The histochemistry and ultrastructure of the clitellum of the enchytraeid Lumbricittus rivalis (Oligochaeta: Annelida)

The monostratified clitellar epidermis of Lumbricillus rivalis consists of supporting cells, granular secretory cells, and globular secretory cells, together with the acid mucous cells typical of normal skin in the anterior and posterior transition zones. The secretion of the granular cells is a neutral glycoprotein with low levels of bound lipid, and that of the globular cells is a sulphated acid mucopolysaccharide lacking detectable protein or lipid.
Ultrastructurally the granular cells possess 1 μm, membrane-bound granules of variable electron density suggestive of maturation changes. The formative granules arise from Golgi vesicles and are moderately electron translucent. They contain parallel-aligned, tubule-like inclusions of 14 nm diameter, observed also in the mature regions of the Golgi. Granules in the mid or apical part of the cell show increased electron density, the formative pattern being wholly or partially obscured and the subunit alignment, when discernable, no longer uniform. Moiré fringe patterns are evident in some granules. The patterns and electron density are lost after pronase digestion. The globular cells contain electron translucent, membrane-bound globules of 2–3 μm diameter with finely fibrous contents. They arise from mature Golgi vesicles and are unaffected by pronase treatment.
The results suggest that the granular cells secrete the cocoon wall and the globular cells the material that surrounds the developing embryos, and are compared with published accounts of other microdrile clitella and with those of the clitellum of lumbricid earthworms.     

Ultrastructural and histochemical studies on the epithelium revestment layer in the tube feet of the starfish Asterina stellifera

The “cuticle,” which revests the starfish tube foot, has been studied by electron microscopy and the findings correlated with histochemical observations. The “cuticle” is composed by two distinct zones; an outer zone including numerous microvilli, which extend from the inner zone into and through a fibrillar substance distinctly organized in two layers. These microvilli protrude slightly beyond the outer surface, where their tips give rise tonumerous extremely delicate fibrils. The second inner zone, of quite variable thickness and condensation of material, presents a coarser fibrous matrix where organelles and inclusions can be found. The whole cuticular complex does not derive from the majority of the epithelial cells, but is probably an extension of a special kind of T-shaped cells appearing at intervals, the “cuticle” forming a syncytial surface. Histochemical investigations indicate that the “cuticle” contains a combination of neutral and acid mucopolysaccharide, with a marked neutral predominance, the outer one displaying also an extremely thin coat of acid mucopolysaccharide with the sulfate group. The ordered arrangement of the microvilli suggests that this situation is imposed by the strong bond existing between the microvilli and the ouble mucopolysaccharide layers which would act as a cementing substance stabilizing the entire apical surface of the cell.     

Scanning electron microscopy of Treponema pallidum (Nichols strain) attached to cultured mammalian cells.

This paper describes the attachment of Treponema pallidum (Nichols strain) to cultured mammalian cells as a visualized by scanning electron microscopy. Treponemes were incubated for 3 hr with cultured cells derived from normal rabbit testes or human skin epithelium, then fixed, processed with critical-point drying, and examined with a Cambridge Mark 2A scanning electron microscope. Large numbers of treponemes became attached to the cultured cells without altering the morphological integrity of the cultured cells. Attachment appeared to involve a very close physical proximity of treponemes to the cultured cells; at the site of attachment, no changes such as swelling or indentation of the cultured cell surface were observed. The addition of ruthenium red to the fixatives produced a treponemal-associated surface precipitate. This material, which is probably mucopolysaccharide and/or phospholipid, may be important in protecting the organisms against host defense mechanisms; in addition, it may be involved in the serological unresponsiveness of freshly prepared suspensions of T. pallidum.     

