Prevention of fertilization by exposure of hamster eggs to soluble acrosin.

Incubation of hamster eggs with trypsin or chymotrypsin, but not with a series of glycosidases or lipases, prevents capacitated spermatozoa from binding to the zona pellucida so that fertilization is prevented (Hartmann & Gwatkin, 1971 ; Gwatkin, Williams & Andersen, 1973). Inactivation of the binding sites for spermatozoa on the zona pellucida by a trypsin-like protease released by discharge of cortical granules also appears to be responsible for the zona reaction which prevents polyspermy (Gwatkin, Williams, Hartmann & Kniazuk, 1973). Acrosin is also a trypsin-like protease and it can be extracted from sperm acrosomes. We therefore investigated whether the fertilization of hamster eggs is blocked by incubation with soluble acrosin. Eggs were collected from hamsters induced to superovulate and the cumuli oophori were removed with hyaluronidase as described previously (Gwatkin, Andersen & Hutchison, 1972). Groups of such cumulus-free eggs were incubated under oil in 20-ul drops of PBS (pH 7-3 ; Dulbecco & Vogt, 1954) containing various concentrations of trypsin or acrosin for 2 h at 37-5°C on a rocker (5-6 oscillations/min). The eggs were then washed in medium 199M2 (Gwatkin & Andersen, 1973) and added to 40-ul drops of a suspension of epididymal spermatozoa previously capacitated by incubation for 5 h in medium 199M2 containing cumulus cells (Gwatkin et a l . , 1972). After incubation for an additional 90 min, the number of spermatozoa bound to the eggs and the penetration of eggs by spermatozoa were recorded under a phase-contrast microscope. Preliminary experiments were carried out with purified boar acrosin (Fritz et a l . , 1975). At a protein concentration of approximately 10 ug (240 pmol acrosin)/ml, this enzyme completely inhibited sperm binding and penetration. In the remaining experiments, a range of molar concentrations of hamster and ram acrosin was used. These enzymes also blocked sperm binding, but for convenience only penetration was determined quantitatively. The molar concentration of the enzyme preparations was determined by fluorometric titration using 4-methylumbelliferyl-/?-guanidinobenzoate (Brown, Andani & Hartree, 1975a). Soluble ram acrosin was isolated from washed ejaculated ram spermatozoa as the fraction T D ' of Brown et a l . (1975b). This material was subjected to affinity chromatography on a column of Sepharose-4B linked to 4(4'-aminophenoxypropoxy)benzamidine (Jameson & Elmore, 1974). Acrosin was obtained as a single protein that contained 12 nmol enzyme/mg. Soluble hamster acrosin was obtained by acid extraction of washed epididymal spermatozoa, followed by chromatography of the extract, first on Sephadex G-100 and then on /j-benzamidine linked with a spacer arm to an insoluble matrix, according to the procedures of Fritz et a l . (1975). The hamster acrosin preparation contained 0-52 nmol enzyme/mg. The activities of these acrosin preparations were compared with that of bovine pancreatic trypsin (crystalline: Worthington Freehold), which contained 19 nmol enzyme/mg. The enzymes were dissolved in 0-5 mM-HCl and kept at 5°C overnight before being diluted to the required concentrations. As shown in Text-fig. 1 pretreatment of eggs with pancreatic trypsin for 2 h completely suppressed sperm penetration, confirming earlier results (Hartmann & Gwatkin, 1971): an inhibition of 50% was obtained with 0-2 pmol trypsin/ml. The acrosin preparations also suppressed sperm penetration, although higher enzyme concentrations were required and the dose-response curves were different. For 50% inhibition, 7 pmol hamster acrosin/ml and 20 pmol ram acrosin/ml were required. However, the amount of protease available to the eggs may have been lower than these values