The amoebocytic corpuscles in the circulating fluid of the lamelli-branches, Indonaia caerulia and Parreysia (Parreysia) favidens.

The circulating amoebocytes of the freshwater mussels, Indonaia caerulia and Parreysia favidens have been studied. The total number of amoebocytes is low and variable, and the individual to individual variation in total counts is particularly significant. The amoebocytes are of 3 main types: Acidophils, Large Basophils and Small Basophils. Acidophils contain large acidophilic granules and basophils fine basophilic granules within the cytoplasm. Acidophils are the largest and small basophils the smallest. The greater volume of the cell is occupied by cytoplasm in acidophils and large basophils and by the nucleus in small basophils. Large basophils are the most and small basophils the least abundant. Amoebocytes clump together by means of filipodia. Large basophils possess these protoplasmic processes in the greatest abundance, and partake most actively and in largest numbers in clumping; while small basophils lack filipodia and play an absolutely passive role. Clumping progressively becomes more extensive with passage of time following cardiac injury inflicted during blood collection. I. caerulia appears to be generally more efficient than P. favidens with regard to clumping reaction. The different structural, numerical and behavioural aspects of the amoebocytes have been considered with reference to their probable causative factors and functional import.     

Possible sequential transformation of a series of acidophils in the pituitary autografts in the renal capsule of male rats in association with ACTH cell.

In our electron microscopy, acidiphils in the pituitary autografts placed in the renal capsules of immature male rats underwent a sequential transformation with the lapse of time: Within 3 and 6 days, all the somaotrophs packed with the large granules of about 350 mmu diameter dispersed. The size and number of the granules in somatotrophs were quickly and markedly reduced with severe modification of cell shape. There was evidence during this time course that Siperstein's or Moriarty's corticotrophs might be synonymous with the stellate shape of acidophils with the arrangement of small granules 150-200 mmu in diameter along the cell acidophils. The "acidophils of the small granule type" possibly related to ACTH production according to Yoshimura et al. (1974) were frequently detected in the grafts as elongated or irregularly shaped cells. Their minute granules 100-150 mmu in diameter were also distributed in row in the cytoplasmic peripheral area. Gradual loss of the minute granules below 100 mmu in diameter eventually made the acidophils to transform into agranular cells. Our own idea that ACTH secretion might correlate with a series of cells transforming along the acidophil-axis was indirectly supported by the present observation on pituitary grafts. On the other hand, basophils rapidly degenerated and died away. Ten and 20 days after autografting, the graft cells which might be principally composed of the cells of acidophil origin enormously proliferated through mitotic division, showing the homologous fine structure, without the normal cell individuality. They always contained three different size and shape of granules simulataneously. Significance of such a rapid and strong response of acidophils to the ectopic replacement in the immature male rats was discussed from the view-point of hypothalamic regulation to simple protein hormones.     

Cytochemical observations of coelomocytes from the earthworm, Lumbricus terrestris.

Synopsis Coelomocytes of the earthworm,Lumbricus terrestris, were stained by cytochemical techniques to determine the biochemical composition of the seven different cell types and subtypes. The enzymes acid phosphatase and -glucuronidase are present in all types of coelomocytes, but are especially abundant in basophils and neutrophils; the differences in enzyme amounts correlate well with the differences in phagocytic activity of the various cell types. No peroxidase is present. The cytoplasmic basophilia of basophils is due primarily to ribonucleic acid. Basophils also contain large deposits of glycogen, with neutrophils and chloragogen cells containing somewhat lesser amounts. The predominant granules of the two types of acidophils and of granulocytes are composed of a basic protein and a neutral mucopolysaccharide or glycoprotein. A second granule population, present in low numbers in acidophils and granulocytes, but in larger numbers in basophils and neutrophils, is small in size and lipid-positive and may, in part, represent lysosomes.Lipid is especially abundant in the vesicles and granules of the two types of chloragogen cells. Some granules of chloragogen cells also contain ferrous and ferric iron and a substance with pseudoperoxidase activity. The cytoplasm contains protein, glycogen, and a neutral mucopolysaccharide. In addition, acid mucopolysaccharides are variably present in the cytoplasm of chloragogen cells, the only coelomocytes to contain this class of substances.     

