Changes in cell surface and intracellular glycoproteins of trophoblastic giant cells during mouse placentation

Changes in lectin bindings of mouse trophoblastic giant cells (TGCs) were examined by light and electron microscopy. Neither Griffonia simplicifolia agglutinin (GS)-II nor succinyl-wheat germ agglutinin (s-WGA) bound to the 1st and 2nd TGCs on day 6.5 post coitum (p.c.), but did so from days 8.5 to 12.5 p.c. Positive reactions with s-WGA were localized in the perinuclear region and cell surface of both 1st and 2nd TGCs; while GS-II bound only to the perinuclear region, where it appeared as network-like deposits. This region was identified as well-developed Golgi lamellae by electron microscopy. Moreover, SDS-PAGE and lectin-blot analysis of the 1st TGCs indicated that the intensity of s-WGA and GS-II bindings increased in the glycoproteins of approximately 43, 40, 37, and 26 kDa and in those of 43 and 38 kDa, respectively, during the 8.5th to 10.5th day p.c. The reaction with GS-I was detected on cell surface of both the 1st and 2nd TGCs on day 6.5 p.c. The reaction in the 1st TGCs was intensely positive throughout their development, whereas the reactivity decreased in the 2nd TGCs on day 10.5 p.c. and completely disappeared on day 12.5 p.c. The GS-I reaction in TGCs was more intense at the maternal side than at the embryonic side. These results suggest that certain Gal and/or GlcNAc glycoproteins on the cell surface and in Golgi lamellae of TGCs dynamically change from the 8.5th to 10.5th day p.c. in association with mouse placentation.     

Changes in lectin binding pattern of gonalds of developing mice

Summary Changes in lectin binding of developing fetal mouse testes and ovaries were examined by light and electron microscopy, with much attention paid particularly to those in carbohydrates of germ cells. Characteristic binding patterns were observed with three lectins (BPA, GS-I, and GS-II) in the germ cells and the somatic cells during the process of testicular and ovarian development. GS-I and BPA, which showed similar binding patterns, preferentially bound to the plasma membrane and small dense bodies (SDB) of germ cells in both testes and ovaries during the 12th to 14th day post coitum (p.c.). In the fetal testes on day 16 p.c., the reaction with both GS-I and BPA completely disappeared. While, in the ovaries, a weak reaction with these lectins was retained as it was in germ cells until the 16th day p.c. The reaction with GS-II was restricted to Sertoli cells in the fetal testes during the 12th to 14th day p.c., and thereafter disappeared on day 16 p.c. The distribution of GS-II binding sites was in agreement with that of the glycogen granules. No positive staining with GS-II was seen in the ovaries throughout their development. These results indicate that certain glycoconjugates containing d-galactose and N-acetyl-d-galactosamine residues are expressed on the cell surface and in the SDB of germ cells during the period of the 12th to 14th day p.c., and that striking changes in function as well as in structure may take place in both germ cells and somatic cells during the 14th to 16th day p.c. in association with testicular and ovarjan development.     

Changes in lectin binding pattern of gonads of developing mice

Changes in lectin binding of developing fetal mouse testes and ovaries were examined by light and electron microscopy, with much attention paid particularly to those in carbohydrates of germ cells. Characteristic binding patterns were observed with three lectins (BPA, GS-I, and GS-II) in the germ cells and the somatic cells during the process of testicular and ovarian development. GS-I and BPA, which showed similar binding patterns, preferentially bound to the plasma membrane and small dense bodies (SDB) of germ cells in both testes and ovaries during the 12th to 14th day post coitum (p.c.). In the fetal testes on day 16 p.c., the reaction with both GS-I and BPA completely disappeared. While, in the ovaries, a weak reaction with these lectins was retained as it was in germ cells until the 16th day p.c. The reaction with GS-II was restricted to Sertoli cells in the fetal testes during the 12th to 14th day p.c., and thereafter disappeared on day 16 p.c. The distribution of GS-II binding sites was in agreement with that of the glycogen granules. No positive staining with GS-II was seen in the ovaries throughout their development. These results indicate that certain glycoconjugates containing D-galactose and N-acetyl-D-galactosamine residues are expressed on the cell surface and in the SDB of germ cells during the period of the 12th to 14th day p.c., and that striking changes in function as well as in structure may take place in both germ cells and somatic cells during the 14th to 16th day p.c. in association with testicular and ovarian development.     

