 mutants could not be inherited (Liontis et al., 2026), in contrast to the decrease in LEA-1 levels (Figure 1H). In both cases, the dysregulation of these genes in alg-3/4 mutants cannot be simply reasoned as disruptions of ALG-3/4 in paternal sperm. Instead, our results suggest that impairments causing LEA-1 downregulation in alg-3/4 mutant males and hermaphrodites are likely inherited throughout development and adulthood, whereas those causing DAF-18 elevation are present only in the generation where both copies of alg-3/4 are mutated. It is therefore likely that DAF-18 and LEA-1 are regulated by separate pathways controlled by ALG-3/4. Overall, our studies of ALG-3/4 reveal multiple cell non-autonomous gene regulatory mechanisms.
","references":[{"reference":"Charlesworth AG, Seroussi U, Lehrbach NJ, Renaud MS, Sundby AE, Molnar RI, et al., Claycomb JM. 2021. Two isoforms of the essential C. elegans Argonaute CSR-1 differentially regulate sperm and oocyte fertility. Nucleic Acids Research. 49: 8836.","pubmedId":"","doi":"10.1093/nar/gkab619"},{"reference":"Chen ATY, Guo C, Itani OA, Budaitis BG, Williams TW, Hopkins CE, et al., Hu PJ. 2015. Longevity Genes Revealed by Integrative Analysis of Isoform-Specific daf-16/FoxO Mutants of Caenorhabditis elegans. Genetics. 201: 613.","pubmedId":"","doi":"10.1534/genetics.115.177998"},{"reference":"Conine CC, Batista PJ, Gu W, Claycomb JM, Chaves DA, Shirayama M, Mello CC. 2010. Argonautes ALG-3 and ALG-4 are required for spermatogenesis-specific 26G-RNAs and thermotolerant sperm in Caenorhabditis elegans. Proceedings of the National Academy of Sciences. 107: 3588.","pubmedId":"","doi":"10.1073/pnas.0911685107"},{"reference":"Conine CC, Moresco JJ, Gu W, Shirayama M, Conte D, Yates JR, Mello CC. 2013. Argonautes Promote Male Fertility and Provide a Paternal Memory of Germline Gene Expression in C. elegans. Cell. 155: 1532.","pubmedId":"","doi":"10.1016/j.cell.2013.11.032"},{"reference":"Duchaine TF, Wohlschlegel JA, Kennedy S, Bei Y, Conte D, Pang K, et al., Mello CC. 2006. Functional proteomics reveals the biochemical niche of C. elegans DCR-1 in multiple small-RNA-mediated pathways. Cell. 124: 343.","pubmedId":"","doi":"10.1016/j.cell.2005.11.036"},{"reference":"Han T, Manoharan AP, Harkins TT, Bouffard P, Fitzpatrick C, Chu DS, et al., Kim JK. 2009. 26G endo-siRNAs regulate spermatogenic and zygotic gene expression in Caenorhabditis elegans. Proceedings of the National Academy of Sciences. 106: 18674.","pubmedId":"","doi":"10.1073/pnas.0906378106"},{"reference":"Hibshman JD, Goldstein B. 2021. LEA motifs promote desiccation tolerance in vivo. BMC Biology. 19: 263.","pubmedId":"","doi":"10.1186/s12915-021-01176-0"},{"reference":"Kenyon C, Chang J, Gensch E, Rudner A, Tabtiang R. 1993. A C. elegans mutant that lives twice as long as wild type. Nature. 366: 461.","pubmedId":"","doi":"10.1038/366461a0"},{"reference":"Klass MR. 1983. A method for the isolation of longevity mutants in the nematode Caenorhabditis elegans and initial results. Mechanisms of Ageing and Development. 22: 279.","pubmedId":"","doi":"10.1016/0047-6374(83)90082-9"},{"reference":"Lee RC, Hammell CM, Ambros V. 2006. Interacting endogenous and exogenous RNAi pathways in Caenorhabditis elegans. RNA. 12: 589.","pubmedId":"","doi":"10.1261/rna.2231506"},{"reference":"Liontis T, Pannarale VT, Mansisidor AR, Pathiranage SK, Patel JY, Grishok A. 2026. Mutual regulation of spermatogenesis-specific Argonaute proteins and Insulin/IGF-1 signaling in aging control. EMBO Reports. 27: 1437-1462.
","pubmedId":"","doi":"10.1038/s44319-025-00682-4"},{"reference":"Murakami S, Johnson TE. 1996. A genetic pathway conferring life extension and resistance to UV stress in Caenorhabditis elegans. Genetics. 143: 1207.","pubmedId":"","doi":"10.1093/genetics/143.3.1207"},{"reference":"Parker GS, Eckert DM, Bass BL. 2006. RDE-4 preferentially binds long dsRNA and its dimerization is necessary for cleavage of dsRNA to siRNA. RNA. 12: 807.","pubmedId":"","doi":"10.1261/rna.2338706"},{"reference":"Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al., Cardona A. 2012. Fiji: an open-source platform for biological-image analysis. Nature Methods. 9: 676-682.
