Christopher W. Stackpole

Characterization of intercellular junctions in the preimplantation mouse embryo by freeze-fracture and thin-section electron microscopy

Abstract Intercellular junction formation in preimplantation mouse embryos was investigated with thin-section and freeze-fracture electron microscopy. At the four-cell stage, regions of close membrane apposition with focal points of membrane contact and occasional underlying cytoplasmic densities were observed between blastomeres of thin-sectioned embryos. Corresponding intramembrane specializations were not, however, observed in freeze-fractured embryos. At the 8- to 16-cell stage, small gap and macula occludens junctions and complexes of these junctions were observed at all levels between blastomeres of freeze-fractured embryos. As development progressed from the early to mid 8- to 16-cell stage, the size of the occludens/gap junction complexes increased, forming fascia occludens/gap junction complexes. At the morula stage, gap junctions and occludens/gap junction complexes were observed on both presumptive trophoblast and inner cell-mass cells. Zonula occludens junctions were first observed at the morula stage on presumptive trophoblast cells of freeze-fractured embryos. The number of embryos possessing zonula occludens junctions increased at the mid compared to the early morula stage. At the blastocyst stage, junctional complexes consisting of zonula occludens, macula adherens, and gap junctions were observed between trophoblast cells of freeze-fractured and thin-sectioned embryos. Isolated gap and occludens junctions, adherens junctions, and occludens/gap junction complexes were observed on trophoblast and inner cell-mass cells.

Relationship between intercellular permeability and junction organization in the preimplantation mouse embryo

Abstract Preimplantation mouse embryos were examined for intercellular permeability to molecules of different molecular weights. Using immunosurgery followed by immunofluorescence, none of the eight-cell embryos, approximately 10% of the early morulae, 50% of the late morulae, and 90% of the early blastocysts were found to block nonspecific anti-mouse thymocyte serum from diffusing into intercellular spaces. Diffusion of horseradish peroxidase and microperoxidase into intercellular spaces of viable embryos was also impeded by some morulae and by all early blastocysts maintained on ice. Peroxidase tracers found within the blastocoel cavity of some early blastocysts examined at 37°C appear to be a result of pinocytosis and transcellular movement. Intercellular diffusion of lanthanum into glutaraldehyde-fixed embryos was impeded only by early blastocysts. These results suggest that a permeability seal is established between external cells of the early mouse embryo prior to blastocoel formation. In addition, freeze-fracture electron microscopy revealed a correlated change in zonula occludens junction organization, indicating that formation of an intercellular permeability barrier and subsequent blastocoel formation may depend upon completion of assembly of these junctions.

Membrane organization in mouse spermatozoa revealed by freeze-etching

Freeze-cleavage and deep-etching have been used to examine the ultrastructural organization of mouse spermatozoon membranes. Within the cleaved plasma membrane, particles are distributed randomly in the head and aggregated in the tail, forming paracrystalline arrays in the middle piece. Cleavage faces of the acrosomal membranes are characterized by a regular arrangement of tightly packed particles, as well as other types of particles. Large particles are concentrated over the thickened apical portion of the acrosome and within the nuclear envelope directly beneath the acrosome. The organization of particles within the nuclear envelope at the junction with the redundant nuclear envelope and at the head-tail junction suggests a functional role for these particles in membrane attachment. The external surface of the plasma membrane of the tail possesses circumferentially oriented strands in the middle piece, and larger longitudinally oriented “zippers” in the principal piece. The function of these elaborations is unclear.

Lectin-mediated agglutination of preimplantation mouse embryos

Abstract Plant lectins were used to monitor qualitative changes in carbohydrate-containing receptors during preimplantation mouse development. Beginning at the morula stage, an age-related decline was observed in agglutination of early mouse embryos by concanavalin A (ConA). In contrast, wheat germ agglutinin (WGA) and Ricinus communis agglutinin (RCA) agglutinated embryos strongly throughout preimplantation development.

Cell surface membrane organization revealed by freeze-drying.