Changes in lectin binding patterns of Leydig cells during fetal and postnatal development in mice

Summary Changes in the lectin binding of mouse Leydig cells during fetal and postnatal development were examined by light- and electron-microscopy using eight different biotinylated lectins (ConA, WGA, RCA-I, UEA-I, GS-I, PNA, SBA and GS-II). At the light-microscopic level, ConA, WGA, RCA-I, UEA-I and GS-I showed the same binding pattern in which all five lectins bound to the plasma membrane and cytoplasm of Leydig cells from the 13th day post coitum (p.c.) to the 8th postnatal week. PNA, SBA and GS-II reactions were positive in the plasma membrane and cytoplasm of Leydig cells from the 13th day p.c. to 15th day post partum (p.p.) but disappeared completely by day 20. At the electron-microscopic level, gold particles representing the GS-I or GS-II binding sites were distributed primarily along the cell surface membrane, including that of microvilli, as well as in the cytoplasm. These results indicate that certain glycoconjugates bearingD-galactose,N-acetyl-D-galactosamine, andN-acetyl-D-glucosamine residues are expressed on the cell surface and in the cytoplasm of Leydig cells during the period from the 13th day p.c. to around the 20th day p.p. The results suggest that these glycoconjugates might play some role in modulating hormone-receptor interaction in the Leydig cells before the 20th day. Furthermore, these results may indicate that sugar residues expressed on the cell surface and in the cytoplasm of Leydig cells are different from those in the fetal-neonatal and adult phases.     

Characterization and Solubilization of β-Glucan Synthases from Cultured Rice Cells

Two types of β-glucan synthases, GS-I and GS-II, were found in cultured rice cells (Oryza sativa L.). In glycerol density gradient centrifugation, GS-I activity peak co-migrated with a marker enzyme of the Golgi membrane, while GS-II co-migrated with the plasma membrane. Analysis of the reaction products of GS-I and GS-II, suggested that GS-I and GS-II were mainly β-1,4,- and β-1,3-glucosyltransferases, respectively. GS-I had a higher substrate affinity for UDP-glucose than GS-II, and needed divalent cations for its activity. Effects of nucleotides on the activity were also considerably different between GS-I and GS-II. GS-I was solubilized well with CHAPS and digitonin, and GS-II was solubilized effectively with sucrose monolaurate.     

Localization of forssman glycolipid and GM1 ganglioside intracellularly and on the surface of germ cells during fetal testicular and ovarian development of mice

Summary Changes in the expression pattern and intracellular localization of Forssman glycolipid (FA) and GM1 ganglioside (GM1) in fetal mouse gonads were examined during germ cell differentiation by immunofluorescence microscopy and immunoelectron microscopy.In male germ cells from the 12th to 14th day p.c., anti-FA binding was localized in granular structures aggregated on one side of the cytoplasm and/or in the plasma membrane. On day 16 p.c., some germ cells still showed patch-like positive reactions in the plasma membrane, but by day 18 p.c., positive reactions for FA had completely disappeared. The female germ cells showed granular bindings of anti-FA scattered throughout their cytoplasm during the 13th to 16th day p.c., although the positive reactions in female germ cells on day 12 p.c. tended to be found in one side of cytoplasm and/or plasma membrane similar to those in male germ cells from 12th to 14th day p.c. On day 18 p.c., positive reactions remained in the plasma membrane of some germ cells, but these positive reactions disappeared before birth. Immunoelectron microscopic observation showed that the sites of anti-FA bindings were equivalent to the small dense bodies (SDB) and the Golgi lamellae both in male and female germ cells. On the other hand, GM1 was not detected in male germ cells at any time during fetal testicular development, whereas an anti-GM1 reaction was detected in the plasma membrane of female germ cells from the 16th to 18th day p.c. (oocytes in the first meiotic prophase).Therefore, these results indicate that kinetic translocation of FA between the plasma membrane and the SDB and Golgi lamellae takes place during germ cell differentiation in fetal gonads. Moreover, the ganglioside composition containing GM1 seems to change in association with the first meiotic prophase.     

