The effect of `Protease' on the zona pellucida of fertilized and unfertilized sheep eggs was examined, and the effects of the treatment and of mechanical removal of part of the zona on the subsequent development of fertilized eggs was assessed in vitro and in vivo.
The zonae of follicular eggs were digested by `Protease' within 25 min of exposure. In ovulated eggs, the resistance of the zona to digestion decreased with age. Zonae of eggs collected 2 days after oestrus (Day 2) were extremely resistant whereas nearly all the Day-5 eggs lost their zonae within 10 min. Fertilization had little effect upon the susceptibility of zonae to digestion by `Protease'.
Neither age of egg nor method of treating the zona had any effect on subsequent development of fertilized Day-2 and Day-6 eggs in culture, but there were effects of both factors on the proportion of eggs which developed to normal Day-25 embryos. `Protease' treatment of Day-2 and Day-3 eggs failed to digest the zona completely and a high proportion of such eggs developed to normal embryos whereas only a few eggs of similar age, in which part of the zona was removed mechanically, developed normally. The zonae of Day-4 to Day-6 eggs were invariably removed by `Protease' and the method of treatment had no effect on the proportion of eggs which developed to normal embryos. Irrespective of method of treatment, more Day-6 than Day-4 eggs developed to normal embryos. Culture for 2 days following treatment did not increase the survival of Day-6 eggs when they were transferred to recipients.
Ovulation and ageing of eggs appear to be associated with changes in the zona pellucida which influence its susceptibility to digestion by proteolytic enzymes. In early cleavage stage eggs, the major rôle of the zona may be protection of the inner cell mass from the uterine environment.
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