Cavitation in the mouse preimplantation embryo: Na/K-ATPase and the origin of nascent blastocoele fluid

This study tested the proposition that Na/K-ATPase activity could be involved in the morphogenetic aspects of mouse blastocyst formation by facilitating the localization of certain organelles to apposed borders, the production of nascent blastocoele fluid, and cavitation. It was assumed that such Na/K-ATPase activity would be sensitive to varying concentrations of external K (Ko)--which would alter plasma membrane potentials--and to ouabain--which would directly alter Na/K-ATPase function. Morulae were cultured for 40 hr in varying concentrations of Ko and/or ouabain and were observed for their ability to form blastocoeles (cavitate) and to localize mitochondria to apposed cell borders. Cavitation was accelerated when Ko was decreased from the control value of 6.0 to 0.6 mM and was delayed when Ko was increased to 25 mM. With Ko at 6.0 mM, 10(-5) M ouabain accelerated cavitation while 10(-4) M ouabain delayed cavitation and reduced the total number of embryos that cavitated by the end of the 40-hr culture period. With Ko at 0.6 mM, 10(-5) M ouabain now delayed cavitation while 10(-4) M ouabain almost completely inhibited it. When Ko was increased to 25 mM, 10(-5) M ouabain again accelerated cavitation while 10(-4) M ouabain delayed-rather than inhibited--cavitation. Morphometric analyses at the electron microscopic level showed changes in the distances of mitochondria from apposed cell borders with conditions that accelerated or delayed cavitation and these changes differed for inside and outside cells of the morula. These observations are consistent with the proposition that Na/K-ATPase activity could be involved in the localization of organelles to apposed cell borders, the production of nascent blastocoele fluid, and in cavitation during mouse blastocyst development.     

Intracellular localization and surface expression of a stage-specific embryonic glycoprotein

The intracellular and cell surface localization of an embryonic glycoprotein antigen (BL) has been investigated in preimplantation mouse embryos using ultrastructural immunocytochemistry. Several interesting points have emerged: (1) BL antigens are exclusively localized subjacent to the plasma membrane in the cortical region of cells, whereas antigens detected by a control antibody against mouse L cells are distributed throughout the embryo. (2) The distribution of BL antigens is polarized beginning with the first cleavage, with expression confined to the cortex underlying the free or apical portions of cells. No antigen is present underlying regions of cell contact. (3) Although embryonic synthesis of BL antigens does not begin until the two-cell stage, BL antigens are observed in unfertilized eggs, a fact verified by immunoblotting.     

Establishment and maintenance of a regionalized glycoprotein distribution during early mouse development

The regionalization of the cell membranes of the mouse embryo into apical and basolateral zones has been studied using antibodies to a pair of glycoproteins expressed during the two-cell to early blastocyst stage. These antigens are found on the outer, free surface and in the underlying cortical cytoplasm, but are not detectable at areas of cell contact. In the early blastocyst stage, antigen also appears at the free surfaces of cells bordering the blastocoel. Antigen regionalization is also reestablished after experimental manipulation and appears to be a direct consequence of cell contact. Thus, blastomeres examined 4 hr after dissociation from four- and eight-cell stage embryos express antigen in cortical areas underlying newly exposed surfaces and new sites of contact between embryos in multiple-embryo aggregates lose detectable antigen within 2 to 4 hr of the formation of the contacts. Microfilaments are involved in controlling the regional expression of these glycoproteins. Incubation of embryos from the two-cell stage in medium containing cytochalasin B interferes with antigen targeting, resulting in abnormal expression of the antigens both on the surface and in the cytoplasm of the embryos. Cytochalasin B treatment of later stage embryos results in an uneven distribution of the antigen in cortical cytoplasm and prevents the complete removal of antigen from new sites of cell contact in multiple-embryo aggregates. The presence of nocodozole, which inhibits the polymerization of microtubules, had no detectable effect on the expression of the antigens. Interference with the glycosylation of these proteins, by incubation of embryos in the presence of tunicamycin, did not alter the regionalized pattern of expression.     

