Drosophila is emerging as a model organism to investigate egg fertilization in insects and the possible conservation of molecular mechanisms of gamete interactions demonstrated in higher organisms. This study shows that the spermatozoa of several species of Drosophila belonging to the melanogaster group have a plasma membrane associated alpha-L-fucosidase with features in common with alpha-L-fucosidases from sperm of other animals, including mammals. The enzyme has been purified and completely characterized in D. ananassae, because of its stability in this species. The sperm alpha-L-fucosidase is an integral protein terminally mannosylated, with the catalytic site oriented toward the extracellular space. It has a M(r) of 256 kDa and a multimeric structure made up by subunits of 48 and 55 kDa. Enzyme characterization included kinetic properties, pI, optimal pH, and thermal stability. A soluble form of the enzyme similar to the sperm associated alpha-L-fucosidase is secreted by the seminal vesicles. Synthetic peptides designed from the deduced product of the D. melanogaster gene encoding an alpha-L-fucosidase were used to raise a specific polyclonal antibody. Immunofluorescence labeling of spermatozoa showed that the enzyme is present in the sperm plasma membrane overlying the acrosome and the tail. Lectin cytochemistry analysis of the egg surface indicated that alpha-L-fucose terminal residues are present on the chorion with a strongly polarized localization on the micropyle. The alpha-L-fucosidase of Drosophila sperm plasma membrane appears to be potentially involved in gamete recognition by interacting with its glycoside ligands present on the egg surface at the site of sperm entry.

We have studied the presence of four sperm glycosidases, alpha-mannosidase, alpha-L- fucosidase and two beta-hexosaminidase isoforms, in 11 species of the genus Drosophila spanning approximately an evolutionary 60 MY period, and in Scaptodrosophila lebanonensis, belonging to the ancestor genus Scaptodrosophila. These enzymes had been previously identified in Drosophila melanogaster as putative receptors for glycoconjugates of the egg surface. Alpha-mannosidase and beta-hexosaminidases are intrinsic proteins of the sperm plasma membrane in species closely related to D. melanogaster as well as in the divergent species D. willistoni, D. hydei, D. virilis, and S. lebanonensis. Alpha-L-fucosidase is restricted to the species of the genus Drosophila. Alpha-mannosidase and beta-hexosaminidases have been purified and characterized in all species. Their molecular masses and optimal pHs are similar in all the species, whereas interspecific differences in enzyme activities were detected. Cross-species comparison of kinetic parameters indicated a relationship between enzyme efficiency and phylogenetic relatedness. Beta-hexosaminidases were the most efficient enzymes. Lectin cytochemistry suggested the presence of carbohydrate residues complementary to the glycosidases on the eggshell at the site of sperm entry in all species. Bioinformatic analysis of the coding sequences of beta-hexosaminases and alpha-L-fucosidase and of their predicted products showed no evidence of positive selection of the genes coding for these enzymes and a high degree of sequence identity of the predicted polypeptides among the species of the genus Drosophila. Collectively, our findings indicate that the Drosophila sperm glycosidases are structurally and functionally conserved and strengthen the hypothesis of their involvement in the interactions with the egg surface.