Fig. 4. Arrested granulosa cell proliferation and reduced FSH receptor expression in Zp3-Foxo3a Tg mice. Twenty-five-day-old Tg or WT mice were used for ovarian staining of PCNA and FSH receptor, or for the BrdU incorporation assay. (A,B) Immunostaining of PCNA expression in ovarian sections from WT (A, arrow) and Tg (B, arrow) mice. Signals appear in brown using DAB as substrate. (C,D) Reduced incorporation of BrdU into granulosa cells of Tg mice (D, arrow) as compared with granulosa cells of WT littermates (C, arrow). Signals appear reddish-brown using AEC as substrate. (E,F) Reduced expression of FSH receptor mRNA in ovaries of Tg mice (F, arrows) as compared with that in ovaries of WT mice (E, arrows), as measured by in situ hybridization. Signals appear in black.