Seasonal changes in the pituitary gland of the feral hawaiian mongoose (Herpestes auropunctatus)

The Hawaiian mongoose (Herpestes auropunctatus) is a seasonally breeding mammal whose pituitary gland resembles that of other Viverridae. Certain features, such as a prominent pars tuberalis interna and a double-layered pars intermedia forming a cup for the neurohypophysis, are unique. With the light microscope, five different cell types can be recognized in the pars distalis after staining with periodic acid-Schiff (PAS)-orange G. Two types of acidophils are seen, a small yellow-staining cell and a large angular orange cell. Two basophilic cells are also seen, one with fine PAS-positive cytoplasmic granules and the other with coarse PAS-positive granules in the cytoplasm. The last cell type seen is the chromophobe. Differential cell counts indicated an altered distribution of chromophils in the ventral pars distalis of the female mongoose with changing season and reproductive status, but the most striking change was a decreased percentage of basophils in the pars distalis during the nonbreeding season.     

The pituitary gland of the coelacanth fish Latimeria chalumnae Smith: General structure and adenohypophysial cell types

The pituitary gland of Latimeria chalumnae is situated rostroventral to the telencephalon. The hollow pituitary stalk is bent forward and is ventrally connected to a saccus-vasculosus-like organ, rostrally to a neurointermediate lobe. The infundibular lumen protrudes far into the neurohypophysial lobules. The elongated principal part (pars cerebralis) of the pars distalis is partly embedded in a dorsal depression of the pars intermedia and caudally invaded by the neurohypophysis. It may be divided into rostral and proximal pars distalis and includes a ramified hypophysial cleft, which continues rostrally as a duct with adjacent islets of pars distalis tissue (parts of a pars buccalis). The adenohypophysis consists of cell cords and follicles. Eight tinctorial cell types can be distinguished: in the rostral islets: large basophils with acidophil globules, in the rostral pars distalis: small basophils, large basophils with amphiphil characters and erythrosin-, orange G-positive acidophils; in the proximal pars distalis: orange G-positive acidophils and small and large basophils, having similar staining properties; in the pars intermedia: one amphiphil cell type.     

Cytodifferentiation of the adenohypophysis of the domestic fowl

Summary In the chick embryo the first membrane-bound secretory granules occur in the cytoplasm of occasional cells in the cephalic lobe of pars distalis at the 7th day of incubation. On the 8th day most of the cells in both the cephalic and caudal lobes contain secretory granules that are variable in size, form and density.On the 9th day at least two types of glandular cells are distinguishable in the cephalic and in the caudal lobes; however, these cells are not comparable with those of the adult gland. Differentiation of acidophils and basophils occurs, apparently simultaneously, in 11-day embryos.The cells of the cephalic and caudal lobes are morphologically distinct from their first appearance. Thus it is concluded that these two lobes develop independently and differently from an early stage of ontogenesis.The secretory granules are formed in the Golgi area of the hypophysial cells after the 8th day of incubation. However, secretory material may be synthesized also by a process not involving the Golgi apparatus.Nerve fibers containing granules first appear in the superficial layer of the median eminence on the 8th embryonic day and by the 12th day three types of granules and two types of clear vesicles are identifiable.The investigation reported herein was supported by grant from Japan-U.S. Cooperative Science Program of Japan Association for Science Promotion to Professor Mikami and by U.S.-Japan Cooperative Science Program Grant No. GF-33334 to Professor Farner.     

