Ultrastructural study of concanavalin-A binding to the surface of preimplantation mouse embryos

Receptors for Con-A were labelled (using the peroxidase-diaminobenzidine technique) on the plasma membrane of unfertilized and fertilized mouse eggs, cleavage stage embryos, trophoblast and inner cell mass (ICM) of the blastocyst. Embryos were exposed to Con-A concentrations of 10 microgram/ml, 50 microgram/ml, or 1,000 microgram/ml and the lowest concentration was observed to be the most suitable for discerning differences between stages of embryonic development. On the surface of unfertilized and fertilized eggs and 2-cell embryos, reaction product appeared as a thin, discontinuous layer. The surface of 4- and 16-cell stage embryos had a thicker, continuous, although non-uniform, layer of the reaction product. On the surface of the cells of the late morula, and on the trophoblastic cells of the blastocyst, clustering of reaction product was observed. Cells of ICM of intact blastocyst were free of the reaction product, showing that either Con-A and/or peroxidase cannot penetrate tight junctions between trophoblastic cells. Reaction product in the form of a thin, uniform layer covered the free surface of the cells of the ICM after they had been isolated (using immunosurgery) and exposed to 50 microgram/ml of Con-A. The amount and distribution of Con-A receptors is discussed, along with their redistribution and mobility in relation to the agglutinability of preimplantation mouse embryos.     

Role of the extracellular matrix protein thrombospondin in the early development of the mouse embryo

The distribution of the extracellular matrix protein thrombospondin (TSP) in cleavage to egg cylinder staged mouse embryos and its role in trophoblast outgrowth from cultured blastocysts were examined. TSP was present within the cytoplasm of unfertilized eggs; in fertilized one- to four-cell embryos; by the eight-cell stage, TSP was also densely deposited at cell-cell borders. In the blastocyst, although TSP was present in all three cell types; trophectoderm, endoderm, and inner cell mass (ICM), it was enriched in the ICM and at the surface of trophectoderm cells. Hatched blastocysts grown on matrix-coated coverslips formed extensive trophoblast outgrowths on TSP, grew slightly less avidly on laminin, or on a 140-kD fragment of TSP containing its COOH terminus and putative cell binding domains. There was little outgrowth on the NH2 terminus heparin-binding domain. Addition of anti-TSP antibodies (but not GRGDS) to blastocysts growing on TSP strikingly inhibited outgrowth. Consistent with its early appearance and presence in trophoblast cells during implantation, TSP may play an important role in the early events involved in mammalian embryogenesis.     

Serological characterization of a nervous system/sperm antigen (NS-52): demonstration of its presence on murine preimplantation embryos.

Anti-NS-5 antiserum raised in C3H.SW/Sn mice against cerebellum of 4-day-old C57BL/6J mice could be shown to recognize two cell surface antigens on cerebellar cells, NS-5₁ and NS-5₂, the latter antigen being shared with mouse and rat but not rabbit sperm. An antigen operationally identical to NS-5₂ was detected using indirect immunofluorescence staining on mouse preimplantation stages of development. While the unfertilized ova did not express detectable antigen on the cell surface, the fertilized egg expressed antigen shortly before the first cleavage division. From that stage onward, the anti-NS-5 antiserum stained the blastomeres of all stages, including the trophoblast cells and inner cell mass cells of the blastocyst. No difference in staining activity was observed for preimplantation embryos of various mouse strains analyzed: C57BL/6J, BALB/c, 129/J, C3H/DiSn, CKB × BALB.K, C3H.SW/Sn, and Swiss Webster mice. The staining activity was removed when the antiserum was preabsorbed with cerebellum or sperm from any of these mouse strains or with cerebellum and sperm of rats. Lymphocytes, thymocytes, liver, kidney, and skeletal muscle from early postnatal and adult mice and heart from early postnatal mice did not absorb the staining activity and neither did rabbit sperm nor cerebellum.     

Ultrastructural cytochemistry of membrane-bound phosphatases in preimplantation mouse embryos.

