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Identification of hydrolase binding activities of the acrosomal matrix of hamster spermatozoa.

The interior of the mammalian sperm acrosome contains a structural framework, the acrosomal matrix, that may regulate both the distribution of hydrolases within the acrosome and their release during the acrosome reaction. To define the biochemical basis of this interaction, we examined the binding of two acrosomal hydrolase, proacrosin and N-acetylglucosaminidase (NAGA), to a purified acrosomal matrix fraction of hamster spermatozoa. Proacrosin-acrosin was chromatographically purified from acid extracts of hamster spermatozoa and consisted of four size variants of 50 kDa, 49 kDa, 45 kDa, and 43 kDa. Each of the four isoforms exhibited the same N-terminal amino acid sequence through 16 residues, suggesting that they may be modified by cleavage at the C-terminus. Polyclonal antiserum against the proacrosin isoforms specifically binds the acrosomal cap as shown by immunofluorescence microscopy. Neither proacrosin nor NAGA were solubilized when sperm were permeabilized with Triton X-100 under low ionic strength conditions; however, both hydrolases were releases by extraction with Triton X-100 containing 0.5 M NaCl. An acrosomal matrix fraction isolated under low ionic strength conditions retained bound proacrosin-acrosin and NAGA, and both hydrolases were released from the matrix by subsequent high-salt extraction. After high-salt treatment, the acrosomal matrix retained specific binding sites for both proacrosin and NAGA. In a blot overlay assay, a set of acrosomal matrix polypeptides between 29 kDa and 24 kDa specifically bound proacrosin. These data suggest that specific interactions between acrosomal matrix polypeptides and hydrolases represent a mechanism to sequester hydrolases within the acrosome and to regulate their release during the acrosome reaction.