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In the process of fertilization, most mammalian spermatozoa must penetrate through a series of egg investments before reaching the vitelline membrane. By contrast, cock spermatozoa have only to penetrate the vitelline membrane of the hen's ovum. The composition of the vitelline membrane of the hen's ovum at the time of ovulation has been described by Bellairs, Harkness & Harkness (1963) as a single layer of connective tissue-like protein. Sperm penetration of the investments surrounding mammalian ova is aided by hydrolytic enzymes contained within the sperm acrosome. One of these enzymes is a trypsin-like enzyme (TLE) or protease which has been demon- strated by Stambaugh & Buckley (1969) to be active in the dissolution of the zona pellucida of rabbit ova. Cock spermatozoa have also been shown to con- tain a high trypsin-like enzymatic activity (Buruiana, 1956; Ho & Meizel, 1970). More recently, Polakoski (1972) has shown that the high trypsin-like activity is present in the acrosomes (acrosomal extracts) of cock spermatozoa. Since the avian ovum is surrounded by a vitelline membrane and no other investments at the time of ovulation and fertilization, penetration of the vitelline membrane by a trypsin-like acrosomal enzyme was evaluated using an in-vitro test system. Recently ovulated ova were collected from the infundibulum of White Leghorn hens. Prediction of the time of ovulation was based on know¬ ledge of the time of oviposition. On average, ovulation of the succeeding ovum usually occurs 30 min after oviposition (Olsen & Neher, 1948). After their recovery, all ova were placed individually in plastic shells (PL 1, Fig. la) and suspended in 15 ml modified Ringer's solution (Olsen & Nehr, 1948). Recently collected, undiluted pooled semen (0-05 ml) was added to each of fifteen ova (referred to as the untreated control group). Of the fifteen ova, twelve were collected from the infundibulum and three from the magnum. Pooled semen samples were collected by the method of Burrows & Quinn (1937). A second group of ten ova (referred to as the treated group) was exposed to 005 ml pooled semen incubated with tosyl-L-lysine chloromethyl ketone (TLCK), a synthetic irreversible trypsin inhibitor. The inhibitor was dissolved in modified Ringer's solution at a concentration of 1000 µg/ml. Each 005 ml semen was incubated for 20 min at 37° C with 0-10 ml TLCK solution (100/tg TLCK/ 0-05 ml semen). Sperm motility was observed before and after incubation with

EVIDENCE FOR THE PENETRATION OF THE VITELLINE MEMBRANE OF THE HEN'S OVUM BY A TRYPSIN-LIKE ACROSOMAL ENZYME