Mitochondrial GTP Metabolism Controls Reproductive Aging in C. elegans

Yi-Tang Lee; Marzia Savini; Tao Chen; Jin Yang; Qian Zhao; Lang Ding; Shihong Max Gao; Mumine Senturk; Jessica Sowa; Jue D Wang; Meng C Wang

doi:10.1016/j.devcel.2023.08.019

. Author manuscript; available in PMC: 2024 Dec 4.

Published in final edited form as: Dev Cell. 2023 Sep 13;58(23):2718–2731.e7. doi: 10.1016/j.devcel.2023.08.019

Mitochondrial GTP Metabolism Controls Reproductive Aging in C. elegans

Yi-Tang Lee ^1,^2,³, Marzia Savini ^2,^3,⁴, Tao Chen ⁵, Jin Yang ⁶, Qian Zhao ⁵, Lang Ding ^5,⁷, Shihong Max Gao ^4,⁵, Mumine Senturk ^2,⁸, Jessica Sowa ⁹, Jue D Wang ⁶, Meng C Wang ^5,^8,^*

PMCID: PMC10842941 NIHMSID: NIHMS1931616 PMID: 37708895

SUMMARY

Healthy mitochondria are critical for reproduction. During aging, both reproductive fitness and mitochondrial homeostasis decline. Mitochondrial metabolism and dynamics are key factors in supporting mitochondrial homeostasis. However, how they are coupled to control reproductive health remains unclear. We report that mitochondrial GTP metabolism acts through mitochondrial dynamics factors to regulate reproductive aging. We discovered that germline-only inactivation of GTP- but not ATP-specific succinyl-CoA synthetase (SCS), promotes reproductive longevity in Caenorhabditis elegans. We further identified an age-associated increase in mitochondrial clustering surrounding oocyte nuclei, which is attenuated by GTP-specific SCS inactivation. Germline-only induction of mitochondrial fission factors sufficiently promotes mitochondrial dispersion and reproductive longevity. Moreover, we discovered that bacterial inputs affect mitochondrial GTP levels and dynamics factors to modulate reproductive aging. These results demonstrate the significance of mitochondrial GTP metabolism in regulating oocyte mitochondrial homeostasis and reproductive longevity and identify mitochondrial fission induction as an effective strategy to improve reproductive health.

Graphical Abstract

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eTOC Blurb

Lee et al. identify a signaling nexus between mitochondrial GTP metabolism and mitochondrial dynamics in regulating C. elegans reproductive aging. They further demonstrate that bacterial metabolites act through this signaling mechanism to modulate the host’s reproductive health during aging.

INTRODUCTION

As one of the earliest signs of age-associated decline, reproductive senescence has a strong impact on society due to the trend of increased average maternal age at first birth¹. Aged women exhibit decreased fertility and increased rates of birth defects and miscarriages². It is estimated that fertility decline occurs on an average of 10 years prior to menopause, and an age-associated decrease in oocyte quality is the major cause for this decline³. Diverse factors can influence oocyte quality, and one of the main contributors is mitochondrial activity⁴. Oocytes have the largest number of mitochondria among all the cells in an organism⁵. Changes in mitochondrial ATP production, membrane potential, and DNA copy numbers have been reported to influence oocyte development, maturation and fertility^4,6–8. Meanwhile, mitochondria exhibit highly dynamic morphology and constantly undergo organellar fission and fusion, leading to changes in their shape, size, and distribution⁹. Specific types of protein machinery are required to maintain mitochondrial fission-fusion dynamics, including the mitochondrial fission GTPase DRP1, mitochondrial outer membrane fusion GTPases MFN1 and MFN2, and mitochondrial inner membrane fusion GTPase OPA1⁹. These regulators of mitochondrial dynamics also modulate mitochondrial distribution within the cell, especially in the oocyte. In mice with Drp1 knockout, the oocyte mitochondrial network is aggregated toward the perinuclear region¹⁰. Similarly, in mouse oocytes overexpressing Mfn1 or Mfn2, the mitochondrial network exhibits perinuclear accumulation without increasing tubular elongation¹¹. Mitochondrial fission-fusion factors have also been linked with oocyte development and maturation^10,12,13. Drp1 knockout in mice oocytes results in abnormal follicular maturation and fertility decline¹⁰. Oocyte-specific knockout of mouse Mfn1 causes defective folliculogenesis, apoptotic cell loss and complete infertility^12,13. These findings indicate the importance of mitochondrial fission-fusion factors in oocyte quality control during development.

On the other hand, in Caenorhabditis elegans (C. elegans), mitochondrial fission-fusion factors have been linked with the regulation of somatic aging. Selectively overexpressing the C. elegans DRP1 homolog drp-1 in the intestine prolongs lifespan¹⁴, and whole-body knockout of drp-1 together with fzo-1, the C. elegans MFN homolog, leads to lifespan extension in C. elegans¹⁵. Besides being a well-established model organism for studying somatic aging, C. elegans share similarities with humans regarding reproductive aging¹⁶. Both genetic factors and environmental cues including bacterial inputs are known to regulate reproductive aging in C. elegans¹⁷. Through a full-genome RNA interference (RNAi) screen, we identified two subunits of mitochondrial Succinyl-CoA Synthetase (SCS) as regulators of reproductive aging¹⁸. SCS is a key mitochondrial enzyme in the TCA cycle converting succinyl-CoA to succinate with a production of GTP or ATP¹⁹. A functional SCS enzyme comprises one alpha subunit and one beta subunit. Two interchangeable beta subunits of SCS determine the GTP/ATP specificity by forming a complex with the constant alpha subunit^20,21.

In this study, we discovered that GTP-specific SCS in the germline regulates reproductive aging through tuning mitochondrial positioning in the oocyte, and that increasing mitochondrial fission selectively in the germline prevents age-associated perinuclear accumulation of oocyte mitochondria and promotes reproductive longevity in C. elegans. Furthermore, we found that the regulation of reproductive aging by GTP-specific SCS and mitochondrial fission-fusion factors responds to the level of vitamin B12 in bacteria. These findings suggest a previously unknown function of mitochondrial GTP metabolism in the germline and its significance in the regulation of mitochondrial homeostasis and oocyte quality during aging.

RESULTS

GTP-specific SCS regulates reproductive aging

In C. elegans, sucl-2 and sucg-1 encode the alpha and the GTP-specific beta subunit of SCS, respectively (Figure 1A). We found that inactivating either sucl-2 or sucg-1 by RNAi not only extends reproductive lifespan (RLS), but also improves fertility in aged hermaphrodites (late fertility) (Figures 1B–D, Supplementary Table 1). As the age of hermaphrodites increased from 1-day-old to 7-day-old and 9-day-old, the percentage of individuals capable of reproducing decreased from 100% to less than 50% and 30%, respectively, when they were mated with 2-day-old young males (Figure 1D). With sucg-1 or sucl-2 RNAi knockdown, the percentage of aged hermaphrodites capable of reproducing increased to more than 70% or 90% on day 7, and more than 50% or 70% on day 9, respectively (Figure 1D).

Next, we examined suca-1 that encodes the ATP-specific beta subunit and found that its RNAi knockdown does not extend RLS or improve late fertility (Figures 1D and 1E, Supplementary Table 1). These results suggest that the SCS complex formed by the alpha subunit SUCL-2 and the beta subunit SUCG-1 is specifically involved in regulating RLS and late fertility. Given that SUCG-1 is responsible for converting GDP to GTP in mitochondria, these results indicate a possible role of mitochondrial GTP metabolism in modulating reproductive aging.

Germline GTP-specific SCS regulates mitochondria and reproductive aging

To understand how the GTP-specific SCS regulates reproductive aging, we first examined the expression pattern of sucg-1 using a CRISPR knock-in line in which endogenous SUCG-1 is tagged with eGFP at the C-terminus. SUCG-1::eGFP expresses predominantly in the germline, with weaker signals in the pharynx, intestine, hypodermis, and muscle (Figure 2A). Using CRISPR knock-in, endogenous SUCA-1 was also tagged with eGFP at the C-terminus, which showed the predominant expression in the pharynx, neuron, intestine, hypodermis, and muscle while a very weak but detectable signal in the germline (Figure S1A). Moreover, we found that the GFP intensity in the germline of the SUCG-1::eGFP worms is increased at day 5 compared to day 1 (Figures 2B and 2C), suggesting an elevation of germline SUCG-1 levels with age. These findings suggest that mitochondrial SUCG-1 may function in the germline to regulate reproductive aging cell-autonomously.

Figure 2. — (A) Confocal imaging of the SUCG-1::eGFP knock-in line, in which the endogenous *sucg-1* is tagged with *egfp,* shows its predominant expression in the germline but weak expression in the intestine, pharynx, muscle, hypodermis and neurons (Scale bar: 100μm; Dashed white line: germline). (B) The SUCG-1::eGFP level in the germline is increased at day 5 compared to day 1 (Scale bar: 100μm). (C) Quantification of SUCG-1::eGFP level in the germline at day 1 and day 5. (AU: arbitrary unit) (D) Germline-specific RNAi inactivation of *sucg-1* extends RLS. (E) Germline-specific RNAi inactivation of *sucl-2* extends RLS. (F) SUCG-1::eGFP colocalizes with mitochondrial TOMM-20::mKate2 in the germline (Scale bar: 30μm for the images with lower magnification; 5μm for the images with higher magnification). (G) Overexpression of mitochondria-targeted *ndk-1*(*mito::ndk-1*) in the germline suppresses the RLS extension caused by *sucg-1* RNAi knockdown. (H) Representative images show that oocyte mitochondria are largely dispersed at day 1 while experiencing increasing perinuclear distribution at day 5 (Scale bar: 5μm; Dashed white line: oocyte outline; N: nucleus). (I) Perinuclear clustering of oocyte mitochondria is increased from day 1 to day 5. (J) The increase in perinuclear distribution of oocyte mitochondria at day 5 is suppressed by *sucg-1* or *sucl-2*, but not *suca-1* RNAi knockdown.

