CLEAVAGES AND GENOME ACTIVATION

In the zygote, an S-phase is completed during the first post-fertilization cell cycle. Thus, when the zygote cleaves to the 2-cell embryo at the first mitosis, each of the two cells, referred to as blastomeres, obtains its full copy of the embryonic genome (Figs. 2-1, 6-1). The embryo is still surrounded by the zona pellucida and remains so for some days. A number of mitotic divisions follow. During this phase of development, the mitotic divisions are special in that they occur almost without cellular growth; the cells become smaller and smaller as the original cytoplasm of the zygote is divided into smaller and smaller portions. These cell divisions are referred to as cleavages. The blastomeres may be of unequal sizes because of asynchrony in cleavage. This asynchrony becomes apparent from the outset; even between the 2- and the 4-cell stages, it results in temporary 3-cell embryos. At least in the mouse, the point of sperm entry may position the plane of the first cleavage division. Furthermore, the 2-cell-stage blastomere inheriting the sperm entry site tends to divide earlier than the other and is more likely to give rise to cells that become positioned internally in the growing ball of cells.

Fig. 6-1: Sections of a bovine zygote (A) with two pronuclei (arrows) and of a 2-cell embryo (B) with nuclei (arrows)

Cleavages begin during the transport of the embryo through the oviduct but, at a species-specific stage of development, the embryo enters the uterus (Table 6-1). The mare is very unusual with regard to passage through the oviduct; only embryos are allowed to enter the uterus while unfertilized oocytes, by an unknown mechanism, are retained in the oviduct.

Table 6-1: Times of passage of the embryo from the oviduct into the uterus, and of blastocyst formation, in different species

During the growth of the oocyte, transcripts and proteins are stored in this specialized cell for later use (Fig. 6-2). At the end of the growth phase transcription diminishes. However, by then the oocyte is more or less loaded with the transcripts and proteins required for driving initial embryonic development and these govern at least the first cleavage. The transcripts and proteins are gradually degraded after fertilization and, at a certain stage of development, activity of the embryonic genome is required. The embryonic genome is activated gradually; transcription is very limited initially but increases later, at species-specific stages, in two phases – those of minor and major activation of the embryonic genome (Table 6-2).

Fig. 6-2: Maternal versus embryonic control of initial embryonic development. During the oocyte growth phase in the follicles, transcripts and proteins are accumulated in the oocyte. With development, these components are gradually used and degraded. In parallel, the embryonic genome undergoes first a minor and later a major activation. The latter occurs at the 4-cell stage in pig and the 8-cell stage in cattle.

Table 6-2: Timing of the minor and major activation of the embryonic genome in different species

After a few cell divisions, the embryo takes the shape of a small ball of cells referred to as a morula after the Latin name for mulberry.

COMPACTION

Individual cells of the morula all look identical to start with, their spherical shapes giving the morula its typical mulberry-like appearance. Later, however, the outer cells differentiate into an epithelium, attach firmly to each other, and give the embryo a smoother surface. This process is referred to as compaction (Fig. 6-3). The outer cells constitute the trophectoderm or trophoblast. In the present book, the term trophectoderm will be used before placentation and the term trophoblast when these cells become engaged in placental formation. The firm attachment between neighbouring cells of the trophectoderm results from specialized intercellular junctions including tight junctions and desmosomes. Hence, a typical epithelium with apical and basolateral cell compartments is formed. There are some species differences in the timing of compaction: in the pig it occurs very early in development, around the 8-cell stage, whereas in cattle it happens later, around the 16- to 32-cell stage.

Fig. 6-3: Sections of pig embryos. A: 4-cell embryo presenting three blastomeres and one nucleus (arrow) in the section. 1: Zona pellucida. B: Morula in the process of compaction. Note the close connections (arrow) between the outer cells. 1: Zona pellucida. C: Blastocyst. 1: Zona pellucida; 2: Trophectoderm; 3: ICM. D: Expanded blastocyst. 1: Zona pellucida; 2: Trophectoderm; 3: ICM.

At the molecular level, differentiation of outer blastomeres, at least in the mouse, appears to rely on down-regulation of the transcription factor Oct4, followed by an up-regulation of other transcription factors such as Cdx2 and Eomesodermin (Fig. 6-4). The inner cells, meanwhile, retain expression of Oct4.

Fig. 6-4: The sequential action of the transcription factors Oct4, Nanog, Cdx2, and GATA-6 during initial cell differentiation in the mouse embryo. At compaction two cell lineages are derived from the totipotent blastomeres: the pluripotent inner cell mass (ICM) and the trophectoderm. The epiblast later differentiates into hypoblast and epiblast.