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.     

An electron microscopic study of the adrenal cortical tissue of the domestic fowl

Summary The fine structure of the adrenal cortex of the domestic fowl has been studied from the 14th day of embryonic life to 980 days after hatching, using the electron microscope.Many mitochondria and vacuoles, round or oval, are observed in all cortical cells. The cristae mitochondriales are not laminar but villous. In the embryo, the mitochondria, whose cristae are not as well developed as those in chick and hen, are very low in electron density. Cytoplasmic vacuoles, either rough or smooth-surfaced, and with a homogeneous content of low electron density, are enclosed by a similar limiting membrane. Some of them are formed by the outer nuclear membrane and are characterized by many small particles on their outer surfaces. These vacuolated structures are perhaps parts of the endoplasmic reticulum. In the 980 day old hen, a number of osmiophilic droplets, round or irregularly shaped, are seen. Some of these are formed by mitochondria, while others accumulate within or around cytoplasmic vacuoles. In the embryo and in the chick, such droplets are never seen. It is assumed that the droplets are substances related to ageing, and that the lipid implicated in cortical hormone is not osmiophilic in the young chick and the embryo. It is perhaps produced in the cytoplasmic vacuoles with the aid of mitochondria. A perisinusoidal space and interparenchymatous cell spaces similar to those described for mammals by other workers, are observed in the adrenal gland of the domestic fowl. I assume that the secretory substance of the cortical and the medullary cell is first secreted into these spaces and then infiltrates into the capillary lumen.     

Serological analysis of early mouse embryo with rat monoclonal antibodies produced against mouse teratocarcinoma cells

Rat-mouse hybridoma antibodies were produced against mouse teratocarcinoma F9 or PCC4 aza1 cells, and four clones were established. Both the F11 (IgM) and F20 (IgG2c) antibodies showed a similar specificity, reacting only with nullipotential teratocarcinoma cells. They were also found to agglutinate sheep red blood cells. Solid-phase enzyme-linked immunofluorescence assay showed that, among the neutral glycolipids studied, they only reacted with the Forssman antigen. P2 antibody (IgG2b) reacted with the undifferentiated-type and embryonal endodermtype teratocarcinoma cells. During the preimplantation stage, this antibody did not stain mouse embryos, but it reacted very weakly with the inner cell mass of blastocysts cultured in vitro. In the 5th-day embryo, the embryonic ectoderm as well as the visceral and parietal endoderm were positive, but the extraembryonic ectoderm was not. Mesoderm of the 7.5th-day embryo also reacted with this antibody. However, P2 antigen was not observed in the 16th-day embryo or in adult tissues. F2 antibody (IgG2a), which was reactive with all of the cultured cell lines tested, showed an immunoreaction with mouse embryos throughout the preimplantation stage. However, in the 7.5th-day embryo, the presence of F2 was limited to the cells forming the parietal endoderm. This antigen was present in some epithelial tissues of the 16th-day embryo and adult mouse. Of these antigens, P2 and F2 are probably novel differentiation antigens of the early mouse embryo. Together with the Forssman antigen, these will be important markers for analyzing cell-surface antigens of mouse teratocarcinoma cells as well as embryos.     

The role of the nucleocytoplasmic ratio in development regulation of the early mouse embryo

The hypothesis suggesting that the blastocoele is able to form only at a definite nucleocytoplasmic ratio was tested. We compared the development of preimplantation mouse embryos under different conditions. The results demonstrated that the start of cavitation is not dependent on the number of cell divisions. Thus, a definite nucleocytoplasmic ratio is not required for blastocoele formation to start. Our studies on embryos with microsurgically altered cytoplasm content provided evidence for the following biological clock mechanism: a change in the cell program of morphogenesis needs definite concentration of the products of a previous genetic program.     