Haemocytes of the cockle Cerastoderma glaucum: morphological characterisation and involvement in immune responses

For the first time, morpho-functional characterisation of haemocytes from the cockle Cerastoderma glaucum was performed to identify circulating cell types and to study their involvement in immune responses. Haemocyte mean number was 5.5 (x 10(5)) cells/mL haemolymph. Two main haemocyte types were found in haemolymph: granulocytes (85%), about 10 microm in diameter and with evident cytoplasmic granules, and hyalinocytes (15%), 8 to 14 microm in diameter, with a few or no granules. Most of the cytoplasmic granules stained in vivo with Neutral Red, indicating that they were lysosomes. On the basis of haemocyte staining properties, granulocytes and hyalinocytes were further classified as basophils and acidophils. Acidophil hyalinocytes were the largest haemocyte type (about 14 microm in diameter) and had an eccentric nucleus and a large cytoplasmic vacuole. Both granulocytes and hyalinocytes (except acidophils) were able to phagocytise yeast cells, although the basal phagocytic index was very low (about 2%). It increased significantly (up to 26%) after pre-incubation of yeast in cell-free haemolymph, suggesting that haemolymph has opsonising properties. Haemocytes also produced superoxide anion. Moreover, both granulocytes and hyalinocytes (except acidophils) were positive for some important hydrolytic and oxidative enzymes. Lysozyme-like activity was recorded in both cell-free haemolymph and haemocyte lysate, although enzyme activity in cell lysate was significantly higher. Results indicate that haemocytes from C. glaucum are effective cells in immune responses.     

The coelomocytes of the earthworm Lumbricus terrestris: morphology and phagocytic properties.

The coelomocytes of Lumbricus terrestris have been classified and described, based on Wright's stained preparations and on living cells. The five major categories consist of basophils, acidophils, neutrophils, granulocytes and chloragogen cells. Both the acidophil and chloragogen cell groups contain two subgroups. Granulocytes also exhibit heterogeneity with respect to staining properties of granules. Some possess acidophilic granules, some basophilic granules, and others contain both types. Granules of acidophils have been observed to be occasionally excreted from the cells. All cell types, with the exception of chloragogen cells, produce pseudopodia and are capable of phagocytosis, a vital component of the earthworm's immune response.     

Development of Haemogregarina boueti in the toad Bufo regularis*

SYNOPSIS. Haemogregarina boueti França, 1910, was found to be the commonest blood parasite in the common toad, Bufo regularis Reuss, in Egypt. The rate of infection was about 30% (of 689 toads examined). In properly fixed blood films, the parasites were almost exclusively intraerythrocytic. Most characteristic was the encapsulated “elongate” form averaging 22.3 by 6 μ with a more-or-less central nucleus and a pointed, slightly bent, posterior end. Infected red cells were conspicuously hypertrophied and their nuclei were markedly displaced and frequently broken into 2-4 parts. Young and growing blood forms as well as two types of hepatic schizonts are described for the first time. Schizonts of the first type develop in hepatic cells, are 28–30 μ in diameter and produce numerous elongate oval merozoites about 8 × 2.2 μ radially arranged around a residual body about 10 μ in diameter. Schizonts of the second type start their growth in erythrocytes but later complete their development as free bodies in the liver sinusoids. When mature, they are 32–35 μ in diameter and produce a larger number of thin merozoites about 8 × 1.5 μ, surrounding a larger residual body about 19 μ in diameter.     

The fine structure of the granular amebocytes of the Pacific oyster, Crassostrea gigas

Oyster granular amebocytes were characterized by electron microscopy to be of two types. Acidophiles contained relatively small, spherical, amorphous, osmiophilic cytoplasmic granules with a maximum dimension of 0.4–0.56 μ. Basophiles contained large, cytoplasmic granules, approximately 0.7–1.2 μ in diameter. The granules resembled hollow, spherical structures in which an outer membrane and a layer of closely associated granular material enclosed an electron-transparent interior.     