(C) *** *p < 0.001* by Student’s t-test; n = 31 (day 1), n = 32 (day 5). (D, E, G) ** *p < 0.01,* *** *p < 0.001* by log-rank test; n = 4 (D) or 3 (E, G) biological independent replicates, ~20 worms per replicate, see Supplementary Table 1 for full RLS Data. (I) n = 43 (day 1), n = 40 (day 5); *** *p < 0.001* by Chi-squared test. (J) n= 45 (EV, D1), n = 45 (*sucg-1*, D1), n = 45 (*sucl-2*, D1), n = 45 (*suca-1*, D1), n = 42 (EV, D5), n = 45 (*sucg-1*, D5), n = 44 (*sucl-2*, D5), n = 48 (*suca-1*, D5); RNAi vs. EV, n.s. *p > 0.05,* *** *p < 0.001* by Chi-squared test adjusted with the Holm–Bonferroni method for multiple comparisons.

To further confirm this cell-autonomous regulation, we utilized a tissue-specific RNAi strain, in which the expression of the RNAi-induced silencing complex component RDE-1 is restored specifically in the germline of the rde-1 null mutant²². We knocked down either sucg-1 or sucl-2 by RNAi selectively in the germline and found that germline-specific knockdown of sucg-1 extends RLS compared to control worms treated with the empty vector (Figure 2D, Supplementary Table 1). Germline-specific knockdown of sucl-2 led to similar RLS-extending effects (Figure 2E, Supplementary Table 1).

These results suggest that sucg-1 and sucl-2 act in the germline to regulate reproductive longevity. We also measured the progeny number in those worms and observed 7% or 11% reduction associated with the sucg-1 or sucl-2 germline-specific RNAi knockdown, respectively (Figures S1B and S1C). The decrease in the progeny number has been previously observed in other interventions leading to RLS extension, such as the loss-of-function mutant of daf-2, eat-2, and sma-2^23–26. In addition, the daf-2 and the eat-2 mutants not only prolong RLS but also extend lifespan^27,28. We found that the whole-body RNAi knockdown of either sucg-1 or sucl-2 leads to a mild lifespan extension (~15%), but suca-1 knockdown does not affect lifespan (Figure S1D, Supplementary Table 2). Upon germline-specific RNAi knockdown, the result was similar except that sucg-1 showed no lifespan extension in one out of three trials, and suca-1 slightly shortened lifespan in one out of three trials (Figure S1E, Supplementary Table 2).

Both GTP- and ATP-specific SCS catalyze succinate production from succinyl-CoA, and their losses lead to increased succinyl-CoA and decreased succinate levels. However, RNAi knockdown of suca-1 and sucg-1 exerted distinctive effects on RLS, suggesting that the change in either succinate or succinyl-CoA level is unlikely linked with the observed reproductive longevity phenotype. In support of this idea, we found that dietary supplementation of sodium succinate or succinic acid does not alter RLS (Figure S1F, Supplementary Table 1). Additionally, germline-specific knockdown of ogdh-1, which encodes a subunit of α-ketoglutarate dehydrogenase (upstream of SCS), led to sterility and production of dead eggs that failed to hatch despite having an intact germline. Meanwhile, germline-specific knockdown of mev-1 or sdhb-1 encoding subunits of succinate dehydrogenase (downstream of SCS) resulted in a very short reproductive time window (Figure S1G, Supplementary Table 1). Together, these results suggest that GTP-specific SCS reduction in the germline promotes reproductive longevity, which is unlikely due to altered succinate or succinyl-CoA levels.

Next, we crossed the sucg-1::egfp line with the transgenic strain that expresses mKate2-tagged TOMM-20 on the outer mitochondrial membrane in the germline²⁹. The co-localization between SUCG-1::eGFP and TOMM-20::mKate2 (Figure 2F) confirms the mitochondrial localization of SUCG-1. To test whether SUCG-1 regulates reproductive longevity through affecting mitochondrial GTP (mtGTP) levels in the germline, we made a transgenic strain expressing mitochondria-matrix-targeting-sequence tagged ndk-1 specifically in the germline. ndk-1 encodes the nucleoside diphosphate kinase that catalyzes GTP synthesis from ATP, and thus its overexpression would increase GTP levels³⁰. We found that ndk-1 overexpression in germline mitochondria is sufficient to suppress the RLS extension caused by sucg-1 knockdown (Figure 2G, Supplementary Table 1), suggesting that GTP-specific SCS regulates reproductive aging through modulating mtGTP levels in the germline.

To test whether the loss of SCS affects germline mitochondrial homeostasis, we utilized the transgenic strain expressing GFP-tagged TOMM-20 in the germline²⁹ and imaged mitochondrial morphology at day 1 and day 5. We found that mitochondrial fragmentation and tubulation morphology exhibits high variations between individuals of the same genotype, which prevented us to draw an explicit conclusion. On the other hand, we observed that the mitochondrial network of oocytes increases perinuclear distribution in day-5 aged worms, while being largely dispersed in day-1 young worms (Figure 2H). We wrote an imaging analysis script to quantify mitochondrial signals in five rings of oocyte cells and classified mitochondrial distribution into three categories – dispersed, intermediate, and perinuclear (Figure S2A). We found that mitochondrial GFP signals are evenly distributed throughout the five rings in the oocyte of day-1 worms (Figure S2B), while in the oocyte of day-5 worms, the percentage of the mitochondrial GFP signal derived from the perinuclear ring 1 is increased (Figures S2B and S2C). Further categorization analysis identified that the perinuclear distribution of oocyte mitochondria is increased in day-5 worms (Figure 2I). To test whether this change in mitochondrial distribution is associated with a decrease in mitochondrial content, we first measured mitochondrial DNA (mtDNA) levels in the dissected germline using quantitative PCR (qPCR). The result showed that the mtDNA level is 60% higher in the germline of day-5 worms than in day-1 worms (Figure S2D). Next, we measured mtDNA copy numbers in isolated oocytes using droplet digital PCR (ddPCR) and found no difference between oocytes of day-1 and day-5 worms (Figure S2E). These results indicate that the age-associated perinuclear accumulation of oocyte mitochondria is unlikely due to a decline in mitochondrial numbers.

Interestingly, we found that RNAi knockdown of sucg-1 or sucl-2 suppresses the age-associated increase in mitochondrial clustering around the nucleus, while RNAi knockdown of suca-1 shows no such effect (Figures 2J and S3A), which are consistent with their effects on RLS and late fertility (Figures 1B–E). Furthermore, sucg-1, sucl-2, or suca-1 germline-specific RNAi knockdown did not affect the germline mtDNA level at day 1 (Figure S2F). At day 5, worms with sucg-1, sucl-2, or suca-1 germline-specific RNAi knockdown exhibited similar germline mtDNA levels, which were ~30% higher than those of control worms (Figure S2F). Thus, the loss of either SCS isoform increases mitochondrial content in the germline with aging, which is not specific to sucg-1 knockdown and thus unlikely related to its effect on oocyte mitochondria positioning. Together, we found that GTP-specific SCS works specifically in the germline to regulate oocyte mitochondrial distribution during reproductive aging.

Mitochondrial fission drives reproductive longevity

Mitochondrial distribution is modulated by the dynamin family of GTPases⁹. To determine whether these GTPases regulate reproductive aging, we examined EAT-3, FZO-1 and DRP-1, which are C. elegans homologs of human OPA1, MFN1/2 and DRP1, respectively³¹. EAT-3 drives inner mitochondrial membrane fusion while FZO-1 is responsible for the fusion of the outer mitochondrial membrane (Figure 3A)^32,33. We found that germline-specific RNAi knockdown of eat-3 increases RLS and late fertility (Figures 3B and 3C, Supplementary Table 1). Meanwhile, fzo-1 knockdown selectively in the germline did not affect late fertility (Figure 3C), and only showed slight RLS extension (11.5%) in one out of three trials (Figure 3D, Supplementary Table 1). Thus, in the germline, EAT-3-mediated inner mitochondrial membrane fusion is involved in regulating reproductive aging. The eat-3 mutant was originally discovered showing abnormal pharyngeal pumping and food intake, like the eat-2 mutant³⁴. The eat-2 mutant is known to slow down reproductive aging as a result of caloric restriction^27,34. To test whether the effect of eat-3 on reproductive aging is also due to a reduction in food intake, we measured the pharyngeal pumping rate and the body size in worms with germline-specific eat-3 RNAi knockdown and found no alterations (Figures S4A–C), suggesting that the RLS extension does not result from caloric restriction.

In contrast to EAT-3 and FZO-1, DRP-1 drives mitochondrial fission (Figure 3A)^35,36. When we knocked down drp-1 by RNAi selectively in the germline, we found that RLS either remains unchanged (in two replicates) or is slightly decreased (in one replicate), and late fertility is not altered in these worms (Figures 3C and 3E, Supplementary Table 1). Conversely, when we overexpressed drp-1 selectively in the germline, transgenic worms showed an extremely long RLS compared to controls (Figure 3F, Supplementary Table 1). Together, these results show that increasing mitochondrial fission factors and decreasing inner mitochondrial fusion factors in the germline are both sufficient to promote reproductive longevity.

Next, we examined whether these mitochondrial fission-fusion factors regulate oocyte mitochondrial distribution. We found that in the drp-1 germline-specific overexpression transgenic strain, the age-associated perinuclear accumulation of oocyte mitochondria is greatly suppressed in day-5 aged worms (Figures 3G and S3B). RNAi knockdown of eat-3 also decreased the perinuclear accumulation of oocyte mitochondria at day 5 (Figures 3H and S3A). However, RNAi knockdown of fzo-1 did not affect oocyte mitochondrial distribution in either day-1 or day-5 worms (Figures 3H and S3A). Upon drp-1 RNAi knockdown, we observed an increase in the perinuclear distribution of oocyte mitochondria in day-1 worms, which however did not reach statistical significance (Figures 3H and S3A). In day-5 worms, drp-1 RNAi knockdown caused disruption in oocyte organization, and mitochondrial morphology became largely unscorable (Figures 3H and S3C). In the few oocytes that still had recognizable cell boundaries, we observed one-sided perinuclear aggregation of mitochondria (Figure S3A). These results suggest that mitochondrial fission-fusion factors modulate mitochondrial distribution in oocytes, which correlates with their regulatory effects on reproductive aging.

