Expression and distribution of cell adhesion molecule uvomorulin in mouse preimplantation embryos

DEVELOPMENTAL

BIOLOGY

124,451-456 (1987)

Expression and Distribution of Cell Adhesion Molecule Uvomorulin in Mouse Preimplantation Embryos Friedrich-Miescher-L&orator&m

der Max-Planck-Gesellschqft, Spemannstrasse 37-39,D-?‘@OTiibingen, West Germany Received April 22, 1981;’accepted August

10, 1987

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 contact 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. 0 1987 Academic PWS, IK INTRODUCTION

Uvomorulin is a cell adhesion molecule (CAM) involved in cell-cell contact of mouse preimplantation embryos, embryonal carcinoma (EC) cells, embryonal mouse hepatocytes, and adult epithelial cells (Vestweber and Kemler, 1984a, 1984b, 1985). It is a membrane glycoprotein with an apparent molecular weight of 120 kDa (gp120) (Peyrieras et al., 1983, Vestweber and Kemler, 1984a), and a structure and tissue distribution very similar to chicken L-CAM (Gallin et ab, 1983) and human cell-CAM 120/80 (Damsky et al, 1983). Other CAMS are structurally identical to uvomorulin, including E-cadherin (Yoshida-Noro et al, 1984), the arc-l antigen (Behrens et aZ., 1985), and the rr-1 antigen (Gumbiner and Simmons 1986). In early mouse development the process of compaction at the eight-cell stage is a prerequisite for the establishment of the trophectodermal and inner cell mass (ICM) cell lineages (Johnson and Ziomek, 1981). The blastomeres of early eight-cell embryos are spherical, evenly covered with microvilli, and rather loosely assoi Present address: Biozentrum der Universitlt Base1 Klingelbergstr. 70, CH-4056 Basel, Switzerland. ‘Present address: Mount Sinai Hospital Research Institute, 600 University Ave., Toronto, Ontario M5G 1X5, Canada. a Present address: Universite d’Aix-Marseille, Centre de MarseilleLuminy, Institut de Cytologie et de Biologie Cellulaire, 70, Route Leon Lachamp, F-13288 Marseille CQdex 2, France. ’ To whom correspondence should be sent: Max-Planck-Institut fur Immunbiologie, Stiibewej 51, D-7800 Freiburg, West Germany.

ciated. During compaction the outer blastomeres maximize their cell contacts and establish junctional complexes including desmosomes and tight junctions. After compaction the outer cell wall is composed of polarized cells which show distinct basolateral and apical membrane domains, the first being smooth, and the latter bearing numerous microvilli on their surfaces (Ducibella and Anderson, 1975; Handyside, 1980; Reeve and Ziomek, 1981). Thus, after compaction an epitheliumlike cell layer of polarized cells surrounds the unpolarized inner cells of the embryo. The process of compaction is disturbed by anti-uvomorulin antibodies. Twocell embryos cultured in the presence of antibodies or Fab fragments develop to a loosely attached grape-like cell mass which after removing antibodies compact and develop into blastocysts. Although the involvement of uvomorulin in the compaction process is well established it is presently less clear whether or to what degree antibodies also interfere with the polarization of eight-cell stage blastomeres (Shirayoshi et al., 1983; Johnson et al., 1986a). The polarization of blastomeres is the first sign of the onset of compaction and occurs not only at the surfaces of these cells but also with cytoplasmic components as microfilaments, microtubules, coated vesicles, and endosomes (for review, see Johnson et ah, 1986b). Although uvomorulin is found on all cells of eight-cell stages or morulae its expression in later stages of development and in adult tissues is exclusively restricted to the epithelial cell lineage (Peyrieras et aL, 1983; Vestweber and Kemler, 1984a). In adult intestinal epithelial

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0 1987 by Academic Press, Inc. of reproduction in any form reserved.

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cells uvomorulin is localized predominantly in the intermediate junctions and to a lesser extent at the basolateral membrane but is not found at the apical membrane domain of these cells (Boller et al, 1985). The present report deals with the subcellular localization and synthesis of uvomorulin in the preimplantation embryo, a unique model for the transition of a nonpolarized cell type of the early eight-cell stage blastomeres to polarized epithelial-like cells of the trophectoderm. MATERIALS

