SPERM-SURFACE CHYMOTRYPSIN-LIKE PROTEASE ACTIVITY REQUIRED FOR FERTILIZATION IN ASCIDIANS

During fertilization, species-specific gamete binding must be followed by sperm penetration of egg vestments before gamete fusion can occur. Sperm proteases, called lysins, aid this process. Sperm from Ascidia ceratodes, Ascidia callosa, and Ascidia paratropa were found to have a surface-mounted chymotrypsin-like protease when studied by enzymology , biotinylation, immunolabeling, and histochemistry. Chymotrypsin subs trates and inhibitors blocked fertilization in a concentration-depende nt manner in A. ceratodes and decreased the number of sperm heads whic h penetrated the egg's vitelline coat, but had no effect on sperm bind ing to follicle cells. Sperm bound to agarose beads coated with the ch ymotrypsin inhibitor alpha2-macroglobulin. Chymotrypsin-like enzyme ac tivity, assayed fluorimetrically using ucinyl-valinyl-tyrosinyl-7-amid o-4-methyl-coumarin as the substrate, was associated with head fractio ns prepared by differential centrifugation. Biotinylation of live sper m followed by detergent extraction showed that chymotrypsin-like activ ity could be removed from the detergent extract using avidin-agarose b eads. Indirect immunofluorescence of unreacted and reacted sperm heavi ly labeled membrane domains overlying the mitochondrion and at the bas e of the head with occasional labeling of the sperm tip. Histochemical studies, which used anyl-alanyl-prolyl-phenylalanyl-beta-naphthylamid e as the substrate, colocalized enzyme activity in head regions of unr eacted and near the mitochondrion of reacted sperm. Thus, we conclude that in ascidian sperm a chymotrypsin-like protease is exposed on the external surface of the plasma membrane of the head, is required for f ertilization, and plays a role in sperm penetration but not binding. ( C) 1994 Academic Press, Inc.