Immunofluorescent localization of a monoclonally defined carbohydrate cell surface antigen (IIC3) during mouse development

A monoclonal antibody (anti-IIC3), raised against F9 embryonal carcinoma cells, detects an antigen which is first expressed at the compacted morula stage and segregates with the trophectoderm of the mouse blastocyst. We have further examined the expression of this antigen during embryonic development. Immunofluorescence experiments on sectioned embryos demonstrate that IIC3 expression is associated with the differentiation of extra-embryonic cell types. It is expressed at the cell surface of the trophectoderm of the attaching blastocyst and differentially by the two derivatives of this layer. The primary and secondary trophoblastic giant cells label intracellularly, whereas the cells of the ectoplacental cone and labyrinth placenta label at the cell surface. IIC3 is also expressed by the primitive endoderm of the blastocyst and subsequently by the visceral endoderm. The parietal endoderm does not express IIC3. Partial characterization of the IIC3 antigen with sugar hapten inhibition and glycosidase digestion experiments, suggests that the antigen is a lactosaminoglycan-like molecule, with galactose and N-acetylgalactosamine residues representing part of the antigenic determinant. Neuraminidase and fucosidase treatment exposed additional anti-IIC3-antigenic sites on the extra-embryonic ectoderm and chorion. A possible role for IIC3 in normal embryonic-uterine interactions is discussed.     

Trophectoderm cell subpopulations in the periimplantation mouse blastocyst

Trophectoderm of the preimplantation mouse blastocyst is composed of two cell subpopulations relative to their proximity to the inner cell mass. The polar trophectoderm overlying the inner cell mass proliferates to form the ectoplacental cone, and the mural trophectoderm endoreplicates and gives rise to giant cells. We examined specific differences in the two trophectoderm cell populations using a lectin (Dolichos biflorus) to detect cell surface characteristics and a simple sugar (D-Gal) to detect differences in incorporation. During the first day of delayed implantation, the mural trophectoderm presented twice as many lectin binding sites as did the polar trophectoderm. The mural trophectoderm of both nondelaying and delayed implantation blastocysts showed a greater rate of incorporation of the tritiated sugar by presenting more reduced silver grains in radioautograms. These results indicate that the mural trophectoderm and polar trophectoderm are two distinct cell types in the periimplantation blastocyst.     

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.     

Ability of outside cells from preimplantation mouse embryos to form inner cell mass derivatives

Previous studies have shown that inside cells in the preimplantation mouse embryo do not become committed to the formation of inner cell mass until after blastocyst formation. However, it is not yet clear whether outside cells are also labile late in preimplantation development or whether they become restricted to trophectoderm development at an earlier stage. The present study investigates the potency of outside cells isolated from late morulae just prior to blastocyst formation and shows that some, if not all, outside cells retain the potential to form inner cell mass derivatives in vitro and in vivo. This suggests that trophectoderm cells are not restricted in potential earlier than ICM cells and that all cells of the early embryo may be labile at least until blastulation.     

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 de-compaction 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.     

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.     

Cell fate in the polar trophectoderm of mouse blastocysts as studied by microinjection of cell lineage tracers

Horseradish peroxidase (HRP), together with Fast Green or rhodamine-conjugated dextran (RDX), was used as an intracellular lineage tracer to determine cell fate in the polar trophectoderm of 3.5-day-old mouse embryos. In HRP-injected midstage (approximately 39-cell) and expanded (approximately 65-cell) blastocysts incubated for 24 hr, the central polar trophectoderm cell was displaced from the embryonic pole an average of 20 micron (5% of blastocyst circumference) and 29 micron (6% of blastocyst circumference), respectively. Expanded blastocysts injected with HRP + Fast Green and incubated for 24 hr or with HRP + RDX and incubated for 48 hr showed a displacement of 24 micron (4% of blastocyst circumference) and 88 micron (14% of blastocyst circumference), respectively. Up to 10 HRP-positive trophectoderm cells were observed among embryos incubated for 48 hr, indicating that in those cases, the labeled progenitor cells had divided at least three times. Our observations show that the central polar trophectoderm cell divides in the plane of the trophectoderm in expanded blastocysts and, along with its descendants, is displaced toward the mural trophectoderm. The systematic tandem displacement of labeled cells and their descendants toward the abembryonic pole suggests the presence of a proliferative area at the embryonic pole of the blastocyst. Large shifts in inner cell mass (ICM) position in relation to the trophectoderm do not occur during blastocyst expansion. Furthermore, random movements within the polar trophectoderm population do not account for the replacement of labeled cells by unlabeled polar trophectoderm cells. Rather, we propose the hypothesis that the ICM contributes these replacement cells to the polar trophectoderm during blastocyst expansion.     

