Open camera or QR reader and scan code to access this article and other resources online. Antioxidant Effect of Melatonin on Proliferation, Apoptosis, and Oxidative Stress Variables in Frozen-Thawed Neonatal Mice Spermatogonial Stem Cells Tahoora Nazeri, 1 Azim Hedayatpour, 2 Shokoofeh Kazemzadeh, 2 Mahmoud Safari, 2 Samiullah Safi, 2 and Maryam Khanehzad 3 Cryopreservation of spermatogonial stem cells (SSCs) is an important method to restore and maintain fertility in preadolescent children suffering from cancer. For protection of SSCs from cryoinjury, various antioxidant agents have been used. The aim of this study was to assess the antiapoptotic and antioxidant effects of mela- tonin in frozen-thawed SSCs. SSCs were isolated from testes of neonatal mice (3–6 days old) and their purities were measured by flow cytometry with promyelocytic leukemia zinc finger protein. After culturing, the cells were frozen in two groups (1) control and (2) melatonin (100 mM) and stored for 1 month. Finally, the cell via- bility, colonization rate, expression of Bcl-2 and BAX gene, and intracellular reactive oxygen species (ROS) were evaluated after freezing-thawing. Melatonin increased the viability and colonization of SSCs and Bcl-2 gene expression. It also diminished BAX gene expression and intracellular ROS. The results of this study show that melatonin with antioxidant and antiapoptotic effects can be used as an additive for freezing and long-term storage of cells and infertility treatment in the clinic. Keywords: melatonin, antioxidant, spermatogonial stem cells, apoptosis Introduction T reatment success has recently increased considerably in young boys who have cancer before puberty and receive high doses of chemotherapy and radiotherapy. Since spermatogonial stem cells (SSCs) are very sensitive to the toxic effects of chemotherapy and radiotherapy agents, 35% of the children with cancer become infertile later in life. 1,2 Therefore, it is necessary to use methods to restore and main- tain fertility in these children. Cryopreservation is one of the most common methods. However, since children, contrary to adults, have no sperm for cryopreservation, it seems that extraction of SSCs from the testis tissue before treatment, cryopreservation, and long-term preservation of these cells and finally transplantation of SSCs after recovery can initi- ate spermatogenesis, sperm production, and fertility in these patients. 3–5 Spermatogenesis is a highly coordinated and complex pro- cess that starts immediately after puberty in the seminiferous tubules with the aim of correct and accurate transfer of genetic and epigenetic information to the next generation and contin- ues throughout life. 6,7 Initiation and continuation of sperma- togenesis are controlled and protected by self-renewal and differentiation of SSCs. SSCs are undifferentiated cells that, in addition to the ability of self-renewal and differentiation into daughter cells, have a significant role in producing the next generation and transferring genetic information to it, and are therefore considered unique cells among human stem cells. 8–10 1 Department of Biology, Islamic Azad University of SariBranch, Mazandaran, Iran. 2 Department of Anatomy, School of Medicine, Tehran University of Medical Science, Tehran, Iran. 3 Department of Anatomical Sciences and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran. BIOPRESERVATION AND BIOBANKING Volume 20, Number 4, 2022 ª Mary Ann Liebert, Inc. DOI: 10.1089/bio.2021.0128 374 Downloaded by 51.15.78.81 from www.liebertpub.com at 09/24/22. For personal use only.