Figure 6. NEDD4 is required for thermotolerance of mouse testes.

(a) A schematic illustration showing long-term heat stress in vivo. WT or Nedd4 cKO mice at 12–16 weeks of age were anaesthetized and incubated in a 42 °C water bath for 20 min per day. After 7 days of this treatment, the mice were allowed to recover for 1 day before euthanasia. (b) Representative pictures showing the staining for EIF4E (SG marker) and CDH1 (SPC marker) after the treatment described in a. A large number of stable SGs persisted only in Nedd4 cKO testes. Arrowheads indicate SPCs with clear-cut staining of SG foci. Scale bar, 50 μm. (c–e) Increased apoptosis was observed in Nedd4 cKO SPCs. Costaining for CDH1 and c-PARP was performed and representative pictures are shown (c). Open arrowheads indicate c-PARP⁻CDH1⁺ SPCs; filled arrowheads indicate c-PARP⁺CDH1⁺ SPCs. Statistical analysis is shown for mean±s.d. in d. CDH1⁺ SPCs were counted and shown as mean±s.d. in e; ***P<0.001, t-test. Scale bar, 50 μm. (f,g) Representative pictures showing a greater extent of c-PARP staining (apoptosis) during the 42–33 °C recovery (4 h) in Nedd4-cKD GSCs than in the control group. Arrowheads indicate apoptotic cells. Statistical analysis is shown for mean±s.d. in g (n=3), t-test. Scale bar, 10 μm.