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SIR-2.1 integrates metabolic homeostasis with the reproductive neuromuscular excitability in early aging male Caenorhabditis elegans

  1. Xiaoyan Guo
  2. L René García  Is a corresponding author
  1. Texas A&M University, United States
  2. Howard Hughes Medical Institute, Texas A&M University, United States
Research Article
Cite this article as: eLife 2014;3:e01730 doi: 10.7554/eLife.01730
9 figures, 5 videos and 2 additional files

Figures

Figure 1 with 1 supplement
sir-2.1(0) males have altered lipid content and their mating ability deteriorates prematurely.

(A) 1-day-old sir-2.1(0) and 2-day-old wild-type males have more lipid content than 1-day-old wild type. Left: quantification of fat staining, mean ± SEM, unpaired t-test. Right: representative images of fat staining. (B) Mating potency of wild-type and sir-2.1(0) males. Copulations were allowed to occur for 5 hr (i) or for an unlimited time (ii). The number of males in each assay is listed at the bottom of each bar. The numerical percentage of wild-type males that mated on day 1 was normalized to 100%. The normalization factor was then applied to the other experimental conditions. The normalized percentages for each day are listed on the top. Fisher’s exact test was used to compare the mating potency prior to normalization. (C) Transient starvation reduces sir-2.1(0) mating deficiency. (D) Mating potency of sir-2.1(0) and rescued strain sir-2.1(0); rgEX399 [Psir-2.1:sir-2.1::yfp]. ns, not significant. Asterisks *, ** and *** indicate the p<0.05, 0.01, and 0.0001 in this paper, respectively.

https://doi.org/10.7554/eLife.01730.003
Figure 1—figure supplement 1
(A) Tissue specific expression of sir-2.1 does not rescue the reduced mating potency of sir-2.1(0) males at day 2.

The lev-11 promoter expresses sir-2.1 in all body wall and sex muscles. The ges-1 promoter expresses sir-2.1 in the intestine. The aex-3 promoter expresses sir-2.1 in all neurons. (B) Adult lifespan of wild-type (circles) and sir-2.1(0) (squares) males (n = 50 for wild-type males; n = 76 for sir-2.1(0) males) (Log-rank [Mantel-Cox] Test). (C) Muscle fiber organization in the genital muscles of 1-day-old and 2-day-old sir-2.1(0) males. Asterisks indicate the diagonal muscles, arrow head indicates the oblique muscles. (D) In vitro sperm activation assays of 2-day-old wild-type and sir-2.1(0) males. Representative images are shown on the top of percentage bars. Arrow indicates the activated sperm with pseudopod, and solid arrow head indicate the inactivated sperm. No significant differences were observed between wild type and sir-2.1(0) males (unpaired t-test, N = 5 trials). In each trial, 50–60 sperm cells were analyzed.

https://doi.org/10.7554/eLife.01730.004
Figure 2 with 1 supplement
sir-2.1(0) males’ sex circuitry becomes more excitable during aging, and those males display ejaculation defects.

(A) 1-day-old wild-type and sir-2.1(0) males (n = 30) have similar response to the ACh agonist arecoline (ARE). The time required for those males to protrude their spicules out in 50 μM ARE solution are not significantly different (i) (unpaired t-test), whereas 2-day-old sir-2.1(0) males require significantly less time to respond to ARE (ii) (unpaired t-test). Mean and SEM are indicated. (B) 1-day-old wild-type and sir-2.1(0) males (n = 30) have similar response to the ACh agonist levamisole (LEV) (i). However, 2-day-old sir-2.1(0) males are more sensitive to LEV (ii). (Fisher’s exact test). (C, D) 2-day-old sir-2.1(0) males have an ejaculation defect. (C) The percentages of 2-day-old wild-type and sir-2.1(0) males that ejaculated during copulation. (Fisher’s exact test). (D) The numbers of cross progeny produced by individual 2-day-old wild-type and sir-2.1(0) with unc-64(e240) hermaphrodites. Mean and SEM are indicated (unpaired t-test).

https://doi.org/10.7554/eLife.01730.005
Figure 2—figure supplement 1
(A) A cartoon illustration of C. elegans male mating behavior.

(B) 2-day-old sir-2.1(0) males have no defect in turning behavior, (C) sensing the vulva of the hermaphrodite and (D) staying at the vulva.

https://doi.org/10.7554/eLife.01730.006
Figure 3 with 1 supplement
Ca2+ imaging of spicule-associated muscles in mating males.

Pseudo-colored images of Ca2+ in the spicule muscles of 2-day-old wild-type and sir-2.1(0) males during mating (A) and (B) are representative frames to show the Ca2+ levels of the spicule-associated muscles during spicule insertion attempts, penetration and the start of sperm transfer (∼10 s after insertion for wild type) or 19 s after insertion (for sir-2.1(0) males). The asterisks indicate the hermaphrodite vulva. Below the images, the Ca2+ transients in the protractor and anal depressor muscles (indicated by the black rectangle in A and B) are plotted for 5 individual wild-type (A) and sir-2.1(0) males (B), respectively.

https://doi.org/10.7554/eLife.01730.010
Figure 3—figure supplement 1
A cartoon illustrating the contractile changes of the spicule-associated muscles during intromission and ejaculation behaviors of a 2-day-old wild-type and sir-2.1(0) male, respectively.

Muscles displaying warmer colors signify the increasing intensity of the contractile events. The square box refers to the region of interest used for calcium imaging measurements shown in Figure 4.

https://doi.org/10.7554/eLife.01730.011
ROS contributes to the mating deterioration.

