Fu Lijie, Wang Chao, Li Wenfu, Dong Hao, Yang Qian, Chang Guilin, Liu Jianping
Department of Urological Surgery No.2, First Affiliated Hospital of Kunming Medical University, No. 295 Xichang Road, Kunming, 650032, Yunnan Province, China.
Reprod Sci. 2025 Mar;32(3):783-791. doi: 10.1007/s43032-024-01723-4. Epub 2024 Oct 24.
The purpose of this study was to explore the mechanism of action of Piceatannol (PIC) in attenuating oxidative damage to sperm during cryopreservation. Semen samples were collected and homogenized into six equal parts for freeze-thawing experiments. Four different concentrations of PIC were utilized as cryoprotectants during the freeze-thawing process, maintaing a semen to PIC ratio of 1:1, while sperm motility after freezing and thawing was analyzed using computer-assisted sperm analysis (CASA). Sperm plasma membrane integrity was assessed via the hypo-osmotic swelling (HOS) test. The levels of reactive oxygen species (ROS), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) activities, long with the ability to scavenge sperm malondialdehyde (MDA), were examined in sperm following the addition of PIC. Quantitative real-time PCR (qRT-PCR) was performed to detect the expression levels of Keap1, Nrf2, GCLC, GCLM, and HMOX1 in sperm. The mechanism by which PIC protects sperm during cryopreservation from oxidative stress damage was further verified. Treatment with PIC at a dose of 5.0 μmol/L significantly improved both sperm motility and viability while effectively reducing ROS levels in frozen sperm. Additionally, the integrity of the sperm plasma membrane was significantly enhanced. Furthermore, the expression level of Keap1 was significantly reduced, whereas the expression levels of GCLC, GCLM, HMOX1, and Nrf2 were significantly increased (p < 0.05) after the addition of PIC. Notably, a significant attenuation of sperm motility and viability was observed in this treatment group when PIC treatment was accompanied by the addition of an Nrf2 inhibitor, resulting in a significant elevation of ROS levels. The finding that PIC modulates ROS in frozen sperm via the Keap1-Nrf2/ARE pathway thereby enhancing sperm viability levels after freezing and thawing provides a novel approach to optimize semen cryopreservation.
本研究旨在探讨白皮杉醇（PIC）在减轻精子冷冻保存过程中氧化损伤的作用机制。收集精液样本并匀浆成六等份用于冻融实验。在冻融过程中使用四种不同浓度的PIC作为冷冻保护剂，保持精液与PIC的比例为1:1，同时使用计算机辅助精子分析（CASA）分析冻融后的精子活力。通过低渗肿胀（HOS）试验评估精子质膜完整性。在添加PIC后，检测精子中活性氧（ROS）、超氧化物歧化酶（SOD）和总抗氧化能力（T-AOC）活性水平以及清除精子丙二醛（MDA）的能力。进行定量实时PCR（qRT-PCR）以检测精子中Keap1、Nrf2、GCLC、GCLM和HMOX1的表达水平。进一步验证了PIC在冷冻保存过程中保护精子免受氧化应激损伤的机制。5.0 μmol/L剂量的PIC处理显著提高了精子活力和存活率，同时有效降低了冷冻精子中的ROS水平。此外，精子质膜的完整性显著增强。此外，添加PIC后，Keap1的表达水平显著降低，而GCLC、GCLM、HMOX1和Nrf2的表达水平显著升高（p < 0.05）。值得注意的是，当PIC处理同时添加Nrf2抑制剂时，该治疗组中观察到精子活力和存活率显著降低，导致ROS水平显著升高。PIC通过Keap1-Nrf2/ARE途径调节冷冻精子中的ROS，从而提高冻融后精子活力水平，这一发现为优化精液冷冻保存提供了一种新方法。
