Influences of in vitro oocyte aging on microfertilization in the mouse with reference to zona hardening

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Abstract
Our purpose was to investigate the influence of in vitro oocyte aging on fertilization and subsequent embryonic development following subzonal sperm injection with reference to spontaneous zona hardening in the mouse. First cleavage rate was significantly higher in long vs short (69 vs 41%, P < 0.001). However, significantly higher blastocyst formation and hatching were observed in short than long (50 vs 7%, P < 0.001, and 86 vs 50%, P < 0.001, respectively). No significant differences were found for two pronuclei formation between short and long (30 vs 27%). Sham injection revealed a significantly higher parthenogenetic activation in long than short (19 vs 2%, P < 0.001). Zona digestion required a significantly longer time for long compared to short (trypsin—37 vs 29 min, P < 0.01; pronase—17 vs 14 min, P < 0.001). Prolonged culture of mature mouse oocytes in vitro alters the zona pellucida, increases the rate of parthenogenetic activation by subzonal sperm injection, and impairs subsequent embryonic development. Zona hardening appears to be an indicator of oocyte aging.