Significance of the Number of Embryonic Cells and the State of the Zona Pellucida for Hatching of Mouse Blastocysts in Vitro Versus in Vivo

We investigated the course of mouse blastocyst hatching in vitro after experimental modulation of the hatching process by growth hormone or by laser treatment and compared it to embryos grown in vivo. When embryos were grown in vitro, successful hatching was dependent on blastocyst expansion and was based on a minimum number of embryonic cells. Embryos grown in the presence of growth hormone were more advanced in their development and hatched earlier. When an artificial opening was laser-drilled into the zona pellucida, hatching occurred at lower numbers of embryonic cells. In vivo, escape from the zona pellucida occurred earlier and independent of blastocyst expansion. However, when we isolated in vivo-grown blastocysts with intact zonae that had developed in vivo and then cultured them in vitro, blastocysts started to expand and hatched the following day when a sufficiently high number of embryonic cells was present. Our data show that successful hatching in vitro is dependent on a sufficiently high number of embryonic cells, which enables blastocyst expansion and zona shedding. In vivo, the lower number of embryonic cells detected in zona-free blastocysts indicates that the underlying mechanism of zona escape is different, does not depend on blastocyst expansion, and presumably involves lytic factors from the uterus.