The ovum is surrounded by the cumulus, the corona, the zona and vitelline membrane. Capacitated rabbit sperm undergo the acrosome reaction during the passage through the cumulus. Enzymes released following the acrosome reaction or enzymes still bound to the vesicles composed of the plasma (PM) and the outer acrosomal membranes (OAM) hydrolyse the cumulus matrix and allow sperm to reach the corona cells and sperm which are left with a limiting inner acrosomal membrane (IAM) encounter the zona pellucida. The sperm binding to the zona and its subsequent penetration may involve a sequential action of binding to the zona and its subsequent penetration may involve a sequential action of acrosomal hydrolases bound to IAM. The fusion of spermatozoa with vitelline membrane probably involves phospholipases of the persistent PM and OAM on the equatorial segment (ES) and the post acrosomal region (PAR). The subsequent exocytosis of corticle granules may also be triggered by sperm enzymes. This sequence of events provides the rationale for this proposal. The overall objective of this proposal is to elucidate further the role of acrosomal enzymes in fertilization. Specifically, we will isolate and compare enzymes associated with acrosomes; determine the localization of key enzymes involved in sperm-egg binding; isolate acrosomal enzymes from rabbit spermatozoa and determine their effects on rabbit ova. After the extraction of acrosomal enzymes, sperm still contain nearly one-half of neuraminidase, one third of arylsulfatase, and one third of hyaluronidase. these enzymes can be extracted by further extraction of the denuded sperm with 1% Triton X-100 and 0.1 M CaCl2. This treatment also solubilizes the equatorial segment. We will follow this extraction procedure by studying simultaneous changes in sperm morphology at the ultrastructural level. In view of increasing importance of glycosyltransferase and sialic acid in gamete interactions, we will attempt localization of these and other acrosomal enzymes by immunofluorescence, radiolabeling, and cytochemical techniques. The enzyme purification will employ affinity chromatography on hydrophobic columns, high pressure liquid chromatography coupled with conventional methods. The role of acrosomal enzymes in sperm-egg binding, and the ovum penetration will be studied. Enzymes will also be tested on isolated zona.
