Ascorbic acid regulates mouse spermatogonial stem cell proliferation in a Wnt/β-catenin/ROS signaling dependent manner

Spermatogonial stem cells (SSCs) provide a foundation for spermatogenesis, but the mechanism of SSC proliferation is still poorly understood. To investigate whether and how ascorbic acid (AA) regulates the growth of mouse SSCs in vitro, the SSCs were cultured in different concentration AA medium for 14 days. The proliferation, apoptosis and the reactive oxygen species (ROS) levels of SSCs in different AA groups were respectively detected. Moreover, the SSC activity in 40 μg/mL AA group and the control was tested by a transplantation assay. To explore the mechanism of AA regulating mouse SSCs proliferation, the dishevelled homolog 2 (DVL2) and nucleoredoxin (NRX) protein levels, the expression of axis inhibition protein 2 (Axin2), leucine-rich G-protein coupled receptor 5 (Lgr5), B-cell lymphoma-2 (Bcl-2), BCL2-Associated X (Bax), c-myc and cyclin D1 genes in Wnt/β-catenin pathway were respectively confirmed. The results showed that the adding concentration of AA did not affect the main shape of SSCs. A 40 μg/mL AA in culture medium promoted the proliferation, and decreased the ROS production and apoptosis rate of SSCs. Moreover, colonization efficiency in the seminiferous tubules of the recipient testis in 40 μg/mL AA group was higher compared with the control group by a transplantation assay. Finally, the appropriate ROS in the 40 μg/mL AA group further adjust the levels of DVL2 and NRX protein in the Wnt/β-catenin pathway to maintain the nuclear intensity of β-catenin, in turn, the expression of apoptosis gene Bax decreased, while the expression of Bcl2, Axin2, Lgr5, c-myc and cyclin D1 genes increased. The study confirmed that AA adjusts the endogenous ROS level to impact on SSC proliferation in a dose-dependent manner by Wnt/β-catenin signaling pathway.