Fig 1: External genitalia and gonad development in adult XY and XX Rspo1KO Sox8KO double mutant mice.External genitalia from adult P40 mice (a–b, k–m), macroscopic view of gonads (c–d, n–p) (Scale bars 1.5 mm), histology as revealed by PAS staining on gonadal sections (e–f, q–s) (Scale bars 100 µm), and immunostaining of SOX9 (Sertoli cell marker, in red) (g–h, t–v), FOXL2 (granulosa cell marker, in green) (g–j, t–y), DMRT1 (Sertoli and germ cell marker, in red) (i–j, w–y), TRA98 (germ cell marker, in white) (i–j, w–y) and DAPI (nuclear marker, in blue) (g–j, t–y) on gonadal sections (Scale bars 50 µm). Inactivation of both Rspo1 and Sox8 in XY Rspo1KO Sox8KO double mutant mice did not cause a sex reversal (a–j). XY Rspo1KO Sox8KO gonads developed as testes with seminiferous tubules (f) containing SOX9 and DMRT1 positive Sertoli cells (h–j), as in control testes (e, g, i). As shown, XX control ovaries developed follicles (s) containing FOXL2-positive granulosa cells (v, y). Adult ovotestes in XX Rspo1KO Sox8KO mice (n, q) were indistinguishable from XX Rspo1KO mice (o, r). These gonads contained an ovarian ‘O’ compartment with follicles and a testicular ‘T’ compartment with seminiferous tubule-like structures, as indicated by arrowheads (q, r). The seminiferous tubule-like structures in XX Rspo1KO Sox8KO and XX Rspo1KO ovotestes contained SOX9 and DMRT1 positive Sertoli cells (t–u, w–x), as in control testes (g, i), but lacked TRA98-positive germ cells (w, x).
Fig 2: Precocious granulosa cell differentiation in XX Rspo1KO Sox8KO Sox9cKO triple mutant fetuses at E17.5.Immunofluorescence of CDKN1B (P27) (mitotic arrest marker, in red) (a–e''), AMH (Sertoli marker and mature granulosa cell marker, in green) (a–e''), DMRT1 (Sertoli and germ cell marker, in red) (f–j''), FOXL2 (granulosa cell marker, in green) (f–j''), TRA98 (germ cell marker, in white) (a–j''), and DAPI (nuclear marker, in blue) (a–j'') on gonadal sections from E17.5 fetuses (main panels scale bar 100 µm). The anterior ‘a’ and posterior ‘p’ axis is shown below each column. For main panels (a–j), highlighted anterior and posterior areas are shown in the respective single and double primed letter panels. Yellow arrowheads indicate granulosa cells expressing CDKN1B or FOXL2, asterisks indicate cells expressing AMH, white arrowheads indicate Sertoli cells expressing DMRT1, and plus symbols indicate germ cells expressing DMRT1 and TRA98. Gonads in XX control fetuses developed as ovaries, as shown by FOXL2 and CDKN1B expression in pre-granulosa cells throughout the gonad (a', a'', f', f'', yellow arrowheads). These fetal ovaries lacked mature granulosa cells expressing AMH (a). In contrast, XX Rspo1-/- (Rspo1KO), XX Rspo1-/-; Sox8-/- (Rspo1KO Sox8KO), XX Rspo1-/-; Sox9flox/flox; Sf1:creTg/+ (Rspo1KO Sox9cKO), and XX Rspo1KO Sox8KO Sox9cKO gonads exhibited down-regulation of CDKN1B (b–e) and ectopic AMH expression in the anterior area (b’-e’, asterisks), indicating Sertoli cells or mature granulosa cells. However, while XX Rspo1KO gonads contained Sertoli cells expressing DMRT1 (g', white arrowhead), these cells were rare in XX double and triple mutants (h–j) (for XX triple mutants, 1 out of 8 gonads studied from n = 4 fetuses). Note that some DMRT1-positive cells are germ cells expressing TRA98 (g'', h'', j'', plus symbols). Thus, while granulosa cells differentiate precociously in XX Rspo1KO gonads lacking Sox8 and/or Sox9 at E17.5, these cells have not yet reprogrammed as Sertoli cells in XX Rspo1KO Sox8KO and XX Rspo1KO Sox9cKO mice. In XX triple mutant fetuses, granulosa cell reprogramming as Sertoli cells may be delayed, or blocked.
