Abstract:
Fertile adults and occasionally twins have been derived from murine blastomeres at the 2-cell stage, indicating that such blastomeres may be equivalently totipotent, but there are conflicting reports that individual blastomeres from 2-cell stage murine conceptuses make different contributions to the embryonic and abembryonic regions of the blastocyst, implying that they differ in developmental potential. Here, we have re-examined this subject using 2 mouse strains, CF1 and NIH Swiss (SW), and 2 experimental approaches, random blastomere destruction at the 2-cell stage by repeated insertion of a needle into its nucleus and lineage tracing with the dye, DiI-CM. The manipulated conceptuses and untreated controls were cultured in KSOM-AA to morula and blastocyst stages (84 or 108 h pc, respectively), fixed, and immunostained for Oct4 and Cdx2. Antigen distribution, number of nuclei (stained by 42,6-diamidino-2-phenylindole), and cell progeny labeled with DiI-CM were examined by confocal laser scanning microscopy. Cell numbers are means � SD and were analyzed by a Student t-test. Cells positive for Cdx2 were assumed to represent trophectoderm or trophectoderm precursors, ones positive for Oct4 but negative for Cdx2 (Oct+Cdx-) inner cell mass. Ablation of a blastomere failed to prevent developmental progression in either strain, but the total number of cells at both morula (SW 11.4 � 3.3 v. 19.2 � 7.1; CF1 10.1 � 2.5 v. 22.1 � 6.4) and blastocyst (SW 48.6 � 7.4 v. 69.4 � 9.9; CF1 24.8 � 6.2 v. 53.8 � 13.5) was significantly reduced. In SW, the average fraction of Oct+Cdx- cells after blastomere ablation was significantly lower (P < 0.05) than in controls in morulae (0.47 � 0.2 v. 0.65 � 0.1) but not in blastocysts (0.33 � 0.1 and 0.34 � 0.1). In CF1, the fraction of Oct+Cdx- cells was lower (P < 0.05) than controls in both morulae and blastocysts (0.31 � 0.2 v. 0.58 � 0.2 and 0.18 � 0.1 v. 0.27 � 0.04, respectively). The CF1 morulae fell mainly into 2 groups, one low fraction (≤0.3, 54%) of Oct+Cdx- cells and the other with a more normal fraction (0.3 to 0.8, 43%) relative to controls. A majority of NIH Swiss morulae had an Oct+Cdx- cell fraction >0.4 and in this respect resembled controls. We then examined these strain differences by lineage tracing. The majority of SW blastocysts (65%, n = 34) demonstrated a random localization of DiI-labeled cell progeny (i.e., there was no preferential distribution of labeled cells to either the embryonic or abembryonic poles). By contrast, in CF1 (n = 38), 32% of blastocysts had labeled cells confined to their embryonic end and 42% with DiI-labeled, Cdx2-positive cells clustered at the abembryonic locale. A random localization was observed in 26% of blastocysts. In conclusion, these data confirm that there is plasticity in early mouse development but also suggest that in CF1, but not in SW conceptuses, blastomeres at the 2-cell stage differ in their abilities to contribute to the embryonic pole. Similar strain differences may explain the disagreements among studies on lineage tracing in early cleavage stage conceptuses.