Localization of Forssman glycolipid and GM1 ganglioside intracellularly and on the surface of germ cells during fetal testicular and ovarian development of mice

Changes in the expression pattern and intracellular localization of Forssman glycolipid (FA) and GM1 ganglioside (GM1) in fetal mouse gonads were examined during germ cell differentiation by immunofluorescence microscopy and immunoelectron microscopy. In male germ cells from the 12th to 14th day p.c., anti-FA binding was localized in granular structures aggregated on one side of the cytoplasm and/or in the plasma membrane. On day 16 p.c., some germ cells still showed patch-like positive reactions in the plasma membrane, but by day 18 p.c., positive reactions for FA had completely disappeared. The female germ cells showed granular bindings of anti-FA scattered throughout their cytoplasm during the 13th to 16th day p.c., although the positive reactions in female germ cells on day 12 p.c. tended to be found in one side of cytoplasm and/or plasma membrane similar to those in male germ cells from 12th to 14th day p.c. On day 18 p.c., positive reactions remained in the plasma membrane of some germ cells, but these positive reactions disappeared before birth. Immunoelectron microscopic observation showed that the sites of anti-FA bindings were equivalent to the "small dense bodies" (SDB) and the Golgi lamellae both in male and female germ cells. On the other hand, GM1 was not detected in male germ cells at any time during fetal testicular development, whereas an anti-GM1 reaction was detected in the plasma membrane of female germ cells from the 16th to 18th day p.c. (oocytes in the first meiotic prophase).(ABSTRACT TRUNCATED AT 250 WORDS)     

Lectin bindings and diethylstilbestrol effects on the recognition of mullerian inhibiting substance (MIS) on chick mullerian ducts by MIS-antiserum

A high intensity of lectin bindings was demonstrated on the epithelial cells and serosa cells of the regressing right Mullerian ducts (Mds) in the female chick embryos. The strong lectin bindings occurs on, or in the regressing Md cells along with marked surface MIS bindings at the age of day 13. However, at the age of days 5-7 1/2, bindings of lectins were weak. Neither Wheat-germ agglutinin (WGA) or Concanavalin A (Con-A) labelings before MIS-antiserum (MIS-Ab) incubation can block antibody recognitions to the antigens, including MIS and growth hormone at the age of day 13. Our previous studies indicated that after WGA labeling on the surfaces of Md epithelial cells prior to the incubation of MIS-Ab at day 10 did not prevent the recognition of MIS-Ab (Wang 1989). On the contrary, at day 7 1/2, the specific binding of MIS was eliminated after preincubations with lectins and prenatal diethylstilbestrol (DES) treatment at the age of day 5. It is suggested that DES provides a protection to the Mds from MIS-induced regression by preventing the MIS binding to its specific membrane receptors. An increase of extra- and intracellular glycoproteins or carbohydrates of regressing Md epithelial cells were suggested. Internalization of WGA but not MIS molecules was found in Md epithelial cells. The Golgi saccules were negative of lectin bindings.     

Purification and Characterization of Griffonia simplicifolia Leaf Lectins

Leaves from mature Griffonia simplicifolia plants were examined for the presence of leaf lectins possessing sugar binding specificities similar to the four known seed lectins (GS-I, GS-II, GS-III, GS-IV). Three (GS-I, -II, -IV) of the four known G. simplicifolia seed lectins were present in the leaves. Leaf G. simplicifolia lectins I and IV were similar to the respective seed lectins. Leaf GS-II, however, was composed of two types of subunits (M_r = 33,000 and 19,000), whereas the seed lectin consists of only one type of subunit (M_r 32,500). Seed and leaf GS-II lectins also had different isoelectric points. All leaf and seed lectins were similar with respect to their hemagglutination and glycoconjugate precipitation properties and all subunits contained covalently bound carbohydrate. Leaf GS-IV appeared slightly under-glycosylated compared to seed GS-IV.

The fate of GS-I and GS-II seed lectins in aging cotyledons was investigated. GS-I isolectins usually contain isolectin subtypes associated with each main isolectin. Upon inbibition and germination, these GS-I isolectin subtypes disappeared. Over time, GS-II lectin did not change its disc gel electrophoretic properties.

