Morula - an overview | ScienceDirect Topics (2024)

Morula Develops Fluid-Filled Cavity and Is Transformed Into Blastocyst

By 4 days of development, the morula, consisting now of about 30 cells, begins to absorb fluid. Several processes seem to be involved. First, as the trophoblast differentiates, it assembles into an epithelium in which adjacent cells are tightly adherent to one another. This adhesion results from the deposition on lateral cell surfaces ofE-cadherin, a calcium-dependent cell adhesion molecule, and the formation of intercellular junctions, specifically,tight junctions, gap junctions, adherens junctions, anddesmosomes. Second, forming trophoblast cells express a basally polarized membrane sodium/potassium ATPase (an energy-dependent ion-exchange pump), allowing them to transport and regulate the exchange of metabolites between the outside of the morula (i.e., the maternal environment of the oviduct) and the inside of the morula (i.e., toward the inner cell mass). The sodium/potassium ATPase pumps sodium into the interior of the morula, and water follows through osmosis to become blastocoelic fluid. As the hydrostatic pressure of the fluid increases, a large cavity called theblastocyst cavity (blastocoel) forms within the morula (seeFig. 1.16). The embryoblast cells (inner cell mass) then form a compact mass at one side of this cavity, and the trophoblast organizes into a thin, single-layered epithelium. The embryo is now called ablastocyst. The side of the blastocyst containing the inner cell mass is called theembryonic pole of the blastocyst, and the opposite side is called theabembryonic pole.

Implantation

As the trophoblasts proliferate, they form an inner cytotrophoblast layer of vigorously dividing cells and an outer layer of nonmitotic syncytiotrophoblasts. As cells of the cytotrophoblasts divide, the newly formed cells are incorporated into the syncytiotrophoblast layer, which enlarges, becomes vacuolated forming interconnected lacunae, and penetrates the endometrial lining. By the end of the 11th day postfertilization, the embryo and its layers have become embedded into the vascularized endometrium (Fig. 20.10; see Fig. 20.9).

Cleavage, Morula, Blastocyst

Following fertilization, cleavage occurs. This consists of a rapid succession of mitotic divisions that produce a mulberry-like mass known as a morula.Fluid is secreted by the outer cells of the morula, and a single fluid-filled cavity develops, known as the blastocyst cavity. An inner-cell mass can be defined, attached eccentrically to the outer layer of flattened cells; the latter becomes the trophoblast. The embryo at this stage of development is called ablastocyst, and the zona pellucida disappears at about this time. A blastocyst cell can be removed and tested for genetic imperfections without harming further development of the conceptus.

5.2 Energy Metabolism of ICM and TE

During the morula stage, prior to blastocoel formation, the first differentiation event takes place to produce the ICM and TE (Fig. 1A). It has been shown previously that the two major cell types, TE and ICM, have distinct energy requirements and metabolic pathways suited for their respective functions (Gopichandran & Leese, 2003; Hewitson & Leese, 1993; Houghton, 2006). For example, ICM and its in vitro derivative, ESCs, have high proliferative potential and use “aerobic” glycolysis for increasing biomass (Jang, Yang, Lee, & Cheong, 2015; Vander Heiden et al., 2009). TE, a columnar epithelium, requires copious amount of ATP for energy-expensive Na⁺/K⁺ ATPase pump to form and expand the blastocoel cavity. Hence, TE has much higher O₂ consumption than ICM presumably due to using OXPHOS to metabolize glucose, the most efficient pathway for producing ATP. In fact, TE has much higher number of mitochondria with morphologies that are associated with active OXPHOS, whereas mitochondria in ICM as well as in ESC exist in more spherical shape with little cristae as compared to those in TE (Cogliati et al., 2013; Houghton, 2006; Stern, Biggers, & Anderson, 1971). Whether changes in TE's mitochondrial morphology are induced after the initiation of TE differentiation or it is integral to the initiation of differentiation is yet to be determined.

