Hatef Behnaz, Taromchi Amirhossein, Nejatbakhsh Reza, Farrokhi Ahmad, Shokri Saeed
Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Zanjan University of Medical Sciences (ZUMS), Zanjan, Iran.
Department of Medical Biotechnology and Nanotechnology, Faculty of Medicine, Zanjan University of Medical Sciences (ZUMS), Zanjan, Iran.
Cryobiology. 2017 Dec;79:37-42. doi: 10.1016/j.cryobiol.2017.09.004. Epub 2017 Sep 22.
The destructive effects of sperm cryopreservation result in reduced sperm motility and increased apoptosis. Oocytes, endometrium, and follicular fluid express stromal cell-derived factor-1 alpha (SDF-1α) or C-X-C motif chemokine ligand 12 (CXCL12) while its specific receptor chemokine, CXC motif receptor 4 (CXCR4) is expressed in the head of sperm. SDF-1α can increase sperm motility and preserve normal mitochondrial status. The present study intends to investigate whether the addition of SDF-1α to freezing extender can facilitate cryosurvival of spermatozoa and how SDF-1α protects spermatozoa against damages during cryopreservation. In this study, we collected 22 semen samples from healthy donors and treated them with different concentrations of SDF-1α, followed by cryopreservation for one month. We measured sperm motility by CASA, mitochondrial ROS generation by flow cytometry using the probe MitoSOX Red™ (MSR) to measure mitochondrial superoxide anion (O2•), DNA fragmentation by flow cytometry according to the TUNEL kit, and expressions of Bcl-2 and Bax by RT-qPCR in freeze-thawed sperm. The results showed that SDF-1α attenuated mitochondrial ROS generation at different doses, particularly the 250 ng/ml treated samples which, in turn, reduced the expressions of pro-apoptotic genes such as Bax. Eventually, SDF-1α reduced DNA fragmentation and ameliorated sperm motility in the 1-100 ng/ml treated samples during cryopreservation. The present study, for the first time, demonstrated that SDF-1α dose-dependently moderated oxidative stress injury in human sperm by reduction of mitochondrial ROS generation. It could subsequently cause a decrease in apoptosis during freeze-thawing and protect human spermatozoa from cryodamage.
精子冷冻保存的破坏作用会导致精子活力降低和细胞凋亡增加。卵母细胞、子宫内膜和卵泡液表达基质细胞衍生因子-1α（SDF-1α）或C-X-C基序趋化因子配体12（CXCL12），而其特异性受体趋化因子CXC基序受体4（CXCR4）在精子头部表达。SDF-1α可提高精子活力并维持正常的线粒体状态。本研究旨在探讨在冷冻稀释液中添加SDF-1α是否能促进精子的冷冻存活，以及SDF-1α如何保护精子在冷冻保存过程中免受损伤。在本研究中，我们从健康供体收集了22份精液样本，用不同浓度的SDF-1α处理，然后冷冻保存一个月。我们通过计算机辅助精子分析（CASA）测量精子活力，使用MitoSOX Red™探针通过流式细胞术测量线粒体活性氧（ROS）生成以检测线粒体超氧阴离子（O2•），根据TUNEL试剂盒通过流式细胞术检测DNA片段化，并通过实时定量聚合酶链反应（RT-qPCR）检测冻融精子中Bcl-2和Bax的表达。结果表明，SDF-1α在不同剂量下均能减弱线粒体ROS生成，尤其是250 ng/ml处理的样本，这反过来又降低了促凋亡基因如Bax的表达。最终，SDF-1α在1-100 ng/ml处理的样本冷冻保存过程中减少了DNA片段化并改善了精子活力。本研究首次证明，SDF-1α通过减少线粒体ROS生成剂量依赖性地减轻人精子的氧化应激损伤。随后它可导致冻融过程中细胞凋亡减少，并保护人精子免受冷冻损伤。
