College of Animal Science, Shanxi Agricultural University, Taigu, 030801, PR China; Lab of Animal Reproductive Biotechnology, Shanxi Agricultural University, Taigu, 030801, PR China.
Lab of Animal Reproductive Biotechnology, Shanxi Agricultural University, Taigu, 030801, PR China.
Theriogenology. 2023 Jul 15;205:27-39. doi: 10.1016/j.theriogenology.2023.04.015. Epub 2023 Apr 16.
The aim of this study was to investigate the effects of mitochondria-targeted antioxidant Mito-TEMPO on the protein profile of ram sperm during cryopreservation and evaluate the cryoprotective roles of Mito-TEMPO on ram sperm quality and fertilization capacity. Semen collected from 8 Dorper rams was cryopreserved in TCG-egg yolk extender supplemented with various concentrations of Mito-TEMPO (0, 20, 40 and 60 μM). After thawing, sperm characteristics, antioxidant status and the abundance of hexose transporters (GLUT 3 and 8) were analyzed. The cervical artificial insemination (AI) was performed to evaluate the fertilization ability of cryopreserved ram sperm. The alterations of sperm proteomic profile between the control and MT groups were determined using iTRAQ-coupled LC-MS. Supplementation with 40 μM of Mito-TEMPO resulted in the highest post-thaw sperm motility and kinematics. Sperm quality, antioxidant capacity and glucose transporter abundance of frozen-thawed ram sperm were elevated in the MT group. The inclusion of 40 μM Mito-TEMPO in freezing extender also resulted in the higher pregnancy rate of ewes. A total of 457 proteins including 179 upregulated proteins and 278 downregulated proteins were defied as differentially expressed proteins (DEPs) using fold change (FC) > 1.2 with P < 0.05. Sixty-one DEPs with (FC > 1.5) were dramatically regulated by Mito-TEMPO. These DEPs are mainly involved in sperm motility, energy metabolism and capacitation. Our data suggest that the beneficial effects of Mito-TEMPO on sperm motility and fertility potential of cryopreserved ram semen are achieved by regulating sperm antioxidant capacity and sperm proteins related to energy metabolism and fertility.
本研究旨在探讨线粒体靶向抗氧化剂 Mito-TEMPO 对冷冻保存过程中公羊精子蛋白质谱的影响，并评估 Mito-TEMPO 对公羊精子质量和受精能力的保护作用。从 8 只多伯羊中采集精液，用 TCG-卵黄稀释液冷冻保存，其中添加了不同浓度的 Mito-TEMPO（0、20、40 和 60 μM）。解冻后，分析精子特性、抗氧化状态以及己糖转运体（GLUT3 和 GLUT8）的丰度。通过颈管人工授精（AI）评估冷冻公羊精子的受精能力。使用 iTRAQ 结合 LC-MS 确定控制组和 MT 组之间精子蛋白质组图谱的变化。添加 40 μM 的 Mito-TEMPO 可提高解冻后精子的活力和运动学特性。MT 组冷冻解冻公羊精子的质量、抗氧化能力和葡萄糖转运体丰度提高。冷冻液中添加 40 μM 的 Mito-TEMPO 还可提高母羊的妊娠率。使用 fold change (FC) > 1.2 且 P < 0.05，共定义了 457 种蛋白质，包括 179 种上调蛋白和 278 种下调蛋白作为差异表达蛋白 (DEPs)。有 61 种 DEPs 的 FC > 1.5，其表达受 Mito-TEMPO 的显著调控。这些 DEPs 主要涉及精子活力、能量代谢和获能。我们的数据表明，Mito-TEMPO 通过调节精子抗氧化能力和与能量代谢和生育能力相关的精子蛋白，对冷冻保存的公羊精液的精子活力和生育潜力产生有益影响。
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