Evidence for perforin-like activity associated with earthworm leukocytes

Earthworm (Eisenia fetida) coelomic fluid contains several leukocytes (coelomocytes): basophils, acidophils and neutrophils as well as chloragocytes. Small coelomocytes and coelomocyte lysate are cytotoxic for the tumor cell target K562. The expression of a lytic factor was investigated by immunocytochemistry using light and transmission electron microscopy. A rat-anti-mouse-perforin-mAb labeled mainly small coelomocytes (nearly 20%) as visualized by light microscopy. TEM analysis using immunogold showed a homogenous labeling in the cytoplasm of small coelomocytes. The highest number of immunogold particles was estimated in coelomocytes with many small cytoplasmic granules. Coelomocytes with large lysosomal granules were also labeled but less intensely. No antibody binding was observed for chloragocytes either in light or electron microscopy. This suggests that the perforin-like activity is associated with only one cell type and that chloragocytes are responsible for other lytic activities. MALDI-MS revealed calreticulin usually associated with perforin in mammalian cells that mediate lysis (e.g. NK, CTL). Together, results strongly suggest the presence of putative perforin in earthworms. This in turn supports the hypothesis that perforin is a conserved component important in immune defense during evolution.     

Eimeria cicaki sp. n. and Isospora thavari sp. n. from the House Lizard Gehyra mutilata Boulenger in Malaysia*

SYNOPSIS. Oocysts and endogenous stages of new species of Eimeria and Isospora from the house lizard, Gehyra mutilata, are described. The ellipsoid to subspherical 2-layered oocysts of E. cicaki averaged 24.0 × 21.0 μm. Polar granules are present. Micropyle and oocyst residuum are absent. Ellipsoid sporocysts average 12.2 × 9.0 μm. A sporocyst residuum is present, but the Stieda body is absent. Endogenous stages are in epithelial cells of the small intestine. The subspherical single-layered oocysts of I. thavari average 23.8 × 22.8 μm. The polar granule is present; micropyle and oocyst residuum are absent. Ellipsoid sporocysts average 12.8 × 9.4 μm. Stieda body and sporocyst residuum are present. There are endogenous stages in epithelial cells of the small intestine.     

Origin of prolactin cells in the pituitary autografts in the renal capsule of young male and female rats.

Origin of the prolactin cells in the pituitary grafts autotransplanted into the renal capsule was electron microscopically investigated in young male and female rats. Prolactin cells may mainly originate from the completely degranulated acidophils after grafting in both sexes. The comprehensive degranulation of acidophils may be essential condition for the development of prolactin cells. Formation of initial prolactin granules is related to the Golgi apparatus in 3 and 6 days in the males. Even prolonged transplantation for the duration of 10 and 20 days failed to develop the typical prolactin cells storing large polymorphic granules in the grafts in the males. In the females, however, the population of prolactin cells became higher (20-30%) and their granulation was more rapidly advanced than in the male. Nine and 16 days after grafting quite numerous typical prolactin cells, characterized by the pronounced vesiculation of ER stored large polymorphic granules in the females. Progressive vesiculation of ER in degranulated acidophils may be the second necessary condition for establishing the prolactin cells. Thus, the entire course of cell conversion of acidophils into prolactin cells was observed in this study. All the graft cells including prolactin cells finally turned into the cells losing the respective individuality (10 days in the males; 32 days in the females). This fine structural similarity was striking in the males. We could not, however, deny the possibility of origination of prolactin cells from the follicular cells or their adjoining undifferentiated cells.     

Trans-epidermal Transport and Storage of Calcium in Holthuisana transversa (Brachyura; Sundathelphusidae) During Premoult

Holthuisana transversa reabsorbs much of its exoskeletal calcium in the last 3 days before ecdysis and stores it in circulating granules in the haemocoel and in non-circulating granules in the subepidermal connective tissue. Calcium enters the epidermal cells from the moulting fluid, probably through their apical microvilli and is either incorporated into intracellular calcium granules or exits the cell via the basolateral membranes to be used in formation of two other granule types. Intracellular granules (0.4–2 μm long) form in large masses in the apical cytoplasm of the epidermal cells. They are formed as membrane-bound vesicles by the Golgi, and calcium and organic matrix material are added from the surrounding cytoplasm. As development proceeds, lamellae appear and calcium carbonate is deposited in the matrix. Granule masses move basally and are stored in the connective tissue. Calcium is also incorporated into extracellular large granules (0.8–3.8 μm long) which are formed in narrow intercellular channels between epidermal cells. A third granule type (small granules, 0.26 μm diameter) is formed in subepidermal connective tissue cells and released into the haemolymph in very large numbers. Calcium was identified in the two larger granule types using X-ray microanalysis and significant amounts of phosphorus and potassium were also present in the large granules. A model for ion cycling between the exoskeleton and granules is presented.     