     

Lectin bindings and diethylstilbestrol effects on the recognition of mullerian inhibiting substance (MIS) on chick mullerian ducts by MIS-antiserum

Summary A high intensity of lectin bindings was demonstrated on the epithelial cells and serosa cells of the regressing right Mullerian ducts (Mds) in the female chick embryos. The strong lectin bindings occurs on, or in the regressing Md cells along with marked surface MIS bindings at the age of day 13. However, at the age of days 5–7 1/2, bindings of lectins were weak. Neither Wheat-germ agglutinin (WGA) or Concanavalin A (Con-A) labelings before MIS-antiserum (MIS-Ab) incubation can block antibody recognitions to the antigens, including MIS and growth hormone at the age of day 13. Our previous studies indicated that after WGA labeling on the surfaces of Md epithelial cells prior to the incubation of MIS-Ab at day 10 did not prevent the recognition of MIS-Ab (Wang 1989). On the contrary, at day 7 1/2, the specific binding of MIS was eliminated after preincubations with lectins and prenatal diethylstilbestrol (DES) treatment at the age of day 5. It is suggested that DES provides a protection to the Mds from MIS-induced regression by preventing the MIS binding to its specific membrane receptors. An increase of extra- and intracellular glycoproteins or carbohydrates of regressing Md epithelial cells were suggested. Internalization of WGA but not MIS molecules was found in Md epithelial cells. The Golgi saccules were negative of lectin bindings.     

Postnatal differentiation of the Golgi apparatus and the dendrites of Purkinje cells of the rat cerebellum

Summary The morphology of postnatal differentiation of the Golgi apparatus, the nucleus, the perikaryon, and the dendrites was studied in Purkinje cells of the rat cerebellum for 30 days after birth using histochemical, histological, and electron microscopic methods.The Golgi apparatus during differentiation undergoes morphological and positional changes. From the 1st to 7th postnatal day, the Golgi apparatus is found in a supranuclear position, and is connected with the axes of differentiating primary dendrites by beam-like processes. From days 8 to 11 this connection disappears, and most of the Golgi apparatus assumes a lateronuclear and infranuclear position. After the 11th or 12th day, the Golgi apparatus is found in perinuclear and peripheral cytoplasmic positions. The formation of granular endoplasmic reticulum occurs in the vicinity of the perinuclear Golgi apparatus. The differentiation of cell and nuclear forms requires approximately 20 days. The morphological changes of differentiation are discussed in relation to the participation of the Golgi apparatus in the differentiation of dendrites and in the formation of the granular endoplasmic reticulum.     

A comparison of lectin binding in rat and human peripheral nerve

Eleven different fluorescein- or peroxidase-conjugated lectins with different sugar-binding affinities were employed to analyze and compare glycoconjugates of rat and human peripheral nerves at the light microscopic level. A majority of lectins showed a distinct binding pattern in different structures of the nerve. Lectin binding was similar but not identical in rat and human nerves. Limulus polyhemus agglutinin did not stain any structures in rat or human nerves. In both species, all other lectins bound to the perineurium. Perineurial staining was intense with Canavalia ensiformis (Con A), Triticum vulgaris (WGA), Maclura pomifera (MPA); moderate with Glycine max (SBA), Griffonia simplicifolia-I (GS-I) and GS-II; weak with Ulex europaeus (UEA), Dolichos biflorus (DBA), and Ricinus communis (RCA). In the endoneurium of both species, ConA staining was intense, MPA and WGA moderate, SBA, GS-II, PNA, and RCA weak, and UEA and DBA absent. Interestingly, GS-I stained rat but not human endoneurium. Most lectins bound to blood vessels. GS-I bound to rat but not human, whereas UEA bound to human but not rat vessels. The results show that lectins can be used to reveal heterogeneity in sugar residues of glycoconjugates within neural and vascular components of nerves. They may therefore be potentially useful in detecting changes in glycoconjugates during nerve degeneration and subsequent regeneration after trauma or in pathological states.     