","pubmedId":"","doi":"10.1038/nmeth.2019"},{"reference":"Tabara H, Yigit E, Siomi H, Mello CC. 2002. The dsRNA Binding Protein RDE-4 Interacts with RDE-1, DCR-1, and a DExH-Box Helicase to Direct RNAi in C. elegans. Cell. 109: 861.","pubmedId":"","doi":"10.1016/S0092-8674(02)00793-6"},{"reference":"Thivierge C, Makil N, Flamand M, Vasale JJ, Mello CC, Wohlschlegel J, Conte D, Duchaine TF. 2011. Tudor domain ERI-5 tethers an RNA-dependent RNA polymerase to DCR-1 to potentiate endo-RNAi. Nature Structural & Molecular Biology. 19: 90.","pubmedId":"","doi":"10.1038/nsmb.2186"},{"reference":"Vasale JJ, Gu W, Thivierge C, Batista PJ, Claycomb JM, Youngman EM, et al., Conte D. 2010. Sequential rounds of RNA-dependent RNA transcription drive endogenous small-RNA biogenesis in the ERGO-1/Argonaute pathway. Proceedings of the National Academy of Sciences of the United States of America. 107: 3582.","pubmedId":"","doi":"10.1073/pnas.0911908107"},{"reference":"Yigit E, Batista PJ, Bei Y, Pang KM, Chen CCG, Tolia NH, et al., Mello CC. 2006. Analysis of the C. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi. Cell. 127: 747.","pubmedId":"","doi":"10.1016/j.cell.2006.09.033"},{"reference":"Zečić A, Dhondt I, Braeckman BP. 2022. Accumulation of Glycogen and Upregulation of LEA-1 in C. elegans daf-2(e1370) Support Stress Resistance, Not Longevity. Cells. 11: 245.
","pubmedId":"","doi":"10.3390/cells11020245"}],"title":"Spermatogenesis-specific Argonautes ALG-3/4 promote LEA-1 expression in oocytes
","reviews":[{"reviewer":{"displayName":"Colin Conine"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[{"curator":{"displayName":"KJ Yook"},"openAcknowledgement":false,"submitted":null}]},{"id":"8b11110b-789a-42f8-af56-11143a412027","decision":"accept","abstract":"Small interfering RNAs bound to Argonautes ALG-3 and ALG-4 (ALG-3/4) in spermatocytes regulate fertility and aging in C. elegans. ALG-3/4 cell non-autonomously repress DAF-18/PTEN in the oocyte, thereby limiting the activity of DAF-16/FOXO and longevity. We find that the DAF-16 and ALG-3/4 target gene lea-1 is downregulated in precursor germ cells and oocytes of alg-3/4 mutants while being upregulated in age-1(hx546) PI3K mutants, where DAF-16 is activated. The downregulation of lea-1 in alg-3/4 mutants is inherited in progeny, unlike the elevation in DAF-18 levels. We therefore propose that LEA-1 and DAF-18 are regulated by ALG-3/4 through two distinct non-autonomous mechanisms.
","acknowledgements":"","authors":[{"affiliations":["Department of Biochemistry & Cell Biology, Chobanian & Avedisian School of Medicine, Boston University, 72 East Concord Street, Boston, MA, 02118, USA","Graduate Program in Genetics and Genomics, Chobanian & Avedisian School of Medicine, Boston University, Boston, MA, 02118, USA"],"departments":["",""],"credit":["conceptualization","formalAnalysis","investigation","visualization","writing_originalDraft","writing_reviewEditing"],"email":"tliontis@bu.edu","firstName":"Thomas","lastName":"Liontis","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0003-3376-1511"},{"affiliations":["Department of Biochemistry & Cell Biology, Chobanian & Avedisian School of Medicine, Boston University, 72 East Concord Street, Boston, MA, 02118, USA","Genome Science Institute, Boston University, Boston, MA, 02118, USA"],"departments":["",""],"credit":["conceptualization","fundingAcquisition","project","supervision","writing_originalDraft","writing_reviewEditing"],"email":"agrishok@bu.edu","firstName":"Alla","lastName":"Grishok","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-8922-7575"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was supported by National Institutes of Health grants [R01 GM135199] to AG and [P40 OD010440] to the Caenorhabditis Genetics Center, and a Hevolution Foundation grant [HF-AGE-23-1268260-52] to AG.
","image":{"url":"https://portal.micropublication.org/uploads/4ffea47629ae9deea4b2a8192a66b585.png"},"imageCaption":"(A) Representative images of endogenously-tagged mNeonGreen::3xFLAG::AID::LEA-1 (mNG::LEA-1) and differential interference contrast (DIC) microscopy of L4 male WT and alg‑3(tm1155); alg‑4(ok1041) mutant worms. Yellow arrow indicates spermatocytes. (B) Quantification of mean mNG::LEA-1 in spermatocytes from panel A. (C) Representative mNG::LEA-1 and DIC images of WT, alg-3/4 mutants, age-1(hx546), and triple mutant age-1; alg-3/4 young adult hermaphrodites. Red arrows indicate pachytene-stage germ cells, blue arrows indicate the −1 oocyte. (D – E) Quantification of mNG::LEA-1 in pachytene-stage germ cells from panel C. (G) Quantification of mNG::LEA-1 in −1 oocytes from panel C. (H) Mean mNG::LEA-1 fluorescence in the −1 oocyte of young adult F1 progeny arising from the indicated crosses. alg-3/4(-): alg-3; alg-4 double mutants. (I) Model of the regulation of LEA-1 by IIS and ALG-3/4. In spermatocytes, ALG-3/4 bind to siRNAs that target mRNAs, promoting lea-1 and repressing a subset of msp genes (Liontis et al., 2026) while promoting others (Conine et al., 2013). Mutating alg-3/4 causes a decrease in lea-1 mRNA and protein levels in spermatocytes, resulting in decreased LEA-1 protein in the oocyte through an unknown mechanism (arrow with \"?\"), likely separate from the derepressed MSP activity that results in disrupted EphR (VAB-1) localization and increased DAF-18 levels (Liontis et al., 2026). DAF-18 is a phosphatase that dephosphorylates PIP3. In contrast, DAF-2 promotes the activation of AGE-1, which phosphorylates PIP2 to PIP3, resulting in repressed DAF-16 activity. In daf-2 or age-1 mutants, DAF-16 is activated and upregulates lea-1 mRNA and protein levels. Scale bars: 50 μm.
