Signaling through the TGF beta-activin receptors ALK4/5/7 regulates testis formation and male germ cell development

Abstract

The developing testis provides an environment that nurtures germ cell development, ultimately ensuring spermatogenesis and fertility. Impacts on this environment are considered to underlie aberrant germ cell development and formation of germ cell tumour precursors. The signaling events involved in testis formation and male fetal germ cell development remain largely unknown. Analysis of knockout mice lacking single Tgfβ family members has indicated that Tgfβ's are not required for sex determination. However, due to functional redundancy, it is possible that additional functions for these ligands in gonad development remain to be discovered. Using FACS purified gonadal cells, in this study we show that the genes encoding Activin's, TGFβ's, Nodal and their respective receptors, are expressed in sex and cell type specific patterns suggesting particular roles in testis and germ cell development. Inhibition of signaling through the receptors ALK4, ALK5 and ALK7, and ALK5 alone, demonstrated that TGFβ signaling is required for testis cord formation during the critical testis-determining period. We also show that signaling through the Activin/NODAL receptors, ALK4 and ALK7 is required for promoting differentiation of male germ cells and their entry into mitotic arrest. Finally, our data demonstrate that Nodal is specifically expressed in male germ cells and expression of the key pluripotency gene, Nanog was significantly reduced when signaling through ALK4/5/7 was blocked. Our strategy of inhibiting multiple Activin/NODAL/TGFβ receptors reduces the functional redundancy between these signaling pathways, thereby revealing new and essential roles for TGFβ and Activin signaling during testis formation and male germ cell development.

Figure 1. Inhba, Inhbb, Tgfb2 and Tgfb3 are expressed in the somatic cells, whilst Nodal is expressed in the germ cells of the developing gonad.

Quantitative real time PCR analysis of: A) ligands Tgfb1, Tgfb2, Tgfb3; B) type I and II Tgf receptors Alk5 and Tgfbr2; C) ligands Inhba, Inhbb and Nodal; D) Activin/Nodal type I receptors Alk4, Alk7, and type II receptors Acvr2a, Acvr2b in FACS purified E12.5–E15.5 XY male germ cells (light blue) and XX female germ cells (light pink) male somatic cells (dark blue) and female somatic cells (dark pink). Expression was normalized to Mapk1 and Canx. Error bars represent standard error of three biological replicates.

Figure 2. E11.5 XY gonads cultured in the presence of ALK4/5/7 inhibitor (SB431542) or ALK5 inhibitor (ALK5i-I) exhibit disrupted testis cord formation.

A) E11.5 (gonad/mesonephos) and E12.5 (gonad only) XY and XX gonad samples cultured for 24 hours in the presence of DMSO, SB431542 or ALK5i-I and analysed using immunoblotting with antibodies specific for SMAD2 and phosphorylated SMAD2 (P-SMAD2). B) Bright field and GFP images of XY and XX gonads collected from E11.5 Oct4-GFP transgenic embryos and cultured for 72 hours in the presence of DMSO, SB431542 or ALK5i-I. C) Immunofluorescence analysis of E11.5 XY and XX gonads cultured for 72 hours in the presence of DMSO, SB431542 or ALK5i-I. SOX9 staining is in red, while MVH (green) staining marks the germ cells. DAPI (blue) marks the nucleus. Scale bars; 200 μm.

Figure 3. Inhibition of ALK4/5/7 decreases Sertoli cell proliferation in E12.5 fetal testes.

A) Bright field and GFP images of male testes collected from E12.5 Oct4-GFP transgenic embryos and cultured in the presence of DMSO, SB431542 or ALK5i-I for 72 hours. Arrows indicate well-developed testis cords marked by GFP positive germ cells in DMSO treated controls. Arrow-heads indicate stunted testis cords marked by GFP positive germ cells in SB431542 treated testes. B) Immunofluorescence analysis of the same testes using SOX9 and MVH antibodies. SOX9 staining is in red, while MVH (green) staining marks the germ cells. DAPI (blue) is included as a nuclear marker. Scale bars; 50 μm. C–D) Flow cytometric analysis of somatic cell (MVH negative cells, Figure 5a) and Sertoli cell (SOX9 positive cells) proliferation based on EdU incorporation of E12.5 testes cultured for 72 hours with DMSO (n = 10, MVH negative cells; n = 3 SOX9 positive cells), ALK5i-I (n = 3) or SB431542 (n = 10, MVH negative cells; n = 3 SOX9 positive cells). In C and D the error bars represent standard error. ** indicates significant difference compared to DMSO (p<0.01). E) Typical example of a flow cytometric analysis of Sertoli cells in E12.5 testes cultured for 72 hours with DMSO or SB431542. SOX9 positive germ cells were gated from somatic cells (top panels) and analysed for active proliferation by measuring incorporation of EdU during S-phase (bottom panels).

