Superoxide anion-induced ferritinophagy is involved in ferroptosis occurrence of cashmere goat sperm during cryopreservation

doi:10.1016/j.jia.2025.12.033

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Erhan Hai^{1, 2}, Boyuan Li^{1, 2}, Yukun Song^{1, 2}, Jian Zhang^{1, 2}, Bingbing Xu^{1, 2}, Yongbin Liu^{1, 2#}, Jiaxin Zhang^{1, 2#}

¹Inner Mongolia Key Laboratory of Sheep & Goat Genetics, Breeding and Reproduction, College of Animal Science, Inner Mongolia Agricultural University, Hohhot, 010018, China

²Key Laboratory of Mutton Sheep & Goat Genetics and Breeding, Ministry of Agriculture and Rural Affairs, Hohhot, 010018, China

Highlights

Ferroptosis initiates at the cooling-equilibration phase, which is associated with the decreased levels of ferroptosis inhibitory proteins.

The occurrence of ferroptosis during sperm cryopreservation is mediated by superoxide anions, and ferritinophagy is involved in this process.

Abstract
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摘要

铁死亡是绒山羊精子冷冻保存过程中主要的细胞调节性死亡类型，该过程会显著制约精液冷冻保存技术的应用，但其具体调控机制仍不明确。本研究发现，铁死亡在降温-平衡阶段就已经发生，且与FTH1等关键铁死亡抑制蛋白的降解相关。冷冻处理会通过上调CYTB表达、降低线粒体抗氧化蛋白水平来导致超氧阴离子累积，而Fer-1无法阻断这一累积过程。超氧阴离子可呈剂量依赖性诱导铁死亡，该效应能被Fer-1缓解；自噬/铁蛋白自噬抑制剂可减轻铁死亡程度，提示铁蛋白自噬参与了这一过程。本研究证实超氧阴离子是绒山羊精子冻融铁死亡的关键介导因子，为优化精子冷冻保存技术提供了新的作用靶点。

Abstract

Ferroptosis is the primary form of regulated cell death in cashmere goat sperm during the freeze-thaw process, which significantly hinders the efficacy and application of frozen semen technology, yet its specific regulatory mechanisms remain unclear. Here, we found it activated during the cooling-equilibration phase, linked to the degradation of critical ferroptosis inhibitory proteins like ferritin heavy chain 1 (FTH1). Freezing causes superoxide anion accumulation via cytochrome b (CYTB) upregulation and reduced mitochondrial antioxidants, unblocked by ferrostatin-1 (Fer-1). Superoxide anions dose-dependently induce ferroptosis, mitigated by Fer-1. Autophagy/ferritinophagy inhibitors alleviate it, implicating ferritinophagy. This identifies superoxide anions as key mediators, offering new targets for sperm cryopreservation.

Keywords: cashmere goat ferroptosis superoxide anion sperm cryoinjury

Online: 19 December 2025

Fund:

This research was funded by grants from Science and Technology Plan of Inner Mongolia Autonomous Region (2025KYPT0035), Inner Mongolia Education Department Special Research Project for First Class Disciplines (YLXKZX-NND-007), Biological Breeding-National Science and Technology Major Project (2023ZD0405104) and the 12th Inner Mongolia "Grassland Talent" High-level Talent Training Project (2023).

About author: Erhan Hai, E-mail: haierhannb@163.com; #Correspondence Yongbin Liu, E-mail: ybliu@imu.edu.cn, Jiaxin Zhang, zjxcau@163.com

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Erhan Hai, Boyuan Li, Yukun Song, Jian Zhang, Bingbing Xu, Yongbin Liu, Jiaxin Zhang. 2025. Superoxide anion-induced ferritinophagy is involved in ferroptosis occurrence of cashmere goat sperm during cryopreservation. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2025.12.033

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