Hai Erhan, Li Boyuan, Song Yukun, Zhang Jian, Zhang Jiaxin
Inner Mongolia Key Laboratory of Sheep and Goat Genetics Breeding and Reproduction, College of Animal Science, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China.
Front Vet Sci. 2025 Mar 7;12:1526474. doi: 10.3389/fvets.2025.1526474. eCollection 2025.
To evaluate the roles of apoptosis and ferroptosis in cryopreservation-induced damage to sheep sperm, with a focus on assessing the effectiveness of inhibitors targeting these pathways.
Initial analysis compared the expression of apoptotic marker Cleaved-caspase3 (CL-caspase3) and ferroptotic marker Transferrin receptor (TFRC) between fresh and cryopreserved sheep sperm. Elevated CL-caspase3 expression and sustained high TFRC expression post-cryopreservation suggested concurrent occurrence of apoptosis and ferroptosis. Consequently, the study employed Deferoxamine Mesylate (DFO), ferrostatin-1 (Fer-1), liproxstatin-1 (Lip-1), and the apoptosis inhibitor Z-VAD-FMK (Z-VAD) at concentrations ranging from 0 to 10 μM. Post-thaw assessments encompassed plasma membrane integrity, acrosome integrity, and ferroptosis biomarkers. Additional experiments were conducted to measure the expression of GPX4, a key regulator of ferroptosis.
Optimal concentrations (2 μM for DFO, Fer-1, and Lip-1; 5 μM for Z-VAD) significantly improved sperm motility and membrane integrity. Among these, Fer-1 demonstrated the greatest efficacy, reducing reactive oxygen species (ROS), lipid peroxidation, and Fe levels. Z-VAD primarily decreased ROS but was less potent than ferroptosis inhibitors. Notably, Glutathione Peroxidase 4 (GPX4) expression was reduced post-cryopreservation, while Fer-1 supplementation restored its levels to those comparable with fresh sperm.
Both apoptosis and ferroptosis play critical roles in sheep sperm cryopreservation. Fer-1 effectively enhanced cryopreservation outcomes by inhibiting ferroptosis, as evidenced by the restoration of GPX4 expression and improvement in sperm quality indicators. These findings highlight ferroptosis inhibition as a promising strategy for preserving genetic material, with implications for animal breeding and biodiversity conservation.
评估凋亡和铁死亡在绵羊精子冷冻保存损伤中的作用，重点是评估靶向这些途径的抑制剂的有效性。
初步分析比较了新鲜和冷冻保存的绵羊精子中凋亡标志物裂解型半胱天冬酶3（CL - caspase3）和铁死亡标志物转铁蛋白受体（TFRC）的表达。冷冻保存后CL - caspase3表达升高和TFRC表达持续高水平表明凋亡和铁死亡同时发生。因此，该研究使用了浓度范围为0至10 μM的去铁胺甲磺酸盐（DFO）、铁抑素-1（Fer - 1）、脂氧素抑制剂-1（Lip - 1）和凋亡抑制剂Z - VAD - FMK（Z - VAD）。解冻后的评估包括质膜完整性、顶体完整性和铁死亡生物标志物。还进行了额外的实验来测量铁死亡关键调节因子谷胱甘肽过氧化物酶4（GPX4）的表达。
最佳浓度（DFO、Fer - 1和Lip - 1为2 μM；Z - VAD为5 μM）显著提高了精子活力和膜完整性。其中，Fer - 1表现出最大的功效，降低了活性氧（ROS）、脂质过氧化和铁水平。Z - VAD主要降低了ROS，但效力低于铁死亡抑制剂。值得注意的是，冷冻保存后谷胱甘肽过氧化物酶4（GPX4）表达降低，而补充Fer - 1可将其水平恢复到与新鲜精子相当的水平。
凋亡和铁死亡在绵羊精子冷冻保存中都起着关键作用。Fer - 1通过抑制铁死亡有效地提高了冷冻保存效果，GPX4表达的恢复和精子质量指标的改善证明了这一点。这些发现突出了抑制铁死亡作为保存遗传物质的一种有前景的策略，对动物育种和生物多样性保护具有重要意义。
