Shirou Nozawa

Differences in lectin binding patterns of normal human endometrium between proliferative and secretory phases

SummaryLectin binding patterns in normal human endometrium were examined by light and electron microscopy using seven different lectins (ConA, WGA, RCA, PNA, UEA-1, DBA, and SBA). For light microscopic observations, criteria based on the incidence and intensity of cells positive for the lectin staining were adopted to evaluate the different staining patterns of the proliferative and secretory endometria obtained by the avidin-biotin-peroxidase complex (ABC) technique. At the light microscopic level, ConA, WGA, and RCA stained endometrial glandular cells in both phases. The number of PNA-positive cells with the binding sites entirely limited to the apical surface tended to be reduced slightly in the secretory phase. UEA-1 weakly stained the apical surface of glandular cells in the proliferative phase but not in the secretory phase. Among the lectins used in this study, DBA and SBA displayed remarkable changes between the phases. That is, in the proliferative phase they produced only a faint or slight positive stain at the apical surface, but the incidence and intensity of DBA- and SBA-positive glandular cells increased in the secretory phase. By electron microscopy, the reaction product of ConA was observed in the plasma membrane, endplasmic reticulum, nuclear envelope, and the Golgi apparatus, and the binding sites of RCA and DBA were observed in the plasma and, Golgi membranes. Between both phases, the reactivity of ConA and RCA showed almost no change. However, the secretory endometrial cells containing the DBA-positive Golgi apparatus were markedly increased in number compared with the proliferative ones bearing the lectin-positive organelles. These results indicate that DBA and SBA are useful for detection of hormone-mediated changes of normal human endometrium, and suggest that such changes are closely related to the Golgi apparatus.

The immunocytochemical localization of new soluble placental tissue proteins (PP14, 16, 17, 19, 20 and PP21) in human andCynomolgus monkey placentae

SummaryApparently Placenta-specific placental tissue proteins (PP14 and PP17) and solitary tissue proteins (PP16, 19, 20 and PP21) were investigated by avidin-biotin immunoperoxidase technique in the human andcynomolgus monkey placentae, membranes, decidua and umbilical cords. In human early placentae, PP14, 16, 17, 19 and PP21 were localized mainly in the cytoplasm of villous syncytiotrophoblast. PP20 was localized in the cytoplasm of basal chorionic trophoblasts. In human term placentae, positive stainings for PP16, 19 and PP21 were observed mainly in all kinds of trophoblastic cells, while positive stainings for PP14, 17 and PP20 were weakened in the trophoblastic cells. PP20 was clearly localized in the cytoplasm of Hofbauer-like cells in the villous stroma. The membrane of villous syncytiotrophoblast showed strongly positive stainings for PP21. PP21 was also localized in the membrane of amniotic and umbilical epithelium. The umbilical epithelium was cytoplasmically positive for PP14, 16 and PP20. Clear positive stainings for PP14 and PP21 were found in the cytoplasm of fetal polymorphonuclear neutrophils. All of the placental proteins were immunocytochemically positive in the decidual large cells. In thecynomolgus monkey placentae, similar immunostaining results were obtained. The monkey could, thus, serve as a model for the investigation of the placental proteins.

Diplococcal β-galactosidase with a specificity reacting to β1–4 linkage but not to β1–3 linkage as a useful exoglycosidase for the structural elucidation of glycolipids

Diplococcal beta-galactosidase, which is known to be useful for the structural studies of glycoprotein-linked oligosaccharides, was found to show the same substrate specificity in cleaving Gal beta 1-4 linkages of glycolipids as that of the oligosaccharides. The optimum conditions of beta-galactosidase in the 80% ammonium sulfate precipitates of the culture medium of Streptococcus (Diplococcus) pneumoniae were determined with nLcOse4Cer radiolabeled by the galactose oxidase-NaB3H4 procedure. Detergent was required for the highest activity, and different combinations of several buffers and detergents showed different properties in stimulating beta-galactosidase, and in enhancing or suppressing N-acetyl-beta-hexosaminidase which was contaminated in the enzyme preparation. The optimum pH was found to be at 6.5, and specific activity and Km were 8.1 nmol/mg protein/h and 1 nmol, respectively. While more than 70% of beta-galactose was liberated from LacCer and nLcOse4Cer within 1 h under the optimum conditions to form GlcCer and nLcOse3Cer, respectively, none was liberated from LcOse4Cer, GalCer, GgOse4Cer, GbOse3Cer, IV3 alpha GalnLcOse4Cer, and Il3NeuAcGgOse4Cer, showing the substrate specificity solely to Gal beta 1-4 linkage.

