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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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Expression and activity of the Cripto-Nodal signaling pathway in postnatal mouse spermatogonia. (a) Semiquantitative RT-PCR analysis of mRNA expression of the indicated elements of the Cripto-Nodal signaling pathway in Kit+ and Kit- spermatogonia. (b) Western blot analysis of Smad2 phosphorylation in cultured untreated Kit+ spermatogonia and in the same cells treated overnight with recombinant Nodal, in the presence or absence of the Alk4/7 selective inhibitor SB431542. Densitometric analysis of western blots from three independent experiments is shown below. Bars represent the mean ±S.D.
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fig3: Expression and activity of the Cripto-Nodal signaling pathway in postnatal mouse spermatogonia. (a) Semiquantitative RT-PCR analysis of mRNA expression of the indicated elements of the Cripto-Nodal signaling pathway in Kit+ and Kit- spermatogonia. (b) Western blot analysis of Smad2 phosphorylation in cultured untreated Kit+ spermatogonia and in the same cells treated overnight with recombinant Nodal, in the presence or absence of the Alk4/7 selective inhibitor SB431542. Densitometric analysis of western blots from three independent experiments is shown below. Bars represent the mean ±S.D.

Mentions: It has been recently reported that Nodal expression in male fetal germ cells autocrinally prevents them to enter meiosis8 and that Fgf9 induces Cripto, a co-receptor for Nodal, in the same cell type.9 To investigate whether the Cripto/Nodal system was active also in postnatal germ cells, we first analyzed the expression pattern of members of the Nodal signaling pathway. We found that Alk4, ActRIIB and Lefty2 transcripts were expressed in both Kit+ and Kit- spermatogonia, whereas Alk7 and Lefty1 were completely absent (Figure 3a). Cripto and Nodal transcripts were expressed at higher levels in Kit- spermatogonia. Following 1h incubation with 100 ng/ml Nodal, pSmad2 levels were rapidly induced in Kit+ spermatogonia and completely abolished by co-treatment with SB431542, a specific Alk4/5/7 inhibitor (Figure 3b). This result indicates that the Cripto/Nodal system can be potentially activated in premeiotic male germ cells.


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)

Expression and activity of the Cripto-Nodal signaling pathway in postnatal mouse spermatogonia. (a) Semiquantitative RT-PCR analysis of mRNA expression of the indicated elements of the Cripto-Nodal signaling pathway in Kit+ and Kit- spermatogonia. (b) Western blot analysis of Smad2 phosphorylation in cultured untreated Kit+ spermatogonia and in the same cells treated overnight with recombinant Nodal, in the presence or absence of the Alk4/7 selective inhibitor SB431542. Densitometric analysis of western blots from three independent experiments is shown below. Bars represent the mean ±S.D.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Expression and activity of the Cripto-Nodal signaling pathway in postnatal mouse spermatogonia. (a) Semiquantitative RT-PCR analysis of mRNA expression of the indicated elements of the Cripto-Nodal signaling pathway in Kit+ and Kit- spermatogonia. (b) Western blot analysis of Smad2 phosphorylation in cultured untreated Kit+ spermatogonia and in the same cells treated overnight with recombinant Nodal, in the presence or absence of the Alk4/7 selective inhibitor SB431542. Densitometric analysis of western blots from three independent experiments is shown below. Bars represent the mean ±S.D.
Mentions: It has been recently reported that Nodal expression in male fetal germ cells autocrinally prevents them to enter meiosis8 and that Fgf9 induces Cripto, a co-receptor for Nodal, in the same cell type.9 To investigate whether the Cripto/Nodal system was active also in postnatal germ cells, we first analyzed the expression pattern of members of the Nodal signaling pathway. We found that Alk4, ActRIIB and Lefty2 transcripts were expressed in both Kit+ and Kit- spermatogonia, whereas Alk7 and Lefty1 were completely absent (Figure 3a). Cripto and Nodal transcripts were expressed at higher levels in Kit- spermatogonia. Following 1h incubation with 100 ng/ml Nodal, pSmad2 levels were rapidly induced in Kit+ spermatogonia and completely abolished by co-treatment with SB431542, a specific Alk4/5/7 inhibitor (Figure 3b). This result indicates that the Cripto/Nodal system can be potentially activated in premeiotic male germ cells.

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