The time of appearance and the ultrastructural localization of the enzyme activity of alkaline phosphatase (AlkPase), 5′-nucleotidase (5′Nuc), Mg²⁺-ATPase, transport ATPase, cyclic AMP phosphodiesterase (cAMP-PDase), and adenylate cyclase (AC) were investigated in unfertilized eggs and in mouse preimplantation embryos. Enzyme activity was associated only with the plasma membrane. AlkPase activity appeared only in limited areas of the plasma membrane of one-cell embryos and increased in the eight-cell and morula stages. In blastocysts, the enzyme activity was concentrated mainly in the trophoblast cells. 5′Nuc activity appeared first in four- or eight-cell embryos and the highest activity was observed in trophoblast cells in the blastocyst and in plasma membrane between cells forming inner cell mass. Mg²⁺-activated ATPase activity was present in all embryos and in unfertilized egg plasma membrane. Transport (Na⁺K⁺)-ATPase appeared only in the closely apposed membranes of adjacent cells in morulae and blastocysts. A very low cAMP-PDase activity appeared between adjacent cells in two-cell embryos, and the highest activity was observed on the outer surface of the plasma membrane of trophoblasts. AC was the only enzyme whose activity was located on the inner (cytoplasmic) side of the plasma membrane and appeared as early as the one-cell stage embryo. The relation between the time of the appearance of enzyme activity and the preparation of embryos for implantation and upon embryonic proliferative activity is discussed.     

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.     

Qualitative patterns of protein synthesis in the preimplantation mouse embryo: I. Normal pregnancy

Qualitative patterns of protein synthesis in preimplantation mouse embryos were examined by SDS-polyacrylamide-gel electrophoresis followed by autoradiography. The results demonstrate that the qualitative pattern of protein synthesis in newly fertilized eggs (day 1) is very similar to the protein pattern obtained from ovulated, unfertilized eggs. By late day 1 or early day 2, most of these “maternal” proteins are no longer being synthesized by the embryo, and many new autoradiographic bands are apparent. The most intriguing aspect of this study is the observation that all major changes in the qualitative pattern of protein synthesis take place between fertilization and the four- to eight-cell stage (day 3). From early day 3 onward, the qualitative pattern of protein synthesis remains essentially unchanged.Many of the major autoradiographic bands observed in mouse embryos from the four- to eight-cell stage and onward are also observed in protein patterns obtained from blastocyst-stage rabbit embryos. The changing patterns of protein synthesis revealed in this study occur before any gross differentiation of the embryos is evident (delineation of the inner cell mass and trophoblast) and before a marked increase in the relative rate of incorporation of l-[³⁵S]methionine takes place. However, the qualitative changes in the pattern of protein synthesis do coincide with a period of extensive fine structural differentiation.     

The Chinese hamster gene map. Assignment of four genes (DTS, PGM2, 6PGD, EnoI) to chromosome 2

Mouse morulae and blastocysts express cell surface antigens that fortuitously cross-react with antisera to human chorionic gonadotropin (hCG). In the present study, the cell surface and cytoplasmic expression of these antigens was followed in mouse unfertilized oocytes, different stages of preimplantation embryos and in early post-implantation embryos cultured from blastocysts. In addition to their known stage-dependent cell surface expression on morulae and blastocysts, these antigens (1) were already present in the cytoplasm of mature unfertilized oocytes and pre-morula stages of embryos; (2) remained expressed as cell surface antigens on cells of the inner cell mass (ICM), but not on the surface of trophectodermal cells with further blastocyst development although (3) they persisted as cytoplasmic antigens in trophectodermal cells. In addition, these antigens were also detectable by antiserum to the alpha subunit of hCG.     

Expression of teratoma-associated antigens on murine ova and early embryos. Identification of two early differentiation markers.

Specifically absorbed rabbit antisera to a mouse testicular teratoma were used to study the expression of three heteroantigens on cell surfaces of early mouse embryos. This antiserum is known to define three antigens on the surface of murine tumor cells, though it does not bind to cells of normal adult tissue. Antigen I, physically associated with H-2 antigens in L-cell plasma membranes and found on many transplantable mouse tumors (7), is expressed by ova and morulae. Antigen I persists on cells of the inner-cell mass beyond implantation, but is not detected on trophoblast. In contrast, antigen II, found on teratoma and hepatoma cells, is absent from cleavage embryos and is first expressed on differentiation of the trophoblast prior to implantation. Antigen III, which is apparently teratoma specific, is absent from all embryonic material studied.     

