Fig. 3. Time course of SOX9 expression in Sry-induced XX Tg gonads. (A) Immunostaining of sagittal sections of XX Tg gonads initiated at 12-13, 18-20 and 23-24 ts, showing SOX9 expression (brown staining) at 6, 9, 12 and 24 hours (h) after Sry induction. Similar to the SOX9 expression pattern in the XX Tg gonads at 12-13 ts, SOX9-positive cells are detected at 6 hours (arrowheads) and increased at 9 hours in the explants at 18-20 ts. However, in the explants at 18-20 ts, SOX9 signals were decreased at 12 hours after Sry initiation, in contrast to the maintained SOX9 expression seen in many cells of the explants at 12-13 ts. In the explants at 23-24 ts, no appreciable signals were detected throughout the culture period. Scale bars: 100 µm. (B) Real-time RT-PCR analysis showing changes of Sox9 expression levels in the XX Tg genital ridges (18-20 ts) treated with (black circle) or without (white circle) HS over a 12-hour-culture period. Circles represent mean values±s.e.m. (n=5 at each point). Vertical axis represents the Sox9/Gapdh amplicon ratio, whereas the horizontal axis represents the culture period after HS treatment. Asterisk indicates significantly (P<0.01, Student's t-test) higher expression of Sox9 transcripts, as compared with all other values in both SRY-induced and control XX Tg explants. The two dashed lines indicate the Sox9 expression levels in the XX (0.36±0.04, lower line) and XY (0.85±0.04, upper line) wild-type genital ridges isolated at 23-24 ts (n=4). The RT-PCR analysis below shows the HS-dependent Sry expression at 3 hours. (C) Comparative immunohistochemistry for SOX9 and SF1/Ad4Bp were performed using two consecutive sections of one Sry-induced XX gonad at 13 and 18 ts, respectively (9 hours after HS treatment). SOX9-positive cells overlap with presumptive supporting cells expressing SF-1/Ad4Bp at both stages (arrowheads). Asterisk, germ cells. Scale bar: 10 µm.