Fan Yilin, Li Xiaowei, Guo Yu, He Xiaoqiang, Wang Yanwen, Zhao Dan, Ma Yan, Feng Xinxin, Zhang Jiyue, Li Jian, Zi Xiangdong, Xiong Xianrong, Fu Wei, Xiong Yan
College of Animal & Veterinary Sciences, Southwest Minzu University, Chengdu, 610041, China; Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Reservation and Utilization, Ministry of Education, Southwest Minzu University, Chengdu, 610041, China; Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Reservation and Utilization of Sichuan Province, Southwest Minzu University, Chengdu, 610041, China.
Longri Breeding Stock Farm of Sichuan Province, Dujiangyan, 611800, China.
Theriogenology. 2023 Apr 1;200:60-69. doi: 10.1016/j.theriogenology.2023.01.024. Epub 2023 Feb 3.
Sperm cryopreservation is one of the most effective methods for the conservation of germplasm resources and used of superior sires widely. However, the motility of yak (Bos grunniens) sperm was low after thawing and the proteomics changes in sperm cryopreservation remain unknown. Therefore, the aim of this study was to explore the differences between fresh sperm and frozen sperm of yak through the proteomic analysis and thus improve the understanding of sperm cryodamage. The Tandem Mass Tags (TMT) technology was used to screen differentially expressed proteins (DEPs) before and after freezing. Then, GO and KEGG analysis were conducted to analyze the DEPs enriched signaling pathways. Finally, the DEPs, including superoxide dismutase 1 (SOD1) and NADH ubiquinone oxidoreductase core subunit S8 (NDUFS8) were verified by the immunofluorescence technique. The results showed that there were 229 DEPs between fresh and frozen-thawed yak sperm. Compared with the fresh sperm, 120 proteins were up-regulated and 109 proteins were down-regulated in frozen-thawed sperm. The GO annotation showed that the up-regulated proteins enriched in metabolic and cytoskeleton-related processes, including lipoprotein metabolic process, lipid transport, extracellular region and intermediate filament cytoskeleton organization. In contrast, the down-regulated proteins enriched in biological processes including single fertilization, sperm capacitation and response to unfolded protein. KEGG pathway analysis indicated that freezing and thawing affected the oxidative phosphorylation pathway, the fructose and mannose metabolic pathway and the glycerolipid metabolic pathway of yak sperm. Immunofluorescence results showed that the protein expression level of SOD1 protein in the frozen group was significantly lower than that in the fresh group (P < 0.01), and the protein expression level of NDUFS8 protein was significantly higher in frozen group (P < 0.01). This study revealed the DEPs between fresh and frozen-thawed sperm and provides a theoretical basis to further explore the exertion of normal biological functions of yak sperm after freezing and thawing.
精子冷冻保存是种质资源保存和广泛利用优良种公牛的最有效方法之一。然而，牦牛精子解冻后的活力较低，精子冷冻保存过程中的蛋白质组学变化尚不清楚。因此，本研究的目的是通过蛋白质组学分析探索牦牛新鲜精子和冷冻精子之间的差异，从而增进对精子冷冻损伤的理解。采用串联质谱标签（TMT）技术筛选冷冻前后差异表达蛋白（DEPs）。然后，进行基因本体（GO）和京都基因与基因组百科全书（KEGG）分析，以分析DEPs富集的信号通路。最后，通过免疫荧光技术对包括超氧化物歧化酶1（SOD1）和烟酰胺腺嘌呤二核苷酸泛醌氧化还原酶核心亚基S8（NDUFS8）在内的DEPs进行验证。结果表明，新鲜牦牛精子与冻融后精子之间存在229个DEPs。与新鲜精子相比，冻融后精子中有120种蛋白质上调，109种蛋白质下调。GO注释显示，上调的蛋白质富集于代谢和细胞骨架相关过程，包括脂蛋白代谢过程、脂质转运、细胞外区域和中间丝细胞骨架组织。相反，下调的蛋白质富集于包括单受精、精子获能和未折叠蛋白反应在内的生物学过程。KEGG通路分析表明，冻融影响了牦牛精子的氧化磷酸化通路、果糖和甘露糖代谢通路以及甘油脂质代谢通路。免疫荧光结果显示，冷冻组中SOD1蛋白的表达水平显著低于新鲜组（P<0.01），而冷冻组中NDUFS8蛋白的表达水平显著高于新鲜组（P<0.01）。本研究揭示了新鲜精子与冻融后精子之间的DEPs，为进一步探索牦牛精子冻融后正常生物学功能的发挥提供了理论依据。