A number of studies have suggested that the differences in energy metabolism between TE and ICM also reflect their relative pluripotency states. ICM, as pluripotent group of cells, shows relatively “quiet” metabolism driven mostly by glycolytic pathway, whereas TE displays OXPHOS-driven high energy metabolism, consistent with energy metabolism of other differentiated cells (Houghton, 2006; Teslaa & Teitell, 2015; Vander Heiden et al., 2009). During the induction of induced pluripotent stem cell (iPSC), metabolic reprogramming of OXPHOS-dominated “differentiated” energy metabolism reverts to glycolytic metabolism, consistent with association of OXPHOS with differentiated cells and glycolysis with pluripotent cells (Shyh-Chang et al., 2013; Teslaa & Teitell, 2015; Xu et al., 2013). Intriguingly, during iPSC induction of mouse embryo fibroblasts, changes in metabolic reprogramming seemed to occur prior to the full onset of expression of pluripotency-associated transcription factors such as Oct4, Nanog, and Sox2, suggesting that metabolic reprogramming may not just be a consequence of pluripotency state (Folmes et al., 2011). Furthermore, inhibition of glycolysis and OXPHOS reduced and promoted, respectively, the efficiency of pluripotency induction, supporting the hypothesis that metabolic reprogramming is a critical component of establishing pluripotency. Conversely, in order for pluripotent cells to differentiate, maturation of mitochondrial-mediated OXPHOS is required, consistent with the idea that metabolic reprogramming is essential and may precede the induction of differentiation (Chung et al., 2007; Mandal, Lindgren, Srivastava, Clark, & Banerjee, 2011).

Preimplantation Development

After fertilization the zygote moves through the fallopian tube to the endometrial cavity for implantation, usually around the seventh day postfertilization. During this time, many events must occur in both the zygote and the endometrium for successful continuation of the pregnancy. In the zygote the earliest events are controlled by maternally “stored” proteins and messenger RNA. Maternal factors control the earliest cell divisions, the first and likely second cleavage divisions. Zygotic control of cell divisions does not occur until zygotic transcription begins at the four- to eight-cell stage in the human.¹ This is termed the maternal-to-zygotic transition or major zygotic transition. Although the zygotic genome is biparental, the contributions to the early zygote from each parent are not equal. A phenomenon called imprinting² silences transcription from one or the other parent's allele. This process appears to be important in early human development and is discussed further later in this chapter.

The zygote then continues to undergo cleavage divisions, now under zygotic control, and is symmetric up to the morula stage, when there are 16 cells (about 3–4 days postfertilization). During early cleavage divisions these blastomeres are “totipotent” (able to form all embryonic and extraembryonic tissues). (Although they are totipotent, there is still some lineagebias due to heterogeneity of cellular contents and motility of nuclear factors.³) The morula then undergoes compaction in preparation for the next big event in embryogenesis, the formation of the blastocyst. At the compaction stage, cell junctions are formed and polarity to the zygote follows. The symmetric morula compacts and now has positional cell polarity with inner and outer cells. Based on murine literature, this polarized zygote shows the first lineage specification such that the outer cells will become trophectoderm and the inner cells will become the inner cell mass, which becomes the embryo (reviewed in Maltepe and Fisher⁴). (Although the inside-outside model of trophoblast lineage specification is perhaps the most accepted one, there are others that explain the phenomenon as well [for review, see Wennekamp and colleagues⁵]).

An early factor expressed from the zygotic genome is POU homeodomain class 5 transcription factor 1 (POU5F1, also known as octamer-binding transcription factor 4 [OCT4]) (Box 3.1), a critical protein involved in maintaining pluripotency.⁶ In the early human zygote, all cells express POU5F1 (OCT4) up to the blastocyst stage. As cells become determined, they express new specific markers of their lineage. The first lineage specification factor expressed in the outer cells of the compacted morula is caudal-type homeobox transcription factor 2 (CDX2).⁶ POU5F1 (OCT4) and CDX2 are coexpressed in the human morula in the outer cells destined to become trophectoderm.⁷

3 Asymmetric Division and the Establishment of the Outer/Inner Configuration of Polar/Apolar Cells

During the morula stage, there are two rounds of asymmetric division at the fourth (from 8- to 16-cell) and fifth (from 16- to 32-cell) cell division. After asymmetric division, the first two distinct cell populations emerge as cells that inherit the apical domain from their parental 8-cell blastomeres and ones that do not, resulting in polar and apolar cells, respectively. The polar cells take an outer position to become the TE and the apolar cells take an inner position to become the ICM, leading to formation of the outer/inner cells within the embryo.

