TGF-β and insulin signaling regulate reproductive aging via oocyte and germline quality maintenance

Abstract

Reproductive cessation is perhaps the earliest aging phenotype that humans experience. Similarly, reproduction of Caenorhabditis elegans ceases in mid-adulthood. Although somatic aging has been studied in both worms and humans, mechanisms regulating reproductive aging are not yet understood. Here, we show that TGF-β Sma/Mab and Insulin/IGF-1 signaling regulate C. elegans reproductive aging by modulating multiple aspects of the reproductive process, including embryo integrity, oocyte fertilizability, chromosome segregation fidelity, DNA damage resistance, and oocyte and germline morphology. TGF-β activity regulates reproductive span and germline/oocyte quality noncell-autonomously and is temporally and transcriptionally separable from its regulation of growth. Chromosome segregation, cell cycle, and DNA damage response genes are upregulated in TGF-β mutant oocytes, decline in aged mammalian oocytes, and are critical for oocyte quality maintenance. Our data suggest that C. elegans and humans share many aspects of reproductive aging, including the correlation between reproductive aging and declining oocyte quality and mechanisms determining oocyte quality.

Figure 1. TGF-β Sma/Mab and insulin/IGF-1 signaling regulate embryo viability, oocyte fertilizability, and oocyte morphology

(A) Schematic of the C. elegans gonad. (B) Percentage of embryos that fail to hatch (± SEP). (C) Percentage of male progeny (± SEP). (D) Percentage of oocytes with 6 DAPI-stained bodies (± SEP). (E) Number of hatched embryos (inset, left), unhatched embryos, and unfertilized oocytes (inset, right) produced each day after mating with young wild-type(wt) males (mean ± SEM); percentages shown in Figure S1F. (F) Oocyte morphology, with defects in yellow. (G) Oocyte morphology markers scored in mated wt animals that are either reproductive (Repro) or post-reproductive (PR). (H) Oocyte morphology markers scored in day 8 mated worms. p-values for wild-type versus daf-2 or sma-2 indicated. For all figures, * indicates p-value < 0.05, ** < 0.01, *** < 0.001.

Figure 2. TGF-β Sma/Mab and insulin/IGF-1 signaling regulate DNA damage response and distal germline integrity

(A) Distal germline morphology, with defects in yellow. (B) Distal germline morphology scores of day 8 mated worms; p-values compare wt versus daf-2 or sma-2. *p < 0.05, ** < 0.01, *** < 0.001. (C) Percent decrease in mitotic germ cell number with age (raw values in Figure S2I). (D) daf-2 and sma-2 lay significantly more hatched embryos than wild type after γ-irradiation (% ± SEP). Animals were mated with young wt males after irradiation.

Figure 3. TGF-β Sma/Mab signaling regulates reproductive aging non-autonomously in hypodermis

(A) Body length of wt, sma-3(wk30), sma-3(wk30);qcEx26[sma-3 gDNA; sur-5::gfp] animals that have lost or silenced transgenic sma-3 expression in the germline (mean ± SEM). (B) Mated reproductive spans of worms in (A). *high matricide rate due to internal progeny hatching. (All reproductive span statistics are shown in Table S1.) (C and D) Scoring of oocyte (C) and distal germline (D) morphology markers in day 8 mated wt, sma-3, and sma-3 germline-lost (GL) animals. (E) Two independent transgenic lines (sma-3(wk30);Pvha-7::gfp::sma-3) expressing sma-3 in the hypodermis have mated reproductive spans similar to wild type (Table S1). (F) sma-9 RNAi significantly extends mated reproductive span of rrf-3 worms, but does not extend the mated reproductive span of the somatic-gonad-only RNAi strain rrf-3;rde-1;qyIs103[Pfos-1a::rde-1].

