Morpho-functional characterization of haemocytes of the compound ascidian Botrylloides leachi (Tunicata, Ascidiacea)

A morpho‐functional study of the colonial ascidian Botrylloides leachi haemocytes was carried out to propose their classification, relationships and specializations. This characterization was obtained by (i) investigations of both living and aldehyde‐fixed cells by light and electron microscopy; (ii) cytochemical and cytoenzymatic assays; (iii) lectin‐affinity assays; (iv) phagocytosis and haemagglutination assays; and (v) anti‐CD34 immunocytochemical assay for vertebrate haematopoietic stem cells. Results indicate that the haemoblast is a circulating stem cell and there are at least five haemocyte differentiation pathways, the last two of which have never been described in botryllids: (i) phagocytic line (hyaline amoebocytes and macrophage‐like cells) share ultrastructural features, the same hydrolytic enzymes and WGA lectin binding, and are involved in yeast phagocytosis and erythrocyte rosette formation; (ii) cytotoxic line (granular amoebocytes and morula cells) with vacuoles containing oxidative enzymes and polyphenolic substrates; (iii) vacuolated cell line (pigment cells and nephrocytes) involved in catabolite storage; (iv) compartment cell line (compartment amoebocytes and compartment cells) able to agglutinate erythrocytes and characterized by vacuoles with a moderately electron‐dense content, positive to arylsulphatase activity and binding DBA, UEA‐I, HPA lectins; and (v) granular cell line includes trophic cells, able to infiltrate the gut epithelium, showing a cytoplasm filled of PAS‐positive vacuoles with arylsulphatase, chloroacetylesterase and β‐glucuronidase activities.     

Correlative Fine Structural,Behavioral, and Histochemical Analysis of Ascidian Blood Cells

Abstract The blood cells of a solitary ascidian, Halocynthia roretzi, were examined by electron microscopy (EM) with reference to their appearance by light microscopy (LM). In addition, their movement and stainability by vital dyes was observed by phase-contrast microscopy, and their stainability by Giemsa was also examined. Nine cell types were recognized: vacuolated cells, hyaline amoebocytes, small amoebocytes, granular amoebocytes, macrogranular cells, globular cells, lymphocyte-like cells, large basophilic cells and large granular cells. Vacuolated cells were found to possess various numbers of vacuoles containing strongly electron-dense materials and could be divided into at least three subgroups. Granular amoebocytes contained microfilaments and many granules of uniform size. Hyaline amoebocytes and small amoebocytes seemed to be specialized as phagocytes. Macrogranular cells and globular cells were not well characterized. In the blood of adult individuals, hemoblasts were rarely found, although lymphocyte-like cells were present. Each of two large cells, large basophilic cells and large granular cells, possessed novel granules or vacuoles, whose functions remain to be elucidated. The possible functions and relationships of these cells among various ascidian species are discussed.     

The blood cells of the sea squirt, Ciona intestinalis: Morphology,differential counts,and in vitro phagocytic activity

The sea squirt, Ciona intestinalis, contains several types of blood cells: stem cells, hyaline, granular, and refractile amoebocytes, signet ring cells, morula cells, small and large compartment cells, and orange cells. Of these cell types, only the hyaline and granular amoebocytes are capable of phagocytosing formalized sheep erythrocytes in vitro. After the addition of erythrocytes to blood cell monolayers, the attachment and ingestion of these particles occurs rapidly. The interrelationships of the various blood cell types are discussed.     

Amoebocytes in mesoglea of Polypodium hydriforme

Mesogleal amoebocytes of free-living Polypodium hydriforme have been studied with transmission electron microscope. The amoebocytes have numerous processes and contain cytoplasmic vacuoles with fibrous material of different density. The phenomenon of cell death (apoptosis) of mesogleal amoebocytes is described. Chromatin of dying cells becomes condensed forming picnotic "caps" in the nucleus. No mitotic cells were encountered among mesogleal amoebocytes. The origin and functions of mesogleal amoebocytes of P. hydriforme are discussed.     

Ultrastructural studies of regenerating spines of the sea urchin Strongylocentrotus purpuratus. II. Cell types with spherules.

