The cytoskeleton and associated proteins during cleavage, compaction and blastocyst differentiation in the pig

Abstract. The organization of the cytoskeleton during early pig embryogenesis was investigated by using fluorescence and electron microscopy. The early morphogenesis of the pig embryo differed from that of the mouse, the standard model of the early mammalian development. In the pig, both compaction and polarization were gradual, and definitive polarization of cell surface microville occurred first shortly before blastocyst formation; the compaction and polarization of the mouse embryo are completed as early as at the 8 cell stage. Furthermore, the pig morula undergoes cycles of compaction and decompaction throughout its development. Distinct changes in the distribution of actin and the actin-associated proteins α-fodrin, vinculin and E-cadherin coincided with these events. In the pig, all these molecules were evenly distributed at all aspects of the blastomeres during early cleavage and then gradually accumulated in regions of intercellular contacts toward the blastocyst stage; microfilaments in trophectoderm cells formed a cortical meshwork associated with apical microvilli and adherent junctions (zonula adherens). In the mouse, the corresponding changes occur earlier, at the 8 cell stage. Microtubules formed a network-like cortical layer beneath the microvilli at the free outer surfaces of pig blastomeres. Cytokeratin bundles were not observed until the early blastocyst, where they characteristically associated with newly formed desmosomes.
In both species a close correlation between morphologically defined developmental stages and the organization of the cytoskeleton: actin and actin-associated proteins are involved in polarization and compaction, whereas the appearance of intermediate filament bundles coincides with the building of the first epithelium, the trophectoderm; it is in the timing of events that a contrast between species is observed.     

Transitions in trophectoderm cellular shape and cytoskeletal organization in the elongating pig blastocyst

Changes in cellular shape and filamentous actin (f-actin) organization within the trophectoderm of pig embryos have been studied by fluorescence microscopy during the transitions from spherical to filamentous blastocysts. Cells comprising the trophectoderm of spherical, ovoid, tubular, and filamentous blastocysts are distinctive in their shape, size, and organization of membrane-associated f-actin. Trophectodermal cells of spherical and ovoid embryos are both generally circular in shape. However, as the spherical embryo acquires an ovoid shape, uniformally distributed apical cell surface microvilli relocate to the apical intercellular margins of adjoining trophectodermal cells. Transitional modifications in cellular shape and f-actin organization are observed in tubular blastocysts when apical cell surface microvilli reappear. In elongating filamentous blastocysts, trophectodermal cells assume a spindle-shaped morphology. The f-actin associated with the apical surface is diminished whereas the associated with the basolateral membrane predominates, especially in constricted regions of the blastocyst. These observations, in conjunction with morphometric parameters of trophectodermal cells and whole blastocysts, are discussed in relation to the role of the actin cytoskeleton in processes that modify trophectodermal cell shape and function in the elongating pig blastocyst.     

Phosphorylation of ezrin on threonine T567 plays a crucial role during compaction in the mouse early embryo

The preimplantation development of the mouse embryo leads to the divergence of the first two cell lineages, the inner cell mass and the trophectoderm. The formation of a microvillus pole during compaction at the eight-cell stage and its asymmetric inheritance during mitosis are key events in the emergence of these two cell populations. Ezrin, a member of the ERM protein family, seems to be involved in the formation and stabilization of this apical microvillus pole. To further characterize its function in early development, we mutated the key residue T567, which was reported to be essential for regulation of ezrin function through phosphorylation. Here, we show that expression of ezrin mutants in which the COOH-terminal threonine T567 was replaced by an aspartate (to mimic a phosphorylated residue; T567D) or by an alanine (to avoid phosphorylation; T567A) interferes with E-cadherin function and disrupts the first morphogenetic events of development: compaction and cavitation. The active mutant ezrin-T567D induces the formation of numerous and abnormally long microvilli at the surface of blastomeres. Moreover, it localizes all around the cell cortex and inhibits cell-cell adhesion and cell polarization at the eight-cell stage. During the following stages, only half of the embryos are able to compact and finally to cavitate. In those embryos, the amount of ezrin-T567D decreases in the basolateral areas, while the proportion of adherens junctions increases. The reverse inactive mutant ezrin-T567A is mainly cytoplasmic and does not perturb compaction at the eight-cell stage. However, at the 16-cell stage, it relocalizes at the basolateral cortex, leading to a strong decrease in the surface of adherens junctions, and finally, embryos abort development. Our results show that ezrin is directly involved in the formation of microvilli in the early mouse embryo. Moreover, they indicate that maintenance of ezrin in basolateral areas prevents microvilli breakdown and inhibits the formation of normal cell-cell contacts mediated by E-cadherin, thereby impairing blastomeres polarization and morphogenesis of the blastocyst.     

