Testicular phosphoproteome in perfluorododecanoic acid-exposed rats

Perfluorododecanoic acid (PFDoA) is a common environmental pollutant, which has been detected in human sera and has adverse effects on testicular function in animal models. Exploring phosphorylation events in testes helps elucidate the specific phosphorylation signals involved in testicular toxicity of PFDoA. Combining efficient prefractionation of tryptic peptide mixtures using self-packed reversed phase C18 columns with TiO2 and IMAC phosphopeptide enrichment techniques followed by 2D-LC-MS/MS, we identified 4077 unique phosphopeptides from 1777 proteins with a false discovery rate below 1.0% in the testes of rats exposed to PFDoA for 110 days. In addition, 937 novel phosphorylation sites were discovered in testicular proteins. Hundreds of phosphorylated proteins identified might be involved in spermatogenesis and sperm function. With increasing PFDoA dosage, the number of casein kinase 2 kinase-modified peptides significantly increased. Pathway analysis suggested that the mitogen-activated protein kinase pathway and cell division cycle protein 2 (CDC2) may have contributed to sperm activity and testicular function. By in vitro experiments, CDC2 phosphorylation activity was found to be likely involved in PFDoA-induced toxicity in Leydig cells. This study provides the first examination of the whole proteins' phosphorylation profile in rat testis and suggests that the MAPK pathway and CDC2 protein phosphorylation are critical for PFDoA testicular toxicity.