The fine structure of spherulecytes, cell types with large, intracellular membrane-bound vacuoles termed spherules, was investigated in regenerating tips of spines of the sea urchin Strongylocentrotus purpuratus. Two categories of cell types were observed: red spherulecytes and colorless spherulecytes. Red spherulecytes were represented by a single cell type, the eleocyte, while colorless spherulecytes consisted of three morphologically distinct cell types termed morula cells, granulocytes, and vacuolecytes. Eleocytes and morula cells were distributed in both the epidermis and dermis, while granulocytes and vacuolecytes were present only in the dermis. After processing for light and electron microscopy, the spherules of eleocytes typically appeared empty, having lost their content of the red pigment, echinochrome. In contrast, the spherules of morula cell, granulocytes, and vacuolecytes enclosed a variety of granular and other material. The cell types reported in this paper resembled, to various degrees, spherulecytes in the coelomic fluid of echinoids described by other investigators.     

Coelomocyte Morphology in the Holothurians Apostichopus japonicus (Aspidochirota: Stichopodidae) and Cucumaria japonica (Dendrochirota: Cucumariidae)

The cellular composition of the coelomic fluid of the Far Eastern holothurinans Apostichopus japonicus and Cucumaria japonica was studied using light and transmission electron microscopy and histochemistry. In the coelomic fluid of A. japonicus, the following types of coelomocytes were distinguished: progenitor cells; amoebocytes; vacuolated cells; small (or young) morula cells; morula cells of type I, type II, and type III; crystal cells; and vibratile cells. In the coelomic fluid of C. japonicawere found progenitor cells, amoebocytes, vacuolated cells, morula cells of type I and type II, crystal cells, and hemocytes containing a respiratory pigment. The issue of stem cell type, which gives rise to coelomocytes, is discussed.     

Ultrastructures and classification of circulating hemocytes in 9 botryllid ascidians (chordata: ascidiacea)

Ultrastructures of circulating hemocytes were studied in 9 botryllid ascidians. The hemocytes are classified into five types: hemoblasts, phagocytes, granulocytes, morula cells, and pigment cells. These five types are always found in the 9 species. They should represent the major hemocyte types of the circulating cells in the blood. Hemoblasts are small hemocytes having a high nucleus/cytoplasm ratio. There are few granular or vacuolar inclusions in the cytoplasm. Phagocytes have phagocytic activity and their shape is variable depending on the amount of engulfed materials. In granulocytes, shape and size of granules are different among the species. Morula cells are characterized by several vacuoles filled with electron dense materials. In pigment cells, the bulk of the cytoplasm is occupied by one or a few vacuoles containing pigment granules. We also described some other hemocyte types found in particular species. Furthermore, we encountered free oocytes circulating in the blood in two species, Botryllus primigenus and Botrylloides lentus.     

Differences between blood cells of juvenile and adult specimens of the pond snail Lymnaea stagnalis

Summary Blood cells (amoebocytes) of juvenile and adult specimens of the pond snail Lymnaea stagnalis were compared. Juvenile snails contain fewer circulating amoebocytes per l haemolymph. However, a higher percentage of these cells shows mitotic activity, as determined by incorporation of ³H-thymidine in vitro. Relatively more amoebocytes of juvenile snails have the characteristics of less differentiated cells: they are small and round with few inclusions, a high nucleus-to-cytoplasm ratio, and a high pyronin stainability. Enzyme cytochemical studies showed that acid phosphatase (AcP), non-specific esterase (NSE), and alkaline phosphatase (AlP) are present in all amoebocytes of juvenile and adult snails. AcP activity is relatively weak. NSE activity is dispersed throughout the cytoplasm and occasionally found in granules, whereas AlP is clearly localized in granules. Differences between the two age groups were found only for the enzyme peroxidase (PO). In juvenile snails a lower percentage of the cells is positive and the granules that contain the activity are less abundant than in amoebocytes of adults. It is suggested that, due to the above-mentioned characteristics of the amoebocytes, the activity of the internal defence system in juvenile L. stagnalis is on a lower level than that in adult snails. This might be an explanation for the fact that juvenile L. stagnalis are highly susceptible to infection by the schistosome Trichobilharzia ocellata, whereas adult snails are less susceptible.     

