Ultrastructural localization of lectin receptors in the preimplantation ovine embryo.код для вставкиСкачать
THE ANATOMICAL RECORD 240:537-544 (1994) Ultrastructural Localization of Lectin Receptors in the Preimplant ation Ovine Embryo P. DE PAZ, A.J. SANCHEZ, J.G. FERNANDEZ, C. GARCIA, C.A. CHAMORRO, AND L. ANEL Departamento de Biologia Celular y Anatomia (P.D.P., A.J.S., J.G.F., C.G., C.A.C.) and Departamento de Patologia Animal (Sanidad) (L.A.), Facultad de Veterinaria, Universidad de Leon, Leon, Spain ABSTRACT Background: Preimplantation development of mammalia is characterized by cell surface changes functioning in intercellular communication and adhesion. The glycoconjugate role in cellular interactions has been analysed for several groups but not in sheep embryos. The binding patterns of eleven lectins during sheep preimplantation development were investigated and the role of glycoconjugates in early development was discussed. Methods: Ultrathin sections from preimplantation ovine embryos (3-7 days) were incubated with different colloidal gold conjugated lectins and the frequency of gold particles on the cell membrane, some organelles, and the zona pellucida was evaluated. Results and Conclusions: We observed a higher staining of WGA, DBA, and SBA lectins in the intercellular contact zone with respect to the free cell surface of blastomeres during cleavage. This indicates that the N-acetyl galactosamine and N-acetyl glucosamine residues may be involved in sheep morula compaction. In contrast, the trophoblast cell displays an increase of staining of some lectins previously identified during cleavage (LcH, WGA, SBA, MPA, and PNA) on the free membrane, and a lack of sugar residues in the intercellular surface. This polarization of the trophoblast cell surface is not observed in the inner cell mass and could provide a mechanism for differentiation within the blastocyst. Intracytoplasmic vesicles show a cytochemical identity with lysosomes in the blastocyst (abundant GlcNAc and Man/Glc residues) that may reflect a functional relationship between both organelles in an intracellular cycle. The zona pellucida presents abundant GalNAc, GlcNAc, and Gal residues during preimplantation ovine development. 0 1994 Wiley-Liss, Inc. Key words: Lectins, Sheep, Preimplantation embryo The nature of the molecules present in the cell membrane and its more extrinsic component is of considerable interest because of the unique position of the cell surface a t the interface between a cell and its external environment. In particular it is important in the understanding of morphogenesis to characterize the changes occurring in cell surface components during development. In most instances, efforts have focussed on the protein components, though it has been suspected for some time that cellular interactions are dependent upon complex carbohydrates. The critical role of glycoconjugates in cellular interactions has been clearly defined by different investigators (Gooi et al., 1981; Sharon and Lis, 1989; Peacock et al., 1990; Brandley, 1991). Cell surface changes in cells and tissue including the preimplantation mammalian embryo have been investigated with lectins (Lis and Sharon, 1986; Dealtry and Sellens, 1987; Spicer and Schulte, 1992). The charac0 1994 WILEY-LISS, INC. terization of the glycoconjugate composition of blastomeres during early development may provide clues a s to the macromolecules functioning in intercellular communication and changes in cell behavior (Shur, 1989; Fenderson and Eddy, 1990). Early development of mammalian embryo is characterized primarily by a major morphological event termed compaction that causes a major reorganization among the cells of the morula. Compaction is a complex process in which polarization of blastomeres occurs, cell-cell contacts are maximized, and specialized junctions formed (Rastan el al., 1985; Wiley, 1988; Watson, 1992). This process is a prerequisite for blastocyst for- Received February 1, 1994; accepted June 22, 1994. Address reprint requests to Paulino De Paz, Departamento de Biologia Celular y Anatomia, Facultad de Veterinaria, Universidad de Leon, 24071 Leon, Spain. 