Guedes de Azevedo Viana Arabela, Gomes Fabio Pereira, Kaya Abdullah, Diedrich Jolene, Topper Einko, Di Silvestre Dario, Machado-Neves Mariana, Memili Erdogan, Moura Arlindo Alencar, Yates John R
Reproduction. 2025 Jan 11;169(2). doi: 10.1530/REP-24-0051. Print 2025 Feb 1.
Bovine sperm and seminal plasma (SP) proteoform atlas was characterized using top-down proteomics. Specific post-translational modifications and protein truncations correlated with semen freezability, with potential links to sperm functional processes.
Top-down proteomics was employed to construct a proteoform atlas of sperm and SP from bulls with low semen freezability (LF) and high semen freezability (HF). Sperm and seminal proteins were fractionated by tandem size exclusion chromatography (<30 kDa) and analyzed by reversed-phase liquid chromatography-tandem mass spectrometry. This approach enabled identification of 299 SP (from 46 families) and 267 sperm proteoforms (from 139 families). Seventy proteoforms belonging to beta-defensin 10, c-type natriuretic peptide (NPPC), caltrin, seminal ribonuclease, osteopontin and binder of sperm protein (BSP) 3 families were unique to HF bulls' SP. LF seminal proteins had 77 unique proteoforms, including caltrin, NPPC, osteopontin, BSP3, serpin family A member 5 and β-NGF families. Proteoform families of SP in HF and LF bulls were related to Ca2+ uptake, capacitation, acrosome reaction, sperm protection, fertilization and proteolytic processes. Thirty-three proteoforms of NPPC, caltrin and cylicin-2 families were upregulated in HF sperm. Twenty-two proteoforms of caltrin, cylicin-2, adenosine triphosphate (ATP) synthases and malate dehydrogenase families were among those upregulated in LF sperm. Truncated and acetylated histone H2A and non-truncated and acetylated c-Myc-binding protein were prevalent in LF sperm. Cylicin-2 proteoforms were observed in HF and LF sperm, and truncated glyceraldehyde-3-phosphate dehydrogenases were observed only in HF sperm. In silico analyses indicated the enrichment of mitochondrial metabolic pathways in HF sperm, including fatty acid metabolism and TCA cycle. Our study provides an unprecedented description of the bovine SP and sperm proteoforms. Post-translational processing appears to define the bioproperties of semen proteins and their associations with sperm cryoresistance.
使用自上而下的蛋白质组学对牛精子和精浆（SP）蛋白质异构体图谱进行了表征。特定的翻译后修饰和蛋白质截短与精液冷冻保存能力相关，与精子功能过程存在潜在联系。
采用自上而下的蛋白质组学构建了精液冷冻保存能力低（LF）和高（HF）的公牛精子和SP的蛋白质异构体图谱。精子和精浆蛋白通过串联尺寸排阻色谱法（<30 kDa）进行分离，并通过反相液相色谱 - 串联质谱法进行分析。这种方法能够鉴定出299种SP（来自46个家族）和267种精子蛋白质异构体（来自139个家族）。属于β - 防御素10、C型利钠肽（NPPC）、钙调素、精浆核糖核酸酶、骨桥蛋白和精子蛋白结合蛋白（BSP）3个家族的70种蛋白质异构体是HF公牛SP所特有的。LF精浆蛋白有77种独特的蛋白质异构体，包括钙调素、NPPC、骨桥蛋白、BSP3、丝氨酸蛋白酶抑制剂家族A成员5和β - 神经生长因子家族。HF和LF公牛SP的蛋白质异构体家族与钙离子摄取、获能、顶体反应、精子保护、受精和蛋白水解过程相关。NPPC、钙调素和环化素 - 2家族的33种蛋白质异构体在HF精子中上调。钙调素、环化素 - 2、三磷酸腺苷（ATP）合酶和苹果酸脱氢酶家族的22种蛋白质异构体在LF精子中上调。截短和乙酰化的组蛋白H2A以及未截短和乙酰化的c - Myc结合蛋白在LF精子中普遍存在。环化素 - 2蛋白质异构体在HF和LF精子中均有观察到，而截短的甘油醛 - 3 - 磷酸脱氢酶仅在HF精子中观察到。计算机分析表明HF精子中线粒体代谢途径富集，包括脂肪酸代谢和三羧酸循环。我们的研究提供了对牛SP和精子蛋白质异构体前所未有的描述。翻译后加工似乎决定了精液蛋白质的生物学特性及其与精子冷冻抗性的关联。
