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Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines
Published on: February 25, 2013
SPRY4-dependent ERK negative feedback demarcates functional adult stem cells in the male mouse germline†
Yanyun Luo1, Makiko Yamada1, Thierry N'Tumba-Byn1
1Department of Surgery, Weill Cornell Medicine, New York, NY, USA.
Abstract:
Niche-derived growth factors support self-renewal of mouse spermatogonial stem and progenitor cells through ERK MAPK signaling and other pathways. At the same time, dysregulated growth factor-dependent signaling has been associated with loss of stem cell activity and aberrant differentiation. We hypothesized that growth factor signaling through the ERK MAPK pathway in spermatogonial stem cells is tightly regulated within a narrow range through distinct intracellular negative feedback regulators. Evaluation of candidate extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK)-responsive genes known to dampen downstream signaling revealed robust induction of specific negative feedback regulators, including Spry4, in cultured mouse spermatogonial stem cells in response to glial cell line-derived neurotrophic factor or fibroblast growth factor 2. Undifferentiated spermatogonia in vivo exhibited high levels of Spry4 mRNA. Quantitative single-cell analysis of ERK MAPK signaling in spermatogonial stem cell cultures revealed both dynamic signaling patterns in response to growth factors and disruption of such effects when Spry4 was ablated, due to dysregulation of ERK MAPK downstream of RAS. Whereas negative feedback regulator expression decreased during differentiation, loss of Spry4 shifted cell fate toward early differentiation with concomitant loss of stem cell activity. Finally, a mouse Spry4 reporter line revealed that the adult spermatogonial stem cell population in vivo is demarcated by strong Spry4 promoter activity. Collectively, our data suggest that negative feedback-dependent regulation of ERK MAPK is critical for preservation of spermatogonial stem cell fate within the mammalian testis.
Insights
Negative feedback regulators, like Spry4, tightly control ERK MAPK signaling essential for maintaining mouse spermatogonial stem cell self-renewal and preventing premature differentiation.
Area of Science:
- Reproductive biology
- Stem cell biology
- Molecular signaling
Background:
- Growth factors regulate spermatogonial stem cell (SSC) self-renewal via ERK MAPK signaling.
- Dysregulated signaling can lead to stem cell loss and abnormal differentiation.
Purpose of the Study:
- To investigate the role of intracellular negative feedback regulators in controlling ERK MAPK signaling in SSCs.
- To determine if Spry4 is a key regulator of SSC fate.
Main Methods:
- Cultured mouse SSCs treated with growth factors (GDNF, FGF2).
- Quantitative single-cell analysis of ERK MAPK signaling.
- Spry4 gene ablation in SSCs.
- Analysis of a Spry4 reporter mouse line.
Main Results:
- Spry4 expression was induced by growth factors in cultured SSCs and high in undifferentiated spermatogonia in vivo.
- Spry4 ablation disrupted ERK MAPK signaling and promoted SSC differentiation.
- Spry4 promoter activity marked adult SSC populations in vivo.
Conclusions:
- Negative feedback regulation of ERK MAPK signaling by Spry4 is crucial for maintaining SSC fate.
- Spry4 preserves SSC self-renewal and prevents aberrant differentiation in the mammalian testis.
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