The stimulating effect of recombinant cytokine LIF on the mouse blastocysts during implantation

The effect of recombinant LIF cytokine (Leukemia inhibitory factor) on the isolated mouse embryos at the stages of middle and late blastocyst has been investigated. We have demonstrated here that this agent is necessary in vitro at the stage of normal trophoblast formation after the blastocysts hatch from zona pellucida. This cytokine (10 ng/ml) caused intensification of adhesion and proliferative activity of the trophoblast cells. This is important for intercellular interactions with endometrium and for invasion of embryos into the uterus. The recombinant LIF insignificantly influenced cells of the inner cell mass.     

Development of Cleaving Mouse Embryos under Pressure

A chamber for applying mechanical pressure to developing mouse eggs is described.
Morphologically normal blastocysts developed from 4-cell eggs cultured under pressure initially near to the point of cell rupture. Four-cell, 8-cell eggs and late morulae, developing with the pressure regularly adjusted to this point, formed trophoblast-like cells internally and peripherally to the cell mass. These observations are consistent with the idea that cells will become trophoblast unless subjected to a specific intercellular environment. A glass plate closely apposed to the cells cannot substitute for this environment. Total enclosure by other cells seems necessary for inner cell mass differentiation.     

Involvement of Glycoproteins in the Development of Early Mouse Embryos: Effect of Tunicamycin and α, α' Dipyridyl in vitro

Early mouse embryos grown in tissue culture were treated with tunicamycin, an inhibitor of protein glycosylation or with αα' dipyridyl, an inhibitor of collagen secretion. Neither treatment blocked development of cleavage stage embryos nor did either interfere with blastocyst formation, hatching, or adhesion to the substratum at low concentrations. However, both treatments caused marked and specific changes in the morphology of the blastocyst outgrowth. Treatment of embryos with tunicamycin caused severe deterioration of the trophoblast layer and subsequent disintegration of the inner cell mass. Tunicamycin completely inhibited the incorporation of mannose into proteins. Treatment with αα' dipyridyl caused dose dependent retardation of the inner cell mass while the trophoblast cells were virtually unaffected. These alterations in morphogenesis occurred only in embryos treated at the blastocyst stage or later in development. Changes caused by α,α' dipyridyl could be partially reversed by addition of collagen to the culture. These findings might indicate the involvement of extracellular matrix macromolecules in embryonic organization.     

Differential inhibition of trophoblast outgrowth and inner cell mass growth by actinomycin D in cultured mouse embryos.

Treatment of preimplantation mouse embryos in vitro with 10(-3) to 10(-1) mug actinomycin D/ml for 2 hr showed that (i) postimplantation development in vitro was inhibited most when embryos were treated at the morula stage and (ii) after the morula stage actinomycin D inhibited trophoblast outgrowth less than inner cell mass development.     

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.     

Autoradiographic studies on the distribution of labeled maternal RNA in early mouse embryos

Maternal RNA of mouse eggs and embryos was labeled by exposure of growing ovarian oocytes to 3H-uridine in vivo 8 to 16 days before ovulation and fertilization. Labeled embryos from the 1-cell stage to the blastocyst stage were collected, fixed, and autoradiographs of plastic sections prepared. The observed grain density was similar in the pronuclei and in the cytoplasm of 1-cell embryos. Knowing the volumes of nucleus and cytoplasm, it was determined that 3% of the maternal RNA was found in the pronuclei. It is suggested that some of this nuclear RNA may be stable small nuclear RNAs (e.g. U1 RNA) retained from the germinal vesicle stage through meiotic maturation. During the 2-cell stage and beyond, maternal RNA is degraded and labeled precursor is reincorporated into nuclear RNA, making it difficult to accurately quantitate the amount of nuclear maternal RNA. It is known that about one third of the total maternal RNA is lost between the 8-cell and blastocyst stages. It was found that cytoplasmic grain densities in inner and outer cells of the morula and blastocyst were not significantly different. Thus, the loss of maternal RNA does not proceed more rapidly in the differentiating trophoblast than in the inner cell mass.     

