Insulin Signaling Regulates Oocyte Quality Maintenance with Age via Cathepsin B Activity

Abstract

A decline in female reproduction is one of the earliest hallmarks of aging in many animals, including invertebrates and mammals [1-4]. The insulin/insulin-like growth factor-1 signaling (IIS) pathway has a conserved role in regulating longevity [5] and also controls reproductive aging [2, 6]. Although IIS transcriptional targets that regulate somatic aging have been characterized [7, 8], it was not known whether the same mechanisms influence reproductive aging. We previously showed that Caenorhabditis elegans daf-2 IIS receptor mutants extend reproductive span by maintaining oocyte quality with age [6], but IIS targets in oocytes had not been identified. Here, we compared the transcriptomes of aged daf-2(-) and wild-type oocytes, and distinguished IIS targets in oocytes from soma-specific targets. Remarkably, IIS appears to regulate reproductive and somatic aging through largely distinct mechanisms, although the binding motif for longevity factor PQM-1 [8] was also overrepresented in oocyte targets. Reduction of oocyte-specific IIS targets decreased reproductive span extension and oocyte viability of daf-2(-) worms, and pqm-1 is required for daf-2(-)'s long reproductive span. Cathepsin-B-like gene expression and activity levels were reduced in aged daf-2(-) oocytes, and RNAi against cathepsin-B-like W07B8.4 improved oocyte quality maintenance and extended reproductive span. Importantly, adult-only pharmacological inhibition of cathepsin B proteases reduced age-dependent deterioration in oocyte quality, even when treatment was initiated in mid-reproduction. This suggests that it is possible to pharmacologically slow age-related reproductive decline through mid-life intervention. Oocyte-specific IIS target genes thereby revealed potential therapeutic targets for maintaining reproductive health with age.

Keywords: aging; cysteine protease; daf-2; insulin signaling; oocyte quality; oocyte-specific transcriptome; pqm-1; reproductive aging.

Figure 1. Characterization of daf-2(−) oocyte gene expression

(A) daf-2 RNAi extends fem-1(hc17) mated reproductive span (n = 59–60, p < 0.0001). Eight independent expression comparisons of Day 8 daf-2(RNAi);fem-1(hc17) vs. fem-1(hc17) oocytes reveal 126 upregulated genes (B) and 30 downregulated genes (C); (FDR = 1%; ranked by significance (SAM)). See also Data S1. Overlap between oocyte-specific and whole worm gene sets that are (D) upregulated with daf-2 RNAi and (E) downregulated with daf-2 RNAi (FDR = 1%; see also Figures S1D,E, and Data S2). (F) Percentages unhatched embryos (orange) and unfertilized oocytes (green) produced daily from mated daf-2(e1370) mutant worms treated with RNAis against top-scoring genes upregulated in daf-2(RNAi) oocytes. Normal progeny are not displayed. P values: differences in unhatched embryo production with age between target gene RNAi-treated group vs. control RNAi-treated group. See also Figure S2. (G) Loss of pqm-1 decreases daf-2(e1370) self-fertilized reproductive span by 50% (n = 40, **** p < 0.0001).

Figure 2. Cathepsin B-like cysteine proteases inhibit oocyte quality maintenance with age

(A) Unlike other members of the clan CA of cysteine proteases, a group of cathepsin B-like genes are significantly downregulated in day 8 daf-2(RNAi);fem-1(hc17) oocytes (FDR = 1%; ranked by SAM significance score). (B) Cathepsin B protease activity (Magic Red Cathepsin B Kit) of the most mature unfertilized oocyte of day 1 and mated day 7 wild-type and daf-2(1370) (± SEM; n = 18–22 individuals; see Figure S3B for representative images). (C) Representative images and (D) scored oocyte morphology defects show significant improvements in oocyte quality for day 5 mated rrf-3(pk1426) worms with life-long feeding of W07B8.4(RNAi) vs. control (L4440 vector) RNAi (n = 73–88). For reference, a young (day 2) mated rrf-3(pk1426) worm on control RNAi is shown; no obvious differences were noted in the oocyte morphology of day 2 W07B8.4(RNAi) vs. control RNAi worms. * p ≤ 0.05.

Figure 3. Inhibition of cathepsin B-like cysteine proteases reduces age-dependent oocyte quality deterioration

(A) Representative images and (B) oocyte morphology defect scoring show that MDL-28170 treatment after day 1 of adulthood significantly improves day 5 mated wild type oocyte quality, compared to DMSO-exposed controls (n = 72–75). (C) Representative images and (D) oocyte morphology defect scoring show that MDL-28170 after day 3 of adulthood significantly improves oocyte quality of day 7 mated wild-type worms, compared to DMSO-exposed controls (n = 45–49). White arrows indicate oocytes. (E) Oocyte morphology scoring of Day 7 mated wild-type worms treated from day 3 of adulthood onward with DMSO control (n = 39), the cathepsin B + calpain inhibitor MDL-28170 (n = 30), or the calpain inhibitor PD-150606 (n = 41) indicates that MDL-28170 treatment in mid-reproduction significantly improves oocyte quality, unlike PD-150606. * p ≤ 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 in comparison with DMSO control group.