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A sucrose density gradient centrifugation procedure for separating the subcellular components of washed and sonicated rabbit epididymal spermatozoa has been developed and applied to the subcellular localization of several sperm enzymes, including the acrosomal enzymes involved in penetrationofthe zona pellucida by the spermatozoon. These zona penetration enzymes were identified as hyaluronidase and a trypsin-like enzyme. They were extracted as a single complex, which sedimented in a sucrose gradient in the vicinity of proteins with molecular weights of 59,000. The combined action of these two enzymes in the acrosomal extract produces a rapid and complete dissolution of the

IDENTIFICATION AND SUBCELLULAR LOCALIZATION OF THE ENZYMES EFFECTING PENETRATION OF THE ZONA PELLUCIDA BY RABBIT SPERMATOZOA