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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.
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