Replicas of freeze-dried cells from various murine monolayer and suspension cultures revealed an external cell surface morphology characterized by randomly distributed heterogeneous particles when viewed by transmission electron microscopy. On the surfaces of Rauscher leukemia virus-infected JLSV 9 (JLSV 9 -RLV) cells, a population of prominent particles can be attributed to virus-specific membrane alterations on the basis of: (i) the large number of particles similar in size to projections on the virus envelope surface; (ii) the paucity of similar particles on the surfaces of uninfected JLSV 9 cells; and (iii) specific reduction of this particle population by antibody-induced displacement of the major viral glycoprotein (gp69/71) on the JLSV 9 -RLV cell surface. Cell surface particulation was demonstrated on various monolayer-and suspension-cultured cells as well as on freshly isolated mouse thymocytes. Particulation appears somewhat less prominent on thymocytes and suspension-cultured cells than on cells grown in monolayers, and the particulation on primary isolates of HSFS/N mouse fibroblasts in monolayer becomes progressively enhanced with extended culturing. Particulation was negligible on erythrocyte membranes and could not be induced by treatments that resulted in lateral aggregation of intramembranous particles. Analysis of the surfaces of protein-free membrane liposomes and of unfixed and trypsin-treated cells and comparison of the effects of glutaraldehyde and osmium tetroxide as fixatives suggest that the observed particulation is related to cell surface membrane proteins and may represent molecular oligomers.

Metrizamide gradient purification of mouse tumor cells

A one-step isopycnic density gradient centrifugation procedure is described for purification of highly viable and homogeneous tumor cells from a variety of solid mouse tumors. Mechanically suspended cells are layered onto preformed continuous gradients of medium 199-buffered 7--33% metrizamide (density range 1.05--1.20 g/cu. cm) isoosmotic with mouse plasma and centrifuged for 30 min. Large numbers of tumor cells, generally 85--95% viable and free from 80--95% of contaminating host lymphoid and phagocytic cells and erythrocytes, were consistently recovered from fractionated thymomas, melanomas, and fibrosarcomas. By a variety of criteria, cell surface and other biological properties of gradient-purified tumor cells were normal.

Assembly of a temperature-sensitive mutant of Rauscher murine leukemia virus at the cell surface induced by low temperature and by ligands☆

Abstract NIH/3T3 mouse fibroblasts, infected with a Rauscher murine leukemia virus temperature-sensitive mutant (ts25) defective in assembly of budding particles at 39°, produce virus at the cell surface when the temperature is shifted rapidly to 0°. Virus buds are not assembled within the first 10 min at 0° and gradually increase in number and degree of development over a 2-hr period. However, release of infectious virus could not be demonstrated at 0° and might, therefore, be an energy-dependent process. Significant budding activity was also induced at the nonpermissive temperature by incubating cells with 0.25% glutaraldehyde or with antiserum to the major virus envelope glycoprotein, gp70. Anti-gp70 serum may induce budding by promoting aggregation of gp70-containing molecular assemblies and, consequently, association of core components in some transmembrane fashion. Induction of virus assembly with glutaraldehyde might occur as a results of nonspecific cross-linking of membrane proteins. These results suggest that procedures commonly used to minimize ligand-induced redistribution of cell surface molecules, i.e., labeling at low temperatures or after mild aldehyde fixation, may not immobilize certain membrane-associated molecules.

Surface structure of virions budding from L1210(V) gln− mouse leukemia cells

Abstract The surface structure of virions budding from L1210(V) gln− murine leukemia cells was studied by freeze-drying intact cells, and two types of virus particles were detected. One type possessed random 10-nm surface projections, similar to budding viruses on cells producing only murine leukemia virus (MuLV). The other type possessed regularly arranged 5-nm projections, similar to budding viruses on cells producing only murine mammary tumor virus (MuMTV). Results of double antibody labeling are in agreement with the indication that individual viral envelopes on L1210(V) gln− cell viruses are homogeneous in their surface structure.