PROJECT SUMMARY Mitochondria are increasingly recognized as key players in a wide range of cellular and developmental processes, such as cell signaling, stem cell self-renewal, and lineage commitment, beyond their canonical roles in metabolism and oxidative phosphorylation. Such functional diversity is largely determined by the cell type- specific mitochondrial features, including mitochondrial number, architecture, distribution, and interaction with other subcellular organelles. These mitochondrial features are tightly regulated via mitochondrial fusion and fission, collectively known as mitochondrial dynamics, the alteration of which will lead to changing mitochondrial functions and activities in a cell type-specific manner, and thus impacts cell fate decision, particularly during development and in stem cell differentiation. We found that upregulated mitochondrial respiration accompanied by increased reactive oxygen species (ROS) is required for postnatal spermatogonial stem cell differentiation. However, such elevated ROS can increase mitochondrial DNA mutations that are deleterious to mitochondrial fitness and cell functions. It remains elusive how stem cells properly balance mitochondrial activities to meet the competing need for increased mitochondrial respiration during differentiation while maintaining mitochondrial fitness. Our pilot data suggest that mitochondria fusion and fission are both upregulated in spermatogonial differentiation, which is essential for sustaining proper male fertility. We thus propose to reveal a novel functional mechanism of how spermatogonial differentiation and germ cell mitochondrial fitness are regulated by concurrently accelerated and properly balanced mitochondrial fusion and fission. To achieve this goal, we will integrate a series of genetically modified mouse models with in vitro spermatogonial differentiation and transplantation approaches. Study findings will fundamentally advance research in both reproductive medicine and mitochondrial biology by explaining how spermatogonial differentiation is regulated via stage-specific mitofusion and fission, thereby unlocking a new area of discovery, namely, how mitochondrial function and health are maintained so in order to support critical and unique events of mammalian development. In addition, by revealing the impacts of mitochondrial dynamics on germ cell mitochondrial fitness, this project will critically inform a novel strategy to treat impaired male fertility due to mitochondrial dysfunctions.