Light and electron microscopy of the leucocytes of Crassostrea virginica (Mollusca: Pelecypoda)

Summary The fine structure of oyster leucocytes resembles to a great extent, that of typical eucaryotic cells. Organelles which have been described for the first time in this report are light granules, dense granules, protocentriole and X structure. Light microscopy reveals two morphological types of oyster leucocytes: agranular and granular. Based upon nuclear morphology and cytoplasmic compositions revealed in electron microscopy, at least three types of agranular and one type of granular cells are recognized.In the Giemsa-stained preparations, granular leucocytes exhibit three distinct types of cytoplasmic granules: refractile, dark blue, and pink, which presumably correspond to light granules Type A, B, and C seen in the electron micrographs. A granular leucocyte may contain one or more types of granules. Cytochemical investigations show that oyster leucocytes contain at least three hydrolytic enzymes: non-specific esterases, acid, and alkaline phosphatase. The latter two enzymes constitute 63% of the enzyme activity detected. These intracellular enzymes may be associated with the light granules and/or lysosome-like bodies.It is also demonstrated that the granular leucocyte population is significantly higher (P<0.001) in the oysters experimentally infected with Bacillus mycoides (72.19±4.71%) as contrasted with that of the controls (37.18±4.48%).Leucocytes in progressive stages of degeneration are also described.Contribution No. 71 from Marine Research Laboratory, University of Connecticut.The initial phase of this investigation was carried out at the Department of Zoology, Rutgers, The State University, New Brunswick, New Jersey, and supported by Public Health Service Research Grant AI-00781 from the National Institute of Allergy and Infectious Diseases of the National Institute of Health, awarded to Dr. L. A. Stauber. Supported by a grant from the University of Connecticut Research Foundation and Faculty Summer Fellowship to S. Y. Feng.     

Comparative analysis of circulating hemocytes of the freshwater snail Pomacea canaliculata

Molluscs are invertebrates of great relevance for economy, environment and public health. The numerous studies on molluscan immunity and physiology registered an impressive variability of circulating hemocytes. This study is focused on the first characterization of the circulating hemocytes of the freshwater gastropod Pomacea canaliculata, a model for several eco-toxicological and parasitological researches.Flow cytometry analysis identified two populations of hemocytes on the basis of differences in size and internal organization. The first population contains small and agranular cells. The second one displays major size and a more articulated internal organization. Light microscopy evidenced two principal morphologies, categorized as Group I (small) and II (large) hemocytes. Group I hemocytes present the characteristics of blast-like cells, with an agranular and basophilic cytoplasm. Group I hemocytes can adhere onto a glass surface but seem unable to phagocytize heat-inactivated Escherichia coli. The majority of Group II hemocytes displays an agranular cytoplasm, while a minority presents numerous granules. Agranular cytoplasm may be basophilic or acidophilic. Granules are positive to neutral red staining and therefore acidic. Independently from their morphology, Group II hemocytes are able to adhere and to engulf heat-inactivated E. coli. Transmission electron microscopy analysis clearly distinguished between agranular and granular hemocytes and highlighted the electron dense content of the granules. After hemolymph collection, time-course analysis indicated that the Group II hemocytes are subjected to an evident dynamism with changes in the percentage of agranular and granular hemocytes. The ability of circulating hemocytes to quickly modify their morphology and stainability suggests that P. canaliculata is endowed with highly dynamic hemocyte populations able to cope with rapid environmental changes as well as fast growing pathogens.     

