Glucose 6-phosphate dehydrogenase deficiency enhances germ cell apoptosis and causes defective embryogenesis in Caenorhabditis elegans

Yang, H-C; Chen, T-L; Wu, Y-H; Cheng, K-P; Lin, Y-H; Cheng, M-L; Ho, H-Y; Lo, S J; Chiu, D T-Y

Quantification of germ cell apoptosis. Germ cell apoptosis was visualized based on CED-1::GFP, a specific reporter, which highlights the somatic sheath cell surrounded apoptotic germ cells upon engulfment by using bcIs39 strain (P lim-7 ced-1::gfp). 25 Staged young adult hermaphrodites of mock and G6PDknockdown C. elegans grown at 20 1C were harvested from RNAi NGM plate. These worms were washed with PBS and anesthetized with 2% levamisole followed by mounting on 2% agarose pad on glass slides. Fluorescent images were taken by using epifluorescence microscope (Leica DM 2500; Leica, Wetzlar, Germany) coupled with CCD camera (Photometrics, Coolsnap K4, Tucson, AZ, USA) and analyzed by imaging software (Metamorph 6.1r0; Molecular Devices, Sunnyvale, CA, USA). The number of apoptotic germ cells was determined by counting green fluorescent germ cells in each gonad. For short-term oxidant treatment, bcIs39 strain was pretreated with H 2 O 2 at young adult stage as previously described.