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 penetration ofthe 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.
Enzymes which dissolve the zona pellucida have been extracted from the rabbit sperm head and characterized as having the enzymic properties of hyaluronidase and trypsin. Their combined actions produce a rapid and complete dissolution of the zona pellucida, but the vitellus and its membrane are unaffected by prolonged exposure to this enzyme complex. Inhibition of the proteolytic component with lima bean or soybean inhibitors of trypsin prevents dissolution of the zona pellucida by the extract.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.