","imageTitle":"ALG-3/4 promote lea-1 expression cell non-autonomously in pachytene germ cells and oocytes
","methods":"C. elegans strains and maintenance
Worms were cultured and assayed at 20 °C on solid nematode growth media (NGM) seeded with E. coli OP50. The mNG::LEA-1 strain LP858 was outcrossed once to eliminate an unexpected dumpy (dpy)-like phenotype. The resulting strain was then crossed to the age-1 and alg-3; alg-4 mutant strains, which have already been outcrossed six times.
Fluorescence microscopy
Compound microscopy was performed using the Zeiss AxioImager Z1. Worms were mounted on 2% agarose pads, paralyzed with 10–20 mM levamisole in M9 buffer, placed under a thin glass coverslip, and immediately imaged. A constant exposure time was always used for images shown in the same figure panel. In addition, controls were always present on the exact same agarose pad as experimental animals (e.g., mutants). The ImageJ (Fiji) software was used for quantification of mNeonGreen::LEA-1 fluorescence and this analysis was performed while blinded to the strains' genotype. Mean fluorescence was normalized to controls in each independent experimental replicate, and results were pooled to produce the graphs shown in the figure. Random counterbalancing of the order in which strains were imaged was done between each independent replicate. Regions of interest were delimited manually: spermatocytes for L4 males; pachytene-stage germ cells in the proximal Rachis region and the proximal −1 oocyte's cytoplasmic region for young Day 1 adult hermaphrodites. At this stage, oocytes were large and mature but no more than 1 embryo was present.
Statistical analyses and graphs
Statistics were analyzed as was done previously (Liontis et al., 2026). Briefly, biological replicates (n) represent the number of animals, whereas independent experimental replicates (N) represent the number of independent experiments started on a different day with independent populations of animals. When comparing means, the F-test for variance and Anderson-Darling, D'Agostino-Pearson, Shapiro-Wilk, or Kolmogorov-Smirnov tests for normality were computed first. The resulting appropriate unpaired Student's t-test (equal vs. unequal variance) or Mann-Whitney test (nonparametric) was then used to assess a two-tailed significant difference between two groups. Tests were computed and graphs were generated using GraphPad Prism 10.
Error bars represent the standard error of the mean.
Parental effects on LEA-1
All strains were homozygous for mNeonGreen::LEA-1. Three males and one hermaphrodite at the L3 – L4 stage were transferred to each plate, with several plates for each cross. Hermaphrodites were considered to have mated when a significant number of progeny (F1) were males (up to 50%). F1 late L4 hermaphrodites that were identified as cross-progeny, i.e. not older than their sibling F1 males, were transferred to a new plate to prevent new crossing as a confounding factor. These F1 animals were imaged 7 hours after being transferred, at which point they reached young adulthood.
","reagents":"Strain name | Genotype | Source |
|---|---|---|
C. elegans wild type | Caenorhabditis Genetics Center | |
Caenorhabditis Genetics Center | ||
Caenorhabditis Genetics Center | ||
Caenorhabditis Genetics Center | ||
AGK lab | ||
AGK lab | ||
age-1(hx546) II; alg-4(ok1041) III; alg-3(tm1155) IV; lea-1(cp431) V | AGK lab | |
E. coli OP50 | Caenorhabditis Genetics Center |
In C. elegans, exogenous and endogenous double-stranded RNAs are recognized and bound by RDE-4, which directs them to Dicer for cleavage (Boyle et al., 2026; Parker et al., 2006; Tabara et al., 2002; Thivierge et al., 2011). Subsequent processing of these RNAs results in double-stranded primary small interfering RNAs (siRNAs) (Duchaine et al., 2006; Lee et al., 2006; Yigit et al., 2006), which bind to Argonaute proteins. The Argonautes ALG-3 and ALG-4 (ALG-3/4) are redundant and expressed in spermatocytes during spermatogenesis (Conine et al., 2010; Han et al., 2009; Vasale et al., 2010). Their disruption causes defects in spermatocyte maturation and sperm function (Conine et al., 2010). ALG-3/4 can repress or promote the expression of their targets, with the latter process being enhanced at 25 °C (Conine et al., 2010, 2013). One gene with decreased levels of corresponding endo-siRNA, mRNA, and protein in alg-3/4 mutant males, i.e. a direct target of ALG-3/4, is lea-1 (Conine et al., 2013).
The lea-1 gene is robustly upregulated in long-lived mutants of the insulin/IGF-1 (IIS) pathway such as the insulin/IGF-1 receptor daf-2 mutant (Chen et al., 2015) and PI3K catalytic subunit age-1 mutant (Liontis et al., 2026). As is the case for the lifespan extension of IIS mutants (Kenyon et al., 1993; Klass, 1983; Murakami & Johnson, 1996), the upregulation of lea-1 in daf-2 mutants requires the downstream activation of the forkhead box O (FOXO) transcription factor DAF-16 (Chen et al., 2015). However, lea-1 does not appear to play a role in aging and is dispensable for the longevity of daf-2 mutants (Hibshman & Goldstein, 2021; Zečić et al., 2022).
We previously found that alg-3/4 mutations cause the disruption of Eph receptor (EphR) perimembrane localization and the elevation of PTEN protein levels in the neighboring mature oocyte (Liontis et al., 2026). These effects caused by alg-3/4 mutations were associated with the enhancement of the lifespan and healthspan of age-1(hx546) mutant hermaphrodites, but not males (Liontis et al., 2026). In addition, paternal sperm carrying the alg-3/4 mutations was not sufficient to elevate oocyte PTEN levels in progeny, and paternal sperm carrying wild-type alg-3/4 was also not sufficient to rescue the particularly low brood size of age-1; alg-3/4 mutants (Liontis et al., 2026). Taken together, our previous findings are consistent with spermatogenically-expressed ALG-3/4 regulating the oocyte in the same generation in a cell non-autonomous manner.
