Institute of Biotechnology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 94976 Nitra, Slovakia.
AgroBioTech Research Centre, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 94976 Nitra, Slovakia.
Int J Mol Sci. 2024 Apr 8;25(7):4129. doi: 10.3390/ijms25074129.
Kaempferol (KAE) is a natural flavonoid with powerful reactive oxygen species (ROS) scavenging properties and beneficial effects on ex vivo sperm functionality. In this paper, we studied the ability of KAE to prevent or ameliorate structural, functional or oxidative damage to frozen-thawed bovine spermatozoa. The analysis focused on conventional sperm quality characteristics prior to or following thermoresistance tests, namely the oxidative profile of semen alongside sperm capacitation patterns, and the levels of key proteins involved in capacitation signaling. Semen samples obtained from 30 stud bulls were frozen in the presence of 12.5, 25 or 50 μM KAE and compared to native ejaculates (negative control-Ctrl) as well as semen samples cryopreserved in the absence of KAE (positive control-Ctrl). A significant post-thermoresistance test maintenance of the sperm motility ( < 0.001), membrane ( < 0.001) and acrosome integrity ( < 0.001), mitochondrial activity ( < 0.001) and DNA integrity ( < 0.001) was observed following supplementation with all KAE doses in comparison to Ctrl. Experimental groups supplemented with all KAE doses presented a significantly lower proportion of prematurely capacitated spermatozoa ( < 0.001) when compared with Ctrl. A significant decrease in the levels of the superoxide radical was recorded following administration of 12.5 ( < 0.05) and 25 μM KAE ( < 0.01). At the same time, supplementation with 25 μM KAE in the cryopreservation medium led to a significant stabilization of the activity of Mg-ATPase ( < 0.05) and Na/K-ATPase ( < 0.0001) in comparison to Ctrl. Western blot analysis revealed that supplementation with 25 μM KAE in the cryopreservation medium prevented the loss of the protein kinase A (PKA) and protein kinase C (PKC), which are intricately involved in the process of sperm activation. In conclusion, we may speculate that KAE is particularly efficient in the protection of sperm metabolism during the cryopreservation process through its ability to promote energy synthesis while quenching excessive ROS and to protect enzymes involved in the process of sperm capacitation and hyperactivation. These properties may provide supplementary protection to spermatozoa undergoing the freeze-thaw process.
山奈酚（KAE）是一种天然类黄酮，具有强大的活性氧（ROS）清除特性，并对体外精子功能有益。在本文中，我们研究了 KAE 预防或改善冷冻-解冻牛精子结构、功能或氧化损伤的能力。分析重点是在耐热性测试之前或之后对常规精子质量特征进行分析，即精液的氧化谱以及精子顶体反应模式和参与顶体反应信号转导的关键蛋白水平。从 30 头种公牛获得的精液样本在存在 12.5、25 或 50 μM KAE 的情况下冷冻，并与天然精液（阴性对照-Ctrl）以及不含 KAE 的冷冻精液（阳性对照-Ctrl）进行比较。与 Ctrl 相比，所有 KAE 剂量均能显著维持精子活力（<0.001）、膜（<0.001）和顶体完整性（<0.001）、线粒体活性（<0.001）和 DNA 完整性（<0.001）在耐热性测试后。与 Ctrl 相比，用所有 KAE 剂量处理的实验组中，过早顶体反应的精子比例显著降低（<0.001）。给予 12.5（<0.05）和 25 μM KAE 后，超氧自由基水平显著降低（<0.01）。同时，在冷冻保存培养基中添加 25 μM KAE 可显著稳定 Mg-ATPase（<0.05）和 Na/K-ATPase（<0.0001）的活性与 Ctrl 相比。Western blot 分析表明，在冷冻保存培养基中添加 25 μM KAE 可防止蛋白激酶 A（PKA）和蛋白激酶 C（PKC）的丢失，PKA 和 PKC 在内的精子激活过程中起着重要作用。总之，我们可以推测，KAE 能够促进能量合成，同时清除过多的 ROS，并保护参与精子顶体反应和超激活过程的酶，从而在冷冻保存过程中特别有效地保护精子代谢。这些特性可能为经历冷冻-解冻过程的精子提供额外的保护。
