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Case Reports
, 121 (1), 328-41

Identification of SOX3 as an XX Male Sex Reversal Gene in Mice and Humans

Case Reports

Identification of SOX3 as an XX Male Sex Reversal Gene in Mice and Humans

Edwina Sutton et al. J Clin Invest.


Sex in mammals is genetically determined and is defined at the cellular level by sex chromosome complement (XY males and XX females). The Y chromosome-linked gene sex-determining region Y (SRY) is believed to be the master initiator of male sex determination in almost all eutherian and metatherian mammals, functioning to upregulate expression of its direct target gene Sry-related HMG box-containing gene 9 (SOX9). Data suggest that SRY evolved from SOX3, although there is no direct functional evidence to support this hypothesis. Indeed, loss-of-function mutations in SOX3 do not affect sex determination in mice or humans. To further investigate Sox3 function in vivo, we generated transgenic mice overexpressing Sox3. Here, we report that in one of these transgenic lines, Sox3 was ectopically expressed in the bipotential gonad and that this led to frequent complete XX male sex reversal. Further analysis indicated that Sox3 induced testis differentiation in this particular line of mice by upregulating expression of Sox9 via a similar mechanism to Sry. Importantly, we also identified genomic rearrangements within the SOX3 regulatory region in three patients with XX male sex reversal. Together, these data suggest that SOX3 and SRY are functionally interchangeable in sex determination and support the notion that SRY evolved from SOX3 via a regulatory mutation that led to its de novo expression in the early gonad.