To further understand the spatial dynamics of genes regulated by ALG-3/4, we aimed here to measure the non-autonomous effects of ALG-3/4 on the expression of their target gene LEA-1. In addition, we were interested in assessing the interaction between the ALG-3/4 and IIS pathways in regulating a common gene target. Although LEA-1 does not appear to play a role in aging, it uniquely satisfies the conditions of being expressed in various tissues, including spermatocytes and oocytes, being regulated by both ALG-3/4 and IIS, and having a readily available reporter strain, LP858, where its coding region was endogenously tagged with a fluorescent protein using CRISPR (mNeonGreen::LEA-1) (Hibshman & Goldstein, 2021).
We confirmed that ALG-3/4 promote LEA-1 protein levels in males by observing a strong decrease in mNeonGreen::LEA-1 levels in the spermatocytes of alg-3(tm1155); alg-4(ok1041) mutant males (Figure 1A, B). We then assessed LEA-1 levels in hermaphrodites (Figure 1C–G). Intriguingly, we found a strong downregulation of mNeonGreen::LEA-1 in the pachytene stage germ cells and proximal (−1) oocyte of alg-3/4 mutant hermaphrodites (Figure 1C, D, G). Given that ALG-3 and ALG-4 are specifically expressed in spermatocytes (Charlesworth et al., 2021; Conine et al., 2010), this finding is consistent with non-autonomous effects of ALG-3/4. Consistent with lea-1 being the most statistically significantly upregulated gene in age-1(hx546) mutants according to our RNA-sequencing data (Liontis et al., 2026), mNeonGreen::LEA-1 levels were considerably increased throughout the germline of age-1 mutant hermaphrodites (Figure 1C, E, G). In these age-1 mutants, the alg-3/4 mutations caused a statistically significant but markedly attenuated reduction in mNeonGreen::LEA-1 levels (Figure 1C, F, G). In other words, the age-1(hx546) background largely (but not entirely) suppressed the effect of alg-3/4 mutations in reducing LEA-1 levels in pachytene germ cells and the proximal oocyte.
Finally, we crossed wild-type (WT) and alg-3/4 mutant worms to observe the effects of maternal or paternal loss of alg-3/4 on the LEA-1 levels of progeny. Cross-progeny of alg-3/4 mutant parents displayed significantly decreased mNeonGreen::LEA-1 levels in their proximal oocytes, compared to cross-progeny of WT worms, as expected from these control conditions (Figure 1H). Surprisingly, progeny of WT males crossed to alg-3/4 mutant hermaphrodites, like those of alg-3/4 mutant males crossed to WT hermaphrodites, exhibited a decrease of similar magnitude in mNeonGreen::LEA-1 levels in their proximal oocyte (Figure 1H). This suggests that the process resulting in the downregulation of lea-1 in alg-3/4 mutants can be inherited both maternally and paternally. Given that these progeny animals are heterozygous for the alg-3/4 mutations, an alternative explanation is that alg-3/4 are haploinsufficient, at least with respect to their regulation of lea-1. This interpretation is unlikely because alg-3/4 heterozygotes do not phenocopy the sterility at 25 °C of alg-3/4 homozygous mutants (Conine et al., 2010, 2013). In addition, it was shown that male alg-3/4 heterozygotes do not display a decrease in lea-1 pre-mRNA levels compared to WT, unless they descend directly from a homozygous alg-3/4 mutant parent (Conine et al., 2013). Overall, although it is unclear how ALG-3/4 promote LEA-1 expression outside of spermatocytes, ALG-3/4 must be functional in both the father and hermaphrodite mother to sustain LEA-1 expression in the oocyte of their progeny.
In this work, we found that ALG-3/4 not only promotes LEA-1 levels in male spermatocytes but also in the oocyte and precursor germ cells of hermaphrodites (Figure 1I). Given that LEA-1 is repressed by IIS, this is a system where ALG-3/4 and AGE-1 have opposite effects on gene expression. In contrast, ALG-3/4 and AGE-1 both inhibit DAF-16 activity in their regulation of lifespan (Liontis et al., 2026). The downregulation of LEA-1 despite the increased activation of DAF-16 in alg-3/4 mutants is paradoxical, suggesting that there is likely an additional signaling pathway regulated by ALG-3/4, separate from the MSP/EphR/PTEN system (Liontis et al., 2026), at play (Figure 1I). This is further supported by the fact that the increase in DAF-18 (PTEN) levels in the oocyte of alg-3/4 mutants could not be inherited (Liontis et al., 2026), in contrast to the decrease in LEA-1 levels (Figure 1H). In both cases, the dysregulation of these genes in alg-3/4 mutants cannot be simply reasoned as disruptions of ALG-3/4 in paternal sperm. Instead, our results suggest that impairments causing LEA-1 downregulation in alg-3/4 mutant males and hermaphrodites are likely inherited throughout development and adulthood, whereas those causing DAF-18 elevation are present only in the generation where both copies of alg-3/4 are mutated. It is therefore likely that DAF-18 and LEA-1 are regulated by separate pathways controlled by ALG-3/4. Overall, our studies of ALG-3/4 reveal multiple cell non-autonomous gene regulatory mechanisms.
","references":[{"reference":"Boyle EP, Aruscavage PJ, Consalvo CD, Bass BL. 2026. Reconstitution of antiviral Dicer activity in vitro reveals distinct contributions of RDE-4 dsRNA-binding motifs. RNA 32: 1328-1342.
