Abstract
Reproductive ageing is one of the earliest human ageing phenotypes, and mitochondrial dysfunction has been linked to oocyte quality decline; however, it is not known which mitochondrial metabolic processes are critical for oocyte quality maintenance with age. To understand how mitochondrial processes contribute to Caenorhabditis elegans oocyte quality, we characterized the mitochondrial proteomes of young and aged wild-type and long-reproductive daf-2 mutants. Here we show that the mitochondrial proteomic profiles of young wild-type and daf-2 worms are similar and share upregulation of branched-chain amino acid (BCAA) metabolism pathway enzymes. Reduction of the BCAA catabolism enzyme BCAT-1 shortens reproduction, elevates mitochondrial reactive oxygen species levels, and shifts mitochondrial localization. Moreover, bcat-1 knockdown decreases oocyte quality in daf-2 worms and reduces reproductive capability, indicating the role of this pathway in the maintenance of oocyte quality with age. Notably, oocyte quality deterioration can be delayed, and reproduction can be extended in wild-type animals both by bcat-1 overexpression and by supplementing with vitamin B1, a cofactor needed for BCAA metabolism.
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Data availability
All RNA-seq data are deposited under the PRJNA966212 BioProject. Microarray data are available on the PUMA database at http://puma.princeton.edu as experiment set 7,368. Proteomics data are available on PRIDE under accession code PXD048296. Source data are provided with this paper.
Code availability
No custom code was used for this study.
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Acknowledgements
We thank H. Shwe and S. Kyin (Princeton Proteomics and Mass Spectrometry Core) for their assistance in sample processing and MS instrumentation, J. Miller and J. Volmar at the Genomics Core Facility of Princeton University for performing library preparation and next-generation sequencing, G. Laevsky and S. Wang (Confocal Imaging Facility, a Nikon Center of Excellence, in the Department of Molecular Biology at Princeton University), The De Botton Protein Profiling Institute of the Nancy and Stephen Grand Israel National Center for Personalized Medicine Weizmann Institute of Science (Y. Levin), the Petry Laboratory and the Gitai Laboratory (the Department of Molecular Biology at Princeton University) for help with equipment, the CGC for strains and Murphy Laboratory members for comments on the manuscript. The study was supported by funding to C.T.M. from the Global Consortium for Reproductive Longevity and Equality (AWD1006679), The Simons Foundation (811235SPI), The Glenn Foundation for Medical Research and Pioneer (DP1AG077430) and Transformative (R01AT011963) grants from the National Institutes of Health.
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Conceptualization was the responsibility of C.L., R.K. and C.T.M. Methodology was the responsibility of C.L., R.K. and C.T.M. Investigation was the responsibility of C.L., J.M.A., R.K., S.S., V.C., T.S., W.K. and S.L. Writing was the responsibility of C.L., R.K. and C.T.M. Funding acquisition was the responsibility of C.T.M.
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Extended data
Extended Data Fig. 1 Highly purified mitochondria are isolated from C. elegans.
Representative western blot of the different cellular fractions. The mitochondrial marker ATP5A is prominently enriched in the membranous fraction, unlike the cytosolic marker αTUB. The membranous fraction was then loaded on a gradient to highly purify the mitochondrial fraction. T- total homogenate, C- cytosolic fraction, M- crude membranes fraction. αTUB- αtubulin, HISH3- histone H3. The asterisk represents a non-specific band detected when using the anti-ATP5A antibody. Dashed lines mark where membranes were cut prior to incubation with the indicated antibody. 3 biological replicates were performed.
Extended Data Fig. 2 Differentially expressed oocyte genes in day 8 daf-2;control and daf-2;bcat-1(RNAi) worms.
Volcano plot of differentially expressed genes identified between daf-2;control and daf-2;bcat-1(RNAi) Day 8 oocytes. Red dots denote upregulated genes in daf-2;control oocytes, and orange dots denote genes downregulated in daf-2;control oocytes relative to daf-2;bcat-1(RNAi) (0.5 < log2FC < −0.5).
Extended Data Fig. 3 Controls for mitochondrial assays.
(a) mtROS levels increase in oocytes after paraquat treatment. A white dashed outline marks the most mature oocytes (−1 oocyte). Right: mtROS levels quantified in mature oocytes either with or without paraquat treatment. Control n = 20, Paraquat n = 24. (b) Left: FCCP treatment reduces mitochondrial membrane potential in oocytes. A white dashed outline marks the most mature oocytes (−1 oocyte). Right: TMRE levels quantified in mature oocytes under control or FCCP treatment conditions. Control n = 15, FCCP n = 22. (a, b) Two-tailed, unpaired t-test. ****p ≤ 0.0001. Dashed lines on the graphs represent mean intensity of autofluorescence. (a, b) Box plots: centre line, median; box range, 25–75th percentiles; whiskers denote minimum–maximum values. 3 biological replicates were performed.
Extended Data Fig. 4 Deletion of acdh-1 in daf-2 or wild-type worms does not affect mitochondrial function.
