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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
Glutamine protects mouse spermatogonial stem cells against NOX1-derived ROS for sustaining self-renewal division in
Takehiro Miyazaki1, Mito Kanatsu-Shinohara1, Narumi Ogonuki2
1Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
Abstract:
Reactive oxygen species (ROS) are generated from NADPH oxidases and mitochondria; they are generally harmful for stem cells. Spermatogonial stem cells (SSCs) are unique among tissue-stem cells because they undergo ROS-dependent self-renewal via NOX1 activation. However, the mechanism by which SSCs are protected from ROS remains unknown. Here, we demonstrate a crucial role for Gln in ROS protection using cultured SSCs derived from immature testes. Measurements of amino acids required for SSC cultures revealed the indispensable role of Gln in SSC survival. Gln induced Myc expression to drive SSC self-renewal in vitro, whereas Gln deprivation triggered Trp53-dependent apoptosis and impaired SSC activity. However, apoptosis was attenuated in cultured SSCs that lacked NOX1. In contrast, cultured SSCs lacking Top1mt mitochondria-specific topoisomerase exhibited poor mitochondrial ROS production and underwent apoptosis. Gln deprivation reduced glutathione production; supra-molar Asn supplementation allowed offspring production from SSCs cultured without Gln. Therefore, Gln ensures ROS-dependent SSC-self-renewal by providing protection against NOX1 and inducing Myc.
Insights
Glutamine (Gln) protects unique spermatogonial stem cells (SSCs) from harmful reactive oxygen species (ROS) by inducing Myc and supporting self-renewal. Gln also provides protection against NOX1, crucial for SSC survival.
Area of Science:
- Stem cell biology
- Reproductive biology
- Cellular biochemistry
Background:
- Reactive oxygen species (ROS) are typically harmful to stem cells.
- Spermatogonial stem cells (SSCs) uniquely rely on ROS for self-renewal, mediated by NOX1.
- The protective mechanisms for SSCs against ROS remain largely unknown.
Purpose of the Study:
- To investigate the role of glutamine (Gln) in protecting spermatogonial stem cells (SSCs) from reactive oxygen species (ROS).
- To elucidate the molecular mechanisms underlying Gln-mediated ROS protection and SSC self-renewal.
Main Methods:
- Culturing of SSCs from immature testes.
- Amino acid measurements in SSC cultures.
- Analysis of gene expression (Myc, Trp53) and apoptosis.
- Assessment of mitochondrial ROS production and glutathione levels.
- Supplementation with asparagine (Asn) to evaluate rescue effects.
Main Results:
- Glutamine (Gln) is essential for SSC survival and self-renewal in vitro.
- Gln induces Myc expression, promoting SSC self-renewal.
- Gln deprivation leads to Trp53-dependent apoptosis, but this is reduced in NOX1-deficient SSCs.
- Mitochondrial dysfunction (lacking Top1mt) impairs ROS production and induces apoptosis.
- Gln deprivation decreases glutathione; Asn supplementation rescues SSC function and allows offspring production.
Conclusions:
- Glutamine (Gln) plays a critical role in protecting SSCs against ROS, ensuring their self-renewal.
- Gln supports ROS-dependent SSC self-renewal by providing protection against NOX1 and inducing Myc.
- Gln is indispensable for SSC survival, acting through pathways involving Myc, Trp53, and glutathione synthesis.
