Abstract
The presence of protein in the zona pellucida of the mouse and rabbit ovum has been established by removal of the zona with Pronase (1) and trypsin (2, 3). The zona pellucida of the mouse (4-6), rat and rabbit was shown to be more resistant to digestion by trypsin after fertilization than before (2).
Sialic acid has been demonstrated in the zona pellucida of the rabbit ovum (7) and treatment of rabbit ova with bacterial neuraminidase reduced the number of penetrating sperm (8). The vitelline coat of invertebrate ova can be dissolved with cysteine or mercaptoethanol (9, 10). The zona pellucida is the analogous layer in mammalian ova and is also soluble in dilute mercaptoethanol solutions.
The sperm acrosome contains various enzymes, such as a trypsin-like enzyme (TLE) (11, 12), a neuraminidase (13), an enzyme that disperses the corona radiata (CPE) (14), and hyaluronidase.
The present study concerns biochemical changes in the zona pellucida at the time of fertilization as indicated by a change in the solubility of the zona pellucida in mercaptoethanol and by a change in susceptibility to digestion by trypsin. These changes occur at the time of fertilization and are induced in unfertilized ova by treatment with preparations of certain sperm acrosomal enzymes.
Materials and Methods. Preparation of zona pellucida solutions for protein estimation and electrophoresis. Mature New Zealand white rabbits were superovulated by four subcutaneous injections of 0.25 ml of FSH (Armour) at 12-hr intervals, followed by an intravenous injection of 125 units of HCG 12 hr after the last injection of FSH. Ova were flushed from the oviducts 12 hr after administration of HCG and suspended in sterile physiological saline. The cumulus and corona layers were removed by agitation, and the ova were washed with 1 to 2 ml of either physiological saline or glass distilled water.
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