Colony specificity and chemotaxis in the compound ascidian Botryllus schlosseri

We re-investigated the behavior of hemocytes during the non-fusion (rejection) reaction between genetically incompatible colonies of the ascidian Botryllus schlosseri. In the course of the reaction, hemocytes - mainly morula cells - crowd inside the blind ends of marginal vascular vessels (known as ampullae) of the colonial leading edge (LE) facing the foreign colony which suggests the occurrence of chemotactic attraction of circulating hemocytes towards the ampullar lumen. Then, cells migrate, through the ampullar tips, into the partially fused tunics and contribute to the formation of the necrotic spots along the contact borders which characterize the reaction. Studies on histological sections clearly indicate that, although morula cell concentration is always higher in ampullae of the LE than in those of the lateral (L) part of the colony, their frequency significantly increases in LE ampullae of rejecting colonies with respect to LE ampullae of both fusing and isolated colonies. In addition, in vitro chemotaxis experiments demonstrated that blood plasma from incompatible colonies can stimulate morula cell migration through polycarbonate filters and this passage is inhibited by antibodies raised against mammalian pro-inflammatory cytokines. The possible nature and role of molecules recognized by anti-cytokine antibodies in hemocyte migration are discussed.     

Quantitative X-ray microanalysis of the morula cell of the blood of the ascidian Halocynthia papillosa (Stolidobranchiata)

Summary The elemental composition of the morula cell of Halocynthia papillosa blood was studied by X-ray microanalysis with respect to the possible iron accumulation in this cell type. We found various amounts of Na, Mg, P, S, Cl, K, Ca, Fe and Br in the cytoplasm, nucleus and vacuoles. With the exception of a few cells, Ca, Fe and Br were not detected. Thus, the morula cells of the studied species are not iron-rich cells.     

The localization of inorganic elements,particularly vanadium and sulphur,in haemolymph from the ascidians Ascidia mentula (Müller) and Ascidiella aspersa (Müller)

Vanadium and sulphur were found by X-ray microanalysis in three different cell types, morula cells, signet-ring cells, and granular amoebocytes, from each of the ascidians Ascidia mentula (Müller) and Ascidiella aspersa (Müller). In addition to the cells containing vanadium and sulphur a few vacuolar cells containing high concentrations of iron, magnesium and calcium were also found. After fixation in the presence of strontium or barium chlorides another cell type containing large amounts of precipitated sulphur was also found, confirming our previous findings that many haemolymph cells in these species contain large amounts of sulphate only. The presence of these various cell types is discussed in relation to the question of the acidity of ascidian blood cells.     

Ultrastructural cytochemistry of the sex pheromone glands of Lutzomyia cruzi male sand flies (Diptera: Psychodidae: Phlebotominae)

The sex pheromone glands of Lutzomyia cruzi male sand flies (Diptera: Psychodidae) were analyzed by cytochemical techniques. In adult males, the epithelium at the fourth abdominal tergite is modified into a glandular epithelium, with large columnar gland cells located side by side. The gland cell cytoplasm contains a large number of mitochondria and peroxisomes, the latter with positive (electron-dense) reaction for catalase, a typical peroxisomal enzyme marker. The gland cell cytoplasm also contains a central vacuolated area, with a large number of electron-lucent vacuoles, not limited by a unit membrane. In well-preserved preparations such vacuoles present a homogenous and slightly electron-dense content, typical of lipid droplets. Indeed, incubation of the tergites with imidazole-buffered osmium tetroxide (to detect lipids) resulted in positive reaction in these vacuoles, as well as in between the microvilli of the gland cells. Use of the osmium–potassium iodide (Os–KI) technique allowed to demonstrate the presence of several endoplasmic reticulum (ER) profiles, as expected in secretory cells. Our data suggest that ER, lipid droplets and peroxisomes are involved in the sand fly pheromone biosynthesis.     

Coelomic Cells in the Connective Tissue Spaces of the Clitellar Epithelium of Lumbricus friendi Cognetti, 1904 (Oligochaeta): an Ultrastructural Study

The ultrastructure of the connective tissue spaces in the clitellar epithelium has been studied in the earthworm Lumbricus friendi. Four morphological types of coelomic cells are described: amoebocytes, mucocyte-like cells, pigment cells and crystal-containing cells. The amoebocytes are characterized by the presence of spherical to oval electron-dense granules, phagocytic vacuoles and numerous microtubules located in the Golgi areas. The mucocyte-like cells show the features of the mucocytes reported in enchytraeid worms (globular inclusions with filamentous and homogeneous, moderately electron-dense material, as well as a filopodous process). The pigment cells contain typical spindle-shaped osmiophilic granules, microtubules (not reported before) and glycogen particles. The crystal-containing cells show inclusions which are polygonal in section with a striated substructure (periodicity of about 4.5 nm). Apart from the mucocyte-like cells, the coelomocytes showed cytoplasmic processes attached to the basement membrane of the spaces. The possible functions of these cells are discussed and a common peritoneal origin is postulated on the base of their morphological and cytological features.     

