Abstract
Classical evolutionary theories propose tradeoffs among reproduction, damage repair and lifespan. However, the specific role of the germline in shaping vertebrate aging remains largely unknown. In this study, we used the turquoise killifish (Nothobranchius furzeri) to genetically arrest germline development at discrete stages and examine how different modes of infertility impact life history. We first constructed a comprehensive single-cell gonadal atlas, providing cell-type-specific markers for downstream phenotypic analysis. We show here that germline depletion—but not arresting germline differentiation—enhances damage repair in female killifish. Conversely, germline-depleted males instead showed an extension in lifespan and rejuvenated metabolic functions. Through further transcriptomic analysis, we highlight enrichment of pro-longevity pathways and genes in germline-depleted male killifish and demonstrate functional conservation of how these factors may regulate longevity in germline-depleted Caenorhabditis elegans. Our results, therefore, demonstrate that different germline manipulation paradigms can yield pronounced sexually dimorphic phenotypes, implying alternative responses to classical evolutionary tradeoffs.
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Data availability
All raw RNA sequencing data (bulk and single-cell) and Hi-C data, as well as processed datasets, can be found in the Gene Expression Omnibus database under accession numbers GSE248741 and GSE218971, respectively. Metabolomics data are available in Supplementary Table 9. All other data are available from the corresponding author upon reasonable request. Individual supplementary tables are available in Mendeley Data (https://data.mendeley.com/datasets/ggys689v6x).
Code availability
The code supporting the current study is available in the following GitHub repository: https://github.com/Harel-lab/germline-regulation-of-the-vertebrate-lifespan. The Hi-C code is available at https://gitlab.com/mcfrith/last/-/blob/main/doc/last-papers.rst.
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Acknowledgements
We thank A. Zaslaver, E. Meshorer, A. Brunet, Y. Tzur, N. E. Stroustrup, M. Nitzan and the Harel laboratory for stimulating discussion and feedback on the manuscript. We thank A. Velan, E. Yanay, A. Abu-tair, Y. Birenbaum, F. Idrees and R. Barakat for help with killifish maintenance and N. Melamed-Book from the imaging facility (HUJI) and S. Malitsky and M. Itkin from the Life Sciences Core Facilities, Metabolic Profiling Unit (Weizmann Institute of Science). This study was supported by ERC StG no. 101078188 (I.H.), the Zuckerman Program (I.H.), the Abisch-Frenkel Foundation 19/HU04 (I.H.), ISF 2178/19 (I.H.), Israeli Ministry of Science 3-17631 (I.H.) and 3-16872 (I.H.), the Moore Foundation GBMF9341 (I.H.), BSF-NSF 2020611 (I.H.), the Israeli Ministry of Agriculture 12-16-0010 (I.H.), the Levi Eshkol Scholarship of the Israeli Ministry of Science (E.M.), the Pamela and Paul Austin Research Center on Aging fellowship (T.A.), the Czech Science Foundation (no. 22-01781O), the Ministry of Education, Youth and Sports of the Czech Republic (no. CZ.02.1.01/0.0/0.0/16_025/0007370) (R.F.), Chan Zuckerberg Initiative 2017-174468 and 2018-182817 (W.J.G.) and National Institutes of Health grants P50HG007735, UM1HG009442 and 1UM1HG009436 (W.J.G.).
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E.M., T.A. and I.H. designed the study. E.M. and R.F. performed experiments. E.M. generated the HPG and dnd1 mutant killifish lines. X.S. and E.M. prepared the single-cell RNA libraries, under the supervision of O.R. and I.H., respectively. T.A. designed and performed the analysis of RNA sequencing, under the supervision of I.H. A.S. performed worm lifespan experiments, under the supervision of E.C. T.A. and E.M. performed statistical analyses, with help from S.S. and D.M.Z. A.O.-G., G.K.M. and W.J.G. performed and analyzed the Hi-C data. T.A., E.M. and I.H. wrote the manuscript. All authors commented on the manuscript.
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Extended data
Extended Data Fig. 1 Filtering germ cell clusters.
(a) Gene expression UMAP plot of piwil1, a germ cell marker gene. (b) Sub-clustering of the germ cells, performed according to Fig. 1c, color-coded by sex (left). Within this cluster, a marker of erythroid cells (top right) and previtellogenic oocytes (bottom right) is also indicated. A full list of erythroid marker genes can be found in Supplementary Table S3.
Extended Data Fig. 2 Pseudotime of germ cell clusters.
(a) Sub-clustering of the male germ cells excluding the female germ cells and erythroid cells. Cells are color-coded and labeled pseudo-time.