Functions of the IGFs in early mammalian development

The identification of growth factors and/or receptors produced by mammalian embryos or present in the maternal reproductive tract is of basic interest, as well as having practical application. Early studies established that receptors binding insulin and the insulin-like growth factors (IGFs) are expressed by preimplantation mouse embryos. These studies have been confirmed at the molecular level using RT-PCR techniques. In addition, high resolution electron microscopy has shown that insulin is internalized by the cells of the blastocyst stage mouse embryo, and that immunologically intact insulin is detectable in the cells of the trophectoderm and inner cell mass. Similar studies with gold labelled IGF-I have shown that this ligand is also bound and internalized by mouse blastocysts. However, although all blastocysts express receptors that bind IGF-I on the basolateral cell surface of the trophectoderm, only 30% exhibit apically located receptors. In order to elucidate the functions of IGFs in early mouse development, we are in the process of constructing protein databases for embryos at the eight-cell and blastocyst stage. By the use of the database, it should prove possible to elucidate targets of growth factor action. © 1993 Wiley-Liss, Inc.     

Ultrastructure of mouse blastocysts during blastocoele expansion

Summary The ultrastructure of mouse blastocysts with nascent and expanded blastocoele is described. In the early blastocyst cells adhere tightly and the blastocoele is often limited at its apex by cells containing a midbody. The expanding blastocyst exhibits a loose cell arrangement due to the presence of intercellular spaces and a cortical layer of filaments develops in cells enclosing the expanded blastocoele. When the blastocoele exceeds 1/2 the embryo diameter desmosomes appear between trophectoderm cells. Possible factors essential for blastocoele formation are discussed.     

The segregation of inner and outer cells in porcine embryos follows a different pattern compared to the segregation in mouse embryos

The mammalian blastocyst consists of an inner cell mass (ICM) enclosed by the trophectoderm. The origin of these two cell populations lies in the segregation of inner and outer cells in the early morula. In the present study, the segregation of inner and outer cells has been studied in porcine embryos and is compared with segregation in mouse embryos. For this, nuclei of inner and outer cells were differentially labelled with two fluorochromes after partial complement-mediated lysis of the outer cells. In porcine and mouse embryos compaction and the first appearance of inner cells occur at different stages of development. In porcine embryos compaction was observed as early as the 4-cell stage, while in mouse embryos compaction occurred in the 8-cell stage. The first inner cells segregated in porcine embryos which were in the transition from four to eight cells and inner cells were added during two subsequent cell cycles. In mouse embryos inner cells segregated predominantly during the fourth cleavage division. From the results obtained we conclude that the segregation of inner and outer cells follows a different pattern in mouse and in porcine embryos.     

Monoclonal antibody to murine embryos defines a stage-specific embryonic antigen expressed on mouse embryos and human teratocarcinoma cells

A murine stage-specific embryonic antigen (SSEA3) is defined by reactivity with a monoclonal antibody prepared by immunization of a rat with 4- to 8-cell-stage mouse embryos. This antigenic determinant, present on oocytes, becomes restricted first to the inner cell mass at the blastocyst stage, and later to the primitive endoderm. Murine teratocarcinoma stem cells do not react with this antibody, whereas human teratocarcinoma stem cells are SSEA3-positive. This antigenic determinant is not expressed on a variety of other human and murine cell lines, but is found on the surface of human erythrocytes. It is a carbohydrate and is present on both cell-surface glycolipids and glycopeptides. These results demonstrate the feasibility of identifying stage-specific antigenic determinants with monoclonal antibody prepared against embryos. The need for thorough screening on a variety of cell types to establish developmentally important cross-reactivities is also emphasized.     

Epidermal growth factor receptor function in early mammalian development

We review here the data indicating a role for epidermal growth factor receptor (EGF receptor) signalling in early mouse development. Embryonic development of the metazoan embryo generally begins with the formation of a cystic structure and epithelial layers that subsequently form anlagen of the definitive body parts and organs. For the mammalian embryo, this cystic structure is a blastocyst whose wall consists of trophectoderm, the first epithelium to develop during mammalian embryogenesis. The onset of expression and function of EGF receptors is coincident with the onset of trophectoderm development. Modulating EGF receptor expression and function modulates trophectoderm differentiation, leading to the hypothesis that functional EGF receptors participate in the induction of trophectoderm development and perhaps of other embryonic epithelial derivatives such as nervous tissues.     

