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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-induced increase of Nanos2 expression depends on Nodal signaling and is inhibited by Kl. (a) Left panel: Semiquantitative RT-PCR analysis of mRNA expression of Nanos2 in untreated Kit+ spermatogonia and in the same cells cultured overnight with Fgf9, in the presence or absence of the Alk4/7 selective inhibitor SB431542. In this experiment, we used Nanos2 primers '1' listed in Supplementary Table 1. Right panel: Semiquantitative RT-PCR analysis of mRNA expression of Nanos2 in untreated Kit+ spermatogonia and in the same cells cultured overnight with Kl, with Fgf9 or with both growth factors. In this experiment, we used Nanos2 primers ‘2' listed in Supplementary Table 1. For both panels, the results of densitometric analysis from three independent experiments is shown below. Bars represent the mean ±S.D. (b) Schematic representation which summarizes the main findings of our present work: Fgf9 maintains differentiating (Kit+) spermatogonia in the mitotic condition and prevent meiosis (Stra8 and Scp3 expression) through a cascade involving sustained Erks activation and the consequent stimulation of the Cripto-Nodal-pSmad2/3 signaling, which leads to expression of the meiotic gatekeeper Nanos2. On the opposite, retinoic acid (RA) and Kl/Kit signaling inhibit Nodal expression and stimulate Pi3k-Akt signaling, which is required for Stra8 and Scp3 expression, with the consequent mitotic-meiotic switch
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fig6: Fgf9-induced increase of Nanos2 expression depends on Nodal signaling and is inhibited by Kl. (a) Left panel: Semiquantitative RT-PCR analysis of mRNA expression of Nanos2 in untreated Kit+ spermatogonia and in the same cells cultured overnight with Fgf9, in the presence or absence of the Alk4/7 selective inhibitor SB431542. In this experiment, we used Nanos2 primers '1' listed in Supplementary Table 1. Right panel: Semiquantitative RT-PCR analysis of mRNA expression of Nanos2 in untreated Kit+ spermatogonia and in the same cells cultured overnight with Kl, with Fgf9 or with both growth factors. In this experiment, we used Nanos2 primers ‘2' listed in Supplementary Table 1. For both panels, the results of densitometric analysis from three independent experiments is shown below. Bars represent the mean ±S.D. (b) Schematic representation which summarizes the main findings of our present work: Fgf9 maintains differentiating (Kit+) spermatogonia in the mitotic condition and prevent meiosis (Stra8 and Scp3 expression) through a cascade involving sustained Erks activation and the consequent stimulation of the Cripto-Nodal-pSmad2/3 signaling, which leads to expression of the meiotic gatekeeper Nanos2. On the opposite, retinoic acid (RA) and Kl/Kit signaling inhibit Nodal expression and stimulate Pi3k-Akt signaling, which is required for Stra8 and Scp3 expression, with the consequent mitotic-meiotic switch

Mentions: One of the downstream effectors of Fgf9 anti-meiotic activity is the upregulation of the RNA-binding protein Nanos2.4, 7 We confirmed that Fgf9 treatment increases Nanos2 expression in Kit+ spermatogonia, and found that this effect also required Nodal activity, as it was abolished by SB431542 treatment (Figure 6a, left panel and Supplementary Figure 3A). Interestingly, Kl co-treatment strongly decreased the Fgf9-mediated induction of Nanos2 mRNA levels (Figure 6a, right panel and Supplementary Figure 3B).


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-induced increase of Nanos2 expression depends on Nodal signaling and is inhibited by Kl. (a) Left panel: Semiquantitative RT-PCR analysis of mRNA expression of Nanos2 in untreated Kit+ spermatogonia and in the same cells cultured overnight with Fgf9, in the presence or absence of the Alk4/7 selective inhibitor SB431542. In this experiment, we used Nanos2 primers '1' listed in Supplementary Table 1. Right panel: Semiquantitative RT-PCR analysis of mRNA expression of Nanos2 in untreated Kit+ spermatogonia and in the same cells cultured overnight with Kl, with Fgf9 or with both growth factors. In this experiment, we used Nanos2 primers ‘2' listed in Supplementary Table 1. For both panels, the results of densitometric analysis from three independent experiments is shown below. Bars represent the mean ±S.D. (b) Schematic representation which summarizes the main findings of our present work: Fgf9 maintains differentiating (Kit+) spermatogonia in the mitotic condition and prevent meiosis (Stra8 and Scp3 expression) through a cascade involving sustained Erks activation and the consequent stimulation of the Cripto-Nodal-pSmad2/3 signaling, which leads to expression of the meiotic gatekeeper Nanos2. On the opposite, retinoic acid (RA) and Kl/Kit signaling inhibit Nodal expression and stimulate Pi3k-Akt signaling, which is required for Stra8 and Scp3 expression, with the consequent mitotic-meiotic switch
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385934&req=5

fig6: Fgf9-induced increase of Nanos2 expression depends on Nodal signaling and is inhibited by Kl. (a) Left panel: Semiquantitative RT-PCR analysis of mRNA expression of Nanos2 in untreated Kit+ spermatogonia and in the same cells cultured overnight with Fgf9, in the presence or absence of the Alk4/7 selective inhibitor SB431542. In this experiment, we used Nanos2 primers '1' listed in Supplementary Table 1. Right panel: Semiquantitative RT-PCR analysis of mRNA expression of Nanos2 in untreated Kit+ spermatogonia and in the same cells cultured overnight with Kl, with Fgf9 or with both growth factors. In this experiment, we used Nanos2 primers ‘2' listed in Supplementary Table 1. For both panels, the results of densitometric analysis from three independent experiments is shown below. Bars represent the mean ±S.D. (b) Schematic representation which summarizes the main findings of our present work: Fgf9 maintains differentiating (Kit+) spermatogonia in the mitotic condition and prevent meiosis (Stra8 and Scp3 expression) through a cascade involving sustained Erks activation and the consequent stimulation of the Cripto-Nodal-pSmad2/3 signaling, which leads to expression of the meiotic gatekeeper Nanos2. On the opposite, retinoic acid (RA) and Kl/Kit signaling inhibit Nodal expression and stimulate Pi3k-Akt signaling, which is required for Stra8 and Scp3 expression, with the consequent mitotic-meiotic switch
Mentions: One of the downstream effectors of Fgf9 anti-meiotic activity is the upregulation of the RNA-binding protein Nanos2.4, 7 We confirmed that Fgf9 treatment increases Nanos2 expression in Kit+ spermatogonia, and found that this effect also required Nodal activity, as it was abolished by SB431542 treatment (Figure 6a, left panel and Supplementary Figure 3A). Interestingly, Kl co-treatment strongly decreased the Fgf9-mediated induction of Nanos2 mRNA levels (Figure 6a, right panel and Supplementary Figure 3B).

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