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Zona-free embryos at the 4-cell stage were classified into four types according to the total points of contact between the 4 blastomeres. Types A, B, C, and D had 3, 4, 5, and 6 points of contact, respectively. Embryos of these types were cultured individually in 20 microliters of Whitten's medium. Blastocysts derived from types A and D embryos at the 4-cell stage were transferred and observed on Day 17 of gestation. The number of live fetuses was significantly lower for type A than for type D embryos (p &lt; 0.05). In spite of the removal of the zone pellucida, both types A and D blastocysts had cell numbers similar to those in zona-intact control embryos at 96 h after insemination. However, type A embryos had fewer (p &lt; 0.05) inner cell mass (ICM) cells than did either the zona-intact control or type D embryos. Type D embryos did not have significantly fewer ICM than did the zona-intact control. Our data show that the cell association of zona-free embryos at the 4-cell stage influences differentiation of cells within the ICM and thus affects subsequent embryonic development. We suggest that one role of the zona pellucida after fertilization, in addition to protecting the preimplantation embryo, is to maintain the cell arrangement of 4-cell stage embryos to ensure subsequent successful cell lineage.

Developmental Ability of Zona-Free Mouse Embryos is Influenced by Cell Association at the 4-Cell Stage