Abstract
Male differentiation of primordial germ cells (PGCs) is initiated by the inhibition of entry into meiosis and exposure to male-inducing factor(s), which are regulated by somatic elements of the developing gonad. Fibroblast growth factor 9 (FGF9) produced by pre-Sertoli cells is essential for male gonadal differentiation and also contributes to survival and male differentiation of XY PGCs. However, it is not clear how FGF9 regulates PGC fate. Using a PGC culture system, we identified dose-dependent, fate-determining functions of FGF9 in XY PGCs. Treatment with low levels of FGF9 (0.2 ng/ml) increased expression of male-specific Dnmt3L and Nanos2 in XY PGCs. Conversely, treatment with high levels of FGF9 (25 ng/ml) suppressed male-specific gene expression and stimulated proliferation of XY PGCs. Western blotting showed that low FGF9 treatment enhanced p38 MAPK (mitogen-activated protein kinase) phosphorylation in the same cells. In contrast, high FGF9 treatment significantly stimulated the ERK (extracellular signal-regulated kinase)1/2 signaling pathway in XY PGCs. We investigated the relationship between the ERK1/2 signaling pathway stimulated by high FGF9 and regulation of PGC proliferation. An ERK1/2 inhibitor (U0126) suppressed the PGC proliferation that would otherwise be stimulated by high FGF9 treatment, and increased Nanos2 expression in XY PGCs. Conversely, a p38 MAPK inhibitor (SB202190) significantly suppressed Nanos2 expression that would otherwise be stimulated by low FGF9 in XY PGCs. Taken together, our results suggest that stage-specific expression of FGF9 in XY gonads regulates the balance between proliferation and differentiation of XY PGCs in a dose-dependent manner.
Highlights
The sex-specific phenotypic fate of mammalian primordial germ cells (PGCs) is induced extrinsically by the gonadal environment rather than intrinsically by sex chromosome constitution (XX, XY) [1]
To clarify direct effects of Fibroblast growth factor 9 (FGF9) on male differentiation of PGCs, 12.5 dpc XY PGCs were cultured with different concentrations of FGF9 (0, 0.2, 1, 5, 25, and 100 ng/ml) for 2 days and male-specific Dnmt3l mRNA expression was examined (Figure 1A)
In the presence of 25 ng/ml FGF9 Dnmt3l expression was significantly suppressed in XY PGCs to a level less than half that seen in the control (P < 0.05) (Figure 1A)
Summary
The sex-specific phenotypic fate of mammalian primordial germ cells (PGCs) is induced extrinsically by the gonadal environment rather than intrinsically by sex chromosome constitution (XX, XY) [1]. PGCs initiate differentiation as either prospermatogonia or oogonia, depending on the sexual phenotype of the surrounding gonadal somatic cells [2]. Wingless-related MMTV integration site (Wnt) signaling and retinoic acid (RA) are essential for oogenesis. Wnt signaling promotes female differentiation and suppresses male differentiation pathway [3,4,5]. Retinoic acid signaling plays an essential role in meiotic initiation in female gonads. Expression of sex determining region Y (Sry) occurs in Sertoli cell precursors to upregulate Sox (Sry-box containing gene 9) and fibroblast growth factor 9 (Fgf9) to promote Sertoli cell differentiation and testis development [10,11,12,13,14]
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