Ultrastructural Study of Concanavalin-A Binding to the Surface of Preimplantation Mouse Embryos 1 MARIAN KONWINSKI,2 ANDRZEJ VORBRODT,a DAVOR SOLTER AND HILARY KOPROWSKI The Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, Pennsylvania 19104
ABSTRACT Receptors for Con-A were labelled (using the peroxidasediaminobenzidine 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 pgl ml, 50 pglml, or 1,000pglml 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 andlor 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 pgiml 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. Lectin binding to specific carbohydrate residues of the cell membrane has been utilized to investigate the surface properties of embryonic cells. Concanavalin A (Con-A) causes agglutination of fertilized and parthenogenetically activated eggs and cleavage stage embryos; however, unfertilized eggs are only agglutinated using a 200 times higher concentration (Pienkowski, '74). These results contrast with those obtained using lectin binding assays. The same amount of either lZ5I-Con-Abinds to the surface of mouse eggs (Pienkowski, '74) or fluorescein-conjugated (FITC) Con-A to hamster eggs (Yanagimachi and Nicolson, '74)prior to and after fertilization. On the contrary, as a result of fertilization, a great increase in Con-A-peroxidase product on the prefixed rabbit eggs was observed by electron microscopy (Gordon et al., '75). Johnson et al. ('75),using FITC-conjugated J. EXP. ZOOL., 200: 311-324.
Con-A, observed that the surface area overlying the mitotic spindle of secondary mouse oocyte does not bind Con-A. Since this part of the surface is sequestered with the second polar body, the entire membrane of newly fertilized eggs is positive. The Con-A-mediated agglutinability of preimplantation mouse embryos gradually decreases during development with dramatic reduction at the blastocyst stage (Rowinski et al., '76). Even after trypsin treatment, blastocysts were agglutinable only with a Con-A concentration 500 times higher than that which agglutinated cleavage stages. This loss ' This work was supported by USPHS research grants CA-10815 and CA-17546 from the National Cancer Institute. RR-05540 from the Division of Research Resources, PDT-26 from the American Cancer Society and funds from the World Health Organization. Fellow at The Wistar Institute from the Institute of Biostructure, Medical School, Warsaw, Poland. Fellow at The Wistar Institute from the Institute of Oncology, Gliwice, Poland recipient of a World Health Organization Exchange of Research Workers grant.
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of agglutinability a t the blastocyst stage was the sole property of external trophoblast cells because isolated inner cell masses were agglutinated by Con-A just as well as cleavage stages (Rowinski et al., '76).However, Con-A binding sites were demonstrated on the trophoblast surface of prefixed mouse blastocysts by the peroxidase method with an electron microscope (Enders and Schlafke,
'74). Using FIK-Con-A, the binding sites on the surface of sea urchin embryo micromeres were found to be randomly distributed on prefixed cells and clustered on live cells (Roberson et al., '75).By means of ferritin-lectin conjugate, lectin binding sites were visualized by electron microscopy on the surface of unfertilized mouse, rat, and hamster eggs (Nicolson et al., '75).Lectin binding sites were randomly distributed if the eggs were prefixed in aldehyde or labelled a t 0°C. Eggs labelled at 25°C showed aggregated wheat germ agglutinin (WGA) and Con-A binding sites and uniform distribution of Ricinus communis agglutinin (RCA),-binding sites on their plasma membranes. In this work we investigate whether the previously observed differences in agglutinability of preimplantation mouse embryos (Pienkowski, '74;Rowinski et al., '76) could be correlated with the distribution of Con-A binding sites labelled with the product of the peroxidase reaction.
of streptomycin, and buffered to pH 7.4 with 5 mM Hepes (Sigma) were used (Pienkowski et al., '74).Hyaluronidase and pronase were dissolved in the same media but the bovine serum albumin was omitted. After removal of the zonae pellucidae, the embryos were incubated for four hours a t 37°C in 5% CO, in air or in 5% COz, 5% 0,and 90% N,. (No appreciable difference was observed between embryos handled in different media and different air mixtures and they are considered together.) Inner cell masses were isolated immunosurgically from blastocysts (Solter and Knowles, '75).
Labelling of embryos To detect Con-A binding sites, the method used was essentially that described by Bernhard and Avrameas ('71) with further modifications related to the use of unfixed cells as described by Bretton et al. ('72) and MartinezPalomo et al. ('72). In the first series of experiments, unfixed embryos were rinsed carefully with phosphate buffered saline (PBS), treated for 15 minutes a t 37°C with Con-A in PBS (1,000pg/ml), washed three times in PBS, exposed to peroxidase in PBS (1,000pg/ml) for 15 minutes a t room temperature (22"C),again washed with PBS and fixed in ice-cold 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2 for 15 minutes, and washed with the same buffer. After fixation, the cells were exposed to 3'3'diaminobenzidine (DAB) 2.5 mg in 0.05 M MATERIALS AND METHODS Tris-HC1 buffer (pH 7.6)containing H,O, for Preparation of embryos 15 minutes a t room temperature, washed and Virgin ICR female mice were superovulated postfixed in ice-cold 1.5% OsO, in 0.1 M cacodyby intraperitoneal injections of 5 IU of preg- late buffer (pH 7.2)for one hour. After washnant mare serum (Gonadin, Cutter) and, 48 ing, the embryos were embedded in 1.5% agar, hours later, with 5 IU of human chorionic dehydrated in ethanol and finally embedded gonadotropin (Pregnyl, Organon) Cumuli oo- in Epon 812 mixture (Konwinski et al., '74). phori were released from oviducts 14 hours In one experiment, the embryos were exafter the second injection; cells of the cumu- posed to Con-A after fixation in 2.5% glutarallus oophorus were removed with 100 IU/ml of dehyde for 30 minutes, according to the orighyaluronidase (Sigma) and secondary oocytes inal technique of Bernhard and Avrameas were collected in handling medium. Fertilized ('71). In other experiments, the embryos were eggs, cleavage stages, and blastocysts were exposed to lower concentrations of Con-A in flushed a t the appropriate time after fertiliza- PBS (10pg/ml and 50 pg/ml) a t 16°C for 30 tion from oviducts or uteri of spontaneously minutes, and processed as described above. ovulating ICR females. Zonae pellucidae were The control medium contained, in addition to removed with 0.2% pronase (Calbiochem).For Con-A, 0.05M a-methyl-D-mannoside. collection and handling of embryos, either The surface coat was also visualized in emWhitten's medium (Whitten, '71)or Eagle's bryos using the Ruthenium Red technique of minimal essential medium supplemented Luft ('64) with modifications described by with 0.3% bovine serum albumin, 10 mM glu- Martinez-Palomo and Brailovsky ('68). tamine, 100,000U/1 of penicillin and 50 mg/l Ultrathin sections unstained or stained