","pubmedId":"","doi":"10.1261/rna.080917.125"},{"reference":"Charlesworth AG, Seroussi U, Lehrbach NJ, Renaud MS, Sundby AE, Molnar RI, et al., Claycomb JM. 2021. Two isoforms of the essential C. elegans Argonaute CSR-1 differentially regulate sperm and oocyte fertility. Nucleic Acids Research. 49: 8836.","pubmedId":"","doi":"10.1093/nar/gkab619"},{"reference":"Chen ATY, Guo C, Itani OA, Budaitis BG, Williams TW, Hopkins CE, et al., Hu PJ. 2015. Longevity Genes Revealed by Integrative Analysis of Isoform-Specific daf-16/FoxO Mutants of Caenorhabditis elegans. Genetics. 201: 613.","pubmedId":"","doi":"10.1534/genetics.115.177998"},{"reference":"Conine CC, Batista PJ, Gu W, Claycomb JM, Chaves DA, Shirayama M, Mello CC. 2010. Argonautes ALG-3 and ALG-4 are required for spermatogenesis-specific 26G-RNAs and thermotolerant sperm in Caenorhabditis elegans. Proceedings of the National Academy of Sciences. 107: 3588.","pubmedId":"","doi":"10.1073/pnas.0911685107"},{"reference":"Conine CC, Moresco JJ, Gu W, Shirayama M, Conte D, Yates JR, Mello CC. 2013. Argonautes Promote Male Fertility and Provide a Paternal Memory of Germline Gene Expression in C. elegans. Cell. 155: 1532.","pubmedId":"","doi":"10.1016/j.cell.2013.11.032"},{"reference":"Duchaine TF, Wohlschlegel JA, Kennedy S, Bei Y, Conte D, Pang K, et al., Mello CC. 2006. Functional proteomics reveals the biochemical niche of C. elegans DCR-1 in multiple small-RNA-mediated pathways. Cell. 124: 343.","pubmedId":"","doi":"10.1016/j.cell.2005.11.036"},{"reference":"Han T, Manoharan AP, Harkins TT, Bouffard P, Fitzpatrick C, Chu DS, et al., Kim JK. 2009. 26G endo-siRNAs regulate spermatogenic and zygotic gene expression in Caenorhabditis elegans. Proceedings of the National Academy of Sciences. 106: 18674.","pubmedId":"","doi":"10.1073/pnas.0906378106"},{"reference":"Hibshman JD, Goldstein B. 2021. LEA motifs promote desiccation tolerance in vivo. BMC Biology. 19: 263.","pubmedId":"","doi":"10.1186/s12915-021-01176-0"},{"reference":"Kenyon C, Chang J, Gensch E, Rudner A, Tabtiang R. 1993. A C. elegans mutant that lives twice as long as wild type. Nature. 366: 461.","pubmedId":"","doi":"10.1038/366461a0"},{"reference":"Klass MR. 1983. A method for the isolation of longevity mutants in the nematode Caenorhabditis elegans and initial results. Mechanisms of Ageing and Development. 22: 279.","pubmedId":"","doi":"10.1016/0047-6374(83)90082-9"},{"reference":"Lee RC, Hammell CM, Ambros V. 2006. Interacting endogenous and exogenous RNAi pathways in Caenorhabditis elegans. RNA. 12: 589.","pubmedId":"","doi":"10.1261/rna.2231506"},{"reference":"Liontis T, Pannarale VT, Mansisidor AR, Pathiranage SK, Patel JY, Grishok A. 2026. Mutual regulation of spermatogenesis-specific Argonaute proteins and Insulin/IGF-1 signaling in aging control. EMBO Reports. 27: 1437-1462.
","pubmedId":"","doi":"10.1038/s44319-025-00682-4"},{"reference":"Murakami S, Johnson TE. 1996. A genetic pathway conferring life extension and resistance to UV stress in Caenorhabditis elegans. Genetics. 143: 1207.","pubmedId":"","doi":"10.1093/genetics/143.3.1207"},{"reference":"Parker GS, Eckert DM, Bass BL. 2006. RDE-4 preferentially binds long dsRNA and its dimerization is necessary for cleavage of dsRNA to siRNA. RNA. 12: 807.","pubmedId":"","doi":"10.1261/rna.2338706"},{"reference":"Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al., Cardona A. 2012. Fiji: an open-source platform for biological-image analysis. Nature Methods. 9: 676-682.
","pubmedId":"","doi":"10.1038/nmeth.2019"},{"reference":"Tabara H, Yigit E, Siomi H, Mello CC. 2002. The dsRNA Binding Protein RDE-4 Interacts with RDE-1, DCR-1, and a DExH-Box Helicase to Direct RNAi in C. elegans. Cell. 109: 861.","pubmedId":"","doi":"10.1016/S0092-8674(02)00793-6"},{"reference":"Thivierge C, Makil N, Flamand M, Vasale JJ, Mello CC, Wohlschlegel J, Conte D, Duchaine TF. 2011. Tudor domain ERI-5 tethers an RNA-dependent RNA polymerase to DCR-1 to potentiate endo-RNAi. Nature Structural & Molecular Biology. 19: 90.","pubmedId":"","doi":"10.1038/nsmb.2186"},{"reference":"Vasale JJ, Gu W, Thivierge C, Batista PJ, Claycomb JM, Youngman EM, et al., Conte D. 2010. Sequential rounds of RNA-dependent RNA transcription drive endogenous small-RNA biogenesis in the ERGO-1/Argonaute pathway. Proceedings of the National Academy of Sciences of the United States of America. 107: 3582.","pubmedId":"","doi":"10.1073/pnas.0911908107"},{"reference":"Yigit E, Batista PJ, Bei Y, Pang KM, Chen CCG, Tolia NH, et al., Mello CC. 2006. Analysis of the C. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi. Cell. 127: 747.","pubmedId":"","doi":"10.1016/j.cell.2006.09.033"},{"reference":"Zečić A, Dhondt I, Braeckman BP. 2022. Accumulation of Glycogen and Upregulation of LEA-1 in C. elegans daf-2(e1370) Support Stress Resistance, Not Longevity. Cells. 11: 245.
