Zhang Renzheng, Wang Xiuyuan, Liu Ruili, Mei Yanfang, Miao Xiuping, Ma Jiaxu, Zou Lei, Zhao Qiuyue, Bai Xuejin, Dong Yajuan
College of Animal Science and Technology, Qingdao Agricultural University, Qingdao, 266109, China.
Laboratory of Animal Molecular Shandong Black Cattle Breeding Engineering Technology Center, Qingdao, 266109, China.
BMC Genomics. 2025 Jan 22;26(1):63. doi: 10.1186/s12864-025-11258-w.
The cryoinjury of semen during cryopreservation reduces sperm motility, constraining the application of artificial insemination (AI) in bovine reproduction. Some fertility markers, related to sperm motility before and after freezing have been identified. However, little is known about the biological mechanism through which freezing reduces sperm motility. This study investigated the selective effects of cryoinjury on high-motility sperm (HMS) and low-motility sperm (LMS) in frozen-thawed from the perspectives of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and ATP levels. The molecular mechanism of decreased sperm motility caused by cryoinjury was explored through a joint analysis of 4D-label free quantitative proteomics and non-targeted metabolomics.
The results indicate that low levels of ROS and high degrees of MMP and ATP play a critical role in the survival of HMS during the freezing process. The sperm samples from the frozen-thawed HMS and LMS were analysed for proteomics and metabolomics, 2,465 proteins and 4,135 metabolites were detected in bovine sperm samples. In contrast to LMS, HMS have 106 proteins and 106 metabolites with high abundance expression, and 79 proteins and 223 metabolites with low abundance expression. Proteomics and metabolomics data exhibit that highly expressed antioxidant enzymes and metabolites in HMS can maintain sperm motility by regulating the ROS produced during freezing to prevent sperm from oxidative stress and apoptosis. Furthermore, the KEGG analysis of differential proteins and metabolites during the freezing process implies that the significant enrichment of glycolysis and cAMP in HMS can guarantee energy supply.
The results provided that during the process of bovine sperm freezing, highly expressed antioxidant enzymes can regulate the reactive oxygen species levels to avoid oxidative stress and the glycolysis signalling pathway ensures ATP production can sustain frozen-thawed sperm motility.
精液在冷冻保存过程中的冷冻损伤会降低精子活力，限制人工授精（AI）在牛繁殖中的应用。一些与冷冻前后精子活力相关的生育力标志物已被确定。然而，关于冷冻降低精子活力的生物学机制知之甚少。本研究从活性氧（ROS）、线粒体膜电位（MMP）和ATP水平的角度，研究了冷冻损伤对冻融后高活力精子（HMS）和低活力精子（LMS）的选择性影响。通过4D标记无标记定量蛋白质组学和非靶向代谢组学的联合分析，探索了冷冻损伤导致精子活力下降的分子机制。
结果表明，低水平的ROS、高程度的MMP和ATP在冷冻过程中对HMS的存活起着关键作用。对冻融后的HMS和LMS精子样本进行蛋白质组学和代谢组学分析，在牛精子样本中检测到2465种蛋白质和4135种代谢物。与LMS相比，HMS有106种蛋白质和106种代谢物高丰度表达，79种蛋白质和223种代谢物低丰度表达。蛋白质组学和代谢组学数据表明，HMS中高表达的抗氧化酶和代谢物可通过调节冷冻过程中产生的ROS来维持精子活力，防止精子氧化应激和凋亡。此外，冷冻过程中差异蛋白质和代谢物的KEGG分析表明，HMS中糖酵解和cAMP的显著富集可保证能量供应。
结果表明，在牛精子冷冻过程中，高表达的抗氧化酶可调节活性氧水平以避免氧化应激，糖酵解信号通路确保ATP产生以维持冻融后精子活力。
