Fig. 5. Defective spermatogenesis in Ret^DN/+ mice is due to maturation delay and reduced germ cell number. (A) Representative picture depicting increased apoptosis by TUNEL staining (brown nuclei) in Ret^DN/+ seminiferous tubules at 3 to 4 weeks of age. (B) Increased apoptosis (TUNEL positive) in Ret^DN/+ seminiferous tubules was noted as early as P17 (n=3 for each time point, ^*=P<0.01, mean±s.e.m.). (C) Ret^DN/+ mice had a reduced number of germ cells by GCNA-immunostaining (left, n=3, ^*=P<0.01, mean±s.e.m.), and reduced cell proliferation (measured by BrdU incorporation, right, n=2, mean±s.d.), relative to wild-type (wt) mice at P10. Note the similar germ cell number between Ret^DN/+ and wild-type testes at birth. (D) Representative cell ploidy analysis of postnatal (P10) testes in Ret^DN/+ mice shows a higher 2n:4n ratio than in wild-type mice, indicating a delay in sperm maturation in Ret^DN/+mice. In the left panel, the 2n and 4n peaks are indicated, the numbers on the peaks represent the percentage of cells in each peak. The bar graph on right summarizes the ploidy results, which indicate a spermatogenesis defect at P10.