Figure 6. The cellular machinery for TAp63α activation in murine oocytes is always present and ready to act upon genotoxic insults.

(A) WB of CHX treatment of nonirradiated (NIRR) and γ-irradiated (IRR) murine ovary samples. The signals of p63, the oocyte marker Msy2 and β-actin are displayed for each time point after NIRR/IRR. The asterisk marks phosphorylated p63. (B) WB of SDS-PAGE loaded with the ovary samples of the Native PAGE in (C). The asterisk marks phosphorylated p63. (C) WB of Native PAGE from (un-)treated and either NIRR or IRR murine ovaries. The p63 signal in the range from 20 kDa to 1,236 kDa is shown. (D) Intensity projection of the Native PAGE p63 signal from (C). The molecular weight range of the p63 dimer and tetramer is colored in green and red, respectively. (E) Quantitative Real-Time PCR of isolated murine oocytes. The bar diagram shows the fold induction of p21, Puma, Mdm2 and Msy2 mRNA after γ-irradiation. Error bars show the standard deviation of the biological duplicates. Brackets above the bars display the p-test results showing no significance (n.s.) between untreated and CHX treated oocytes for all targets. (F) Inhibition of Chk2 suppresses the DNA-damage induced phosphorylation of TAp63α in γ-irradiated ovaries. Chk2 inhibitor II at concentrations of 5 and 25 µM was added 2 hr before irradiation with 1.5 Gy. Ovaries were harvested 4 hr after irradiation and analyzed by SDS PAGE and Western Blot. Activated TAp63α gets degraded fast while preventing activation via inhibition of Chk2 preserves the original cellular concentration. (G) Native PAGE analysis of the same samples used as in (F). Inhibition of Chk2 prevents tetramerization and keeps TAp63α in a closed and dimeric state.

DOI: http://dx.doi.org/10.7554/eLife.13909.021

Figure 6—figure supplement 1. p63 is responsible for inducing apoptosis in oocytes.

(A) Verification of CHX activity in ovary culture. WB of CHX and MG132 treatment of mouse ovaries. After overnight culture with either DMSO or CHX, ovaries were incubated with DMSO, CHX, MG132 or a combination of the latter two for additional 8 hr. The signal of ubiquitin (mono- and poly-ubiquitin bands) and β-actin as a loading control are displayed. (B) Immunohistochemistry staining of P8 mouse ovaries either non-irradiated (NIRR) or 8h after γ-irradiation (IRR) for Msy, p53, p63 or p73. Stainings for Msy and p63 were developed with a 30 s exposure time and then stopped due to high signal intensity. Stainings for p53 and p73 were exposed for 5 min. The red arrows indicate primordial follicles, which express high amount of TAp63α and are responsive to a low dosage of γ-irradiation. Scale bar: 50 µm.

DOI: http://dx.doi.org/10.7554/eLife.13909.022