小鼠胚胎致密化起始的调控机制

植入前小鼠胚胎的发育事件包括第一次卵裂、胚胎基因组激活、桑椹胚致密、囊胚形成。小鼠受精卵胚胎的致密化发生在8-细胞阶段晚期, 致密过程中, 胚胎卵裂球本身以及卵裂球之间发生了一系列的变化。这些变化包括卵裂球微绒毛以及胞质成分的极性化分布, 卵裂球之间形成特殊的胞间连接。致密化是哺乳动物胚胎发育过程中的第一个细胞分化事件, 即导致了内细胞团以及滋养外胚层的产生。植入后, 内细胞团将发育成为胚体, 滋养外胚层将发育成为胎盘等胚外组织。细胞粘附分子E-cadherin介导的胞间粘附起始了致密化。卵裂球发生粘附所需的组分在致密前已经存在, 但是直至8-细胞阶段晚期连接复合体才表现出明显的粘附活性。敲除E-cadherin基因, 发现母源性的E-cadherin足以介导致密。E-cadherin介导的胞间粘附是细胞粘附的第一步。文章综述了E-cadherin介导胞间粘附的具体过程以及蛋白激酶C(Protein kinase C, PKC)调控该过程的相关 机制。     

Cell specific loss of polarity-inducing ability by later stage mouse preimplantation embryos

The individual blastomeres of the preimplantation mouse embryo become polarized during the 8-cell stage. Microvilli become restricted to the free surface of the embryo and this region of the membrane shows increased labeling with FITC-Con A and trinitrobenzenesulfonate (TNBS). Previous studies have shown that this polarity develops in response to asymmetric cell-cell contact with stage specific induction competent blastomeres. In the present study, the ability of later stage embryos to induce 8-cell polarization has been investigated. Newly-formed, nonpolar 8-cell stage blastomeres (1/8 cells) were isolated, then aggregated with morulae, inner cell clusters (from morulae), blastocysts, or inner cell masses (ICM) and cultured for 8 hr. Aggregates were then assayed for polarity. The results show a hierarchy of inducing ability, with the ICM and IC cluster possessing greater activity than the morula and polar trophectoderm of the early blastocyst, while the mural trophectoderm shows very little inducing activity. Furthermore, the inducing ability of the polar trophectoderm decreases with complete expansion and hatching of the blastocyst. These results indicate that the ability to induce 8-cell blastomere polarization is retained by the embryo beyond the 8-cell stage and that this ability is lost with further differentiation.     

Involvement of LIMK1/2 in actin assembly during mouse embryo development

LIMKs (LIMK1 and LIMK2) are serine/threonine protein kinases that involve in various cellular activities such as cell migration, morphogenesis and cytokinesis. However, its roles during mammalian early embryo development are still unclear. In the present study, we disrupted LIMK1/2 activity to explore the functions of LIMK1/2 during mouse early embryo development. We found that p-LIMK1/2 mainly located at the cortex of each blastomeres from 2-cell to 8-cell stage, and p-LIMK1/2 also expressed at morula and blastocyst stage in mouse embryos. Inhibition of LIMK1/2 activity by LIMKi 3 (BMS-5) at the zygote stage caused the failure of embryo early cleavage, and the disruption of LIMK1/2 activity at 8-cell stage caused the defects of embryo compaction and blastocyst formation. Fluorescence staining and intensity analysis results demonstrated that the inhibition of LIMK1/2 activity caused aberrant cortex actin expression and the decrease of phosphorylated cofilin in mouse embryos. Taken together, we identified LIMK1/2 as an important regulator for cofilin phosphorylation and actin assembly during mouse early embryo development.     

