Cytochemical and functional characterization of blood and inflammatory cells from the lizard Ameiva ameiva

The fine structure and differential cell count of blood and coelomic exudate leukocytes were studied with the aim to identify granulocytes from Ameiva ameiva, a lizard distributed in the tropical regions of the Americas. Blood leukocytes were separated with a Percoll cushion and coelomic exudate cells were obtained 24 h after intracoelomic thioglycollate injection. In the blood, erythrocytes, monocytes, thrombocytes, lymphocytes, plasma cells and four types of granulocytes were identified based on their morphology and cytochemistry. Types I and III granulocytes had round intracytoplasmic granules with the same basic morphology; however, type III granulocyte had a bilobued nucleus and higher amounts of heterochromatin suggesting an advance stage of maturation. Type II granulocytes had fusiformic granules and more mitochondria. Type IV granulocytes were classified as the basophil mammalian counterpart based on their morphology and relative number. Macrophages and granulocytes type III were found in the normal coelomic cavity. However, after the thioglycollate injection the number of type III granulocyte increased. Granulocytes found in the coelomic cavity were related to type III blood granulocyte based on the morphology and cytochemical localization of alkaline phosphatase and basic proteins in their intracytoplasmic granules. Differential blood leukocyte counts showed a predominance of type III granulocyte followed by lymphocyte, type I granulocyte, type II granulocyte, monocyte and type IV granulocyte. Taken together, these results indicate that types I and III granulocytes correspond to the mammalian neutrophils/heterophils and type II to the eosinophil granulocytes.     

Cytochemical aspects of Mercenaria mercenaria hemocytes.

The hemocytes of the hard clam M. mercenaria were of three types: an agranulocyte, a small, and a large granulocyte. The agranulocyte, with only a thin periphery of cytoplasm surrounding the nucleus, had no visible cytoplasmic granules in living preparations but did exhibit a few centers of nonspecific esterase activity. This cell type represented 2% of the hemocyte population. The small granulocyte possessed four distinct granule types and comprised 61% of the total cell population. Large granulocytes accounted fro 37% of all hemocytes. While they contained the same four granule types identified in the small granulocyte, only one-third the total number were present. The nucleus of all three hemocyte types appeared morphologically similar. The four types of granules observed were a blunt, dot-like, a refractile and a filamentous granule. Blunt granules were identified as mitochondria, based on their ability to reduce Janus Green B to diethyl safranin, the presence of NADH dehydrogenase activity and boundary staining with Sudan black B. Dot-like granules were identified as lysosomes on the basis of neutral red staining, localization of acid phosphatase and nonspecific esterase activity and staining with Sudan black B. Refractile granules were demonstrated to be membrane-bound, lipid-filled structures that reacted positively with Sudan black B and Oil red O, respectively; these granules act as lipid storage centers. Nuclear similarity of the three cell types suggest that these cells might represent different stages of maturity, rather than three distinct cell lines. This was also indicated by the similar yet graded cytochemical reactions and the varying degree of motility and phagocytic activity demonstrated by hemocyte types.     

Granulation staining and cytochemistry of peripheral blood leucocytes in healthy carp (Cyprinus carpio L.) I. Granulocytes

The cytochemistry and staining of granula in peripheral blood granulocytes in healthy carp ( Cyprinus carpio L.) are described. Blood smears were stained for periodic acid-Schiff (PAS), peroxidase, oxidase, alkaline and acid phosphatase, α-naphthyl-acetate esterase, α-naphthyl-butyrate esterase, naphthol-AS-chloroacetate esterase (AS-D), naphthol-AS-acetate esterase and β-glucuronidase. Different granula types were shown by triazid-staining (eosinophil and neutrophil granula) and methylenblue-staining for basophil granulation. Toluidinblue-staining was used for basophil granulocytes. Lipids were shown by the Sudan-black-reaction. Four granulocyte subpopulations are described: neutrophil, heterophil, basophil and eosinophil granulocytes. Neutrophils possess all tested granula types, whereas heterophil and basophil granulocytes show only basophil granula. Neutrophils and heterophils show no activity of the tested esterases with the exception of AS-D. Only neutrophils were peroxidase-positive. Alkaline phosphatase and β-glucuronidase were not detected in granulocytes. Basophils and especially eosinophils were rarely found in peripheral blood.     