The ultrastructure of the innervation of the intestinal wall in the teleosts Myoxocephalus scorpius and Pleuronectes platessa

Summary The innervation of the intestinal wall in the teleosts Myoxocephalus and Pleuronectes was examined electron microscopically. Two classes of axons can be identified. The first, which is in the majority, contains numerous 50–150 nm granular vesicles as well as some 40–50 nm agranular vesicles while the second contains predominantly the 40–50 nm agranular vesicles. Chromate/dichromate staining methods suggest that the first type is aminergic. Both types lie in close association with the perikarya of intrinsic myenteric neurons but only axons containing predominantly agranular vesicles have synaptic membrane specialisations. No axon bundles pass into the longitudinal muscle layer in Myoxocephalus gut and though some do in Pleuronectes, they do not closely approach the smooth muscle cells. Axons containing large granular vesicles lie in intimate contact with the myocytes of the circular muscle layer. Both axon types pass through the submucosa to form a plexus underneath the mucosal epithelium. Varicosities containing agranular or granular vesicles are separated from the epithelial cells by a gap of about 200 nm in which lies a basal lamina.     

On the ultrastructure of follicles and isolated follicular granulosa cells of porcine ovary

Summary The endoplasmic reticulum in granulosa cells of primary, secondary, and small tertiary follicles of the porcine ovary is sparse and largely of the granular type.In granulosa cells of large tertiary follicles the endoplasmic reticulum shows distinct signs of proliferation. Some cells even contain whorls of endoplasmic reticulum membranes, essentially of the agranular variety.Direct continuity between endoplasmic reticulum membranes of the granular and agranular type as well as the continuous increase in agranular membranes suggest that these membranes may originate from the granular membranes.Granulosa cells isolated from large tertiary follicles by microdissection and keptin vitro show essentially the same ultrastructure as granulosa cells of intact large tertiary follicles.Some lipid droplets appear to be localized in cavities of the endoplasmic reticulum. It is suggested that the droplets contain precursor material for steroid hormone synthesis.Finally, the development of the agranular endoplasmic reticulum including the appearance of whorls in some granulosa cells of large tertiary follicles indicates that steroid synthesis may occur in such follicular granulosa cells.Read at the Meeting of the Swedish Society for Pathology in Umeå, September 25, 1965 (Bjersing, 1966).This investigation was supported by grants from the Swedish Medical Research Council (Projects No. 13 X-78-01, 12 X-78-02, and 12 X-78-03).     

Morphological changes in gland cells and axons resulting from stimulation of the salivary nerves of the Cockroach, Nauphoeta cinerea

The salivary glands of the cockroach, Nauphoeta cinerea (Olivier, 1789), are innervated and there is considerable evidence to suggest that dopamine is the neurotransmitter at the neuroglandular junction. As the gland is a bilaterally symmetrical structure it was possible to electrically stimulate the salivary nerve supplying the ipsilateral side of the gland whilst the contralateral side of the gland served as a convenient control. Saliva elicited from the glands by electrical stimulation of these nerves was collected and used to monitor the physiological state of the tissue. Glands were fixed for light and electron microscopy during secretion and it was observed that the ductules in peripheral acinar cells were distended in stimulated sides of the glands but not in contralateral unstimulated sides. This evidence implies that peripheral cells are responsible for the initiation of salivary fluid secretion. Changes were also observed in the catecholamine containing axons that innervate the glands. In stimulated axons a statistically significant reduction in numbers of small agranular vesicles was observed when compared with contralateral unstimulated controls and freshly fixed tissue. This was not the case with the larger granular vesicles of the same axons which showed no reduction in number as a result of stimulation. In addition it was also noted that the small agranular vesicles tended to aggregate and change their shapes in response to nerve stimulation. These results imply that the small agranular vesicles play a role in transmitter release.     

中华绒螯蟹血细胞的显微、亚显微形态结构及其分类

通过相差显微镜和电镜观察,根据中华绒螯蟹血细胞胞质内有无颗粒以及颗粒大小、染色反应、折光性和形成方式的特点,血细胞分为胞质内无颗粒的无颗粒细胞、胞质内只有具折光性和呈淡红色反应大颗粒的大颗粒细胞、胞质内只有无折光性和呈淡蓝色染色反应小颗粒的小颗粒细胞以及胞质内同时具有大颗粒和小颗粒二种颗粒特性的大小颗粒中间型细胞.小颗粒的形成方式是高尔基体成熟面小泡脱离后直接成为小颗粒,而大颗粒的形成方式是高尔基体成熟面小泡脱离后,数个小泡逐渐聚集成蜂窝状大颗粒,进一步发育成熟为均质大颗粒.实验结果表明:三种有颗粒的细胞是互相独立的,可能分别由无颗粒细胞分化而成.       

