Department of Anatomy, School of Medicine, Tehran University of Medical Science, Tehran, Iran.
Department of Veterinary Medicine, Faculty of Veterinary Medicine, Islamic Azad University, Shabestar, Iran.
Biopreserv Biobank. 2024 Feb;22(1):68-81. doi: 10.1089/bio.2022.0178. Epub 2023 Aug 15.
Cryopreservation and transplantation of spermatogonial stem cells (SSCs) may serve as a new method to restore male fertility in patients undergoing chemotherapy or radiotherapy. However, SSCs may be damaged during cryopreservation due to the production of reactive oxygen species (ROS). Therefore, different antioxidants have been used as protective agents. Studies have shown that metformin (MET) has antioxidant activity. The aim of this study was to assess the antioxidant and antiapoptotic effects of MET in frozen-thawed SSCs. In addition, the effect of MET on the proliferation and differentiation of SSCs was evaluated. To this end, SSCs were isolated from mouse pups aged 3-6 days old, cultured, identified by flow cytometry (ID4, INTEGRIN β1), and finally evaluated for survival and ROS rate. SSCs were transplanted after busulfan and cadmium treatment. Cryopreserved SSCs with and without MET were transplanted after 1 month of cryopreservation. Eight weeks after transplantation, the recipient testes were evaluated for the expression of apoptosis (BAX, BCL2), proliferation (PLZF), and differentiation (SCP3, TP1, TP2, PRM1) markers using immunohistochemistry, Western blot, and quantitative real-time polymerase chain reaction. The findings revealed that the survival rate of SSCs was higher in the 500 μm/mL MET group compared to the other groups (50 and 5000 μm/mL). MET significantly decreased the intracellular ROS production. Transplantation of SSCs increased the expression level of proliferation (PLZF) and differentiation (SCP3, TP1, TP2, PRM1) markers compared to azoospermia group, and their levels were significantly higher in the MET group compared to the cryopreservation group containing basic freezing medium ( < 0.05). MET increased the survival rate of SSCs, proliferation, and differentiation and decreased the ROS production and the apoptosis rate. Cryopreservation by MET seems to be effective in treating infertility.
精原干细胞（SSC）的冷冻保存和移植可能成为化疗或放疗患者恢复生育能力的新方法。然而，由于活性氧（ROS）的产生，SSC 在冷冻保存过程中可能会受到损伤。因此，不同的抗氧化剂被用作保护剂。研究表明，二甲双胍（MET）具有抗氧化活性。本研究旨在评估 MET 对冻融 SSC 的抗氧化和抗凋亡作用。此外，还评估了 MET 对 SSC 增殖和分化的影响。为此，从 3-6 天大的幼鼠中分离 SSC，进行培养，流式细胞术（ID4、INTEGRIN β1）鉴定，最后评估其存活率和 ROS 率。在接受白消安和镉处理后进行 SSC 移植。经过 1 个月的冷冻保存后，移植含有和不含有 MET 的冷冻 SSC。移植后 8 周，通过免疫组织化学、Western blot 和实时定量聚合酶链反应评估受体睾丸中凋亡（BAX、BCL2）、增殖（PLZF）和分化（SCP3、TP1、TP2、PRM1）标志物的表达。结果表明，500μm/ml MET 组的 SSC 存活率高于其他组（50μm/ml 和 5000μm/ml）。MET 显著降低了细胞内 ROS 的产生。与无精子症组相比，SSC 移植增加了增殖（PLZF）和分化（SCP3、TP1、TP2、PRM1）标志物的表达水平，与含有基础冷冻培养基的冷冻保存组相比，MET 组的表达水平显著更高（<0.05）。MET 提高了 SSC 的存活率、增殖和分化，降低了 ROS 的产生和凋亡率。MET 冷冻保存似乎可以有效治疗不育症。