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with lead and uranyl acetate were examined Two-cell embryos under a Hitachi HS-8 or Hitachi HV-11E elecThe distribution of the surface layer reactron microscope. tion product after treatment with the lowest (10 pglml) concentration of Con-A is irreguRESULTS lar and discontinuous, the same as in oocytes Unfertilized eggs and zygotes. Only short segments of surface Mouse oocytes treated with the lowest con- layer show a noticeable concentration of eleccentration of Con-A (10 pg/ml) show a very tron-dense material (fig. 9). The pinocytotic thin layer of discontinuous, moderately elec- channels and vesicles contain densely packed tron-dense material covering the external reaction product. Exposure to higher concensurface of the cell (fig. 1).This layer is simi- trations of Con-A (50 pg/ml and 1,000 pglml) larly thin on the surfaces of long processes result in a thicker and continuous layer of the and microvilli (fig. 1) and on the smoother reaction product. segments of the cell surface (fig. 2). In cell Four- and eight-cell embryos membrane invaginations and inside pinocyAt this stage of development, even if the totic vesicles, the electron-dense reaction product is more concentrated (fig. 1). Some lowest concentration of Con-A (10 pglml) is variations in labelling of individual oocytes used, the reaction product forms a thin, but may exist, but a predominant pattern can be continuous layer situated on the external surdiscerned after examining several sections of face of cell membrane (figs. 10, 13). Under higher magnification, however, one can oba few cells. The surfaces of oocytes treated with a high- serve that the concentration of electron-dense er concentration of Con-A (50 pglml) are material is uneven, and more dense and covered with a relatively thick layer of elec- thicker short segments are interspersed tron-dense product (fig. 3). This layer, how- among thinner and lighter areas (fig. 11).At ever, is still discontinuous and of a non- higher concentrations of Con-A (50 pglml), the surface layer is quite dense but also of a uniform thickness. After treatment with the highest concen- nonuniform thickness (fig. 12). A very similar tration of Con-A (1,000 pglml) the electron- pattern is observed after treatment with the dense reaction product forms a continuous, highest concentration (1,000pg/ml) of Con-A. rather uniformly distributed layer covering Generally, the differences in thickness and both smooth and undulated segments of the density of surface labelling between embryos a t different stages are more noticeable a t the cell surface (fig. 4). lowest (10 pg/ml) rather than the higher (50 or 1,000 pg/ml) concentrations of Con-A. This Fertilized eggs is clearly visible when figures 1,2,5,9, 10 and After fertilization, the overall pattern of 13, all representing labelling with 10 pg/ml distribution of the reaction product is very Con-A, are cotnpared with electron microsimilar to that observed in oocytes. Exposure graphs showing embryos treated with higher to the lowest concentration of Con-A (10 p g l concentrations (50or 1,000 pg/ml) of Con-A ml) gives a very thin, discontinuous layer of (figs. 3, 4, 6,7, 12). material of a low electron density (fig. 5 ) . In Morulae caveolae and pinocytotic vesicles the electrondense material is always more concentrated The surface of morulae show numerous prothan on the free cell surface. trusions, microvilli, and caveolae. After treatThe appearance of fertilized eggs (fig. 6 ) ment with a low concentration of Con-A (10 treated with 50 pg/ml of Con-A is very similar pglml), the surface layer appears thinner and to that of unfertilized eggs. Treatment with more uneven than in previous stages (comthe highest doses of Con-A (1,000 pglml) pare figs. 13 and 14). On the flat segments of leads to the appearance of an almost entirely plasma membrane the reaction product is continuous, relatively thick surface layer (fig. dense, while on microvilli i t becomes thin and 7). The pattern of the surface layer revealed discontinuous (fig. 14). In pinocytotic vacby the Ruthenium Red technique (fig. 8) is uoles or in deep caveolae, high concentration almost identical to that obtained after treat- of electron-dense material is frequently obment with the highest concentration of served. The reaction product penetrates beCon-A. tween cells only to the level of the apical junc-
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The use of low concentrations of Con-A and the incubation of unfixed embryos a t 16'C further allows observation of the lectin-induced receptors redistribution occurring as a result of their mobility, as suggested by Collard and Temmink ('74). The concentration of Con-A a t 50 pg/ml recommended by Bretton et al. ('72) seems too high for such purposes because the Blastocysts differences in the distribution of Con-A bindAfter treatment with low concentrations ing sites on the surfaces of embryos were not (10 pg/ml) of Con-A, a relatively thin and dis- as easily perceived. Differences in the intensity and character continuous layer of reaction product forms on the surface of most trophoblastic cells (fig. of surface labelling were more distinct be17). This layer is of uneven thickness and it is tween concentrations of 10 pg/ml and 50 pg/ composed of alternatively distributed less or ml than between 50 pg/ml and 1,000pg/ml of more electron-dense segments. The similarly Con-A. Only the comparison of distributions uneven, discontinuous but thicker layer with of the reaction product a t a concentration of few clusters is present on the external surface 10 pg/ml allowed the observation that the relof trophoblastic cells treated with a more con- ative amount of Con-A bound to the cell surcentrated (50 pg/ml) solution of Con-A (fig. face is very similar on the surfaces of oocytes, 18).In deep cavities, in marginal gulfs, and in zygotes, and 2-cell embryos. Con-A receptors pinocytotic vacuoles (fig. 18: arrow) the elec- are a t these stages of development few and tron-dense product is more concentrated than non-uniformly distributed, possibly as the result of Con-A-induced redistribution (Collard on the surface. After treatment with the highest concen- and Temmink, '74). Our results conflict with tration of Con-A (1,000 pg/ml) the superficial those of Gordon et al. ('75) who found, also by layer is thin but continuous, with very numer- Con-A binding, a distinct increase in the ous, large clusters of darkly stained material thickness of the glycoprotein surface layer on (fig. 21: arrowheads). These clusters are pres- the rabbit eggs following fertilization. The ent on the external surface of the tropho- use of different species might explain these blastic cells. On the contrary, the surface of differences. Pienkowski ('74) observed a trophoblastic cells facing the inner cell mass marked increase in agglutinability after the (EM) or blastocoele is always free of the reac- fertilization of mouse oocytes, although he did not find a difference in the binding capacity of tion product (fig. 2 1: arrows). The exposure of glutaraldehyde-fixed cells 1251-labelledCon-A between