Ginkgolides induce apoptosis and decrease cell numbers in mouse blastocysts

In this report, we examine the cytotoxic effect of ginkgolides, the major components of Ginkgo biloba extracts, on the blastocyst stage of mouse embryos and on subsequent early postimplantation embryonic development in vitro. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay revealed that blastocysts treated with 5 or 10muM ginkgolide A or ginkgolide B showed increased apoptosis versus untreated controls. This could be correlated with the observation that ginkgolide-treated blastocysts showed a significant reduction in the average number of total cells in the blastocyst and trophectoderm/inner cell mass lineage versus controls. In addition, ginkgolide-pretreated blastocysts showed normal levels of implantation on culture dishes in vitro, but significantly fewer embryos reached the later stages of embryonic development in the treatment groups versus the controls, instead dying at relatively early stages of development. Our results collectively indicate that ginkgolide treatment of mouse blastocysts induces apoptosis, decreases cell numbers, retards early postimplantation blastocyst development, and increases early-stage blastocyst death. These novel findings provide important new insights into the effect of Ginkgo biloba extracts on mouse blastocysts.     

Cytokeratin filament assembly in the preimplantation mouse embryo

The timing, spatial distribution and control of cytokeratin assembly during mouse early development has been studied using a monoclonal antibody, TROMA-1, which recognizes a 55,000 Mr trophectodermal cytokeratin (ENDO A). This protein was first detected in immunoblots at the 4-cell stage, and became more abundant at the 16-cell stage and later. Immunofluorescence analysis revealed assembled cytokeratin filaments in some 8-cell blastomeres, but not at earlier stages. At the 16-cell stage, filaments were found in both polarized (presumptive trophectoderm; TE) and apolar (presumptive inner cell mass; ICM) cells in similar proportions, although polarized cells possessed more filaments than apolar cells. By the late 32-cell, early blastocyst, stage, all polarized (TE) cells contained extensive filament networks whereas cells positioned inside the embryo tended to have lost their filaments. The presence of filaments in inside cells at the 16-cell stage and in ICM cells was confirmed by immunoelectron microscopy. Lineage tracing techniques demonstrated that those cells in the ICM of early blastocysts which did possess filaments were almost exclusively the progeny of polar 16-cell blastomeres, suggesting that these filaments were directly inherited from outside cells at the 16- to 32-cell transition. Inhibitor studies revealed that proximate protein synthesis but not mRNA synthesis is required for filament assembly at the 8-cell stage. These results demonstrate that there are quantitative rather than qualitative differences in the expression of cytokeratin filaments in the inner cell mass and trophectoderm cells of the mouse embryo.     

Maintenance of the inner cell mass in human blastocysts from fragmented embryos

The degree of fragmentation during early cleavage is universally used as an indicator of embryo quality during human in vitro fertilization treatment. Extensive fragmentation has been associated with reduced blastocyst formation and implantation. We examined the relationship between early fragmentation and subsequent allocation of cells to the trophectoderm and inner cell mass in the human blastocyst. We retrospectively analyzed data from 363 monospermic human embryos that exhibited varying degrees of fragmentation on Day 2. Embryos were cultured from Day 2 to Day 6 in Earle balanced salt solution with 1 mM glucose and human serum albumin. Rates of development and blastocyst formation were measured. The number of cells in the trophectoderm and inner cell mass and the incidence of apoptosis were assessed following differential labeling with polynucleotide-specific fluorochromes. Increasing fragmentation resulted in reduced blastocyst formation and lower blastocyst cell numbers. For minimal and moderate levels of fragmentation, the reduction in cell numbers was confined largely to the trophectoderm and a steady number of inner cell mass cells was maintained. However, with extensive fragmentation of more than 25%, cell numbers in both lineages were reduced in the few embryos that formed blastocysts. Apoptotic nuclei were present in both the trophectoderm and inner cell mass, with the lowest incidence in blastocysts that had developed from embryos with minor (5-10%) fragmentation. Paradoxically, higher levels of apoptosis were seen in embryos of excellent morphology, suggesting a possible role in regulation of cell number.     

