Fucosidases of Sperm and Milt in Darters (Percidae: Etheostomatini)

Darters (Percidae: Etheostomatini) offer attractive opportunities for analysis of the evolution of reproductive barriers and the molecular basis of fertilization. Here we report some basic characteristics of the sperm of darters, and, to our knowledge, the first report of sperm associated � -L-fucosidase in fishes. Fluorometric enzyme assays using 4- methylumbelliferyl-alpha-L-fucopyranoside (4-MU-fuc) as a substrate provided direct quantification of enzyme activity in both the soluble and cellular fractions of milt from all six species tested. Further analysis using SDS-PAGE and Western blotting revealed that fish sperm and milt contain � -L-fucosidase isoforms with molecular weights similar to those reported for human semen. Fertilization of darter ova by conspecific sperm, as assessed by development of the embryo to the 4 cell stage, was inhibited by pretreatment of sperm with the fucosidase-specific competitive inhibitor DFJ, implicating the sperm fucosidase in fertilization and/or early embryogenesis. Identification of sperm associated � -L- fucosidase in darters is consistent with the importance of carbohydrates during fertilization.