Cellular heterogeneity in normal human urothelium: Quantitative studies of lectin binding

Summary A panel of 10 FITC-labelled lectins (MPA, PNA, ConA, DBA, SBA, RCA-120, WGA, UEA, GS-I, GS-II) was applied to cryosections of seven specimens of normal urothelium. Seven of the lectins (MPA, ConA, RCA, WGA, UEA, GS-I and GS-II) showed a pattern of increasing fluorescence intensity from basal to superficial cells of the urothelium whereas PNA, DBA and SBA showed more uniform binding throughout the urothelium. Urothelial cell suspensions labelled with FITC-lectins were studied by flow cytometry to quantify the variation in binding to different cells types. Three cellular subpopulations were identified in normal urothelium on the basis of their optical properties. Fluorescence intensity due to specific lectin binding was then measured separately for each subpopulation. Although there was some variation among individual cases, a general pattern emerged in this small series. WGA, RCA, and GS-II bind in large quantities to all urothelial cells while PNA, SBA, ConA and DBA show little binding. MPA, RCA, UEA and GS-I showed the most marked increase in fluorescence intensity from basal to superficial cells as observed microscopically and quantified by flow cytometry.     

The glycoprotein-bound large carbohydrates from embryonal carcinoma cells carry receptors for several lectins recognizing N-acetylgalactosamine and galactose

When various lectins were mixed with radioactively labeled embryoglycan (polylactosamine-type glycoprotein-bound carbohydrates from early embryonic cells) isolated from F9 embryonal carcinoma cells and the resulting complex was precipitated with ammonium sulfate, the glycan was found to react with the following lectins: Helix pomatia agglutinin (HPA), soybean agglutinin (SBA), Sophora japonica agglutinin (SJA), and Ricinus communis agglutinin-1 (RCA-1). Furthermore, affinity chromatography on lectin-agarose revealed that receptors for Griffonia simplicifolia agglutinin-I (GS-I) were also carried by the glycan. Together with the previous finding that the glycan carries receptors for Dolichos biflorus agglutinin (DBA) and peanut agglutinin (PNA), the present result established that the glycan has receptors for a variety of lectins recognizing N-acetylgalactosamine and/or galactose in teratocarcinoma cells. Intact molecules carrying GS-1 receptors and SJA receptors were isolated from F9 cells and teratocarcinoma OTT6050 and were shown to be high-molecular weight glycoproteins similar to DBA receptors isolated from the same sources.     

Subcellular compartmentalization of saccharide moieties in cultured normal and malignant cells

We studied subcellular localization of saccharide moieties in cultured normal and malignant cells fixed in paraformaldehyde and treated with a nonionic detergent, using lectins specific for various surgar residues as probes in fluorescence microscopy. In normal cells, concanavalin A and Lens culinaris agglutinin, specific for mannose-rich carbohydrate cores in glycoproteins, labeled the endoplasmic reticulum as a wide perinuclear region. Other lectins, on the other hand, stained the Golgi apparatus as a juxtanuclear reticular structure. A similar compartmentalization was also seen in all malignant cells studied, although the Golgi apparatus in these cells was distinctly vesicular in appearance. Our results indicate that saccharide moieties in both normal and malignant cells are similarly compartmentalized, and thus speak in favor of a unidirectional subcellular flow for both membrane and secreted glycoconjugates.     

Establishment and differentiation of murine EG cell lines derived from primordial germ cells]

Primordial germ cells (PGC) were isolated from 8.5, 10.5, 12.5 days post coitum (dpc) embryos of F1 (Balb/c x ICR), C57BL/6J, 129/svJ, 129/sv-ter mice, and cultured on mitotically inactive MEF or STO feeder layer cells with addition of leukemia inhibitory factor, stem cell factor and basic fibroblast growth factor in cultures. PGCs formed densely packed and AKP positive colonies with pluripotential marker gene (oct-4) expression resembling undifferentiated ES cells in morphology and growth pattern. Five EG cell lines derived from PGCs were established: EG1(8.5 dpc, F1), EG2 and EG3 (8.5 dpc, C57BL/6J), EG4 (10.5 dpc, 129/svJ), EG5 (10.5 dpc, 129/sv-ter). No long term culture was obtained from 12.5 dpc PGCs of 29 embryos. All five EG cell lines cultured on feeder layer cells or in LIF containing medium still remain undifferentiated state at 15 th passage. Under appropriate conditions, EG cells formed embryoid bodies in suspension culture and multiple types of differentiated cells in monolayer culture. When these EG cells were injected in nude mice, they formed teratocacinomas containing differentiated cells such as cartilage, neural tissue and epithelium. These results show that EG1-5 cell lines derived from 8.5, 10.5 dpc embryos are pluripotential.     

