Jung Sang-Eun, Oh Hui-Jo, Ahn Jin-Seop, Kim Yong-Hee, Kim Bang-Jin, Ryu Buom-Yong
Department of Animal Science and Technology, Chung-Ang University, Anseong-si 17546, Gyeonggi-do, Korea.
Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Antioxidants (Basel). 2021 May 10;10(5):754. doi: 10.3390/antiox10050754.
We postulated that supplementation of antioxidant or apoptosis inhibitor in post-thaw culture media of spermatogonial stem cells (SSCs) alleviates reactive oxygen species (ROS) generation and apoptosis. Our aim was to develop an effective culture media for improving post-thaw recovery of SSCs. To determine the efficacy of supplementation with hypotaurine (HTU), α-tocopherol (α-TCP), and Z-DEVD-FMK (ZDF), we assessed the relative proliferation rate and SSC functional activity and performed a ROS generation assay, apoptosis assay, and western blotting for determination of the Bax/Bcl-xL ratio, as well as immunocytochemistry and real-time quantitative polymerase chain reaction (RT-qPCR) for SSC characterization. The relative proliferation rates with HTU 400 μM (133.7 ± 3.2%), α-TCP 400 μM (158.9 ± 3.6%), and ZDF 200 μM (133.1 ± 7.6%) supplementation were higher than that in the DMSO control (100 ± 3.6%). ROS generation was reduced with α-TCP 400 μM (0.8-fold) supplementation in comparison with the control (1.0-fold). Early apoptosis and Bax/Bcl-xL were lower with α-TCP 400 μM (2.4 ± 0.4% and 0.5-fold) and ZDF 200 μM (1.8 ± 0.4% and 0.3-fold) supplementation in comparison with the control (5.3 ± 1.4% and 1.0-fold) with normal characterization and functional activity. Supplementation of post-thaw culture media with α-TCP 400 μM and ZDF 200 μM improved post-thaw recovery of frozen SSCs via protection from ROS generation and apoptosis after cryo-thawing.
我们推测，在精原干细胞（SSCs）解冻后的培养基中添加抗氧化剂或凋亡抑制剂可减轻活性氧（ROS）的产生并减少细胞凋亡。我们的目的是开发一种有效的培养基，以提高SSCs解冻后的复苏率。为了确定补充亚牛磺酸（HTU）、α-生育酚（α-TCP）和Z-DEVD-FMK（ZDF）的效果，我们评估了相对增殖率和SSC功能活性，并进行了ROS生成测定、凋亡测定以及用于确定Bax/Bcl-xL比值的蛋白质印迹分析，同时进行了免疫细胞化学和实时定量聚合酶链反应（RT-qPCR）以对SSCs进行表征。添加400 μM HTU（133.7 ± 3.2%）、400 μM α-TCP（158.9 ± 3.6%）和200 μM ZDF（133.1 ± 7.6%）时的相对增殖率高于二甲基亚砜（DMSO）对照组（100 ± 3.6%）。与对照组（1.0倍）相比，添加400 μM α-TCP时ROS生成减少（0.8倍）。与对照组（5.3 ± 1.4%和1.0倍）相比，添加400 μM α-TCP（2.4 ± 0.4%和0.5倍）和200 μM ZDF（1.8 ± 0.4%和0.3倍）时早期凋亡和Bax/Bcl-xL较低，且具有正常的表征和功能活性。解冻后的培养基中添加400 μM α-TCP和200 μM ZDF可通过保护冷冻解冻后的SSCs免受过氧化氢生成和细胞凋亡的影响，从而提高冷冻SSCs解冻后的复苏率。