Hemocytes of Bulinus truncatus rohlfsi (Mollusca: Gastropoda)

Two basic cell types occur in the hemolymph of Bulinus truncatus rohlfsi: granulocytes and hyalinocytes. Granulocytes are divided into three subtypes: (1) Granulocytes I, which account for 19% of the hemocytes, are small, young amoebocytes with 1–20 filopodia and small numbers of cytoplasmic granules, including some lysosomes; (2) granulocytes II, which account for 78% of the cells, are large, fully developed amoebocytes that possess 1–20 filopodia and many granules, both acidophilic and basophilic, including numerous lysosomes, phagosomes, and mitochondria; and (3) spent granulocytes, which are rare, have few filopodia, large accumulations of glycogen granules and prominent vacuoles in addition to lysosomes in the cytoplasm. These three subtypes of granulocytes probably represent ontogenetic stages within a single cell line. In addition, granulocytes with 40 or more filopodia and little ectoplasm, found in only 1 of 45 snails examined, probably reflect a pathologic condition. Hyalinocytes, which account for 3% of all hemocytes, are similar in size to mature granulocytes, but have few or no cytoplasmic granules and lack filopodia and glycogen granules. Total hemocyte concentration in hemolymph is 328,000 ± 188,000 cells/ml.     

Lysosomal Activities of the Vacuole in Damaged and Recovering Plant Cells

ON the evidence from cytochemical studies it seems that a wide range of lysosomal enzymes is present in the vacuole of plant cells^1,2. Cytological studies have also suggested that biochemically identified lysosomal particles are equivalent to small vacuoles and because membranous material has been seen within these vacuoles it was suggested that the plant cell vacuole is functionally equivalent to the secondary lysosome of the animal cell³. In this role it would be engaged in the. breakdown of cytoplasmic constituents and, using light microscope cinematography, it has been shown that after certain experimental environmental changes, structures appeared and then disappeared in the vacuoles of yeast cells⁴. Generally, electron micrographs of plant cells have remnants of disorganized membranes within their vacuoles, which have consequently come to be regarded as repositories of waste materials.     

Identification of the anti-CD3-unresponsive subpopulation of CD4+, CD45RA+ peripheral T lymphocytes.

The majority of peripheral CD4+ T lymphocytes proliferate in vitro in response to anti-CD3 in presence of autologous APC. The present study describes a subpopulation of CD4+ T cells that cannot be activated and progress into cell cycle by stimulation with anti-CD3 plus APC or with mitogenic combinations of anti-CD2. The in vitro responses of these anti-CD3-unresponsive CD4+ T cells were investigated with a panel of mAb to CD2, CD3, and CD28, and found to be similar to those previously observed for mature thymocytes: only the combination of anti-CD2 plus anti-CD28 produced cell proliferation. Anti-CD3-unresponsive T cells were CD45RA+, but represented only 14 to 22% of the CD4+, CD45RA+ T cell population. Activation with anti-CD2 plus anti-CD28 mAb resulted in major changes in the cell surface phenotype and functional properties: a loss of CD45RA+ occurred and an increased expression of CD45RO, CD29, and CD58 (LFA3), as well as a gain in responsiveness to anti-CD3 and anti-CD2. This change in CD45 phenotype from CD45RA to CD45RO occurs in both the anti-CD3-responsive and in the anti-CD3-unresponsive subsets of the CD45RA+, CD4+ cells after cell proliferation. The anti-CD3-unresponsive subset may represent a pool of not yet fully differentiated peripheral T cells. The acquisition of anti-CD3 responsiveness could occur as a consequence of Ag priming or by an Ag-independent mechanism. Involvement of the CD28 Ag in this process is suggested from the present study.     

