Fig. 4. Defective induction of early non-cell-autonomous morphogenic events caused by delayed SRY activation. (A) Immunohistochemistry for SOX9 and PAS staining in the Sry-induced (HS+) and non-induced (HS-) XX Tg explants at 18-19 ts (12-hour culture). PAS reaction and anti-SOX9 staining of two consecutive sections show that testis-specific glycogenesis is induced in the SOX9-positive cells of the Sry-induced XX Tg gonads (HS+). Insets show higher magnified images of the positive cells. (B) Mesonephric cell migration assay using the XX Tg gonad and GFP-positive XY mesonephros isolated at 13 ts and 18-19 ts. Gonads were isolated from HS-treated or non-treated genital ridges, assembled with GFP-positive mesonephros, and then cultured for 48 hours. No contribution of GFP mesonephric cells is detectable in both Sry-induced (HS+) and non-induced (HS-) explants at 18 ts, in contrast to the higher contribution of GFP-positive cells in both SRY-induced XX Tg explants at 13 ts (13ts, HS+) and control XY wild-type explants at 18 ts (18 ts, XY wt). Dashed lines indicate the gonadal surface area of reconstituted explants. (C,D) Cell proliferation assay of double immunohistochemistry using anti-BrdU (green) and anti-SF1/Ad4Bp (red) antibodies in SRY-induced (HS+) and non-induced (HS-) XX Tg explants at 12-13 ts and 18-19 ts (12-hour culture). The gonadal explants were labeled for BrdU (green) for 3 hours after the 9-hour pre-culture. The BrdU-positive cells are highly detected in the SF1-positive cells (red) in and near the coelomic epithelium in all explants (C). However, in explants at 18-19 ts, there was no significant difference between SRY-induced and non-induced XX Tg gonads in their mitotic indices (D; the mean percentage of BrdU-positive cells in coelomic epithelium ±s.e.m.; n=5). The mitotic index of coelomic epithelial cells in the Sry-induced explants at 12-13 ts is significantly higher, when compared with those in the other three explants (^**P<0.01). In D, the two dashed lines indicate the mitotic index of the coelomic epithelial cells in the XX (43.3±1.0%, lower line) and XY (57.8±1.5%, upper line) wild-type explants at 12-13 ts (n=5), respectively. ms, mesonephros. Scale bars: 100 µm.