GTP-specific SCS regulates reproductive aging through tuning mitochondrial distribution

We then asked whether the change in mitochondrial distribution is responsible for the reproductive longevity-promoting effect conferred by sucg-1 knockdown. To answer this question, we utilized an auxin-inducible degron (AID) system to deplete the DRP-1 protein specifically in the germline upon the auxin treatment (Figure 4A). We first generated a CRISPR knock-in line (gfp::degron::drp-1) in which endogenous DRP-1 is tagged with GFP and degron at the N-terminus³⁷. This line was next crossed with the single-copy integrated transgenic strain where the auxin-inducible F-box protein TIR1 is selectively expressed in the germline (sun-1p::TIR1::mRuby)³⁸. Using this system, auxin administration led to TIR1-mediated degradation of the degron-tagged DRP-1 protein in the germline but not in other tissues (Figure 4B). We found that the auxin-induced DRP-1 depletion in the germline causes no significant change in RLS (Figure 4C, Supplementary Table 1), recapitulating the finding from germline-specific RNAi knockdown of drp-1 (Figure 3E). More importantly, although the germline-specific DRP-1 depletion did not affect RLS on its own, it fully suppressed the RLS extension caused by sucg-1 RNAi knockdown (Figure 4D, Supplementary Table 1).

Figure 4. — (A) A diagram demonstrating auxin-induced degradation of endogenous DRP-1 tagged with GFP and Degron. (B) Confocal imaging of GFP shows that the endogenous DRP-1 protein is specifically depleted in the germline upon the auxin treatment (Scale bar: 100μm for the images with lower magnification; 30μm for the images with higher magnification). (C) Auxin-induced germline-specific depletion of DRP-1 does not affect RLS. (D) Auxin-induced germline-specific depletion of DRP-1 abrogates the RLS extension caused by *sucg-1* RNAi. (E) The *drp-1* loss-of-function mutant increases the perinuclear clustering of oocyte mitochondria at day 1, which is not suppressed by *sucg-1* RNAi knockdown.

(C, D) n.s. *p > 0.05,* *** *p < 0.001* by log-rank test; n = 3 biological independent replicates, ~20 worms per replicate, see Supplementary Table 1 for full RLS Data. (E) n= 38 (WT, EV RNAi, D1), n = 41 (*drp-1(tm1108),* EV RNAi, D1), n = 41 (WT, *sucg-1* RNAi, D1), n = 46 (*drp-1(tm1108), sucg-1* RNAi, D1); RNAi vs EV and WT vs *drp-1* mutant, n.s. *p > 0.05,* * *p < 0.05,* *** *p < 0.001 by* Chi-squared test adjusted with the Holm–Bonferroni method for multiple comparisons.

Furthermore, the drp-1 loss-of-function mutant increased perinuclear clustering of oocyte mitochondria at day 1, and sucg-1 RNAi knockdown failed to suppress this increase (Figures 4E and S3D), which suggests the requirement of DRP-1 for the loss of SUCG-1 to drive oocyte mitochondrial dispersion. Therefore, mitochondrial GTP metabolism can regulate reproductive longevity by affecting mitochondrial positioning in the germline through a DRP-1-mediated mechanism.

GTP-specific SCS regulates reproductive aging in response to bacterial inputs

To confirm the difference between sucg-1 and suca-1 in regulating reproductive aging, we generated their CRISPR knockout lines (Figure S5A). suca-1 knockout worms were phenotypically wild type, and similarly to the RNAi knockdown worms, did not show an RLS change (Figures S5B and S5C, Supplementary Table 1). On the other hand, while sucg-1 homozygous knockout worms appeared wild-type in the parental generation, their progeny exhibited delayed development because of maternal sucg-1 deficiency. To avoid this maternal effect, we generated a heterozygous parental line by crossing the sucg-1 knockout line (KO) with the sucg-1::egfp knock-in line (GFP) (Figure 5A). This way, we could examine the reproductive phenotype of the progeny that carries the following genotypes, KO/KO, KO/GFP, and GFP/GFP, on the sucg-1 locus (Figure 5A). We found that the sucg-1 homozygous KO/KO worms have extended RLS compared to either KO/GFP heterozygous or GFP/GFP homozygous worms (Figures 5B and S5D, Supplementary Table 1). These results confirm the specificity of GTP-specific SCS in regulating reproductive aging.

Figure 5. — (A) A diagram showing the strategy to obtain *sucg-1* homozygous knockout (KO) mutants from heterozygous mutants with *sucg-1* KO at one locus and *sucg-1::egfp* (GFP) at the other locus. (B) *sucg-1* KO/KO mutants show a significant increase in RLS compared to *sucg-1* GFP/GFP and *sucg-1* KO/GFP worms. (C) With OP50 bacteria, *sucg-1* KO/KO mutants show no significant differences in RLS compared to *sucg-1* GFP/GFP or *sucg-1* KO/GFP worms. (D) Germline mitochondrial GTP (mtGTP) level is increased by 9.4-fold in day 5 aged worms compared to day 1 young worms on HT115 bacteria. With OP50 bacteria, the germline mtGTP level increase from day 1 to day 5 is 3-fold. The germline mtGTP level is higher in worms on HT115 bacteria than those on OP50 bacteria at day 5, but not at day 1. (E) Germline mitochondrial ATP (mtATP level) is not significantly different in worms of different ages and on different bacteria.

(B, C) n.s. *p > 0.05,* *** *p < 0.001* by log-rank test; n = 3 biological independent replicates, ~80 worms per replicate split into 3 genotypes, see Supplementary Table 1 and Supplementary Table 3 (C) for full RLS Data. (D, E) Error bars represent mean ± s.e.m., n = 4 biologically independent samples, n.s. *p > 0.05*, * *p < 0.05,* ** *p < 0.01,* *** *p < 0.001* by Student’s t-test adjusted with the Holm–Bonferroni method for multiple comparisons.

When examining these knockout mutant worms, we also had an interesting observation that on OP50 E. coli, neither the sucg-1 nor the suca-1 homozygous knockout caused an RLS extension (Figures 5C and S5E–G, Supplementary Table 3). Previous findings in our lab identified that C. elegans show distinct reproductive strategies when exposed to different bacteria. Wild-type worms that host OP50 E. coli have a longer RLS and improved late fertility than those on HB101 E. coli, while wild-type worms on HT115 E. coli had similar RLS and late fertility to those on HB101¹⁷ (Figures S6A and S6B, Supplementary Table 1). For the germline-specific RNAi knockdown of sucg-1, sucl-2 or suca-1, the experiments were conducted in the background of HT115 E. coli (Figures 2D, 2E, and S6C, Supplementary Table 1). When we examined their effects in the background of OP50 E. coli, we found that none of them enhances the RLS extension caused by OP50 (Figures S6D–F, Supplementary Table 3). These results suggest that different E. coli may affect mitochondrial GTP to exert different effects on worm reproductive aging.

To examine whether bacterial inputs affect mtGTP levels in the germline, we utilized the transgenic strain where germline mitochondria were tagged with GFP and triple HA and purified mitochondria using anti-HA magnetic beads via immunoprecipitation. We then measured germline mtGTP using liquid chromatograph coupled with mass spectrometry. We found that in the germline of day-5 worms on HT115 E. coli, the mtGTP level is increased by nearly 10-fold compared to day-1 worms, but the mtGTP induction level is only around 3-fold in the germline of worms on OP50 E. coli (Figure 5D). Moreover, day-5 worms on HT115 E. coli had a higher germline mtGTP level compared to worms on OP50 E. coli, while no difference in the germline mtGTP level was observed in day-1 worms (Figure 5D). On the other hand, the germline mitochondrial ATP (mtATP) levels were comparable between worms at different ages or on different bacteria (Figure 5E). These results suggest that reproductive longevity conferred by OP50 E. coli may be linked to an attenuation in the age-related increase in GTP production.

Bacteria modulate mitochondrial distribution during reproductive aging

Next, we examined whether OP50 E. coli causes changes in oocyte mitochondrial distribution using the TOMM-20::GFP strain. When compared to worms on HT115 E. coli, worms on OP50 E. coli attenuated the age-associated increase in the perinuclear clustering of oocyte mitochondria (Figures 6A and S3E). Moreover, germline-specific depletion of the DRP-1 protein or the germline-specific RNAi knockdown of drp-1 fully suppressed the RLS extension in worms on OP50 E. coli (Figures 6B and S6G, Supplementary Table 3). In addition, with the AID system, we could apply the auxin treatment selectively during adulthood, leading to DRP-1 loss after the germline completes development and switches from spermatogenesis to oogenesis. We found that this adult-only depletion of DRP-1 in the germline suppresses the RLS extension in worms on OP50 E. coli (Figure 6C, Supplementary Table 3), supporting the significance of oocyte mitochondrial distribution in regulating reproductive aging. Furthermore, germline-specific overexpression of drp-1 increases RLS in worms on either HT115 (Figure 3F, Supplementary Table 1) or OP50 E. coli (Figure 6D, Supplementary Table 3); while germline-specific RNAi knockdown of eat-3 failed to further enhance the RLS extension in worms on OP50 E. coli (Figure 6E, Supplementary Table 3). Like in worms on HT115 E. coli, germline-specific RNAi knockdown of fzo-1 did not alter RLS in worms on OP50 E. coli (Figure S6H, Supplementary Table 3).

Figure 6. — (A) The perinuclear clustering of oocyte mitochondria is decreased in day 5 worms on OP50 compared to those on HT115 bacteria. (B) Auxin-induced germline-specific depletion of DRP-1 reduces RLS in worms on OP50 bacteria. (C) Adult-only germline-specific depletion of DRP-1 reduces RLS in worms on OP50 bacteria. (D) Germline-specific overexpression of *drp-1* prolongs RLS in worms on OP50 bacteria. (E) Germline-specific RNAi inactivation of *eat-3* fails to extend RLS in worms on OP50 bacteria. (F) With OP50 bacteria, the distribution of oocyte mitochondria is not significantly different between EV control worms and those subjected to *eat-3* or *fzo-1* RNAi knockdown at day 5. With *drp-1* RNAi knockdown, oocyte mitochondrial distribution becomes unscorable due to distorted germline.

