Apoptotic inhibitor Y-27632 improves post-thaw viability and stemness of sheep spermatogonial stem cells

Spermatogonial stem cells (SSC) are of high significance in animal reproduction, breeding, and regenerative medicine. Cryopreservation of putative SSC is a prerequisite for long-term storage and future applications. However, dissociated putative SSC are very susceptible to cryostress and undergo apoptosis, thereby reducing their functional competence. To minimize the dissociation-induced apoptosis during cryopreservation and post-thaw culture, the role of anti-apoptotic molecule, Y-27632, was evaluated in sheep putative SSC.

The putative SSC were isolated, enriched, and cultured from prepubertal sheep testis. Y-27632 (0, 5,10, and 15 µM) was added in putative SSC cryomedia (Cy group) and in both freezing and post-thaw culture media (Cy + y group). The effects were compared with non-treated control (C group). One month after cryopreservation, the putative SSC were assessed for viability, ROS production, metabolic activity, stemness associated undifferentiated status, colony characteristics, and apoptosis associated pathway/genes.

The viability of the dissociated post-thaw putative SSC was significantly higher > 90% when cryopreserved in presence of Y-27632, compared to control (C; 82.5 ± 1.7%). The metabolic activity and stemness associated undifferentiated status were also significantly (p < 0.05) improved in 10 and 15 Cy + y as compared to C. ROCK1 and p53 expression was significantly (p < 0.05) downregulated in 10 Cy + y compared to the C. Also, a significantly (p < 0.01) higher apoptotic cells were observed in C in comparison to 10 Cy and 10 Cy + y (3.9 ± 0.8% vs 1.9 ± 0.2% and 0.35 ± 0.05%).

These findings suggest that 10 µM Y-27632, when added to cryomedia and post-thaw culture, reduces apoptosis and thereby improves viability of cells during cryopreservation. It also helps in maintaining undifferentiated status and metabolic activity of sheep putative SSC.