男性不育与全氟烷基和多氟烷基物质：牛精子蛋白磷酸化和与生育相关的功能特征改变的证据†。
Male infertility and perfluoroalkyl and poly-fluoroalkyl substances: evidence for alterations in phosphorylation of proteins and fertility-related functional attributes in bull spermatozoa†.
机构信息
Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI 53706, USA.
Theriogenology Laboratory, Southern Regional Station of ICAR National Dairy Research Institute, Bengaluru, Karnataka 560030, India.
出版信息
Biol Reprod. 2024 Sep 14;111(3):723-739. doi: 10.1093/biolre/ioae089.
BACKGROUND
Perfluoroalkyl and poly-fluoroalkyl substances (PFAS) are pervasive environmental pollutants and potential threats to reproductive health. Epidemiological studies have established an association between PFAS and male infertility, but the underlying mechanisms are unclear.
OBJECTIVES
Investigate the effect of perfluorooctane sulfonic acid (PFOS), the most prevalent and representative PFAS, on bull sperm protein phosphorylation and function.
METHODS
We exposed bull sperm to PFOS at 10 (average population exposure) and 100 μM (high-exposure scenario), and analyzed global proteomic and phosphoproteomic analysis by TMT labeling and Nano LC-MS/MS. We also measured sperm fertility functions by flow cytometry.
RESULTS
PFOS at 10-μM altered sperm proteins linked to spermatogenesis and chromatin condensation, while at 100 μM, PFOS affected proteins associated with motility and fertility. We detected 299 phosphopeptides from 116 proteins, with 45 exhibiting differential expression between control and PFOS groups. PFOS dysregulated phosphorylation of key proteins (ACRBP, PRKAR2A, RAB2B, SPAG8, TUBB4B, ZPBP, and C2CD6) involved in sperm capacitation, acrosome reaction, sperm-egg interaction, and fertilization. PFOS also affected phosphorylation of other proteins (AQP7, HSBP9, IL4I1, PRKAR1A, and CCT8L2) related to sperm stress resistance and cryotolerance. Notably, four proteins (PRM1, ACRBP, TSSK1B, and CFAP45) exhibited differential regulation at both proteomic and phosphoproteomic levels. Flow cytometric analysis confirmed that PFOS increased protein phosphorylation in sperm and also decreased sperm motility, viability, calcium, and mitochondrial membrane potential and increased mitochondrial ROS in a dose-dependent manner.
CONCLUSIONS
This study demonstrates that PFOS exposure negatively affects phosphorylation of proteins vital for bull sperm function and fertilization.
背景
全氟烷基和多氟烷基物质（PFAS）是普遍存在的环境污染物，也是生殖健康的潜在威胁。流行病学研究已经证实 PFAS 与男性不育之间存在关联，但潜在机制尚不清楚。
目的
研究最普遍和最具代表性的全氟辛烷磺酸（PFOS）对公牛精子蛋白磷酸化和功能的影响。
方法
我们将公牛精子暴露于 10（人群平均暴露）和 100μM（高暴露情况）的 PFOS 中，并通过 TMT 标记和纳升液相色谱-串联质谱（Nano LC-MS/MS）进行了全局蛋白质组学和磷酸蛋白质组学分析。我们还通过流式细胞术测量了精子的生育功能。
结果
10μM 的 PFOS 改变了与精子发生和染色质浓缩相关的精子蛋白，而 100μM 的 PFOS 则影响了与运动和生育能力相关的蛋白质。我们从 116 种蛋白质中检测到 299 个磷酸肽，其中 45 个在对照组和 PFOS 组之间表现出差异表达。PFOS 失调了关键蛋白（ACRBP、PRKAR2A、RAB2B、SPAG8、TUBB4B、ZPBP 和 C2CD6）的磷酸化，这些蛋白参与精子获能、顶体反应、精卵相互作用和受精。PFOS 还影响了其他与精子应激抵抗和抗冻能力相关的蛋白（AQP7、HSBP9、IL4I1、PRKAR1A 和 CCT8L2）的磷酸化。值得注意的是，有四种蛋白（PRM1、ACRBP、TSSK1B 和 CFAP45）在蛋白质组学和磷酸蛋白质组学水平上均表现出差异调节。流式细胞术分析证实，PFOS 增加了精子中的蛋白磷酸化，且呈剂量依赖性地降低了精子的活力、活率、钙和线粒体膜电位，并增加了线粒体 ROS。
结论
本研究表明，PFOS 暴露会对公牛精子功能和受精所需的蛋白磷酸化产生负面影响。
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