The functional morphology of the alimentary tract of barnacles (Cirripedia: Thoracica)

The alimentary tract of barnacles is made up of cuticle-lined foregut and hindgut with an intervening U-shaped midgut associated anteriorly with a pair of pancreatic glands and perhaps midgut caeca. Epithelial salivary glands secrete acid mucopolysaccharide, glycoprotein or both. Cells of all the midgut regions are capable of absorption which is carried out mainly by the anterior midgut and caeca. Midgut cells of Balanus balanoides (L.) show a seasonal variation in the distribution of intracellular lipid droplets. Midgut cells rest on an elastic basal lamina and secrete a peritrophic membrane which contains mucopolysaccharide and protein. Cells of the stratum perintestinale connect with the midgut epithelial cells via cell processes which probably translocate absorbed materials. Glycoprotein globules and lipid droplets accumulate in the body parenchyma of B. balanoides and are transported to the ovaries to form yolk (glycolipovitellin). The pancreatic gland cells of all barnacles are active secretory cells secreting proteinaceous material (probably digestive enzymes).     

Cytochemistry of the skin of patients with mucopolysaccharidoses.

The distribution of complex carbohydrates has been investigated at the light and electron microscope levels in sweat glands of normal subjects and patients with Hurler's or Hunter's disease. Normal sweat glands examined with a battery of light microscopic histochemical methods revealed sulphated complex carbohydrate in secretory granules of the dark cells. These granules lacked affinity for dialysed iron (DI) at the light and electron microscope levels. The DI method demonstrated acid complex carbohydrates ultrastructurally on the surface of the intercellular canaliculi and central lumen in normal sweat glands. DI-reactive acidic material, presumably of mucopolysaccharide nature, surrounded and extended between collagen bundles in the stroma of normal skin, but was absent from the band which ensheathed the sweat gland and consisted of individual rather than bundled collagen fibrils. DI-reactive mucopolysaccharide lined and partially filled vacuoles of dark cells showing a laminar distribution in vacuoles of clear cells in sweat glands of a Hunter patient. The DI method also visualized mucopolysaccharide distributed throughout vacuoles in fibroblasts of this patient. DI-reactive acid material covered the luminal surface of the sweat gland, coated collagen bundles in the stroma and spared the periglandular collagenous sheath in skin from Hurler and Hunter patients as in that from normal controls. Acid phosphatase was localized ultrastructually in vacuoles and nearby cytoplasm and on plasmalemmae of clear cells, dark cells and myoepithelial cells of sweat glands from Hurler and Hunter patients. Vacuoles of dermal fibroblasts and Schwann cells in these specimens also exhibited strong acid phosphatase activity.     

DEPENDENCE OF SALIVARY EPITHELIAL MORPHOLOGY AND BRANCHING MORPHOGENESIS UPON ACID MUCOPOLYSACCHARIDE-PROTEIN (PROTEOGLYCAN) AT THE EPITHELIAL SURFACE

The morphogenetic role of the acid mucopolysaccharide (glycosaminoglycan) at the epithelial surface of mouse embryo submandibular glands has been studied by comparing the in vitro morphogenesis of epithelia from which the mucopolysaccharide was removed with that of those that retained the mucopolysaccharide. Epithelia isolated free of mesenchyme by procedures which retain the bulk of surface mucopolysaccharide maintain their lobular shape and undergo uninterrupted branching morphogenesis in culture in direct combination with fresh mesenchyme. Under identical culture conditions, epithelia from which surface mucopolysaccharide was removed lose their lobules and become spherical masses of tissue. During continued culture, the spherical epithelia produce outgrowths from which branching morphogenesis resumes. The morphogenetically active mucopolysaccharide is localized within the basal lamina of the epithelial basement membrane and appears to be bound to protein. During culture in combination with mesenchyme, epithelia undergoing uninterrupted morphogenesis show maximal accumulation of newly synthesized surface mucopolysaccharide at the distal ends of the lobules, the sites of incipient branching. In contrast, the material accumulates nearly equivalently over the surface of the spherical epithelia, with the exception that there is greater accumulation of the material at the surfaces of the budding outgrowths, the sites where morphogenesis will resume. Rapidly proliferating cells are localized within the lobules of epithelia undergoing uninterrupted morphogenesis, but are distributed uniformly in the cortex of the spherical epithelia, except for the outgrowths which show a greater localization of proliferating cells. It is concluded that normal salivary epithelial morphology and branching morphegenesis require the presence of acid mucopolysaccharide-protein within the epithelial basal lamina.     