AND METHODS

Cells and embryos. The embryonal carcinoma (EC) cell line PCC4azal was cultured as described (Vestweber and Kemler, 1984a). Mouse preimplantation embryos and unfertilized eggs were collected from superovulated NMRI mice mated with Swiss albino males and cultured in Whitten’s medium (Whitten, 1971) in a 5% CO2 atmosphere at 37°C as described (Kemler et ah, 1977). If necessary embryos were freed off their zona pellucidae by treatment with Tyrode’s acid solution. Antibodies. Rabbit anti-uvomorulin antibodies were affinity purified on the 84-kDa uvomorulin fragment coupled to Sepharose 4B (Pharmacia) as described (Schuh et al., 1986). These antibodies recognize only uvomorulin (gp120) in immunoblot but additional proteins of 102, 92, and 88 kDa in immunoprecipitation experiments. Immunoprecipitation and immunoblot. PCC4azal cells were metabolically labeled overnight with 20 &i/ ml [35S]methionine (sp act 1100 Ci/mmole) in DMEM (10% fetal calf serum) without methionine. Embryos (between 75 and 350 per immunoprecipitation) were labeled for 10 hr in 100 ~1 Whitten’s medium containing 100 &i[35S]methionine. One-cell embryos (18 or 30 hr post HCG) were treated with 10 PM a-amanitin (Sigma) for 4,14, or 24 hr and subsequently labeled for 10 hr in the presence of the drug. Immunoprecipitations with affinity-purified anti-uvomorulin antibodies from detergent extracts of cells and embryos were analyzed as described earlier (Vestweber and Kemler, 1984a). Immunoblots from cell lysates of about lo3 oocytes or 2 X lo5 EC cells were carried out as described previously (Vestweber and Kemler, 1985). Controls included IgG from preimmune sera and cell lysates plus Staph A without antibodies. Electron microscopy. Embryos were fixed for 2 hr in a mixture of 2% formaldehyde and 0.2% glutardialdehyde in phosphate-buffered saline (PBS), pH 7.4. Fixed embryos were embedded in 2% agarose, immersed in 1.8 M sucrose for cryosectioning, frozen in liquid nitrogen, and cut on a microtome with cryoattachment (FC4, Reichert, Vienna, Austria) at temperatures between -70

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and -100°C. Sections of about 100 nm were treated in a drop of 2.3 M sucrose according to the procedure of Tokuyasu and Singer (Tokuyasu and Singer, 1976). The melted sections were washed in PBS to remove the sucrose and incubated with affinity-purified antibodies (15 pg/ml) and rabbit antibodies with unrelated specificities for controls. Bound antibodies were detected by protein A bound to 8-nm colloidal gold particles (Miihlpfordt, 1983), as described (Roth et al., 1978). After postfixation with 0.5% osmium tetroxide and staining with 0.5% acidic uranyl acetate, the sections were dehydrated in a graded series of ethanol and embedded in a film of Epon 812 (2% in ethanol) as described (Keller et ak, 1984). After staining with 1% lead citrate, preparations were examined in a Siemens Elmiscope Ia at 80 kV and micrographs were taken on Agfa Scientia films. RESULTS

In order to study uvomorulin synthesis during early embryogenesis, cell lysates of metabolically labeled embryos from different cleavage stages were subjected to immunoprecipitation experiments using affinity-purified antibodies. No synthesis of uvomorulin (gp120) was detected in unfertilized eggs or one-cell stage embryos (Fig. lA, lanes b and c). Uvomorulin synthesis started at the late two-cell stage (Fig. lA, lanes d and e). In contrast, in unfertilized and fertilized eggs antiuvomorulin antibodies precipitated a 102-kDa protein

9P120, P102,

- ” , I’-

a

‘IF

f

44

b

f

FIG. 1. Synthesis of uvomorulin during mouse preimplantation development. (A) Immunoprecipitation with affinity-purified anti-uvomorulin antibodies (15 pg/ml). Uvomorulin (gp120) is first synthesized in two- to four-cell embryos. Lane (a) PCC4azal cells (control); (b) 350 unfertilized eggs; (c) 180 one-cell embryos; (d) 150 two- to four-cell embryos; (e) 85 four- to eight-cell embryos; (f) 110 four- to eight-cell embryos with control IgG. (B) In immunoblots gp120 is detected in cell lysates from 800 unfertilized eggs (lane (g)). Lane (h) 1.2 X lo5 PCC4azal cells (control). (C) One-cell embryos (30 hr post HCG) were treated with 10 &f or-amanitin for 14 hr (lane (i)), 24 hr (lane (k)), and 34 hr (lane (1)) and subsequently immunoprecipitated. Lane (m) one-cell embryos (24 hr) with control IgG. Each immunoprecipitation was performed with about 350 embryos. No gp120 synthesis is detectable.

VESTWEBER ET AL.