oocytes and to a high concentration of Con-A (1,000 p g l zygotes. We also did not observe the differml) leads to the formation of prominent ences in the distribution and amount of surclusters of the reaction product, especially on face labelling with Con-A on unfertilized and fertilized mouse eggs. These observations sugthe surface of the blastocysts (fig. 20). The isolated ICM exposed to Con-A shows gest that agglutinability is not directly rethe presence on their surface of a thin, but lated to the amount of bound Con-A, but clearly visible, layer of dense material (fig. 22: reflects some additional changes in cell SUParrowheads). This electron-dense material is face. not present on the plasma membrane of interAn increase of receptor number, leading to nally located cells (fig. 22: arrows). After the formation of a thicker and continuous treatment of embryos with control medium layer of the reaction product, was noted in 4containing both Con-A (50 pglml) and a- and 8-cell embryos. Concomitant with the methyl-D-mannoside (0.05M) the cell surface differentiation of trophoblastic cells, changes remains completely unstained (fig. 19). in the distribution of Con-A receptors and the formation of clusters is observed. These DISCUSSION clusters might result from aggregation of Our results suggest that the lowest concen- Con-A binding sites, which would be indicatrations of Con-A (10 pg/ml) best demon- tive of the increased mobility of surface recepstrate the subtle differences in the amounts of tors. In the blastocyst stage the distribution Con-A bound to the cell surface between of surface receptors on trophoblastic cells is different stages of embryonic development. similar to that observed on the surface of nortional complex (fig. 14). After exposures to higher concentrations of Con-A (50 pg/ml) the surface layer becomes thicker, and a few dense clusters appear (fig. 15). These clusters are more numerous and easily noticeable after treatment with Con-A a t a concentration of 1,000 pg/ml (fig. 16, arrows).
CON-A BINDING SITES OF PREIMPLANTATION EMBRYOS
mal, differentiated cells (Bretton et al., '72; Enders and Schlafke, '74; Temmink et al., '75). In mouse blastocyst, tight junctions forming zonae occludentes are formed between trophoblastic cells (Ducibella et al., '75; Enders and Schlafke, '74); therefore, the components of incubation medium and the reaction products are not able to penetrate between the trophoblastic cells and, consequently, no reaction product would be noted on the surfaces of cells of ICM. The distribution of Con-A receptors on the free surface of immunosurgically (Solter and Knowles, '75) isolated ICMs was very similar to that observed in 4- or 8-cell embryos. Thus, the distribution of Con-A receptors on the ICM is of an early embryonic type, while the distribution of receptors on the surface of trophoblastic cells compares to that observed in adult cells. This change of surface property of trophoblastic cells may be the reason for diminished agglutinability of blastocysts as noted by Rowinski et al. ('76). In accordance with the suggestion of Temmink et al. ('75) we can suppose that the differences in clustering of Con-A binding sites between early embryos and trophoblastic cells are responsible for differences in the agglutinability by Con-A. The lack of clustering observed on the surface of isolated ICM is accompanied by their agglutinability with low concentrations of Con-A, whereas the whole blastocyst is not agglutinable (Rowinski et al., '76). Our results suggest that the agglutinability of early preimplantation mouse embryos depends not only on the number and distribution of Con-A receptors, but probably on other physico-chemical properties of cell surface which can influence the mobility of surface layer and the binding sites of Con-A. Fluidity of the plasma membrane and possible redistribution and clustering of Con-A receptors probably influence to a large extent agglutinability of early embryos. LITERATURE CITED Bernhard, W., and S. Avrameas 1971 Ultrastructural visualization of cellular carbohydrate components by means of Concanavalin A. Exp. Cell Res., 64: 232-236. Bretton, R., R. Wicker and W. Bernhard 1972 Ultrastructural localization of Concanavalin A receptors in normal
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and SV40-transformed hamster and rat cells. Int. J. Cancer, 10: 397-410. Collard, J. G., and J. H. M. Temmink 1974 Binding and cytochemical detection of cell-bound Concanavalin A. Exp. Cell Res., 86: 81-86. Ducibella, T., D. F. Albertini, E. Anderson and J. D. Biggers 1975 The preimplantation mammalian embryo: characterization of intercellular junctions and their appearance during development. Develop. Biol., 45: 231-250. Enders, A. C., and S. Schlafke 1974 Surface coats of the mouse blastocyst and uterus during the preimplantation period. Anat. Rec., 180: 31-46. Gordon, M., L. R. Fraser and P. V. Dandekar 1975 The effect of ruthenium red and concanavalin A on the vitelline surface of fertilized and unfertilized rabbit ova. Anat. Rec., 181: 95-112. Johnson, M. H., D. Eager, A. Muggleton-Harris and H. M. Grave 1975 Mosaicism in organization of cancanavalin A receptors on surface membrane of mouse eggs. Nature, 257: 321-322. Konwinski, M., J. Abramczuk, W. Baranska and W. Szymkowiak 1974 Size changes of mouse ova during preparation for morphometric studies in the electron microscope. Histochem., 42: 315-322. Luft, J. M. 1964 Electron microscopy of cell extraneous coats a s revealed by Ruthenium Red staining. J. Cell. Biol., 23: 53A-54A. Martinez-Palomo, A,, and C. Brailovsky 1968 Surface layer in tumor cells transformed by Adeno-12 and SV40 viruses. Virology, 34: 379-382. Martinez-Palomo,A,, R. Wicker and W. Bernhard 1972 U1trastructural detection of concanavalin surface receptors in normal and in polyoma-transformed cells. Int. J. Cancer, 9: 676-684. Nicolson, G. L., R. Yanagimachi and H. Yanagimachi 1975 Ultrastructural localization of lectin-binding sites on the zonae pellucidae and plasma membranes of mammalian eggs. J. Cell. Biol., 66: 263-274. Pienkowski, M. 1974 Study of the growth regulation of preimplantation mouse embryos using concanavalin A. Proc. Soc. Exp. Biol. Med., 145: 464-469. Roberson, N., A. Neri and S. B. Oppenheimer 1975 Distribution of Concanavalin A receptor sites on specific populations of embryonic cells. Science, 189: 639-640. Rowinski, J., D. Solter and H. Koprowski 1976 Changes of Concanavalin A induced agglutinability during preimplantation mouse development. Exp. Cell Res., 100: 404408. Solter D., and B. B. Knowles 1975 Immunosurgery of mouse blastocyst. Proc. Nat. Acad. Sci. (USA.), 72:50995102. Temmink, J. H. M., J. G. Collard, H. Spits and E. Roos 1975 A comparative study of four cytochemical detection methods of concanavalin A binding sites on the cell membrane. Exp. Cell Res., 92: 307-322. Whitten, W. K. 1971 Nutrient requirements for the culture of preimplantation embryos in vitro. Adv. Biosci., 6: 129-141. Yanagimachi, R., and G. L. Nicolson 1974 Changes in lectin-binding to the plasma membranes of hamster eggs during maturation and preimplantation development. J. Cell. Biol., 63: 381a.
