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We previously discovered that  Caenorhabditis elegans  synthesizes Cox-independent F-series prostaglandins (PGs). To delineate the Cox-independent prostaglandin pathways and evaluate their role in sperm motility in  C .  elegans , we developed a novel biochemical method for the rapid production of F-series PGs using arachidonic acid as the substrate and worm lysate as source of enzyme(s). Among the four F2-series PGs produced in the reaction, three of them were identified as 8-isoPGF2α, 5iPF2 VI, and PGF2α based on their retention times and MS/MS spectral comparison with standards using LC-MS/MS. PG production was not markedly affected by specific antioxidants, or Cox, Lox, and Cyp inhibitors, suggesting that these PGs are formed through a novel, biologically regulated mechanism in  C .  elegans . This study also assessed the ability of 8-isoPGF2α, 5iPF2 VI, PGF2α, and a mixture containing these PGs in a 0.5/0.08/1 ratio that reflects their synthetic composition to modulate sperm motility in  fat- 2 mutants. PGF2α and the PG mixture at 25 μM concentration significantly stimulated sperm velocity by 28% and 38%, whereas 8-isoPGF2α and 5iPF2 VI reduced the velocity by 21% and 30%, respectively, compared to vehicle control. These results indicate that the sperm motility effects of PGs are structure- and composition-dependent in  C .  elegans .

Signature profile of cyclooxygenase-independent F2 series prostaglandins in C. elegans and their role in sperm motility