Fig 3: Lack of testis cords in XY Rspo1KO Sox8KO Sox9cKO triple mutant fetuses at E17.5.Immunofluorescence of CDKN1B (P27) (mitotic arrest marker, in red) (a–d''), AMH (Sertoli marker and mature granulosa cell marker, in green) (a–d''), DMRT1 (Sertoli and germ cell marker, in red) (e–h'''), FOXL2 (granulosa cell marker, in green) (e–h'''), TRA98 (germ cell marker, in white) (a–h'''), and DAPI (nuclear marker, in blue) (a–h) on gonadal sections from E17.5 fetuses (main panel scale bar 100 µm). For gonads in panels (c–d), the anterior ‘a’ and posterior ‘p’ axis is shown below each column. Below each main panels (a–h), highlighted areas are shown in respective primed letter panels. Yellow arrowheads indicate granulosa cells expressing CDKN1B or FOXL2, asterisks indicate cells expressing AMH, white arrowheads indicate Sertoli cells expressing DMRT1, and plus symbols indicate germ cells expressing DMRT1 and TRA98. Gonads in XY Rspo1-/-; Sox8-/- (Rspo1KO Sox8KO) fetuses exhibited AMH and DMRT1 positive Sertoli cells organized as testis cords (b, f) and lacked FOXL2-positive granulosa cells (f), as in control testes (a, e). Cells expressing AMH were found in XY Rspo1-/-; Sox9flox/flox; Sf1:creTg/+ (Rspo1KO Sox9cKO) and XY Rspo1KO Sox8KO Sox9cKO gonads (c’ and d’, asterisks), indicating Sertoli cells or mature granulosa cells. Indeed, both exhibited granulosa cells expressing CDKN1B and FOXL2 (c', d', g', h', h''', yellow arrowheads). However, while XY Rspo1KO Sox9cKO gonads exhibited DRMT1-positive, TRA98-negative Sertoli cells (g', white arrowhead), these cells were scarce in XY triple mutant gonads (h) (6 of 6 XY triple mutant gonads studied from n = 3 fetuses). Note that some DMRT1 expressing cells in XY Rspo1KO Sox9cKO and XY triple mutant gonads are germ cells expressing TRA98 (g', g'', h'', h''', plus symbols). Thus, although XY Rspo1KO Sox9cKO and XY triple mutant gonads contain mature granulosa cells at E17.5, these cells do not reprogram as Sertoli cells in XY triple mutant fetuses.
Fig 4: Absence of seminiferous tubules in XY and XX Rspo1KO Sox8KO Sox9cKO triple mutant adult mice.External genitalia from adult P40 mice (a–c, p–r), macroscopic view of gonads (d–f, s–u) (Scale bars 1.5 mm), histology as revealed by PAS staining on gonadal sections (g–i, v–x) (Scale bars 100 µm), and immunostaining of SOX9 (Sertoli cell marker, in red) (j–l, y–a'), FOXL2 (granulosa cell marker, in green) (j–o, y–d'), DMRT1 (Sertoli and germ cell marker, in red) (m–o, b'–d'), TRA98 (germ cell marker, in white) (m–o, b'–d'), and DAPI (nuclear marker, in blue) (j–o, y–d') on gonadal sections (Scale bars 50 µm). As shown, though adult XY Rspo1-/-; Sox9fl/fl; Sf1:creTg/+ (Rspo1KO Sox9cKO) double mutant gonads developed a short anogenital distance (b), internally these mice developed hypoplastic testes (compare e with d). XX Rspo1KO Sox9cKO gonads developed as ovotestes (t), as in XX Rspo1KO single mutants and XX Rspo1KO Sox8KO double mutants (see Figure 2r, q). Although Sox9fl/fl is inactivated by Sf1:creTg/+ in Rspo1KO Sox9cKO mice (k, z), XY double mutant gonads exhibited seminiferous tubules (h) containing DMRT1-positive Sertoli cells which are TRA98-negative (n), as in control testes (g, m). XX double mutant gonads contained an ovarian compartment ‘O’ with follicles and a testicular ‘T’ compartment with seminiferous tubule-like structures, as indicated by black arrowheads (w). The seminiferous tubule-like structures contained DMRT1-positive Sertoli cells (c'), as in control testes (m), but lacked TRA98-positive germ cells (c'). Both the XY and XX Rspo1KO Sox8KO Sox9cKO triple mutant mice develop externally as female with a short anogenital distance (c, p) as in the double mutants and control female (b, q–r). Despite this, the triple mutants gonads (f, s) developed as atrophied ovaries (i, v), which were smaller than control ovaries (u, x). XY and XX triple mutant gonads exhibited an ovarian ‘O’ compartment and a distinct interstitial compartment, as indicated by asterisks (i, v). The gonads contained follicles up to the antral stage, though some exhibited irregular granulosa cell organization, as indicated by blue arrowheads (i, v). Notably, XY and XX triple mutants lacked testicular sex cords (i, v) that were present in XY and XX Rspo1KO Sox9cKO gonads (h, w). Immunostaining on XY and XX Rspo1KO Sox8KO Sox9cKO gonads confirmed the absence of SOX9 and DMRT1 positive Sertoli cells and the presence of ovarian follicles with granulosa cells expressing FOXL2 (l, o, y, b'), as in control ovaries (a', d'). For these analyses, n = 3 XY and n = 3 XX triple mutant mice were examined.
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