Short-term starvation stress at young adult stages enhances meiotic activity of germ cells to maintain spermatogenesis in aged male Caenorhabditis elegans

Abstract

To survive and reproduce, living organisms must evolve numerous mechanisms to re-adjust their physiology when encountering adverse conditions that subject them to severe stress. We found that short-term starvation (STS) stress in young adult male Caenorhabditis elegans can significantly improve their vitality (relative to nonstressed males) when they are aged. In addition, we found that stress-treated aged males maintained reproductive activity equivalent to young males, whereas nonstressed aged males quickly lost reproductive ability. STS stress can preserve sperm number and quality in aged male worms. Spermatogenesis involves germ cell mitosis and meiosis. We found that germ cell meiotic activity is more sensitive to aging than mitotic activity and is declining rapidly with age. We examined the role of numerous factors important for spermatogenesis on STS-preserved spermatogenesis during aging. Our results show that mutant strains deficient in anaphase-promoting complex/cyclosome (APC/C) function fail to exhibit the STS stress-enhanced spermatogenesis found in wild-type N2 worms, suggesting that the mechanism underlying starvation-induced spermatogenesis involves the APC/C complex, a conserved ubiquitin-protein ligase E3 complex. Furthermore, transgenic expression of FZY-1/CDC-20, a coactivator of APC/C, ameliorated the age-associated decline of meiosis, similar to the hormetic effect of STS.

Keywords: CDC-20; anaphase-promoting complex/cyclosome (APC/C); meiosis; short-term starvation; spermatogenesis; stress response hormesis.

Figure 1

Short‐term starvation (STS) stress preserves viability and vitality in adult male C. elegans during aging. (a) Schematic of STS treatments in adult male C. elegans maintained at 22°C. The first day of adulthood is denoted D1. Y: young‐age; M: mid‐age; S: starved. (b) Survival curves of N2 adult male worms maintained at either 15 or 22°C and that received various STS treatments as shown in Figure 1a. Data represent mean of n = 4 (15°C, middle panel) and n = 5 (22°C, upper panel), respectively. The survival curves for all the replicates are shown in Supporting Information Figure S1. For mean lifespan (bottom panel), data represent mean ± SD. Difference between two indicated groups: *, p < 0.05, **, p < 0.01. (c) Physical activity of STS stress‐treated adult male worms. D10 adult male worms subjected to various STS stress treatments as shown in Figure 1a were assayed for their thrashing activity. Data represent mean ± SD, n ≥ 10. Difference between two indicated groups: *, p ≤ 0.05; **, p ≤ 0.001. (d) qRT‐PCR analysis of hsp‐70A mRNA levels in D10 adult male worms subjected to various STS stress treatments as shown in Figure 1a and treated at 30°C for 3 hr to induce hsp‐70Aexpression. Data represent mean ± SD, n = 4. *Different from fed control (F) value of same group, p ≤ 0.001. (e) qRT‐PCR analysis of sod‐3 mRNA levels in D10 adult male worms subjected to various STS stress treatments as shown in Figure 1a. Data represent mean ± SD, n = 4. Difference between two indicated groups: *, p ≤ 0.001. (f) Body ATP levels of D10 adult male worms subjected to various STS stress treatments as shown in Figure 1a. Data represent mean ± SD, n = 3. Difference between two indicated groups: *, p ≤ 0.001

Figure 2

Short‐term starvation (STS) stress‐preserved vitality is coupled to reproductive fitness in adult male C. elegans during aging. (a) Total sperm numbers in N2 adult male worms of different ages maintained at 22°C, and of different STS stress‐treated worms as shown in Figure 1a. Data represent mean ± SD, n ≥ 10. Left panel, *different from day 2 level, p ≤ 0.05; **different from day 2 level, p ≤ 0.001. Right panel, difference between two indicated groups: *, p ≤ 0.05; **, p ≤ 0.001. (b) Sperm quality assayed in an in vitro sperm activation system using triethanolamine (TEA). Data represent mean ± SD, n ≥ 10. Left panel, *different from day 2 level, p ≤ 0.001. Right panel, difference between two indicated groups: *, p ≤ 0.001. (c) Relative mRNA levels of msp‐3 and try‐5 by qRT‐PCR analysis.Data represent mean ± SD, n = 4. Difference between two indicated groups: *, p ≤ 0.001. (d) Copulatory activity in D10 adult male worms. Data represent mean ± SD, n = 4. Difference between the two indicated groups: *, p ≤ 0.001. (e) Fertilization ability in D10 adult male worms. Data represent mean ± SD, n ≥ 30. Difference between the two indicated groups: *, p ≤ 0.05. **, p ≤ 0.001

