Fig. 1: Cyclophosphamide triggers apoptosis in pre-pubertal mouse ovary in a dose-dependent manner.

P7 mice were injected with vehicle (PBS) or increasing concentrations of cyclophosphamide (75, 100, and 200 mg/kg) and sacrificed within 16 h from injection. Ovarian sections were analyzed by in situ TdT-mediated dUTP nick-end labelling (TUNEL) assay. a A small area of each image was selected and zoomed in (magnification box). The graph shows the quantification of TUNEL-positive cells. Quantification of TUNEL-positive cells was performed by counting six different middle ovarian sections derived from three distinct ovaries. Follicle apoptosis was assessed by IF assay with two specific antibodies against cleaved PARP (green) and Msy2 (red), a cytoplasmic marker of germ cells. b Yellow arrows at the bottom indicate positive oocytes, while the dashed box illustrates PARP positive-granulosa cells from large growing follicle. Quantification of cleaved PARP-positive cells was performed by counting several (6 < x < 10) middle ovarian sections derived from three distinct ovaries. Scale bar magnification, 100 μm for TUNEL assay and 25 μm for IF assay. c Follicle apoptosis was assessed by IF assay with two specific antibodies against cleaved PARP (green) and p63 (red), a nuclear marker of germ cells. We also monitored γH2AX, a marker of DDR, in the reserve oocytes (yellow arrows). d The pro-apoptotic PUMA and NOXA genes expression was assessed by RT-qPCR; GAPDH was used as reference control; data are shown as fold change vs PBS. e Mouse images were obtained 7 days after cyclophosphamide injection. a, b, d Bar column represents mean ± s.d.; statistical significance was determined using one-way analysis of variance (ANOVA) (**P < 0.01; ***P < 0.001 as compared with PBS-treated group)