Although asymmetric division is essential for the first lineage specification, the frequency of asymmetric division of 8-cell blastomeres appears to be context dependent. Isolated single 8-cell blastomeres exhibit a higher frequency of asymmetric division, approximately 82%, while couplets of 8-cell blastomeres display a frequency of only 50% (Johnson & Ziomek, 1981b). In intact embryos, the average asymmetric division frequency during the 8–16 cell division is approximately 60%–70% in total (Anani et al., 2014; Fleming, McConnell, Johnson, & Stevenson, 1989). This suggests that on average five or six blastomeres of the 8-cell embryo divide asymmetrically. However, in individual embryos, the frequency of asymmetric division is quite variable, ranging from only one blastomere to all eight dividing asymmetrically. What controls the decision between symmetric or asymmetric division in individual blastomeres is not fully known yet. In an early study by Pickering et al., they suggested that the size of the apical domain and the position of cell–cell contact influence the decision for asymmetric division (Johnson, 2009; Pickering, Maro, Johnson, & Skepper, 1988). In their model, the larger apical domain has a higher chance to divide symmetrically while the smaller one has a higher chance to divide asymmetrically. On the other hand, more recent studies suggest that cellular characteristics such as the cell shape during division (Dard, Le, Maro, & Louvet-Vallée, 2009) and the nuclear position before division (Ajduk, Biswas Shivhare, & Zernicka-Goetz, 2014) show some correlation with divisional orientation in intact embryos.

A recent study analyzing the orientation of mitotic spindles during the 8–16 cell division in isolated blastomeres demonstrated that the majority of cells aligned their mitotic spindle to the apico-basal axis regardless of the apical area encompassing the cell surface (Korotkevich et al., 2017). Destabilization of the apical domain in Cdc42^−/− mutant embryos leads to randomization of their orientation. In conjunction with this, the live imaging analysis of SAS4-GFP transgenic mouse embryos, which visualize microtubule-organizing centers (MTOC) localization, shows that acentrosomal spindle assembly occurs subapically during de novo apical domain formation. This suggests that the apical domain somehow recruits MTOC to regulate spindle orientation to facilitate asymmetric division, ensuring the production of the two cell populations at the 16-cell stage (Korotkevich et al., 2017). This notion is interesting because there are no astral microtubules in mitotic spindles of the early mouse embryo (Hiraoka, Golden, & Magnuson, 1989; Szollosi, Calarco, & Donahue, 1972). Astral microtubules are an essential component to reorient the mitotic spindle and align its orientation with cell polarity during asymmetric division in other systems (Bergstralh, Dawney, & St Johnston, 2017). Reorientation of the mitotic spindles has not yet been observed in early mouse embryos. Are mitotic spindles formed in an oriented manner without reorientation? High-resolution time-lapse analysis of spindle formation in early embryos will answer this question in the near future.

