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First published online 26 November 2008
doi: 10.1242/dev.029587

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A critical time window of Sry action in gonadal sex determination in mice

¹ Department of Veterinary Anatomy, The University of Tokyo, Yayoi 1-1-1, Bunkyoku, Tokyo 113-8657, Japan.
² Department of Anatomy, Kyorin University School of Medicine, Shinkawa 6-20-2, Mitaka, Tokyo 181-8611, Japan.
³ Department of Aging Intervention, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Gengo 36-3, Morioka-cho, Obu, Aichi 474-8511, Japan.
⁴ Department of Molecular Biology, Graduate School of Medicine, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan.
⁵ Institute for Molecular Bioscience, The University of Queensland, St Lucia, Brisbane, QLD 4072, Australia.

View larger version (60K): [in this window] [in a new window]   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).

View larger version (113K): [in this window] [in a new window]   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.

View larger version (56K): [in this window] [in a new window]   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.

View larger version (65K): [in this window] [in a new window]   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.

View larger version (87K): [in this window] [in a new window]   Fig. 5. Female-specific patterns of Fgf9/Wnt4 signaling states lead to the defective testis induction caused by delayed Sry expression. (A) Whole-mount in situ hybridization analyses showing Fgf9 and Wnt4 expression in Sry-induced (HS+) and non-induced (HS-) XX Tg gonads at 13 and 18 ts (18-hour culture). The gonadal area is indicated by a dashed line. (B) Real-time RT-PCR analysis showing Fgf9 and Wnt4 transcript levels in the Sry-induced and non-induced XX Tg explants initiated at 18 ts (18-hour culture). The transcript levels in the wild-type XY (black bar) and XX (white bar) explants (HS+) of the same littermates at the same stage are also shown. Vertical axis represents Fgf9 or Wnt4 expression level relative Gapdh. The data represent mean values ±s.e.m. (n=4). (C-E) Time-course immunohistochemical analyses with anti-SOX9 (C,D) and anti-3βHSD (E) antibodies, showing effects of exogenous FGF9 (100 ng/ml) and sFRP2 (1.5 µg/ml) on the initiation (9-hour culture) and maintenance (24-hour and 72-hour culture) of Sertoli cells (C,D) and subsequent Leydig cell differentiation (72-hour culture; E) in XX Tg explants Sry-induced at 18-19 ts. (D) Quantification of the relative number per gonadal area of SOX9-positive cells in the immunostained sections (C) of these XX Tg explants cultured in the presence (black circle) or absence (white circle) of FGF9 and sFRP2 (mean values of cell number ±s.e.m.; n=4). The value in the XY wild-type explants of the same littermates (18-19 ts; 72-hour culture) is set as 100% (18.3±1.5x103 cell number per mm2). Asterisks indicate a significant difference (**P<0.01) as compared with the data from the FGF9/sFRP2-treated XX Tg explants initiated at 18-19 ts. The relative numbers of SOX9-positive cells in the XX Tg explants Sry-induced at 12-13 ts are also plotted in D (white square). (E) The addition of FGF9 and sFRP2 together appears to induce an increase of 3βHSD-positive cell number in the Sry-induced XX Tg explants (18-19 ts) at 72 hours in culture, as compared with those of the Sry-induced explants non-treated (None) or treated with FGF9 alone (+FGF9). Scale bars: 100 µm.

View larger version (64K): [in this window] [in a new window]   Fig. 6. Delayed Sry expression can induce testis development in XX Wnt4+/- genital ridges. (A,B) The genital ridges were isolated from the XX Tg Wnt4+/+ (wild type) or Wnt4+/- embryos at 18 ts. The left genital ridges were treated with HS (HS+), while the right ones were used as non-treated control (HS-). After culture for 72 hours, sagittal serial sections of these XX Tg explants were comparatively analyzed by anti-SOX9 (A) and anti-laminin (B) staining (brown staining). Both maintenance of SOX9 expression and testis cord formation are properly induced in the XX Wnt4+/- explant Sry-induced at 18 ts (right upper panels in A,B). In the non-treated (HS-) Wnt4+/- explant from the same embryo, neither SOX9 expression nor cord formation is detectable (right lower panels in A,B), which is similar to non-treated and Sry-induced XX Tg explants of the Wnt4+/+ wild-type littermate. Scale bars: 100 µm.

View larger version (31K): [in this window] [in a new window]   Fig. 7. Schematic showing two distinct critical time windows of SRY action for the initiation and maintenance of Sox9 expression in developing XX gonads. The horizontal bar represents the bipotential period (blue), and the early (purple) and late (red) ovarian differentiation phases of developing XX gonads. The initiation and maintenance patterns of endogenous Sox9 expression in Sry-induced XX Tg gonads at each developmental stage are shown in the upper part (XX Tg). The timings of Sry and Sox9 expression and testis cord formation in XY wild-type gonads are shown in the lower part (XY). In XX Tg gonads at 12-14 ts, artificial Sry induction cell-autonomously induces initial Sox9 activation and glycogenesis in pre-Sertoli cells. Such SRY/SOX9 expression leads to high-FGF9/low-WNT4 expression patterns in the gonadal area, which results in the maintenance of Sox9 expression and the testis-specific induction of early morphogenic events [CE (coelomic epithelial cell) proliferation and mesonephric cell migration; blue arrows]. In XX Tg gonads at 16-21 ts, the delayed Sry induction is capable of promoting initial Sox9 activation and glycogenesis in pre-Sertoli cells. However, a lack of SRY action before 16 ts results in the female-specific high Wnt4 expression that leads to failure of the maintenance of SOX9/FGF9 expression, resulting in the ovarian development at later stages (purple arrows). Beyond 22 ts, neither testis formation nor transient Sox9 activation was detected in Sry-induced XX gonads (red arrows).

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