【摘要】 【目的】研究施氏鲟精子冷冻损伤分子机理，从遗传水平分析影响施氏鲟精子冷冻复苏后质量的关键因子。【方法】实验采用4D Label-free定量蛋白组学技术筛选施氏鲟精子超低温保存前后的差异蛋白。施氏鲟精子经过24 h冻存后复苏检测，以差异倍数FC>1.5，显著性差异值P<0.05为标准筛选差异蛋白质，对得到的蛋白进行亚细胞定位、结构域预测、GO富集分析、KEEG富集分析。【结果】共鉴定得到1 859个蛋白，差异1.5倍以上的蛋白200个，其中下调蛋白92个、上调蛋白108个。差异蛋白定位到6个条目上，分别为细胞质120个、细胞核100个、细胞外36个、线粒体35个、质膜29个、溶酶体2个。结构域预测结果显示，胰蛋白酶、脂质运载蛋白/胞浆脂肪酸结合蛋白家族、醛/酮还原酶家族、微管蛋白C端结合域及Nup53/35/40-型RNA识别基序是参与精子冻融损伤应答的相关结构域。GO富集分析结果显示，差异蛋白主要参与686种生物过程、199种细胞成分以及394类分子功能，主要富集到激素结合、镁离子结合、脂质代谢、核膜等条目。KEGG富集分析结果显示，差异蛋白注释到63条信号通路，主要涉及糖代谢、氨基酸代谢和细胞核膜运输等信号通路。【结论】施氏鲟冻精与鲜精的蛋白质组分差异明显，这些差异蛋白质可能对精子冷冻效果产生影响，可作为施氏鲟精子冻融的候选标志蛋白。本研究可为后续进一步优化施氏鲟精子冷冻技术提供理论基础与研究思路。更多还原

【Abstract】 Acipenser schrenckii, commonly known as the Amur sturgeon, is a vital species in China’s sturgeon industry due to its significant economic value. Cryopreservation of sperm is a crucial technique for conserving and utilizing sturgeon germplasm resources, facilitating long-term storage of viable sperm. Previous studies have optimized the extender formulas,assessed antioxidant activities, examined enzyme functions related to energy metabolism, and compared sperm ultrastructure.However, there remains a paucity of research on the molecular changes occuring during sturgeon sperm cryopreservation.Addressing this gap, a recent study utilized 4D label-free quantitative proteomics to discern differentially abundant proteins between fresh and freeze-thawing A. schrenckii semen, post-thawing at 24 h. Proteins were extracted using SDT lysis buffer(4% SDS, 100 mmol/L Tris-HCl pH 7.6, 0.1 mol/L DTT) and sonication, followed by centrifugation for collection, identifying a total of 1 859 proteins. Employing a threshold of a 1.5-fold difference factor(FC) and a significance level(P-value) less than0.05, 200 differentially abundant proteins were delineated, including 92 down-regulated and 108 up-regulated. Subcellular mapping revealed protein localization across various cellular compartments, while GO and KEGG enrichment analyses unveiled involvement in diverse biological processes, cellular components, molecular functions, and signaling pathways. Notably, Fasactivated serine/threonine phosphoprotein kinase 1(FASTK1) was up-regulated in freeze-thawing semen for 48.81-fold, indicating a role in cell proliferation regulation for self-protection. Conversely, the down-regulation cold inducible RNA-binding protein(CIRP) suggested its involvement in cell proliferation, which ultimately affected the motility of free-thawing semen. Upregulated Nup proteins potentially contributed to membrane protein conformation changes, destabilizing intracellular environments. Down-regulated DNAH-9 was linked to flagellum membrane disruption and fracture. Changes in proteins associated with energy metabolism and antioxidant response, including CK, SDHC, 26S proteasome, CAT and SOD, may elucidate the decreased motility of sturgeon semen after freeze-thawing process. These molecular alternations may impact sperm viability and function, possibly explain reduced fertilization successpost-cryopreservation. The identified proteins could serve as candidate markers for A. schrenckii semen quality during the freeze-thawing process, guiding the development of improved cryopreservation protocols. This research lays a valuable foundation for deeper exploration into the molecular mechanisms of sturgeon sperm cryopreservation, crucial for the conservation and sustainable utilization of this vital fish resource.更多还原