Fig. 1. Characterization of the inducible Sry Tg mouse line. (A,B) Whole-mount in situ hybridization (A) and immunohistochemical (B) analyses showing HS-dependent Sry induction at both mRNA and protein levels in HSP-Sry Tg mice 2 hours after HS treatment (43°C for 10 minutes; right of each set in both A and B); control non-heat-shocked HSP-Sry Tg mice are shown on the left. (A) Ectopic Sry induction was detected in 10.5 dpc whole embryos and 16.5 dpc organs, such as ovary (Ov), testis (Ts) pancreas (Pc), adrenal (Ad) and kidney (Kd; right). (B) Anti-SRY immunohistochemical staining confirmed the ubiquitous expression of SRY proteins in the HS-treated somatic cells of the 16.5 dpc organs (note nuclear localization; asterisks, tubular lumens; arrowheads, germ cells). No signal is detectable in non-treated samples (left). Scale bar: 50 µm. (C) Whole-mount in situ hybridization, showing Sry expression patterns in organ cultures [0-24 hours (h) after Sry induction] of XY wild-type (upper) and XX Tg (lower) genital ridges at 12-13 ts (tail somite stage; approximately 11.0 dpc). Anterior/posterior edges of the gonadal area are indicated by arrowheads. (D,E) Four-day cultured explants of XY and XX wild-type genital ridges (left in D) and the XX Tg genital ridges with or without HS treatment (right in D; E) at 12-13 ts. Testis cords are indicated by dashed lines. Hematoxylin and Eosin (HE) staining shows testis cords in the left genital ridge (HS+) and the presence of the meiotic germ cells in the right genital ridge (HS-) of the same XX Tg embryo (arrows in insets in D indicate germ cells). Immunohistochemical staining with anti-SOX9, anti-3βHSD and anti-SCP3 antibodies demonstrates the differentiation of testicular Sertoli and Leydig cells in the HS-treated XX explants. Scale bars: 100 µm (bars in insets in D, 10 µm).