538 P. DE PAZ ET AL TABLE 1. Description of the lectins used in this study and their specificities' Lectin Concanavalia ensiformis Abbreviation ConA Specificity' Man a1,2 Man a1,2 Man > Man a1,2 Man > a Man > a Glc > a GlcNAc con^.^ 1:15 Sugar (0.2 M) D-Man 1:15 D-Man 1:lO D-GlcNAc Source Sigma Chemical co. (St. Louis, MO) E-Y Laboratories (San Mateo, CAI E-Y Laboratories 1:lO D-GlcNAc Lens culinaris Lch a Man > a Glc > a GlcNAc Griffonia simplicifolia 11 Triticum vulgare GS-I1 a GlcNAc = WGA Dolichus biflorus DBA Glycine max SBA GlcNAc p1,4 GlcNAc p1,4GlcNAc > GlcNAc p1,4 GlcNAc >> GlcNAc GalNAc a1,3 GalNAc > a GalNAc >> a Gal a GalNAc = p GalNAc > a Gal GalNAc > Gal Gal p1,3 GalNAc > GaINH, > Gal a-L-Fucosa a-L-Fucosa Maclura pomifera MPA Arachis hypogaea PNA Ulex europaeus I Lotus tetranoglo bus Limulus polyphemus UEA-I Lotus LPA 13 GlcNAc NeurGc a 2,3 GalNAc > NeurAc a 2,6 GalNAc > NeurAc = NeurGc Diluent Buffer pH 0.01 M PBS 7-8 0.15 M NaCl 0.1 mM Ca2+ and Mn2+ 0.05 M TRIS 7.2 0.15 M NaCl 0.01 M PBS 7.45 E-Y Laboratories 0.01 M PBS 7.45 1:lO D-GalNAc E-Y Laboratories 0.01 M PBS 7.45 1:lO D-GalNAc E-Y Laboratories 0.01 M PBS 7.45 1:lO 1:lO D-Gal D-Gal E-Y Laboratories E-Y Laboratories 0.01 M PBS 0.01 M PBS 7.45 7.45 1:lO 1:lO L-FUC L-FUC E-Y Laboratories E-Y Laboratories 0.01 M PBS 0.01 M PBS 7.45 7.45 NeurNAc E-Y Laboratories 0.05 M T R I S 0.15 M NaCl 0.1 mM Ca'+ 8 1:5 'For each lectin the dilution (Conc.),inhibiting sugar, and diluent solution are indicated. The buffers used are the ones recommended by the commercial laboratories. 'After Goldstein and Poretz (1986). 3Referred to commercial solutions. mation in which two distinct cell populations can be catheter. The embryos were classified morphologically distinguished: the trophectoderm and the inner cell as excellent to poor according to Lidner and Wright mass. There is abundant evidence that glycoconjugates (1983) and only those with a quality grade of 1 or 2 are involved in these processes (Gooi et al., 1981; Kim- were analyzed. ber and Bagley, 1987; Bayna et al., 1988; Hathaway et al., 1989; Bukers a t al., 1990). L ectin Staining In this paper we have investigated the binding pattern of 11 lectins during preimplantation development The embryos were fixed with a mixture of formaldeof sheep embryos and discussed the role of glycoconju- hyde (4%) and glutaraldehyde (0.25%) in PBS for 1 hr gates in early development. a t 4°C and immersed in 0.5 M ammonium chloride for another 30 min. After dehydration in a graded series of MATERIALS AND METHODS ethanol (30, 50, 70, 90, and 100%) a t decreasing temEmbryo Collection peratures (0, -20, -30, -30, and -3O"C, respectively) Adult Churra ewes were pretreated with fluoroge- the pieces were infiltrated and embedded in Lowicryl stone acetate (FGA; Chronogest, Intervet, Holland) 30 K4M at -25°C for 24 h r and 20°C for 72 hr. mg intravaginal pessaries for 12 days. Sixty hours beThe sections were then cut with glass knives on an fore sponge removal donors were superovulated with ultratome LKB V and mounted on 150 mesh nickel 12 mg of follicle stimulating hormone-pituitary extract grids having a formvar film. Grids were preincubated (FSH-P,Shering