FIG 4.

PI3K-like kinase inhibitor wortmannin and ATM inhibitor KU55933 block IR-induced TAp63α phosphorylation and IR-induced primordial follicle oocyte death. (A) Immunoblots showing the levels of ATM Ser1981phosphorylation using phospho-specific MAb (upper panels) and TAp63α phosphorylation shift (middle panels) in nonirradiated or 4.5-Gy-irradiated P5 ovaries without or with wortmannin (WMn) or KU55933 (KU) pretreatments using the indicated doses. Wortmannin pretreatments were for 30 min on dissected ovaries, followed by washing before IR treatment. KU55933 pretreatments were for 2 h prior to IR treatment. Ovaries were examined 2 h after IR treatment. Contralateral ovary pairs, denoted by brackets, were used and loaded adjacently. Arrow, nonshifted TAp63α; double arrowheads, high shift. Tubulin, loading control. (B) Immunohistochemistry sections of nonirradiated versus 0.45-Gy-irradiated P5 ovaries examined at P7. Ovaries were either untreated or pretreated with wortmannin in DMSO (Wmn/DMSO) or pretreated with only DMSO (DMSO) as outlined for 30 min, washed, and irradiated. The ovaries were examined at P7. Red arrowheads indicate primordial follicle oocytes; blue arrowheads indicate primary or secondary follicle oocytes undergoing growth and maturation; the arrows point to representative oocytes magnified in the insets. Scale bars, 50 μm. (C) Quantification of total number of oocytes in P5 ovaries treated as outlined. Ovaries were examined 2 days after IR treatment at P7 (n = 3 mice); error bars indicate the SEM. **, P ≤ 0.002 (Kruskal-Wallis test). (D) Immunoblots analysis of TAp63α levels remaining, which is indicative of oocyte survival, in nonirradiated or 4.5-Gy-irradiated ovaries without (−Wmn) or with (+Wmn) wortmannin pretreatment 2 or 24 h after IR treatment, as outlined.