Contribution to the exodermis in the rootlets ofFraxinus excelsior L.

The exodermis of ash roots is initiated early in the apical meristem. When fully differentiated, it is composed of alternating “long” and “short” cells measuring approx. 70×25×25 μm and 25×28×25 μm respectively. At a short distance from the apex, the long cells undergo structural and histochemical changes from a “primary” towards a “secondary” stage: an impermeable suberized lamella is formed, cellulose lamellae become impregnated by lignin, and the protoplast dies off. The short cells show a distinctly thickened outer wall (“cap”) which is composed exclusively of cellulose, and possess abundant cytoplasm with a large nucleus. In the process of ageing the structure of short cells is not markedly affected. Following the early disintegration of rhizodermis, the exodermis works as the external protective layer of tho fragile end-root. Its long cells fulfil the mechanical protection, the short cells serve as passage cells for solutions and gases, during the whole life-span of the rootlet.     

Ultrastructural and cytochemical observations on the granulocytes of the sturgeon, Acipenser brevirostrum (Chondrostei)

Granulocytes from cranial granulopoietic tissue were studied under the electron microscope, and cytochemistry carried out oncranial and peripheral blood granulocytes of two sturgeons, Acipenser brevirostrum . Ultrastructurally, eosinophils and basophils had homogeneous electron-dense granules similar to those of teleosts and some higher vertebrates. Neutrophils contained two granule types: small elongated fibrillar granules and large (<3.8μm long) usually homogeneous granules.
Neutrophil fibrillar granules were positive for alkaline phosphatase (ALP), acid phosphatase (ACP), α-naphthyl acetate esterase (ANAE), acetyl-l-tyrosine-α-naphthyl esterase (ATNE) and periodic acid Schiff (PAS) reaction. The large homogeneous granules were negative for all enzymes, and were only PAS positive. Eosinophils had granular, cyanide-, azide- and aminotriazole-resistant peroxidase (PO) and were ACP, ATNE, tosyl-l-lysine-α-naphthyl esterase (TLNE) and Luxol fast blue positive.
Ultrastructure and cytochemistry are discussed in relation to other vertebrates, and eosinophils identified as the main phagocytic leucocyte.     

Haemogregarina faiyumensis n. sp. in the Toad Bufo regularis in Egypt*

SYNOPSIS. Haemogregarina faiyumensis n. sp., a parasite of toads, Bufo regularis, in Faiyuni Province, Egypt, U.A.R., is described. In a survey of 689 toads from various localities in Cairo, Giza and Faiyurn provinces, only 3 out of 13 toads from Kom O Shim near Faiyum were infected. This species, known only by blood forms (most probably gametocytes) of two different staining reactions, is 13-17 μ long and 4-5 μ wide, with an average of 15.5 × 4.5 μ. The nucleus is typically subcircular and 3-5 × 3–5 μ, with an average of 4.5 × 3.9 μ.     

Morphological and histochemical studies on a PAS-positive granular leukocyte in blood and connective tissues of Catostomus commersonii Lacépède (teleostei:pisces).

This study was undertaken to identify an ubiquitous granular leukocyte found in Catostomus commersonni Lacépède. The cell contains large, numerous, strongly PAS-positive cytoplasmic granules, an eccentric nucleus and prominent, persistent juxtanuclear space. It develops in the hemopoietic tissue of the kidney, and mature cells are found not only in kidney and peripheral blood but also in areas of connective tissue where mast cells are usually located. Electron microscopy confirms the presence of a large Golgi apparatus, unlamellated cytoplasmic granules and extensive rough-surfaced endoplasmic reticulum. Histochemical studies show that the cytoplasmic granules are alcianophobic, non-metachromatic and unstained by acridine orange. Histamine is detectable spectrophotometrically in kidney tissue, but the PAS-positive granular leukocyte does not consistently degranulate after treatment with histamine liberator 48/80. The authors suggest that while the PAS-positive granular leukocyte is not identical with classical basophils/mast cells, which are absent in C. commersonnii, it may represent an evolutionary precursor of these cells.     

