During ejaculation, a large amount of seminal plasma proteins interact with the sperm membrane, leading to a series of biochemical and structural changes implicated in sperm function and gamete interaction. However, the roles of the majority of these proteins remain unknown. This study aimed to investigate the proteome and functionality of the major equine proteins of seminal plasma and the sperm membrane. Seminal plasma and enriched-membrane proteins (150 μg) were separated by two-dimensional gel electrophoresis, and the respective maps were analyzed. Protein identification was performed by in-gel digestion and tandem mass spectrometry (GeLC-MS/MS). Samples were also submitted to in-solution digestion (complex protein mixture) and identified by shotgun analysis by LC-MS/MS; bioinformatic tools were used to investigate protein functions. Seminal plasma and sperm membrane extract maps contained 91.0 ± 8.2 spots and 245.3 ± 11.3 spots, respectively, within the 3-10 pH range. In total, the most abundant proteins identified in 2D maps and in complex protein mixtures included 24 proteins for seminal plasma and 33 for sperm membrane extract, with a high degree of confidence (P < 0.05). Of these, HSP1, CRISP3 and KLK1E2 were the most abundant in seminal plasma; HSP1 was highly abundant in sperm membrane extract, in many isoforms, which is related to membrane destabilization and may compromise sperm preservation. HSP1-polybromo-1 interactions suggested a role in DNA stabilization. Prosaposin was identified in seminal plasma and may play a role in the fertilization process. IZUMO4, a member of the IgSF family involved in the prefertilization stages, was identified in 2D gel and MS/MS analysis of sperm membrane extract. Ten proteins of seminal plasma were found to interact with the sperm membrane and were related to binding and catalytic activities (clusterin, CRISP3, epididymal sperm-binding protein 1, kallikrein1E2, seminal plasma protein A3, and HSP1). Additionally, other identified proteins were associated with DNA integrity, capacitation and recognition of pregnancy. These findings indicate that the binding of specific proteins to the plasma membrane during ejaculation may influence sperm survival after cryopreservation and may play a role in decreasing the quality in stallions with toxic seminal plasma. Elucidation of these interactions is an important step in understanding the biological processes related to equine fertility and facilitates future investigations on the selection and application of low freezability semen strategies.

中文翻译：

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马精浆和精子膜：功能蛋白质组学评估

在射精过程中，大量精浆蛋白与精子膜相互作用，导致一系列与精子功能和配子相互作用有关的生化和结构变化。然而，大多数这些蛋白质的作用仍然未知。本研究旨在研究精浆和精子膜的主要马蛋白质的蛋白质组和功能。通过二维凝胶电泳分离精浆和富集膜蛋白（150 μg），并分析各自的图谱。通过凝胶内消化和串联质谱法 (GeLC-MS/MS) 进行蛋白质鉴定。样品也进行溶液内消化（复杂的蛋白质混合物），并通过 LC-MS/MS 的鸟枪分析进行鉴定；生物信息学工具被用于研究蛋白质功能。在 3-10 pH 范围内，精浆和精子膜提取物图分别包含 91.0 ± 8.2 个点和 245.3 ± 11.3 个点。总的来说，在二维图和复杂蛋白质混合物中鉴定的最丰富的蛋白质包括 24 种用于精浆的蛋白质和 33 种用于精膜提取物的蛋白质，具有很高的置信度 (P < 0.05)。其中，HSP1、CRISP3 和 KLK1E2 在精浆中含量最高；HSP1 在精子膜提取物中的含量很高，以许多同种型的形式存在，这与膜不稳定有关，可能会影响精子的保存。HSP1-polybromo-1 相互作用表明在 DNA 稳定中起作用。Prosaposin 在精浆中被鉴定，并且可能在受精过程中起作用。IZUMO4，参与优选阶段的 IgSF 家族的成员，在精子膜提取物的 2D 凝胶和 MS/MS 分析中鉴定出。发现精浆的 10 种蛋白质与精子膜相互作用，并与结合和催化活性有关（聚集蛋白、CRISP3、附睾精子结合蛋白 1、激肽释放酶 1E2、精浆蛋白 A3 和 HSP1）。此外，其他已鉴定的蛋白质与 DNA 完整性、获能和妊娠识别相关。这些发现表明，射精过程中特定蛋白质与质膜的结合可能会影响冷冻保存后精子的存活率，并可能在降低具有有毒精浆的种马的质量方面发挥作用。