Figure 4. Insulin/IGF-1 signaling regulates reproductive aging non-autonomously in intestine and muscle

(A) daf-16 germline silent worms (daf-16(mu86);daf-2(e1370);muIs105 [Pdaf-16::gfp::daf-16 +rol-6(su1006)], Figure S4) with only somatic daf-16 activity have a reproductive span similar to daf-2 mutants (statistics in Table S1). (B-D) daf-16 activity in intestine (B and D) and muscle (C and D) significantly restores reproductive span extension, while neuronal daf-16 activity (C and D) does not. (E and F) Oocyte (E) and distal germline (F) morphology scores of day 8 mated daf-16;daf-2, daf-2, endogenous-promoter-driven and tissue-specific promoter-driven daf-16 transgenic animals. *p < 0.05, ** < 0.01, *** < 0.001 for daf-16;daf-2 versus other genotypes.

Figure 5. TGF-β Sma/Mab signaling regulates oocyte quality and body size through distinct sets of downstream targets

(A) sma-9 RNAi adult-only treatment reduces body size significantly (p<0.001, 14% decrease), while adult-only treatment does not (mean ± SEM). (B) Mated reproductive spans of rrf-3 animals treated with control RNAi whole-life, with sma-9 RNAi whole-life, or with sma-9 RNAi in adulthood only (Table S1). (C) Expression heatmap of 386 genes significantly upregulated in sma-2;fem-1 oocytes (FDR=0%). (D) Enriched GO terms for genes in (C). Example genes from this study (worm) and genes up-regulated in young vs. old mouse (Hamatani et al., 2004) or human (Steuerwald et al., 2007) oocytes are listed, with highly homologous or important interacting genes in bold. (Expanded gene list in Table S3.) GO terms also enriched in younger human (H), mouse (W), or worm (W) oocytes are labeled with corresponding superscript letters. * indicates a gene involved in corresponding GO function but failed to be recognized by DAVID (not included in gene counts). (E) GO terms enriched in TGF-β Sma/Mab mutant oocytes are largely distinct from those enriched in Sma/Mab L4 and L2 (Liang et al., 2007) larvae.

Figure 6. TGF-β Sma/Mab signaling regulates genes essential for embryonic viability in oocytes

(A) RNAi knockdown of many sma-2-regulated oocyte targets increase sma-2 mutant’s embryonic lethality (mean ± SEM). (B) RNAi knockdown of smc-4, cyb-3, and E03H4.8 have early and severe effects on reproductive span (Table S1). (C-F) RNAi knockdown of smc-4, cyb-3, and E03H4.8 greatly increase the percentage of unhatched embryos (orange) in mated sma-2 mutants (compare D-F with C). Wild type treated with RNAis shown in Figure S6D-F. (G-J) smc-4, cyb-3, and E03H4.8 RNAi-treated sma-2 animals exhibit severely degraded germlines at day 8 (compare H-J with G). Contours of gonads shown in yellow, visible oocytes outlined by dotted lines in (G).

Figure 7. TGF-β Sma/Mab signaling regulates genes important for age-associated oocyte quality maintenance

(A-F) RNAi treatments of TGF-β target genes accelerate oocyte quality decline, increasing the percentage of unhatched embryos (orange) and/or unfertilized oocytes (yellow) earlier in life (compare with Figure 6C). mlh-1, math-33, and F47G4.4 RNAis have greater effects (A-C), while F52D10.2, uev-2, and pme-5 have milder effects (D-F). Wild type treated with RNAis shown in Figure S7C-F, I-J). (G) uev-2 RNAi treatment significantly increases sma-2’s production of unhatched embryos after γ-irradiation, while pme-5 and mlh-1 do not. Animals were mated with young wt males after irradiation. (H) Model of reproductive aging regulation by the TGF-β Sma/Mab (pink) and insulin/IGF-1 signaling (red) pathways. Ligands (Insulin-Like Peptides, TGF-β DBL-1) are secreted neuronally and mediate signaling to the soma (hypodermis, intestine, and muscle), generating as yet unidentified secondary signals to regulate reproduction. These secondary signals block distal germline and oocyte integrity maintenance with age, resulting in germline morphology decline, slowed germ cell proliferation, and a decline in oocyte quality. Downstream effectors in oocytes include chromosome segregation, cell cycle, and DNA damage response/repair genes, etc. Declines in embryonic viability and infertility mark reproductive cessation. The germline and somatic gonad regulate somatic aging (Hsin and Kenyon, 1999), suggesting a bi-directional signaling flow in the coordination of somatic and germline aging. (Photo courtesy of Ian Chin-Sang.)