Development of Monozygotic Twin Mouse Embryos from the Time of Blastomere Separation at the Two-Cell Stage to Blastocyst

The development of blastomeres separated from two-cell stage murine embryos has been compared. Blastomeres were removed from the zona pellucida (ZP) and cultured individually; the twin embryos were compared during their progression to blastocyst in terms of development rate, cell number, morphology, conformation at the four-cell stage, and CDX2 and POU5F1 (also known as OCT4) expression. In general, twin embryos, whether obtained from superovulated or normally bred dams, displayed comparable cell numbers as they advanced. They formed morulae and blastocysts more or less synchronously with each other and with control embryos, although possessing about half of the latter's cell number. Despite this apparent synchrony, the majority of twin blastocysts differed in terms of their relative complements of POU5F1+/CDX2- cells, which represent inner cell mass (ICM), and POU5F1+/CDX2+ cells, which identify trophectoderm (TE). Many, but not all, exhibited a disproportionately small ICM. By contrast, demiembryos retained within their ZP and created by randomly damaging one of the two blastomeres in two-cell stage embryos exhibited a more normal ratio of ICM to TE cells at blastocyst and significantly less variance in ICM cell number. One possible explanation is that ZP-free demiembryos only infrequently adopt the same conformation as their partners, including the favorable tetrahedral form, at the four-cell stage, suggesting that such embryos exhibit a high degree of plasticity with regard to the orientation of their first two cleavage planes and that a significant number likely deviate from paths that provide an optimal geometric progression to blastocyst. These data could explain the difficulty of creating monozygotic twins from two-cell stage embryos.