Cell polarity and cell diversification during early mouse embryogenesis.

At the eight-cell stage of mouse development, the organization of blastomeres changes from radially symmetrical to polarized. This acquisition of cell polarity, followed by asymmetric divisions, leads to the formation of two phenotypically different cell types, which give rise to the first two cell lineages of the mouse blastocyst embryo, trophectoderm and the inner cell mass. Cell fate, controlled by positional information, is not irreversibly fixed during differentiation, providing the embryo with considerable developmental flexibility.     

Changes in the organization of the actin cytoskeleton during preimplantation development of the pig embryo

The organization of the actin cytoskeleton was studied in unfertilized porcine oocytes and preimplantation stage embryos from Day 1 through Day 8 of development. Fixed and detergent-extracted oocytes and embryos were analyzed by fluorescence microscopy after staining with either rhodamine-phalloidin to localize filamentous actin or with affinity-purified anti-actin antibodies to localize the total immunodetectable actin. Whereas unfertilized oocytes contain immunoreactive cytoplasmic actin, rhodamine-phalloidin binding is not detected until fertilization when a prominent cortical staining pattern becomes apparent. In early cleavage stage embryos, filamentous actin is concentrated in the cell cortex of blastomeres especially at sites of cell-cell contact. Compacting morulae exhibit a marked accumulation of actin at the margins of blastomeres where numerous interdigitating cell processes are located. The predominantly pericellular distribution of actin becomes a distinguishing feature of trophectodermal cells in the expanding blastocyst at Day 6 of development; these cells form a prominent actin-limited zone circumscribing the inner cell mass. In Day 8 blastocysts, three cell types are present that are readily distinguishable based upon their actin displays among other cytological features. Trophectodermal cells exhibit continuous actin-rich lateral borders and stress fibers along their basal surface. Inner cell mass cells contain a discontinuous actin boundary and prominent foci of actin along their blastocoelic surface. Lining the blastocoel are patches of endodermal cells in which the actin is exclusively cortical. The data are discussed with respect to differences between species and the chronology of actin rearrangements during preimplantation development of the porcine embryo.     

Influence of early fate decisions at the two-cell stage on the derivation of mouse embryonic stem cell lines

The first event of differentiation in mammalian embryogenesis is the segregation of the inner cell mass and trophectoderm lineages in the blastocyst. Cellular and molecular events related to this process are still a controversial issue. During the years it was thought that first allocation of blastomeres before the blastocyst stage was done in the late eight-cell stage with the formation of inner and outer cells. Lately, many studies have pointed out that individual blastomeres at the four-cell stage differ in their developmental properties according to their position within the embryo. In this report, we wanted to elucidate whether these early decisions influence the production of mouse embryonic stem cell lines, so that a selective isolation of blastomeres at the four-cell stage to derive the lines could improve the efficiency of the derivation process. Results from blastomere tracking experiments support the idea of a different developmental potential of blastomeres within the four-cell stage embryo. However, we also show a high plasticity in the developmental pattern of blastomeres once isolated from the embryo, thus making all four-cell stage blastomeres equally competent to derive ESC lines.     

In vivo functional analysis of ezrin during mouse blastocyst formation

During mouse blastocyst formation, a layer of outer cells differentiates in less than 48 h into a functional epithelium (the trophectoderm). Ezrin, an actin-binding structural component of microvilli in epithelial cells, is also involved in signal transduction and ionic pump control. In the mouse embryo, ezrin becomes restricted to the apical cortex of all blastomeres at compaction and of outer cells at later stages. Here we investigated the function of ezrin in living embryos during epithelial differentiation using mutant forms of ezrin tagged with green fluorescent protein (GFP). GFP-tagged wild-type ezrin (Ez/GFPc) behaved like endogenous ezrin and did not interfere with development. Deletion of the last 53 amino acids (Delta53/GFP) changed the localization of ezrin: after compaction, Delta53/GFP remained associated with the apical and basolateral cortex in all blastomeres, and its expression slightly disturbed the cavitation process. Finally, full-length ezrin with GFP inserted at position 234 (Ez/GFPi) was localized all around the cortex throughout development, although it was concentrated at the apical pole after compaction. In embryos expressing Ez/GFPi, the duration of the 16-cell stage was reduced, while the onset of cavitation was delayed. Moreover, cavitation was abnormal, and the blastocoele was small and retracted almost completely several times as if there were major leakages of blastocoelic fluid. Our results suggest that, in addition to its role in microvilli organization, ezrin is involved in the formation of a functional epithelium through a still unknown mechanism.     