(A) n = 48 (HT115, D1), n = 53 (OP50, D1), n = 47 (HT115, D5), and n = 48 (OP50, D5); HT115 vs. OP50, n.s. *p > 0.05*, *** p < 0.001 by Chi-squared test. (B, C, D, E) n.s. *p > 0.05*, ** *p < 0.01, *** p < 0.001* by log-rank test; n = 3 (B, C, D) or 4 (E) biological independent replicates, ~20 worms per replicate, see Supplementary Table 3 for full RLS Data. (F) n= 44 (EV, D1), n = 43 (*eat-3*, D1), n = 41 (*fzo-1*, D1), n = 42 (*drp-1*, D1), n = 43 (EV, D5), n = 43 (*eat-3*, D5), n = 43 (*fzo-1*, D5); OP50 condition; RNAi vs. EV, n.s. *p > 0.05* by Chi-squared test adjusted with the Holm–Bonferroni method for multiple comparisons.

Furthermore, we found that drp-1 RNAi knockdown largely disturbs oocyte organization and mitochondrial distribution in day-5 worms on OP50 E. coli (Figures 6F and S3G), and in the small percentage of oocytes with recognizable cell boundaries, one-sided perinuclear aggregation of mitochondria was observed (Figure S3F). On the other hand, RNAi knockdown of either eat-3 or fzo-1 had no effects on oocyte mitochondrial distribution in worms on OP50 E. coli (Figures 6F and S3F). RNAi knockdown of sucg-1, sucl-2, or suca-1 could not alter oocyte mitochondrial distribution in worms on OP50 E. coli either (Figures S6I and S3F). Together, these results suggest that like GTP-specific SCS, OP50 bacterial inputs modulate mitochondrial distribution and reproductive longevity via mitochondrial fission-fusion factors.

Vitamin B12 deficiency in OP50 E. coli contributes to reproductive longevity

Our previous study identified that the trace amount of HB101 mixing in OP50 E. coli is sufficient to shorten RLS, suggesting the involvement of bioactive metabolites in regulating reproductive aging. Interestingly, it has been shown that OP50 E. coli is low in vitamin B12 (VB12), and the VB12 level affects mitochondrial dynamics in worm muscle^39–42. We monitored VB12 levels using the transgenic strain expressing the acdh-1p::gfp reporter^39,40, and found that GFP intensity is ~2.5-fold higher in worms on OP50 than those on HT115 E. coli (Figures 7A and 7B). To test whether VB12 deficiency could contribute to reproductive longevity, we supplied two different forms of VB12, methylcobalamin (meCbl) and adenosylcobalamin (adoCbl) to worms on OP50 and HT115 E. coli. We discovered that supplementation of either meCbl or adoCbl reduces the RLS extension in worms on OP50 E. coli but does not affect RLS in worms on HT115 E. coli (Figures 7C, S7A, S7B, and S7C Supplementary Tables 1 and 3). In addition, meCbl supplementation increased the perinuclear accumulation of oocyte mitochondria in day-5 worms on OP50 E. coli (Figures 7D and S3H), to a level similar with worms on VB12 sufficient HT115 E. coli. These results suggest that bacteria-derived VB12 plays a crucial role in regulating oocyte mitochondrial distribution and reproductive aging.

Figure 7. — (A, B) As a VB12-deficiency reporter, the *acdh-1:gfp* signal level is higher in day-1 worms on OP50 than those on HT115 bacteria (Scale bar: 100μm in A). GFP signal quantification is shown in B (AU: arbitrary unit). (C) Supplementation of meCbl shortens RLS of WT worms on OP50 bacteria. (D) Supplementation of meCbl increases the perinuclear clustering of oocyte mitochondria in WT worms on OP50 bacteria at day 5. (E) Supplementation of meCbl does not shorten RLS of the *sucg-1* knockout worms on OP50 bacteria. (F) Summary model representing mitochondrial GTP metabolism and mitochondrial fission-fusion couple in the oocyte to regulate reproductive longevity, which is modulated by metabolic inputs from bacteria.

(B) n = 15 (HT115), n = 15 (OP50); *** *p < 0.001* by Student’s t-test. (C) *** p < 0.001 by log-rank test; n = 3 biological independent replicates, ~20 worms per replicate, see Supplementary Table 3 for full RLS Data. (D) n= 40 (EV, D1), n = 45 (128nM meCbl, D1), n = 42 (EV, D5), n = 38 (128nM meCbl, D5); OP50 condition; 128nM meCbl vs EV, n.s. *p > 0.05, *** p < 0.001* by Chi-squared test. (E) n.s. *p > 0.05* by log-rank test; n = 3 biological independent replicates, ~80 worms per replicate split into 3 genotypes, see Supplementary Table 3 for full RLS Data.

We further examined whether VB12 signals through GTP-specific SCS to modulate reproductive aging. We found that although the sucg-1 heterozygous mutant (KO/GFP) and gfp homozygous (GFP/GFP) worms on OP50 E. coli experience a decrease in RLS when supplied with meCbl, this was not observed in the sucg-1 homozygous (KO/KO) mutant worms (Figures 7E, S7D, and S7E, Supplementary Table 3). This result suggests that SUCG-1 is required for VB12 to regulate reproductive aging. Two enzymes utilize VB12 as the cofactor for their functions, namely MMCM-1, a mitochondrial enzyme that converts methymalonyl-CoA to succinyl-CoA, and METR-1, the methionine synthase (MTR) that converts homocysteine to methionine. We discovered that mmcm-1 RNAi knockdown does not affect RLS in worms on either OP50 or HT115 E. coli (Figures S7F and S7G, Supplementary Tables 1 and 3). These results further support that succinyl-CoA is not involved in the regulation of reproductive aging. On the other hand, metr-1 RNAi knockdown increased RLS in worms on HT115 but not OP50 E. coli (Figures S7F and S7G, Supplementary Tables 1 and 3), suggesting that the VB12-methionine synthase branch, which controls purine synthesis^43,44, mediates the bacterial effect on reproductive aging.

DISCUSSION

In summary, our work discovered mitochondrial GTP-specific SCS as a key regulator of oocyte mitochondrial distribution and reproductive health, and further identified how bacterial inputs act through mitochondrial factors to modulate reproductive longevity (Figure 7F). We found that mitochondria exhibit dispersed structure in young oocytes but undergo perinuclear clustering in aged oocytes. Interestingly, a similar age-associated change in oocyte mitochondrial distribution was also observed in mice, which has been linked to decreased Drp1 activity¹⁰. In our studies, we found that germline-specific overexpression of drp-1 is sufficient to prolong RLS. In addition to its requirement for the RLS extension conferred by OP50 E. coli and the loss of GTP-specific SCS, DRP-1 is reported in a recent study to be necessary for the RLS extension in the daf-2 mutant⁴⁵. These findings together suggest that the mitochondrial fission factor DRP1 may act downstream of multiple mechanisms and plays an evolutionally conserved role in regulating reproductive health during aging.

It is important to note that although most studies related to mitochondrial fission-fusion factors focus on mitochondrial morphology (tubular vs fragmented), their regulation of mitochondrial distribution has been observed in both oocytes and somatic cells^{10,11,46–51}. Now, our data support that the key role of mitochondrial fission-fusion factors in regulating reproductive aging is predominantly attributed to their control of mitochondrial positioning in oocytes. Perinuclear clustered mitochondria have been associated with various cellular stress^52–58. Transient perinuclear clustering may help elicit transcriptional responses⁵² and sequester damaged mitochondria⁵⁹, to restore mitochondrial homeostasis. However, prolonged perinuclear clustering of oocyte mitochondria in aged worms and mice could block mitophagy-mediated clearance of damaged mitochondria, increase ER-mitochondria aggregation to impair calcium homeostasis, as well as disrupt mitochondrial segregation required for cell division upon fertilization. Our studies provide direct evidence that the dietary and genetic interventions that drive mitochondrial dispersion from perinuclear clustering sufficiently promote reproductive longevity in worms. It would be interesting to test whether similar mechanisms could help improve reproductive health during aging in mammals.

Our studies also identified that mitochondrial metabolism may directly signal through mitochondrial fission-fusion factors. SCS locates in the mitochondrial matrix, likely to be very close to the mitochondrial inner membrane. It is reported that various enzymes in the TCA cycle interact closely and form a metabolon to facilitate their reactions⁶⁰. One of these enzymes, succinate dehydrogenase, is anchored in the mitochondrial inner membrane⁶¹. Given that SCS provides succinate for succinate dehydrogenase as a substrate, the interaction between these two enzymes may recruit SCS close to the inner membrane, leading to a high local GTP level when the TCA cycle is active. Interestingly, it was reported that inner mitochondrial membrane fusion requires a higher concentration of GTP than outer mitochondrial fusion⁶². Furthermore, members of another family of GTP-producing enzymes, nucleoside diphosphate kinases, have been shown to directly interact with OPA1 in the mitochondrial inner membrane to regulate mitochondrial membrane dynamics in human cells^30,63. Our studies found that the germline-loss of EAT-3/OPA1, but not FZO-1/MFN1/2 recapitulates the effect of the germline-loss of SUCG-1 in promoting reproductive longevity. Considering the age-associated increase in the germline SUCG-1 level, it is possible that an increase in GTP production close to the inner membrane drives mitochondrial fusion via EAT-3 during reproductive aging, and this imbalance of mitochondrial dynamics consequently contributes to the decline of oocyte quality.

Two recent studies discovered that MTR loss results in decreased GTP and ATP levels^43,44. Thus, we speculate that high VB12 in HT115 E. coli could lead to MTR-mediated induction of GTP synthesis in the cytosol and, in turn, increased mtGTP levels. Consistently, the germline mtGTP level is significantly greater in worms on HT115 E. coli. At present, we do not have direct evidence on how bacteria-derived VB12 modulates GTP-specific SCS in the reproductive system, aside from genetic analysis confirming the requirement of SUCG-1 for the effect of VB12. No mRNA or protein level difference was detected between OP50 and HT115 conditions. It is possible that VB12 influences the activity and/or substrate availability of GTP-specific SCS in oocyte mitochondria. In addition, MTR reduction also affects the level of methionine and/or homocysteine, which may also act through mitochondria to regulate reproductive aging.