Extended Data Fig. 3 Perturbation and rescue of the reproductive axis in killifish.
(a) Histological sections from one-month-old males. n ≥ 4 individuals from each genotype (except for lhb, which was sex-linked, n = 1). Scale bar: 50 µm. Sperm developmental stages according to102: SG: spermatogonia; SC: spermatocytes; ST: spermatids; SZ: spermatozoa. (b) Quantification of sperm maturation, examples in (a). Data presented as proportion of each developmental stage. n ≥ 4 individuals for each experimental group (except for lhb). Significance was measured by a two-sided χ2 test with the WT proportion as the expected model. (c) Distribution of genotype progeny from heterozygous pairs (stratified by sex). n = 30-130 individuals, per genotype/sex. Significance was measured by a two-sided χ2 test with Mendelian proportions (25:50:25) as the expected model and FDR correction. (d) Quantification of somatic growth by calculating the standard length of one-month-old males (left) and females (right) of the indicated genotypes. n = 8-12 individuals for all experimental groups. Error bars indicate mean ± SEM. Significance was calculated using one-way ANOVA. (e) Quantification of male fertility. Each dot represents the number of eggs per breeding pair, per week of egg collection (except for lhb, which was sex-linked). n = 4-6 pairs for each group, over 4 collections. Error bars indicate mean ± SEM with individual points. Significance was calculated using one-way ANOVA. (f) Schematic illustration (left). Representative of n = 3 females. Quantification of fertility output (right) in lhbΔ8/Δ8 mutant females following plasmid electroporation. Each dot represents the number of eggs per breeding pair per week. n = 3-6 pairs over 4 collections. Error bars show mean ± SEM. Significance was calculated using a two-sided Student’s t-test. (g) smFISH in the ovaries of the indicated genetic models. Marker for immature germ-cells (ddx4) in red, and for fshr and lhr in green. Representative of n ≥ 6 individuals. Scale bar: 50 µm. (h) Oxford Grid plots (left) showing correspondence between the Hi-C linkage groups (Y-axis) and previously predicted chromosomes (X-axis). Positive strand in blue, and negative strand matches in red. Cumulative fraction of genes (right) located on Hi-C pseudochromosomes and on contigs in the original assembly (gray). In the Hi-C scaffolded assembly (black), most small contigs containing genes are placed into the 19 main pseudochromosomes.
Extended Data Fig. 4 Physiological characterization germline-depleted fish.
(a) Quantification of somatic growth (standard length) of WT or dnd1Δ4/Δ4 one-month-old mutants: males (top) and females (bottom), n = 8-12 individuals for all experimental groups. Error bars show mean ± SEM. Significance was measured using a two-sided Student’s t-test. (b) Representative images of adult (2.5-month-old) males (top) and females (bottom) WT or dnd1Δ4/Δ4 mutant fish. Black arrowheads highlight the presence of eggs in the female representative of n ≥ 4 individuals. Scale bar: 3.5 mm. (c) Distribution of genotype progeny from dnd1Δ4/+ heterozygous pairs (stratified by sex). Significance was measured by two-sided χ2 test with Mendelian proportions (25:50:25) as the expected model and FDR correction. n ≥ 500 fish for each group and p-values are indicated. (d) Tail-fin regeneration following whole-body irradiation in males. Genotypes and treatments are indicated (n = 4 individuals for non-irradiated WT and 6 individuals for WT and dnd1). The length of the outgrowth was calculated as the percentage of the original fin size (before the amputation). Error bars indicate mean ± SEM. Significance was calculated using repeated-measures two-way ANOVA with a Dunnet post-hoc compared to the irradiated WT, and p-values are indicated. (e) Tail-fin regeneration during aging, in males (left) and females (right). Fish sex, genotypes, and age are indicated (n = 5-7 individuals for all experimental groups). Error bars indicate mean ± SEM. Significance was calculated using repeated measures two-way ANOVA with a Tukey post-hoc, and p-values of the comparison between young and old fish of each genotype are indicated by color-coding.
Extended Data Fig. 5 Calibration of DNA damage detection.
(a) Representative images of apoptosis detected by the TUNEL assay (green and green arrows) and DNA (PI, in red). Assay was performed in gut sections of non-irradiated (left) or irradiated fish (right). n = 6 individuals in each experimental group. Scale bar: 50 µm.
Extended Data Fig. 6 Characterization of cell type markers and differential gene expression in dnd1Δ4/Δ4 mutant gonads.
