Sperm surface beta-N-acetylhexosaminidases are among the molecules mediating early gamete interactions in invertebrates and vertebrates, including man. The plasma membrane of Drosophila spermatozoa contains two beta-N-acetylhexosaminidases, DmHEXA and DmHEXB, which are required for egg fertilization. Here, we demonstrate that three putative Drosophila melanogaster genes predicted to code for beta-N-acetylhexosaminidases, Hexo1, Hexo2, and fdl, are all expressed in the male germ line. fdl codes for a homolog of the alpha-subunit of the mammalian lysosomal beta-N-acetylhexosaminidase Hex A. Hexo1 and Hexo2 encode two homologs of the beta-subunit of all known beta-N-acetylhexosaminidases, which we have named beta(1) and beta(2), respectively. Immunoblot analysis of sperm proteins indicated that the gene products associate in different heterodimeric combinations forming DmHEXA, with an alphabeta(2) structure, and DmHEXB, with a beta(1)beta(2) structure. Immunofluorescence demonstrated that all the gene products localized to the sperm plasma membrane. Although none of the genes was testis-specific, fdl was highly and preferentially expressed in the testis, whereas Hexo1 and Hexo2 showed broader tissue expression. Enzyme assays carried out on testis and on a variety of somatic tissues corroborated the results of gene expression analysis. These findings for the first time show the in vivo expression in insects of genes encoding beta-N-acetylhexosaminidases, the only molecules so far identified as involved in sperm/egg recognition in this class, whereas in mammals, the organisms where these enzymes have been best studied, only two types of polypeptide chains forming dimeric functional beta-N-acetylhexosaminidases are present in Drosophila three different gene products are available that might generate numerous dimeric isoforms.

An infertile man presented a spermiogram in which 100% of the spermatozoa displayed separation of head from tail at the level of the proximal centriole. Most tails were normally structured and ended anteriorly with the proximal centriole covered by a continuous plasma membrane. In a small percentage of tails a rudimentary connecting piece was surrounded by a minute cytoplasmic mass and the middle piece was missing, whereas the chromatoid body and the spindle-shaped body were still present. Finally, a few tails had a large cytoplasmic mass surrounding either regular connecting and middle pieces or a rudimentary connecting piece continuous with the main piece. Tails of the first type had good forward motility, although the pattern of movement appeared altered. The other types were immotile or motile but without forward progression. In the loose heads the implantation fossa had failed to differentiate. The separation of heads from tails appeared to be the result of a specific morphogenetic defect and took place at different stages of spermatid differentiation, giving rise to the structurally different types of tails.

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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.

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