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Calculation of the number of Cdx2 expressing cells versus total number of blastomeres within each of the 19 sets of embryo fragments showed that 78.3% blastomeres in experimental embryo fragments expressed Cdx2 (Table 2), which was higher and statistically significant (pQuinapyramine cells per embryo in experimental embryo fragments was higher than in intact embryos (23.8 versus 19; Tables 2 and S2). To obtain apolar 1/8 blastomeres described in the previous section we separated them from the pairs of 2/8 resulting from single 1/4 blastomere division. As the procedure of separation takes about 15�C30?min, we had to be sure that during that time the pair of 2/8 blastomeres did not polarize. We used the FITC-ConA staining, which detects formation of the cap of microvilli on the apical part of polar cell which is the early sign of polarization. We found that 2/8 couplets resulting from single 1/4 blastomeres cultured for 15?min and 30?min, remained apolar (Fig. 3B). The first signs of polarization were detected after 7?h of culture, and distinct cap of microvili was noticeable after 9?h. Similar sign of polarization was observed in control intact 8-cell embryos that developed from 4-cell stage embryos (Fig. GDC-0449 3B). This indicates that the 1/8 blastomeres separated from the 2/8 pairs within 15�C30?min after their formation, were apolar (Figs. 2A and MS-275 clinical trial 3C). However we found that during the next 10�C12?h, when the 1/8 divided and the embryo fragments were composed of two 1/16 blastomeres (2/16 pair), both of the cells became polarized, as was confirmed by ConA labeling (Fig. 3B and C). Taken together, we found that when embryo fragments were obtained from apolar 1/8 blastomeres, the polarization of blastomeres was delayed to the 16-cell stage. Because in the embryo fragments (in contrast to the intact embryos) all of the 1/16 blastomeres were polarized, this results in increase of number of Cdx2-positive cells In full sets of fragments obtained from both polar 1/8 blastomeres (Fig. 1A, Table 1) and apolar 1/8 blastomeres (Fig. 2A, Table 2), the 4-cell fragments (4/32 quartets) were the most frequent (82% and 80%, respectively). We divided all of the 4-cell embryo fragments into three groups depending on Cdx2 expression pattern �� those in which 2 out of 4 (4/2), 3 out of 4 (4/3) or all 4 (4/4) cells were Cdx2 positive. We found that among all of the 4-cells fragments obtained from polar 1/8 blastomeres, 62.8% of fragments showed the 4/2 pattern of Cdx2 expression, while the patterns 4/3 and 4/4 were observed in 15.2% and 22% of fragments, respectively (Fig. 4A and B).
