ARTICLE |
Correspondence to: Nicolai Miosge, Abteil. Histologie, Zentrum Anatomie, Georg-August-Universität Göttingen, Kreuzbergring 36, 37075 Göttingen, Germany.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In biological tissues, specific carbohydrate moieties of the oligosaccharide chains of glycoproteins can be localized by lectin binding. Such carbohydrate moieties are among the factors that mediate cell-cell or cell-matrix interactions during pre- and postimplantation embryonic development. Binding sites for the lectins RCA I, WGA, and LTA were localized in preimplantation mouse embryos at the ultrastructural level with the help of postembedding lectin gold cytochemistry. WGA and RCA I binding sites, but no LTA binding sites, were present in the zona pellucida. WGA and RCA I binding sites were found at cell surfaces of morulae and in cells of the inner cell mass of blastocysts, suggesting that N-acetylglucosamine-, terminal ß-galactosyl-, and N-acetylgalactosamine-rich glycoproteins might be involved in cell-cell and cell-matrix interactions. WGA binding sites were found predominantly in electron lucid vesicles of the blastomeres, whereas RCA I was detected in electron dense vesicles of the compacted morula and later in the polar trophoblast cells. This allows early identification of blastomere cells that later differentiate into the polar trophoblast. (J Histochem Cytochem 45:447-453, 1997)
Key Words: preimplantation mouse embryo, ultrastructural localization, lectins RCA I, WGA, LTA
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Glycosylation is an important post-translational modification of proteins involved in cell-cell or cell-matrix interactions during pre- and postimplantation embryonic development (
Lectin binding sites have been localized in preimplantation embryos, mainly to elucidate the role of carbohydrates in the fertilization and implantation process. Chavez and Enders (1981) found RCA I bound to Day 5 mouse embryos.
Most researchers, have thus far relied on pre-embedding lectin histochemistry on whole embryos at the light microscopic level: WGA, RCA I, RCA II, and ConA have been demonstrated to bind to preimplantation embryos (
Although some authors have localized glycoconjugates in the zona pellucida of postovolatory oocytes in the hamster (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals
Female NMRI mice were kept on a normal day/night cycle and received Altromin commercial food and water ad libitum. The day on which a vaginal plug was detected at 1100 hr after a mating period of 3 hr was designated Day 0 of gestation.
Tissue Preparation
On Days 2-4 of gestation (from the four-cell stage to blastocyst stages), the mice were sacrificed by cervical dislocation and the uteri with the oviducts were removed. These were transferred to 0.1 M PBS, pH 7.2, at 4C, the oviducts were sealed with a clamp, and buffer solution was injected into the cavum uteri from the vaginal side. When pressure had built up, the clamp was quickly opened and the embryos flushed out through the oviduct into the buffer solution.
LR Gold Embedding
The embryos were next fixed for 15 min in 4% formaldehyde and 0.5% glutaraldehyde in 0.1 M PBS. After incubation for 30 min in 10 mM ammonium chloride, the embryos were dehydrated in a series of 30%, 50%, and 70% ethanol and transferred to gelatin capsules containing LR Gold (London Resin; Reading, UK) supplemented with 0.8% of the light-sensitive accelerator benzil. The gelatin capsules containing the embryos were sealed air-tight and the LR Gold was then polymerized at -25C with the light of a halogen lamp (15 V, 150 W).
Lectin-Gold Cytochemistry
For this study, five morulae of the eight-cell stage, five morulae of the 16-cell stage, five blastocysts with a zona pellucida, and five blastocysts with no zona pellucida were employed.
For orientation purposes and for determination of the developmental stages of the embryos, 1-µm-thick sections were cut from the embedded embryos and stained with toluidine blue. Thereafter, ultrathin sections were cut with a Reichert ultramicrotome and collected on formvar-coated nickel grids. The nickel grids were preincubated for 10 min at room temperature (RT) with 0.01 M PBS (pH 7.2). The sections were then incubated for 1 hr at RT with 30-nm gold-labeled RCA I, 15-nm gold-labeled WGA, or with 20-nm gold-labeled LTA (all from EY Laboratories; San Mateo, CA) diluted 1:20 in PBS buffer. After having been rinsed in PBS, the sections were stained with 2% osmium tetroxide in 0.01 M PBS for 10 min, 1% uranyl acetate for 15 min, and 1% lead citrate for 5 min and were examined with a Zeiss EM 109 electron microscope.
Controls
The control for the specificity of the staining reactions was carried out on sections incubated for 1 hr at RT with the gold-labeled lectins, the carbohydrate binding sites of which had been blocked by preincubation with the corresponding inhibiting sugars for 1 hr at RT.
WGA was incubated with N,N'-diacetylchitobiose (0.1 mM), N,N',N''-triacetylchitotriose (0.1 mM), and N-acetyl-D-glucosamine (0.5 mM), RCA I with ß-D-galactose (0.5 mM), and -lactose (0.5 mM) and LTA with
-fucose (0.5 mM).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Morula Stage
WGA.