Localization and Characterization of an Integral Membrane Protein Antigen Expressed by Pigment Cells in Embryos of the Sea Urchin Strongylocentrotus purpuratus

Chromogenic mesenchymal cells in plutei of the sea urchin Strongylocentrotus purpuratus express a tissue specific epitope recognized by the monoclonal antibody Sp–1. In transmission electron micrographs of pre-embed immunoperoxidase labelled plutei, the epitope is localized to pigment cell surfaces. Cell membranes of epidermal cells apposed to pigment cells are also immunoreactive, but endodermal cells apposed to pigment cells are not. Separation of Sp–1 immunoreactive material into the aqueous phase after embryo extraction in butanol indicates that the antigen is a protein or glycoprotein, and other solubilization characteristics suggest that it is an integral membrane constituent. The epitope is destroyed by general proteases and treatment with guanidine hydrochloride, and is resistant to oxidation by periodate and glycosyloxidases, suggesting that it is peptide rather than carbohydrate. On immunoblots of whole embryo extracts, or after SDS-PAGE analysis of ³⁵S-methionine-labelled embryos immunoprecipitated with Sp–1, a band showing an apparent molecular mass of 110 kD is seen at all stages from mid-gastrula to 26-day pluteus. We conclude that the Sp–1 antigen is a 110 kD integral membrane protein which may interact with the epidermal cells over which the pigment cells migrate.     

Normal thymic cortical epithelial cells developmentally regulate the expression of a B-lineage transformation-associated antigen

Nonlymphoid, stromal cells in the mouse thymus are believed to be important in T cell maturation and have been proposed to play a central role in the acquisition of major histocompatibility complex (MHC) restriction and self-tolerance by maturing thymocytes. Both cortical and medullary epithelial cells in the thymus express high levels of class II (A) major histocompatibility antigens (MHC Ags). We show here that a specific subset of these A^– epithelial cells express a transformation-associated antigen (6C3Ag) found previously on the surfaces of Abelson murine leukemia virus-transformed pre-B cells and on those bone marrow-derived stromal cell clones which support normal and preneoplastic pre-B cell proliferation. Among solid lymphoid organs, only the thymus contains 6C3Ag¹ cells and within the thymus, this antigen is found exclusively on A^– epithelial cells in cortical regions. It is striking that the expression of the 6C3Ag on thymic epithelium is developmentally regulated, suggesting a role for this lymphostromal antigen in the maturation of the thymic microenvironment.     

Antibodies to a renal Na+/glucose cotransport system localize to the apical plasma membrane domain of polar mouse embryo blastomeres.

Mouse preimplantation embryos were examined for the cell surface expression of epitopes that cross-react with antibodies to a 75-kDa subunit of a purified porcine renal brush border Na+/glucose cotransport system. A Na+ cotransport system is hypothesized to reside in the apical plasma membrane domain of mouse polar blastomeres and to be associated with the induction of their apical-basal polarity. Western blot analysis showed that unfertilized oocytes as well as preimplantation embryos contain a cross-reacting antigen with an apparent molecular weight of about 75,000. Embryos and their isolated blastomeres were double-labeled and assayed by indirect immunofluorescence (IIF) for the expression of epitopes (visualized by labeling with rabbit antiserum or mouse monoclonal IgG to cotransporter followed by the appropriate rhodamine-conjugated second antibodies) and for the development of cell surface polarity (visualized by the apical restriction of fluoresceinated succinylated concanavalin A binding; FS Con A). IIF did not detect these epitopes until after the second cleavage when 4-cell embryos expressed low-to-moderate levels. Although epitopes were expressed on all surfaces of 4-cell blastomeres, some blastomeres expressed more epitopes on their apical surfaces than on their basolateral ones. All precompaction 8-cell embryos expressed epitopes, with expression being greater apically on some blastomeres. The level of expression appeared to reach a maximum on morulae and to decline on cavitating embryos. Assays performed on isolated blastomeres from postcompaction embryos showed that by the 16-cell stage epitope expression appeared to become restricted to FS Con A-labeled apical plasma membrane domains and was no longer evident on basolateral domains. This apparent apical restriction of epitope expression was confirmed by electron microscopic examination of immunogold-labeled isolated polar 16-cell blastomeres. These results demonstrate that preimplantation mouse embryos contain an antigen(s) that is immunologically and structurally similar to a 75-kDa renal Na+/glucose cotransporter. The onset of cell surface expression of this antigen precedes development of the stable polar phenotype.     