Figure 3

Meiotic, not mitotic, activity of spermatogenesis declines rapidly in young adult male C. elegans. (a) Anatomy of the C. elegans male testis and germ cell nuclei morphology. Right panel, DIC image of an intact testis isolated from a D2 N2adult male worm. Left panel, nuclei visualized by Hoechst 33342 staining. Regions of the testis are indicated. (b) Left panel, EdU incorporation rate in testes of N2 adult male worms of different ages maintained at 22°C. More than 50 testes from each age‐group were examined for the presence of EdU signal after labeling for 24 hr. Right panel, fluorescent images of EdU‐labeled and Hoechst 333‐stained testes. Arrow indicates EdU‐labeled nuclei. (c) Right panel, meiotic division activity in the testes of N2 adult male worms of different ages maintained at 22°C. More than 50 testes from each age‐group were analyzed. Left panel, immunofluorescent staining of meiotic α‐tubulin in testes of D2 and D5 N2 adult males. Arrow indicates an α‐tubulin spindle

Figure 4

Short‐term starvation (STS) abolishes meiotic activity during stress, but markedly enhances this activity during poststress ages in adult male C. elegans. (a) EdU incorporation rate in testes of D4 adult males that had been starved for 2 days at 22°C. More than 50 testes from each age‐group were examined for the presence of EdU‐labeled nuclei after labeling for 6 hr. (b) Meiotic activity in testes of adult males of different ages maintained at 22°C. In the starved group, adult males were starved from D2 to D4. More than 50 testes from each age‐group were analyzed. (c) Meiotic division activity in testes of D10 adult males subjected to STS treatments earlier as shown in Figure 1a and maintained at 22°C. More than 50 testes from each age‐group were analyzed. (d) Translocation efficiency of EdU‐labeled nuclei in testes of D10 adult males previously subjected to different STS stress treatments as shown in Figure 1a. More than 50 testes from each age‐group were examined for the localization of EdU‐labeled nuclei after labeling for 24 hr

Figure 5

Transcriptional expression of fzy‐1/cdc‐20 is a target of aging and STS stress. (a) RNA‐seq analysis of expression levels of APC/C subunits in D4 N2 adult male worms starved for 2 days. Data represent mean ± SD, n = 3. (b) RNA‐seq analysis of expression levels of APC/C subunits in D4 N2 adult male worms starved for 2 days (D4S2). The ratio of mRNA levels from starved worms (D4S2) over levels before STS stress treatment (D4) was calculated. Data represent mean ± SD, n = 3. (c) qRT‐PCR analysis of mRNA levels of APC/C subunits in testes isolated from D10 adult males previously subjected to an STS stress treatment as shown in Figure 1a. qRT‐PCR analysis of the indicated genes was normalized against sgo‐1 expression levels. Data represent mean ± SD, n = 4. Difference between two indicated groups: *, p ≤ 0.05. **, p ≤ 0.01

Figure 6

Transgenic expression of fzy‐1in gonads retards age‐associated decline in meiosis and spermatogenesis in adult male C. elegans. (a) Upper panel, western blotting analysis of IFY‐GFP fusion protein degradation in adult males of fzy‐1transgenic worms at each age and after repeated STS stress treatment (2S) as described in Figure 1a. White arrowheads indicate the super‐shifted bands of GFP signal. Lower panel, Coomassie blue staining of the same blots. (b) Meiotic activities in testes of α‐tubulin/fzy‐1transgenic adult male worms of different ages at 22°C, and of different STS stress‐treated as shown in Figure 1a. More than 50 worms from each age‐group or treatment group were live‐imaged. Upper panel, fluorescent live images of the division zone of adult male testes. Arrows indicate examples of α‐tubulin spindles. Lower panel, comparison of the percentage of meiotically active testes at each age and after STS stress treatment. (c) Sperm counts in transgenic adult male worms of different ages maintained at 22°C, and of different STS stress‐treated worms as shown in Figure 1a. Data represent mean ± SD, n = 10–12. Difference between two indicated groups: *, p ≤ 0.01