Strongest staining for WGA binding sites was seen in the zona pellucida (Figure 1) and on all cell surfaces of the blastomeres (Figure 2). In the cytoplasm, many labeled membrane vesicles were seen. Strong gold labeling was also observed in association with the limiting membrane of vesicles of the blastomeres, which appeared to contain electron-lucid material in their interior. These stained vesicles were seen in all blastomeres of both early and compacted morula. In the cytoplasm of the flat blastomeres located in the outer zone of the compacted morula, a few solitary gold-labeled electron-dense vesicles were present (Figure 3).
|
RCA I. The staining pattern in the morula for RCA I binding sites was similar to that described for WGA binding sites. Again, strong labeling was seen in the zona pellucida. However, this staining was weaker than that seen for WGA binding sites (Figure 4). Intense staining for RCA I of those vesicles within the cytoplasm of the blastomeres that contained electron- lucid material in their interior was visible. Again, stained membrane vesicles were seen in the cytoplasm of the blastomeres. The electron-dense vesicles within the flat cells of the compacted morula showed staining for RCA I. Intense staining of the cell surfaces was the same as that described for WGA (Figure 5).
LTA. No staining for LTA binding sites was seen in the zona pellucida or the blastomeres. Only a few contact zones between single blastomeres showed gold labeling for LTA.
Controls for Morulae. The control experiments were carried out under the same conditions as described in Materials and Methods. They showed no reaction. A morula incubated with WGA diluted 1:20 in PBS buffer after preincubation with its inhibiting sugars is shown in Figure 6.
Blastula Stage
WGA.
Again, strong staining for WGA binding sites was seen in the zona pellucida of early blastocysts. The cell surfaces of the cells of the inner cell mass (ICM) were labeled, as well as the cisternae and vesicles of Golgi complexes. Only a few stained membrane vesicles were seen in the cytoplasm of the ICM cells. Strongest staining of cells of the blastocysts was seen in the polar trophoblast in both early and late stages of development (Figure 7). Labeling was located in electron-dense vesicles within the cytoplasm, predominantly oriented towards the outer surface of the blastocysts. Interdigitate cell contacts of the adjacent polar trophoblast cells, as well as a few microvilli on their outer surfaces, were also stained. The inner surfaces of these cells were always labeled when the cells were in contact with the ICM.
|
In the mural trophoblast, only a few gold-labeled dense vesicles were seen in the cytoplasm. In contrast, many stained vesicles with electron-lucid material in their interior, again with preference for the limiting membrane, and stained vacuoles were observed (Figure 8). In some but not all microvilli oriented towards the exterior, as well as in microvilli oriented towards the inner cavity of the blastocysts, WGA binding sites were seen.
RCA I. In general, in the blastocyst stages also, the staining pattern for RCA I binding sites was similar to that for WGA binding sites. In those blastocysts with a zona pellucida, staining of this structure was weaker than that seen for WGA. Plasma membranes of the cells of the ICM were strongly labeled. Strong staining was again found in electron-dense vesicles in the cytoplasm of polar trophoblast cells. Areas at which cells from the ICM were in contact with the polar trophoblast cells (Figure 9), as well as interdigitate contact zones (Figure 10) of the polar trophoblast cells, were positive for RCA I. Corresponding to the staining pattern for WGA binding sites, staining of vacuoles and vesicles, located in the cytoplasm and oriented towards the outer surface of the blastocysts with electron-lucid material in their interior, occurred within the trophoblast cells. Staining for RCA I binding sites was seen in some microvilli of the mural trophoblast cells located at the outer surface of the blastocysts and on the inner surface oriented towards the blastocyst cavity.
LTA. Staining for LTA binding sites was restricted to a few electron-dense vesicles located in the polar trophoblast cells of all blastocysts (Figure 11). In contrast to the morula stages, no staining of solitary intercellular contact zones or cilia-like structures was seen in the blastocysts, whether or not they possessed a zona pellucida.
Controls for Blastocysts. Control experiments were carried out as described for morulae. They showed no reactions.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Lectins are elegant markers for detection of sugar moieties of glycoproteins or glycolipids. With their help, it is possible to recognize different carbohydrates in the various developmental stages of embryogenesis. In the work presented here, we used the postembedding lectin-gold method on LR Gold embedded embryos.