Long-term in vitro generation of amoebocytes from the Indian horseshoe crab Tachypleus gigas (Müller)

Amoebocyte is the single type of cell circulating in the horseshoe crab hemolymph, which plays a major role in the defense system of the animal. Granules present in these cells are sensitive to nanogram quantities of bacterial endotoxins, which form the basis of the Limulus amoebocyte lysate (LAL) test. Normally, amoebocytes for the production of the LAL are collected by cardiac puncture; hence, development of the in vitro culture system for amoebocytes will reduce the variability of the lysate and help to conserve the 400 million-yr-old living fossil. In the present investigation we have attempted organ culture of gill flaps that have been shown to be the source of amoebocytes. The gill flaps were cultured at 28 degrees C on a rocker platform in a modified L-15 medium supplemented with 10% v/v horseshoe crab serum. This led to the release of amoebocytes outside the gill flaps for a period of 6-8 wk with a more or less steady number of amoebocytes during the weekly harvest. No significant difference was seen in the yield of amoebocytes from male and female horseshoe crabs. Confocal laser microscopy studies revealed significant difference in the size of amoebocytes released in vitro as compared with those obtained in vivo. Thus, we have optimized the culture conditions for the long-term generation of amoebocytes in vitro from the Indian horseshoe crab Tachypleus gigas by reducing the incidence of contamination, simulating in vivo conditions for the organ culture of gill flaps, and improvising the nutritional status using the modified L-15 medium, providing the desired osmolarity and pH.     

Morula cells as the major immunomodulatory hemocytes in ascidians: evidences from the colonial species Botryllus schlosseri

Immunocytochemical methods were used to study the presence and distribution of IL-1-alpha- and TNF-alpha-like molecules in the hemocytes of the colonial ascidian Botryllus schlosseri. Only a few unstimulated hemocytes were positive to both the antibodies used. When the hemocytes were stimulated with either mannan or phorbol 12-mono-myristate, the phagocytes were not significantly changed in their number, staining intensity, or cell morphology. In contrast, stimulated morula cells were intensely labeled, indicating that these cells play an important immunomodulatory role.     

Tight junctions and cavitation in the human pre-embryo.

In the human morula, tight junctions are found between all cell pairs, at all levels of cellular apposition, associated with underlying masses of microfilaments. In cavitating morula, lanthanum tracer gained access to the intracellular spaces, except at the intersections with nascent extracellular cavities, marking the first assembly of zonulae occludentes. Presumptive trophectoderm cells contained vacuoles and larger cavities often associated with secondary lysosome-like bodies. Since the vacuoles and intracellular and extracellular cavities contain electron-dense polygranules of about 23 nm diameter, they may have common origins. In trophectoderm cells of the early blastocytes, the large intracellular vacuoles and cavities were absent, and the zonulae occludentes were located apically. Mechanisms for nascent blastocoele formation are discussed.     

Fine structural changes and lysosomal phosphatase cytochemistry of ameloblasts associated with the transitional stage of enamel formation in the rat incisor.

Trimetaphosphatase (TMPase) and cytidine-5'-monophosphatase (CMPase) were used as lysosomal markers in the transitional ameloblasts (TA) to investigate the distribution of lysosomal structures and to correlate the cytochemical findings with the ultrastructural features of these cells. Of particular interest were the cytochemical and morphological changes which occur as the ameloblasts approach the maturation stage of enamel formation. The sequence of changes observed provides a basis for designation of three regions of the transitional zone (early and late TA and modulating ameloblasts). In the early TA region, the cells decreased in height and contained phagic vacuoles as well as numerous TMPase and CMPase reactive structures. Late transitional ameloblasts had invaginations at their distal ends as well as membrane-bound structures, both filled with fine granular material. Dense bodies, phagic vacuoles, and other elements of the lysosomal system were enzyme reactive. Modulating ameloblasts lacked the phagic vacuoles but exhibited large numbers of multivesicular bodies, vesicles, and secretory granules. Their distal ends were morphologically altered indicating a change towards ruffle- or smooth-ended varieties of maturation ameloblast. In the former, increased granular material was observed within cell membrane invaginations and associated membrane-bound structures. In the latter, intercellular spaces widened and were filled with granular material. The present cytochemical findings of an extensive lyosomal system in transitional ameloblasts confirm the function of those cells in reducing the secretory ameloblast population and in the selective elimination of their protein-synthesizing organelles. Furthermore, this extensive lysosmal system and the present morphological findings are consistent with a potential role for transitional ameloblasts in contributing to the marked loss of enamel protein known to occur during maturation.