Figure 6. FOXO4-DRI alleviates testosterone secretion insufficiency in naturally aged mice. (A) Immunohistochemical staining showing that FOXO4 was rarely expressed in young (3 months of age) mice testes, but expressed typically in the Leydig cells of naturally aged (20-24 months of age) mice. In aged mice testes, FOXO4 was predominantly expressed in cytoplasm and showed nuclear localization in some Leydig cells (arrows). Scale bar: 50 μm. (B) Serum testosterone levels in naturally aged (20-24 months of age) male mice are lower than in young adult (3 months of age) male mice, but are significantly increased 30 days after FOXO4-DRI treatment (intraperitoneal injection of 5 mg/kg every other day for three administrations). (C–E) Western blots revealing that FOXO4-DRI treatment increases protein levels of the testosterone synthesis-related proteins 3β-HSD and CYP11A1 in the testes of aged mice. Data depict the mean ± SD; n=6. *P<0.05.