It was originally considered that the outer/inner configuration of the polar/apolar cells is regulated by divisional orientation (Johnson, 2009). When cell division is planar, both daughter cells are polar and placed at an outer position, while when cell division is orthogonal, one daughter cell inherits the whole surface apical domain to become an outer polar cell and the other daughter cell is deposited directly inside of the embryo, becoming an apolar inner cell. This simple idea was proposed based on the results of isolated blastomeres without direct observation of intact embryos. Apart from orthogonal and planar divisions, the existence of frequent oblique divisions was noticed in an early time-lapse study (Sutherland, Speed, & Calarco, 1990). This early observation was fully confirmed in recent studies using more sophisticated live-imaging techniques (Anani et al., 2014; McDole, Xiong, Iglesias, & Zheng, 2011; Samarage et al., 2015; Watanabe, Biggins, Tannan, & Srinivas, 2014; Yamanaka, Lanner, & Rossant, 2010). In intact embryos, many 8-cell blastomeres divide in an oblique orientation with respect to the embryo surface. Careful positional analysis revealed that in 16-cell stage embryos, only one or two cells fully take an inner position (Anani et al., 2014; Dietrich & Hiiragi, 2007). Additionally, there is a unique population that appears to occupy the intermediate nuclear position between the outer and inner positions (Anani et al., 2014; McDole et al., 2011; Watanabe et al., 2014). Many of these cells internalize into the inner position before the next division cycle (16–32 cell division). This suggests that divisional orientation is not a good predictor of their final cell allocation (Anani et al., 2014; Dard et al., 2009; Samarage et al., 2015; Watanabe et al., 2014). On the other hand, many divisions in an oblique orientation are asymmetric in terms of the inheritance of the apical domain (Anani et al., 2014; Yamanaka et al., 2010). Both daughter cells take an outer position soon after division, despite one of them being an apolar cell. This outer apolar cell later internalizes to take an inner position (Anani et al., 2014; Korotkevich et al., 2017; Maître et al., 2016). Interestingly, this is not fully deterministic; as some cells can occasionally repolarize and stay at an outer position to adopt the TE fate.

Cytotoxicity

In B. schlosseri, MCs degranulate and release their granular content upon the recognition of foreign molecules: this results in the induction of cytotoxicity. The effect is directly related to the presence, in the medium, of active PO released by MCs.^39,40 Experimental evidences indicate that cytotoxicity is related to the production of reactive oxygen species (ROS) by PO and the consequent induction of oxidative stress related to the depletion of reduced glutathione and total thiols.⁴¹ In agreement with this assumption, ROS scavengers (superoxide dismutase, catalase, and sorbitol) and PO inhibitors (sodium benzoate, phenylthiourea, and diethyldithiocarbamate) can significantly reduce the extent of cytotoxicity observed invitro when hemocytes are challenged with foreign molecules, such as those in the plasma from genetically incompatible colonies, on the surface of yeast (Saccharomyces cerevisiae) or gram-positive bacterial (Bacillus clausii) cells.^40,41

Diagnosis

Unlike the rarity of morulae in circulating mononuclear cells in HME, 20% to 80% of patients with HGA have morulae identified in peripheral blood neutrophils (Fig. 194-2).^60,153 Culture of A. phagocytophilum requires 1 week or longer and is not routinely available, whereas PCR amplification of A. phagocytophilum nucleic acids from blood is 54% to 100% sensitive and highly specific and can be performed in a timely manner.^140,152 Serologic diagnosis is most often achieved retrospectively by detection of immunoglobulin G (IgG) antibodies reactive with A. phagocytophilum in infected tissue culture cells.^160,161 By current criteria, an IgG titer of at least 64 is considered significant, but a fourfold rise provides more definitive evidence for infection because of the fact that 15% to 16% of the population in the upper Midwest and New York State have preexisting serologic reactions.⁷² Immunoglobulin M testing could be useful because reactions are demonstrated only during the first 45 to 60 days, but this test lacks sensitivity and is not generally advocated.¹⁶⁰ A role for Western immunoblot confirmation or use of recombinant antigens for serodiagnosis is not currently defined for humans.¹⁶²

The blastocyst

The blastocyst forms as the morula continues to divide and enters the uterus and its cells begin to absorb fluid, probably first in intracellular vacuoles that later accumulates between blastomeres (Figure 4(a) and 4(b)). The blastomeres, especially those in the outer cell mass, develop tight junctions, and, as the morula continues to absorb fluid, it collects between blastomeres of the inner cell mass. The process results in the formation of a tiny fluid-filled cavity, and the blastocyst bores a hole through and emerges from its zona pellucida. The blastocyst, with its larger component and shell of outer cell mass-derived trophoblast and its much smaller disc of inner cell mass, is now a structure consisting of 107–256 cells. The loss of the zona pellucida and descent of the blastocyst enables the blastocyst to lie, in direct apposition, to the endometrial epithelium. The blastocyst becomes tightly adherent to the endometrium via cell adhesion molecules and penetrates the endometrial surface epithelium, and implantation begins as trophoblast infiltrates the interstitium between endometrial glands (Figure 4(c)). At about day 7, some cells within the superficial layer of the inner mass of the embryonic disc differentiate into a layer of amnioblasts; the most superficial and centrally located amnioblasts lose their attachments to the underlying population of amnioblasts. This process results in the formation of a dome-like cavity lined by amniotic epithelium that begins to accumulate fluid and completely overlies the discoid inner cell mass.

