Abstract
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. Investigate the effect of perfluorooctane sulfonic acid (PFOS), the most prevalent and representative PFAS, on bull sperm protein phosphorylation and function. We exposed bull sperm to PFOS at 10 (average population exposure) and 100μM (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. PFOS at 10-μM altered sperm proteins linked to spermatogenesis and chromatin condensation, while at 100μM, 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. This study demonstrates that PFOS exposure negatively affects phosphorylation of proteins vital for bull sperm function and fertilization.
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