Observations on the cytochemistry of the hemocytes of an insect, Galleria mellonella

A number of cytochemical parameters of the hemocytes of larval Galleria mellonella, an insect frequently used as a model by comparative cellular immunologists, are described. Cytochemical methods were used to quantify hemocyte granule-associated components, the results are compared to those obtained for leukocytes from higher animals. Granulocytes contained a population of nonlysosomal granules rich in mucopolysaccharide not seen in plasmatocytes. The numbers and dimensions of these granules showed a positive correlation to cell size, probably reflecting a developmental sequence in granulocyte maturation. Both granulocytes and plasmatocytes had other granules containing the typical lysosomal enzymes, acid phosphatase, beta-glucuronidase, esterase, and lysozyme. The nonlysosomal enzyme alkaline phosphatase was not found in Galleria hemocytes; it is also absent from vertebrate monocytes, macrophages, and immature polymorphonuclear leukocytes. Insect hemocytes appear to lack certain components of antibacterial systems typical of mammalian blood cells, such as H2O2-generating systems, cationic proteins, and myeloperoxidase. The bactericidal mechanisms of hemocytes probably involve lysozyme, as well as other biologically active cellular and humoral factors unique to insects.     

In exocrine pancreas, the basolateral endocytic pathway converges with the autophagic pathway immediately after the early endosome

Intracisternal granules (ICGs) are insoluble aggregates of pancreatic digestive enzymes and proenzymes that develop within the lumen of the rough endoplasmic reticulum of exocrine pancreatic cells, especially in guinea pigs. These ICGs are eliminated by autophagy. By morphological criteria, we identified three distinct and sequential classes of autophagic compartments, which we refer to as phagophores, Type I autophagic vacuoles, and Type II autophagic vacuoles. Lobules of guinea pig pancreas were incubated in media containing HRP for periods of 5-120 min to determine the relationship between the endocytic and autophagic pathways. Incubations with HRP of 15 min or less labeled early endosomes at the cell periphery that were not involved in autophagy of ICGs, but after these short incubations none of the autophagic compartments were HRP positive. After 30-min incubation with HRP, early endosomes at the cell periphery, late endosomes in the pericentriolar region, and, in addition, Type I autophagic vacuoles containing ICGs were all labeled by the tracer. Type II autophagic vacuoles were not labeled after 30-min incubation with HRP but were labeled after incubations of 60-120 min. Phagophores did not receive HRP even after 120 min incubations. We concluded that the autophagic and endocytic pathways converge immediately after the early endosome level and that Type I autophagic vacuoles precede Type II autophagic vacuoles on the endocytic pathway. We studied the distribution of acid phosphatase, lysosomal proteases and cation-independent-mannose-6-phosphate receptor (CI-M6PR) in the three classes of autophagic compartments by histochemical and immunocytochemical methods. Phagophores, the earliest autophagic compartment, contained none of these markers. Type I autophagic vacuoles contained acid phosphatase but, at most, only very low levels of cathepsin D and CI-M6PR. Type II autophagic vacuoles, by contrast, are enriched for acid phosphatase, cathepsin D, and other lysosomal enzymes, and they are also enriched for CI-M6PR. Moreover, soluble fragments of bovine CI-M6PR conjugated to colloidal gold particles heavily labeled Type II but not Type I autophagic vacuoles, and this labeling was specifically blocked by mannose-6-phosphate. This indicates that the lysosomal enzymes present in Type II autophagic vacuoles carry mannose-6-phosphate monoester residues. Using 3-C2, 4-dinitroanilino-3'-amino-N-methyldipropylamine (DAMP), we showed that Type II autophagic vacuoles are acidic. We interpret these findings as indicating that Type II autophagic vacuoles are a prelysosomal compartment in which the already combined endocytic and autophagic pathways meet the delivery pathway of lysosomal enzymes.     

