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

摘要

Darters (Percidae: Etheostomatini) offer attractive opportunities for analysis of the evolution of reproductivebarriers and the molecular basis of fertilization. Here we report some basic characteristics of the sperm of darters, and, toour 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 inboth the soluble and cellular fractions of milt from all six species tested. Further analysis using SDS-PAGE and Westernblotting revealed that fish sperm and milt contain-L-fucosidase isoforms with molecular weights similar to thosereported for human semen. Fertilization of darter ova by conspecific sperm, as assessed by development of the embryo tothe 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-Lfucosidasein darters is consistent with the importance of carbohydrates during fertilization.