Cytoplasmic projections of trophectoderm distinguish implanting from preimplanting and implantation-delayed mouse blastocytes

A scoring scheme was devised to characterize visually the morphological differentiation of whole-mount, unfixed mouse blastocysts. Embryos were recovered from groups of intact mice (implanting embryos) and mice ovariectomized on Day 3 of pregnancy (implantation-delayed embryos) every 3 h from 18:00 h on Day 4 until 12:00 h on Day 5. Blastocyst differentiation was assessed according to the presence of a zona pellucida, the appearance of the outer margin of trophectoderm cells, the visibility of the blastocoele and the relative size of the inner cell mass. The results obtained indicate that, during this period, implanting and implantation-delayed mouse blastocysts lose the zona as well as exhibit rounded trophectoderm cells, an enlarged inner cell mass and an increasing opacity of the blastocoele. In contrast, the trophectoderm cells of implanting blastocysts only exhibit extensive cytoplasmic projections, probably due to remodelling of the intracellular cytoskeleton. Growth of the inner cell mass appeared to precede the other morphological changes in the majority of blastocysts, and thus might be a prerequisite for further differentiation. The rate of blastocyst differentiation and the survival of embryos were adversely affected by the condition of delayed implantation, induced by ovariectomy. This study suggests that the appearance of cytoplasmic projections from trophectoderm cells is central to the control of blastocyst implantation.     

Trophoblast-specific expression and function of the integrin alpha 7 subunit in the peri-implantation mouse embryo.

For implantation and placentation to occur, mouse embryo trophoblast cells must penetrate the uterine stroma to make contact with maternal blood vessels. A major component of the uterine epithelial basement membrane and underlying stromal matrix with which they interact is the extracellular matrix protein laminin. We have identified integrin alpha 7 beta 1 as a major receptor for trophoblast-laminin interactions during implantation and yolk sac placenta formation. It is first expressed by trophectoderm cells of the late blastocyst and by all trophectoderm descendants in the early postimplantation embryo through E8.5, then disappears except in cells at the interface between the allantois and the ectoplacental plate. Integrin alpha 7 expression is a general characteristic of the early differentiation stages of rodent trophoblast, given that two different cultured trophoblast cell lines also express this integrin. Trophoblast cells interact with at least three different laminin isoforms (laminins 1, 2/4, and 10/11) in the blastocyst and in the uterus at the time of implantation. Outgrowth assays using function-blocking antibodies show that alpha 7 beta 1 is the major trophoblast receptor for laminin 1 and a functional receptor for laminins 2/4 and 10/11. When trophoblast cells are cultured on substrates of these three laminins, they attach and spread on all three, but show decreased proliferation on laminin 1. These results show that the alpha 7 beta 1 integrin is expressed by trophoblast cells and acts as receptor for several isoforms of laminin during implantation. These interactions are not only important for trophoblast adhesion and spreading but may also play a role in regulating trophectoderm proliferation and differentiation.     

Potential of isolated mouse inner cell masses to form trophectoderm derivatives in vivo

Recent in vitro experiments on immunosurgically isolated mouse inner cell masses (ICMs) have suggested that some ICM cells may retain the potential to form trophectoderm after initial blastocyst formation. These experiments relied almost solely on in vitro morphology for identification of trophectoderm derivatives and provided no proof that the putative trophectoderm cells were capable of functioning in utero. We present clear in vivo evidence that at least some cells in ICMs isolated from early blastocysts do retain the potential to form postimplantation trophectoderm derivatives. Early ICMs occasionally contributed to trophoblast fractions in ICM/morula aggregation chimeras. More strikingly, when aggregated with each other, these ICMs were capable of implanting in the uterus, a property of trophectoderm cells alone. Indeed, some aggregates reconstituted complete egg cylinders. However, ICMs isolated from later blastocysts rarely produced in vivo trophoblast, and it appears that the ability to form trophectoderm is lost around the 16–19 cell ICM stage. These results are discussed in relation to changing patterns of gene activity in early development.     

Oct4 protein remains in trophectoderm until late stages of mouse blastocyst development

Oct-4, the marker of pluripotent cells, is crucial for murine preimplantation development. During the formation of the blastocyst Oct-4 is downregulated in the trophectoderm (TE) and its expression becomes restricted to the inner cell mass (ICM). In order to determine the exact timing of the disappearance of Oct-4 protein from TE we analyzed the localization and level of Oct-4 at different stages of blastocyst development. The presence of Oct-4 protein was determined by immunohistochemistry using confocal microscopy. We found that the downregulation of Oct-4 protein in TE of mouse blastocysts progresses gradually during development, and Oct-4 protein persists in some of the TE cells at least until the expanded blastocyst (120-140 cells) stage. Our findings indicate that the switching-off of the Oct-4 expression is not necessary for the trophectoderm formation. The complete elimination of Oct-4 protein from TE occurs at the period of blastocyst implantation, when lack of Oct-4 is required for the proper functioning of the trophectoderm.