Reprograming of murine blastocoele formation

The present study shows that there is communication between reaggregated asynchronous cleavage stage blastomeres that regulates blastocoele formation. Individual blastomeres from eight-cell murine embryos were transferred to empty zonae pellucidae, intact two-cell embryos, or enucleated two-cell embryos, and were examined over a period of 75 hours for development of cavitation. It was found that the isolated blastomeres cavitated concurrently with intact control eight-cell embryos, while intact control two-cell embryos cavitated 24 hours later. However, the embryos resulting from combining a two-cell embryo and a blastomere from an eight-cell embryo cavitated at a time in between the eight- and two-cell controls.     

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.     

Production of free estrogens and estrogen conjugates by the preimplantation equine embryo

In vitro production of free estrogens and estrogen conjugates by intact Day 12.5, 13.5 and 14.5 equine embryos was measured at 2-h intervals over a 24-h culture period. Production of free estrogens was higher for Day 14.5 than Day 12.5 embryos. Differences in production of conjugated estrogens were not significant, but a trend toward increased production with increased age of embryo was apparent. No trend toward increased free and conjugated estrogen production per cell was observed with age. Embryo diameter and number of cells increased with age but varied considerably within groups. The amount of free and conjugated estrogens measured in blastocoelic fluid did not decrease over the 24-h culture period, suggesting that estrogens detected in culture medium were produced by the embryo and not the result of leakage of maternal estrogen from the blastocoele. The results of this study support previous results that estrogen production increases with development of equine embryos. This increase in estrogen production appears to be more closely associated with the diameter of the embryo, and hence its number of cells, than with increased intracellular steroidogenic activity.     

Distribution of the major histocompatibility complex antigens on different cellular components of human liver

Monoclonal mouse antibodies to the "framework" determinants of the class I and II molecules of the major histocompatibility complex (MHC) were used to demonstrate the presence of the MHC antigens in human liver. First, the localization of these antigens was demonstrated from frozen section histology with indirect FITC immunofluorescence and the cell component(s) binding the mouse antibody were identified by rabbit marker antisera and indirect TRITC immunofluorescence. Second, the antigen expression on the cell surface was analyzed by the Staphylococcus aureus rosette method from cytological cell smears. All antibodies reacted with cells in the liver sinusoids, both with the Kupffer cells and at least partially with the sinusoidal endothelial cells. The same antisera reacted also with the bile duct cells, though weaker, and with some stromal cells in close proximity of the blood vessels. The vascular endothelial cells of hepatic artery, hepatic vein, and portal vein displayed no reaction. Thus human liver differs strikingly from, e.g., human kidney, where the vascular endothelial cells contain large amounts of MHC antigens on the cell surface. This difference may be one explanation to why liver allografts are less promptly rejected than renal allografts in man.     

The stage-specific expression of TEC-1, -2, -3, and -4 antigens on bovine preimplantation embryos