Ultrastructure of salivary glands of Ornithodoros (Ornithodoros) moubata (Ixodoidea: Argasidae)

The paired salivary glands of unfed adult Ornithodoros (Ornithodoros) moubata are composed of type I (agranular) and type II (granular) alveoli. Type I alveoli consis of one large central cell surrounded by peripheral cells having the morphology of fluid-transporting epithelia. Type II alveoli contain granular and agranular cells; the former are comprised of morphologically distinct types of cells (a, b, and c) containing granules of different structures and chemical composition with respect to polysaccharide and protein. The agranular cells are the interstitial and cap cells. Golgi bodies and rough endoplasmic reticulum (RER) are found in all granular cells and apparently are involved in granule formation. No appreciable structural changes were observed in type I alveoli during or after feeding. Type c cell granules are released before granules from types a and b cells and may contain anticoagulant substances that promote the blood flow of the host during the tick feeding. Although the cap cells are not structurally affected by feeding, interstitial cells are developed into transporting epithelia.     

Structural and functional characterisation of the blood cells of the bivalve mollusc,Scrobicularia plana

Light and electron microscopical studies were carried out in order to characterise the blood cells of the bivalve mollusc, Scrobicularia plana. Three types of haemocytes were recognised: eosinophilic granular haemocytes, basophilic granular haemocytes and basophilic agranular haemocytes. The eosinophilic granulocytes were vesicular and contained large granules whereas the basophilic granulocytes were found to contain small granules and glycogen 'lakes'. The basophilic agranular haemocytes were significantly smaller than the granular haemocytes and had a high nucleus to cytoplasm ratio. Functional characterisation of the blood cells identified activity for the lysosomal enzymes: acid phosphatase, beta-glucuronidase, non-specific esterase and arylsulphatase. There was also a weak staining reaction for phenoloxidase and peroxidase activities. Phagocytosis of Gram-positive bacteria was demonstrated by the haemocytes and antibacterial activity was shown by cell-free haemolymph. Assays to determine release of reactive oxygen species from the haemocytes did not detect any reactive oxygen generation.     

Fine-structural study of leucocytes in the goldfish, Carassius auratus

An electron microscopic study was performed on leucocytes from circulating blood of the goldfish. The leucocytes were divided into eight types: neutrophil, eosinophil, large granular leucocyte (LGL), medium-sized granular leucocyte (MGL), small granular leucocyte (SGL), fine granular leucocyte (FGL), lymphocyte, and monocyte. In this report the thrombocyte was excluded from leucocytes, and LGL, MGL, SGL and FGL were tentatively classified based on the size of intracytoplasmic granules possessed by each cell. The existence of goldfish monocytes was electron microscopically demonstrated for the first time in the present report.     

Microscopical studies on the hemocytes of bivalves and their phagocytic interaction with selected bacteria

Hemocytes represent one of the most important defense mechanisms against foreign material in Mollusca. The morphology, hematological parameters and behaviour of hemolymph cells were studied in the southern quahogMercenaria campechiensis, the eastern oysterCrassostrea virginica, and the blood arkAnadara ovalis challenged with the bacteriaVibrio vulnificus andV. anguillarum. Two general classes of hemocytes (granular and agranular) exist inC. virginica andM. campechiensis. In contrast,A. ovalis possesses 3 general classes (granular, agranular and erythrocytes). Three types of granules were identified by light microscopy. When hemolymph cells were studied by transmission electron microscopy, the cytoplasm of hemolymph cells was noted to contain many organelles, including electron dense granules. Both agranular and granular hemolymph cells were capable of colchicine-sensitive pseudopodial movement and spreading. The results indicate that marine bivalves possess hemolymph blood cells which may play a role in the internal defense paralleling mammalian phagocytes. The morphology of these cells, as determined by light, scanning and transmission electron microscopy, showed some similarity to mammalian-mononuclear phagocytes. The sub-cellular events of molluscan hemocyte phagocytosis ofV. vulnificus andV. anguillarum were studied by both scanning and transmission electron microscopy. The role of these cells and the factors which govern their behavior are of economic and public health importance.     