Rabbit antiserum against a purified surface glycoprotein decompacts mouse preimplantation embryos and reacts with specific adult tissues

A rabbit antiserum against a purified embryonal carcinoma (EC) cell surface glycoprotein interferes with cell-cell interaction in mouse preimplantation embryos. The 123 kD glycoprotein seems not to be an integral membrane component. The reactivity pattern of the antiserum was studied by immunofluorescence on cryostat sections of post-implantation embryos and of adult tissues. During embryonic development positive reactions were found on all epithelial cells, irrespective of their germ layer origin. Epithelial cells of adult tissues—tongue, uterus, gut, kidney, trachea and liver—react with the antibodies. The results are compared with cell-adhesive molecules previously described on EC cells and preimplantation embryos.     

LANTHANUM STAINING OF THE SURFACE COAT OF CELLS : Its Enhancement by the Use of Fixatives Containing Alcian Blue or Cetylpyridinium Chloride

Among the techniques which have been reported to stain the surface coat of cells, for electron microscopy, is lanthanum staining en bloc. Similarly, the presence of the cationic dye, Alcian blue 8GX, in a primary glutaraldehyde fixative has been reported to improve the preservation of the surface coat of cells of many types; however, the preserved coat is not very electron opaque unless thin sections are counterstained. The present paper shows that for several rat tissues lanthanum staining en bloc is an effective electron stain for the cell surface, giving excellent contrast, if combined sequentially with prefixation in an aldehyde fixative containing Alcian blue. The cationic substance cetylpyridinium chloride was found to have a similar effect to that of Alcian blue in enhancing the lanthanum staining of the surface coat material of the brush border of intestinal epithelial cells. The patterns of lanthanum staining obtained for the tissues studied strikingly resemble those reported in the literature where tissues are stained by several standard methods for demonstrating mucosubstances at the ultrastructural level. This fact and the reproduction of the effect of Alcian blue by cetylpyridinium chloride constitute a persuasive empirical argument that the material visualized is a mucopolysaccharide or mucopolysaccharide-protein complex.     

A fine structural analysis of the epidermis and cuticle of the oligochaete aeolosoma Bengalense stephenson

The fine structure of the epidermis and cuticle has been described for the oligochaete Aeolosoma bengalense. The epidermis is a pseudostratified epithelium and consists of the following cell types: ciliated and nonciliated supportive cells, pigment cells and associated satellite cells, mucous cells, basal cells, and ciliated non-supportive columnar cells. Overlying and restricted to the supportive cells is a delicate cuticle composed of: (a) a discontinuous layer of membrane-bounded surface particles; (b) a thin filamentous layer of moderate electron density just under the surface particles; (c) a thicker inner filamentous layer of low electron density. Digestion with pronase effectively removes the cuticle. This, together with the fact that it stains with alcian blue and ruthenium red, indicates that the cuticle contains an acid mucopolysaccharide. Regeneration of the cuticle, following pronase treatment, is marked by the elaboration of numerous microvilli by the supportive cells. Most of the microvilli are transitory and evidence supports a microvillar origin for the cuticular surface particles. The presence of cuticular surface particles may be a characteristic shared in common by all oligochaetes and, perhaps, some polychaetes.     