Corp., Kenilworth, NJ, USA) with the for 15 min on a drop of 0.5 M glycine in PBS and incufollowing dose: 4, 4,3, 3, 2, and 2 mglanimal at 12 hr bated on a drop of the gold labelled lectin for 6 hr in a intervals. At the time of fifth dosage, 400 IU pregnant moist chamber a t room temperature according to Table mare serum gonadotrophin (PMSG, Foligon, Intervet, 1. At the end of the labelling procedure the sections Holland) were administered. Ewes were both naturally were washed with PBS, rinsed in doubly-distilled wamated and artificially inseminated 12-24 h r after the ter, and dried. The grids were stained with uranyl aconset of estrus. etate and lead citrate. The embryos were recovered surgically 3 to 7 days The colloidal gold-conjugated lectins and the inhibafter sponge removal. The uterine horns or oviduct iting sugars used in this study are shown in Table 1. were flushed with DPBS + 15% FCS using a Foley Preliminary studies with sugar a t different concentra- 539 LECTIN RECEPTORS IN EARLY OVINE EMBRYO TABLE 2. Staining patterns (mean 2 s.d.) showed by the lectins used in this study in the different stages of ovine Dreimdantation develoDment' Cell membrane (uipm) Cleavage Lectin ConA LcH GS-I1 Free Interc 10 (2.1) 4 (2.4) 12 (2.6) - - - WGA 25(6.4) 42 (12.3) DBA 10 (2) 35 (5.9) SBA 8 (2.9) 40 (8) MPA 4(0.5) 4 (0.3) PNA 2(0.5) 4 (0.4) UEA-I 611.6) Lotus LPA - Organelles (uipm2) BICM Cleavage B Troph BICM Zona pellucida ( d p m 2 ) Free Interc Interc Vesic Lysos Vesic Lysos Vesic Lysos Cleav Blast 8 (2.1) 6 (1.9) 72 (17.9) 18 (3.8) 80 (26.1) 14 (6.4) 78 (13.3) 16 (9.2) 32 (11.3) 30 (3.7) 69(11.2) 4(1.4) 4(1.5) 26(9.1) 29 (13.6) 26 (4.4) B Troph - - - - 2 (0.5) 8(1.4) 86(18.3) - - - - - - 18 (2) 210 (10) 34 (2) 40 (4) 36 (4.2) 40 (3.3) 20 (8) 230 (16) 14 (3.1) 6 (2) 28 (6.8) 34 (2.3) - - - - 6(1.2) 6(1.1) 4(0.5) 4(1.6) - - - - - - 4 (1.4) 4(1.2) 4(1.3) 8(1.1) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 16(5.2) - - - - - - - - - - - ~ 32 (11.8) 120 (13) 28 (13.5) 140 (7.2) 74(15.9) 8 (3.4) 37 (7.2) 30(6) 28(4.1) 8(0.5) - 'These data refer to the free and intercellular (Interc) membrane (colloidal gold particlesipm), the cell organelles (gold colloidal particlesipm'), and the zona pellucida (gold colloidal particlesipm'). > 100 particlesiu = very heavy; 50-100 = heavy; 25-50 = light; < 25 = sparse;-= 0. Vesic = intracytoplasmic vesicles; Lysos = lysosomes; Cleav = cleavage; B Troph = blastocyst trophectoderm; BICM = blastocyst inner cell mass; Blast = blastocyst. tions were carried out in order to assess the carbohydrate specificity of the lectin to the embryonic cells. In the whole series of experiments we used grids with sections from the same embryo a s a control for the specific binding of the lectins. These sections were incubated with the lectin-gold complex previously mixed with the corresponding specific sugar for 1 h r before use (see Table 1). Six embryos from each stage were used, six ultrathin sections per embryo were incubated with lectin and six micrographs per section were obtained by random sampling. The frequency of gold particles per square micron (organelle and zona pellucida) or per micron (plasma membrane) was determined by counting on the electron micrographs. A double square lattice ((264) is superimposed on each micrograph to estimate the area of cellular organelles and the length of membrane profiles (Weibel, 1979). The gold particle number of each structure was counted and this count was standardised to 1 pm2 (organelle) or 1 pm (plasma membrane) and the means and standard error of means calculated. RESULTS We analyzed several developmental stages of ovine embryos up to blastula stage by means