","pubmedId":"","doi":"10.3390/cells11020245"}],"title":"Spermatogenesis-specific Argonautes ALG-3/4 promote LEA-1 expression in oocytes
","reviews":[],"curatorReviews":[{"curator":{"displayName":"KJ Yook"},"openAcknowledgement":false,"submitted":"1786749178505"}]},{"id":"6e02738d-cd27-4a2f-babb-5f55d890c6e6","decision":"publish","abstract":"Small interfering RNAs bound to Argonautes ALG-3 and ALG-4 (ALG-3/4) in spermatocytes regulate fertility and aging in C. elegans. ALG-3/4 cell non-autonomously repress DAF-18/PTEN in the oocyte, thereby limiting the activity of DAF-16/FOXO and longevity. We find that the DAF-16 and ALG-3/4 target gene lea-1 is downregulated in precursor germ cells and oocytes of alg-3/4 mutants while being upregulated in age-1(hx546) PI3K mutants, where DAF-16 is activated. The downregulation of lea-1 in alg-3/4 mutants is inherited, unlike the elevation in DAF-18 levels. We therefore propose that ALG-3/4 regulate LEA-1 and DAF-18 through distinct non-autonomous mechanisms.
","acknowledgements":"","authors":[{"affiliations":["Department of Biochemistry & Cell Biology, Chobanian & Avedisian School of Medicine, Boston University, 72 East Concord Street, Boston, MA, 02118, USA","Graduate Program in Genetics and Genomics, Chobanian & Avedisian School of Medicine, Boston University, Boston, MA, 02118, USA"],"departments":["",""],"credit":["conceptualization","formalAnalysis","investigation","visualization","writing_originalDraft","writing_reviewEditing"],"email":"tliontis@bu.edu","firstName":"Thomas","lastName":"Liontis","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0003-3376-1511"},{"affiliations":["Department of Biochemistry & Cell Biology, Chobanian & Avedisian School of Medicine, Boston University, 72 East Concord Street, Boston, MA, 02118, USA","Genome Science Institute, Boston University, Boston, MA, 02118, USA"],"departments":["",""],"credit":["conceptualization","fundingAcquisition","project","supervision","writing_originalDraft","writing_reviewEditing"],"email":"agrishok@bu.edu","firstName":"Alla","lastName":"Grishok","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0000-0001-8922-7575"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"This work was supported by National Institutes of Health grants [R01 GM135199] to AG and [P40 OD010440] to the Caenorhabditis Genetics Center, and a Hevolution Foundation grant [HF-AGE-23-1268260-52] to AG.
","image":{"url":"https://portal.micropublication.org/uploads/4ffea47629ae9deea4b2a8192a66b585.png"},"imageCaption":"(A) Representative images of endogenously-tagged mNeonGreen::3xFLAG::AID::LEA-1 (mNG::LEA-1) and differential interference contrast (DIC) microscopy of L4 male WT and alg‑3(tm1155); alg‑4(ok1041) mutant worms. Yellow arrow indicates spermatocytes. (B) Quantification of mean mNG::LEA-1 in spermatocytes from panel A. (C) Representative mNG::LEA-1 and DIC images of WT, alg-3/4 mutants, age-1(hx546), and triple mutant age-1; alg-3/4 young adult hermaphrodites. Red arrows indicate pachytene-stage germ cells, blue arrows indicate the −1 oocyte. (D – F) Quantification of mNG::LEA-1 in pachytene-stage germ cells indicated in panel C. (G) Quantification of mNG::LEA-1 in −1 oocytes indicated in panel C. (H) Mean mNG::LEA-1 fluorescence in the −1 oocyte of young adult F1 progeny arising from the indicated crosses. alg-3/4(-): alg-3; alg-4 double mutants. (I) Model of the regulation of LEA-1 by IIS and ALG-3/4. In spermatocytes, ALG-3/4 bind to siRNAs that target mRNAs, promoting lea-1 and repressing a subset of msp genes (Liontis et al., 2026) while promoting others (Conine et al., 2013). Mutating alg-3/4 causes a decrease in lea-1 mRNA and protein levels in spermatocytes, resulting in decreased LEA-1 protein in the oocyte through an unknown mechanism (arrow with \"?\"), likely separate from the derepressed MSP activity that results in disrupted EphR (VAB-1) localization and increased DAF-18 levels (Liontis et al., 2026). DAF-18 is a phosphatase that dephosphorylates PIP3. In contrast, DAF-2 promotes the activation of AGE-1, which phosphorylates PIP2 to PIP3, resulting in repressed DAF-16 activity. In daf-2 or age-1 mutants, DAF-16 is activated and upregulates lea-1 mRNA and protein levels. Scale bars: 50 μm.
","imageTitle":"ALG-3/4 promote lea-1 expression cell non-autonomously in pachytene germ cells and oocytes
","methods":"C. elegans strains and maintenance
Worms were cultured and assayed at 20 °C on solid nematode growth media (NGM) seeded with E. coli OP50. The mNG::LEA-1 strain LP858 was outcrossed once to eliminate an unexpected dumpy (dpy)-like phenotype. The resulting strain was then crossed to the age-1 and alg-3; alg-4 mutant strains, which have already been outcrossed six times.
Fluorescence microscopy
Compound microscopy was performed using the Zeiss AxioImager Z1. Worms were mounted on 2% agarose pads, paralyzed with 10–20 mM levamisole in M9 buffer, placed under a thin glass coverslip, and immediately imaged. A constant exposure time was always used for images shown in the same figure panel. In addition, controls were always present on the exact same agarose pad as experimental animals (e.g., mutants). The ImageJ (Fiji) software was used for quantification of mNeonGreen::LEA-1 fluorescence and this analysis was performed while blinded to the strains' genotype. Mean fluorescence was normalized to controls in each independent experimental replicate, and results were pooled to produce the graphs shown in the figure. Random counterbalancing of the order in which strains were imaged was done between each independent replicate. Regions of interest were delimited manually: spermatocytes for L4 males; pachytene-stage germ cells in the proximal Rachis region and the proximal −1 oocyte's cytoplasmic region for young Day 1 adult hermaphrodites. At this stage, oocytes were large and mature but no more than 1 embryo was present.