Glycoprotein synthesis in developing sea urchin embryos

Surface membrane glycoproteins have been postulated in many mammalian cells to be involved in external surface membrane functions such as cell adhesion, cell-cell recognition, and cell movement. In developing echinoderm embryos, cell adhesion, recognition, and movement of individual cell types have been attributed to differences in the external surface membranes of these cells. Results reported here suggest that the three cell types of 16-cell sea urchin embryos have a mechanism that could establish differences in the carbohydrate portion of glycoproteins located in the external surface membrane. The results demonstrate 1) that glycoproteins are synthesized during early sea urchin development and 2) that slightly different rates of glycoprotein synthesis exist for the three types of blastomeres from 16-cell sea urchin embryos.     

Role of mitochondrial ribosome-dependent translation in germline formation in Drosophila embryos

In Drosophila, mitochondrially encoded ribosomal RNAs (mtrRNAs) form mitochondrial-type ribosomes on the polar granules, distinctive organelles of the germ plasm. Since a reduction in the amount of mtrRNA results in the failure of embryos to produce germline progenitors, or pole cells, it has been proposed that translation by mitochondrial-type ribosomes is required for germline formation. Here, we report that injection of kasugamycin (KA) and chloramphenicol (CH), inhibitors for prokaryotic-type translation, disrupted pole cell formation in early embryos. The number of mitochondrial-type ribosomes on polar granules was significantly decreased by KA treatment, as shown by electron microscopy. In contrast, ribosomes in the mitochondria and mitochondrial activity were unaffected by KA and CH. We further found that injection of KA and CH impairs production of Germ cell-less (Gcl) protein, which is required for pole cell formation. The above observations suggest that mitochondrial-type translation is required for pole cell formation, and Gcl is a probable candidate for the protein produced by this translation system.     

Identification and purification of a cell surface glycoprotein mediating intercellular adhesion in embryonic and adult tissue

An antiserum against material shed into serum-free medium by MCF-7 human mammary carcinoma cells (anti-SFM II) disrupts cell-cell interactions in murine mammary tumor epithelial cells (MMTE). We now report purification of an 80 kd glycoprotein (GP80) from SFM of MCF-7 mammary carcinoma cells that blocks the activity of anti-SFM II. Anti-SFM II also inhibits compaction of eight-cell mouse embryos, and purified GP80 blocks this reaction. An antiserum against purified GP80 (anti-GP80) has all adhesion-disrupting activities displayed by anti-SFM II. It recognizes one band at 80 kd in SFM and a 120 kd band in detergent extracts of epithelial but not fibroblastic cells. In immunofluorescence studies it is restricted to sites of cell-cell interaction in cultured epithelial cells. Thus a cell surface glycoprotein of 120 kd, the medium form of which is approximately 80 kd, which is neither species nor tissue specific, is expressed at early stages of mammalian development and is found on epithelia.     

The pig yolk sac I

Summary Yolk sacs from pig embryos ranging between 18 mm and 55 mm in length were investigated by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and histochemistry. The organ was no longer present in embryos of 70 mm length. The endoderm proliferates in embryos of about 20 mm length with gland-like endodermal cell columns and finally becomes stratified, representing over 90% of the yolk sac mass. The endodermal cells show a high activity of oxidoreductases and lysosomal enzymes; their luminal surface bears few absorptive specializations. The mesothelium is inert, as judged from its surface ultrastructure, organelle composition and enzyme content. TEM reveals the endodermal cells to be polarized even in stratified areas. They resemble liver parenchymal cells with respect to their basal villi, which are exposed to capillaries with discontinuous or fenestrated endothelium. Giant mitochondria with crystalline inclusions in the mature endodermal cytoplasm are outnumbered by large stacks of the rough ER, which can amount to 60% of the cytoplasm. This conspicuous RER is suspected to be the production site of serum proteins which are discharged into the vascular bed. Close to the time of the organ's regression, an unusual storage of material in terminal buds of the ER was found. Intercellular canaliculi and the endocytic apparatus of the endoderm are thought to serve regression.     

