Research Paper Volume 12, Issue 3 pp 2680—2697

Methionine increases yolk production to offset the negative effect of caloric restriction on reproduction without affecting longevity in C. elegans

Figure 5. The yolk proteins vitellogenin are required for methionine to mitigate the negative effect of CR on reproduction. (A) Calorie restriction reduced yolk protein production. Animals expressing tdimer2 -tagged vitellogenin YP170 were raised under ad libido (AL) and calorie restricted (CR) conditions as shown in Figure 1A and imaged by using Leica microscope. Representative images of 2 independent experiments were shown. (B) Methionine reversed the decreased YP170::tdimer2 expression caused by CR Animals expressing tdimer2-tagged vitellogenin YP170 were raised under ad libido (AL) and calorie restricted (CR) conditions as shown in Figure 1A and imaged by using Leica microscope. 10 worms from 2 independent experiments were quantified by using ImageJ software and plotted by using Graphpad Prism software. P values were obtained by using student’s t-test: ns, not significant; **, P<0.001. (C) Blocking oocyte uptake of yolk proteins vitellogenin prevents methionine’s beneficial effects on CR-compromised egg production. CR were achieved by using the eat-2(ad1116) mutant worms. N2 wild-type and eat-2 mutant worms were raised on HT115 bacteria expressing vector only control (L4440) or rme-2 double stranded interference RNA (RNAi) to knock down rme-2 gene expression. Day-1 adult worms were examined for egg production by counting the total eggs within 3 hours. Data were collected from 3 independent experiment and plotted to show egg from per worm per hour (egg/worm/h). P values were obtained by t-test: ns, not significant; **, P<0.001. (D) Blocking oocyte uptake of yolk proteins vitellogenin prevents methionine’s beneficial effects on CR-compromised embryo survival. CR and RNAi of N-2 wild-type and eat-2 animals were conducted as in E and synchronized eggs (n>100) were obtained by allowing day-1 adult worms to lay eggs for 2 hours. Survival were measured by counting the dead and viable embryos. Data from 3 independent experiments were collect for analysis. P values were obtained by t-test: ns, not significant; **, P<0.001.