Development of monozygotic twin mouse embryos from the time of blastomere separation at the two-cell stage to blastocyst

Abstract

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.

FIG. 1.

Embryo development after splitting two-cell stage embryos into individual blastomeres. Two-cell blastomeres were bisected microsurgically from a single two-cell stage embryo retrieved at 24 h pc and removed from the enclosing ZP. The two sister embryos were cultured individually as demiembryo A (A1–A4) and demiembryo B (B1–B4). At 60 h pc (24 h after bisection), both sisters had cleaved once (A1 and B1). By 72 h pc, the embryos were at the four-cell stage (A2 and B2) and showed signs of early compaction but with different conformations (see also Fig. 3). By 84 h, both embryos (A3 and B3) were around the eight-cell stage and had compacted fully even though they did not have the usual close-to-spherical shape of control morulae. Instead, the contour of the embryos was irregular, and the conformation of individual blastomeres was visible. By 108 h pc, one sister had formed a blastocyst (A4) whereas the other (B4) possessed a compacted cluster of cells and three outer cells (arrowed), one of which possessed a large vacuole. The B4 abnormal blastocyst would be classified as Cav in Tables 1 and 2. Bar = 50 μm.

FIG. 2.

Kinds of cavity observed in embryos at 108 h pc after development from bisected two-cell stage embryos. At 108 h pc, embryos derived from two-cell blastomere displayed a range of morphologies although the majority had advanced to form normal appearing blastocysts, often similar to the expanded blastocyst shown here (D) and in Figure 1A4. Some embryos with few cells displayed more than one vacuole (A), single blastomeres with an apparent vacuole (B), or a single small cavity (C). Embryos resembling A to C were categorized as Cav in Tables 1 and 2. Bar = 50 μm.

FIG. 3.

The main kinds of conformations adopted by zona-free, four-cell stage, murine embryos after development from blastomeres derived from bisected two-cell stage embryos. 3-1) These images of typical four-cell stage embryos were observed at 72 h pc and resemble the four conformations reported by others [40] for four-cell stage, zona-free mouse embryos. Image A represents an embryo with a flat conformation; B, although compacting, has a tetrahedral conformation; C and D have an open conformation never seen in control embryos enclosed in a ZP. Because the conformations were classified retrospectively from stored images, it was often difficult to distinguish the two classes with the open configuration, that is, type C from type D, because of their orientations in the images, particularly after compaction had begun. Bar = 50 μm. 3-2) The proportion of zona-free demiembryos that assumed the different conformations A–D shown in 3-1. 3-3) Types of four-cell conformations observed within pairs of demiembryos.

FIG. 4.

Numbers of cells in demiembryo pairs at 84 and 108 h pc. Embryos were derived by bisection at the two-cell stage and twin partners cultured separately for either 84 h (A and B) or 108 h (C and D). Bisected embryos were derived either from normally bred dams (N; A and C) or superovulated dams (S; B and D). At 84 h when the embryos were mainly morulae and at 108 h when most were blastocysts, groups of embryos were fixed, immunostained for CDX2 and POU5F1, and counterstained with DAPI. The total number of cells in each embryo was then counted by using confocal microscopy to provide optical sections through the entire structure [35]. The numbers on the x-axis for each bar graph refers to a pair of demiembryos that were arbitrarily designated demiembryo 1 and demiembryo 2. The plots illustrate the relative similarity in cell number within twin pairs of embryos at both morula and blastocyst.

FIG. 5.

The number of ICM (POU5F1+/CDX2−; black bars) and TE (POU5F1+/CDX2+; grey bars) cells in cultured embryos at 108 h pc. A) Values for 31 pairs of twin embryos derived by bisection at the two-cell stage of development. In each case, both partners (a and b) had 15 or more cells and at least 27 had a well-defined blastocoel cavity. The data are derived from 13 N embryo pairs and 18 S embryo pairs. B) Values for 33 control (nonmanipulated) CF-1 embryos. C) Values from 20 zona-enclosed demiembryos in which the partner blastomere had been damaged at the two-cell stage of development to prevent it from dividing. In all the treatments, dividing cells that failed to stain for either antigen are not included in the analyses.

FIG. 6.

Expression of POU5F1 and CDX2 in sister blastocysts at 108 h pc after development from bisected two-cell stage embryos. A) These are typical examples of sister blastocysts at 108 h pc and show projection images (derived from optical sections obtained at 3 μm intervals) for CDX2 (green, upper left), POU5F1 (red, upper right), DAPI staining (blue, lower left), and a merged image of the three projection images and a differential interference contrast image (lower right) of two sister blastocysts derived from the same two-cell stage embryo after blastomere separation. The blastocyst on the upper right of each panel has 5 POU5F1+/CDX2− positive cells (ICM) and 28 POU5F1+/CDX2+ positive cells (TE). The arrows in the panels show a cell at mitotic metaphase within that blastocyst; the arrow in the DAPI image shows the alignment of chromosomes within that cell, while the arrows in the POU5F1 and CDX2 images show diffuse, cytoplasmic staining for POU5F1 and CDX2, respectively, within that cell. Because it is impossible to judge the nuclear association of the POU5F1 and CDX2 signals, the cells in mitosis were eliminated from further assessment of ICM/TE cell numbers. The blastocyst on the lower left of each panel has 9 POU5F1+/CDX2− nuclei (ICM) and 29 POU5F1+/CDX2+ nuclei (TE). The fraction of POU5F1+/CDX2− cells for the upper embryo is 0.15 (5/33) and for the lower embryo 0.24 (9/38). Bar = 50 μm. B) This panel shows a more extreme case in which the ICM is unusually small in both twins. The blastocyst on the upper right has 4 POU5F1+/CDX2− nuclei (ICM) and 33 CDX2+ nuclei (TE) in which only very weak POU5F1 staining can be detected, probably because the blastocyst is well expanded. The blastocyst on the lower left has 2 POU5F1+/CDX2− nuclei (ICM) and 29 POU5F1+/CDX2+ nuclei (TE). Bar = 50 μm.