Studies on the surface of chick blastoderm cells. II. Electron microscopy of surface binding characteristics

The surfaces of cells from the early embryo of the chick were examined with the electron microscope using histochemical techniques in order to determine the distribution of cell surface material. The use of lanthanum nitrate as a stain for protein-polysaccharide showed that in whole embryos the surface layer was present in areas of close membrane apposition, but relatively sparse where the membranes bounded large intercellular spaces. On cells freshly dissociated with EDTA, this technique demonstrated a very uniform layer over the surface, possibly indicating some redistribution. Close examination of the lanthanum stained material in whole embryos revealed the presence of periodicities about 180 Å in diameter. In correlation with a progressive reduction of electrophoretic mobility, demonstrated previously, from embryonic stage 1 to stage 5, some decrease in the affinity of the surface for lanthanum and colloidal iron was observed.     

A ruthenium red-toluidine blue procedure for staining epoxy sections in the light microscopy.

Ruthenium red (RR) has been widely used as a fixation additive for electron microscopy on the basis of its capacity to retain acid mucopolysaccharide residues of the cell surface coat. Little is known about the properties of this compound as a direct staining agent for epoxy-resin embedded material. In this study, semithin sections of Epon-infiltrated muscle tissue samples have been treated with 1% RR followed by counterstaining with 1% toluidine blue. This 2 step staining procedure has proven to be simple, rapid, and reliable and to give a dramatic improvement in image resolution and contrast. Thus, we believe that histochemical procedures employing RR may find in the future interesting applications for the direct staining of epoxy-resin embedded tissues.     

A new embryonic antigen,p67, present during mouse preimplantation development

An antibody prepared against nullipotential teratocarcinoma stem cells (A-N₁) detects cell surface antigens expressed by early mouse embryos and inhibits in vitro development of embryos in the absence of complement [Calarco and Banka, 1979]. Here we report the immunoprecipitation and electrophoretic characterization of A-N₁-detected antigens from preimplantation mouse embryos. Predominant antibody activity is directed against a 67,000-dalton glycoprotein (p67) with a mean pI of 5.3, which has not been previously described. This protein is not detected, at least as p67, after culture of embryos in tunicamycin. The p67 antigen is also expressed by pluripotential PSA1 teratocarcinoma cells but not by several different differentiated mouse cell types.     

Cell surface proteins during early Xenopus development: analysis of cell surface proteins and total glycoproteins provides evidence for a maternal glycoprotein pool

Summary The populations of cell surface proteins and total glycoproteins were investigated in early Xenopus embryos through lectin staining, affinity binding of glycoproteins to lectins, and use of a succinimide ester to biotinylate cell surface molecules. Lectin staining shows that the egg is endowed with a thick layer of surface glycoprotein, and that glycoprotein is immediately detected on the newly formed membranes of nascent blastomeres. The amount of glycoprotein found in eggs and early embryos remains constant, and electrophoretic analysis reveals no changes in abundant lectin-binding glycoproteins through the neurula stage. In contrast, the amount of cell surface protein increases dramatically from the 2-cell to the gastrula stages. Despite this quantiative increase, only a small number of differences in cell surface proteins were detected during this period. A series of bands was detected which appears to be specific to the outer surface of the embryo. Because the populations of surface proteins and of total glycoproteins overlap to a great extent, the increase in cell surface protein, in the absence of a change in total glycoprotein, indicates the presence of a maternal glycoprotein pool in the Xenopus egg, from which the cell surface proteins of embryonic blastomeres are recruited.     

ACID MUCOPOLYSACCHARIDE (GLYCOSAMINOGLYCAN) AT THE EPITHELIAL-MESENCHYMAL INTERFACE OF MOUSE EMBRYO SALIVARY GLANDS

Acid mucopolysaccharide (glycosaminoglycan) has been demostrated at the epithelial-mesenchymal interface of mouse embryo submandibular glands by (a) specific staining for polymeric sulfate with Alcian blue 8 GX at various magnesium concentrations, (b) specific staining for polymeric uronic acid by selective oxidation of these residues to Schiff-reactive compounds, (c) electron microscope localization of ruthenium red staining, (d) radioautographic localization of glucosamine-³H and ³⁵SO₄, and (e) by susceptibility of the glucosamine radioactivity at the interface to digestion with protease-free hyaluronidase. Moreover, material labeled with glucosamine-³H and ³⁵SO₄ and with chemical characteristics identical with those of acid mucopolysaccharide were isolated from the glands. Acid mucopolysaccharide is distributed over the entire epithelial surface. The amount of acid mucopolysaccharide, as revealed by the staining procedures, is nearly equivalent at all sites. In contrast, the rate of accumulation of glucosamine-labeled mucopolysaccharide is greater at the surface of the distal ends of the growing and branching lobules. This distribution of newly synthesized acid mucopolysaccharide at the sites of incipient cleft formation suggests that surface-associated acid mucopolysaccharide is involved in the morphogenetic process. A mechanism of branching morphogenesis is proposed which accounts for the distribution of collagen fibers and total and newly synthesized acid mucopolysaccharide at the epithelial surface.     

Immunologic detection of retina cognin on the surface of embryonic cells

The retina cell-aggregating glycoprotein, referred to as the retina cognin, has been demonstrated to be located at the surface of embryonic neural retina cells. The term cognin is used to indicate its postulated role in the mechanism of mutual recognition and morphogenetic association of embryonic cells. Antiserum was prepared to the highly purified retina cognin derived from isolated cell membranes of chick embryo retina, and it was used to detect the cognin on cells from chick embryos by means of complement-mediated cell lysis. Retina cells (from 10-day embryos) freshly dissociated with trypsin showed little—if any—lysis by the cognin antiserum; this is consistent with the sensitivity of the cognin to trypsin. However, the cells became susceptible to immunolysis after a period of incubation at 37 °C, which indicates regeneration of the cognin at the cell surface during the recovery period. This regeneration required protein synthesis. Immunofluorescence tests showed binding of the antiserum to the surface of the recovered cells, thereby further demonstrating the surface location of the cognin. The presence, availability or ability to regenerate the cognin, as assayed here, declined sharply with the embryonic age of the cells. Addition of exogenous cognin to freshly trypsin-dissociated retina cells (from 10-day embryos) markedly increased their susceptibility to immunolysis by the cognin antiserum, which indicates that the added cognin becomes associated with the surface of these cells. In contrast, addition of retina cognin to cells freshly trypsinized from 10-day embryo optic tectum and cerebrum, or from 14-day retina did not increase their susceptibility to immunolysis by the cognin antiserum. These results are consistent with earlier findings that enhancement of cell aggregation by the retina cognin is tissue-specific and stage-specific. Cells from non-neural tissues of the chick embryo were not lysed by the retina cognin antiserum. However, neural tissues, such as optic tectum, were found to contain cells which showed surface cross-reaction with the retina cognin antiserum.     

THE ULTRASTRUCTURE AND HISTOPHYSIOLOGY OF HUMAN ECCRINE SWEAT GLANDS

The electron microscopy of human eccrine sweat glands has been studied before and after stimulation by pilocarpine iontophoresis. The identity of the dark and clear cells in the secretory segment as defined by Montagna et al. (23) was determined by studying serial sections, thin for electron microscopy and thick for light microscopy. Cells with numerous apical secretory vacuoles are termed mucoid (dark) cells, since these vacuoles stain positively for acid mucopolysaccharide. Clear cells are intimately associated with intercellular canaliculi. The "cuticular border" of surface cells of the duct is a condensation of tonofilaments and granules. Numerous mitochondria are concentrated in basal cells of the duct. The presence of mucoid cells in the secretory segment may bear on the interpretation of the pathologic findings in the disease cystic fibrosis of the pancreas, and suggests that this disease may be due to a basic disorder of mucopolysaccharide production. The possible roles of the various cellular components in the elaboration of sweat are discussed.