Membranes

The conversion of the early morula to a blastocyst is accomplished by the formation of a central fluid-filled cavity. This largely separates the primary trophoblastic cell mass, from which the placenta and extraplacental chorion develop, from those cells which give rise to the embryo and contribute to the formation of the yolk sac and amnion; these latter cells form the eccentrically situated inner cell mass which remains in contact with the cytotrophoblast on the inner aspect of the blastocyst wall (Fig. 3.22). During the 8th and 9th postovulatory days the inner cell mass arranges itself into a bilaminar disc, the inner layer (i.e. that facing the blastocyst cavity) forming the primitive embryonic endoderm and the outer, which is in contact with the cytotrophoblast, forming the primitive embryonic ectoderm. The amniotic cavity first appears as a slit-like space between the embryonic ectoderm and the adjacent cytotrophoblast; this enlarges to form, by the 12th postovulatory day, a small cavity, the base of which is formed by embryonic ectoderm and the walls and roof of which are formed of cytotrophoblast (Fig. 3.23). At the same time, endodermal cells migrate out from the deeper layer of the embryonic disc to line the blastocyst cavity and thus form the primary yolk sac. The extraembryonic mesenchyme subsequently appears (Fig. 3.24), possibly derived from the trophoblast, and separates off the primary yolk sac from the blastocyst wall; the extraembryonic mesenchyme also intrudes between, and largely separates off, the roof of the amniotic sac and the trophoblast of the chorion. A connection between the two is, however, maintained for a time by the persistence of a column of cells, the amniotic duct, which provides a pathway for the continuing migration of cells of trophoblastic origin into the amniotic epithelium. Mitotic activity at the margin of the embryonic ectodermal disc suggests that the ectoderm is also a continuing source of supply of amniotic epithelial cells.

The extraembryonic mesenchyme forms a loose reticulum in which small cystic spaces appear; these gradually enlarge and fuse to form the extraembryonic coelom which splits the extraembryonic mesenchyme into two layers, one opposed to the trophoblast and also covering the amnion (the parietal extraembryonic mesenchyme) and the other covering the yolk sac (the visceral extraembryonic mesenchyme) (Fig. 3.25). The progressively enlarging extraembryonic coelom also separates the amnion away from the inner aspect of the chorion, except at the caudal end of the embryo where an attachment of extraembryonic mesenchyme persists to form the body stalk from which the umbilical cord will eventually be derived.

Subsequently, the amniotic space enlarges at the expense of the extraembryonic coelom and the developing embryo bulges into the expanding amniotic cavity (Fig. 3.26). Meanwhile, the yolk sac becomes partially incorporated into the embryo where it gives rise to the gut; that part of the yolk sac remaining outside the embryo communicates with the primitive gut. This communicating channel, however, gradually becomes elongated and attenuated to form the vitelline duct, the extraembryonic yolk sac becoming progressively removed further away from the embryo to be eventually incorporated into the lower end of the body stalk.

Further expansion of the amniotic sac leads to more or less complete obliteration of the extraembryonic coelom with eventual fusion of the extraembryonic mesenchyme covering the amnion with that lining the chorion. At the same time, the extraplacental chorion (the chorion laeve) ceases to produce syncytiotrophoblast and the cytotrophoblastic component undergoes a partial regression. Hence, the single fused amniochorionic membrane is now fully formed and will consist of, from fetal to maternal side, amniotic epithelium, condensed extraembryonic mesenchyme, a loose reticular layer which possibly represents the vestige of the extraembryonic coelom, extraembryonic mesenchyme and trophoblast.