Necrostatin-1 improves the cryopreservation efficiency of murine spermatogonial stem cells via suppression of necroptosis and apoptosis.

Cryopreservation of spermatogonial stem cells (SSCs) is important to preserve the lineages of valuable livestock and produce transgenic animals. Although interest in molecular-based cryopreservation methods have been increasing to improve their efficiency, the issue of necroptosis has not yet been considered. Therefore, the purpose of this study was to understand the role of necroptosis using necrostatin-1 (Nec-1), necroptosis inhibitor, in SSC cryopreservation, and to investigate the potential application of Nec-1 as a cryoprotectant. To determine the cryopreservation efficiency of Nec-1, we assessed recovery rate, proliferation potential, cellular membrane damage, RIP1 protein expression, apoptosis, and its mechanism. Stable characterization and functional activity of SSCs was determined via immunofluorescence, RT-qPCR, and in vivo transplantation of SSCs. Our results showed a higher proliferation potential in 50 μM Nec-1 (146.5 ± 16.8%) than in DMSO controls (100.0 ± 3.4%). Furthermore, the cryoprotective effects of Nec-1 were verified by a decrease in RIP1 expression (3.1 ± 0.2-fold vs. 1.3 ± 0.3-fold) and in early apoptosis (4.3 ± 0.8% vs. 2.6 ± 0.1%) compared to DMSO controls. Normal functional activity was observed in the SSCs after cryopreservation with 50 μM Nec-1. In conclusion, necroptosis could be a cause of cryoinjury, and their inhibitor may serve as potential effective cryoprotectant. This study will contribute to establish a molecular-based cryopreservation method, and thereby expanding the use of SSCs into the domestic livestock industry as well as for clinical applications.