Embedding in the hydrophilic resin LR Gold (
The preimplantation embryos investigated showed WGA and RCA I binding sites in the zona pellucida. This was previously shown by
Our postembedding approach allows the localization of lectin binding sites also within the intracellular structures of the embryos. Strong staining of the cell surfaces of the cells of the blastomeres and of the cells of the ICM of the blastocysts was seen. Therefore, the sugars represented by WGA, i.e., mainly N-acetylglucosamine (GlcNAc) moieties and by RCA I, i.e., mainly terminal ß-D-galactosyl residues (ß-Gal) and secondarily N-acetylgalactosamine (GalNAc) moieties (
The staining of contact zones between the polar trophoblast and the ICM with WGA and RCA I hints at the involvement of GlcNAc- and ß-Gal-rich glycoproteins in cell recognition processes between polar trophoblast cells and the adjacent ICM cells. Furthermore, morulae and blastocysts exhibit quite a number of intracytoplasmic vesicles, some of them electron-lucid and others with electron-dense material in their interior. We found significant differences (see Figure 12 and Figure 13) in the number and staining pattern of these vesicles. One population of blastomeres within the morulae contained large numbers of electron-dense vesicles strongly stained for RCA I, as well as a few dense vesicles stained for WGA binding sites. Corresponding to the pattern described above, cells of the polar trophoblast of the blastocysts also showed electron-dense vesicles stained for WGA and RCA I binding sites. In contrast, another population of blastomeres in the morulae exhibited electron-lucid vesicles stained for WGA. Such vesicles stained for WGA were also found in cells of the mural trophoblast.
|
Our results therefore show that even as early as the morula stage one can identify blastomeres that will later develop into polar trophoblast cells by the presence of large numbers of electron-dense vesicles stained with RCA I, as well as a few dense vesicles stained for WGA binding sites. The cells of the polar trophoblast later differentiate into cells of the extraembryonic ectoderm (
In a previous investigation we found developing basement membranes in the blastocyst at the interior side of the mural trophoblast (
![]() |
Acknowledgments |
---|
We would like to thank Cyrilla Maelicke, B.Sc., for editing the manuscript.
Received for publication May 8, 1996; accepted October 30, 1996.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Avilés M, Martínez-Menárguez JA, Castells MT, Madrid JF, Ballesta J (1994) Cytochemical characterization of oligosaccharide side chains of the glycoproteins of rat zona pellucida: an ultrastructural study. Anat Rec 239:137-149[Medline]
Brownell AG (1977) Cell carbohydrates of preimplantation embryos as assessed by lectin binding. J Supramol Struct 7:223-234[Medline]
Chávez DJ, Enders AC (1981) Temporal changes in lectin binding of peri-implantation mouse blastocysts. Dev Biol 87:267-276[Medline]
Damjanov I (1987) Biology of disease, lectin cytochemistry and histochemistry. Lab Invest 57:5-20[Medline]
Gardner RL (1982) Origin and differentiation of extraembryonic tissues in the mouse. Int Rev Exp Pathol 24:63-133
Herken R, Fussek M, Thies M (1988) Light and electron microscopical postembedding lectin histochemistry for WGA-binding sites in the renal cortex of the mouse embedded in polyhydroxy aromatic resins LR-White and LR-Gold. Histochemistry 89:277-282[Medline]
Herken R, Sander B, Hoffman M (1990) Ultrastructural localization of WGA, RCA I, LFA and SBA binding sites in the seven-day-old mouse embryo. Histochemistry 94:525-530[Medline]
Konwinski M, Vorbrodt A, Solter D, Koprowski H (1977) Ultrastructural study of concanavalin-A binding to the surface of preimplantation mouse embryos. J Exp Zool 200:311-324[Medline]
Muramatsu T (1988) Developmentally regulated expression of cell surface carbohydrates during mouse embryogenesis. J Cell Biochem 36:1-14[Medline]
Newman GR, Jansani B, Williams ED (1983) A simple post-embedding system for rapid demonstration of tissue antigens under the electron microscope. Histochem J 15:543-555[Medline]
Nicholson GL, Yanagimachi R, Yanagimachi H (1975) Ultrastructural localization of lectin-binding sites on the zonae pellucidae and plasma membranes of mammalian eggs. J Cell Biol 66:263-274[Abstract]
Roux E, Kan FWK (1991) Changes of glycoconjugate contents of the zona pellucida during oocyte growth and development in the golden hamster: a quantitative cytochemical study. Anat Rec 230:347-360[Medline]
Salamat M, Götz W, Horster A, Janotte B, Herken R (1993) Ultrastructural localization of carbohydrates in Reichert's membrane of the mouse. Cell Tissue Res 272:375-381[Medline]
Salamat M, Miosge N, Herken R (1995) Development of Reichert's membrane in early mouse embryo. Anat Embryol 192:275-281[Medline]
Sato M, Muramatsu T (1985) Reactivity of five N-acetylgalactosamine-recognizing lectins with preimplantation embryos, early postimplantation embryos and teratocarcinoma cells of the mouse. Differentiation 29:29-38[Medline]
Spicer SS, Schulte BA (1992) Diversity of cell glycoconjugates shown histochemically: a perspective. J Histochem Cytochem 40:1-38
Wu T-C, Lee M-C, Damjanov I (1984) Lectin binding sites of the mouse ovary, intraovarian and ovulated ova. Histochemistry 80:527-533[Medline]
Zuzack JS, Tasca RJ (1985) Lectin-induced blockage of developmental processes in preimplantation mouse embryos in vitro. Gamete Res 12:275-290