Acidification of the phagosome in Crassostrea virginica hemocytes following engulfment of zymosan

Phagocytic hemocytes are responsible for engulfing and internally degrading foreign organisms within the hemolymph and tissue of the eastern oyster, Crassostrea virginica. Since rapid acidification of the phagosome lumen is typically essential for activation of hydrolytic and reactive oxygen intermediate (ROI) producing enzymes in vertebrate cells, we measured phagosomal pH in oyster hemocytes by using the emission fluorescence of two fluorescent probes, rhodamine and Oregon Green 488 (OG 488), conjugated to zymosan to determine whether oyster hemocyte phagosomes become acidified after phagocytosis of zymosan. The average pH of 1079 phagosomes within 277 hemocytes 1 h after phagocytosis of zymosan was 3.9 +/- 0.03. Observations of 141 hemocytes with internalized zymosan by light microscopy revealed that, over a 60-min time period, 51% of highly granular hemocytes became partially granular, and 29% became agranular. In addition, 83% of partially granular hemocytes containing zymosan at time = 0 became agranular within 60 min. A comparison revealed that the phagosomes of agranular hemocytes were much more acidic (pH 3.1 +/- 0.02) than those of highly granular hemocytes (4.9 +/- 0.02; P < 0.05). These values are significantly lower than most reported in the literature for blood cells from metazoan organisms.     

Salivary gland ultrastructure of the unfed adult and feeding female of Haemaphysalis (Rhipistoma) leachi (Ixodoidea: Ixodidae)

The paired salivary glands of unfed adult Haemaphysalis (Rhipistoma) leachi contain one type of agranular and three types of granular alveoli connected to a salivary duct system. Type I agranular alveoli consist of one large, central cell surrounded by peripheral cells with numerous basal membrane infoldings indicative of epithelia involved in fluid transport. Glycogen particles, lipid-like droplets, and the parallel pattern of infolded membranes disappeared from the peripheral cells during feeding. Types II, III, and IV granular alveoli contain some agranular interstitial epithelial cells, cap cells, and fundus cells, but are predominantly composed of structurally different granular cell types a, b, c, d, e, and f. Agranular cells develop during the early stages of feeding. Granular a, c, e, and f cells release their granules directly after attachment to the host and possibly are involved in cement secretion required for firm attachment to it. The b cell granules are replaced by b₁ filamentous granules during feeding. Golgi bodies and rough endoplasmic reticulum (RER) participate in the formation of most types of granules. The d cells contain lamella-like structures and condensing vacuoles, probably responsible for lysosome formation. The main salivary duct and all types of alveoli are innervated by neurosecretory axons.     

ULTRASTRUCTURAL OBSERVATIONS OF VITELLOGENESIS IN THE SPIDER CRAB, LIBINIA EMARGINATA L

Ovaries from the spider crab, Libinia emarginata L. were studied to learn more of vitellogenesis in crustaceans. Oogonia and previtellogenic oocytes were found in the core of the ovaries. Vitellogenic oocytes are located more peripherally. Profiles of the endoplasmic reticulum are abundant in the vitellogenic oocytes. The granular and agranular reticulum as well as the Golgi complex are active in yolk synthesis. As vitellogenesis proceeds, yolk precursors are incorporated into the egg by micropinocytosis at the egg surface. Thus, in Libinia, yolk materials appear to be derived from both intra- and extraoocytic sources.     

