The PGD2 pathway, independently of FGF9, amplifies SOX9 activity in Sertoli cells during male sexual differentiation

Abstract

Activation by the Y-encoded testis determining factor SRY and maintenance of expression of the Sox9 gene encoding the central transcription factor of Sertoli cell differentiation are key events in the mammalian sexual differentiation program. In the mouse XY gonad, SOX9 upregulates Fgf9, which initiates a Sox9/Fgf9 feedforward loop, and Sox9 expression is stimulated by the prostaglandin D2 (PGD2) producing lipocalin prostaglandin D synthase (L-PGDS, or PTDGS) enzyme, which accelerates commitment to the male pathway. In an attempt to decipher the genetic relationships between Sox9 and the L-Pgds/PGD2 pathway during mouse testicular organogenesis, we found that ablation of Sox9 at the onset or during the time window of expression in embryonic Sertoli cells abolished L-Pgds transcription. By contrast, L-Pgds(-/-) XY embryonic gonads displayed a reduced level of Sox9 transcript and aberrant SOX9 protein subcellular localization. In this study, we demonstrated genetically that the L-Pgds/PGD2 pathway acts as a second amplification loop of Sox9 expression. Moreover, examination of Fgf9(-/-) and L-Pgds(-/-) XY embryonic gonads demonstrated that the two Sox9 gene activity amplifying pathways work independently. These data suggest that, once activated and maintained by SOX9, production of testicular L-PGDS leads to the accumulation of PGD2, which in turn activates Sox9 transcription and nuclear translocation of SOX9. This mechanism participates together with FGF9 as an amplification system of Sox9 gene expression and activity during mammalian testicular organogenesis.

Fig. 1.

Confocal analysis of co-immunofluorescence experiments on XY 12.5 dpc gonads. Top: frozen sections were stained for L-PGDS (red), together with AMH (left) or VASA (right, green), and analyzed by laser confocal microscopy. Bottom: Hoechst staining (HST, blue) labeled nuclei. Sections (250-500 nm) were routinely visualized and panels that are shown represent a unique z-section. Arrowheads highlight L-PGDS and AMH overlapping staining and arrows indicate L-PGDS within the cytoplasm of germ cells. Scale bars: 10μ m.

Fig. 2.

Initiation of L-Pgds expression is dependent on SOX9. (A) Real-time RT-PCR analysis of L-Pgds expression in 11.5 dpc wild-type male (XY^+/+), female (XX^+/+) and Ck19-Cre; Sox9^flox/flox mutant male (XY^-/-) gonads. Datasets (n=5) were normalized to Gapdh expression and averaged. Normalized relative expression revealed that L-Pgds expression is completely abolished in mutant (XY^-/-) compared with wild-type (XY^+/+) gonads (^*, P value <0.05). Error bars indicate s.d. of triplicate experiments. (B) mRNA in situ hybridization for L-Pgds (left) and germ cell marker Oct4 (right) on frozen sections from wild-type (Sox9^+/+, top) and mutant (Sox9^-/-, bottom) 12.0 dpc male gonads. L-Pgds antisense riboprobe revealed that L-Pgds expression within the testis cords (arrows) of wild-type (Sox9^+/+) gonads is abolished in mutant (Sox9^-/-) gonads, whereas Oct4 expression is similar in both Sox9 genotypes. Scale bars: 50 μm.

Fig. 3.

Maintenance of L-Pgds expression is dependent on SOX9. (A-H) mRNA in situ hybridization for L-Pgds was performed on 13.5 dpc (A,C) and 14.5 dpc (E,G) gonads from wild-type (A,E) and Amh-Cre; Sox9^flox/flox mutant (C,G) XY embryos. In parallel to L-Pgds in situ hybridization, co-immunofluorescence staining for SOX9 (green) and AMH (red) was performed on wild-type (B) and mutant (D) 13.5 dpc XY gonads, and wild-type (F) and mutant (H) 14.5 dpc XY gonads. TC, testis cords. Scale bars: 75 μm in A,C,E,G; 50μ m in B,D,F,H. (I) Real-time RT-PCR analysis of L-Pgds and Sox9 expression in 15.5 dpc wild-type male (XY^+/+), female (XX^+/+) and Amh-Cre; Sox9^flox/flox mutant male (XY^-/-) gonads. Normalized relative expression revealed that L-Pgds and Sox9 expression is significantly reduced in mutant (XY^-/-) compared with wild-type (XY^+/+) gonads (^***, P<0.01). Error bars indicate s.d. of triplicate experiments.

Fig. 4.

