Abstract

Background: Cryostorage of spermatogonial stem cells (SSCs) is an appropriate procedure for long-term storage of SSCs for fertility preservation. However, it causes damage to cellular structures through overproduction of ROS and oxidative stress. In this study, we examined the protective effect of melatonin as a potent antioxidant in the basic freezing medium to establish an optimal cryopreservation method for SSCs. Methods: SSCs were obtained from the testes of neonatal male mice aged 3–6 days. Then, 100 μM melatonin was added to the basic freezing medium containing DMSO for cryopreservation of SSCs. Viability, apoptosis-related markers (BAX and BCL2), and intracellular ROS generation level were measured in frozen–thawed SSCs before transplantation using the MTT assay, immunocytochemistry, and flow cytometry, respectively. In addition, Western blotting and immunofluorescence were used to evaluate the expression of proliferation (PLZF and GFRα1) and differentiation (Stra8 and SCP3) proteins in frozen–thawed SSCs after transplantation into recipient testes. Results: The data showed that adding melatonin to the cryopreservation medium markedly increased the viability and reduced intracellular ROS generation and apoptosis (by decreasing BAX and increasing BCL2) in the frozen–thawed SSCs (p < 0.05). The expression levels of proliferation (PLZF and GFRα1) and differentiation (Stra8 and SCP3) proteins and resumption of spermatogenesis from frozen–thawed SSCs followed the same pattern after transplantation. Conclusions: The results of this study revealed that adding melatonin as an antioxidant to the cryopreservation medium containing DMSO could be a promising strategy for cryopreservation of SSCs to maintain fertility in prepubertal male children who suffer from cancer. © 2022, The Author(s).