Localization of type I interferon in murine trophoblast and decidua during decidual formation.

Mouse trophoblast and decidua were examined by means of immunohistochemistry to define the localization of type I interferon. The decidua were stained for type I interferon at the time of implantation. The strong reaction was first observed in the primary decidual zone on day 5 and subsequently in the secondary decidual zone on day 6. After day 10, the decidua basalis and decidua capsularis showed a strong reaction. At the one-cell stage, embryos were weakly labelled, but a positive reaction was recognized in compacted morulae. Blastocysts on days 3 and 4 were positive in trophoblast and inner cell mass and a strong reaction was observed in the primitive endoderm on day 4. The visceral endoderm on day 5 and the trophoblast on day 6 were positive. After day 10, the trophoblast giant cells, labyrinth, visceral yolk sac and fetal blood cells gave a positive reaction. This study is the first demonstration of type I interferon localization in situ in mouse trophoblast and decidua during decidual formation.     

Gap junctional communication in the preimplantation mouse embryo.

In this study, we examined cell-to-cell communication via gap junctional channels between the cells of the early mouse embryo from the 2-cell stage to the preimplantation blastocyst stage. The extent of communication was examined by monitoring for the presence of ionic coupling, the transfer of injected fluorescein (molecular weight 330) and the transfer of injected horseradish peroxidase (molecular weight 40,000). In the 2-cell, 4-cell and precompaction 8-cell embryos, cytoplasmic bridges between sister blastomeres were responsible for ionic coupling and the transfer of injected fluorescein as well as the transfer of injected horseradish peroxidase.In contrast, no communication was observed between blastomeres from different sister pairs. Junction-mediated intercellular communication was unequivocably detected for the first time in the embryo at the early compaction stage (late 8-cell embryo). At that stage, ionic coupling was present and fluorescein injected into one cell spread to all eight cells of the embryo. Injected horseradish peroxidase was passed to only one other cell, however, again indicating the presence of cytoplasmic bridges between sister blastomeres. Junctional communication with respect to both ionic coupling and dye transfer was retained between all the cells throughout compaction. At the blastocyst stage, trophoblast cells of the blastocyst were linked by junctional channels to other trophoblast cells as well as to cells of the inner cell mass, as indicated by the spread of injected fluorescein. In addition, the extent of communication between the cells of the inner cell mass was examined in inner cell masses isolated by immunosurgery; both ionic coupling and the complete spread of injected fluorescein were observed.     

Trophoblast regeneration by inner cell masses isolated from cultured mouse embryos

The developmental potential of the inner cell mass (ICM) of the cultured mouse embryo was determined by testing the ability of the ICM to regenerate trophoblast in vitro. ICM's isolated by immunosurgery from either single or chimeric embryos were able to regenerate trophoblast when they were isolated at 69 hours of culture from the 2-cell stage, but they had lost this capacity by 93 hours of culture. Trophoblast regeneration by isolated ICM's did not appear to require either a critical cell mass at the time of isolation or cell proliferation during regeneration.     

Expression of a glucose-regulated cell surface protein in early mouse embryos

A 100,000-Da glucose-regulated surface protein (100K-GRP) has previously been isolated from the cell surface and culture medium of human fibroblasts. A rabbit antiserum directed against this protein reacts with the cell surface of both human and murine cultured cells and with a broad spectrum of mammalian tissues. It is shown, via indirect immunofluorescence, that this protein is also present on cells of the developing mouse embryo and can be detected as early as the 4-cell stage. The 8-cell embryo and morula show positive surface labeling; the inner cell masses of both the pre- and postimplantation blastocysts are also positive but the trophectoderm is not. At the 6-day egg cylinder stage, the embryonic and extra-embryonic ectoderm label intensely with the antiserum and visceral endoderm shows faint labeling. No labeling can be detected on parietal endoderm or on the trophoblastic giant cells invading the uterine decidua. However, the internal cells of the ectoplacental cone exhibit bright fluorescence. The same pattern is observed on 7- to 8.5-day embryos, except that at this stage no label is associated with the visceral endoderm. In addition, mesodermal cells emerging from the primitive streak are also labeled.