of colloidal gold conjugated lectins. The results refer to the frequency of the gold particles on the cell membrane (the free cell surface and the intercellular membrane were distinguished), some organelles (intracytoplasmic vesicles and lysosomes), and the zona pellucida. Cleavage The results obtained during the cleavage period have been analyzed as a whole although we carried out a complete study of 8-cell stage, early morula and compact morula. Throughout these stages neither qualitative nor quantitative differences were found, and thus the data are summarized a s a whole in Table 2. On the free cell surface a sparse staining appeared for LcH, DBA, SBA, MPA, and PNA lectins and a light staining for WGA lectin. The intercellular contact surface of the ovine embryonic cells during cleavage (Fig. la,b) showed light quantity of GalNAc (DBA and SBA) and GlcNAc (WGA)residues but sialic acid was absent. Intracytoplasmic vesicles (Fig. lc,d) were very heavily stained with Con A (MadGlc) and WGA (GlcNAc) lectins. In contrast, lysosomes sparsely showed Man/Glc (Con A) residues. Concerning the zona pellucida (Fig. 2) we were able to detect a very heavy reaction with WGA lectin, as well as a heavy reaction with DBA (GalNAc), SBA (GalNAc), MPA (Gal), and PNA (Gal) lectins. Blasfocyst When the embryo reached the blastocyst stage the lectin labelling shown by the two cell populations, trophectodermal cells and inner cell mass, was separately evaluated. The lectin binding profile of the blastocyst zona pellucida (Fig. 2) is similar to the patterns shown during cleavage stages: very heavy staining for WGA, light for MPA and PNA and sparse for DBA and GS-11. Only SBA and DBA lectins decreased their reactions in this phase, which means that GalNAc residues were lost. Trophectoderm The outer cell surface of the trophectodermal cells appeared remarkably marked with WGA and LcH lectins (Fig. 3a,c) and lightly stained by SBA (Fig. 3b), MPA and PNA lectins (Fig. 3d). However, the reaction shown by DBA, LPA (Fig. 3e), and UEA-I lectins was sparse. A poor labelling of the intercellular zones was evidenced except for Con A and UEA-1 lectins. The reaction to lectins of the free cell surface of the ovine blastocyst varied qualitatively and quantitatively with respect to cleavage period. There was a n increase in staining for LcH, SBA, WGA, MPA, and PNA and new binding sites for UEA-I and LPA. Concerning the intercellular membrane, there was a specific loss of staining due to the lack of DBA and to the decrease of SBA and WGA staining. P. DE PAZ ET AL. 540 Fig. 1. Sections of cleavage-stages sheep embryos. a: DBA lectin. A light staining of intercellular surfaces is shown ( x 84,000). b: WGA lectin. The intercellular membrane area shows a light labelling ( x 84,000). c: ConA lectin. A specific staining is observed in intracytoplasmic vesicles from the 8 cell embryo ( X 84,000). d WGA lectin. Intracytoplasmic vesicles with a heavy labelling ( X 84,000). A heavy reaction to the Con A lectin (Fig. 3f) and a light one to LcH and WGA lectins was found in the intracytoplasmic vesicles from ovine blastulae. Lysosomes were also very heavily marked by WGA (Fig. 3g), lightly by Con A (Fig. 3h), and sparsely by LcH. The specific labelling pattern shown by cell organelles at the blastula stage was quite different. Intracytoplasmic vesicles globally reduced their staining with WGA with respect to cleavage stages. In contrast, lysosomes increased their staining with WGA and LcH lectins. Inner Cell Mass There is no specific polarity in the cell membrane of the blastocyst internal mass. This cell membrane showed a sparse histochemical reaction against the colloidal