We discovered that low bacterial VB12 levels are associated with reproductive longevity. There are significant variations in VB12 levels among different bacterial species. Bacteria with high or low levels of VB12 have been associated with decreased fertility in C. elegans^39,40,64. In humans, a high maternal VB12 level at birth is associated with an increased risk of developing autism spectrum disorder in children⁶⁵. However, VB12 deficiency can lead to adverse maternal and child health problems^66,67. Thus, there may be an antagonistic pleiotropy-like effect at the nutrient level, wherein VB12 is essential for appropriate development of the germline and progenies, but later accelerates reproductive decline during aging. Our study suggests that environmental inputs from the microbiota should also be taken into account when considering this antagonistic pleiotropic effect.

LIMITATIONS OF THE STUDY

We utilized the strain DCL569 to perform germline-specific RNAi knockdown and the pie-1 promoter to drive germline-specific overexpression, which did not specifically target oocytes and therefore limited our investigation into oocyte-specific processes. Additionally, due to technical limitations, we were unable to track mitochondrial distribution at all stages of oocyte maturation and capture detailed images of mitochondrial outer and inner membrane dynamics at high spatial resolution.

STAR★METHODS

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Meng C. Wang (mengwang@janelia.hhmi.org).

Materials availability

Materials including C. elegans strains, sequences, and plasmids in this study are available upon request.

Data and code availability

All reproductive lifespan and lifespan data are available in the paper’s supplemental information. All other data reported in this paper will be shared by the lead contact upon request. All original code is available in this paper’s supplemental information. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Strains and maintenance

C. elegans strains N2, DCL569, EGD629, EGD623, EU2917, CA1472, CU6372, and VL749 were obtained from the Caenorhabditis Genetics Center. PHX3617 and PHX4685 were acquired from Suny Biotech. MCW618, MCW1220, MCW1315, MCW1325, MCW1326, MCW1329, MCW1330, MCW1331, MW1357, MCW1373, MCW1375, MCW1385, MCW1408, MCW1473, MCW1550, MCW1581, MCW1584 were made in our lab. All C. elegans strains were kept at 20°C for both maintenance and experiment. All C. elegans were non-starved for at least 2 generations on NGM plates seeded with OP50 bacteria before any experiment. The detailed genotypes of each strain are listed in the KEY RESOURCES TABLE.

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Bacterial and Virus Strains
Escherichia coli HT115(DE3)	Caenorhabditis Genetics Center	HT115
Escherichia coli OP50	Caenorhabditis Genetics Center	OP50
Escherichia coli HB101	Caenorhabditis Genetics Center	HB101
Vidal RNAi library	Open Biosystems	ORF RNAi Collection V1.1
Ahringer RNAi library	Source BioScience	C. elegans RNAi Collection (Ahringer)
RNA interference competent OP50 strain (rnc:14::DTn10; laczgΑ::T7pol camFRT)	Neve et al. 2020 ⁷²
Experimental Models: Organisms/Strains
C. elegans N2 Wild-type	Caenorhabditis Genetics Center	N2 (RRID:WB-STRAIN:WBStrain00000001)
sucg-1(syb3617[sucg-1::eGFP]) IV	Suny Biotech	PHX3617
suca-1(syb4685[suca-1::eGFP]) X	Suny Biotech	PHX4685
mkcSi13[sun-1p::rde-1::sun-1 3’UTR + unc-119(+)] II; rde-1(mkc36) V	Caenorhabditis Genetics Center	DCL569 (RRID:WB-STRAIN:WBStrain00005607)
egxSi155[mex-5p::tomm-20::mKate2::pie-1 3’UTR + unc-119(+)] II; unc-119(ed3) III	Caenorhabditis Genetics Center	EGD629
egxSi155[mex-5p::tomm-20::mKate2::pie-1 3’UTR + unc-119(+)] II; unc-119(ed3) III; sucg-1(syb3617[sucg-1::eGFP]) IV	This paper	MCW1373
raxEx618[pie-1p::cox8(MTS)::ndk-1::3xHA::pie-1 3’UTR + myo-2p::GFP]	This paper	MCW1581
egxSi152[mex-5p: tomm-20::gfp::pie-1 3’UTR + unc-119(+)] II; unc-119(ed3) III	Caenorhabditis Genetics Center	EGD623
raxEx190[pie-1p::drp-1::tbb-2 3’UTR + myo-2p::GFP]	This paper	MCW618
raxIs141[pie-1p::drp-1::tbb-2 3’UTR + myo-2p::GFP]	This paper	MCW1220
raxIs141[pie-1p::drp-1.b::tbb-2 3’UTR + myo-2p::GFP]; egxSi152[mex5p::tomm-20::gfp::pie-1 3’UTR + unc-119(+)] II; unc-119(ed3) III	This paper	MCW1357
drp-1(or1941[GFP::drp-1]) IV	Caenorhabditis Genetics Center	EU2917 (RRID:WB-STRAIN:WBStrain00007414)
drp-1(rax82[GFP::Degron::drp-1]) IV	This paper	MCW1315
ieSi68[sun-1p::TIR1::mRuby::htp-1 3’UTR + Cbr-unc-119(+)] II; unc-119(ed3) III	Caenorhabditis Genetics Center	CA1472 (RRID:WB-STRAIN:WBStrain00004073)
ieSi68[sun-1p::TIR1::mRuby::htp-1 3’UTR + Cbr-unc-119(+)] II; unc-119(ed3) III; drp-1(rax82[GFP::Degron::drp-1]) IV	This paper	MCW1326
drp-1(tm1108) IV	Caenorhabditis Genetics Center	CU6372 (RRID:WB-STRAIN:WBStrain00005196)
egxSi152[mex5p::tomm-20::GFP::pie-1 3’UTR + unc-119(+)] II; unc-119(ed3) III; drp-1(tm1108) IV	This paper	MCW1584
sucg-1(rax83) IV	This paper	MCW1325
sucg-1(rax86) IV	This paper	MCW1331
suca-1(rax84) X	This paper	MCW1329
suca-1(rax85) X	This paper	MCW1330
sucg-1(syb3617[sucg-1::eGFP]); sucg-1(rax83) IV	This paper	MCW1375
sucg-1(syb3617[sucg-1::eGFP]); sucg-1(rax86) IV	This paper	MCW1385
raxIs89[sun-1p::eGFP::sun-1 3’UTR] III)	This paper	MCW1408
raxIs98[sun-1p::eGFP::3xHA::sun-1 3’UTR] III)	This paper	MCW1473
raxIs109[sun-1p::tomm-20(1–55aa)::eGFP::3xHA::sun-1 3’UTR] III	This paper	MCW1550
wwIs24 [acdh-1p::GFP + unc-119(+)]	Caenorhabditis Genetics Center	VL749 (RRID:WB-STRAIN:WBStrain00040155)
Oligonucleotides
See Table S4 for primers used in the study	Integrated DNA Technologies
See Table S5 for crRNA and tracrRNA used in the study	Horizon Discovery Ltd.
Recombinant DNA
pDONR221	Invitrogen	Cat# 12536017
pCM1.36	Addgene	RRID:Addgene_17249
pCM1.127	Addgene	RRID:Addgene_21384
pCFJ150	Addgene	RRID:Addgene_19329
pPK605	Addgene	RRID:Addgene_38148
Software and Algorithms
ImageJ v1.52p	http://fiji.sc/	RRID:SCR_003070
Illustrator CC 2022	Adobe	RRID:SCR_010279
PRISM v9	GraphPad Software	RRID:SCR_002798
SPSS v24.0	IBM	RRID:SCR_016479
BioRender	https://biorender.com/	RRID:SCR_018361
MATLAB R2020a	MathWorks	RRID:SCR_001622

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The E. coli strain HT115 (DE3) was obtained from the Ahringer RNAi library. The E. coli strains OP50 and HB101 were obtained from the Caenorhabditis Genetics Center.

Strain generation – Extrachromosomal array

MCW618 (raxEx190 [pie-1p::drp-1::tbb-2 3’UTR + myo-2p::GFP]) was generated by microinjecting the pie-1p::drp-1::tbb-2 3’UTR linearized PCR product and myo-2p::GFP plasmids into the gonad of young adults. MCW1581 (raxEx618[pie-1p:: cox8(mitochondrial targeting sequence)::ndk-1::3xHA::pie-1 3’UTR + myo-2p::GFP]) was generated by microinjecting pie-1p::cox8(mitochondrial targeting sequence)::ndk-1::3xHA::pie-1 3’UTR linearized PCR product and myo-2p::GFP plasmid into the gonad of young adults.

Strain Generation – Integration of extrachromosomal array

MCW1220 (raxIs141 [pie-1p::drp-1::tbb-2 3’UTR + myo-2p::GFP]) was generated by the integration of extrachromosomal array in MCW618 which is induced by gamma irradiation exposures (4500rad, 5.9min) at the L4 stage. Later, the integrated progenies were backcrossed to N2 five times.

Strain Generation – CRISPR-Cas9 mediated insertion and deletion

MCW1315 (drp-1(rax82[GFP::Degron::drp-1]) IV) was generated by inserting the Degron sequence into the GFP::drp-1 locus of EU2917 between GFP and drp-1 following the protocol from Dokshin et al. 2018 with some modifications⁶⁸. In short, a mixture of Cas9 protein (1.25μg/μl), tracrRNA (1μg/μl), target crRNA (0.4μg/μl), dpy-10 crRNA (0.16μg/μl), and partially single-stranded DNA donor (300nM final concentration for each PCR product) was microinjected into the gonad of young adults. The partially single-stranded DNA donor was generated by mixing 2 PCR products – Degron sequence with 30 or 100 base pair homology arms on each side, and heat to 95°C then gradually cooling back to 20°C for melting and reannealing. After 3 days, the plates that have worms with Dpy phenotype were carefully chosen as jackpot plates for individualization of non-Dpy worms. These worms were subjected to pooled and then individual genotyping PCR after they reproduced to ensure passage of the genotype.

The progenies (F2) of the specific F1 worm with the desired genotype were further individualized for identification of homozygosity using genotyping PCR and then Sanger sequencing.