L-Pgds expression is independent of FGF9 signaling. (A) mRNA in situ hybridization for L-Pgds (left) was performed on frozen sections from wild-type (Fgf9^+/+, top) and mutant (Fgf9^-/-, bottom) 12 dpc male gonads. Following L-Pgds in situ hybridization, the sections were submitted to immunofluorescence for SOX9 (red) and L-Pgds in situ hybridization is represented in green (right). Arrows highlight SOX9 and L-Pgds expressing cells. g, gonad; m, mesonephros. Scale bars: 50 μm in left, 10 μm in right. (B) Real-time RT-PCR analysis of Fgf9 expression was performed on 12.5 dpc male heterozygous (XY^+/-) and homozygous (XY^-/-) L-Pgds mutant gonads. For each point, datasets (n=5) were normalized to Gapdh expression and averaged. Normalized relative expression shows that Fgf9 expression is similar in both genotypes. Error bars indicate s.d. of triplicate experiments. (C) Real-time RT-PCR analysis of Sox9 expression was performed on NT2/D1 cells treated with PGD2, FGF9, PGD2+FGF9 or with vehicle (ethanol, nt) for 20-120 minutes. Datasets (n=5) were normalized to 18S expression and averaged. Normalized Sox9 relative expression revealed that FGF9 and PGD2 signaling molecules cooperate to additively upregulate expression of Sox9.

Fig. 5.

Sertoli cell differentiation and testis cord formation is delayed in the absence of L-Pgds. (A) Seven to nine L-Pgds^-/- male gonads were analyzed at developmental stages 11.5 dpc, 12.5 dpc, 13.5 dpc and 14.5 dpc in terms of their SOX9 phenotype (SOX9 subcellular localization and number of SOX9-expressing cells) by immunofluorescence. `Normal' (gray) corresponds to a phenotype with exclusively nuclear SOX9 and a number of SOX9-expressing cells identical to that observed in L-Pgds<sup>+/-</sup> gonads. `Abnormal' (black) corresponds to the presence of cytoplasmic and nuclear SOX9 and/or a number of SOX9-expressing cells decreased by 25% compared with that observed in L-Pgds^+/- gonads. (B,C) Immunofluorescence of frozen sections from 11.5 dpc and 12.5 dpc L-Pgds^+/- (top) and L-Pgds^-/- (bottom) male gonads, stained for SOX9 (red; B,C) and laminin (green, C), and with Hoechst dye (HST, blue; B) to label nuclei. In B, enlarged panels (right) highlight cytoplasmic SOX9 (arrows) in L-Pgds^-/- XY gonads compared with exclusive nuclear SOX9 (asterisks) in L-Pgds^+/- gonads. In C, SOX9 and laminin staining revealed a low level of SOX9 expression and impaired testis cord formation (long arrows) in L-Pgds^-/- gonads, compared with L-Pgds^+/- gonads. Short arrows indicate SOX9-positive cells lying outside testis cords and TC indicates completely formed testis cords. Scale bars: 50 μm in B (left), 20 μm in B (right), 50 μm in C.

Fig. 6.

Phenotype of 13.5 dpc XY L-Pgds^-/- gonads. (A) Sagittal cryosections of 13.5 dpc XY L-Pgds^+/- (left) and L-Pgds^-/- (right) gonads were submitted for SOX9 (red) and laminin (green) co-immunofluorescence. Long arrows show impaired testis cord organization, short arrows indicate SOX9-positive cells lying outside testis cords in L-Pgds^-/- gonads, normal testis cords are highlighted by TC. (B) Two 13.5 dpc XY L-Pgds^-/- gonads (KO1, left and KO2, right) with different abnormal phenotypes are shown. Frozen sections were stained for SOX9 (red) and laminin (left) or tubulin (right, green). Top: XY L-Pgds^+/- gonads. Asterisks and arrows indicate nuclear and cytoplasmic SOX9, respectively. Bottom panels are enlarged from middle panels. Scale bars: 100 μm in A, 50 μm in B (KO1), 30 μm in B (KO2).

Fig. 7.

Quantification of Sox9 and Amh transcript levels in L-Pgds^-/- gonads and phenotype of 17.5 dpc L-Pgds^-/- gonads. (A,B,D) Relative expression of Sox9 (black bars; A,D) and Amh (gray bars; B,D) mRNA in male (XY^+/-) and female (XX^+/-) heterozygous and in male homozyous (XY^-/-) mutant gonads collected from 12.5 dpc and 17.5 dpc embryos, normalized to Gapdh mRNA levels. Error bars indicate s.d. of triplicate experiments performed on 10 individual gonads. (^*, P<0.05 in A,B and ^***, P<0.025 in D). (C) SOX9 immunostaining (red) was performed on frozen sections from 17.5 dpc L-Pgds^+/- (top) and L-Pgds^-/- (bottom) male gonads. Testis cords (TC) are highlighted by dashed lines. Scale bars: 50 μm.