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

Tassinari V, Campolo F, Cesarini V, Todaro F, Dolci S, Rossi P - Cell Death Dis (2015)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biomedicina e Prevenzione, Università degli Studi di Roma Tor Vergata, Rome, Italy.

ABSTRACT
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.

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Fgf9-mediated meiotic inhibition in Kit+ spermatogonia is mediated by activation of Nodal signaling. (a) Quantitative RT-PCR analysis of Cripto and Nodal mRNA expression in untreated Kit+ spermatogonia and in the same cells treated overnight with Kl or Fgf9. Data represent the mean±S.D. from five independent experiments. (b) Representative western blot analysis of Smad2 phosphorylation and Cripto expression in Kit+ control spermatogonia and in the same cells treated overnight with Fgf9. (c) Quantitative RT-PCR analysis of Cripto mRNA expression in untreated Kit+ spermatogonia and in the same cells treated overnight with Kl or with Fgf9, in the presence or absence of the Alk4/7 selective inhibitor SB431542. Data represent the mean±S.D. from four independent experiments. (d) Quantitative RT-PCR analysis of Nodal mRNA expression in untreated Kit+ spermatogonia and in the same cells treated overnight the Alk4/7 selective inhibitor SB431542. Data represent the mean±S.D. from three independent experiments. (e) Western blot analysis of Smad2 phosphorylation and expression of meiotic markers (Stra8 and Scp3) in cultured untreated Kit+ spermatogonia and in the same cells treated overnight with Fgf9, in the presence or absence of the Alk4/7 selective inhibitor SB431542. Densitometric analysis of western blots from four independent experiments is shown on the right. Bars represent the mean±S.D.
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fig4: Fgf9-mediated meiotic inhibition in Kit+ spermatogonia is mediated by activation of Nodal signaling. (a) Quantitative RT-PCR analysis of Cripto and Nodal mRNA expression in untreated Kit+ spermatogonia and in the same cells treated overnight with Kl or Fgf9. Data represent the mean±S.D. from five independent experiments. (b) Representative western blot analysis of Smad2 phosphorylation and Cripto expression in Kit+ control spermatogonia and in the same cells treated overnight with Fgf9. (c) Quantitative RT-PCR analysis of Cripto mRNA expression in untreated Kit+ spermatogonia and in the same cells treated overnight with Kl or with Fgf9, in the presence or absence of the Alk4/7 selective inhibitor SB431542. Data represent the mean±S.D. from four independent experiments. (d) Quantitative RT-PCR analysis of Nodal mRNA expression in untreated Kit+ spermatogonia and in the same cells treated overnight the Alk4/7 selective inhibitor SB431542. Data represent the mean±S.D. from three independent experiments. (e) Western blot analysis of Smad2 phosphorylation and expression of meiotic markers (Stra8 and Scp3) in cultured untreated Kit+ spermatogonia and in the same cells treated overnight with Fgf9, in the presence or absence of the Alk4/7 selective inhibitor SB431542. Densitometric analysis of western blots from four independent experiments is shown on the right. Bars represent the mean±S.D.

Mentions: As Fgf9 treatment for 24 h inhibits meiosis both in fetal and postnatal male germ cells in vitro,4, 7 we investigated whether this stimulation might influence Cripto and/or Nodal levels. To dissect the mechanisms of anti-meiotic effect of Fgf9, we focused on Kit+ spermatogonia. The peculiarity of these cells is that they can proliferate or enter the meiotic process in vivo and in vitro depending on the environmental stimuli, whereas Kit- cells are not able to enter meiosis before the onset of Kit expression.1, 4, 19 As shown in Figures 4a and b, Fgf9 induced a strong increase of Cripto levels both at mRNA and protein levels, together with the induction of Smad2 phosphorylation (Figure 4b), while Nodal expression was unaffected (Figure 4a). On the contrary, Kl did not enhance Cripto mRNA levels, but it inhibited Nodal expression (Figure 4a). Inhibition of Nodal signaling by SB431542 treatment completely blocked Fgf9 induction of Cripto expression (Figure 4c) and decreased endogenous Nodal mRNA levels (Figure 4d). These results demonstrate the existence of a positive Cripto/Nodal autocrine loop in postnatal spermatogonia. Activation of the Cripto/Nodal signaling pathway was required, at least in part, for mediating Fgf9 anti-meiotic effects in Kit+ spermatogonia, as suggested by the full recovery of Stra8 and Scp3 expression following the addition of SB431542 to Fgf9-treated cells (Figure 4e).