Statistical analyses and graphs
Statistics were analyzed as was done previously (Liontis et al., 2026). Briefly, biological replicates (n) represent the number of animals, whereas independent experimental replicates (N) represent the number of independent experiments started on a different day with independent populations of animals. When comparing means, the F-test for variance and Anderson-Darling, D'Agostino-Pearson, Shapiro-Wilk, or Kolmogorov-Smirnov tests for normality were computed first. The resulting appropriate unpaired Student's t-test (equal vs. unequal variance) or Mann-Whitney test (nonparametric) was then used to assess a two-tailed significant difference between two groups. Tests were computed and graphs were generated using GraphPad Prism 10.
Error bars represent the standard error of the mean.
Parental effects on LEA-1
All strains were homozygous for mNeonGreen::LEA-1. Three males and one hermaphrodite at the L3 – L4 stage were transferred to each plate, with several plates for each cross. Hermaphrodites were considered to have mated when a significant number of progeny (F1) were males (up to 50%). F1 late L4 hermaphrodites that were identified as cross-progeny, i.e. not older than their sibling F1 males, were transferred to a new plate to prevent new crossing as a confounding factor. These F1 animals were imaged 7 hours after being transferred, at which point they reached young adulthood.
","reagents":"Strain name | Genotype | Source |
|---|---|---|
C. elegans wild type | Caenorhabditis Genetics Center | |
Caenorhabditis Genetics Center | ||
Caenorhabditis Genetics Center | ||
Caenorhabditis Genetics Center | ||
AGK lab | ||
AGK lab | ||
age-1(hx546) II; alg-4(ok1041) III; alg-3(tm1155) IV; lea-1(cp431) V | AGK lab | |
E. coli OP50 | Caenorhabditis Genetics Center |
In C. elegans, exogenous and endogenous double-stranded RNAs are recognized and bound by RDE-4, which directs them to Dicer for cleavage (Boyle et al., 2026; Parker et al., 2006; Tabara et al., 2002; Thivierge et al., 2011). Subsequent processing of these RNAs results in double-stranded primary small interfering RNAs (siRNAs) (Duchaine et al., 2006; Lee et al., 2006; Yigit et al., 2006), which bind to Argonaute proteins. The Argonautes ALG-3 and ALG-4 (ALG-3/4) are redundant and expressed in spermatocytes during spermatogenesis (Conine et al., 2010; Han et al., 2009; Vasale et al., 2010). Their disruption causes defects in spermatocyte maturation and sperm function (Conine et al., 2010). ALG-3/4 can repress or promote the expression of their targets, with the latter process being enhanced at 25 °C (Conine et al., 2010, 2013). One gene with decreased levels of corresponding endo-siRNA, mRNA, and protein in alg-3/4 mutant males, i.e. a direct target of ALG-3/4, is lea-1 (Conine et al., 2013).
The lea-1 gene is robustly upregulated in long-lived mutants of the insulin/IGF-1 (IIS) pathway such as the insulin/IGF-1 receptor daf-2 mutant (Chen et al., 2015) and PI3K catalytic subunit age-1 mutant (Liontis et al., 2026). As is the case for the lifespan extension of IIS mutants (Kenyon et al., 1993; Klass, 1983; Murakami & Johnson, 1996), the upregulation of lea-1 in daf-2 mutants requires the downstream activation of the forkhead box O (FOXO) transcription factor DAF-16 (Chen et al., 2015). However, lea-1 does not appear to play a role in aging and is dispensable for the longevity of daf-2 mutants (Hibshman & Goldstein, 2021; Zečić et al., 2022).
We previously found that alg-3/4 mutations cause the disruption of Eph receptor (EphR) perimembrane localization and the elevation of PTEN protein levels in the neighboring mature oocyte (Liontis et al., 2026). These effects caused by alg-3/4 mutations were associated with the enhancement of the lifespan and healthspan of age-1(hx546) mutant hermaphrodites, but not males (Liontis et al., 2026). In addition, paternal sperm carrying the alg-3/4 mutations was not sufficient to elevate oocyte PTEN levels in progeny, and paternal sperm carrying wild-type alg-3/4 was also not sufficient to rescue the particularly low brood size of age-1; alg-3/4 mutants (Liontis et al., 2026). Taken together, our previous findings are consistent with spermatogenically-expressed ALG-3/4 regulating the oocyte in the same generation in a cell non-autonomous manner.
To further understand the spatial dynamics of genes regulated by ALG-3/4, we aimed here to measure the non-autonomous effects of ALG-3/4 on the expression of their target gene LEA-1. In addition, we were interested in assessing the interaction between the ALG-3/4 and IIS pathways in regulating a common gene target. Although LEA-1 does not appear to play a role in aging, it uniquely satisfies the conditions of being expressed in various tissues, including spermatocytes and oocytes, being regulated by both ALG-3/4 and IIS, and having a readily available reporter strain, LP858, where its coding region was endogenously tagged with a fluorescent protein using CRISPR (mNeonGreen::LEA-1) (Hibshman & Goldstein, 2021).
We confirmed that ALG-3/4 promote LEA-1 protein levels in males by observing a strong decrease in mNeonGreen::LEA-1 levels in the spermatocytes of alg-3(tm1155); alg-4(ok1041) mutant males (Figure 1A, B). We then assessed LEA-1 levels in hermaphrodites (Figure 1C–G). Intriguingly, we found a strong downregulation of mNeonGreen::LEA-1 in the pachytene stage germ cells and proximal (−1) oocyte of alg-3/4 mutant hermaphrodites (Figure 1C, D, G). Given that ALG-3 and ALG-4 are specifically expressed in spermatocytes (Charlesworth et al., 2021; Conine et al., 2010), this finding is consistent with non-autonomous effects of ALG-3/4. Consistent with lea-1 being the most statistically significantly upregulated gene in age-1(hx546) mutants according to our RNA-sequencing data (Liontis et al., 2026), mNeonGreen::LEA-1 levels were considerably increased throughout the germline of age-1 mutant hermaphrodites (Figure 1C, E, G). In these age-1 mutants, the alg-3/4 mutations caused a statistically significant but markedly attenuated reduction in mNeonGreen::LEA-1 levels (Figure 1C, F, G). In other words, the age-1(hx546) background largely (but not entirely) suppressed the effect of alg-3/4 mutations in reducing LEA-1 levels in pachytene germ cells and the proximal oocyte.