(a-b) Dot-plot (top left, a, left, b) and UMAPs presenting the expression of select marker genes for males (a) and females (b). Dot-plot clusters are color-coded according to Fig. 6a, b (for males and females separately). Note that most of the markers overlap with Fig. 1c. A full list of markers can be found in Supplementary Tables S5, S6. (c) smFISH in ovaries for selected markers in dnd1Δ4/Δ4 mutants. Amh (red), a marker for Pre- and mature Granulosa cells, and cyp19a1 (green), a specific marker for mature Granulosa. Note that cyp19a1 is expressed in the adjacent adipose tissue. Representative of n ≥ 4 individuals. Scale bar: 20 µm. (d) Log2 fold-change heatmap represents the gene expression ratio between WT and dnd1Δ4/Δ4 fish of the indicated cell-types. The genes were selected as they were similarly altered in several cell types. (e) smFISH in WT fish and dnd1Δ4/Δ4 ovaries. Eef1a1, a marker for translation initiation (green), and ptgds, a marker for ovarian epithelium (in red). Representative of n ≥ 4 individuals. Scale bar: 20 µm.
Extended Data Fig. 7 Germline depletion extends male maximal lifespan.
(a) Quantile-Quantile (Q-Q) plots for the survival data of WT and dnd1Δ4/Δ4 fish (shown in Fig. 7b), assessed separately for males (left) and females (right). (b) Heatmap for normalized enrichment scores (NESs) in the male liver, comparing the response to gonadal and hormonal treatments in mice with germline-depleted young fish (left, selected pathways highlighted on the right). A full list of pathways can be found in Supplementary Table S8. Esr1: estrogen receptor KO; E2: estrogen treatment; T: testosterone treatment. (c) Lifespan of C. elegans from the glp-1(e2144) mutants, grown either at the permissive 15 °C (fertile, left) or restrictive 25 °C (germline depleted, right), and fed with either EV or eef1a1 RNAi. P-values were calculated according to log-rank. Worm numbers and raw data can be found Supplementary Table S4.
Extended Data Fig. 8 Germline depletion enhances regeneration under several stressors.
(a) PCA for hepatic transcript levels. Groups include male (blue) or female (red), WT (dark shades) or dnd1Δ4/Δ4 (light shades), either young (circles) or old (triangles). n = 3-4 samples per condition, and each symbol represents an individual fish. (b) Top, left: gene expression UMAP plot of dnd1 gene. Right and bottom: tail-fin regeneration of WT and dnd1morphant fish following whole-body irradiation. n = 4 individuals for WT and dnd1morphant males, 7 for WT and dnd1morphant females and 10 for non-irradiated males and females. Error bars indicate mean ± SEM. Significance was calculated using repeated-measures two-way ANOVA with a Dunnet post-hoc, compared to WT irradiated fish, and p-values are indicated. (c) Tail-fin regeneration of WT fish following treatment by chloroquine or paraquat. Treatments and sexes are indicated. n = 5 individuals for untreated females, 11 for chloroquine treated females, 12 for untreated and chloroquine treated males, and paraquat treated females and 15 for paraquat treated males. The same untreated fish were used as control for both treatments. Error bars indicate mean ± SEM. Significance was calculated using repeated-measures two-way ANOVA with a Sidak post-hoc, and p-values are indicated. (d) Tail-fin regeneration of WT and dnd1morphant fish following treatment by chloroquine or paraquat. Treatments and sexes are indicated. n = 5 individuals for chloroquine treated dnd1morphant males, 7 for paraquat treated dnd1morphant males and chloroquine treated dnd1morphant females, 8 for chloroquine treated WT and paraquat treated WT and dnd1morphant females, 9 for paraquat treated WT males and 10 for chloroquine treated WT males. Error bars indicate mean ± SEM. Significance was calculated using repeated-measures two-way ANOVA with a Sidak post-hoc, and p-values are indicated. (e) Right: Representative images of proliferation detected in the livers of male fish of the indicated experimental groups, following a 3-day treatment of EdU (green, see Methods) with DAPI (blue) for detecting nuclei. Representative of n = 5-7 individuals per group. Scale bar: 50 μm. Left: Quantification of the percentage of proliferating cells. Error bars indicate mean ± SEM. Significance was calculated using one-way ANOVA with a Tukey post-hoc, and p-values are indicated.
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Moses, E., Atlan, T., Sun, X. et al. The killifish germline regulates longevity and somatic repair in a sex-specific manner. Nat Aging 4, 791–813 (2024). https://doi.org/10.1038/s43587-024-00632-0
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DOI: https://doi.org/10.1038/s43587-024-00632-0
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