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

Decreased egg production and hatching by G6PD knockdown in C. elegans. In the first generation of Gi C. elegans, no significant phenotypic changes including morphology, pharyngeal pumping, mobility, and growth rate were observed (data not shown). The lack of major phenotypes upon G6PD knockdown indicated that the residual G6PD activity in Gi C. elegans is sufficient to maintain basic cell functions in C. elegans (Figure 2b) . Nevertheless, the brood size derived from the first generation of Gi hermaphrodite was significantly reduced (65% of mock, P ¼ 0.011, n ¼ 60) compared with mock control (Figure 4a ). This finding indicates that G6PD is required for maintaining normal egg production. In addition to the decreased egg production upon G6PD knockdown, a majority of eggs produced by Gi C. elegans were unable to hatch compared with mock (10% of mock, Po0.005, n ¼ 60) (Figure 4b ). This dramatic observation is consistent with a previous report that G6PD knockdown induces embryonic lethality in C. elegans. 24 Enhanced germ cell apoptosis by G6PD knockdown in C. elegans. In order to investigate whether decreased egg production induced by G6PD knockdown is the consequence of enhanced germ cell death, the germ cell apoptosis was determined in G6PD-deficient C. elegans using GFP reporter strain bcIs39 (P lim-7 ced-1::gfp), which highlights somatic sheath cell around apoptotic germ cells upon oogenesis. 25 Under basal condition, the number of apoptotic germ cells in bcIs39 Gi C. elegans was significantly increased by 2-fold (7.4 per gonad, P ¼ 0.04, n ¼ 80) compared with bcIs39 mock (3.7 per gonad, n ¼ 60) (Figure 4c ). This result clearly demonstrates that reduction of G6PD increases germ cell apoptosis. Involvement of high oxidative stress in altered germ cell apoptosis and egg production by G6PD knockdown in C. elegans. Increased oxidative stress has been associated with reduced brood size in C. elegans. [26] [27] [28] To test whether high oxidative stress was involved in altered germ cell apoptosis and egg production caused by G6PD knockdown, a short-term H 2 O 2 treatment was exogenously applied to C. elegans adult followed by scoring the apoptotic germ cells (Figures 5a and b) and egg production (Figure 5c ). At 5 mM and 10 mM H 2 O 2 , the apoptotic germ cells were increased in mock C. elegans (6.2±1.8 cells per gonad, P ¼ 0.109 and 7.5 ± 1.9 cells per gonad, Po0.005, respectively) compared with untreated control (3.7±1.3 cells per gonad) ( Figure 5a ). However, the apoptotic germ cells in H 2 O 2 -treated Gi C. elegans showed no discernible difference (5 mM H 2 O 2 : 7.8 ± 2.3 cells per gonad, P ¼ 0.276 and 10 mM H 2 O 2 : 7.2±1.7 cells per gonad, P ¼ 0.652) compared with untreated Gi control (7.4 ± 1.9 cells per gonad) ( Figure 5a) . Furthermore, at 5 mM H 2 O 2 , the total egg production of both mock and Gi C. elegans were not significantly affected compared with untreated control (P ¼ 0.924 and P ¼ 0.832, respectively) ( Figure 5c and Supplementary Table S1 ).