CG8005 Mediates Transit-Amplifying Spermatogonial Divisions via Oxidative Stress in Drosophila Testes

Abstract

The generation of reactive oxygen species (ROS) widely occurs in metabolic reactions and affects stem cell activity by participating in stem cell self-renewal. However, the mechanisms of transit-amplifying (TA) spermatogonial divisions mediated by oxidative stress are not fully understood. Through genetic manipulation of Drosophila testes, we demonstrated that CG8005 regulated TA spermatogonial divisions and redox homeostasis. Using in vitro approaches, we showed that the knockdown of CG8005 increased ROS levels in S2 cells; the induced ROS generation was inhibited by NAC and exacerbated by H₂O₂ pretreatments. Furthermore, the silencing of CG8005 increased the mRNA expression of oxidation-promoting factors Keap1, GstD1, and Mal-A6 and decreased the mRNA expression of antioxidant factors cnc, Gclm, maf-S, ND-42, and ND-75. We further investigated the functions of the antioxidant factor cnc, a key factor in the Keap1-cnc signaling pathway, and showed that cnc mimicked the phenotype of CG8005 in both Drosophila testes and S2 cells. Our results indicated that CG8005, together with cnc, controlled TA spermatogonial divisions by regulating oxidative stress in Drosophila.

Figure 1

Knockdown of CG8005 in spermatogonia resulted in differentiation defects. (a) DNA staining in WT, Bam>CG8005 RNAi, and Bam>CG8005 RNAi;Δ86/+ testes. Yellow double arrows label undifferentiated cells at the apex of the testis. (b) The distance of undifferentiated cells at the apex of the testis. (c) The number of pointed fusomes. (d) The number of branched fusomes. (e, f) Immunostaining of WT, Bam>CG8005 RNAi, and Bam>CG8005 RNAi;Δ86/+ testes. Representative pointed fusomes are indicated with yellow arrowheads, and branched fusomes are indicated with white arrowheads. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001. Scale bar: 20 μm.

Figure 2

ROS generation in CG8005 RNAi testes. (a) DHE staining in testes with H₂O₂ treatment. (b) DHE fluorescence intensity of testes with H₂O₂ treatment. (c) DHE staining in WT, Bam>CG8005 RNAi, and Bam>CG8005 RNAi;Δ86/+ testes. (d) DHE fluorescence intensity of WT, Bam>CG8005 RNAi, and Bam>CG8005 RNAi;Δ86/+ testes. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001. Scale bar: 20 μm.

Figure 3

Knockdown of CG8005 induced ROS accumulation in S2 cells. (a) RNAi efficiency verification of CG8005 in control and siCG8005 (siCG8005-222 and siCG8005-419) S2 cells. (b) DHE staining in control and siCG8005 S2 cells. (c) DHE fluorescence intensity in control and siCG8005 S2 cells. (d) DCF staining in control and siCG8005 S2 cells. (e) DCF fluorescence intensity in control and siCG8005 S2 cells. (f) Relative mRNA levels of oxidation-promoting factors (Keap1, GstD1, and Mal-A6) in control and siCG8005 S2 cells. (g) Relative mRNA levels of antioxidant factors (cnc, Gclm, maf-S, ND-42, and ND-75) in control and siCG8005 S2 cells. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001. Scale bar: 30 μm.

Figure 4

Pretreatment with NAC in siCG8005-mediated S2 cells. (a, b) DHE (a) and DCF (b) staining in control, 1.25 mM NAC, 2.5 mM NAC, and 5.0 mM NAC-treated S2 cells. (c, d) DHE (c) and DCF (d) fluorescence intensities in control, 1.25 mM NAC-, 2.5 mM NAC-, and 5.0 mM NAC-treated S2 cells. (e, f) DHE (e) and DCF (f) fluorescence intensities of pretreatment with NAC in siCG8005-treated S2 cells. (g, h) DHE (g) and DCF (h) staining of pretreatment with NAC in siCG8005-treated S2 cells. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001. Scale bar: 30 μm.

Figure 5

Pretreatment with H₂O₂ in siCG8005-mediated S2 cells. (a, b) DHE (a) and DCF (b) staining in control, 100 μM H₂O₂-treated, and 200 μM H₂O₂-treated S2 cells. (c, d) DHE (c) and DCF (d) fluorescence intensities in control, 100 μM H₂O₂-treated, and 200 μM H₂O₂-treated S2 cells. (e, f) DHE (e) and DCF (f) fluorescence intensities of pretreatment with H₂O₂ in siCG8005-treated S2 cells. (g, h) DHE (g) and DCF (h) staining of pretreatment with H₂O₂ in siCG8005-treated S2 cells. ^∗∗P < 0.01 and ^∗∗∗P < 0.001. Scale bar: 30 μm.

Figure 6

Knockdown of cnc in spermatogonia led to differentiation defects and ROS accumulation in Drosophila testes. (a, b) Immunostaining of WT, Bam>cnc RNAi, and Bam>cnc RNAi;Δ86/+ testes. Two independent UAS-cnc RNAi lines were used for the functional analysis of cnc. Yellow double arrows label undifferentiated cells at the apex of the testis. (c) The distance of undifferentiated cells at the apex of the testis. (d) DHE staining in WT, Bam>cnc RNAi, and Bam>cnc RNAi;Δ86/+ testes. (e) DHE fluorescence intensity of WT, Bam>cnc RNAi, and Bam>cnc RNAi;Δ86/+ testes. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001. Scale bar: 20 μm.

Figure 7

Effects of ROS accumulation in sicnc-treated S2 cells. (a) RNAi efficiency verification of cnc in control and cnc-siRNA (sicnc-725 and sicnc-61) S2 cells. (b) Relative mRNA levels of antioxidant factors (Gclm, maf-S, ND-42, and ND-75) in control and sicnc S2 cells. (c) Relative mRNA levels of oxidation-promoting factors (Keap1, GstD1, and Mal-A6). (d, e) DHE staining (d) and DHE fluorescence intensity (e) of pretreatment with NAC in sicnc-treated S2 cells. (f, g) DHE staining (f) and DHE fluorescence intensity (g) of pretreatment with H₂O₂ in sicnc-treated S2 cells. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001. Scale bar: 30 μm.