MCW1325(sucg-1(rax83) IV), MCW1331(sucg-1(rax86) IV), MCW1329(suca-1(rax84) X), and MCW1330(suca-1(rax85) X) knockout or partial knockout strains were generated using methodologies described in Chen et al. 2014 with modifications⁶⁹. A mixture of Cas9 protein (1.25μg/μl), tracrRNA (1μg/μl), 2 target crRNAs (0.4μg/μl each) on 5’ and 3’ of a gene, and dpy-10 crRNA (0.16μg/μl), were microinjected into the gonad of young adults. The screening process was the same as described for the knock-in strain MCW1315. MCW1329 and MCW1330 were backcrossed to N2 three times.

MCW1408 (raxIs89[sun-1p::eGFP::sun-1 3’UTR] III) was generated by inserting sun-1p::eGFP::sun-1 3’UTR into ChrIII 7007.6 position. A mixture of Cas9 protein (1.25μg/μl), tracrRNA (1μg/μl), target crRNA (0.4μg/μl), and partially single-stranded DNA donor (10nM final concentration for each PCR product) was microinjected into the gonad of young adults. The partially single-stranded DNA donor was generated by mixing 2 PCR products - sun-1p::eGFP::sun-1 3’UTR sequence with 150bp of flanking homology arms on each side and the plain sun-1p::eGFP::sun-1 3’UTR sequence (both amplified using pYT17 plasmid as template), and heat to 95°C then gradually cool back to 20°C for melting and reannealing. Each injected worms were individualized post-injection. After 4 days, F1s were screened under fluorescence scope for green fluorescence in the germline. The progenies (F2) of the specific F1 worm with the desired genotype were further individualized for identification of homozygosity using fluorescence scope and then genotyping PCR followed by Sanger sequencing.

MCW1473 (raxIs98[sun-1p::eGFP::3xHA::sun-1 3’UTR] III) was generated by inserting triple HA sequence between eGFP and sun-1 3’UTR at ChrIII 7007.6 position; sun-1p::eGFP::sun-1 3’UTR genetic locus in MCW1408. The experiment procedure was the same as generating MCW1315 except for the usage of single-strand oligodeoxynucleotides (with 30~40nt homology arms on each side; 250ng/μl final concentration) instead of partially single-stranded DNA donor as the repair template, and melting and reannealing step by heating and cooling was not performed.

MCW1550 (raxIs109[sun-1p::tomm-20(1–55aa)::eGFP::3xHA::sun-1 3’UTR] III) as generated by inserting the first 165 nucleotides of tomm-20 gene between sun-1p and eGFP at ChrIII 7007.6 position; sun-1p::eGFP::3xHA::sun-1 3’UTR genetic locus in MCW1473. The experiment procedure was the same as generating MCW1473. Later, MCW1550 was backcrossed to N2 five times.

Genotyping PCR was performed using spanning primers for MCW1315, MCW1325, MCW1331, MCW1329, MCW1330, and MCW1408, and then followed by confirmation with Sanger sequencing. For MCW1473 and MCW1550, genotyping PCR screen was performed using spanning primer on the 5’ and internal primer on the 3’, and the candidates were further verified using genotyping PCR by spanning primers followed by confirmation with Sanger sequencing.

All primers used for genotyping are listed in Supplementary Table 4. Sequences of all crRNAs and the tracrRNA used for generating strains by CRISPR-Cas9 are listed in Supplementary Table 5.

Strain Generation – Crossing

MCW1373 (egxSi155 [mex-5p::tomm-20::mKate2::pie-1 3’UTR + unc-119(+)] II; unc-119(ed3) III; sucg-1(syb3617[sucg-1::eGFP]) IV) was generated by crossing PHX3617 male to EGD629 hermaphrodite. eGFP⁺ F1s were selected to a population plate under the fluorescent scope, and the eGFP⁺ F2s on the population plate were then picked into individual plates. The F3s were later examined for green fluorescence, and individual plates with all eGFP⁺ (homozygous) F3 worms were then selected. Confocal imaging was then used to screen for the tomm-20::mKate2 homozygous genotype, and genotyping PCR followed by Sanger sequencing were used to examine the unc-119 genotype.

MCW1326 (ieSi68 [sun-1p::TIR1::mRuby::htp-1 3’UTR + Cbr-unc-119(+)] II; unc-119(ed3) III; drp-1(rax82[GFP::Degron::drp-1]) IV) was generated by crossing MCW1315 male to CA1472 hermaphrodite. F1s were picked into individual plates, and then the GFP::Degron::drp-1; TIR-1::mRuby (heterozygous) genotype was inspected by confocal imaging after egg laying. The F2s from F1 with the correct heterozygous genotype were then picked into individual plates. Later, F3s were later used to screen for the correct homozygous genotype of GFP::Degron::drp-1; TIR-1::mRuby by confocal imaging. Lastly, genotyping PCR followed by Sanger sequencing were used to examine the unc-119 genotype.

MCW1357 (raxIs141[pie-1p::drp-1.b::tbb-2 UTR + myo-2p::GFP]; egxSi152[mex5p::tomm-20::GFP::pie-1 3’UTR + unc-119(+)] II; unc-119(ed3) III) was generated by crossing EGD623 male to MCW1220 hermaphrodite. F1s were inspected for the mex5p::tomm-20::GFP::pie-1 3’UTR by the fluorescent microscope, and the worms with the correct (heterozygous) genotype were individualized. Later, the myo-2p::GFP⁺ F2s from F1 with the correct mex5p::tomm-20::GFP::pie-1 3’UTR heterozygous genotype were then picked into individual plates. Later, F3s were later used to screen for the correct homozygous genotype of myo-2p::GFP and mex5p::tomm-20::GFP::pie-1 3’UTR by fluorescence scope. Lastly, genotyping PCR followed by Sanger sequencing were used to examine the unc-119 genotype.

MCW1584 (egxSi152[mex5p::tomm-20::GFP::pie-1 3’UTR + unc-119(+)] II; unc-119(ed3) III; drp-1(tm1108) IV) was generated by crossing EGD623 male to CU6372 hermaphrodite. F1s were then inspected for the mex5p::tomm-20::GFP::pie-1 3’UTR by the fluorescent microscope, and the ones with the correct (heterozygous) genotype were individualized. The F2s from F1 with the correct mex5p::tomm-20::GFP::pie-1 3’UTR heterozygous genotype were then picked into individual plates, and single worm lysed for drp-1(tm1108) PCR genotyping after egg laying. Later, F3s were used to screen for the correct homozygous genotype of mex5p::tomm-20::GFP::pie-1 3’UTR by fluorescent microscope. Lastly, genotyping PCR followed by Sanger sequencing were used to examine the unc-119 genotype.

Genotyping PCR of for drp-1(tm1108) and unc-119 was performed using spanning primers followed by confirmation with Sanger sequencing. The primers used for drp-1(tm1108) and unc-119 genotyping are listed in Supplementary Table 4.

MCW1375 (sucg-1(syb3617[sucg-1::eGFP]); sucg-1(rax83) IV) and MCW1385 (sucg-1(syb3617[sucg-1::eGFP]); sucg-1(rax86) IV) were obtained by crossing PHX3617 male to MCW1325 or MCW1331 hermaphrodites. eGFP⁺ F1s were picked under the fluorescent microscope and picked into individual plates. Later, F2s were used to confirm the sucg-1::gfp/KO heterozygous genotype of the F1 parental worms by fluorescent microscope (eGFP⁺/eGFP^- F2s should be around 3:1). Heterozygous genotypes were maintained by picked eGFP⁺ heterozygous worms (lower eGFP intensity than homozygous) for passage.

METHOD DETAILS

RNA interference (RNAi) experiments

RNAi libraries created by the lab of Dr. Marc Vidal and Dr. Julie Ahringer were used in this study^70,71. sucg-1, mev-1, sdhb-1, ogdh-1, drp-1, eat-3, and mmcm-1 RNAi clones were acquired from the Vidal library while sucl-2, suca-1, and metr-1 RNAi clones were acquired from the Ahringer library. fzo-1 RNAi clone was generated in the lab using L4440 as the vector backbone and full-length fzo-1 transcript as the insert. All RNAi clones were verified by Sanger sequencing. For OP50 RNAi experiments, the genetically modified competent OP50 bacteria [rnc14::DTn10 laczgA::T7pol camFRT] generated by our lab was used and transformed with 50 ng of the RNAi plasmid every time before the experiment⁷². All RNAi colonies were selected in both 50 µg ml⁻¹ carbenicillin and 50 µg ml⁻¹ tetracycline resistance. All RNAi bacteria were cultured for 14 hours in LB with 25 µg ml⁻¹ carbenicillin, and then seeded onto RNAi agar plates that contain 1 mM IPTG and 50 µg ml⁻¹ carbenicillin. The plates were then left at room temperature overnight for induction of dsRNA expression. For the RNAi experiments that require auxin treatment, fresh bacteria were concentrated 4 times before seeding onto the plates, and then left at 4°C overnight before usage.

Construction of plasmid and fusion PCR product

The pie-1p::drp-1::tbb2 3’UTR plasmid was generated by PCR amplifying the complete coding sequence of drp-1.b transcript from N2 cDNA and utilized Gateway BP recombination to clone into pDONR221 which contains Gateway attLR recombination sequences. drp-1.b CDS entry clone was then recombined with the entry clones pCM1.36-tbb-2 3’UTR and pCM1.127-pie-1p into destination vector pCFJ150 using Gateway LR recombination.

The pie-1p::cox8(mitochondrial targeting sequence)::ndk-1::3xHA::pie-1 3’UTR oligonucleotide was generated by 3-fragment fusion PCR using cox8(mitochondrial targeting sequence)::ndk-1::3xHA, pie-1p, and pie-1 3’UTR PCR product. The cox8(mitochondrial targeting sequence)::ndk-1::3xHA oligonucleotide was synthesized by IDT, and utilized as the template for amplification and homology arm tagging (tagged with pie-1p and pie-1 3’UTR homologies on 5’ and 3’ end respectively). Both pie-1p and pie-1 3’UTR PCR products were amplified using pPK605 plasmid (Addgene) as the template.

