E-cadherin (uvomorulin)-mediated cell interactions are essential for preimplantation development in mammals. We observed that E-cadherin is expressed at contact sites between blastomeres of 2-cell mouse embryos of non-blocking genotype (CBA x C57BL F1) explanted at 32 h post human chorionic gonadotrophin (HCG) and cultured in vitro, while blastomere rounding and reduced zones of contact and E-cadherin-staining were observed in embryos of a blocking strain (MF1) arrested at the 2-cell stage. Embryos of MF1 strain can be rescued by aggregation with four 2-cell embryos of the non-blocking genotype. An early event in rescue is E-cadherin expression at contact zones between adjacent embryos of different genotype in aggregation chimeras. E-cadherin-mediated signalling appears important for the rescue (including formation of adherens-like contacts, cell polarization and morphogenetic processes) since there is no rescue when E-cadherin-specific antibodies are present during phytohaemagglutinin-mediated aggregation and subsequent culture. In blocked embryos, the distribution of microtubules is disturbed and concomitantly mitochondria cluster around the nucleus. Rescue by aggregation retains normal mitochondrial distribution in the presence of a dense microtubular lattice in all blastomeres. Therefore, E-cadherin-mediated signalling and its downstream effects on cytoskeletal organization are essential in the rescue of blocking embryos by aggregation. Normal preimplantation development appears to be dependent on the polarized expression of surface E-cadherin and the microtubule-mediated dispersal of mitochondria.
The capacity of sister blastomeres of mouse embryos for induced fusion changed during the 2-cell stage. It was at low level (24%) at the early 2-cell stage, increased and reached 98.5% at the middle 2-cell stage and fell sharply to 31% at the late 2-cell stage. At the time corresponding to the G 2 /Mphase of the cell cycle the blastomeres fused in only 8% of cases. Vital staining of 2-cell embryos by rhodamine 123 showed that the mitochondria were dispersed throughout the cytoplasm with a ringlike (around the nucleus) or spot-like (over the metaphase plate) concentration in the centre of each blastomere. At the periphery of blastomeres the mitochondrial content was low. The behaviour of the mitochondria reflected the subsequent events of structural and functional integration of the sister blastomeres under induced fusion: a discernible boundary between partners during 30min after electrofusion or 1 h after fusion with polyethylene glycol; movement of the two 'rings' to the centre of the blastomere fusion products (BFP) to form one large bright 'spot' over the common metaphase plate; mitochondria outlining the shape of the spindle and connection between sister blastomeres until completion of the first mitosis of BFP. The data obtained suggest that fusion of the blastomeres does not lead to extensive changes in the hybrid cytoplasm and integration of nuclear material is taking place only at metaphase stage. Cytogenetic examination of BFP at the 2-cell stage confirmed reconstruction of the tetraploid embryos and found that sister blastomeres of such embryos could asynchronously enter the next cleavage division similarly to normal diploid 2-cell embryos.
Embryos from certain mouse strains are arrested at the 2-cell stage in cell culture (‘2-cell block’), whereas those from other strains develop to the blastocyst stage under the same conditions. It was previously shown that blocking embryos can be rescued in culture by aggregation with an excess of 2-cell stages of a non-blocking strain such as CBA × C57BL/6 F2. Here we have employed a LacZ transgene in a blocking strain (NMRI) to follow the fate of rescued blastomeres up to the blastocyst stage. We found that rescued blastomeres can participate in both inner cell mass and trophoblast formation, thus completely overcoming the 2-cell block.
SummaryUnder our culture conditions, mouse embryos from the BALB/c inbred mouse strain develop successfully in culture only from the late 2-cell stage onwards (so-called 2-cell block), whether or not EDTA is added to the culture medium. (CBA × C57BL) F2 embryos do not exhibit a 2-cell block. Medium conditioned by culture of non-blocking embryos from the 2-cell to the 8-cell stage did not improve the development of blocking embryos, nor did co-culture of blocking and non-blocking embryos, with or without conditioned medium. On the other hand phytohaemagglutinin (PHA)-assisted aggregation of an early 2-cell BALB/c embryo with five surrounding non-blocking F2 embryos (2-cell or 8-cell) or five BALB/c 8-cell embryos allowed the early 2-cell BALB/c embryos to develop into blastocysts within 72 h. Aggregation of blocking BALB/c 2-cell embryos with each other had no ‘rescue’ effect. When blocking and non-blocking 2-cell embryos were aggregated together, an integrated blastocyst was formed; but when the early 2-cell BALB/c embryos were aggregated with non-blocking 8-cell embryos, the blocking embryos formed a separate small blastocyst, which nonetheless retained adherent contact with the non-blocking embryos throughout the culture period. Ultrastructural analysis showed that 2-cell embryos aggregated with the aid of PHA form close adherent cell contacts up to several micrometres in length.
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