Differences in sperm protein abundance and carbonylation level in bull ejaculates of low and high quality

In breeding and insemination centres, significant variation in bull ejaculate quality is often observed between individuals and also within the same individual. Low-quality semen does not qualify for cryopreservation and is rejected, generating economic loss. The mechanisms underlying the formation of low-quality ejaculates are poorly understood; therefore, the aim of the present study was to investigate the proteomic differences and oxidative modifications (measured as changes in protein carbonylation level) of bull ejaculates of low and high quality. Flow cytometry and computer-assisted sperm analysis were used to assess differences in viability, reactive oxygen species (ROS) level, and sperm motility. To analyse changes in protein abundance, two-dimensional difference gel electrophoresis (2D-DIGE) was performed. Western blotting in conjunction with two-dimensional electrophoresis (2D-oxyblot) was used to quantitate carbonylated sperm proteins. Proteins were identified using matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight spectrometry. High quality ejaculates were characterised by higher sperm motility, viability, concentration, and a lower number of ROS-positive cells (ROS+). We found significant differences in the protein profile between high- and low-quality ejaculates, and identified 14 protein spots corresponding to 10 proteins with differences in abundance. The identified sperm proteins were mainly associated with energetic metabolism, capacitation, fertilisation, motility, and cellular detoxification. High-quality ejaculates were characterised by a high abundance of extracellular sperm surface proteins, likely due to more efficient secretion from accessory sex glands and/or epididymis, and a low abundance of intracellular proteins. Our results show that sperm proteins in low-quality ejaculates are characterised by a high carbonylation level. Moreover, we identified, for the first time, 14 protein spots corresponding to 12 proteins with differences in carbonylation level between low- and high-quality ejaculates. The carbonylated proteins were localised mainly in mitochondria or their immediate surroundings. Oxidative damage to proteins in low-quality semen may be associated with phosphorylation/dephosphorylation disturbances, mitochondrial dysfunction, and motility apparatus disorders. Our results contribute to research regarding the mechanism by which low- and high-quality ejaculates are formed and to the identification of sperm proteins that are particularly sensitive to oxidative damage.在育种与授精中心中，常观察到不同个体间乃至同一公牛个体内，其精液品质存在显著差异。低品质精液无法满足冷冻保存要求而被废弃，由此造成经济损失。目前学界对低品质精液形成的潜在机制尚缺乏深入了解，因此本研究旨在探究高低品质公牛精液的蛋白质组差异，以及以蛋白质羰基化水平变化为衡量指标的氧化修饰情况。本研究采用流式细胞术与计算机辅助精子分析技术，分别检测精子存活率、活性氧（reactive oxygen species，ROS）水平以及精子活力的差异。为分析蛋白质丰度变化，实验采用双向差异凝胶电泳（two-dimensional difference gel electrophoresis，2D-DIGE）；通过结合双向电泳的蛋白质免疫印迹（2D-oxyblot）对羰基化精子蛋白进行定量。采用基质辅助激光解吸电离飞行时间/飞行时间质谱（matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight spectrometry）对蛋白质进行鉴定。高品质精液的特征为精子活力、存活率与浓度更高，ROS阳性细胞数量更少。本研究发现高低品质精液的蛋白质谱存在显著差异，共鉴定出14个蛋白质斑点，对应10种丰度存在差异的精子蛋白。所鉴定的精子蛋白主要参与能量代谢、精子获能、受精过程、精子运动以及细胞解毒等生物学过程。高品质精液富含细胞外精子表面蛋白，这可能源于附属性腺与/或附睾的分泌效率更高；同时其细胞内蛋白丰度相对较低。本研究结果显示，低品质精液中的精子蛋白羰基化水平显著升高。此外，本研究首次鉴定出14个蛋白质斑点，对应12种在高低品质精液中羰基化水平存在差异的蛋白质。这些羰基化蛋白主要定位于线粒体及其邻近区域。低品质精液中蛋白的氧化损伤可能与磷酸化/去磷酸化紊乱、线粒体功能障碍以及精子运动装置异常相关。本研究结果有助于深入解析高低品质精液的形成机制，并可用于筛选对氧化损伤尤为敏感的精子蛋白。