Disruption of the cytokeratin filament network in the preimplantation mouse embryo

The distribution of the cytokeratin network in the intact preimplantation mouse embryo and the role of cytokeratin filaments in trophectoderm differentiation were investigated by means of whole-mount indirect immunofluorescence microscopy and microinjection of anti-cytokeratin antibody. Assembled cytokeratin filaments were detected in some blastomeres as early as the compacted 8-cell stage. The incidence and organization of cytokeratin filaments increased during the morula stage, although individual blastomeres varied in their content of assembled filaments. At the blastocyst stage, each trophectoderm cell contained an intricate network of cytokeratin filaments, and examination of sectioned blastocysts confirmed that extensive arrays of cytokeratin filaments were restricted to cells of the trophectoderm. Microinjection of anticytokeratin antibody into individual mural trophectoderm cells of expanded blastocysts resulted in a dramatic rearrangement of the cytokeratin network in these cells. Moreover, antibody injection into 2-cell embryos inhibited assembly of the cytokeratin network during the next two days of development. Despite this disruption of cytokeratin assembly, the injected embryos compacted and developed into blastocysts with normal morphology and nuclear numbers. These results suggest that formation of an elaborate cytokeratin network in preimplantation mouse embryos is unnecessary for the initial stages of trophectoderm differentiation resulting in blastocyst formation.     

Expression and distribution of cell adhesion molecule uvomorulin in mouse preimplantation embryos

We have examined the synthesis and distribution of the cell adhesion molecule uvomorulin in mouse preimplantation embryos. Uvomorulin can already be detected on the cell surface of unfertilized and fertilized eggs but is not synthesized in these cells. Uvomorulin synthesis starts in late two-cell embryos and seems not to be correlated with the onset of compaction. The first signs of compaction are accompanied by a redistribution of uvomorulin on the surface of blastomeres. During compaction uvomorulin is progressively removed from the apical membrane domains of peripheral blastomeres. In compact morulae uvomorulin is no longer present on the outer surface of the embryo but is localized predominantly in membrane domains involved in cell-cell contacts of adjacent outer blastomeres. On inner blastomeres of compact morulae uvomorulin remains evenly distributed. This uvomorulin distribution once established during compaction is maintained and also found in the blastocyst: on trophectodermal cells uvomorulin localization is very similar to that in adult intestinal epithelial cells while uvomorulin remains evenly distributed on the surface of inner cell mass cells. The possible role of the redistribution of uvomorulin for the generation of trophectoderm and inner cell mass in early mouse embryos is discussed.     

Membrane-cytoskeletal interactions in the early mouse embryo

Differentiation in the early mouse embryo begins at the 8-cell stage when the blastomeres flatten against each other by active spreading movements and surface and cytoplasmic elements become concentrated in the apical (uncontacted) region of the cells. A ring of cortical myosin marks the demarcation between the contacted and the uncontacted cellular domains. The organization of the cortical contractile apparatus in the blastomeres bears a formal resemblance to that of other cells that are engaged in similar motile activities. It has been proposed that a flow of cortical filaments could provide the motor that powers these movements. The applicability of such a cortical flow model to the early embryo and the implications for cell flattening and cell polarization are discussed in this review.     