PTHrP induces changes in cell cytoskeleton and E-cadherin and regulates Eph/Ephrin kinases and RhoGTPases in murine secondary trophoblast cells

The differentiation of murine trophoblast giant cells (TGCs) is well characterised at the molecular level and, to some extent, the cellular level. Currently, there is a rudimentary understanding about factors regulating the cellular differentiation of secondary TGCs. Using day 8.5 p.c.-ectoplacental cone (EPC) explant in serum-free culture, we have found parathyroid hormone-related protein (PTHrP) to regulate cellular changes during TGC differentiation. PTHrP greatly stimulated the formation and organisation of actin stress fibres and actin expression in trophoblast outgrowth. This coincided with changing cell shape into a flattened/fibroblastic morphology, suppression of E-cadherin expression, and increased cell spreading in culture. PTHrP also increased the nuclear staining of beta-catenin and, similar to activator protein-2gamma (AP-2gamma), showed microtubule-dependent nuclear localisation in vitro. These cellular and behavioural changes correlated with changes in the expression of RhoGTPases and in both expression and phosphorylation of Eph/Ephrin kinases. The effects of PTHrP on trophoblast cellular differentiation were abolished after blocking its action. In conclusion, PTHrP provides an excellent example of the extrinsic factors that, through their network of activities, plays an important role in cellular differentiation of secondary TGCs.     

Effect of entomopathogenic fungus Tolypocladium species metabolite efrapeptin on Galleria mellonella agglutinin

In the current study it has been found that efrapeptins, secondary metabolites of entomopathogenic fungi Tolypocladium cylindrosporum, interfere with agglutinin. The effect of efrapeptins on G. mellonella agglutinin was tested using rabbit blood. The results revealed that the end point for control larvae were 12.5 whereas for treated larvae which injected with 5 microl of 5 microg efrapepins ml(-1) or 50 microg efrapepins ml(-1) the end points were 10.5 and 8.5, respectively. Considering that efrapeptins suppress agglutination this study suggest that efrapeptins may interfere with the ligand-receptor interactions that are likely to occur at the plasma membrane of specific haemocytes. It has been suggested that majority of interactions between cellular and humoral components of the insect immune system are receptor-mediated.     

Intracellular features of type II procollagen and chondroitin sulfate proteoglycan synthesis in chondrocytes

The intracellular compartments of chondrocytes involved in the synthesis and processing of type II procollagen and chondroitin sulfate proteoglycan (CSPG) monomer were investigated using simultaneous double immunofluorescence and lectin localization reactions. Type II procollagen was distributed in vesicles throughout the cytoplasm, whereas intracellular precursors of CSPG monomer were accumulated in the perinuclear cytoplasm. In this study, cytoplasmic vesicles that stained intensely with antibodies directed against CSPG monomer but did not react with type II collagen antibodies, also were observed. A monoclonal antibody, 5-D-4, that recognizes keratan sulfate determinants was used to identify the Golgi complex (the site of keratan sulfate chain elongation). Staining with 5-D-4 was restricted to the perinuclear cytoplasm. The vesicles outside the perinuclear cytoplasm that stained intensely with antibodies to CSPG monomer did not react with 5-D-4. Fluorescent lectins were used to characterize further subcellular compartments. Concanavalin A, which reacts with mannose-rich oligosaccharides, did not stain the perinuclear region, but it did stain vesicles throughout the rest of the cytoplasm. Because mannose oligosaccharides are added cotranslationally, the stained vesicles throughout the cytoplasm presumably correspond to the rough endoplasmic reticulum. Wheat germ agglutinin, which recognizes N-acetyl-D-glucosamine and sialic acid (carbohydrates added in the Golgi), stained exclusively the perinuclear cytoplasm. By several criteria (staining with the monoclonal antibody 5-D-4 and with wheat germ agglutinin), the perinuclear cytoplasm seems to correspond to the Golgi complex. The cytoplasmic vesicles that react with anti-CSPG monomer and not with anti-type II collagen contain precursors of CSPG monomer not yet modified by Golgi-mediated oligosaccharide additions (because they are not stained with wheat germ agglutinin or with the anti-keratan sulfate antibody); these vesicles may have a unique function in the processing of CSPG.