Research Paper Volume 12, Issue 3 pp 2680—2697

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.