Abstract
Cryopreservation of male germline stem cells (GSCs) is an essential technique for their long-term preservation and utilization in various fields. However, the specific apoptosis pathways involved in cryoinjury during freezing remain unclear. Therefore, our study sought to identify the pathways involved in cryoinjury-induced apoptosis and thereby to improve freezing efficiency during GSC cryopreservation through the creation of a specific molecular-based cryoprotectant. The activities of caspase-8, caspase-9, caspase-3, and caspase-7 were assessed by Western blot analyses to determine the role of specific apoptosis pathways in GSC cryoinjury. Specifically, the role of a specific caspase was identified by recovery rate, relative proliferation rate, Annexin V/propidium iodide co-staining, and caspase activity using its inhibitor and activator. Moreover, the safety of the cryoprotectant was assessed by immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, the efficacy of the molecular-based cryoprotectant was assessed using frozen cells in the presence of dimethyl sulfoxide (DMSO) (control), trehalose, a caspase-8 inhibitor Z-IETD-FMK [ZIF], or a mixture of the aforementioned compounds, after which the changes in Src signaling were measured. Our results demonstrated that caspase-8 plays a major role in cryoinjury-induced apoptosis and therefore its inhibition improves freezing efficiency. Specifically, a significantly higher relative proliferation rate was observed in the Z-IETD-FMK 0.01 μM-treated cells than in the DMSO control (100% ± 6.2% vs. 189.8% ± 9.5%), with decreases in both early apoptosis (16.6% ± 2.2% vs. 7.5% ± 1.0%) and caspase-8 activity (1.0-fold vs. 0.4-fold). The relative proliferation rate was significantly higher in the cryoprotectant mixture (246.0% ± 12.2%) than other individual treatment groups (trehalose 200 mM, 189.8% ± 9.5%; Z-IETD-FMK 0.01 μM, 189.7% ± 2.2%) with no significant differences in Src signaling. Therefore, our findings provide novel insights into the development of freezing protocols to enhance GSC freezing efficiency, thereby facilitating the wider adoption of GSCs in the livestock industry and/or clinical trials.