Fig. 2. Artificial delay of Sry expression by 6 hours leads to the failure of proper testis formation. (A) Immunohistochemical staining with anti-laminin, SOX9 and anti-SCP3 antibodies, showing 4-day-cultured explants of XX Tg genital ridges HS treated at 13, 15 and 18 ts (tail somite stage). In contrast to the testis formation observed in the XX Tg explants HS treated at 13 ts, some explants HS treated at 15 ts display ovotestis development with a central testicular area. Beyond this stage, HS treatment is not capable of inducing XX/testis sex reversal in the XX Tg genital ridges (18 ts). (B,C) Whole-mount in situ hybridization (B) and immunohistochemical (C) analyses of the XX Tg genital ridges at 13 and 18 ts (3 hours after HS treatment), showing no appreciable difference between the 13 and 18 ts stages in the signal intensities for HS-dependent Sry expression at both mRNA and protein levels. Insets in C show higher magnification, with nuclear localization of SRY protein in the presumptive supporting cells directly associated with germ cells (asterisk) at both stages. In B and C, anterior/posterior edges of the gonadal area are indicated by arrowheads. Scale bars: 100 µm.