Cell Adhesion Molecule Uv omorulin

(Fig. lA, lanes b and c) which was also detected in later stages. This protein has been found to be structurally unrelated to uvomorulin (gp120) (Vestweber and Kemler, 1984b; Peyrieras et ab, 1985) which has been confirmed in a more recent study by analyzing the peptide composition of both proteins after complete tryptic digests (D. Vestweber et al, unpublished observations). Although the gp120-102-kDa protein relation is not clear at present, the latter is always detected in immunoprecipitation experiments with anti-uvomorulin antibodies (Peyrieras et al., 1985; Vestweber and Kemler, 1984b). The early embryo is so far the only cell type where the 102-kDa protein but not gp120 is detected (compare Fig. 1, lanes a, d, and e with b and c). The 102-kDa protein is not recognized by anti-uvomorulin antibodies in immunoblot experiments (Vestweber and Kemler, 1984a; Peyrieras et ak, 1985). The 102-kDa protein recognition could be due either to some crossreactive anti-uvomorulin antibodies (Peyrieras et al., 1985) or to a complex formation with gp120, since immunoblot experiments with cell lysates of unfertilized eggs easily detect gp120 (Fig. lB, lane g). Thus in onecell embryos the newly synthesized 102-kDa protein could be associated with maternally derived gp120. Taken together the immunoprecipitation and immunoblot experiments show that uvomorulin (gp120) is already present but not synthesized in one-cell embryos, and that uvomorulin synthesis starts at the late twocell stage, perhaps at the onset of transcription of the embryonic genome. The structurally unrelated 102-kDa protein is already synthesized in unfertilized eggs. That its synthesis requires stored maternal mRNA is suggested from immunoprecipitations with cy-amanitintreated one-cell embryos where the 102-kDa protein synthesis was unchanged in comparison to control embryos (Fig. lC, lane i). Culturing one-cell embryos with the drug for different times revealed that the 102-kDa protein synthesis from maternal mRNA can still be detected up to an equivalent four-cell stage. On the other hand, a-amanitin blocked the synthesis of gp120 (Fig. lC, lanes k and 1). This, together with the onset of uvomorulin synthesis at the late two-cell stage, supports the idea that gp120 synthesis depends on embryonic gene expression. Uvomorulin is expressed on the cell surface of all preimplantation stage embryos including unfertilized eggs as monitored by indirect immunofluorescence tests (not shown) which confirmed earlier observations (Hafil et ak, 1983; Shirayoshi et al., 1983; Johnson et al., 1986a). Immunoelectron microscopy on ultrathin frozen sections was performed on two-cell and subsequent cleavage stage embryos to study the subcellular localization of uvomorulin. At the two- to four-cell stage the cell surface of blastomeres was uniformly labeled (Fig.

in Mouse Preimplantation

Embryos

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2A). Uniform labeling was also observed on the cell surface of polar bodies (not shown) which supports the finding that uvmorulin is present on the cell surface before the onset of embryonic gene activity. At the four-cell stage, a higher uvomorulin concentration was observed in areas of close membrane apposition (Fig. 2B). In morulae (16- to 32-cell stage) the apical surface of outer cells was essentially free of gold particles (Figs. 3A, 3B). Labeling was restricted to cell membranes between adjacent cells. In areas where zonulae adherens were forming, structures resembling intermediate junctions were heavily labeled (Fig. 3B, arrow), while tight junctions and desmosomes were negative. In contrast to the restricted uvomorulin localization in the outer cells of compact morulae, inner cells exhibited even cell surface labeling (Fig. 3C), though more label was observed over the microvilli (Fig. 3D). These microvilli have been described as basal microvilli in regions of imminent cell-cell contacts (Ducibella et ak, 1977). When ultrathin sections of blastocysts were labeled with anti-uvomorulin antibodies, the staining pattern was similar to that found in compact morulae. The apical surface of trophectodermal cells was nega-

FIG. 2. Immunoeleetron microscopical localization of uvomorulin on four-cell stage mouse embryos. Uvomorulin is evenly distributed on the whole cell surface (A) but concentrated in areas of close membrane apposition (B). Bars represent 0.5 pm, respectively.

