(P-424) Glutamine protects mouse spermatogonial stem cells against NOX1-derived ROS for sustaining self-renewal division

Abstract Authors: Takehiro Miyazaki¹, Mito Kanatsu-Shinohara¹, Narumi Ogonuki², Shogo Matoba²,

Atsuo Ogura², Chihiro Yabe-Nishimura³, Hongliang Zhang⁴, Yves Pommier⁴, Andreas Trumpp⁵, and Takashi Shinohara<sup>1



1</sup>Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto, Japan

²RIKEN, Bioresource Center, Tsukuba, Japan

³Deparment of Pharmacology, Kyoto Prefectural University of Medicine, Kyoto, Japan

⁴Deveopmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institute of Health, Bethesda, Maryland, USA

⁵Division of Stem Cells and Cancer, Deutsches Krebsforshungszentrum (DKFZ), Heidelberg, Germany

Abstract Text: 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 Mycexpression 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.