Fig. 6: Imatinib partially prevents oocyte apoptosis induced by cyclophosphamide. | Cell Death & Disease

Fig. 6: Imatinib partially prevents oocyte apoptosis induced by cyclophosphamide.

From: Kinase-independent inhibition of cyclophosphamide-induced pathways protects the ovarian reserve and prolongs fertility

Fig. 6

P7 mice were injected with vehicle (PBS) or cyclophosphamide (100 mg/kg) with/without increasing concentrations of imatinib (12 and 42 mg/kg) and sacrificed within 16 h from injection. a Ovarian sections were analyzed by in situ TdT-mediated dUTP nick-end labelling (TUNEL) assay. 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. b γH2AX and DNAPK activation was observed with IF assay using phospho-specific antibodies and p63 was used as a nuclear marker for germ cells. Quantification was performed by counting several (6 < x < 8) middle ovarian sections derived from three distinct ovaries. Co-staining for p-DNAPK and γH2AX showed the activation of DNA damage response in reserve oocytes. Quantification was performed by counting several (6 < x < 8) middle ovarian sections derived from three distinct ovaries. c Ovarian reserve apoptosis was assessed by IF assay using antibodies against cleaved PARP (green) and Msy2 (red), a cytoplasmic antigen of germ cells. Quantification of cleaved PARP-positive cells was performed by counting several (6 < x < 8) middle ovarian sections derived from three distinct ovaries. a–c Bar column represents mean ± s.d.; statistical significance was determined using one-way analysis of variance (ANOVA) (***P < 0.001 as compared with the group treated with 100 mg/kg cyclophosphamide). d Ovaries dissected 3 days after injection were analyzed with IHC assay using Msy2 antibody (Fig. 5b). Ovaries from three independent experiments were analyzed; each dot in the box plot represents the average number of follicles (primordial + primary and secondary) per section of each gonad collected. Statistical significance was determined using one-way analysis of variance (ANOVA) (***P < 0.001 as compared with cyclophosphamide-treated group at 100 mg/kg). Scale bar magnification, 100 μm for TUNEL assay and 25 μm for IF assay

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