Finally, we crossed wild-type (WT) and alg-3/4 mutant worms to observe the effects of maternal or paternal loss of alg-3/4 on the LEA-1 levels of progeny. Cross-progeny of alg-3/4 mutant parents displayed significantly decreased mNeonGreen::LEA-1 levels in their proximal oocytes, compared to cross-progeny of WT worms, as expected from these control conditions (Figure 1H). Surprisingly, progeny of WT males crossed to alg-3/4 mutant hermaphrodites, like those of alg-3/4 mutant males crossed to WT hermaphrodites, also exhibited a similar decrease in mNeonGreen::LEA-1 levels in their proximal oocyte (Figure 1H). This suggests that the process resulting in the downregulation of lea-1 in alg-3/4 mutants can be inherited both maternally and paternally. Given that these progeny animals are heterozygous for the alg-3/4 mutations, an alternative explanation is that alg-3/4 are haploinsufficient, at least with respect to their regulation of lea-1. This interpretation is unlikely because alg-3/4 heterozygotes do not phenocopy the sterility at 25 °C of alg-3/4 homozygous mutants (Conine et al., 2010, 2013). In addition, it was shown that male alg-3/4 heterozygotes do not display a decrease in lea-1 pre-mRNA levels compared to WT, unless they descend directly from a homozygous alg-3/4 mutant parent (Conine et al., 2013). Overall, although it is unclear how ALG-3/4 promote LEA-1 expression outside of spermatocytes, ALG-3/4 must be functional in both the father and hermaphrodite mother to sustain LEA-1 expression in the oocyte of their progeny.
In this work, we found that ALG-3/4 not only promotes LEA-1 levels in male spermatocytes but also in the oocyte and precursor germ cells of hermaphrodites (Figure 1I). Given that LEA-1 is repressed by IIS, this is a system where ALG-3/4 and AGE-1 have opposite effects on gene expression. In contrast, ALG-3/4 and AGE-1 both inhibit DAF-16 activity in their regulation of lifespan (Liontis et al., 2026). The downregulation of LEA-1 despite the increased activation of DAF-16 in alg-3/4 mutants is paradoxical, suggesting that there is likely an additional signaling pathway regulated by ALG-3/4, separate from the MSP/EphR/PTEN system (Liontis et al., 2026), at play (Figure 1I). This is further supported by the fact that the increase in DAF-18 (PTEN) levels in the oocyte of alg-3/4 mutants could not be inherited (Liontis et al., 2026), in contrast to the decrease in LEA-1 levels (Figure 1H). In both cases, the dysregulation of these genes in alg-3/4 mutants cannot be simply reasoned as disruptions of ALG-3/4 in paternal sperm. Instead, our results suggest that impairments causing LEA-1 downregulation in alg-3/4 mutant males and hermaphrodites are likely inherited throughout development and adulthood, whereas those causing DAF-18 elevation are present only in the generation where both copies of alg-3/4 are mutated. It is therefore likely that DAF-18 and LEA-1 are regulated by separate pathways controlled by ALG-3/4. Overall, our studies of ALG-3/4 reveal multiple cell non-autonomous gene regulatory mechanisms.
","references":[{"reference":"Boyle EP, Aruscavage PJ, Consalvo CD, Bass BL. 2026. Reconstitution of antiviral Dicer activity in vitro reveals distinct contributions of RDE-4 dsRNA-binding motifs. RNA 32: 1328-1342.
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","pubmedId":"","doi":"10.1038/nmeth.2019"},{"reference":"Tabara H, Yigit E, Siomi H, Mello CC. 2002. The dsRNA Binding Protein RDE-4 Interacts with RDE-1, DCR-1, and a DExH-Box Helicase to Direct RNAi in C. elegans. Cell. 109: 861.","pubmedId":"","doi":"10.1016/S0092-8674(02)00793-6"},{"reference":"Thivierge C, Makil N, Flamand M, Vasale JJ, Mello CC, Wohlschlegel J, Conte D, Duchaine TF. 2011. Tudor domain ERI-5 tethers an RNA-dependent RNA polymerase to DCR-1 to potentiate endo-RNAi. Nature Structural & Molecular Biology. 19: 90.","pubmedId":"","doi":"10.1038/nsmb.2186"},{"reference":"Vasale JJ, Gu W, Thivierge C, Batista PJ, Claycomb JM, Youngman EM, et al., Conte D. 2010. Sequential rounds of RNA-dependent RNA transcription drive endogenous small-RNA biogenesis in the ERGO-1/Argonaute pathway. Proceedings of the National Academy of Sciences of the United States of America. 107: 3582.","pubmedId":"","doi":"10.1073/pnas.0911908107"},{"reference":"Yigit E, Batista PJ, Bei Y, Pang KM, Chen CCG, Tolia NH, et al., Mello CC. 2006. Analysis of the C. elegans Argonaute family reveals that distinct Argonautes act sequentially during RNAi. Cell. 127: 747.","pubmedId":"","doi":"10.1016/j.cell.2006.09.033"},{"reference":"Zečić A, Dhondt I, Braeckman BP. 2022. Accumulation of Glycogen and Upregulation of LEA-1 in C. elegans daf-2(e1370) Support Stress Resistance, Not Longevity. Cells. 11: 245.
","pubmedId":"","doi":"10.3390/cells11020245"}],"title":"Spermatogenesis-specific Argonautes ALG-3/4 promote LEA-1 expression in oocytes
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