gold-conjugated lectins used in this study (Fig. 4a). The intensity of reaction showed by lysosomes varied in WGA (very heavy), LcH (light), and Con A (sparse). Nevertheless the intracytoplasmic vesicles reacted sparsely with LcH and WGA (Fig. 4b,c) and remarkably with Con A (Fig. 4d). Like the trophectodermal cells, the intracytoplasmic vesicles and the lysosomes from ICM blastomeres showed qualitative and quantitative differences with respect to the cleavage period. DISCUSSION The results obtained in the present work demonstrate a specific pattern in the distribution of sugar residues in the preimplantation ovine embryo. During ovine embryo cleavage the zanes that showed most sugar residues detected by colloidal gold conjugated lectins were the zona pellucida (GlcNAc), the intracytoplasmic vesicles (Man/Glc and GlcNAc), and the intercellular contact surface (GlcNAc and GalNAc). In contrast, a t the blastocyst stage, lectin labelling reduced in the intercellular surfaces but sugar residues increased on the free cell membrane (ManiGlc, GlcNAc, and Gal) and lysosomes (GlcNAc). The zona pellucida presents abundant GalNAc, GlcNAc, and Gal residues during preimplantation ovine embryo, but a t the blastocyst stage GalNAc residues are less abundant. LcH and Con A lectins, with Man/Glc affinity mainly bound to free cell surface in the trophoblast cells and to vesicles in the different stages of early ovine develop- LECTIN RECEPTORS IN EARLY OVINE EMBRYO 54 1 Fig. 2. Zona pellucida. a: WGA lectin. A very heavy labelling is observed ( X 24,000). b: DBA lectin 84,000). c: MPA lectin ( x 84,000). d PNA lectin ( x 84,000). (x ment. The changes in the Con A distribution patterns zones. The presence of SBA in the basal membrane of of the cell surface of the preimplantation embryo have the ectoderm and other epithelial structures has been already been described in mouse (Wu, 1980; Chavez, reported in the 7-day mouse embryo (Herken et al., 1990) and rabbit (Chen and Cao, 1992). Handyside 1990). Kimber and Bird (1985) have located DBA re(1980) has experimentally shown a polarization of the ceptors in 8 cell mouse embryos (compaction phase) and Con A binding sites in isolated blastomeres during mu- suggested a relationship between those receptors and rine embryo compaction. This author suggests that the compaction phase. In the same way we can relate such a distribution pattern of Con A receptors could cleavage and the abundant DBA staining observed in provide a molecular mechanism for attaining spatial the ovine intercellular surface. differentiation of trophectoderm and inner cell mass The lectins with high affinity to galactose (MPA and within the blastocyst. Kimber and Bird (1985) have PNA) show a sparse binding pattern throughout the shown that the recovery of the cell surface glycoconju- preimplantation period, except in the trophectodermal gates in the 8-16 cell embryo after enzymatic dissoci- microvilli and zona pellucida. In this sense, Handyside ation occurs at the same time a s recompaction, so these (1980) has described PNA receptors in murine morulae glycoconjugates may be involved in this process. This with a sparse staining a t the early stages, which inobservation agrees with the quantitative polarization creased a t the 16 cell stage after compaction. Rober and in the Man/Glc (LcH and Con A lectins) we found in the Holtz (1988) located galactose and fucose on the emintercellular surface of the trophoblast cells during bryonic membranes during pig embryo implantation ovine embryo cleavage. which disappear once the materno-fetal contact is esDBA and SBA staining patterns indicate a sparse tablished. D-galactose residues