Improving the Efficacy of Cryopreservation of Spermatogonia Stem Cells by Antioxidant Supplements

Abstract

Cryopreservation of spermatogonial stem cells (SSCs) is an applicable method for young males seeking fertility preservation before starting a treatment. It increases reactive oxygen species (ROS) formation and oxidative stress, which damages cellular structures. In this study, we added two antioxidants, catalase and α-tocopherol (α-TCP), to the basic freezing medium to evaluate their effects on the efficiency of SSCs. SSCs were isolated from testes of 3- to 6-day-old male mice using enzymatic digestion. The enrichment of isolated cells was evaluated by flow cytometry and Stra8 antibody. Catalase (40 μg/mL), or α-TCP (200 μg/mL) was added to the basic freezing medium. The cell viability was evaluated by the methylthiazoltetrazolium (MTT) assay. After thawing, cells were cultured for 1 month, and the expression pattern of specific genes of SSCs and the ability of the cells to restore spermatogenesis were used to determine the efficiency of the cryopreservation method. The survival rate of the frozen cells in the presence of catalase or α-TCP was significantly higher than the control group (p < 0.05). The number of colonies and their diameter measured after 1 month were significantly higher in the antioxidant groups than in the control group (p < 0.05). Gene expression and resumption of spermatogenesis also followed the same pattern. Thus, adding antioxidants to the basic freezing medium can be helpful in increasing the quality and viability of SSCs after cryopreservation. This new approach to stem cells cryopreservation can also be a promising strategy for fertility preservation in patients who suffer from malignancy.

The effect of combining vitamin E and C on the viability improvement of transfected ovine spermatogonial stem cells after cryopreservation and thawing

“…However, the procedure does not come without limitations, one of which is the inability to retrieve adequate numbers of functional SSCs following freezing (8). This reduction in the survival rate of SSCs may be caused by the accumulation of toxic products of metabolism and ROS formation (9).…”

“…The applied methods for cryopreservation of SSCs are similar to those of somatic cells (8). However, recent findings on SSC cryopreservation appear not to be satisfactory.…”

Optimization of <i>in vitro</i> culture and transfection condition of bovine primary spermatogonial stem cells

“…On the other hand, vitamin E, which is known as a natural antioxidant, reacts with soluble free radicals in lipid membranes and prevents the process of lipid peroxidation by scavenging reactive oxygen species before they can cause damage to the cells (Moreira et al, 2010). According to Aliakbari et al (2016), using α-tocopherol as a lipid soluble antioxidant not only increased SSCs viability up to 70-80%, but the apoptotic rates of cells were decreased. In addition, analysing the expression of Bax and Bcl2 genes after supplementing culture with antioxidants revealed a falling apoptosis rate (Aliakbari et al, 2016).…”

“…According to Aliakbari et al (2016), using α-tocopherol as a lipid soluble antioxidant not only increased SSCs viability up to 70-80%, but the apoptotic rates of cells were decreased. In addition, analysing the expression of Bax and Bcl2 genes after supplementing culture with antioxidants revealed a falling apoptosis rate (Aliakbari et al, 2016).…”

“…Derivatives of the vitamin E family are shown to have significant effects on cell signalling, regulation of gene expression, inflammation, cell proliferation, apoptosis, and scavenging of ROS and reactive nitrogen species (Aliakbari et al, 2016).…”