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

Tassinari V, Campolo F, Cesarini V, Todaro F, Dolci S, Rossi P - Cell Death Dis (2015)

Fgf9-mediated meiotic inhibition in Kit+ spermatogonia is mediated by activation of Nodal signaling. (a) Quantitative RT-PCR analysis of Cripto and Nodal mRNA expression in untreated Kit+ spermatogonia and in the same cells treated overnight with Kl or Fgf9. Data represent the mean±S.D. from five independent experiments. (b) Representative western blot analysis of Smad2 phosphorylation and Cripto expression in Kit+ control spermatogonia and in the same cells treated overnight with Fgf9. (c) Quantitative RT-PCR analysis of Cripto mRNA expression in untreated Kit+ spermatogonia and in the same cells treated overnight with Kl or with Fgf9, in the presence or absence of the Alk4/7 selective inhibitor SB431542. Data represent the mean±S.D. from four independent experiments. (d) Quantitative RT-PCR analysis of Nodal mRNA expression in untreated Kit+ spermatogonia and in the same cells treated overnight the Alk4/7 selective inhibitor SB431542. Data represent the mean±S.D. from three independent experiments. (e) Western blot analysis of Smad2 phosphorylation and expression of meiotic markers (Stra8 and Scp3) in cultured untreated Kit+ spermatogonia and in the same cells treated overnight with Fgf9, in the presence or absence of the Alk4/7 selective inhibitor SB431542. Densitometric analysis of western blots from four independent experiments is shown on the right. Bars represent the mean±S.D.
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fig4: Fgf9-mediated meiotic inhibition in Kit+ spermatogonia is mediated by activation of Nodal signaling. (a) Quantitative RT-PCR analysis of Cripto and Nodal mRNA expression in untreated Kit+ spermatogonia and in the same cells treated overnight with Kl or Fgf9. Data represent the mean±S.D. from five independent experiments. (b) Representative western blot analysis of Smad2 phosphorylation and Cripto expression in Kit+ control spermatogonia and in the same cells treated overnight with Fgf9. (c) Quantitative RT-PCR analysis of Cripto mRNA expression in untreated Kit+ spermatogonia and in the same cells treated overnight with Kl or with Fgf9, in the presence or absence of the Alk4/7 selective inhibitor SB431542. Data represent the mean±S.D. from four independent experiments. (d) Quantitative RT-PCR analysis of Nodal mRNA expression in untreated Kit+ spermatogonia and in the same cells treated overnight the Alk4/7 selective inhibitor SB431542. Data represent the mean±S.D. from three independent experiments. (e) Western blot analysis of Smad2 phosphorylation and expression of meiotic markers (Stra8 and Scp3) in cultured untreated Kit+ spermatogonia and in the same cells treated overnight with Fgf9, in the presence or absence of the Alk4/7 selective inhibitor SB431542. Densitometric analysis of western blots from four independent experiments is shown on the right. Bars represent the mean±S.D.
Mentions: As Fgf9 treatment for 24 h inhibits meiosis both in fetal and postnatal male germ cells in vitro,4, 7 we investigated whether this stimulation might influence Cripto and/or Nodal levels. To dissect the mechanisms of anti-meiotic effect of Fgf9, we focused on Kit+ spermatogonia. The peculiarity of these cells is that they can proliferate or enter the meiotic process in vivo and in vitro depending on the environmental stimuli, whereas Kit- cells are not able to enter meiosis before the onset of Kit expression.1, 4, 19 As shown in Figures 4a and b, Fgf9 induced a strong increase of Cripto levels both at mRNA and protein levels, together with the induction of Smad2 phosphorylation (Figure 4b), while Nodal expression was unaffected (Figure 4a). On the contrary, Kl did not enhance Cripto mRNA levels, but it inhibited Nodal expression (Figure 4a). Inhibition of Nodal signaling by SB431542 treatment completely blocked Fgf9 induction of Cripto expression (Figure 4c) and decreased endogenous Nodal mRNA levels (Figure 4d). These results demonstrate the existence of a positive Cripto/Nodal autocrine loop in postnatal spermatogonia. Activation of the Cripto/Nodal signaling pathway was required, at least in part, for mediating Fgf9 anti-meiotic effects in Kit+ spermatogonia, as suggested by the full recovery of Stra8 and Scp3 expression following the addition of SB431542 to Fgf9-treated cells (Figure 4e).

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Biomedicina e Prevenzione, Università degli Studi di Roma Tor Vergata, Rome, Italy.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus