Lectin binding sites on human endocervix: a comparison with secretory and proliferative endometrium

Endocervix and corresponding endometrium of women of reproductive age were studied histochemically with 13 fluorescein isothiocyanate-labeled lectins to delineate the differences between the epithelial cells in two anatomical sites. Lectin from Maclura pomifera (MPA), Ulex europaeus (UEA-I), Glycine max (SBA), and Vicia villosa (VVA) bound only to endocervical epithelium and were the only four lectins that distinguished endocervical from endometrial epithelium. These differences were independent of menstrual cyclic changes and blood group antigen secretion. These data show that lectins can be used to histochemically distinguish endocervical from endometrial glands.     

Lectin binding sites in developing mouse limb buds

Summary The binding sites of the following biotinylated lectins were demonstrated in serial paraffin sections of fore- and hindlimb buds from day-9 to day-16 mouse embryos with the Avidin-Biotin-Peroxidase Complex (ABC) procedure: Concanavalin A (Con A), Soybean Agglutinin (SBA), Wheat Germ Agglutinin (WGA), Peanut Agglutinin (PNA), Ricinus Communis Agglutinin I (RCA), Ulex Europaeus I Agglutinin (UEA), and Dolichos Biflorus Agglutinin (DBA). Alternating neighbouring sections were used to compare the distribution of PNA staining, PNA staining after neuraminidase treatment (N-PNA) and the autoradiographic sites of [³⁵S]-sulphate uptake. Unspecific binding sites common to all lectins tested were observed in periderm and chondrocytes. Several lectin affinities were seen in the undifferentiated mesoderm (Con A, WGA, RCA), blood vessels (WGA, PNA, N-PNA, RCA, UEA, DBA) and macrophages (Con A, WGA, N-PNA, RCA). A very selective and mainly extracellular affinity to N-PNA was demonstrated in the condensed preskeletal mesoderm, where it characterizes indistinct prospective chondrogenic, perichondral and pre-articular areas. Comparison with the distribution pattern of [³⁵S]-sulphate uptake and other previously published histochemical data suggests that N-PNA staining occurs at the late blastema stage, i.e. after the stage of cell condensation and before the earliest deposit of stainable matrix in chondrogenic areas. This property later disappears from the chondrifying rudiments, and is maintained in perichondral and pre-articular tissues. Surprisingly, only the pre-articular areas bind PNA without pretreatment with neuraminidase. A transient RCA binding probably related to terminal morphogenesis was detected in the undifferentiated distal part of the predigital columns of day-12 and day-13 limb buds. From the day-13 stage onwards, diverse new lectin affinities appeared in differentiating tissues, such as pretendinous rudiments, perichondrium and prospective periosteum, muscular connective tissue, myotubes, superficial fasciae and prospective dermis. A strong SBA and PNA staining was also detected in the extracellular matrix associated with the epithelial septa separating the roots of the digits in day-15 and day-16 limb buds.     

Lectin binding to the human retina

Formalin-fixed, paraffin-embedded tissue sections of the human retina were stained with different fluorescein isothiocyanate-conjugated lectins. The lectins used were concanavalin A (Con A), Triticum vulgaris (WGA), glycine maximum (SBA), Dolichos biflorus (DBA), Ulex europaeus (UEA I), Arachis hypogaea (PNA), and Ricinus communis (RCA I). Con A stained both the inner and outer segments of the rods and cones, whereas WGA stained the inner and outer segments of the rods and the outer segments of the cones. PNA selectively stained only the inner segments of the cones. In addition, Con A and WGA stained neuron cytoplasm and nerve fibers in different layers of the retina. The results obtained differ in some important aspects from those previously obtained in the frog and monkey retina; this finding may be due to species differences. The results of lectin staining in the normal human retina may form the basis for future studies of retinal diseases.     

T-type Ca2+ channels in spermatogenic cells and sperm

Cell function is importantly regulated by the intracellular concentration of Ca²⁺ ([Ca²⁺]i). Sperm development and function are deeply influenced by [Ca²⁺]i which is modulated amongst other ion transporters by plasma membrane Ca²⁺ permeable channels. The presence and role of voltage-dependent Ca²⁺ channels (Ca_V) of the T-type (Ca_V3) in sperm physiology have become a matter of debate in recent years. Though they are functionally present in later stages of development in spermatogenic cells and testicular sperm and their mRNAs and proteins detected from spermatogenic cells to mature mammalian spermatozoa, their currents have not been recorded in mature spermatozoa. This review critically summarizes the evidence for the involvement of Ca_V3 channels in sperm development and function.     

Lectin binding characteristics of mouse placental cells

The lectin binding characteristics of mouse placental cells were examined. Wax embedded tissue sections of placentae from d 14 pregnant mice were stained with 26 lectins, with a wide range of sugar specificities. Cell cultures prepared from d 14 mouse placentae and cultured for 24 h were stained with 7 of the lectins to determine if they could be used as markers for the different trophoblast cells in culture. In tissue sections all placental cell populations bound lectin but no lectin bound specifically to any single trophoblast population. All the lectins which bound to layer 1 cytotrophoblast lining the maternal blood spaces of the labyrinthine placenta also bound to the fetal endothelium of the labyrinthine placenta. Binding of lectin appeared strongest on the adluminal membrane of these cell populations suggesting a role for the carbohydrate moieties in nutrient transfer. Few lectins bound to junctional zone trophoblast. Overall, the binding of lectin to cultured cells did not correlate exactly with lectin binding to the cell populations in tissue sections. The value of lectins as markers for placental cells in culture was therefore found to be limited. Our findings indicate that carbohydrate expression by at least some placental cells may vary in culture from that expressed by the cells in vivo with obvious concerns for the validity of functional in vitro studies.     

Lectin binding and steroid receptors in human breast carcinomas

A series of breast carcinomas of known steroid receptor status have been examined for evidence of binding of the lectins peanut agglutinin, soy bean agglutinin and wheat germ agglutinin. Correlations were found between oestrogen receptor status and reactivity of carcinomas to peanut agglutinin and soy bean agglutinin but these were not absolute. Wheat germ agglutinin binding was unrelated to the presence of oestrogen receptors. No relationship was evident between progestogen receptors and the binding of any lectin. It therefore seems unlikely that lectin histochemistry can replace steroid receptors as markers of hormone dependence in breast carcinomas.     

Lectin binding patterns of human tissue mast cells indicate marked phenotypical diversity.

Glycoconjugate expression by human tissue mast cells (MCs) from various sources (including lymph nodes with signs of chronic non-specific lymphadenitis, skin lesions of urticaria pigmentosa, and bone marrow infiltrates associated with systemic mastocytosis) was studied histochemically with a broad panel of fluorescein-labelled lectins. Of the 19 lectins applied, 11 (sugar specificities: fucose, N-acetylgalactosamine and neuraminic acid) did not stain any MCs, while 8 (sugar specificities: mannose, N-acetylglucosamine, and galactose) were found to bind to MCs. These lectins exhibited different binding patterns in various disease entities. Only a few of these 8 lectins (in particular, phythaemagglutinin-L) produced strong staining of the MCs in most or all of the cases. Some (e. g. phythemagglutinin-E) produced only weak staining, and this in only a few cases. The lectins used, however, did not distinguish between reactive and tumorous MCs. Although lectins are therefore unlikely to be of use in resolving problems of differential diagnosis concerning proliferation of MCs, our investigation has shown that tissue MCs exhibit marked phenotypical diversity with regard to their lectin-binding properties.     

Ultrastructural localization of a nuclear autoantigenic sperm protein in spermatogenic cells and spermatozoa

This study demonstrates the ultrastructural localization of rabbit nuclear autoantigenic sperm protein (NASP) in spermatogenic cells and spermatozoa. NASP is present in rabbits, rats, mice, and human testes and spermatozoa. It has recently been sequenced in rabbits and humans and characterized as an acidic, histone binding protein. Currently it has been proposed that NASP may play a role in regulating early events of spermatogenesis through its ability to bind and translocate testicular histone variants to nucleosomes. The ultrastructural localization of NASP confirms that it is initially present in primary spermatocytes in their Golgi regions and nucleus. In round spermatids it is present in the nucleus as well as in the acrosome and subacrosomal space. In later spermatids, testicular spermatozoa, and ejaculated spermatozoa, NASP is concentrated over the nucleus, although some is still present in the acrosome. It is likely that NASP would be carried into the ovum with the sperm nucleus at fertilization. © 1993 Wiley-Liss, Inc.     

Lectin binding in normal, keratoconus and cross-linked human corneas

In this study the characterization of various types of sugar residues in normal, keratoconus and cross-linked human corneas was performed using immunohistochemical localization with lectins. Corneal samples were collected and divided into three groups: (1) normal corneas from cadavers; (2) keratoconic corneal buttons; (3) keratoconic corneal buttons treated with cross-linking. A series of lectins including: DBA, SBA, PNA, ConA, WGA, UEA I, GNA, DSA, MAA, SNA, were used in combination with chemical and enzymatic treatments. Compared with the normal corneas, N-acetyl-D-glucosamine increased in the keratoconus corneas. L-fucose increased and/or appeared in the keratoconus and the cross-linked corneas. N-acetyl-D-galactosamine was more abundant in the epithelium of keratoconus corneas, but was lacking in the keratoconus and cross-linked endothelium. D-galactose-(β1-4)-N-acetyl-D-glucosamine was absent in the whole stroma of the keratoconus corneas and in the deep layers of the cross-linked ones. Sialic acids increased in the keratoconus corneas and decreased in the cross-linked ones. These results showed altered glycosylation in the keratoconic corneas and partially similar glycosylation in the cross-linked corneas, compared to the normal ones. This suggests a role played by sugar residues in maintaining the corneal structure. The changes could be related to structural alterations in keratoconus. The present findings contribute to our understanding of the effect of cross-linking treatment of keratoconic corneas in therapeutic attempts to re-establish the normal corneal structure.     

Lectin binding to human gastric adenocarcinomas and adjacent tissues.

The binding of lectins to paraffin sections of nine gastric carcinomas and adjacent mucosa was examined by fluorescence microscopy. A battery of nine lectins was employed, and both intestinal and diffusely infiltrating tumors were tested. Wheat germ agglutinin and Ricinus communis agglutinin I appeared to bind to both mucus and nonmucus glycoproteins; these lectins labeled tumor cells, benign epithelial cells, and nonepithelial tissues strongly and consistently. Peanut agglutin, soybean agglutinin, Dolichos biflorus agglutinin, Bandeiraea simpifolica agglutinin, and Ulex europaeus agglutinin I bound extensively to mucosubstances in vacuoles and apices of benign epithelial cells but often bound to tumor cells focally and in some cases not at all. Neuraminidase digestion enhanced lectin staining in some tumors; but in others, especially those of the diffusely infiltrating type, neuraminidase digestion did not enhance the staining of tumor cells. The results suggest that the decrease in the proportion of tumor cells labeling with lectin relative to superficial epithelial cells can be due either to the oversialylation of mucoproteins or to the loss of glycosylating enzyme activity. Concanavalin A did not bind to mucosubstances in the vacuoles or apices of benign epithelium, but bound to mucus vacuoles of metaplastic epithelium and to coarse cytoplasmic granules in two of the tumors examined. This suggests either the abnormal addition of mannose to mucus glycoprotein or the production of a distinct glycoprotein by some gastric tumors.     

Lectin binding to carcinoma-in situ cells of the testis

Summary Seven patients with carcinoma-in-situ of the testis were studied. Testicular biopsies were treated with eight fluorescein isothiocyanate conjugated lectins, and particular attention was paid to the similarities between CIS germ cells, normal germ cells and seminoma cells. In the cytoplasm of CIS cells a large number of granularly distributed Con A and LCA binding sites was noticed, indicating the presence of mannose and N-acetyl-glucosamine in these cells. The perinuclear fluorescence observed by WGA and RCA I suggests the incorporation of N-acetyl-glucosamine and galactose into glycoproteins in cytoplasmic celt organelles of these cells. The distribution of glycoconjugates in CIS germ cells is similar to that of invasive seminoma cells confirming the malignant nature of CIS cells. However, as there are differences in lectin binding of spermatogenetic cells and CIS cells, no conclusions regarding the origin of CIS cells can be drawn.     

Lectin binding pattern in human retinal pigment epithelium.

A comparative lectin histochemical study of human retinal pigment epithelium (RPE) was performed to investigate the lectin binding pattern of normal, reactive and proliferating RPE. Normal RPE with attached sensory retina was found to bind the lectins Con A, WGA, PNA and RCA I. Reactive and proliferating RPE in retinal detachment and in photocoagulation scars revealed the same lectin binding pattern although its cellular topography changed. RPE-macrophages showed an additional reaction with SBA. In periretinal membranes of human PVR the typical lectin binding pattern of Con A, WGA, PNA and RCA I was found in pigmented and in a subpopulation of non-pigmented cells, suggesting that these lectin-positive elements were of RPE-origin. Additionally, single pigmented cells positive for SBA were found indicating macrophage differentiation. Thus lectin histochemistry provides a tool for cytochemical identification of RPE and its morphologic variants by revealing a specific combination of sugar-binding sites.     

Somatostatin binding sites in human leptomeninx

Specific binding sites for somatostatin (SRIF) have been visualized in the human leptomeninx by means of autoradiographical techniques using the stable SRIF octapeptide analog 125I-204-090 as radioligand. The binding sites are specific for SRIF since only biologically active SRIF analogs compete with the radioligand. The density of the binding sites is comparable to that of the adjacent cortical structure. The presence of SRIF binding sites in the human leptomeninx, together with the recent demonstration of SRIF binding sites in human meningiomas, strongly suggest that this peptide may play a role in the physiology of the leptomeninx.     

Benzodiazepine binding sites in human myometrium

We report that membrane preparations of human myometrium possess binding sites for [3H] RO 5-4864, a specific ligand for the peripheral type of benzodiazepine receptors. Scatchard analysis shows a high-affinity binding site (Kd = 3.1 +/- 1.1 nM) and a class of low-affinity binding sites (Kd greater than 30nM) with positive cooperativity. Displacement and tryptic digestion experiments indicate that both these benzodiazepine binding sites are specific and proteic in nature. So benzodiazepines might exert their relaxant activity on uterine muscle directly at peripheral level through specific binding sites.     

Membrane androgen binding sites are preferentially expressed in human prostate carcinoma cells

Background  

Prostate cancer is one of the most frequent malignancies in males. Nevertheless, to this moment, there is no specific routine diagnostic marker to be used in clinical practice. Recently, the identification of a membrane testosterone binding site involved in the remodeling of actin cytoskeleton structures and PSA secretion, on LNCaP human prostate cancer cells has been reported. We have investigated whether this membrane testosterone binding component could be of value for the identification of prostate cancer.     

Solubilization and characterization of moderate and high affinity histamine binding sites on human blood mononuclear cells

The Triton X-100 solubilized extract of human peripheral blood mononuclear cells, in direct binding studies with 10(-9)-10(-6) M [3H]histamine contained both high and moderate affinity sites whose dissociation constants (Kd 4.4 X 10(-9) and 6.7 X 10(-7) M) were commensurate with basal plasma histamine levels and plasma levels obtained following physiological or mild pathological stimuli, respectively. Binding was enhanced by mM concns of calcium cations and by the protease inhibitor Pepstatin A. It was inhibited by bacitracin, agents interfering with thiol groups, Triton X-100 concns greater than 0.2% and EDTA. Binding was optimal between the pH range of 7.0 and 8.5 and was enriched for in a plasma membrane preparation. Thus the histamine binding sites identified maintained their specific ligand binding properties after solubilization from the cell surface and displayed properties fulfilling the criteria for receptors.     

Developmental potential of human spermatogenic cells co-cultured with Sertoli cells.

BACKGROUND: Development of an in-vitro culture system capable of supporting human early germ cell differentiation would be important for treatment of azoospermic patients. METHODS: Sertoli cells, spermatogonia and spermatocytes were isolated from testicular biopsies of 61 non-obstructive azoospermic patients, and co-cultured using Vero cell conditioned medium only or supplemented with recombinant (r)FSH or rFSH plus testosterone. Germ cell purity was checked by fluorescent in-situ hybridization (FISH) analysis. RESULTS: Best results were achieved with both hormones, which elicited 6.9% of meiosis index and 22.7% of differentiation into normal late spermatids after 2-3 weeks of culture. In-vitro matured spermatids were microinjected into oocytes to study their developmental potential. Round spermatids elicited 37.5% of fertilization and 28.6% blastocyst rates. Abnormal elongating and elongated spermatids enabled 8.3 and 27.3% fertilization rates respectively, but none achieved the blastocyst stage. Normal elongating and elongated spermatids elicited 30.5% fertilization and 42.9% of blastocyst rates. FISH analysis showed sex chromosome anomalies in all embryos, except in the case of morulae from normal late spermatids. CONCLUSIONS: Results suggest that meiosis and spermiogenesis can be resumed in vitro, with normal differentiated spermatids showing a low fertilization potential but regular rates of blastocyst formation. However, most of the embryos did not reach the morula stage and showed major sex chromosome abnormalities.     

人精子结合蛋白HBRP的抗体制备及定位

目的制备人类新的精子结合蛋白HBRP的单克隆抗体,确定其在精子的定位及其与精子功能相关的作用。方法分别利用PCR、Western blot、动物免疫、血清纯化等方法得到HBRP单克隆抗体,用ELISA方法检测抗体滴度;利用间接免疫荧光方法观察HBRP在精子的定位。结果经纯化鉴定获得HBRP融合表达蛋白;首次获得HBRP单克隆抗体;应用制备的单克隆抗体,确定HBRP主要结合在精子尾部的中段和主段。结论 HBRP结合在人成熟精子尾部的中段和主段,提示其参与精子运动功能的调控。     

Lectin binding pattern in the embryonal and early fetal human vertebral column

Summary Paraffin sections from vertebral columns of ten human embryos and fetuses ranging from stage 16 to the 12th week were stained with the FITC-coupled lectins PNA, RCA I, Con A and WGA in order to investigate changes in carbohydrate-binding sites during vertebral development. PNA revealed a specific binding site in the vertebral body blastema in the precartilaginous stage of development. Beginning with the 25-mm CRL embryo, PNA-binding sites occurred in the developing fibrous annulus and the inner zone of the intervertebral discs. The first binding sites for RCA I were seen in the extracellular matrix of vertebral bodies during the cartilaginous stage of vertebral development. During early ossification of the vertebrae, staining for RCA I-binding sites in the cytoplasm of the chondrocytes and the area around the future cartilaginous end-plates was observed. Con A bound to the chondrocyte cytoplasm, and also very strongly to notochordal cells in all developmental stages examined. WGA-binding sites appeared simultaneously with cartilage formation. Connective tissue components, e.g. ligaments, were diffusely stained by WGA. Also this lectin showed an affinity for vertebral body chondrocytes. We discuss the biochemical aspects of these lectin-binding sites, and their possible roles in the differentiation process of the human vertebral column. The results of this first lectin histochemical study on human vertebral development are compared with related results in other species.     

Lectin histochemistry of the embryonic heart: Fucose-specific lectin binding sites in developing rats and chicks

Glycoconjugates, particularly their sugar side chains, play important roles in embryonic development. Changes in cell-surface-associated glyco-conjugates are known to affect cell differentiation, cellular interactions, and other developmental phenomena during embryogenesis. The embryonic heart goes through a series of complicated morphologic events during development. Of particular interest is morphogenesis of the outflow tract. This region of the embryonic heart originates from more than one cell population and undergoes a complex process of septation during formation of the great vessels. Histochemi-cal analysis with a series of fucose-specific lectins conjugated to horseradish peroxidase has revealed the presence of a fucosylated glycoconjugate in the outflow tract of the developing heart. The results reveal further that the expression of the fucosylated glycoconjugate is stage-dependent and thus probably genetically regulated. The timing and distribution of staining with the lectin OFA suggest that this fucosylated glycocoiyu-gate may play a role in directing the migration of neural crest cells into the heart and subsequent formation of the conus septum.