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

Abstract

Summary. 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 enzymesinvolved in penetration of the 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 zona pellucida, which can be completely inhibited by soybean or lima bean trypsin inhibitors. Thus, penetration of the zona pellucida must be an enzymatic process involving mainly the trypsin-like enzyme of the acrosome. The subcellular localization of catalase, carbonic anhydrase, and the two kinetic forms of lactic dehydrogenase are also described to demonstrate the contrast in subcellular localization.