FGF9 promotes mouse spermatogonial stem cell proliferation mediated by p38 MAPK signalling

Abstract

Objectives: Fibroblast growth factor 9 (FGF9) is expressed by somatic cells in the seminiferous tubules, yet little information exists about its role in regulating spermatogonial stem cells (SSCs).

Materials and methods: Fgf9 overexpression lentivirus was injected into mouse testes, and PLZF immunostaining was performed to investigate the effect of FGF9 on spermatogonia in vivo. Effect of FGF9 on SSCs was detected by transplanting cultured germ cells into tubules of testes. RNA-seq of bulk RNA and single cell was performed to explore FGF9 working mechanisms. SB203580 was used to disrupt p38 MAPK pathway. p38 MAPK protein expression was detected by Western blot and qPCR was performed to determine different gene expression. Small interfering RNA (siRNA) was used to knock down Etv5 gene expression in germ cells.

Results: Overexpression of Fgf9 in vivo resulted in arrested spermatogenesis and accumulation of undifferentiated spermatogonia. Exposure of germ cell cultures to FGF9 resulted in larger numbers of SSCs over time. Inhibition of p38 MAPK phosphorylation negated the SSC growth advantage provided by FGF9. Etv5 and Bcl6b gene expressions were enhanced by FGF9 treatment. Gene knockdown of Etv5 disrupted the growth effect of FGF9 in cultured SSCs along with downstream expression of Bcl6b.

Conclusions: Taken together, these data indicate that FGF9 is an important regulator of SSC proliferation, operating through p38 MAPK phosphorylation and upregulating Etv5 and Bcl6b in turn.

Keywords: fibroblast growth factor; niche; self-renewal; spermatogonial stem cell.

FIGURE 1

Effect of Fgf9 and Gdnf overexpression plasmids on mouse testes. A, Schematic of experimental design. Two lentiviral plasmids were created containing cDNA of Gdnf or Fgf9 under the control of the ubiquitous promoter EF1. 293T cells were transfected alongside the packaging vector with one of three treatments: Gdnf vector, Fgf9 vector or empty vector. Lentiviral particles were harvested from the 293T cells and injected into the seminiferous tubules of recipient mice. B, FGF9 IHC staining of seminiferous tubule sections from testes infected with overexpression plasmids for 11 wk. Negative control: sections without primary antibody. Scale bar: 50 µm (top) or 20 µm (bottom). C, 11 wk after injection, testes were collected and weighed (FGF9‐overexpression lentivirus injection, n = 5; GDNF overexpression lentivirus injection, n = 3). *P < .05, **P < .01, Student's t test. D, Histology and PLZF IHC staining of seminiferous tubule sections from testes infected with overexpression plasmids. Scale bar: 50 µm

FIGURE 2

Immunofluorescence of seminiferous tubules exposed to Fgf9 and Gdnf overexpression. A, Cross‐sections of seminiferous tubules stained with antibodies against PLZF (marker for undifferentiated spermatogonia), SYCP3 (essential for meiosis), DAPI (DNA stain) following injection of vector, Fgf9 and Gdnf overexpression plasmids in addition to both overexpression plasmids injected together. Scale bar: 50 µm. B, Whole‐mount staining for PLZF and both PLZF and DAPI of seminiferous tubules injected with vector, Fgf9 overexpression and Gdnf overexpression plasmids. Seminiferous epithelial cycle of all tubules showed here are stages II–VI as determined by transillumination technique. Scale bar: 50 µm

FIGURE 3

Effect of FGF9 on germ cell proliferation and SSC proliferation. A, Effect of FGF9 and FGF2 treatment on THY‐1⁺ germ cell number in culture. Cells were treated with GDNF, GFRα1 and listed concentration of FGF (n = 3). *P < .05, ANOVA of final cell numbers. B, Representative morphology of cultured THY‐1⁺ germ cell clumps after treatment for 7 days. Scale bar: 100 µm. C, Colony number per 10⁴ cultured THY‐1⁺ germ cells following transplantation (n = 3, 6 testes per replicate). *P < .05, ANOVA. D, SSC number following 2 and 4 wk in culture (n = 3). *P < .05, ANOVA of final cell numbers. E, Representative morphology of receipt testes which were transplanted with germ cells after treatment for 4 weeks. Scale bar: 5 mm. All error bars show SEM

FIGURE 4

Regulation of p38 MAPK signalling by FGF9 in germ cells. A, Top signalling pathways from Ingenuity Pathway Analysis (Qiagen) of RNA‐seq data. Germ cells were treated with GDNF, GFRα1 and FGFs for 7 d. Then, cells were harvested for RNA‐seq (n = 3). Pathways ordered by 20ng/mL FGF9 P‐values. Scale shows ‐log P‐value and ‘+’denotes Z‐score of > 1 and ‘‐’ a Z‐score of <−1. B, Effect of SB203580 on germ cell number. Cultured THY‐1⁺ germ cells (ie, heterogeneous cultures of cells including stem cells and spermatogonia) were grown for 7 d in mSFM with GDNF and GFRα1. Cells were additionally treated with 5 μmol/L of SB203580, 20 ng/mL FGF9, neither or both. Percentages show proportional reduction of cell number after 7 d (n = 3). *P < .05, Student's t test between proportional germ cell loss. C, Representative morphology of cultured THY‐1⁺ germ cell clumps after treatment for 7 d. Scale bar: 100 µm. D, Colony number per 10⁴ cultured THY‐1⁺ germ cells following transplantation (n = 4, 5 testes per replicate). *P < .05, ANOVA. E, SSC number following 1 wk in culture. Percentages show proportional reduction of cell number after 7 d (n = 4, 5 testes per replicate). *P < .05, Student's t test between proportional stem cell losses. F, Protein expressions of phosphorylated‐p38 MAPK and p38 MAPK in germ cells with different treatments. Germ cells were cultured with FGF9 (40 ng/mL) for 2 d and protein was extracted for detection. G, Quantitation of phosphorylated‐p38 MAPK and p38 MAPK protein expressions in germ cells with different treatments (n = 3). *P < .05, Student's t test. All error bars show SEM