Immunocytochemical and immuno-electron-microscopical study of growth hormone cells in male and female rats of various ages

Summary Growth hormone (GH) secretory cells were identified by immunogold cytochemistry, and were classified on the basis of the size of secretory granules. Type I cells contained large secretory granules (250\2-350 nm in diameter). Type II cells contained the large secretory granules and small secretory granules (100\2-150 nm in diameter). Type III cells contained the small secretory granules. The percentages of each GH cell type changed with aging in male and female rats of the Wistar/Tw strain. Type I cells predominated throughout development; the proportion of type I cell was highest at 6 months of age, and decreased thereafter. The proportion of type II and type III cells decreased from 1 month to 6 months of age, but then increased at 12 and 18 months of age. The pituitary content of GH was highest at 6 months of age, and decreased thereafter. Estrogen and androgen, which are known to affect GH secretion, caused changes in the proportion of each GH cell type. The results suggest that when GH secretion is more active the proportion of type I GH cell increased, and when GH secretion is less active the proportion of type II and type III cells increased. The type III GH cell may therefore be an immature type of GH cell, and the type I cell the mature type of GH cell. Type II cells may be intermediate between type I and III cells.     

SEQUENTIAL DEGRANULATION OF THE TWO TYPES OF POLYMORPHONUCLEAR LEUKOCYTE GRANULES DURING PHAGOCYTOSIS OF MICROORGANISMS

The sequential discharge of neutrophilic polymorphonuclear leukocyte (PMN) granules—azurophils and specifics—was investigated by electron microscopy and cytochemistry. Thus the enzyme content of PMN phagocytic vacuoles was determined at brief intervals after phagocytosis of bacteria, utilizing peroxidase as a marker enzyme for azurophil granules, and alkaline phosphatase for specifics. At 30 s, approximately half the phagocytic vacuoles were reactive for alkaline phosphatase, whereas none contained peroxidase. Peroxidase-containing vacuoles were rarely seen at 1 min, but by 3 min, vacuoles containing both enzymes were consistently present. Alkaline phosphatase was found in both small and large vacuoles, whereas peroxidase was visible only in large ones. By 10 min, very big phagocytic vacuoles containing considerable amounts of reaction product for both enzymes were evident. These observations indicate that the two types of PMN granules discharge in a sequential manner, specific granules fusing with the vacuole before azurophils. In an earlier paper, we reported that the pH of phagocytic vacuoles drops to 6.5 within 3 min and to ~4 within 7–15 min. Substances known to be present in specific granules (alkaline phosphatase, lysozyme, and lactoferrin) function best at neutral or alkaline pH, whereas most of those contained in azurophil granules (i.e., peroxidase and the lysosomal enzymes) have pH optima in the acid range. Hence the sequence of granule discharge roughly parallels the change in pH, thereby providing optimal conditions for coordinated activity of granule contents.     

A cytochemical, light and electron microscopical study of the leucocytes of the adult river lamprey, Lampetra fluviatilis (L. Gray)

The morphological features of the leucocytes from the blood of the river lamprey, Lampetra fluvimilis , were studied using light and electron microscopy. Four cell types were identified, namely granulocytes, lymphocytes, monocytes and thrombocytes. Enzyme cytochemical tests were also performed for the detection of acid phosphatase, β-glucuronidase and peroxidase. All the leucocyte types were positive for acid phosphatase and β-glucuronidase, to a variable extent, with the greatest activity seen in the granulocytes. None of the leucocyte types however, contained any peroxidase activity.
Only one type of granulocyte was found and this appears to be analogous to the mammalian neutrophil/heterophil. Characteristically, it has a cytoplasm containing a large number of morphologically heterogeneous granules (0.07–0.40 um in diameter). It is suggested that these granules, rather than belonging to several subpopulations, are in fact part of a single maturation series.
The results of this study show that precise identification of lamprey leucocytes can only be achieved using a combination of ultrastructural and cytochemical techniques.     

THE DEVELOPMENT OF PIGMENT GRANULES IN THE EYES OF WILD TYPE AND MUTANT DROSOPHILA MELANOGASTER

The eye pigment system in Drosophila melanogaster has been studied with the electron microscope. Details in the development of pigment granules in wild type flies and in three eye color mutants are described. Four different types of pigment granules have been found. Type I granules, which carry ommochrome pigment and occur in both primary and secondary pigment cells of ommatidia, are believed to develop as vesicular secretions by way of the Golgi apparatus. The formation of Type II granules, which are restricted to the secondary pigment cells and contain drosopterin pigments, involves accumulation of 60- to 80-A fibers producing an elliptical granule. Type III granules appear to be empty vesicles, except for small marginal areas of dense material; they are thought to be abnormal entities containing ommochrome pigment. Type IV granules are characteristic of colorless mutants regardless of genotype, and during the course of development they often contain glycogen, ribosomes, and show acid phosphatase activity; for these reasons and because of their bizarre and variable morphology, they are considered to be autophagic vacuoles. The 300-A particles commonly found in pigment cells are identified as glycogen on the basis of their morphology and their sensitivity to salivary digestion.     

