Surface antigen in early differentiation.

Abstract

Addition of Fab fragments from rabbit antiserum to surface antigen F9 to 2-cell stage mouse embryos in culture does not alter cleavage; however, the addition prevents the formation of compact morulae and blastocysts. A similar effect is observed when Fab fragments are added to already compact 8-cell stage or even older morulae, but disappears at the beginning of blastocoel formation. This effect is reversible: uncompact 30-cell embryos washed free of Fab become compact in a few hours, produce blastocysts, and upon reimplantation into pseudopregnant mothers can produce mice. Development is not altered by divalent anti-F9 antibodies, by Fab fragments from sera directed against other embryo surface antigens, or by succinyl concanavalin A. There are reasons to assume that the cell surface plays an important role in the development of the embryo (1). We have investigated this point by examining the effect of several antisera reacting with surface structures of embryonic cells on the in vitro development of the mouse preimplantation embryo (2). Among these sera, one which proved effective in blocking morula compaction and blastocyst formation without inhibiting cleavage was directed against the nullipotent, embryonal carcinoma (EC) line F9. This line carries the "F9 antigen," which has been shown to be expressed on EC cells, germ line cells, and preimplantation embryos (3, 4). MATERIALS AND METHODSMice. The following stocks were used: 129/Sv, an inbred subline of the original 129 strain; C57BI/6 X CBA, an F1 hybrid of C57BI/6 and CBA. All mice were produced at the Institut Pasteur.Cells. Two cultured cell lines were used: F9-41, a nullipotent EC cell (5); PYS-2, a parietal yolk sac cell (6) devoid of F9 antigen.Mouse Anti-F9 Serum was produced in syngeneic male 129/Sv mice by hyperimmunization with irradiated F9-41 cells (3). Before use anti-F9 serum was absorbed with PYS-2 cells (vol/vol, serum dilution 1:5 in Hank's medium containing 4% -heat-inactivated, gamma globulin-free, fetal calf serum, 1 hr at 40). The absorbed serum retained most of the original activity against F9 cells.Rabbit Anti-F9 Serum. Rabbits were immunized with 5 X 107 F9-41 cells in complete Freund's adjuvant. After 1 month the animals received four booster injections at 2-week intervals with 5 X 107 cells each. Serum was collected and heat inactivated 1 week after the last injection. The cytotoxic titer of the unabsorbed serum against F9-41 cells was 1/6000. Rabbit anti-F9 was massively absorbed against lymphocytes (twice, vol/vol, serum The absorbed conjugates were sampled and stored frozen at -80°. They were used at a 1/25 dilution.IgG and Fab Preparation. The immunoglobulin (IgG) fraction of an unabsorbed rabbit anti-F9 serum was isolated by precipitation with 40% saturated ammonium sulfate, pH 7.0, followed by a chromatography on Whatman DEAE-cellulose (DE52) according to Levy and Sober (7). For Fab preparation, the method of Porter (8) was used. IgG (100 mg) was digested with 1 mg of papain (Worthington). Undigested IgG was separ...

Chromosomal mapping of the structural gene coding for the mouse cell adhesion molecule uvomorulin.

“…The cell adhesion molecule uvomorulin is involved in the compaction process of mouse preimplantation embryos and the aggregation of embryonal carcinoma cells (2,3). In postimplantation embryos and in adult tissues, uvomorulin is exclusively expressed in epithelial cells (4); in particular, it is concentrated in the intermediate junctions of the intestine epithelial cell layer (5).…”

Expression of α-catenin in α-catenin-deficient cells results in a reduced proliferation in three-dimensional multicellular spheroids but not in two-dimensional monolayer cultures

“…All transfectants exhibiting reduced cell growth demonstrated compacted aggregation in multicellular suspension cultures; transfectants showing the same levels of cell growth as control nD cells were loosely adherent, existing as 'grape-like' aggregates in multicellular suspension cultures (see Figure 2a and Matsubara and Ozawa, 2001). Aggregate compaction (Kemler et al, 1977;Takeichi, 1977;Hyafil et al, 1980) results from a morphological change in which cells increase their adherence to each other, maximizing contact areas. This effect is associated with cadherin-mediated cell-cell adhesion (Vestweber and Kemler, 1985), involving actin cytoskeletal forces (Surani et al, 1990).…”

The Wistar Legacy to Embryonic Stem Cell Research

“…A polyclonal antibody to a molecule, originally dubbed uvomorulin, but later renamed Ecadherin, was shown to mark the changing blastomeres of 8-cell embryos, just before they compacted and formed the blastocyst with its outer layer of trophectodermal cells and its inner cell mass. (14) However, this antibody did not mark the stem cells per se. Our first monoclonal antibody to stagespecific embryonic antigen-1 (SSEA1), was made by fusing lymphocytes from mice immunized with the F9 EC cell and the requisite myeloma cell line, and then screening the supernatants from those cells surviving selection on F9 cells and on preimplantation mouse embryos.…”