Figure 1
Figure 1. XX Tg/+ adults develop as males.
(AC) External genitalia of XX, XY, and XX Tg/+ individuals. Note the male phenotype of the XX Tg/+ animal. (DF) XX, XY and XX Tg/+ internal reproductive tracts. Scale bars: 2 mm. (G) XY and XX Tg/+ testes. (H and I) Histological sections of XY and XX Tg/+ testes, respectively, showing the absence of sperm in the latter. Scale bars: 100 μm. All animals used for this analysis were between 20 and 24 weeks of age.
Figure 2
Figure 2. XX Tg/+ gonads have a normal male appearance and express the Sox3 transgene at 13.5 dpc.
(A) XX, XY, and XX Tg/+ 13.5-dpc gonads shown in lateral and medial views and analyzed for Sox9 and Amh expression and EGFP fluorescence in unfixed tissue. Scale bar: 250 μm. (B) XX Tg/+ 13.5-dpc gonad section stained with EGFP and Sox9 antibodies. Lower panel shows higher-magnification view of the boxed region. Arrows indicate EGFP-positive, Sox9-negative cells in the interstitium. Scale bars: 50 μm.
Figure 3
Figure 3. 13.5-dpc XX Tg/+ gonads show variable cord formation and Sox9 expression that coincides with the spatial localization of transgene (EGFP) expression.
(A) Sox9 expression and corresponding EGFP expression in representative XX Tg/+ (strong), and XX Tg/+ (weak) 13.5-dpc gonads. Scale bar: 250 μm. (B) Optical slices of whole mount 12.5-dpc XX Tg/+ and XY gonads stained with EGFP (green), Sox9 (red), and Pecam1 (blue). The dotted lines indicate the outline of the gonads. Scale bar: 150 μm.
Figure 4
Figure 4. Mechanism of transgene-induced sex reversal and its activation in the gonads.
(A) 13 ts XX Tg/+ and XY transverse sections presented as a 6-μm stack stained with Sox3 and Sox9. Enlargements corresponding to the boxed region are shown on the right. Arrows indicate Sox3/Sox9-positive cells. Scale bars: 50 μm. (B) Single optical slices of whole mount 11.5-dpc XX, XY, and XX Tg/+ genital ridges stained with Sox9 and presented as an overlay with corresponding differential interference contrast (DIC) image. Higher-magnification view of the boxed region in the XX Tg/+ genital ridge is shown in the right panel, presented as a 60-μm stack of optical sections and highlighting the extent of colocalization between Sox3 and Sox9. Scale bars: 50 μm. In B, genital ridges are oriented so that anterior is to the left and the coelomic epithelium at the top. (C) Transactivation experiment using the mouse Sox9 TESCO enhancer reporter. Note that Sox3 and Sry exhibit similar synergistic activation of the reporter with SF1, but fail to activate in the absence of SF1. Negative control transfections using a reporter lacking the enhancer are also shown (–TESCO). Western blot analysis indicated that comparable levels of Sox3, Sry, and Sox9 protein were expressed (data not shown). Data are mean ± SEM. ***P < 0.001, **P < 0.01, *P < 0.05.
Figure 5
Figure 5. The Sox3 transgene is not able to mimic Sox9 in the gonad at 12.
dpc. (AF) XY Sox3-SrTg;Sox9fl/fl;R26CreERT2 mice were either treated or not treated with tamoxifen (TM). In the absence of TM, the gonad developed into a normal testis containing testis cords (AC). When Sox9 was ablated after TM treatment, testis cords were not formed (D and E), and Foxl2-positive cells were detected in the gonad (F). (GL) Similar experiments were performed for XX Sox3-SrTg;Sox9fl/fl;R26CreERT2 mice. The mice developed XX sex-reversed testis without TM (GI). In contrast, TM treatment resulted in ovarian morphology and the expression of Foxl2 (JL). Scale bars: 0.5 mm (A, D, G, and J) and 50 μm (B, C, E, F, H, I, K, and L).
Figure 6
Figure 6. The Sr transgene has integrated upstream of Aldh1a1 on chromosome 19.
(A) Schematic representation of transgene insertion site on chromosome 19 illustrating PCR primer position (black arrows), Southern probe (red), and relevant restriction sites. (B) FISH analysis of Tg/+ XY metaphase chromosomes. Sox3 transgene (modified RP23-174O19 BAC) and chromosome 19 (RP23-142D22 BAC) signals are shown in red and green, respectively. The inset shows a magnified view of chromosome 19 into which the transgene has integrated (19*). Scale bars: 2.5 μm; 0.5 μm in inset. (C) PCR assay of genomic DNA using primers that flank the transgene integration site. Product is amplified from Tg/+ DNA and not from +/+ DNA. All genomic DNA samples were shown to be amplifiable using Gapdh primers (data not shown). H2O indicates negative control reaction. (D) Southern blot analysis comparing +/+ and Tg/+ DNA digested with KpnI, PstI, and SpeI. Marker sizes (kb) are shown on the right. Note the additional bands of expected size in each of the Tg/+ tracts. K, KpnI; P, PstI; S, SpeI.
Figure 7
Figure 7. Expression profile of Aldh1a1/Aldh1a1 and Sox3 in XY and XX Tg/+ gonads from 11.5 to 13.5 dpc.
(A) qRT-PCR analysis of 11.5- to 13.5-dpc gonads. Normalized expression levels of each gene are shown relative to β-actin. Two cDNA series were analyzed twice each, and error bars represent SD of the mean of the two series. (B) In situ hybridization showing Aldh1a1 expression in transverse sections of 13.5-dpc XY, XX Tg/+, and XX gonads. No signal was detected using an Aldh1a1 sense probe. (C) Transverse sections of XY, XX Tg/+, and XX 13.5-dpc gonads stained with Aldh1a1, Sox3, and DAPI. Protein expression levels are consistent with the transcript expression analysis shown in B. (D) Confocal micrograph of 13.5-dpc XX Tg/+ gonad showing the extensive overlap in Sox3 and Aldh1a1 expression. Arrows indicate Sox3/Aldh1a1-positive cells lining the testis cords. Scale bars: 100 μm.
Figure 8
Figure 8. Expression of Aldh1a1 and EGFP in the gonadal ridge of XX Tg/+ gonads at 6–17 ts.
Immunohistochemistry and in situ hybridization of serial transverse sections showing EGFP and Aldh1a1 distribution in 6–17 ts embryos. Given the low level of Aldh1a1 protein in the XX Tg/+ GR during these early stages of sex determination, the expression of Aldh1a1 in adjacent sections is included at each stage for comparison. Aldh1a1/Aldh1a1 is first observed at 6 ts (magnified in box inset). Aldh1a1/Aldh1a1 is detected in a diffuse pattern at 12 ts. A rare EGFP-positive cell is found within a population of Aldh1a1-positive cells in the GR at this stage (magnified in box 1). At 17 ts, Aldh1a1/Aldh1a1 becomes more restricted, while the abundance of EGFP within the GR increases (magnified in box 2). Scale bars: 100 μm.
Figure 9
Figure 9. SOX3 rearrangements in 46, XX males.
An image derived from the UCSC genome browser ( showing the location of the rearrangements identified in patients A–C in relation to the SOX3 gene. Also shown are rearrangements previously identified in healthy individuals (as recorded in the Database of Genomic Variants;, including CNVs and an inversion. It is noteworthy that duplication breakpoints in patients A and C are in close proximity to other known CNV/inversion breakpoints, suggesting structural or sequence motifs that may be responsible for recurrent rearrangements. Numbering corresponds to nucleotide position on the X chromosome (chrX), based on reference sequence hg18.
Figure 10
Figure 10. SOX3 activates the hTES.
Human SOX3 (100 ng) activates hTES-luc (1.6 μg) (10–fold) compared with human SRY (100 ng) (5-fold) in CHO cells. In the presence of added SF1 (20 ng), further transactivation occurs with both SOX3 (25-fold) and SRY (20-fold) (n = 2–3). Error bars represent SD. Unless otherwise indicated, statistical comparisons were made between fold activations of expression vector and control empty vector. ***P < 0.001, **P < 0.01.

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