Changes in cell surface and cortical cytoplasmic organization during early embryogenesis in the preimplantation mouse embryo

Membrane topography and organization of cortical cytoskeletal elements and organelles during early embryogenesis of the mouse have been studied by transmission and scanning electron microscopy with improved cellular preservation. At the four- and early eight-cell stages, blastomeres are round, and scanning electron microscopy shows a uniform distribution of microvilli over the cell surface. At the onset of morphogenesis, a reorganization of the blastomere surface is observed in which microvilli becomes restricted to an apical region and the basal zone of intercellular contact. As the blastomeres spread on each other during compaction, many microvilli remain in the basal region of imminent cell-cell contacts, but few are present where the cells have completed spreading on each other. Microvilli on the surface of these embryos contain linear arrays of microfilaments with lateral cross bridges. Microtubules and mitochondria become localized beneath the apposed cell membranes during compaction. Arrays of cortical microtubules are aligned parallel to regions of apposed membranes. During cytokinesis, microtubules become redistributed in the region of the mitotic spindle, and fewer microvilli are present on most of the cell surface. The cell surface and cortical changes initiated during compaction are the first manifestations of cell polarity in embryogenesis. These and previous findings are interpreted as evidence that cell surface changes associated with trophoblast development appear as early as the eight-cell stage. Our observations suggest that morphogenesis involves the activation of a developmental program which coordinately controls cortical cytoplasmic and cell surface organization.     

Changes in the distribution of vinculin during preimplantation mouse development

It has been proposed that vinculin is a microfilament bundle-membrane linking cytoskeletal protein. We used double-fluorescence microscopy to study the distribution of vinculin and F-actin in mouse oocytes and preimplantation embryos. In oocytes and in the cells of cleavage- and blastocyst-stage embryos, vinculin exhibited a diffuse cytoplasmic distribution and was concentrated in a submembranous layer. The presence of vinculin in oocytes was confirmed by immunoblotting. In oocytes, a distinct concentration of actin was observed above the second metaphase spindle. During the 8-cell stage, compacting blastomeres exhibited partial polarization of cortical vinculin and actin toward their outward-facing surfaces. In precompaction-stage blastomeres, the submembranous layer of vinculin contained a ring-like concentration in the most peripheral region of each intercellular contact area. During later development, the amount of vinculin localized in the areas of intercellular contacts became modified. In embryos ranging from the compacted 8-cell stage to the mid-morula stage, the vinculin-specific fluorescence was only intense in some intercellular contacts, being indistinct in most contact areas. In late morulae, the flattened outer cells increasingly exhibited concentration of vinculin in contact areas. In contrast, actin-specific fluorescence was clearly evident in most intercellular contacts throughout the morula stage. At the early blastocyst stage, all contacts of the trophectoderm (TE) cells again regularly exhibited concentration of both components. At the late blastocyst stage, the staining pattern changed once again: the contact-associated concentration of vinculin-specific fluorescence was not observed in polar TE cells, while remaining clear in mural TE cells. In blastocyst outgrowths, TE cells displayed typical vinculin plaques at the peripheries of the cells. The continuous changes in the distribution of vinculin and actin suggest that these components are involved in the control of cellular relationships during early development. Immunoelectron microscopy and experiments using cytochalasin were performed in an attempt to relate the distribution of vinculin to the ultrastructural features of embryo cells.     

The effect of oxygen on the development of preimplantation mouse embryos in vitro

The optimal oxygen tension for development of preimplantation mouse embryos to the blastocyst stage in vitro was found to be between 2.5% and 5%. One- and two-cell embryos had a more sharply defined range of oxygen tension capable of supporting development than 8-cell and morula stages. At all stages of development, more embryos developed to the blastocyst stage under 5% O2 compared to the numbers of developing under higher oxygen tensions (20% and 40% O2). The blastocysts developing under 20% O2 had fewer blastomeres than those which developed under 5% O2. As the time required for development to the blastocyst stage in vitro increased, there were fewer blastomeres present at the blastocyst stage. These results indicate that the cleaving mouse embryo has an optimal oxygen requirement in vitro of about 5%. At higher oxygen tensions, fewer embryos develop to the blastocyst stage and in those which do develop, there are fewer cell divisions. If a gradient of oxygen tension exists across the blastomeres from the outside of the embryo to its centre, the blastomeres might be using this gradient to obtain imformation about their location within the embryo and respond accordingly. Thus blastomeres on the outside at a higher oxygen tension would divide at a slower rate and form trophectoderm whereas those on the inside at a lower oxygen tension would divide more rapidly and contribute to the inner cell mass.     