PLATE 1 EXPLANATION OF FIGURES
Figures 1 to 4 show the surface of oocytes after Con-A-peroxidasereaction. The sections are either unstained or stained with lead citrate and uranyl acetate. All magnifications are X 17,400.
1 The surface of the oocyte exposed to Con-A in a concentration of 10pg/ml at 16°C. The discontinuous, thin layer of reaction product covers the numerous protrusions and microvilli. In surface invaginations and in pinocytotic vesicles the concentration of the reaction product is higher than on microvilli. 2 Another fragment of the oocyte treated as that presented in figure 1. An interrupted, thin surface layer is visible.
3 The oocyte exposed to Con-A at a concentration of 50 fig/ml at 16OC. The surface layer is thicker than in oocytes shown in figures 1 and 2. 4 The oocyte exposed to Con-A at a concentration of 1,00Opg/rnlat 37°C. The surface layer is relatively thick,
similar to that observed at a Con-A concentration of 50 pg/ml. Figures 5 to 8 show the surface of one-cell embryos (zygotes) after Con-A or Ruthenium Red reaction. All magnifications are at X 17,400.
5 The surface of zygote treated similarly to the oocytes presented in figures 1 and 2. The layer of the reaction product is discontinuous and very thin. 6 The surface of a zygote treated with Con-A at a concentration of 50 pg/ml. The reaction product is forming a nonuniformly thick layer. 7 The surface of a zygote treated with Con-A at a concentration of 1,00Opg/ml. The layer of the reaction product is relatively thick and similar to that observed in the oocyte (compare fig. 4). 8 Ruthenium Red stained coat on the surface of the zygote. The thickness of this coat is very similar to the surface coat observed with high concentrations of Con-A (compare figs. 7 and 4).
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PLATE 1
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PLATE 2 EXPLANATION OF FIGURES
9 The surface of a 2-cell embryo incubated with Con-A (10pg/ml). The layer of the reaction product is discontinuous and highly concentrated in pinocytotic vesicles and channels. X 17,400.
10 The surface of a four-cell embryo exposed to Con-A a t a concentration of 10 pg/ml. The surface layer becomes continuous. X 17,400. 11 The surface of the same embryo as presented in fig. 10, shown under higher magnification. The uneven distribution of the reaction product is evident. X 39,000.
12 The reaction product on the surface of a four-cell embryo exposed to Con-A at a concentration of 50 pgiml. X 17,900. 13 The surface of an eight-cell embryo treated with Con-A at a concentration of 10 pg/ml. The continuous, relatively thick layer is well visible on the cell surface. X 17,900. 14 The surface of the morula treated with Con-A at a concentration of 10pg/ml. The thickness of the surface layer is nonuniform (uneven) and some gaps can he observed. Notice that reaction product does not penetrate beyond the junctional complex (arrow). X 17,900. 15 The morula treated with Con-A a t a concentration of 50 pg/ml. The surface layer is thicker than a t a concentration of 10 pg/ml (compare fig. 14); it is continuous and formation of small clusters (caps) can he noted. X 17,900. 16 The morula treated with Con-A a t a Concentration of 1,000pg/ml. The thickness of the surface-layer is similar to that shown in figure 15. Numerous clusters are present (arrows). X 17,900.
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PLATE 2
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PLATE 3 EXPLANATION OF FIGURES
17 The surface of external (trophoblastic) cells of blastocyst exposed to Con-A at a concentration of 10 pg/ml. The reaction product forms a thin, discontinuous layer. X 17,900. 18 The blastocyst exposed to Con-A at a concentration of 50pg/ml. The undulated cell surface is covered by a layer of uneven thickness and nonuniform electron density. The higher concentration of the reaction product is visible in cytoplasmic vacuoles (arrows). X 17,900.
19 The blastocyst exposed to Con-A 50pg/ml and methylmannoside f0.05M). No reaction product is detectable on the cell surface. X 17,900. 20 The blastocyst exposed to Con-A (1,000 pg/ml) after fixation in glutaraldehyde. The large clusters of the reaction product protrude from the cell surface. X 12,300. 21 The surface of a trophoblastic cell from a blastocyst exposed to Con-A (1,000 pg/ml). Numerous clusters and caps of the reaction product are noticeable on the external surface of the cell. The internal surface facing the blastocoele (arrows) displays a lack of reaction product. X 12,300.
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PLATE 3
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PLATE 4 EXPLANATION OF FIGURE
22 The inner cell mass (ICMf after exposure to Con-A at a concentration of 50 wg/ml. The black reaction product is present on the surface of the cell mass (arrowheads) but absent from the surface of internal cells (arrows). X 6,300.