would play a role in the density of GalNAc residues in the free cell membrane implantation process as demonstrated by glycosylglycoconjugates of the blastomeres during cleavage, transferase treatments (Chavez, 1990). The exclusive but in the intercellular contact zones that density is presence of galactose residues at the free membrane higher. At the blastocyst stages, the free cell surface trophoblastic cells agrees with this hypothetic role. was richer in GalNAc residues than intercellular The absence of Lotus staining and the poor density of Fig. 3 LECTIN RECEPTORS IN EARLY OVINE EMBRYO 543 Fig. 4. Inner cell mass of the sheep blastocyst. a: WGA lectin. Lectin receptors are observed in the intercellular membrane ( x 84,000). b LcH lectin. Intracytoplasmic vesicles and lysosomes showing gold colloidal particles ( x 63.000). c: ConA lectin. Secondary lysosome ( x 84,000). d WGA lectin. Lysosome ( x 84,000). colloidal gold UEA-conjugated particles indicates the lack of fucosyl residues in the ovine preimplantation embryo. In contrast, Kimber and Bird (1985) and Dealtry and Sellens (1987) described the presence of receptors to fucose specific lectins in the preimplantation mouse embryo. Likewise, during preimplantation embryonic development we have only sparsely detected sialic acid residues (LPA) in the microvilli of the blastocyst trophectodermal cells. Chavez (1990) reported the presence of sialic acid residues on the trophoblast in the mouse. According to this author a loss of these residues is necessary to accomplish implantation. While GS-I1 receptors were not detected in the ovine Fig. 3. Trophectoderm of the sheep blastocyst. a: LcH lectin. Detail of the cell surface microvilli ( x 84,000).b: SBA lectin. Free surface of the blastomeres shows numerous microvilli i x 84,000).c: WGA lectin. Gold colloidal particles in the microvilli ( x 84,000). d: MPA lectin. Sparse staining in cell membrane ( x 84,000).e: LPA lectin. Cell membrane ( x 84,000). f:ConA lectin. Intracytoplasmic vesicles i x 84,000). g: lectin WGA. Very heavy labelling in a lysosome ( x 63,000). h ConA lectin. Secondary lysosome ( x 84,000). embryo during the stages analyzed, WGA shows the highest staining in our panel of lectin staining, indicating a greater distribution of GlcNAc residues during these development phases. These wide differences in the staining with GS-I1 and WGA lectins (both lectins have the same sugar affinity) may be due to the fact that GS-I1 lectin has high affinity and interacts with a non-reducing terminal GlcNAc group, but WGA has wider binding sites where 2-6 sugar residues can fit (Goldstein and Poretz, 1986). In contrast to our results, Kimber and Bird (1985) located BSL-I1 (GS-11) receptors in the preimplantation mouse embryo. Cell membranes from preimplantation ovine embryo show abundant GlcNAc residues, specially in the microvilli of the trophoblastic cells. Intracytoplasmic vesicles during cleavage and lysosomes in the zonal blastocyst also show abundant GlcNAc residues. The intracytoplasmic vesicles from mammal embryos do not have a definite function. An energy reserve function and a role in the cavitation process within the blastula have been proposed in other models (Mohr and Trounson, 1982) for embryos other than ovine. In the present work, we found a significant cy- 544 P. DE PAZ ET AL. tochemical identity of vesicles and lysosomes during preimplantation development which demonstrated these vesicles could be related to lysosomes in a n intracellular cycle. In this sense, Hyttel et al. (1988) suggest that the intracytoplasmic vesicles from bovine fertilized oocytes are involved in the formation of metabolic cell