Altered Ontogenesis of Muscarinic Receptors in Agranular Cerebellar Cortex

Abstract: The developmental pattern, the agonist binding properties and the cellular origin(s) of muscarinic binding sites were investigated in agranular cerebellum of x-irradiated rats, of Gunn rats with hereditary hyperbilirubinemia, and of staggerer mutant mice. The density of muscarinic binding sites was found to be higher than normal in all of these cerebellar types, indicating that granular neurons do not greatly contribute to binding of acetylcholine in the rodent cerebellum. The total number of muscarinic binding sites as measured by binding of [³H]4NMPB remains unchanged in the agranular cerebellum of x-irradiated rats. However, the number of muscarinic sites is reduced by about 30% in the agranular cerebellum of homozygous Gunn rats (jj), in which fibrous astrocytes and Purkinje cells are also damaged. In the cerebellum of staggerer mice (sg/sg), where a cascade of events leads to massive damage to mossy fibers and Golgi cells in addition to granular neurons and Purkinje cells, the content of muscarinic receptors is reduced by 50%. Thus, the number of muscarinic binding sites in the rodent cerebellum seems to depend on the integrity of the additional cell types and cellular elements, damaged in these agranular models. The ontogenetic variations in the affinity of cerebellar muscarinic sites for binding of carbamylcholine in normal and Gunn rat cerebellum were compared with those observed in x-irradiated and staggerer cerebellum, where elimination of granular neurons induces the formation of ‘heterologous’ synapses. Muscarinic binding affinity increases 10-fold during postnatal development in the cerebellum of normal and Gunn rats. In the immature x-irradiated cerebellum, the affinity of muscarinic binding sites was found to be nearly as high as that detected in the adult normal cerebellum. In contrast, cerebella of 5-month-old staggerer mice display 5-fold lower affinity than their normal counterpart values, as low as that determined in normal immature cerebellum. The characteristic ontogenetic pattern of muscarink binding is therefore indicated to be related to the formation of correct circuitry, but not to the presence of granular neurons, in the developing rat cerebellum.     

Agranular membranes in free polysome preparations and their possible interference in studies of protein biosynthesis

1. Phospholipid-rich membranous contaminants are present in free polysomes from rat liver isolated on discontinuous sucrose gradients. 2. Electron-microscopic studies indicate that the membranous contaminants are mainly agranular with very occasional granular membranes. This is confirmed by the study of their sedimentation behaviour and their initial rate of labelling with radioactive glucosamine in vivo. 3. Conventional ribosome-purification methods fail to remove the contaminants, whereas deoxycholate effectively solubilizes the membranous contaminants with little breakdown of polysomes. 4. Amino acid-incorporation studies show that these membranous contaminants may seriously interfere in assessment of the functional capacity of free polysomes in protein biosynthesis in vivo.     

Ultrastructural evidence for exocytosis in the median eminence of the rat

Summary Exocytosis has been demonstrated by electron microscopy in the external zone of the median eminence of the rat. Exocytotic profiles have been observed in nerve fibres characterized by the presence of granular vesicles with median diameters of 90–103 nm and agranular vesicles of about 50 nm. In addition to the small agranular vesicles, coated vesicles of the same size have been found in many nerve fibres, suggesting that at least part of the agranular vesicles in the median eminence originate by micro-pinocytosis. The nature of the fibres showing exocytosis is discussed. Attention is drawn to the possibility of identifying types of fibres in the median eminence by the occurrence of exocytosis.The technical assistance of Mrs. R. M. Y. Hartsteen is acknowledged with gratitude. We would like to thank Prof. J. Moll for his helpful criticism. We also thank Miss P. Delfos and Mr. W. van den Oudenalder for photographic assistance.     

Interaction of molluscs and foreign substances: The morphology and behavior of hemolymph cells of the American oyster, Crassostrea virginica, in vitro

Hemolymph cells of Crassostrea virginica have been studied in the living state with apochromatic and brightfield-phase contrast microscopy and in the fixed and stained state with brightfield and Nomarski optics. Cells of two size populations have been recognized. The large cells are of two classes: granulocytes and fibrocytes. Granulocytes commonly include mixtures of acidophilic, basophilic, and refractile granules and are believed to be of one general type. Fibrocytes are subcategorized as being of the primary and secondary types, with the first enclosing lobate nuclei and the second enclosing spherical or ovoid nuclei. The small cells, designated as hyalinocytes, are either agranular or slightly granular.     