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PLATE 4
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ABSTRACT Receptors for Con-A were labelled (using the peroxidasediaminobenzidine 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 pgl ml, 50 pglml, or 1,000pglml 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 andlor 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 pgiml 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. Lectin binding to specific carbohydrate residues of the cell membrane has been utilized to investigate the surface properties of embryonic cells. Concanavalin A (Con-A) causes agglutination of fertilized and parthenogenetically activated eggs and cleavage stage embryos; however, unfertilized eggs are only agglutinated using a 200 times higher concentration (Pienkowski, '74). These results contrast with those obtained using lectin binding assays. The same amount of either lZ5I-Con-Abinds to the surface of mouse eggs (Pienkowski, '74) or fluorescein-conjugated (FITC) Con-A to hamster eggs (Yanagimachi and Nicolson, '74)prior to and after fertilization. On the contrary, as a result of fertilization, a great increase in Con-A-peroxidase product on the prefixed rabbit eggs was observed by electron microscopy (Gordon et al., '75). Johnson et al. ('75),using FITC-conjugated J. EXP. ZOOL., 200: 311-324.
Con-A, observed that the surface area overlying the mitotic spindle of secondary mouse oocyte does not bind Con-A. Since this part of the surface is sequestered with the second polar body, the entire membrane of newly fertilized eggs is positive. The Con-A-mediated agglutinability of preimplantation mouse embryos gradually decreases during development with dramatic reduction at the blastocyst stage (Rowinski et al., '76). Even after trypsin treatment, blastocysts were agglutinable only with a Con-A concentration 500 times higher than that which agglutinated cleavage stages. This loss ' This work was supported by USPHS research grants CA-10815 and CA-17546 from the National Cancer Institute. RR-05540 from the Division of Research Resources, PDT-26 from the American Cancer Society and funds from the World Health Organization. Fellow at The Wistar Institute from the Institute of Biostructure, Medical School, Warsaw, Poland. Fellow at The Wistar Institute from the Institute of Oncology, Gliwice, Poland recipient of a World Health Organization Exchange of Research Workers grant.
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of agglutinability a t the blastocyst stage was the sole property of external trophoblast cells because isolated inner cell masses were agglutinated by Con-A just as well as cleavage stages (Rowinski et al., '76).However, Con-A binding sites were demonstrated on the trophoblast surface of prefixed mouse blastocysts by the peroxidase method with an electron microscope (Enders and Schlafke,
'74). Using FIK-Con-A, the binding sites on the surface of sea urchin embryo micromeres were found to be randomly distributed on prefixed cells and clustered on live cells (Roberson et al., '75).By means of ferritin-lectin conjugate, lectin binding sites were visualized by electron microscopy on the surface of unfertilized mouse, rat, and hamster eggs (Nicolson et al., '75).Lectin binding sites were randomly distributed if the eggs were prefixed in aldehyde or labelled a t 0°C. Eggs labelled at 25°C showed aggregated wheat germ agglutinin (WGA) and Con-A binding sites and uniform distribution of Ricinus communis agglutinin (RCA),-binding sites on their plasma membranes. In this work we investigate whether the previously observed differences in agglutinability of preimplantation mouse embryos (Pienkowski, '74;Rowinski et al., '76) could be correlated with the distribution of Con-A binding sites labelled with the product of the peroxidase reaction.
of streptomycin, and buffered to pH 7.4 with 5 mM Hepes (Sigma) were used (Pienkowski et al., '74).Hyaluronidase and pronase were dissolved in the same media but the bovine serum albumin was omitted. After removal of the zonae pellucidae, the embryos were incubated for four hours a t 37°C in 5% CO, in air or in 5% COz, 5% 0,and 90% N,. (No appreciable difference was observed between embryos handled in different media and different air mixtures and they are considered together.) Inner cell masses were isolated immunosurgically from blastocysts (Solter and Knowles, '75).
Labelling of embryos To detect Con-A binding sites, the method used was essentially that described by Bernhard and Avrameas ('71) with further modifications related to the use of unfixed cells as described by Bretton et al. ('72) and MartinezPalomo et al. ('72). In the first series of experiments, unfixed embryos were rinsed carefully with phosphate buffered saline (PBS), treated for 15 minutes a t 37°C with Con-A in PBS (1,000pg/ml), washed three times in PBS, exposed to peroxidase in PBS (1,000pg/ml) for 15 minutes a t room temperature (22"C),again washed with PBS and fixed in ice-cold 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2 for 15 minutes, and washed with the same buffer. After fixation, the cells were exposed to 3'3'diaminobenzidine (DAB) 2.5 mg in 0.05 M MATERIALS AND METHODS Tris-HC1 buffer (pH 7.6)containing H,O, for Preparation of embryos 15 minutes a t room temperature, washed and Virgin ICR female mice were superovulated postfixed in ice-cold 1.5% OsO, in 0.1 M cacodyby intraperitoneal injections of 5 IU of preg- late buffer (pH 7.2)for one hour. After washnant mare serum (Gonadin, Cutter) and, 48 ing, the embryos were embedded in 1.5% agar, hours later, with 5 IU of human chorionic dehydrated in ethanol and finally embedded gonadotropin (Pregnyl, Organon) Cumuli oo- in Epon 812 mixture (Konwinski et al., '74). phori were released from oviducts 14 hours In one experiment, the embryos were exafter the second injection; cells of the cumu- posed to Con-A after fixation in 2.5% glutarallus oophorus were removed with 100 IU/ml of dehyde for 30 minutes, according to the orighyaluronidase (Sigma) and secondary oocytes inal technique of Bernhard and Avrameas were collected in handling medium. Fertilized ('71). In other experiments, the embryos were eggs, cleavage stages, and blastocysts were exposed to lower concentrations of Con-A in flushed a t the appropriate time after fertiliza- PBS (10pg/ml and 50 pg/ml) a t 16°C for 30 tion from oviducts or uteri of spontaneously minutes, and processed as described above. ovulating ICR females. Zonae pellucidae were The control medium contained, in addition to removed with 0.2% pronase (Calbiochem).For Con-A, 0.05M a-methyl-D-mannoside. collection and handling of embryos, either The surface coat was also visualized in emWhitten's medium (Whitten, '71)or Eagle's bryos using the Ruthenium Red technique of minimal essential medium supplemented Luft ('64) with modifications described by with 0.3% bovine serum albumin, 10 mM glu- Martinez-Palomo and Brailovsky ('68). tamine, 100,000U/1 of penicillin and 50 mg/l Ultrathin sections unstained or stained
CON-A BINDING SITES OF PREIMPLANTATION EMBRYOS
