The Cryopreservation Effect of Melatonin on Neonatal Mouse Spermatogonial Stem Cells, Increasing Proliferation and Decreasing Apoptosis

Abstract

Background: Considering the increasing infertility rate and the key role of spermatogonia stem cells (SSCS) in initiating spermatogenesis and maintaining fertility, trying to minimize the damage caused by long-term storage techniques of SSCs is considered as one of the important topics in basic research and clinic. Therefore, this study was designed to investigate the effect of melatonin on neonatal mouse spermatogonial stem cells following freezing and thawing. Methods: Spermatogonial stem cells were isolated from neonatal mouse 3-5 days old BALB / C after enzymatic digestion and identified with IDA marker by flow cytometry. After culture, the cells were frozen in two groups:1) control (basal cryopreservation) and 2) 100 μM melatonin in basal cryopreservation for one month. After freezing-thawing process, the expression level of the apoptotic protein Caspase-3 and the expression of the proliferative protein Gfra1in the cells were evaluated. The significance level was considered to be P ≤ 0.05. Findings: After freezing-thawing process, melatonin significantly reduced the expression of the apoptotic protein Caspase-3. Also, a significant increase in the expression of Gfra1protein was reported in the presence of melatonin compared to the control group. Conclusion: The results of this study indicate that melatonin as a cryoprotectant by reducing apoptosis and increasing proliferation of spermatogonial stem cells can be a suitable option for cryopreservation and long-term storage of cells and infertility treatment in the clinic. © 2026, Isfahan University of Medical Sciences(IUMS). All rights reserved.