Fig. 4.

Proliferation and apoptosis of the MDE cells under PI3K inhibition. (A) E14.5 rat female urogenital ridge stained with whole mount in situ hybridization for Wnt7a with thick transverse lines indicating the positions examined. After incubation with or without 25 μM of LY294002 for 2 or 4 h, ridges were fixed, sectioned through and stained. Five serial sections were selected from the posterior tip of the Müllerian duct (P), anterior portion where the most anterior portion of the ovary is included (A) and the mid level (M). (B) Percentages of divided MDE cells were determined in the different portions of the Müllerian duct after 2-h incubation with or without 25 μM of LY294002. Divided cells were detected by BrdU, PAX8, and DAPI staining. Six urogenital ridges were examined (N=6). Columns represent mean percentages ± SD of BrdU positive cells in the MDE cells with LY294002 (gray columns) and nontreatment (white columns). Significant differences between the proliferating proportion in A, M, and P were determined by single-factor analysis of variance (ANOVA) and multiple comparison (p<0.05). A gradual decrease in the proportion of proliferating cells was observed along the A-P axis, with position A showing a statistically higher proliferation than M and P (p=0.0004 and 0.00002, respectively). Student’s t-test indicated that LY294002 did not affect the rate of proliferation of the MDE at any position. (C) The percentage of apoptotic MDE cells was examined in different portions of the Müllerian duct after 4 h of incubation with 25 μM of LY294002. Apoptotic cells were detected by TUNEL, PAX8, and DAPI staining. Six urogenital ridges were examined (N=6). Columns represent mean percentages ± SD of TUNEL positive cells in the Müllerian duct. Statistical analysis using multiple comparison showed that apoptosis in A was significantly less than M (p=0.04), while P (PL) did not differ significantly from M and A.