FIGURE 5

Single‐cell RNA‐sequencing analysis of FGF9 effect on cultured THY‐1⁺ germ cells in vitro. RNA‐sequencing analysis of 4,143 cells cultured for 48 h in mSFM with 20 ng/mL FGF9 integrated with 4482 control cells cultured with no growth factors for the same time. A, Cells clustered and scored for cell cycle expression (see Figure S7C). B, Clustering of cells after regressing out cell cycle genes for S and G₂M phases. C, Pseudotime trajectory. D, Assignment of unbiased clusters to cell identities indicated by gene expression profile (see Figure S7G for dying cells and STO). Note that ‘SSCs’ are used to designate the cluster containing SSCs, it is likely that not all cells are true SSCs. E, Module scores using sets of marker genes. For SSC: Gfra1, Ret, Etv5, Id4, Tspan8 and Esrp1. For progenitor spermatogonia: Upp1, Lhx1, Nanos3, Sox3 and Galnt12. For differentiating spermatogonia, Kit, Sohlh1, Crabp1 and Lmo1. F, Violin plots of gene expression by cell type and treatment. Three genes were selected as representative markers of gene expression for each of the three cell identities

FIGURE 6

Regulation of Etv5 by FGF9 in germ cells. A, Heat map of selected self‐renewal and differentiation genes. Scale shows log₂‐fold change. *P < .05, **P < .01, ***P < .001. B, qPCR expression values of Etv5 (n = 3). Starvation treatment: cells cultured in mSFM with no growth factors for 2 d. FGF9 treatment: cells treated with mSFM containing 20 ng/mL of FGF9 for 2 d. Withdraw treatment: cells cultured in mSFM with no growth factors for 2 d. **P < .01, ANOVA. C, qPCR analysis of Etv5 expression. Cells were cultured with FGF9 or FGF9 with SB203580 (30 μmol/L) for 3 d and then harvested for qPCR analysis (n = 3). **P < .01, Student's t test. D, Effect of Etv5 siRNA on germ cell number. After 24 h of transfection of Etv5 siRNA, cells were cultured with FGF9 for 6 d (n = 3). *P < .05, Student's t test. E, Representative morphology of cultured THY‐1⁺ germ cell clumps after treatment for 6 d. Scale bar: 100 µm. F, Colony number per 10⁴ cultured THY‐1⁺ germ cells following transplantation (n = 3, 4 testes per replicate). After 24 h of transfection of Etv5 siRNA, cells were cultured with FGF9 for 6 d and then transplanted into recipient animals. *P < .05, Student's t test. G, SSC number recovered from culture (n = 3, 4 testes per replicate). *P < .05, Student's t test. All error bars show SEM

FIGURE 7

Regulation of Bcl6b by Etv5 after FGF9 activation. A, qPCR expression values of Bcl6b (n = 3). Starvation treatment: cells cultured in mSFM with no growth factors for 2 d. FGF9 treatment: cells treated with mSFM containing 20 ng/mL of FGF9 for 2 d. Withdraw treatment: cells cultured in mSFM with no growth factors for 2 d. *P < .05, ANOVA. B, qPCR analysis of Etv5 and Bcl6b expressions in cultured THY‐1⁺ germ cells after 24 h of transfection with Etv5 siRNA (n = 3). *P < .05, Student's t test. C, qPCR analysis of Etv5 and Bcl6b expressions in cultured THY‐1⁺ germ cells. After 24 h of transfection of Etv5 siRNA, cells were starved for 2 d and treated with FGF9 for 2 d (n = 3). *P < .05, **P < .01, ANOVA. D, qPCR analysis of Bcl6b expression. Cells were cultured with FGF9 or FGF9 with SB203580 (30 μmol/L) for 3 d and then harvested for qPCR analysis (n = 3). **P < .01, Student's t test. All error bars show SEM

FIGURE 8

Diagram of proposed action of FGF9. A, Somatic cells provide growth factors to influence cell fate of SSCs. Sertoli cells supply GDNF and FGF2, Leydig cells provide CSF‐1 and FGF9, myoid cells provide GDNF and CSF‐1, blood vessels produce GDNF and FGF2 signalling occurs from differentiating germ cells as well. ² , ⁵⁶ B, FGF2 induces via FGF receptors (FGFR 1c, 3c > 2c, 1b, 4Δ) both p38 MAPK and ERK signalling pathways that independently lead to proliferation of SSCs via Etv5 upregulation. ⁹ , ³⁶ , ⁵¹ FGF9 also stimulates FGF receptors (FGFR 3c > 2c> 1c, 3b» 4Δ) with considerable overlap with FGF2, ³⁶ and we show in this study this leads to p38 MAPK phosphorylation, which activates Etv5 gene expression as well. After Etv5 activation, Bcl6 expression is upregulated, thereby regulating SSC proliferation. Simultaneously, FGF9 exposure inhibits differentiation, ultimately by affecting the expression of pro‐differentiation genes