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tive. Gold particles were localized at lateral membranes (Fig. 4A), in agreement with the observation by Damjanov et al. (1986) that the lateral membrane domains of trophectodermal cells stained with anti-human cell CAM 120/80 antibodies. In addition uvomorulin antibodies reacted with the basal membrane of trophectodermal cells, even on cells facing the blastocyst cavity (not shown). Staining of the ICM resembled that found on inner cells of compact morulae, in that the cell surface was uniformly labeled (Fig. 4B). DISCUSSION

In blastocyst formation the transition from spherical nonpolarized cells to the formation of the epithelial cell layer of the trophectoderm is initiated by close cell-cell contacts and accompanied by the appearance of distinct junctional structures (Ducibella et al., 1977). This transition is the first example of the formation of an epithelial layer, one of the basic morphogenetic events in multicellular organisms. Uvomorulin has been shown to be involved in the compaction process of early embryos (Kemler et ak, 19’77;Hyafil et al., 1980; Vestweber and Kemler, 1984a), and in the maintenance of the epithelial layer of adult epithelial cells (Vestweber and Kemler, 1985; Behrens et ah, 1985; Gumbiner and Simons, 1986), although the molecular mechanism of the uvomorulinmediated adhesion is not known. In this report we examined whether uvomorulin synthesis or cell surface localization could be correlated with the onset of compaction and the formation of trophectodermal cells. We show that uvomorulin synthesis starts at the late twocell stage, when stored maternal mRNA is broken down and the embryonic genome is activated (Bachvarova and DeLeon, 1980; Piko and Clegg, 1982; Clegg and Piko, 1983). Uvomorulin synthesis at this stage is blocked by the RNA polymerase II inhibitor a-amanitin, suggesting that uvomorulin synthesis depends on newly transcribed embryonal mRNA. The start of uvomorulin synthesis coincides with the onset of embryonic gene activity, rather than with the onset of compaction, so it seems unlikely that uvomorulin synthesis triggers compaction. Our immunoblot and immunofluorescence results indicate that uvomorulin is already present in unfertil-

FIG. 3. Immunoelectron microscopical localization of uvomorulin on compact morulae (16- to 3%cell stage). The outer surface of the embryo is essentially free of labeling (A, B). In the zonula adherens significant labeling was found in structures resembling the intermediate junctions (arrow) whereas tight junctions and desmosomes are not labeled (B). Cell surfaces of inner cells are homogeneously labeled (C) with a more prominent labeling over the basal microvilli (D). ds, desmosome; es, embryonic surface, tj, tight junction. Bar represents 0.5 pm.

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Cell Adhesion Molecule Uvomwulin in Mouse Preimpluntation Embryos

are found in four-cell embryos. Uvomorulin becomes more concentrated in the areas of close cell contact described by Magnuson et al. (1977) as focal points of membrane contact with underlying cytoplasmic densities. It is possible that the higher concentration of uvomorulin in these cell contact areas mediates the adhesion of four-cell blastomeres. The uvomorulin redistribution finally leads to the restriction of uvomorulin to the basolateral membrane in trophectodermal cells. A recent hypothesis about the establishment of the occluding barrier during the formation of an epithelial cell layer postulates that uvomorulin redistribution and the assembly of the zona adherens via uvomorulin-mediated interactions are initial events (Gumbiner and Simons, 1987). Clearly the redistribution of uvomorulin is a developmentally regulated event, since it occurs in compact morulae only in cells committed for epithelial cell differentiation. REFERENCES ANDERSON, E., and ALBERTINI, D. F. (1976). Gap junctions between the oocyte and companion follicle cells in the mammalian ovary. J. Cell Biol. 71,680-686. BACHVAROVA, R., and DELEON, V. (1980). Polyadenylated RNA of mouse ova and loss of maternal RNA in early development. Dev. Biol 74, l-8. BEHRENS,

FIG. 4. Immunoelectron microscopical localization of uvomorulin on blastocysts. The outer cell surface of trophectodermal cells is negative while labeling is restricted to the lateral membrane parts (A). Cells of the inner cell mass are uniformly labeled (B). es, embryonic surface; tj, tight junction. Bar represents 0.5 grn.

ized eggs. Since uvomorulin is not synthesized at that stage it is presumably maternally derived, perhaps persisting from oogenesis. During egg growth and maturation, granulosa cells contact the oocytes by cytoplasmic processes through the intervening zona pellucida. Specialized membrane areas such as gap junctions and desmosomes have been reported at these sites of oophorus-oocyte contact (Zamboni, 1974; Anderson and Albertini, 1976; Gilula et aZ.,1978). Uvomorulin was not found in the developing follicle and stroma of ovaries of 16-day-old embryos (Damjanov et ak, 1986), but could play a role later, in the establishment of cell contacts in primordial follicles. Although uvomorulin does not seem to trigger the onset of compaction, our distribution studies show that compaction is accompanied by uvomorulin redistribution on the cell surface. The earliest signs of a change of the uniform uvomorulin distribution on the cell surface

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