Fgf9 inhibition of meiotic differentiation in spermatogonia is mediated by Erk-dependent activation of Nodal-Smad2/3 signaling and is antagonized by Kit Ligand

Both fibroblast growth factor 9 (Fgf9) and Kit Ligand (Kl) signal through tyrosine kinase receptors, yet they exert opposite effects on meiotic differentiation in postnatal spermatogonia, Fgf9 acting as a meiosis-inhibiting substance and Kl acting as a promoter of the differentiation process. To understand the molecular mechanisms that might underlie this difference, we tried to dissect the intracellular signaling elicited by these two growth factors. We found that both Fgf9 and Kl stimulate Erk1/2 activation in Kit+ (differentiating) spermatogonia, even though with different time courses, whereas Kl, but not Fgf9, elicits activation of the Pi3k-Akt pathway. Sustained Erk1/2 activity promoted by Fgf9 is required for induction of the autocrine Cripto-Nodal-Smad2/3 signaling loop in these cells. Nodal signaling, in turn, is essential to mediate Fgf9 suppression of the meiotic program, including inhibition of Stra8 and Scp3 expression and induction of the meiotic gatekeeper Nanos2. On the contrary, sustained activation of the Pi3k-Akt pathway is required for the induction of Stra8 expression elicited by Kl and retinoic acid. Moreover, we found that Kl treatment impairs Nodal mRNA expression and Fgf9-mediated Nanos2 induction, reinforcing the antagonistic effect of these two growth factors on the meiotic fate of male germ cells.In the mouse testis, competence to enter meiosis is acquired during the differentiative stages in which spermatogonia undergo Kit-dependent mitotic divisions, but not in Kit-negative spermatogonial stem cells. Retinoic acid (RA) stimulates Kit expression in spermatogonia and Kit Ligand (Kl) expression in Sertoli cells.¹ Kl is essential to promote the mitotic expansion of Kit+ premeiotic germ cells.² However, the concerted action of both RA and Kl can induce meiotic entry of in vitro cultured Kit+ spermatogonia.¹ Indeed, both RA and Kl increase the expression of genes that are fundamental for the beginning of the meiotic process,^{1, 3} and in particular of Stimulated by Retinoic Acid Gene 8 (Stra8), which is essential for the switch from the mitotic cell cycle to the meiotic program.¹ Selective inhibitors of Kit tyrosine-kinase activity block both RA- and Kl-induced meiotic entry, suggesting that the two factors converge on common, Kit-dependent, signaling pathways.¹ RA- and Kl-induced meiotic entry is mediated, at least in part, by activation of the Pi3k-Akt pathway. RA promotes meiosis also by inhibiting the expression of the meiotic gatekeeper Nanos2, an RNA-binding protein that silences genes essential for spermatogonial differentiation and meiotic entry.⁴ Fibroblast growth factor 9 (Fgf9) secreted by Sertoli cells acts as a meiosis-inhibiting substance by increasing Nanos2 levels in premeiotic spermatogonia, thus opposing the RA/Kl/Kit axis.⁴ In the male fetal gonad, meiotic entry of male fetal gonocytes is inhibited by the action of Cyp26b1, which degrades RA of mesonephric origin.^{5, 6} Fgf9 contributes to prevent meiosis in the male fetal gonad by inducing Nanos2 expression in gonocytes.^{4, 7} Several recent works have shown that Nodal (a Tgfβ superfamily member) plays an important role in the inhibition of the meiotic program of fetal male germ cells.^{8, 9, 10} Moreover, it has been demonstrated that the Fgf9 inhibitory effect on meiotic entry of fetal gonocytes might be mediated, at least in part, by activation of Cripto-Nodal signaling.⁹In the present work, we show that the Fgf9 antimeiotic effect is mediated by activation of the Cripto-Nodal-Smad2/3 signaling in postnatal spermatogonia, and that this activation requires Fgf9-dependent stimulation of the Erk1/2 pathway. On the contrary, Kl promotes meiotic differentiation through the activation of the Pi3k-Akt pathway and inhibition of Nodal expression.