The pYT17-sun-1p::eGFP::sun-1 3’UTR plasmid was generated via 4-fragment Gibson cloning from vector backbone, sun-1p, modified eGFP, and sun-1 3’UTR PCR products. sun-1p and sun-1 3’UTR PCR products were amplified using N2 worm lysate as the template. The modified eGFP PCR product was amplified using PHX3617 worm lysate as the template.

Primers used for the amplification are listed in Supplementary Table 4.

Reproductive lifespan assay and progeny number measurement

Synchronized L1 larvae from egg preparation were plated onto 6cm NGM plates seeded with the specific bacteria (default: HT115) and grew to L4 stage before being individualized into single 3cm NGM plates. The worms were transferred to a new plate every day except for the day right after individualization, which we collectively (L4 + day-1-old adult) count as day 1. The transferring stopped when we observed 2 days of non-reproducing events consecutively or until day 12. After each transfer, plates were stored at room temperature for 2 days before checking the reproductive status, and counting progeny number if necessary. The last day of progeny production was counted as the day of reproductive cessation, and worms that could not be tracked until the day of reproductive cessation due to missing, death, germline protrusion, or internal hatching were counted as censors on the last day which we could determine the reproductive status. The animals were removed from the reproductive lifespan analysis if they died before producing any progeny. For total progeny number measurement, only the worms that are tracked until reproductive cessation are included.

For RLS experiments of MCW1581 (raxEx618[pie-1p:: cox8(mitochondrial targeting sequence)::ndk-1::3xHA::pie-1 3’UTR + myo-2p::GFP]), day 1 myo-2p::GFP⁺ F1s of injected parental worms were individually picked onto EV or sucg-1 RNAi plates. 3 and 4 days later, the plates with myo-2p::GFP⁺ F2s were selected, and the same number of myo-2p::GFP⁺ and myo-2p::GFP^- F2 worms at L4 stage were picked from each population plate into individual EV or sucg-1 RNAi plates. The later part of the RLS methodology follows the protocol above.

For RLS experiments of MCW1375 and MCW1385 strains, heterozygous parental worms were individualized onto the 6cm NGM plates at day 1 adulthood and the plates were kept for 4 days. The genotypes of the parental worms were then examined by the eGFP phenotypes in F1 under the fluorescent scope to ensure heterozygosity (of the parental line), and F1 progenies at L4 stage were randomly picked and individualized onto 3cm NGM plates. The later part of the RLS methodology follows the protocol above, with an additional step of examining the genotype of each F1 worm by observing the eGFP phenotypes in F2s.

Late fertility assay

Synchronized L1 larvae from egg preparation were plated on 6cm NGM plates seeded with the specific bacteria (default: HT115) and transferred every 2 days to new NGM plates from L4 until day 9. Individual hermaphrodites were transferred to 3cm NGM plates seeded with OP50 bacteria together with 2 day-2-old young N2 males for mating. Hermaphrodites were mated for 2 days before the first round of examination, which will exclude the plates with dead hermaphrodites, germline protruded hermaphrodites, or 2 dead males. The plates were then kept for one more day until the second-round examination of progeny production. Unlike RLS, internal hatched worms were not censored but instead considered as a reproduction event in late fertility assay. 15–20 hermaphrodites were used for each experiment, which was repeated at least 3 times independently to reach 60 worms per condition (before exclusion). The results from different trials were then pooled to conduct Fisher’s exact test to determine whether the number of worms that resumed reproduction after mating in each condition significantly differed from the controls.

Confocal imaging

Sample preparations were done by anesthetizing the worms in 1% sodium azide (NaAz) in M9 buffer and mounting them on 2% agarose pads on glass slides, and later covering the pads with coverslips. The worms were then imaged on laser scanning confocal FV3000 (Olympus, US) with water immersion 60x objective (UPLSAPO 60XW, Olympus, US) for SUCG-1 mitochondrial localization in the germline, germline morphology and mitochondrial localization of day 5 worms subjected to drp-1 RNAi knockdown, and oocyte mitochondrial distribution. 20x objective (UPLSAPO 20X, Olympus, US) was used for assessing the expression pattern of SUCG-1::eGFP and SUCA-1::eGFP, and the intensity of SUCG-1::eGFP in the germline on day 1 and day 5. 10X objective (UPlanFL N 10X, Olympus, US) was used to measure the body length of worms subjected to EV or eat-3 germline-specific RNAi knockdown, and intensity of intestinal acdh-1p::GFP on OP50 and HT115 bacteria.

Fluorescent intensity profiling of SUCG-1::eGFP and acdh-1p::GFP

The images of the germline SUCG-1::eGFP were generated by 20x z-stacked confocal imaging of PHX3617 strain, and the images of intestinal acdh-1p::GFP were generated by 10x z-stacked confocal imaging of VL749 strain.

For a given 3D image stack of eGFP labeled germline or intestine, the max intensity at each (x,y) location was projected to a single image, i_max. Multiple polygons p₁, p₂, …, p_m (m is the number of imaged germlines) were manually selected on i_max to outline germlines. A 2D mask m_i was generated for each p_i, with i =1, 2, …, m. m_i was extended to 3D mask vi by multiplying the depth of the stack and then using the vi to select the 3D region for calculation total and average intensity of eGFP. For SUCG-1::eGFP, the region selected spans from the proliferation zone to the mid-point of the U-shaped loop due to technical difficulties in consistently getting quality images of the entire germline and the blurred border between oocyte and spermatheca in aged worms. For acdh-1p::GFP, the entire intestine of the worms was selected for analysis.

All analyses above were done using MATLAB. Student’s test was used to determine whether the SUCG-1::eGFP intensities of day-5-old worms are statistically distinct from the day-1-old worms, and whether the acdh-1p::GFP intensities of worms on OP50 bacteria are statistically distinct from worms on HT115 bacteria. The code for the analyses is provided in Supplementary File 1.

Analysis of oocyte mitochondrial network

The images of the oocyte mitochondrial network were generated by 60x confocal imaging of EGD623 strain or mutant and integrated strains crossed with EGD623, and the position −2 oocytes were used for downstream analysis. Stacked oocytes with little distance between the nuclear membrane and the lateral side of the plasma membrane were excluded from the analysis.

For code-based radial intensity profiling of the oocyte mitochondrial network, mitochondrial distribution as their distance from the nucleus was quantified by generating two masks using manual selection with a polygon on the images - polygon p₁ outlining the nucleus and p₂ outlining the cell body. A set of rays were calculated with their origins at the mass center of p₁. The rays were customized to cover 360° with a step size of 1°. Each ray intersected with p₁ and p₂ and got a line segment. All line segments were divided into 5 equal segments, and labeled as ls₁, ls₂, …, ls₅, starting from the segment closest to the nucleus. All ends of ls₁ were connected to get a ring

shape r₁, and then the same for ls₂ to ls₅ resulting in r₂ to r₅. These rings were used as masks to select regions in an oocyte for mitochondrial intensity calculation leading to the generation of radial mitochondrial distribution. All the above analyses were done using MATLAB. The code for the analyses is provided in Supplementary File 2.

Later, the ring 1 occupancy of each oocyte was converted into one of the three categories using the following cutoffs – dispersed when lower than 23.5%, intermediate when equal or higher than 23.5% but lower than 26.5%, and perinuclear when equal or higher than 26.5%. The cutoffs were defined through double-blind categorization. Chi-squared test was then used to determine whether the oocyte mitochondrial distribution of each condition is significantly different from the control.

mtDNA levels measurement by quantitative PCR (qPCR) and droplet digital PCR (ddPCR)

The protocol from Gervaise et al. 2016 was followed for worm collection and germline dissection⁷³.

For the germline mtDNA levels measurement by qPCR, around 30 germlines were dissected for each condition. After dissection, germlines in M9 solution were collected into a PCR tube with a glass Pasteur pipette and centrifuged at 15000rpm for 2 minutes. Later, the excess M9 solution was removed from the PCR tube, and worm lysis buffer was added. The PCR tube was then placed at −80°C for at least 15 minutes before incubating at 60°C for 60 minutes followed by 95°C for 15 minutes for lysis and DNA release. qPCR was then performed using Power SYBR green master mix (Applied Biosystems #4367659) in a realplex 4 qPCR cycler (Eppendorf). To calculate the relative mtDNA levels, the cycle number of nduo-1 and ctb-1 (both encoded by mitochondrial DNA) were normalized to ant-1.3 (encoded by genomic DNA).

For the oocyte mtDNA copy number measurement by ddPCR, the proximal gonads containing differentiated oocytes (from −1 oocyte to where the loop/U-turn starts) were dissected. After dissection, each proximal gonad in M9 solution was collected into a PCR tube using a P2 pipette with low-retention tips, and worm lysis buffer was added. The PCR tube was then placed at −80°C for at least 15 minutes before incubating at 60°C for 60 minutes followed by 95°C for 15 minutes for lysis and DNA release. ddPCR was then performed using EvaGreen supermix (Bio-Rad #1864034) with droplet generation (Bio-Rad #1864002) followed by PCR in a thermocycler (Bio-Rad #1851196), and signals in each droplet were then detected by the droplet reader (Bio-Rad #1864003). The QX manager software was used for visualization and threshold setting to acquire droplet numbers that are positive and negative for the signal and then followed by copy number calculation using the online tool from Stilla Technologies (https://www.stillatechnologies.com/digital-pcr/statistical-tools/poisson-law-calculation/ ). To calculate the mtDNA copy per oocyte, the copy number of nduo-1 and ctb-1 were normalized to ant-1.3.

Body length measurement

The DIC channel on confocal microscopy was used to image the full body lengths of day 1 worms subjected to EV or eat-3 germline-specific RNAi knockdown side by side. The images were then analyzed using ImageJ by drawing segmented lines spanning the head to tail of the worms, which was then followed by distance measurement.

Pharyngeal pumping measurement

A digital camera (ORCA-Flash4.0 LT, Hamamatsu) attached to the stereoscope was used to record the pharyngeal pumping rate of worms subjected to EV or eat-3 germline-specific RNAi knockdown. After recording, the movies were played at 0.25X speed, and the times of pharyngeal pumping in each second (pumping rate) were counted. For each worm, the average pumping rate in 5–10 seconds was used for analysis.