Light and electron microscopic investigations of six types of glandular cells of the bovine adenohypophysis

Summary Twelve bovine adenohypophyses were prepared for light and electron microscopy of the cell types of pars distalis. Correlation between the light and electron microscopy was effected by use of alternate thin and thick sections. Cytological changes in the experimental animals were used as criteria for the identification of six different types of secretory cells.Two types of acidophils, alpha and epsilon cells, are recognized in peripheral area of the pars distalis by light and electron microscopy. The alpha cells contain orangeophilic secretory granules of a maximum diameter of 400–450 m and correspond to ordinary acidophils (STH cells). The second type, epsilon cells, contains larger, fuchsinophilic granules of 600 to 900 m in diameter, increase in number and granulation after pregnancy and thyroidectomy, and are thought to be prolactin cells (LTH cells).Two types of amphophils, zeta and delta 1 cells, were found in the central area of the pars distalis. The zeta cells contain smaller numbers of amphophilic, cored granules (200 m maximum diameter) and based on the comparison with literature on other species of animals, are designated as ACTH cells. The delta 1 cells are round or oval and contain very dense, spherical granules (250–300 m) which are stained red or reddish purple with PAS, aldehyde thionin and PAS-methyl blue methods. They show extreme enlargement and bizarre cytoplasmic appearance after castration and are designated tentatively as LH gonadotrophs or LH cells.Two types of basophils, beta and delta 2 cells, were also identified by correlative light and electron microscopy. The beta cells are polygonal in outline, distributed exclusively in the zona tuberalis and contain large, less dense secretory granules (300–400 m) which are stained selectively with Gomori's aldehyde fuchsin. After thyroidectomy, they lose their secretory granules and are transformed into large, vacuolated thyroidectomy cells. They are therefore, identified as thyrotrophs or TSH cells. The delta 2 cells are round, oval or polygonal in shape and contain basophilic granules ranging from 220 to 300 m in diameter. They show extreme enlargement and vacuolization due to the dilation of endoplasmic reticulum, after castration, and are designated tentatively as FSH gonadotrophs or FSH cells.The investigation reported herein was supported by a Scientific Research Grant (No. 291049) from the Ministry of Education of Japan.     

Ultrastructure of the bone marrow of the salamander Plethodon glutinosus (Caudata: Plethodontidae)

The unusual lymphogranulopoietic bone marrow of the large lungless salamander Plethodon glutinosus was examined by light and electron microscopy. Developing neutrophils, eosinophils, and fat cells were found in large numbers, while lymphocytes of various sizes, plasma cells, plasmablasts, macrophages, pigment cells, and fibroblasts were present in more moderate numbers. Basophils were observed only rarely. Macrophages were found in extravascular locations and did not appear to be associated directly with the walls of the blood vessels supplying the marrow. Both neutrophils and eosinophils seemed to arise from small precursor cells whose ultrastructural features bore a resemblance in some ways to those of mammalian myeloblasts described by Bainton and Farquhar ('66). Developing neutrophils and eosinophils seemed to produce only single populations of specific cytoplasmic granules, rather than both primary (azurophilic) and secondary (specific) inclusions, as are produced typically by mammalian granulocytes. Both eosinophilic and neutrophilic granules were formed in association with Golgi complexes; and eosinophilic granules were much larger, more densely stained, and more regularly rounded in shape than the inclusions of developing neutrophils. Peroxidase activity was associated with the specific granules of neutrophils but seemed to be lacking in the granules of eosinophils. The specific granules of eosinophils were especially unusual because they contained irregularly shaped, lightly stained cores which occasionally displayed a distinctly crystalline substructural organization. The specific granules of basophils also possessed a prominent crystalline organization. The overall appearance of the marrow of Plethodon suggests that it functions not only as a valuable source of neutrophils, eosinophils, and cells of the lymphoid series, but also as a part of the phagocytic system of the animals and as an important repository for fat.