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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
Restoration of Spermatogenesis is Dependent on Activation of a SPRY4-ERK Checkpoint Following Germline Stem Cell
Ying Liu1,2, Tansol Choi1, Brad Pearson1,2
1Department of Surgery, Weill Cornell Medicine, New York, NY, USA.
Abstract:
Mammalian spermatogonial stem cells (SSCs) sustain male fertility through continuous self-renewal and differentiation, leading to the production of haploid spermatozoa throughout adulthood. However, SSCs are vulnerable to genotoxic drugs, and patients receiving chemotherapy face a high risk of germline instability and infertility. The molecular mechanisms and cellular pathways that choreograph SSC recovery after chemotherapeutic insult remain unknown. Previously, we identified SPRY4 as an ERK-dependent negative feedback regulator of growth factor signaling that is critical for preservation of stem cell activity in cultured mouse SSCs. Here, we demonstrate that following alkylating agent busulfan (BU)-induced injury, germline-specific Spry4 gene deletion (Spry4 G-KO) reduces stem cell regeneration but promotes differentiation with rapidly enhanced nuclear ERK1/2 activity in undifferentiated (Aundiff) spermatogonia (including SSCs) in adult mice. Genes essential for stem cell maintenance, including Id1 and Cxcl12, were dysregulated by loss of Spry4. Furthermore, the MEK1/2 inhibitor PD0325901, but not mTORC1 inhibitor Rapamycin, was sufficient to promote spermatogonial proliferation in Spry4 G-KO testis 10 days post-BU treatment. Notably, the restoration of both spermatogonia pool and fertility was delayed in adult Spry4 G-KO males long-term after injury. In summary, germline-specific deletion of Spry4 results in hyper-activation of the MAPK/ERK pathway in Aundiff spermatogonia, unleashing excessive spermatogenesis after germline damage, and ultimately impairing germline regeneration in adult males. Our study indicates an essential role for SPRY4-ERK signaling as a molecular checkpoint in securing SSC recovery upon chemotherapy drug-induced germline damage, revealing how stem cells normally withstand environmental stress.
Insights
Deleting the SPRY4 gene in male mice impairs spermatogonial stem cell (SSC) recovery after chemotherapy. This leads to hyperactivated ERK signaling, excessive differentiation, and delayed fertility restoration, highlighting SPRY4
Area of Science:
- Reproductive Biology
- Stem Cell Biology
- Molecular Signaling
Background:
- Mammalian spermatogonial stem cells (SSCs) are crucial for male fertility, maintaining sperm production through self-renewal and differentiation.
- Chemotherapy can induce genotoxicity, leading to germline instability and infertility due to SSC vulnerability.
- The mechanisms governing SSC recovery after chemotherapy-induced damage are not fully understood.
Purpose of the Study:
- To investigate the role of SPRY4 (SPRY domain-containing protein 4) in SSC recovery following chemotherapy.
- To elucidate the molecular pathways, particularly MAPK/ERK signaling, involved in SSC response to genotoxic injury.
Main Methods:
- Utilized germline-specific Spry4 gene deletion (Spry4 G-KO) in adult mice.
- Administered busulfan (BU), an alkylating agent, to induce germline injury.
- Assessed SSC regeneration, differentiation, ERK1/2 activity, and gene expression post-treatment.
- Investigated the effects of MEK1/2 and mTORC1 inhibitors on spermatogonial proliferation.
Main Results:
- Germline-specific Spry4 deletion reduced SSC regeneration but increased differentiation post-BU injury.
- Spry4 deletion led to enhanced nuclear ERK1/2 activity in undifferentiated spermatogonia.
- Genes critical for stem cell maintenance (Id1, Cxcl12) were dysregulated in Spry4 G-KO mice.
- MEK1/2 inhibition promoted spermatogonial proliferation in damaged Spry4 G-KO testes, while Rapamycin did not.
- Spermatogonia pool and fertility restoration were significantly delayed in Spry4 G-KO males long-term after injury.
Conclusions:
- SPRY4 acts as a critical negative feedback regulator in the ERK pathway, essential for SSC recovery after chemotherapy.
- Germline deletion of Spry4 results in MAPK/ERK pathway hyperactivation, impairing long-term germline regeneration.
- SPRY4-ERK signaling is a key molecular checkpoint for protecting SSCs against chemotherapy-induced stress and ensuring fertility restoration.
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