(A) Survival rates of wild-type and sir-2.1(0) males on NGM containing 10 mM paraquat at 24 hr and 48 hr post paraquat exposure. (B) Mating potency of 1-day-old wild-type males exposed to 0.01, 0.1, and 1 mM paraquat. (C) The percentages of males with their spicules protruding out (SpOUT) in response to 1 μM levamisole (LEV) after treatment with 1 mM paraquat. (DG) Exposing males to N-acetyl-cystine (NAC) improves mating. The percentages of 3-day-old wild-type (D) and 2-day-old sir-2.1(0) (E) males that protrude their spicules out in response to 100 nM LEV after NAC exposure. Mating potency of 3-day-old wild-type (F) and 2-day-old sir-2.1(0) (G) males after NAC exposure (Fisher’s exact test).

https://doi.org/10.7554/eLife.01730.014
sir-2.1(0) males have altered expression of metabolic genes.

Relative mRNA expression level of genes involved in metabolic processes such as glycolysis (A), TCA cycle (B), fatty acid oxidation(C), Gluconeogenesis/glyceroneogenesis/lipid synthesis (D), Glyoxylate cycle (E), and ETC (F) in 2-day-old wild type, 1-day-old, and 2-day-old sir-2.1(0) males relative to 1-day-old wild type. d1 WT refers to day1 wild type; d2 WT refers to day 2 wild type; d1 s2 refers to day1 sir-2.1(0); d2 s2 refers to day 2 sir-2.1(0) (unpaired t-test compared to 1-day-old wild type).

https://doi.org/10.7554/eLife.01730.015
Figure 6 with 1 supplement
sir-2.1(0) males might have enhanced metabolism.

(A) Schematic illustration of main metabolic enzymes which have altered expression in sir-2.1(0) males. Red arrows indicate catabolic pathways. Blue arrows indicate anabolic pathways. (B) ATP content measured in 1, 2 and 3-day-old wild-type and sir-2.1(0) males. (C) Glycogen staining in 1-day-old wild type and sir-2.1(0). The glycogen staining level is quantified by measuring the mean gray level of the ROI indicated on the top right corner. The mean gray level is inversely correlated with the iodine stain. (D) Oil Red O staining of wild type and mutant C. elegans males. (E) sir-2.1(+) and sir-2.1(0) need pck-2 to maintain their mating at day 2 and day 1 respectively. All percentages of mating potency are normalized to that of 1-day-old wild-type male. (F) sir-2.1(0) requires pck-1 to maintain their mating at day 1 and day 2, while sir-2.1(+) males do not need pck-2 to maintain their mating at either day 1 or day 2. (G) 2% glucose reduces 1-day-old sir-2.1(0) mating potency, but not 2-day-old wild-type males (Fisher’s exact test).

https://doi.org/10.7554/eLife.01730.016
Figure 6—figure supplement 1
The level of glucose content is similar between 1-day-old sir-2.1(0) and wild-type males.
https://doi.org/10.7554/eLife.01730.017
sir-2.1(0) males have compromised expression of anti-oxidant genes.

Relative mRNA expression level of anti-oxidant genes superoxide dismutase (A), catalase (B), and glutathione transferase (C) in 1, 2-day-old wild type and sir-2.1(0) males (unpaired t-test).

https://doi.org/10.7554/eLife.01730.018
Exogenous nicotinamide improves mating during aging.

sir-2.1 overexpression cannot increase mating potency of 3-day-old wild type (A). However, feeding with a NAD+ precursor nicotinamide (NAM) significantly improve the mating potency of 3-day-old wild type (B) but not 2-day-old sir-2.1(0) males (C). Overexpression of sir-2.1 cannot further promote the effect of exogenous NAM (D).

https://doi.org/10.7554/eLife.01730.019
A cartoon of the metabolism and behavior that occurs in wild-type and sir-2.1(0) males during early aging.

For successful reproductive behavior, SIR-2.1 is required to maintain proper carbon flow to meet the male’s energy demands and balance the generation of ROS. In 1-day-old old sir-2.1(0) males, catabolism such as glycolysis and fatty acid oxidation is enhanced, and consequently, oxidative phosphorylation and generation of ROS are also increased. Without SIR-2.1, ROS accumulation by day 2 of adulthood can lead to hyperexcitability of the male’s genital neuromuscular circuitry. This results in blocked ejaculation and impotency. It is possible that in 2- to 3-day-old wild-type males, the NAD+-dependent SIR-2.1 activity declines due to a lower ratio of NAD to NADH; thus older wild-type males might have a similar physiology as 1-day-old sir-2.1(0) males.

https://doi.org/10.7554/eLife.01730.020

Videos

Video 1
Wild-type male’s ejaculation.
https://doi.org/10.7554/eLife.01730.007
Video 2
2-day-old sir-2.1(0) male’s ejaculation.
https://doi.org/10.7554/eLife.01730.008
Video 3
2-day-old sir-2.1(0) male’s ejaculation.
https://doi.org/10.7554/eLife.01730.009
Video 4
Ca2+ transient in a 2-day-old wild-type male.
https://doi.org/10.7554/eLife.01730.012
Video 5
Ca2+ transient in a 2-day-old sir-2.1(0) male.
https://doi.org/10.7554/eLife.01730.013

Additional files

Supplementary file 1

Expression levels of metabolic enzymes though real-time PCR. d1 WT refers to day1 wild type; d2 WT refers to day 2 wild type; d1 s2 refers to day1 sir-2.1(0); d2 s2 refers to day 2 sir-2.1(0).

https://doi.org/10.7554/eLife.01730.021
Supplementary file 2

Primers used in this study.

https://doi.org/10.7554/eLife.01730.022

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