Figure 4. Blocking ALK4/5/7 or ALK5 in E11.5 XY gonads, but not E12.5 testes, permits germ cell entry into meiosis.

Immunofluorescence analysis of: A–C) E11.5 XY and XX gonads cultured for 72 hours or 96 hours in the presence of DMSO, SB431542 or ALK5i-I. STRA8 (A), SYCP3 (B) and γH2AX-P (C) staining is in red, while MVH or DPPA4 (green) staining marks the germ cells. DAPI (blue) is included as a nuclear marker. Scale bars; 200 μm. D) TUNEL staining of E11.5 gonads cultured for 72 hours in the presence of DMSO or SB431542. Red staining marks TUNEL positive cells, while germ cells are marked by DPPA4 staining in green. DAPI (blue) is included as a nuclear marker. Scale bars; 200 μm. E) E12.5 testes cultured for 72 hours in the presence of DMSO or SB431542. γH2AX-P staining is in red, while MVH (green) staining marks the germ cells. DAPI (blue) is included as a nuclear marker. Scale bars 50 μm.

Figure 5. Inhibition of ALK4/5/7 disrupts germ cell cycle arrest in E12.5 testes.

A) Typical example of a flow cytometric analysis of germ cells from E12.5 testes cultured for 72 hours with DMSO or SB431542. MVH positive germ cells were gated from somatic cells (left panels) and analysed for active proliferation by measuring incorporation of EdU during S-phase (middle panels) and for cell cycle stage by measuring DNA content (right panels). B–C) E12.5 testes treated with DMSO (n = 10), ALK5i-I (n = 3) or SB431542 (n = 10) for 72 hours B) Germ cell proliferation based on EdU incorporation C) ModFit analysis of germ cell cycle state, based on propidium iodide staining for DNA content. Error bars represent standard error. D–F) Immunofluorescence analysis of E12.5 testes cultured for 72 hours in the presence of DMSO or the ALK4/5/7 inhibitor SB431542. D) p27^KIP1 (red); E. KI67 (red); F) phosphorylated RB (RB-P) (red). In D–F MVH staining (green) marks the germ cells while DAPI (blue) marks nuclear DNA. Scale bars; 20 μm.

Figure 6. Inhibition of ALK4/5/7 inhibits male fetal germ cell differentiation.

A) qRTPCR analysis of Nodal and Lefty1, and the male germ cell differentiation markers Dppa4 and Dnmt3l in E12.5 testes cultured for 72 hours with DMSO or SB431542. All of these genes are specifically expressed by the germ cells. Relative expression (± SEM, n = 3) was calculated after normalization to Mapk1 and Canx . B–C) Immunofluorescence analysis of E12.5 testes cultured for 72 hours in the presence of DMSO or SB431542. B) DPPA4 is in red, KI67 is in Pacific Blue and MVH is in green. In the SB431542 treated images the arrows indicate germ cells that express low levels of DPPA4 and remain proliferative (KI67 positive), while the arrow-heads indicate germ cells that express high levels of DPPA4 and have exited the cell cycle (KI67 negative); C) MILI (red, left panel); FGFR3 (red right panel); In C–D MVH staining (green) marks the germ cells while DAPI (blue) marks nuclear DNA. Scale bars; 20 μm. D) qRTPCR analysis of Nodal and the pluripotency markers Nanog and Sox2 in E12.5 testes cultured for 72 hours with DMSO or SB431542. Relative expression (± SEM, n = 3) was calculated after normalization to Mapk1 and Canx. E) Immunofluorescence analysis of E12.5 testes cultured for 72 hours in the presence of DMSO or SB431542. SOX2 (red, left panel); OCT4 (red right panel). MVH staining (green) marks the germ cells. DAPI (blue) marks nuclear DNA. Scale bars; 20 μm.

Figure 7. TGFβ/Activin/NODAL signaling in the developing testis.

A–B) TGFβ2and TGFβ3 signaling produced from somatic (Sertoli, Leydig or other) cells, regulates testis cord organization through ALK5, while Activin promotes Sertoli cell proliferation through ALK4/7. Male germ cell development and mitotic arrest is promoted primarily by Activin through ALK4/7. (A, B). Activin signaling through ALK4/7 regulates somatic and germ cell development directly (A) or may indirectly lead regulate germ cell differentiation and mitotic arrest by directing somatic cell differentiation and the production of other, unknown ligands by the somatic cells (B). NODAL produced from germ cells positively regulates Nanog expression in germ cells and thereby regulates germ cell pluripotency (A, B). In this model Activin and NODAL have opposing roles in germ cells, promoting and inhibiting differentiation, respectively.