Figure 4. Calorie restriction-induced reproduction defect is attributed to oocyte but not sperm.(A) Schematic diagram showing the experimental design to study the origins of reproductive defect caused by CR. Shown are different crosses between CR hermaphrodites and males. CR hermaphrodites crossed to CR males is expected cto still have reproductive defect. If normal hermaphrodites crossed to CR males still have reproductive defect, then the defect was traced to sperm. If CR hermaphrodites crossed to normal males still have reproductive defect, then the defect was traced to oocyte. (B) Evaluation of egg production from different crosses shown in A. Males were maintained by crossing equal number of males to hermaphrodites. Ad libido (AL) and CR conditions were achieved by plating 1X1011/mL and 1X108/mL OP-50 bacteria, respectively, on solid nematode growth (NG) medium containing carbenicillin and kanamycin. CR or non-CR males and hermaphrodites (n>20) were crossed for 1 day and hermaphrodites were transferred to new plate to collect synchronized eggs. Egg production were evaluated in per worm per hour (egg/worm/h). Data were collected from 3 independent experiments. P values were obtained by t-test: ns, not significant; **, P<0.001. (C) The hatching rate of different crosses shown in A. CR and crossed were conducted as in B and synchronized eggs were collected from 20 mated hermaphrodites and counted. Eggs were allowed to hatch for 2-3 days and successful hatching were determined by survival worms. Data were collected from 3 independent experiments with each experiment examine n>100 eggs. P values were obtained by t-test: ns, not significant; **, P<0.001.