The preimplantation developmental period is associated with constant changes within the embryo, and some of these changes are apparent on the embryo cell surface. For example, during transition from maternal to embryonic genome control and the compaction and differentiation of embryonic cells, the cell surface undergoes morphologic alterations that reflect changes in gene control. In order to gain insight into the events occurring during embryonic development and cellular differentiation, monoclonal antibodies specific for cell surface antigens (TEC antigens) of embryonic cells have been generated previously and shown to recognise either the carbohydrate moiety of embryoglycan or a developmentally regulated protein epitope. The TEC antigens have been identified on mouse preimplantation embryos, and their expression is specific to particular developmental stages. To determine whether these antigens are conserved in higher mammals, we examined the expression of four TEC antigens (TEC-1 to TEC-4) on in vitro–derived bovine and murine embryos during the preimplantation stage of development. It was found that bovine oocytes and embryos derived from in vitro maturation (IVM) and in vitro fertilisation (IVF) showed stage-specific expression of each of the TEC antigens investigated, with the pattern of expression overlapping but not identical to that seen in the mouse. Immunoprecipitation together with Western blot analysis showed that the TEC monoclonal antibodies recognised a single glycoprotein band with an apparent molecular weight of 70 kDa. Confocal microscopy of immunofluorescence staining of the bovine cells showed this protein to be located on the cell surface. The apparent negative expression of these TEC antigens by immunohistochemistry and immunoprecipitation at particular stages of development appears to be due to the epitopes being inaccessible to the TEC antibodies, since Western blotting revealed the TEC antigens to be present at all stages of development examined. Antibodies identifying stage-specific antigens will provide useful markers to characterise early embryonic cells, monitor normal embryonic development in vitro, and identify cell surface structures having a function in cell-cell interactions during embryogenesis and differentiation. Mol. Reprod. Dev. 49:19–28, 1998. © 1998 Wiley-Liss, Inc.     

Identification of a glycoprotein, gp21, of adult T cell leukemia virus by monoclonal antibody

A mouse hybridoma cell line producing monoclonal antibody, F10, was established from mice hyperimmunized with cells bearing adult T cell leukemia (ATL) virus (ATLV). F10 antibody reacted with an ATLV structural polypeptide ( gp21 ) with a m.w. of 21,000 that was glycosylated on cell surfaces. The gp21 was expressed on cell surfaces of all ATL-associated antigen (ATLA)-positive human cell lines but not on ATLA-negative cell lines nor peripheral blood leukocytes stimulated with mitogens. The gp21 was also detected with anti-ATLA-positive human serum, and the binding of F10 antibody to ATLA-positive cell surfaces was significantly blocked by pretreatment with anti-ATLA-positive human sera. Double immunofluorescence staining with F10 antibody and anti-ATLA-positive human serum caused co-capping on cell surfaces, which suggests that gp21 co-exists with other ATLV antigens expressed on cell surfaces. Immunoprecipitation studies also suggested that the gp21 is a minor component of the ATLV envelope.     

The expression and localization of surface neoantigens in transformed and untransformed cultured cells infected with avian tumor viruses

The presence and localization of neoantigens induced in cultured cells, infected or transformed with avian tumor viruses (ATV), were studied ultrastructurally on carbon platinum replicas of cell surfaces. The use of antibody, labeled with hemocyanin molecules, provided sensitive detection and analysis of cell surface antigen distribution. The subgroup-specific antigens of the viral envelope were found in considerable amount in the plasma membranes of ATV-infected chick embryo fibroblasts. The distribution of these antigens over the cell surface, evaluated on cells which were prefixed with glutaraldehyde, was found to be diffuse with a greater density on the cell processes in some cells. Reaction of antibody to viral envelope antigens with living ATV-infected cells resulted in a number of patterns of redistribution of membrane antigen-antibody complexes (AAC). Redistribution occurred in symmetrical or asymmetrical modes. The former consisted of randomly oriented aggregates (patches) of AAC over the cell surface. The latter included: (a) linear accumulation of AAC at cell margins; and (b) condensation of complexes into one or more centers of coalescence. These observations could be made on chick embryo cells infected (but not transformed) by avian leukosis virus, or on cells oncogenically transformed by avian sarcoma virus. The regions of coalescence were suggestive of the “capping” phenomenon seen in other systems, and their formation was temporally correlated with endocytosis of labeled AAC and the gradual loss of AAC from the surface. The effects of several biologically perturbing substances on the processes of redistribution were investigated in ALV-infected fibroblasts. Sodium azide, puromycin, actinomycin D, and colchicine had no effect on either form of asymmetrical redistribution. Cytochalasin B (CB) and iodoacetic acid (IAA) appeared to have some effect on the marginal redistribution, and to completely prevent the condensation into foci of coalescence (FC). When treated with these compounds, reacted with antibody at low temperature, washed free of unbound antibody, and warmed at 37° C, cells rapidly cleared their surfaces of AAC. This was not accompanied by formation of FC or endocytosis. In some of these cells, a distribution was observed which suggested a possible centrifugal flow of antigenic sites – perhaps an alternate route for disposal of AAC. None of the drugs tested affected symmetrical redistribution. Repeated attempts at detection and topographical analysis of a tumor-specific antigen on the surface of Rous sarcoma virus-transformed chicken and rat cells have provided no evidence for antibody to such an antigen in the serum of immunized animals. Autochthonous, homologous, and heterologous immunizations of chickens and rats did not produce a detectable antibody response to a virus-specific tumor surface antigen. Preliminary results, however, suggest the expression of an individual-specific (unique) tumor antigen on the surface of Rous sarcoma cells.     