Selective terminal α2–3 and α2–6 sialylation of glycosphingolipids with lacto-series type 1 and 2 chains in human meconium

Human meconium was found to contain two kinds of gangliosides with the same carbohydrate sequences belonging to the lacto‐series. They were detected by TLC‐immunostaining with monoclonal antibodies directed to the NeuAcα2–Gal and Lc4Cer structures. One of these two gangliosides, a major one, which migrated on TLC to a position below that of standard IV3NeuAcnLc4Cer from human erythrocytes, reacted with the antibody to NeuAcα2–6Gal. The other minor one, which migrated on TLC to a position corresponding to standard IV3NeuAcnLc4Cer, was detected with the antibody to Lc4Cer only when the plate, on which the individual gangliosides were separated, was subjected to prior treatment with Vibrio cholerae sialidase. The structures of the gangliosides, each identified by means of permethylation analysis and enzyme treatment after isolation with antibody monitoring, were shown to be IV6NeuAcnLc4Cer for the former and IV3NeuAcLc4Cer for the latter, indicating that the lacto‐series type 2 (nLc4Cer) and 1 (Lc4Cer) chains are sialylated at different linkages, α2–6 and α2–3, respectively. IV6NeuAcLc4Cer and IV3NeuAcnLc4Cer were not detected, even in trace amounts, on TLC‐immunostaining with the monoclonal antibodies. The concentrations of IV6NeuAcnLc4Cer and IV3NeuAcLc4Cer were 448 and 18 nmol/g dry wt of human meconium.

Human cervical epidermal carcinoma-associated intracellular localization of glycosphingolipid with blood group A type 3 chain.

A monoclonal antibody, MRG‐1, was produced by immunizing a mouse with a human ovarian mucinous cyst adenocarcinoma‐derived cell line, RMUG‐L. By immunohistochemical staining, the antigen was found to be exclusively localized in the intracellular structures of the cells used as the antigen and of the epithelial cells in normal human cervical glands. However, although the antigen was predominantly detected in the plasma membrane and the intercellular structure of the middle layer of normal human cervical squamous epithelium (92%), it was also contained in the intracellular structure of cervical epidermal carcinoma at a high frequency (80%). The striking difference in the distribution of the MRG‐1 antigen between normal and cancerous tissues was found to be a cervical carcinoma‐associated phenomenon and a useful tumor marker for immunohistochemical examination. Since the antigen was found to be of a blood group A‐related nature by immunohistochemical staining of the tissues and to be a glycosphingolipid, it was purified from human erythrocytes of blood group A, and the structure was concluded to be GalNAcα1–3Gal(2–1αFuc)jβ1–3GalNAcα1–3Gal(2–1αFuc)‐ β1–4GlcNAcβ1–3Galβl‐4Glcβ1–1′Cer, blood group A type 3 chain‐containing glycosphingolipid, by NMR, negative ion FABMS and permethylation analysis. In the subcellular localization analysis of the antigen, type 3‐A glycosphingolipid antigen was detected in the Golgi body and the microsomes of RMUG‐L cells, and the distribution coincided with the finding by immunohistochemical staining. In addition, in cervical epidermal carcinoma, although the blood group A, mainly type 2‐A chain, was localized in the plasma membrane and the intercellular structure, the blood group A type 3 chain was selectively found in the perinuclear structure. Also, the blood group A type 3 chain in cervical dysplasia as well as that in normal cervix was predominant in the plasma membrane. Thus, the selective intracellular localization of blood group A type 3 chain was a phenomenon characteristic of cervical epidermal carcinoma and the carcinoma in situ.

Luteal phase-characteristic induction of I3SO3-GalCer in human cervical epithelia and uterine endometria, and follicular phase-characteristic formation of a ganglioside-derived negative charge gradient in different regions of fallopian tubes

In a series of experiments on the hormone-dependent molecular alteration in the human genital tract during the menstrual cycle, we focused our attention on a change in the negative charge due to the sulfuric acid- and sialic acid-containing glycosphingolipids. Although a ganglioside-derived negative charge was maintained in the cervical epithelia and uterine endometria at a relatively constant concentration throughout the luteal and follicular phases, I3SO3GalCer in both tissues characteristically increased in the luteal phase, indicating that the synthesis of I3SO3-GalCer in both tissues is associated with the menstrual cycle. However, I3SO3-GalCer in mucosae of the fallopian tubes in both phases was present in a concentration similar to that in the uterine endometrium in the luteal phase, and the change in the concentration did not associated with the menstrual cycle. On the other hand, although the concentrations of I3SO3-GalCer and II3NeuAc-LacCer, a major ganglioside, were similar in different regions, that is, the isthmus, ampulla and fimbriae of the fallopian tubes in the luteal phase, II3NeuAc-LacCer was present in a gradually increasing concentration from the isthmus to the fimbriae in the follicular phase, giving a gradually decreasing ratio of I3SO3GalCer to ganglioside from the uterus to the fimbriae. These findings indicate that the metabolism of sulfo- and sialoglycosphingolipids in the human genital tract is strictly controlled by estrogen and progesterone.

Characteristic alteration in the concentration of IV 3NeuAc α-nLc4Cer in the villi of human placenta during the gestational period

A study on the ganglioside composition in the villi isolated from human placenta at various gestational periods was carried out by conventional procedures including thin-layer chromatography (TLC), TLC-immunostaining, negative ion fast-atom bombardment mass spectrometry (FABMS) and exoglycosidase treatment. The major gangliosides in the villi were II3 NeuAc-LacCer (GM3) and IV3NeuAc alpha-nLc4Cer, comprising 60-70% of the total gangliosides. The concentration of IV3NeuAc alpha-nLc4Cer per gram dry weight of tissue in the villi was found to be gradually decreased from the early to the late gestational period and the molecule with 2-hydroxy fatty acids was undetectable after 20 weeks of the gestational period. However, no significant correlation between the concentration of GM3 and the gestational periods was observed. Thus the characteristic alteration in the concentration of IV3NauAc alpha-nLc4Cer in the villi might be related to various functions of human placental villi during the gestational period.