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with lead and uranyl acetate were examined Two-cell embryos under a Hitachi HS-8 or Hitachi HV-11E elecThe distribution of the surface layer reactron microscope. tion product after treatment with the lowest (10 pglml) concentration of Con-A is irreguRESULTS lar and discontinuous, the same as in oocytes Unfertilized eggs and zygotes. Only short segments of surface Mouse oocytes treated with the lowest con- layer show a noticeable concentration of eleccentration of Con-A (10 pg/ml) show a very tron-dense material (fig. 9). The pinocytotic thin layer of discontinuous, moderately elec- channels and vesicles contain densely packed tron-dense material covering the external reaction product. Exposure to higher concensurface of the cell (fig. 1).This layer is simi- trations of Con-A (50 pg/ml and 1,000 pglml) larly thin on the surfaces of long processes result in a thicker and continuous layer of the and microvilli (fig. 1) and on the smoother reaction product. segments of the cell surface (fig. 2). In cell Four- and eight-cell embryos membrane invaginations and inside pinocyAt this stage of development, even if the totic vesicles, the electron-dense reaction product is more concentrated (fig. 1). Some lowest concentration of Con-A (10 pglml) is variations in labelling of individual oocytes used, the reaction product forms a thin, but may exist, but a predominant pattern can be continuous layer situated on the external surdiscerned after examining several sections of face of cell membrane (figs. 10, 13). Under higher magnification, however, one can oba few cells. The surfaces of oocytes treated with a high- serve that the concentration of electron-dense er concentration of Con-A (50 pglml) are material is uneven, and more dense and covered with a relatively thick layer of elec- thicker short segments are interspersed tron-dense product (fig. 3). This layer, how- among thinner and lighter areas (fig. 11).At ever, is still discontinuous and of a non- higher concentrations of Con-A (50 pglml), the surface layer is quite dense but also of a uniform thickness. After treatment with the highest concen- nonuniform thickness (fig. 12). A very similar tration of Con-A (1,000 pglml) the electron- pattern is observed after treatment with the dense reaction product forms a continuous, highest concentration (1,000pg/ml) of Con-A. rather uniformly distributed layer covering Generally, the differences in thickness and both smooth and undulated segments of the density of surface labelling between embryos a t different stages are more noticeable a t the cell surface (fig. 4). lowest (10 pg/ml) rather than the higher (50 or 1,000 pg/ml) concentrations of Con-A. This Fertilized eggs is clearly visible when figures 1,2,5,9, 10 and After fertilization, the overall pattern of 13, all representing labelling with 10 pg/ml distribution of the reaction product is very Con-A, are cotnpared with electron microsimilar to that observed in oocytes. Exposure graphs showing embryos treated with higher to the lowest concentration of Con-A (10 p g l concentrations (50or 1,000 pg/ml) of Con-A ml) gives a very thin, discontinuous layer of (figs. 3, 4, 6,7, 12). material of a low electron density (fig. 5 ) . In Morulae caveolae and pinocytotic vesicles the electrondense material is always more concentrated The surface of morulae show numerous prothan on the free cell surface. trusions, microvilli, and caveolae. After treatThe appearance of fertilized eggs (fig. 6 ) ment with a low concentration of Con-A (10 treated with 50 pg/ml of Con-A is very similar pglml), the surface layer appears thinner and to that of unfertilized eggs. Treatment with more uneven than in previous stages (comthe highest doses of Con-A (1,000 pglml) pare figs. 13 and 14). On the flat segments of leads to the appearance of an almost entirely plasma membrane the reaction product is continuous, relatively thick surface layer (fig. dense, while on microvilli i t becomes thin and 7). The pattern of the surface layer revealed discontinuous (fig. 14). In pinocytotic vacby the Ruthenium Red technique (fig. 8) is uoles or in deep caveolae, high concentration almost identical to that obtained after treat- of electron-dense material is frequently obment with the highest concentration of served. The reaction product penetrates beCon-A. tween cells only to the level of the apical junc-
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M. KONWINSKI, A. VORBRODT, D. SOLTER AND H. KOPROWSKI
The use of low concentrations of Con-A and the incubation of unfixed embryos a t 16'C further allows observation of the lectin-induced receptors redistribution occurring as a result of their mobility, as suggested by Collard and Temmink ('74). The concentration of Con-A a t 50 pg/ml recommended by Bretton et al. ('72) seems too high for such purposes because the Blastocysts differences in the distribution of Con-A bindAfter treatment with low concentrations ing sites on the surfaces of embryos were not (10 pg/ml) of Con-A, a relatively thin and dis- as easily perceived. Differences in the intensity and character continuous layer of reaction product forms on the surface of most trophoblastic cells (fig. of surface labelling were more distinct be17). This layer is of uneven thickness and it is tween concentrations of 10 pg/ml and 50 pg/ composed of alternatively distributed less or ml than between 50 pg/ml and 1,000pg/ml of more electron-dense segments. The similarly Con-A. Only the comparison of distributions uneven, discontinuous but thicker layer with of the reaction product a t a concentration of few clusters is present on the external surface 10 pg/ml allowed the observation that the relof trophoblastic cells treated with a more con- ative amount of Con-A bound to the cell surcentrated (50 pg/ml) solution of Con-A (fig. face is very similar on the surfaces of oocytes, 18).In deep cavities, in marginal gulfs, and in zygotes, and 2-cell embryos. Con-A receptors pinocytotic vacuoles (fig. 18: arrow) the elec- are a t these stages of development few and tron-dense product is more concentrated than non-uniformly distributed, possibly as the result of Con-A-induced redistribution (Collard on the surface. After treatment with the highest concen- and Temmink, '74). Our results conflict with tration of Con-A (1,000 pg/ml) the superficial those of Gordon et al. ('75) who found, also by layer is thin but continuous, with very numer- Con-A binding, a distinct increase in the ous, large clusters of darkly stained material thickness of the glycoprotein surface layer on (fig. 21: arrowheads). These clusters are pres- the rabbit eggs following fertilization. The ent on the external surface of the tropho- use of different species might explain these blastic cells. On the contrary, the surface of differences. Pienkowski ('74) observed a trophoblastic cells facing the inner cell mass marked increase in agglutinability after the (EM) or blastocoele is always free of the reac- fertilization of mouse oocytes, although he did not find a difference in the binding capacity of tion product (fig. 2 1: arrows). The exposure of glutaraldehyde-fixed cells 1251-labelledCon-A between oocytes and to