Hsp72 inhibits apoptosis upstream of the mitochondria and not through interactions with Apaf-1

Hsp72 protects cells against apoptosis in response to various stresses. By simultaneously measuring cytochrome c localization and nuclear morphology in mouse embryo fibroblasts, we have shown that Hsp72 blocks cytochrome c release from mitochondria in response to cytotoxic stress and that permeabilization of the outer mitochondrial membrane is the critical point in deciding the fate of the cell. Hsp72 did not inhibit apoptosis in mouse embryo fibroblasts once cytochrome c had been released from the mitochondria. Recent reports have claimed that Hsp72 can prevent caspase activation by inhibiting the oligomerization of Apaf-1 in the presence of cytochrome c and dATP. We now show that this apparent function of recombinant Hsp72 is due to the presence of salt in the Hsp72 preparation and that the same response can be achieved by the addition of heat-denatured Hsp72 in the same high salt buffer or by the high salt buffer alone. Hsp72 expressed in a range of different cell lines had no inhibitory effect on cytochrome c-stimulated caspase activity of cytosolic extracts. We conclude that the protective effect of Hsp72 occurs upstream of the mitochondria and not through the inhibition of the apoptosome.     

Murine cell surface glycoproteins. Purification of the polymorphic Pgp-1 antigen and analysis of its expression on macrophages and other myeloid cells

We previously reported the initial characterization of a polymorphic major cell surface glycoprotein of about 80,000 daltons from mouse embryo 3T3 cells. This glycoprotein has now been purified 1800-fold to apparent homogeneity by monoclonal antibody affinity chromatography. The purified molecule retained the total antigenic activity of the cell, as determined by antibody binding assays. The quantity of the glycoprotein, 0.06% of the total protein of the crude cell extract, confirmed its presence as a major constituent of the cell plasma membrane. The monoclonal antibody was also used to detect related antigens in cells and tissues of C57BL/6J mice. The antigen was present in high concentration in macrophages and subpopulations of bone marrow and blood polymorphonuclear cells. Much lower concentrations of antigen were detected in spleen cells, thymocytes, and extracts of solid tissues. The apparent Mr of the target antigen of myeloid cells was 92,000. This molecule was a major surface constituent of myeloid cells with 10(6) antibody binding sites per cell containing 10% of total 125I incorporated by the lactoperoxidase procedure. The macrophage glycoprotein labeled on the cell surface with 125I was highly sensitive to trypsin, yielding an antigenically active soluble glycopolypeptide of about 65,000 daltons, that contained all of the incorporated 125I. A similar 65,000-dalton glycopeptide was released from 3T3 cells by trypsin cleavage. These data indicate that a major cell surface constituent of mouse myeloid cells is a 92,000-dalton glycoprotein closely related to the 80,000-dalton glycoprotein of mouse embryo 3T3 cells.