Heat sensitivity of the cortical granule protease from hamster eggs.

Previous studies (Gwatkin, Williams, Hartmann & Kniazuk, 1973) showed that the zona reaction of the hamster ovum is produced by a cortical granule protease, which inactivates the species-specific receptor-for-spermatozoa located in the zona pellucida. This receptor was earlier shown to be sensitive to low concentrations of pancreatic trypsin (Hartmann & Gwatkin, 1971). Several years ago Austin & Braden (1954) showed that exposure of the ovi¬ ducts of oestrous rats to 45°C for 8 to 12 min, just before the expected time of fertilization, increased the proportion of polyspermic eggs. The cause of this loss in the efficiency of the zona reaction was not known. We now report that exposure of hamster ova to heat before fertilization does not increase polyspermy significantly until the temperature is raised to 40°C. At this temperature, cortical granule discharge is inhibited, resulting in massive polyspermy. In contrast to the relative stability of the enzyme confined within the cortical granules, the free protease is sensitive to very slight elevations of temperature, undergoing partial inactivation even at 37°C. This fact has important physio¬ logical implications. Ova were collected from the oviducts ofgolden hamsters {Mesocricetus auratus) either 21 or 24 hr after the induction of ovulation with HCG, and their cumuli oophori were removed with hyaluronidase, as previously described (Gwatkin et al., 1973). The eggs were placed in 20-μ1 drops of medium 199M2 (Gwatkin & Andersen, 1973) on the bottom of plastic Petri dishes (60 mm in diam.) containing 10 ml mineral oil, previously equilibrated with KrebsRinger solution. The dishes were then submerged, up to the level of the oil, in water-baths adjusted to various temperatures between 37°C and 42°C. After 30 min, the eggs were removed, washed twice in 0-5-ml vols of medium 199M2 and exposed at 37CC to capacitated spermatozoa (Gwatkin, Andersen & Hutchison, 1972). Penetration of the eggs by the spermatozoa was recorded 90 min later. The presence of cortical granules in the penetrated eggs was