a high concentration of Con-A (1,000 p g l zygotes. We also did not observe the differml) leads to the formation of prominent ences in the distribution and amount of surclusters of the reaction product, especially on face labelling with Con-A on unfertilized and fertilized mouse eggs. These observations sugthe surface of the blastocysts (fig. 20). The isolated ICM exposed to Con-A shows gest that agglutinability is not directly rethe presence on their surface of a thin, but lated to the amount of bound Con-A, but clearly visible, layer of dense material (fig. 22: reflects some additional changes in cell SUParrowheads). This electron-dense material is face. not present on the plasma membrane of interAn increase of receptor number, leading to nally located cells (fig. 22: arrows). After the formation of a thicker and continuous treatment of embryos with control medium layer of the reaction product, was noted in 4containing both Con-A (50 pglml) and a- and 8-cell embryos. Concomitant with the methyl-D-mannoside (0.05M) the cell surface differentiation of trophoblastic cells, changes remains completely unstained (fig. 19). in the distribution of Con-A receptors and the formation of clusters is observed. These DISCUSSION clusters might result from aggregation of Our results suggest that the lowest concen- Con-A binding sites, which would be indicatrations of Con-A (10 pg/ml) best demon- tive of the increased mobility of surface recepstrate the subtle differences in the amounts of tors. In the blastocyst stage the distribution Con-A bound to the cell surface between of surface receptors on trophoblastic cells is different stages of embryonic development. similar to that observed on the surface of nortional complex (fig. 14). After exposures to higher concentrations of Con-A (50 pg/ml) the surface layer becomes thicker, and a few dense clusters appear (fig. 15). These clusters are more numerous and easily noticeable after treatment with Con-A a t a concentration of 1,000 pg/ml (fig. 16, arrows).
CON-A BINDING SITES OF PREIMPLANTATION EMBRYOS
mal, differentiated cells (Bretton et al., '72; Enders and Schlafke, '74; Temmink et al., '75). In mouse blastocyst, tight junctions forming zonae occludentes are formed between trophoblastic cells (Ducibella et al., '75; Enders and Schlafke, '74); therefore, the components of incubation medium and the reaction products are not able to penetrate between the trophoblastic cells and, consequently, no reaction product would be noted on the surfaces of cells of ICM. The distribution of Con-A receptors on the free surface of immunosurgically (Solter and Knowles, '75) isolated ICMs was very similar to that observed in 4- or 8-cell embryos. Thus, the distribution of Con-A receptors on the ICM is of an early embryonic type, while the distribution of receptors on the surface of trophoblastic cells compares to that observed in adult cells. This change of surface property of trophoblastic cells may be the reason for diminished agglutinability of blastocysts as noted by Rowinski et al. ('76). In accordance with the suggestion of Temmink et al. ('75) we can suppose that the differences in clustering of Con-A binding sites between early embryos and trophoblastic cells are responsible for differences in the agglutinability by Con-A. The lack of clustering observed on the surface of isolated ICM is accompanied by their agglutinability with low concentrations of Con-A, whereas the whole blastocyst is not agglutinable (Rowinski et al., '76). Our results suggest that the agglutinability of early preimplantation mouse embryos depends not only on the number and distribution of Con-A receptors, but probably on other physico-chemical properties of cell surface which can influence the mobility of surface layer and the binding sites of Con-A. Fluidity of the plasma membrane and possible redistribution and clustering of Con-A receptors probably influence to a large extent agglutinability of early embryos. LITERATURE CITED Bernhard, W., and S. Avrameas 1971 Ultrastructural visualization of cellular carbohydrate components by means of Concanavalin A. Exp. Cell Res., 64: 232-236. Bretton, R., R. Wicker and W. Bernhard 1972 Ultrastructural localization of Concanavalin A receptors in normal
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and SV40-transformed hamster and rat cells. Int. J. Cancer, 10: 397-410. Collard, J. G., and J. H. M. Temmink 1974 Binding and cytochemical detection of cell-bound Concanavalin A. Exp. Cell Res., 86: 81-86. Ducibella, T., D. F. Albertini, E. Anderson and J. D. Biggers 1975 The preimplantation mammalian embryo: characterization of intercellular junctions and their appearance during development. Develop. Biol., 45: 231-250. Enders, A. C., and S. Schlafke 1974 Surface coats of the mouse blastocyst and uterus during the preimplantation period. Anat. Rec., 180: 31-46. Gordon, M., L. R. Fraser and P. V. Dandekar 1975 The effect of ruthenium red and concanavalin A on the vitelline surface of fertilized and unfertilized rabbit ova. Anat. Rec., 181: 95-112. Johnson, M. H., D. Eager, A. Muggleton-Harris and H. M. Grave 1975 Mosaicism in organization of cancanavalin A receptors on surface membrane of mouse eggs. Nature, 257: 321-322. Konwinski, M., J. Abramczuk, W. Baranska and W. Szymkowiak 1974 Size changes of mouse ova during preparation for morphometric studies in the electron microscope. Histochem., 42: 315-322. Luft, J. M. 1964 Electron microscopy of cell extraneous coats a s revealed by Ruthenium Red staining. J. Cell. Biol., 23: 53A-54A. Martinez-Palomo, A,, and C. Brailovsky 1968 Surface layer in tumor cells transformed by Adeno-12 and SV40 viruses. Virology, 34: 379-382. Martinez-Palomo,A,, R. Wicker and W. Bernhard 1972 U1trastructural detection of concanavalin surface receptors in normal and in polyoma-transformed cells. Int. J. Cancer, 9: 676-684. Nicolson, G. L., R. Yanagimachi and H. Yanagimachi 1975 Ultrastructural localization of lectin-binding sites on the zonae pellucidae and plasma membranes of mammalian eggs. J. Cell. Biol., 66: 263-274. Pienkowski, M. 1974 Study of the growth regulation of preimplantation mouse embryos using concanavalin A. Proc. Soc. Exp. Biol. Med., 145: 464-469. Roberson, N., A. Neri and S. B. Oppenheimer 1975 Distribution of Concanavalin A receptor sites on specific populations of embryonic cells. Science, 189: 639-640. Rowinski, J., D. Solter and H. Koprowski 1976 Changes of Concanavalin A induced agglutinability during preimplantation mouse development. Exp. Cell Res., 100: 404408. Solter D., and B. B. Knowles 1975 Immunosurgery of mouse blastocyst. Proc. Nat. Acad. Sci. (USA.), 72:50995102. Temmink, J. H. M., J. G. Collard, H. Spits and E. Roos 1975 A comparative study of four cytochemical detection methods of concanavalin A binding sites on the cell membrane. Exp. Cell Res., 92: 307-322. Whitten, W. K. 1971 Nutrient requirements for the culture of preimplantation embryos in vitro. Adv. Biosci., 6: 129-141. Yanagimachi, R., and G. L. Nicolson 1974 Changes in lectin-binding to the plasma membranes of hamster eggs during maturation and preimplantation development. J. Cell. Biol., 63: 381a.