complexes. With respect to the participation of these organelles in the cavitation process, the intracytoplasmic vesicles of morula cell are heavily stained for ConA (MadGlc) a n WGA (GlcNac) but i t is sparse in the intercellular of trophectodermal cells. This could mean that intracytoplasmic vesicles do not suffer exocytosis during the cavitation process or the sugar residues of glicoproteins are modified, since no membranes appear with such a definite staining in the blastocoele. The abundant labelling of the ovine zona pellucida for Gal, GalNac, and GlcNac sugars during the preimplantation period is in agreement with the observations made by several authors (Kan et al., 1990; Shalghi et al., 1991; Miller et al., 1993). In this sense, it is highly significant that the zona pellucida is a distinctive extracellular matrix to which have been attributed the functions of species specificity of fertilization, block to polyspermy, and protection of the embryo during early development (Riddell et al., 1992). Boice et al. (1992) indicated that the bovine oviduct-specific secretory glycoconjugates become intimately associated with early cleavage stage embryos, thus possibly playing a critical role in normal embryonic development. The differences in sugar composition described in the ovine preimplantation embryo should reflect molecular characteristics whose developmental variations may have a functional significance. In the present work we detect a regional polarization of ManIGlc (Con A and LcH), GalNAc (DBA and SBA), and GlcNAc (WGA) residues in the intercellular contact zone of the blastomeres during cleavage when they are compared with the cell free surface. In contrast, the increase in the trophoblast cell membrane of some carbohydrates previously identified during cleavage (Man/Glc, GlcNAc, and Gal) is evidence of cell surface modifications, and so they could play a basic role during the preimplantation period. ACKNOWLEDGMENTS This work was supported by the CICYT grant GAN88-0102. LITERATURE CITED Bayna, E.M., J.H. Shaper, and B.D. Shur 1988 Temporally specific involvement of cell surface p-1,4 galactosyltransferase during mouse embryo morula compaction. Cell, 53t145-157. Boice, M.L., P.A. Mavrogianis, C.N. Murphy, H.G. Verhage, R.S. Prather, and B.N. Day 1992 Immunocytochemical analysis of the association of bovine oviduct. specific secretory glycoconjugates with early embryos. Theriogenology, 7t194. Brandley, B.K. 1991 Cell surface carbohydrates in cell adhesion. Semin. Cell Biol., 2t281-287. Bukers, A,, J. Friedrich, B.P. Nalbach, and H. Denker 1990 Changes in lectin binding patterns in rabbit endometrium during pseudopregnancy, early pregnancy and implantation. In: Trophoblast Research, Val. 4. Plenum Publishing, New York, pp. 285305. Chavez, D.J. 1990 Possible involvement of D-galactose in the implantation process. In: Trophoblast Research, Val. 4. Plenum Publishing, New York, pp. 259-272. Chen, S.Z., and Y. Cao 1992 Variations of lectin-binding glycoproteins in early pregnant rabbit embryo. Sci. China 35t577-584. Dealtry, G.B., and M.H. Sellens 1987 Lectin receptors on the peri- and early post-implantation mouse embryos. Roux Arch. Dev. Biol., 196:59-65. Fenderson, B.A., and E.M. Eddy 1990 Glycoconjugate expression during embryogenesis and its biological significance. Bioessays, 12: 173-179. Goldstein, I.J., and R.D. Poretz 1986 Isolation, physicochemical characterization and carbohydrate-binding specificity of lectins. In: The Lectin. Properties, Functions and Applications in Biology and Medicine. I.E. Liener, N. Sharon & I.J. Goldstein, ed. Academic Press, Orlando, FL, pp. 25-250. Gooi, H.C., T. Feizi, A. Kapadia, B.B. Knowles, D. Salter, and