Analysis of compaction in the preimplantation mouse embryo

An SEM analysis of the effects of tunicamycin, cytochalasin B, and colcemid has yielded insights into the process of compaction in the early mouse embryo. All three reagents block or reverse compaction and decrease the number of microvilli (MV), although some MV polarization is permitted. In addition, tunicamycin is shown to lessen cell adhesion even in compacted embryos. Cytochalasin B causes the formation of MV clumps some of which are preferentially localized to the apex or lateral ring region. Colcemid reverses compaction and, coupled with Pronase treatment, completely blocks compaction of uncompacted 8-cell embryos. Observations also suggest that MV polarization can occur only once but compaction (the close adherance and flattening of blastomeres) can be reversed and reinduced. Evidence is consistent with a three-step compaction process involving (1) cell surface recognition and attachment of a ring of lateral microvilli to adjacent blastomeres, (2) subsequent microfilament shortening in these lateral MV, and (3) maintenance of the compacted and polarized state by microtubules.     

Four-cell stage mouse blastomeres have different developmental properties

Blastomeres of the early mouse embryo are thought to be equivalent in their developmental properties at least until the eight-cell stage. However, the experiments that have led to this conclusion could not have taken into account either the spatial origin of individual blastomeres or the spatial allocation and fate of their progeny. We have therefore readdressed this issue having defined cell lineages in mouse embryos undergoing different patterns of cleavage in their second division cycle. This has enabled us to identify a major group of embryos in which we can predict not only the spatial origin of each given four-cell blastomeres, but also which region of the blastocyst is most likely to be occupied by its progeny. We show that a pattern of second cleavage divisions in which a meridional division is followed by one that is equatorial or oblique allows us to identify blastomeres that differ in their fate and in their developmental properties both from each other and from their cousins. We find that one of these four-cell stage blastomeres that inherits some vegetal membrane marked in the previous cleavage cycle tends to contribute to mural trophectoderm. The progeny of its sister tend to donate cells to part of the ICM lining the blastocyst cavity and its associated trophectoderm. Chimaeras made entirely of these equatorially or obliquely derived blastomeres show developmental abnormalities in both late preimplantation and early postimplantation development. By contrast, chimaeras made from four-cell stage blastomeres from early meridional divisions develop normally. The developmental defects of chimaeras made from the most vegetal blastomeres that result from later second cleavages are the most severe and following transplantation into foster mothers they fail to develop to term. However, when such individual four-cell blastomeres are surrounded by blastomeres from random positions, they are able to contribute to all embryonic lineages. In conclusion, this study shows that while all four-cell blastomeres can have full developmental potential, they differ in their individual developmental properties according to their origin in the embryo from as early as the four-cell stage.     

Delay of polarization event increases the number of Cdx2-positive blastomeres in mouse embryo

During preimplantation mouse embryo development expression of Cdx2 is induced in outer cells, which are the trophectoderm (TE) precursors. The mechanism of Cdx2 upregulation in these cells remains unclear. However, it has been suggested that the cell position and polarization may play a crucial role in this process. In order to elucidate the role of these two parameters in the formation of TE we analyzed the expression pattern of Cdx2 in the embryos in which either the position of cells and the time of polarization or only the position of cells was experimentally disrupted. Such embryos developed from the blastomeres that were isolated from 8-cell embryos either before or after the compaction, i.e. before or after the cell polarization took place. We found that in the embryos developed from polar blastomeres originated from the 8-cell compacted embryo, the experimentally imposed outer position was not sufficient to induce the Cdx2 in these blastomeres which in the intact embryo would form the inner cells. However, when the polarization at the 8-cell stage was disrupted, the embryos developed from such an unpolarized blastomeres showed the increased number of cells expressing Cdx2. We found that in such experimentally obtained embryos the polarization was delayed until the 16-cell stage. These results suggest that the main factor responsible for upregulation of Cdx2 expression in outer blastomeres, i.e. TE precursors, is their polarity.