AN ANALYSIS OF COLLAGEN SECRETION BY ESTABLISHED MOUSE FIBROBLAST LINES

In vitro synthesis of collagen by established mouse fibroblast lines has been examined by electron microscopy. During rapid growth (log phase), when collagen could not be detected in the cultures, the cells lacked a well developed granular ergastoplasm and Golgi system. Upon cessation of growth (stationary phase), collagen accumulated in the cultures and the cells demonstrated highly developed granular and smooth ergastoplasm. Collagen appeared to be synthesized in the rough-surfaced endoplasmic reticulum and to be transported as a soluble protein to the cell surface by vesicular elements of the agranular ergastoplasm. Fusion of the limiting membranes of these vesicles with the cell membrane permitted the discharge of the soluble collagen into the extracellular space, where fibrils of two diameter distributions formed. The secretion of collagen is concluded to be of the merocrine type. Alternative theories of collagen secretion are discussed and the data for established lines compared with the results of other in vitro and in vivo studies of collagen fibrillogenesis.     

Fine structural changes in Sertoli and Leydig cells during the reproductive cycle of the ground squirrel, Citellus lateralis

During spermatogenesis in sexually mature ground squirrels Leydig and Sertoli cells were morphologically well differentiated. For Leydig cells the most prominent organelles were lipid droplets, mitochondria with tubulo-vesicular cristae and abundant agranular reticulum organized as a mass of anastomosing tubules. These morphological criteria suggest that the Leydig cells were steroidogenically active. Sertoli cells exhibited a topographical distribution of certain organelles with basal regions containing stacks of granular reticulum, and large areas of agranular reticulum. The cytoplasm surrounding maturing germ cells contained numerous microtubules, and an adluminal layer of spermatids at a certain stage of spermiogenesis became enveloped by Sertoli cytoplasm containing an enormous proliferation of agranular reticulum. The presence of these organelles in Sertoli cells suggests that during spermatogenesis they are active in the synthesis of proteins and steroids. In particular the mass of agranular reticulum surrounding late stage spermatids indicates that steroids may be required for spermatid maturation and/or spermiation. By contrast Leydig and Sertoli cells observed during testicular regression, when only spermatogonia remain in the seminiferous tubules, had undergone structural changes. Leydig cells were still numerous and large with abundant agranular reticulum that was now organized as a loose assemblage of single unbranched tubules. Sertoli cells were drastically reduced in both cytoplasmic volume and content of organelles.     

Neurotensin immunoreactivity in the central nucleus of the rat amygdala: an ultrastructural approach

Neurotensin (NT) was demonstrated in the central nucleus of the rat amygdala (CNA) using a modification of the avidin-biotin complex immunohistochemical technique. Electron-dense reaction product (particles were 15-25 nm in diameter) was localized in perikarya, dendrites, axons, and axon terminals. It was found also associated with profiles of rough endoplasmic reticulum, mitochondria, microtubules, and small agranular as well as large granular vesicles. In distal dendrites, the reaction product was associated with microtubules, vesicles, and postsynaptic densities. Axon terminals of three types formed synaptic contracts with NT-immunoreactive neurons in the CNA: one was characterized by numerous round or oval agranular vesicles, the second by numerous pleomorphic vesicles, and the third by agranular vesicles that were loosely distributed and pleomorphic. All three types formed symmetric axosomatic and asymmetric axodendritic contacts. NT-immunoreactive axon terminals containing small round agranular vesicles stood out clearly from the intermingling profiles of immunonegative structures. We found numerous glomeruli, each consisting of a central NT-immunoreactive dendrite surrounded by all three types of axon terminals. We observed that some NT-immunoreactive terminals formed symmetric axoaxonal contacts with each other, providing evidence for the presence of local NT-to-NT circuits, whereas many others synapsed with axon terminals devoid of NT immunoreactivity.