Antagonism of the testis- and ovary-determining pathways during ovotestis development in mice

Abstract

Ovotestis development in B6-XY(POS) mice provides a rare opportunity to study the interaction of the testis- and ovary-determining pathways in the same tissue. We studied expression of several markers of mouse fetal testis (SRY, SOX9) or ovary (FOXL2, Rspo1) development in B6-XY(POS) ovotestes by immunofluorescence, using normal testes and ovaries as controls. In ovotestes, SOX9 was expressed only in the central region where SRY is expressed earliest, resulting in testis cord formation. Surprisingly, FOXL2-expressing cells also were found in this region, but individual cells expressed either FOXL2 or SOX9, not both. At the poles, even though SOX9 was not up-regulated, SRY expression was down-regulated normally as in XY testes, and FOXL2 was expressed from an early stage, demonstrating ovarian differentiation in these areas. Our data (1) show that SRY must act within a specific developmental window to activate Sox9; (2) challenge the established view that SOX9 is responsible for down-regulating Sry expression; (3) disprove the concept that testicular and ovarian cells occupy discrete domains in ovotestes; and (4) suggest that FOXL2 is actively suppressed in Sertoli cell precursors by the action of SOX9. Together these findings provide important new insights into the molecular regulation of testis and ovary development.

Figure 1. Histology of B6 XY^POS ovotestes

Sagittal sections of paraffin-embedded B6 XY, B6 XY^POS and B6 XX fetuses at 14.5 dpc were stained with haematoxylin and eosin. (A) B6 XY gonad shows typical testicular morphology with testis cords enclosing the germ cells. (B) The B6 XY^POS ovotestis contained testicular cords in the centre and seemingly undifferentiated tissue at the poles (separated by yellow lines). (C) B6 XX gonad displays ovarian morphology with germ cells that have entered meiosis. (D) Enlargement of the region marked with a rectangle in (A) shows testis cords. (E) Enlargement of the region marked with a rectangle in (B, right) shows testis cords in the central region of the ovotestis. (F) Enlargement of the region marked with a rectangle in (B, left) shows meiotic germ cells (yellow arrowheads) at the anterior pole. (G) Enlargement of the region marked with a rectangular in (C) shows meiotic germ cells. Images are oriented so that anterior pole is at the top (a, anterior, p, posterior). Scale bar, 50 μm.

Figure 2. Temporal and spatial expression of SRY in B6 XY^POS gonads

Immunofluorescence of sagittal sections of paraffin-embedded B6 XY (A–C) and B6 XY^POS (D–F) fetuses from 11.5 dpc to 13.5 dpc with antibodies specific for SRY (green) and E-cadherin (red), which is expressed in germ cells and in the mesonephric tubules of the mesonephros. SRY expression is similar in B6 XY^POS to B6 XY gonads at 11.5 dpc (A and D). At 12.5 dpc the down-regulation of SRY is delayed in B6 XY^POS compared to B6 XY gonads (B and E) and at 13.5 dpc a few SRY-positive cells are detectable only in B6 XY^POS gonads (F, arrowheads). Sections are adjacent sections to those shown in Figures 3 and 4. (G–I) SRY down-regulation is delayed in B6 XY^POS ovaries. Immunofluorescence of sagittal sections of a paraffin-embedded B6 XY^POS fetus at 13.5 dpc to with antibodies specific for SRY (green, G), SOX9 (green, H) and FOXL2 (green, I) together with E-cadherin (red in all three panels), which is expressed in germ cells and in the mesonephric tubules of the mesonephros. FOXL2 is expressed along the whole length of the gonad, whereas no SOX9 expression was detected. SRY expression remains detectable at 13.5 dpc, a stage at which it is normally completely shut down. Pictures are oriented so that anterior pole is at the top. DAPI staining (blue) marks cell nuclei. Scale bar for all panels, 100 μm. Dotted lines encompass gonads.

Figure 3. Temporal and spatial expression of SOX9 in B6 XY^POS gonads

Immunofluorescence of sagittal sections of paraffin-embedded B6 XY (A–C) and B6 XY^POS (D–F) fetuses from 11.5 dpc to 13.5 dpc with antibodies specific for SOX9 (green) and E-cadherin (red). SOX9 is expressed in Sertoli cells in B6 XY gonads throughout development (A–C), whereas SOX9 expression is induced and maintained in the B6 XY^POS gonads only in the centre (D–F). Sections are adjacent to those shown in Figures 2 and 4. (G–I) Double immunofluorescence of 12.5 dpc B6 XY^POS fetus with antibodies specific to SRY (red) and SOX9 (green). Red channel only (G), green channel only (H) and merged channels (I). DAPI staining (blue) marks cell nuclei. Pictures are oriented so that anterior pole is at the top. Scale bar for all panels, 100 μm. Dotted lines encompass gonads.

Figure 4. Analysis of the female marker FOXL2 in B6 XY^POS gonads

Immunofluorescence of sagittal sections of paraffin-embedded B6 XY (A–C), B6 XY^POS (D–F), and B6 XX (G–I) fetuses from 11.5 dpc to 13.5 dpc with antibodies specific for FOXL2 (green) and E-cadherin (red). FOXL2 is expressed in B6 XY^POS gonads from 11.5 dpc until 13.5 dpc (D–F) in an area overlapping with SOX9 expression (E and F, compare with Fig. 3E and F). No FOXL2 expression was detectable in B6 XY gonads (A–C), and strong expression was seen in B6 XX gonads from 11.5 dpc (G–I). Sections are adjacent to those shown in Figures 2 and 3. DAPI staining (blue) marks cell nuclei. Pictures are oriented so that the anterior pole is at the top. Scale bar for all panels, 100 μm. Dotted lines encompass gonads.

Figure 5. FOXL2 and SOX9 are expressed in overlapping regions in B6 XY^POS ovotestes, but are mutually exclusive at the cellular level

(A) Immunofluorescence of adjacent sagittal sections of a paraffin-embedded B6 XY^POS fetus at 14.5 dpc with antibodies specific for SOX9 (green, left panel) and FOXL2 (green, right panel) together with E-cadherin (red in both panels). Dotted lines delineate ovarian tissue at the poles from testicular tissue in the centre of the gonad. FOXL2 is detectable even within morphological testicular tissue (see rectangle). Scale bar, 100 μm. (B) Double immunofluorescence of a sagittal section of B6 XY^POS ovotestis at 14.5 dpc with antibodies specific for SOX9 (green) and FOXL2 (red) indicates that these two genes are not co-expressed in individual cells. DAPI staining (blue) marks cell nuclei; yellow staining is due to autofluorescence of blood cells. Scale bar, 100 μm.

Figure 6. SF1 is expressed by somatic cells throughout B6 XY^POS ovotestes

Immunofluorescence of sagittal sections of paraffin-embedded B6 XY^POS fetuses from 11.5 to 13.5 dpc with antibodies specific for (A–D) SF1 (green) and E-cadherin (red) and adjacent sections (E–H) for FOXL2 (green) and E-caherin (red). SF1 is strongly expressed by somatic cells in the B6 XY^POS gonads along the whole length of the gonad at 11.5 (A) and 12.5 dpc (B) also in regions in which the ovarian pathway was initiated as shown by FOXL2 expression (E and F). From 13.5 dpc onwards SF1 expression is decreased in FOXL2-positive regions (C and D, compare with G and H). DAPI staining (blue) marks cell nuclei. Pictures are oriented so that anterior pole is at the top. Scale bar for all panels, 100 μm. Dotted lines encompass gonads.