Effect of miR-30a-5p on Apoptosis, Colonization, and Oxidative Stress Variables in Frozen-Thawed Neonatal Mice Spermatogonial Stem Cells.

Abstract

Cryopreservation of spermatogonial stem cells (SSCs) is a useful method for fertility preservation in preadolescent children suffering from cancer. However, SSCs may become damaged during cryopreservation due to the generation of reactive oxygen species (ROS). For this reason, various antioxidant agents have been used to protect SSCs from cryopreservation-induced damages. Recently, it has been reported that miR-30a-5p has antiapoptotic and antioxidant activity. The aim of this study was to assess the antiapoptotic and antioxidant effects of miR-30a-5p mimics in frozen-thawed SSCs. To this end, SSCs were isolated from male BALB/C mice (3-6 days old) and cultivated for 14 days. After the detection of optimum concentration, a miR-30a-5p mimic or miR-30a-5p inhibitor with Lipofectamine was transfected into SSCs and, finally, the cell groups were frozen for 1 week. After thawing, different properties, including cell viability (using MTT), colonization of SSCs (number and diameter of colonies), ROS generation (using DCFH-DA assay), levels of malondialdehyde (MDA) and superoxide dismutase (SOD), and gene expression of Bcl-2 and BAXBax (using quantitative real-time PCR), were investigated. The transfection of SSCs with miR-30a-5p mimics before the freezing-thawing process significantly increased the viability, number, and diameter of SSCs colonies. Also, the miR-30a-5p mimic decreased the levels of ROS production and MDA, but it increased the SOD levels. Moreover, the miR-30a-5p mimic decreased BAX and increased Bcl-2 expression in frozen-thawed SSCs. The transfection of SSCs with the miR-30a-5p mimic can increase cell viability and antioxidant defense, and it can decrease apoptosis during the freezing-thawing process. If SSC is able to produce spermatozoa after the transfection of miR-30a-5p and the freezing-thawing process, it can be suggested as a promising strategy for the cryopreservation of SSCs in prepubertal boys suffering from cancer.