(a) Left: mtROS levels do not change in daf-2 mature oocytes on Day 5 upon acdh-1 deletion. Right: quantification of mtROS levels in daf-2;acdh-1 mature oocytes on day 5. N ≥ 45 for each condition. Graph shows results of pooled three biological replicates. (b) Left: acdh-1 deletion does not affect mitochondrial membrane potential in daf-2 mature oocytes on day 5. Right: quantification of TMRE signal in daf-2 mature oocytes on Day 5 upon acdh-1 deletion. N > 10 for all conditions. (c) Left: mtROS levels do not change in wild-type mature oocytes on Day 5 upon acdh-1 deletion. Right: quantification of mtROS levels in wild-type vs. acdh-1 mutant mature oocytes on day 5. N ≥ 17 for each condition. Graph shows results of pooled three biological replicates. (a–c) A white dashed outline marks the most mature oocytes (−1 oocytes). Two-tailed, unpaired t-test. ns- nonsignificant. For all panels, representative images are shown. 3 biological replicates were performed.
Extended Data Fig. 5 Downregulating bcat-1 in wild-type worms does not affect lifespan.
(a) Downregulation of bcat-1 reduces the late-mated reproduction of adult wild-type worms at day 6 of adulthood. n = 69 for control, n = 58 for bcat-1(RNAi). Chi-square test. ****p ≤ 0.0001. 3 biological replicates were performed. (b) GFP expression in control (left) and in the bcat-1-overexpression strain (GFP-BCAT-1 protein fusion). 3 biological replicates were performed. (c) Adult-only knockdown of bcat-1 does not affect the lifespan of wild-type animals. Lifespan data from 6 biological replicates are shown. Results are plotted as Kaplan–Meier survival curves, and the p-values were calculated using Mantel–Cox log-ranking. All replicates are shown. Statistical data is detailed in Table S2.
Extended Data Fig. 6 Differentially expressed oocyte genes in day 8 control and bcat-1 overexpressing worms.
Volcano plot of differentially expressed genes identified between Day 8 oocytes from bcat-1-overexpressing worms compared to the control (DESeq2, padj < 0.05). Pink dots denote upregulated genes in oocytes from bcat-1 overexpressing worms, and gold dots denote downregulated genes (0.5 <log2FC <−0.5).
Supplementary information
Supplementary Tables 1–5
Supplementary Table 1 Proteomic data. LFQ intensity of all peptides detected by LC–MS/MS proteomic analysis in mitochondria fractions isolated from young (day 1 adult) and reproductively aged (day 5 adult) wild-type worms and from daf-2(e1370) mutants. a–c, All peptides detected, and the significant proteins (proteins with more than one peptide, FC > 2 and q value < 0.05) (a) for comparing wild-type proteomic data of day 1 versus day 5 (b), as well as the significant proteins for comparing proteomic data of Supp daf-2 versus wild-type day 5 (c). Supplementary Table 2 Statistical data. a–c, Statistical analysis data of all biological replicates for reproductive span (a), lifespan (b) and late-mated reproduction experiments (c). Supplementary Table 3 RNA sequencing data for day 8 oocytes isolated from daf-2(e1370);control versus daf-2(e1370);bcat-1(RNAi) animals. a,b, Oocyte genes significantly upregulated (a) or downregulated (b) in daf-2(e1370);control(RNAi) versus daf-2(e1370);bcat-1(RNAi) animals. DESeq2 results were filtered for daf-2;control(RNAi) upregulated genes (bcat-1-dependent daf-2 oocyte genes) and daf-2;control(RNAi) downregulated genes (Padj < 0.05). c, Significant Gene Ontology terms are listed for the upregulated and downregulated gene sets. Supplementary Table 4 RNA sequencing data for day 8 oocytes isolated from fem-1(hc17) IV vs fem-1(hc17) IV; bcat-1 overexpressing worms. a,b, Significantly upregulated (a) and downregulated (b) genes in isolated oocytes of fem-1(hc17) IV versus fem-1(hc17) IV; risIs3 worms. DESeq2 results were filtered for differentially expressed genes (Padj < 0.05). Supplementary Table 5 C. elegans strains used in this study. Strain names and genotypes are listed.
Supplementary Video 1
Supplementary Video 1: daf-2 control germline mitochondria. 3D projections of z-slices of germline-labelled mitochondria in daf-2 animals treated with control RNAi. Related to data in Fig. 4a.
Supplementary Video 2
Supplementary Video 2: daf-2;bcat-1(RNAi) germline mitochondria. 3D projections of z-slices of germline-labelled mitochondria in daf-2 animals treated with bcat-1 RNAi. Related to data in Fig. 4a.
Supplementary Video 3
Supplementary Video 3: daf-2;bcat-1(RNAi) germline mitochondria. 3D projections of z-slices of germline-labelled mitochondria in daf-2 animals treated with bcat-1RNAi. Related to data in Fig. 4a.
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Lesnik, C., Kaletsky, R., Ashraf, J.M. et al. Enhanced branched-chain amino acid metabolism improves age-related reproduction in C. elegans. Nat Metab 6, 724–740 (2024). https://doi.org/10.1038/s42255-024-00996-y
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DOI: https://doi.org/10.1038/s42255-024-00996-y
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