M.J. Evans 1981 Stage-specific embryonic antigen involves al-3 fucosylated type 2 blood group chains. Nature, 292t156-158. Handyside, A.H. 1980 Distribution of antibody- and lectin-binding sites on dissociated blastomeres fron mouse morulae: Evidence for polarization a t compaction. J. Embryol. Exp. Morphol., 60:99116. Hathaway, H.J., L.C. Romagnano, and B. Babiarz 1989 Analysis of cell surface galactosyltransferase activity during mouse trophectodermal differentiation. Dev. Biol., 134t351-361. Herken, R., B. Sander, and M. Hofmann 1990 Ultrastructure localization of WGA, RCA I, LFA and SBA binding sites in the sevenday-old mouse embryo. Histochemistry, 94.525-530. Hyttel, P., T. Greve, and N. Callesen 1988 Ultrastructure of in-vitro fertilization in superovulated cattle. J. Reprod. Fertil., 82t1-13. Kan FW, E. Roux, S. STJacques, and G. Bleau 1990 Demonstration by lectin-gold cytochemistry of transfer of glycoconjugates of oviductal origin to the zona pellucida of oocytes after ovulation in hamsters. Anat. Rec., 226:37-47. Kimber, S.J., and P.R. Bagley 1987 Cell-surface enrichment of fucosylated glycoconjugates in the 8- to 16-cell mouse embryo. An autoradiographic study. Roux Arch. Dev. Biol., 196t492-498. Kimber, S.J., and J.M. Bird 1985 Cell surface changes in preimplantation mouse embryos during compaction investigated using FITC conjugated lectins after proteolytic enzymes treatment. Roux Arch. Dev. Biol., 194t470-479. Lindner, G.M., and R.W. Wright 1983 Bovine embryo morphology and evaluation. Theriogenology, 20t407-416. Lis, H., and N. Sharon 1986 Lectins as molecules and as tools. Annu. Rev. Biochem., 55t35-67. Miller, C.C., R.K. Merkie, P.M. Brooks, A.B. Caudle, and R.A. FayrerHosken 1993 Carbohydrate and protein analysis of the equine zona pellucida. Theriogenology, 39t268. Mohr, L., and A.O. Trounson 1982 Comparative ultrastructure of hatched human, mouse and bovine blastocysts. J. Reprod. Fertil., 66:499 -504. Peacock, J.S., AS. Colsky, and V.B. Pinto 1990 Lectins and antibodies as tools for studying cellular interactions. J. Immunol. Methods, 126t147-157. Rastan, S., S.J. Thorpe, P. Scudder, S. Brown, H.C. Gooi, and T. Feizi 1985 Cell interactions in preimplantation embryos: Evidence for involvement of saccharides of the poly-N-acetyllactosamine series. J. Embryol. Exp. Morphol., 87:115-128. Riddell, K.P., D.A. Stringfellow, V.S. Panangala, and J.M. Scodras 1992 Monoclonal antibodies to the bovine zona pellucida. Theriogenology, 37r285. Rober, R.A., and W. Holtz 1988 Membrane-bound glycoconjugates and enzymes a t maternal-embryonal interaction sites during implantation in swine. In: 11th Int. Congr. Anim. Reprod. Artif. Insem., Dublin, vol. 2, pp. 120. Shalgi, R., R. Maymon, B. Barshir, D. Amihal, and E. Shutelsky 1991 Distribution of lectin receptor sites in the zona pellucide of folicullar and ovulated rat oocytes. Mol. Reprod. Dev., 29t365-373. Sharon, N., and H. Lis 1991 Lectins as cell recognition molecules. Science, 246t227-246. Shur, B.D. 1989 Glycoconjugates as mediators of cellular interactions during development. Curr. Opin. Cell Biol., 1:905-912. Spicer, S.S., and B.A. Schulte 1992 Diversity of cell glycoconjugates shown histochemically: A perspective. J. Histochem. Cytochem., 4Ot527-533. Watson, A.J. 1992 The cell biology of blastocyst development. Mol. Reprod. Dev., 33t492-504. Weibel, E.R. 1979 Stereological Methods. Val. 1.New York, Academic Press. Wiley, L.M. 1988 Trophectoderm: the first epithelium to develop in the mammalian embryo. Scanning Microsc., 2t417-426. Wu, J.T. 1980 Concanavalin A binding capacity of preimplantation mouse embryos. J. Reprod. Fertil., 58t455-461.