PLATE 1 EXPLANATION OF FIGURES
Figures 1 to 4 show the surface of oocytes after Con-A-peroxidasereaction. The sections are either unstained or stained with lead citrate and uranyl acetate. All magnifications are X 17,400.
1 The surface of the oocyte exposed to Con-A in a concentration of 10pg/ml at 16°C. The discontinuous, thin layer of reaction product covers the numerous protrusions and microvilli. In surface invaginations and in pinocytotic vesicles the concentration of the reaction product is higher than on microvilli. 2 Another fragment of the oocyte treated as that presented in figure 1. An interrupted, thin surface layer is visible.
3 The oocyte exposed to Con-A at a concentration of 50 fig/ml at 16OC. The surface layer is thicker than in oocytes shown in figures 1 and 2. 4 The oocyte exposed to Con-A at a concentration of 1,00Opg/rnlat 37°C. The surface layer is relatively thick,
similar to that observed at a Con-A concentration of 50 pg/ml. Figures 5 to 8 show the surface of one-cell embryos (zygotes) after Con-A or Ruthenium Red reaction. All magnifications are at X 17,400.
5 The surface of zygote treated similarly to the oocytes presented in figures 1 and 2. The layer of the reaction product is discontinuous and very thin. 6 The surface of a zygote treated with Con-A at a concentration of 50 pg/ml. The reaction product is forming a nonuniformly thick layer. 7 The surface of a zygote treated with Con-A at a concentration of 1,00Opg/ml. The layer of the reaction product is relatively thick and similar to that observed in the oocyte (compare fig. 4). 8 Ruthenium Red stained coat on the surface of the zygote. The thickness of this coat is very similar to the surface coat observed with high concentrations of Con-A (compare figs. 7 and 4).
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CON-A BINDING SITES OF PREIMPLANTATION EMBRYOS M. Konwinski, A. Vorbrodt, D. Solter and H. Koprowski
PLATE 1
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PLATE 2 EXPLANATION OF FIGURES
9 The surface of a 2-cell embryo incubated with Con-A (10pg/ml). The layer of the reaction product is discontinuous and highly concentrated in pinocytotic vesicles and channels. X 17,400.
10 The surface of a four-cell embryo exposed to Con-A a t a concentration of 10 pg/ml. The surface layer becomes continuous. X 17,400. 11 The surface of the same embryo as presented in fig. 10, shown under higher magnification. The uneven distribution of the reaction product is evident. X 39,000.
12 The reaction product on the surface of a four-cell embryo exposed to Con-A at a concentration of 50 pgiml. X 17,900. 13 The surface of an eight-cell embryo treated with Con-A at a concentration of 10 pg/ml. The continuous, relatively thick layer is well visible on the cell surface. X 17,900. 14 The surface of the morula treated with Con-A at a concentration of 10pg/ml. The thickness of the surface layer is nonuniform (uneven) and some gaps can he observed. Notice that reaction product does not penetrate beyond the junctional complex (arrow). X 17,900. 15 The morula treated with Con-A a t a concentration of 50 pg/ml. The surface layer is thicker than a t a concentration of 10 pg/ml (compare fig. 14); it is continuous and formation of small clusters (caps) can he noted. X 17,900. 16 The morula treated with Con-A a t a Concentration of 1,000pg/ml. The thickness of the surface-layer is similar to that shown in figure 15. Numerous clusters are present (arrows). X 17,900.
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CON-A BINDING SITES OF PREIMPLANTATION EMBRYOS M. Konwinski, A. Vorbrcdt, I). Solter and H. Koprowski
PLATE 2
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PLATE 3 EXPLANATION OF FIGURES
17 The surface of external (trophoblastic) cells of blastocyst exposed to Con-A at a concentration of 10 pg/ml. The reaction product forms a thin, discontinuous layer. X 17,900. 18 The blastocyst exposed to Con-A at a concentration of 50pg/ml. The undulated cell surface is covered by a layer of uneven thickness and nonuniform electron density. The higher concentration of the reaction product is visible in cytoplasmic vacuoles (arrows). X 17,900.
19 The blastocyst exposed to Con-A 50pg/ml and methylmannoside f0.05M). No reaction product is detectable on the cell surface. X 17,900. 20 The blastocyst exposed to Con-A (1,000 pg/ml) after fixation in glutaraldehyde. The large clusters of the reaction product protrude from the cell surface. X 12,300. 21 The surface of a trophoblastic cell from a blastocyst exposed to Con-A (1,000 pg/ml). Numerous clusters and caps of the reaction product are noticeable on the external surface of the cell. The internal surface facing the blastocoele (arrows) displays a lack of reaction product. X 12,300.
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CON-A BINDING SITES OF PREIMPLANTATION EMBRYOS M. Konwinski, A. Vorbrodt, D. Solter and H. Koprowski
PLATE 3
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PLATE 4 EXPLANATION OF FIGURE
22 The inner cell mass (ICMf after exposure to Con-A at a concentration of 50 wg/ml. The black reaction product is present on the surface of the cell mass (arrowheads) but absent from the surface of internal cells (arrows). X 6,300.
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CON-A BINDING SITES OF PREIMPLANTATION EMBRYOS M. Konwinski, A. Vorbrcdt. D. Solter and H. Koprowski
PLATE 4
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