Auxin treatment

Auxin (Alfa Aesar #A10556) was administered to the C. elegans using methodologies described in Zhang et al. 2015 with slight modification³⁸. A 400mM auxin stock solution in ethanol was prepared and filtered through a 0.22µm filter, which was stored at 4°C for up to 2 weeks. Auxin stock solution was added into the NGM liquid agar with a concentration of 1 to 100 (1%) after the autoclaved liquid agar dropped below 50°C and then poured into the plates making a final auxin concentration of 4mM. For the control plates, filtered ethanol was added to the NGM liquid agar with a concentration of 1 to 100 (1%). The plates were stored at 4°C inside a box with low photopermeability after the agar solidified. Before usage, fresh bacteria were concentrated by 4X before seeding onto the plates, and the plates that weren’t used immediately were stored at 4°C for up to 5 days.

Germline mitochondrial GTP and ATP measurement

Synchronized MCW1550 L1 larvae from egg preparation were plated onto 15cm NGM plates seeded with the 20X concentrated bacteria and grew to day 1. The worms were then harvested (day 1 sample) or filtered daily (filter out eggs and progenies) using a 40µm cell strainer and seeded onto a new 15cm NGM plate until day 5 before getting harvested (day 5 sample). Approximately 50k worms were used for day 5 sample collection and 100k worms were used for day 1 sample collection.

Germline mitochondria isolation was performed using methodologies described in Ahier et al. 2018 with modification⁷⁴. In short, worms were harvested into a 15cm centrifuge tube and washed 3 times with 10ml M9 buffer and then 2 more times with cold KPBS buffer (136mM KCl, 10mM KH2PO4, pH = 7.2). The worms were then transferred to a dauncer on ice and daunced until most worms were clearly broken. Later, the lysates were transferred into a centrifuge tube for low-speed centrifugation to precipitate large fragments, and the supernatant containing the organelles was then collected and centrifuged again at high speed to precipitate the organelles. The pellet was resuspended in KPBS buffer, anti-HA magnetic beads (Pierce #88837) were added, and the tube was incubated at 4°C for an hour to ensure binding efficiency. The anti-HA magnetic beads were then washed three times with KPBS, portioned out for protein concentration measurement by BCA assay and mitochondrial DNA content detection by qPCR, and the remaining beads were stored at −80°C for later steps of GTP and ATP detection.

For detection of nucleotides, immunoprecipitated mitochondria (with around 100 to 200μg mitochondria protein) were resuspended in pre-chilled water to the concentration of 1μg mitochondria protein per μl water. 500μl pre-chilled chloroform was then immediately added to the resuspended mitochondria samples, followed by vigorous vortexing to quench metabolism and to extract soluble metabolites. The mitochondria extracts were centrifuged at 20000g for 10min at 4°C to remove the organic phase, followed by another centrifugation at 20000g for 10min at 4°C to remove cell debris. The resulting supernatants were diluted 10 times (to 0.1μg mitochondria protein per μl water) and analyzed immediately using HPLC-MS as described previously^75,76.

Data analysis was performed using the Metabolomics Analysis and Visualization Engine (MAVEN) software⁷⁷. For each sample, ion counts of nucleotides were normalized to mitochondrial protein mass followed by mtDNA level. All samples were then normalized to the (HT115 bacteria; D1) condition to indicate fold changes.

Cobalamin treatment

Methylcobalamin (Sigma-Aldrich #M9756) and adenosylcobalamin (Sigma-Aldrich #C0884) were administered to the C. elegans using methodologies similar to auxin treatment. A 1.28mM aqueous stock solution was freshly prepared and filtered through a 0.22µm filter. The stock solution was added into the NGM liquid agar with a concentration of 1 to 10000 (0.01%) after the autoclaved liquid agar dropped below 50°C and then poured into the plates making a final cobalamin concentration of 128nM. For the control plates, filtered double-distilled water was added to the NGM liquid agar instead. The plates were stored at 4°C inside a box with low photopermeability after the agar solidified. Bacteria were seeded before usage, and the plates that weren’t used immediately were stored at 4°C for up to 5 days.

Succinate treatment

Sodium succinate (Sigma Aldrich #S2378) and succinic acid (Thermo Scientific Chemicals #AA3327236) were administered to the C. elegans via supplementation into the NGM plates. Precalculated amounts of sodium succinate and succinic acid were added into the liquid agar right after being taken out from the autoclave to make 10mM final concentration, and the agar was then poured into the plates after cooling down. The plates were stored at 4°C inside a box with low photopermeability after the agar solidified. Bacteria were seeded before usage.

Lifespan assay

Synchronized L1 larvae from egg preparation were plated onto 6cm NGM plates seeded with the bacteria that carry specific RNAi clones. Once reaching the L4 stage, worms were transferred to new 6cm plates, with 25–40 worms per plate and 80–120 worms per condition. Adult worms were transferred to new plates and examined every two days for their responses to gentle touch, to determine their status (alive or dead). The worms that could not be tracked until the day of death due to missing were counted as censors on the last day of valid observation.

QUANTIFICATION AND STATISTICAL ANALYSIS

The reproductive lifespan and lifespan analyses were performed using Kaplan-Meier survival analysis and a log-rank test in the SPSS. Chi-squared tests and Fisher’s exact tests were performed in Graphpad PRISM to compare categorical variables, and Holm-Bonferroni method was used for correction as indicated in the corresponding figure legends. Student’s t-test (unpaired) was performed in Excel to compare the mean of different samples, and Holm-Bonferroni method was used for correction as indicated in the corresponding figure legends. For all figure legends, asterisks indicate statistical significance as follows: n.s. = not significant p>0.05; * p<0.05; ** p<0.01; *** p<0.001. Data were collected from at least three independent biological replicates. Figures and graphs were constructed using BioRender, PRISM, and Illustrator.

Supplementary Material

NIHMS1931616-supplement-2.pdf^{(4.6MB, pdf)}

Highlights.

Mitochondrial GTP-specific succinyl-CoA synthetase regulates reproductive longevity
Oocyte mitochondrial positioning influences reproductive health during aging
Mitochondrial fission in the oocyte promotes reproductive longevity
Bacterial vitamin B12 modulates reproductive aging via GTP succinyl-CoA synthetase

ACKNOWLEDGMENT

This work was supported by NIH grants R01AG045183 (M.C.W.), R01AT009050 (M.C.W.), R01AG062257 (M.C.W.), DP1DK113644 (M.C.W.), R35GM127088 (J.D.W.), March of Dimes Foundation (M.C.W.), Welch Foundation (M.C.W.), HHMI investigator (M.C.W.), American Federation for Aging Research (Y.L.), Louis and Elsa Thomsen Wisconsin Distinguished Graduate Fellowship (J.Y.). We thank P. Svay and C. Huang for maintenance support; Dr. I. Neve for discussion and technical support; Dr. B. Bowerman for providing the drp-1 endogenous locus sequence information of the EU2917 strain; Dr. H. Zoghbi for providing access to ddPCR equipment and Dr. S. Wu and Dr. J. Revelli in her lab for experimental consultation; BioRender for the support on creating the graphical abstract, Fig 1A, Fig 3A, and Fig 4A; the Caenorhabditis Genetics Center (CGC) for C. elegans strains.

Footnotes

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DECLARATION OF INTERESTS

The authors declare no competing interests.

INCLUSION AND DIVERSITY

One or more of the authors of this paper self-identifies as a gender minority in their field of research. While citing references scientifically relevant for this work, we also actively worked to promote gender balance in our reference list.

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Associated Data

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Supplementary Materials

NIHMS1931616-supplement-2.pdf^{(4.6MB, pdf)}

Data Availability Statement

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PERMALINK

Mitochondrial GTP Metabolism Controls Reproductive Aging in C. elegans

Yi-Tang Lee

Marzia Savini

Tao Chen

Jin Yang

Qian Zhao

Lang Ding

Shihong Max Gao

Mumine Senturk

Jessica Sowa

Jue D Wang

Meng C Wang

SUMMARY

Graphical Abstract

eTOC Blurb

INTRODUCTION

RESULTS

GTP-specific SCS regulates reproductive aging

Figure 1. GTP-specific Succinyl-CoA Synthetase (SCS) regulates reproductive aging.

Germline GTP-specific SCS regulates mitochondria and reproductive aging

Figure 2. GTP-specific SCS functions in the germline to regulate oocyte mitochondria during reproductive aging.

Mitochondrial fission drives reproductive longevity

Figure 3. Mitochondrial fission-fusion factors regulate reproductive longevity.

GTP-specific SCS regulates reproductive aging through tuning mitochondrial distribution

Figure 4. GTP-specific SCS regulates reproductive aging through mitochondrial fission factor.

GTP-specific SCS regulates reproductive aging in response to bacterial inputs

Figure 5. Bacterial inputs regulate germline mitochondrial GTP and reproductive aging.

Bacteria modulate mitochondrial distribution during reproductive aging

Figure 6. Mitochondrial fission-fusion factors mediate bacterial effects on reproductive longevity.

Vitamin B12 deficiency in OP50 E. coli contributes to reproductive longevity

Figure 7. Bacterial VB12 regulates oocyte mitochondria and reproductive aging.

DISCUSSION

LIMITATIONS OF THE STUDY

STAR★METHODS

RESOURCE AVAILABILITY

Lead contact

Materials availability

Data and code availability

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Strains and maintenance

Strain generation – Extrachromosomal array

Strain Generation – Integration of extrachromosomal array

Strain Generation – CRISPR-Cas9 mediated insertion and deletion

Strain Generation – Crossing

METHOD DETAILS

RNA interference (RNAi) experiments

Construction of plasmid and fusion PCR product

Reproductive lifespan assay and progeny number measurement

Late fertility assay

Confocal imaging

Fluorescent intensity profiling of SUCG-1::eGFP and acdh-1p::GFP

Analysis of oocyte mitochondrial network

mtDNA levels measurement by quantitative PCR (qPCR) and droplet digital PCR (ddPCR)

Body length measurement

Pharyngeal pumping measurement

Auxin treatment

Germline mitochondrial GTP and ATP measurement

Cobalamin treatment

Succinate treatment

Lifespan assay

QUANTIFICATION AND STATISTICAL ANALYSIS

Supplementary Material

Highlights.

ACKNOWLEDGMENT

Footnotes

REFERENCES

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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