The effect of superovulation on the contributions of individual blastomeres from 2-cell stage CF1 mouse embryos to the blastocyst

Abstract

It remains controversial whether blastomeres of 2-cell stage mouse embryos show bias in their contribution to the blastocyst and whether there is any effect of superovulation. Two-cell stage embryos from CF1 mice were derived by either natural breeding (N) or superovulation (S) and cultured in vitro. At blastocyst, inner cell mass and trophectoderm were distinguished by Cdx2 and Oct4 immunostaining. A fluorescent dye (CM-Dil) was also used to tag individual blastomeres at the 2-cell stage, and the descendant cells identified by their red fluorescence. S and N embryos developed to blastocyst at the same rate and contained a similar number of cells. However, with S embryos, the descendants of the blastomere labeled with CM-DiI contributed predominantly to either the embryonic or abembryonic pole about 70% of the time, whereas most N embryos displayed random patterning, with no restriction to one or other of the poles. In S-embryos, but not N-embryos, the leading blastomere at second cleavage contributed preferentially to the embryonic pole of the blastocyst and the lagging blastomere to the abembryonic pole and hence mural trophectoderm. In addition, a tetrahedral rather than a flat morphology at the 4-cell stage of S-embryos was strongly biased to displaying the embryonic/abembryonic pattern at blastocyst. In contrast, S-embryos lacking a zona pellucida resembled N embryos in their patterning. In CF1 mice, superovulation has little effect on development to blastocyst, but enforces a greater degree of lineage restriction than natural breeding, most likely through constraints imposed by the zona pellucida.

Fig. 1.. Relative localization of Oct4 and Cdx2 in CF1 murine blastocysts.

These are images of embryos (108 h pc) from CF-1 superovulated dams created by projection of series of confocal sections (4–6 μm each) for Oct4 (upper left), Cdx2 (upper right) and DAPI (lower left). (A) early blastocyst; (B) hatching blastocyst. Arrowheads in (A) indicate Oct4+/Cdx2− cells, namely ICM, in an early blastocyst in which Oct4 is expressed relatively uniformly in both ICM and TE. By contrast, in a more advanced, hatching blastocyst (B), Oct4 is expressed much more strongly in ICM than in TE. Scale bars represent 50 μm.

Fig. 2.. Cell fates of blastomeres tagged at the 2-cell stage of development with DiI-CM.

After tagging one randomly chosen blastomere with the membrane dye, DiI-CM (red) at the 2-cell stage of development, embryos were allowed to proceed in their development through various intermediary stages until they reached blastocyst. Each embryo was counter stained with DAPI (blue) and anti-Cdx2 antibody (green). (A) 2-cell stage embryos; (B) 4-cell stage; (C) 8-cell stage; (D) morula. In blastocysts, labeled cell progeny were frequently concentrated towards either the abembryonic pole [(E) red cells located opposite the ICM] or the embryonic pole [(F) red cells associated with the region occupied by the ICM and polar trophectoderm]. Supplementary Fig. S1 A–F provides the original confocal stacks for Fig. 2 A–F, and also for a blastocyst with a typically random distribution of DiI (Supplementary Fig. S1G) in which there was no preferential localization to either the embryonic or abembryonic poles.