Characterization of gelatinase from pig polymorphonuclear leucocytes. A metalloproteinase resembling tumour type IV collagenase.

The metalloproteinase 'gelatinase' stored in the granules of pig polymorphonuclear leucocytes has been purified in the latent form. The enzyme is secreted as an Mr 97,000 proenzyme that can be activated in the presence of 4-aminophenylmercuric acetate (APMA) by self-cleavage to generate lower-Mr species, of which an Mr 88,000 form was the most active. Trypsin-initiated activation generated different Mr gelatinases of much lower specific activity. Activation was slowed but not prevented by the presence of the tissue inhibitor of metalloproteinases, TIMP. The activated gelatinase formed a stable complex (Mr 144,000) with TIMP, in a Zn2+- and Ca2+-dependent manner, and complex formation was inhibited by the presence of the substrate gelatin. Similar to the human granulocyte gelatinase, the organomercurial-activated pig enzyme degraded gelatin and TCA and TCB fragments of type I collagen, as well as elastin and types IV and V collagen. The degradation of type IV collagen was shown, both by polyacrylamide-gel electrophoresis and by electron microscopic analysis, to generate 3/4 and 1/4 fragments as described for mouse tumour type IV collagenase. Furthermore, an antiserum raised to mouse type IV collagenase recognized the pig granulocyte gelatinase. An antiserum to the pig polymorphonuclear leucocyte gelatinase recognized other high-Mr gelatinases, including those from human granulocytes, pig monocytes and rabbit connective tissue cells, but not the Mr 72,000 enzyme from connective tissue cells. These data suggest that there are two distinct major forms of gelatinolytic activity that also cause specific cleavage of type IV collagen. These enzymes are associated with a wide variety of normal connective tissue and haemopoietic cells, as well as many tumour cells.     

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.     

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.     

DIFFERENTIATION OF MONOCYTES : Origin, Nature, and Fate of Their Azurophil Granules

The origin, content, and fate of azurophil granules of blood monocytes were investigated in several species (rabbit, guinea pig, human) by electron microscopy and cytochemistry. The life cycle of monocytes consists of maturation in bone marrow, transit in blood, and migration into tissues where they function as macrophages. Cells were examined from all three phases. It was found that: azurophil granules originate in the Golgi complex of the developing monocyte of bone marrow and blood, and ultimately fuse with phagosomes during phagocytosis upon arrival of monocytes in the tissues. They contain lysosomal enzymes in all species studied and peroxidase in the guinea pig and human. These enzymes are produced by the same pathway as other secretory products (i.e., they are segregated in the rough ER and packaged into granules in the Golgi complex). The findings demonstrate that the azurophil granules of monocytes are primary lysosomes or storage granules comparable to the azurophils of polymorphonuclear leukocytes and the specific granules of eosinophils. Macrophages from peritoneal exudates (72–96 hr after endotoxin injection) contain large quantities of lysosomal enzymes throughout the secretory apparatus (rough ER and Golgi complex), in digestive vacuoles, and in numerous coated vesicles; however, they lack forming or mature azurophil granules. Hence it appears that the monocyte produces two types of primary lysosomes during different phases of its life cycle—azurophil granules made by developing monocytes in bone marrow or blood, and coated vesicles made by macrophages in tissues and body cavities.     

Morphology and ultrastructure of the earthworm Dendrobaena veneta (Lumbricidae) coelomocytes

Microscope techniques, light microscope (LM), transmission electron microscope (TEM), scanning electron microscope (SEM) were employed to describe and classify coelomocytes of the oligochaete Dendrobaena veneta. Three main cell types were distinguished in the coelomic fluid: eleocytes, amoebocytes and granulocytes. Eleocytes are large, oval cells containing characteristic granules called chloragosomes. Amoebocytes are most numerous coelomocytes and have been divided into two types (I and II). Both amoebocytes of the types I and II often form aggregations of a few to about a dozen cells. Granulocytes are oval cells with spherical nuclei and cytoplasm containing polymorphic, electron dense granules. Contrary to the amoebocytes, the granulocytes do not form aggregations. Morphology and ultrastructure of coelomocytes are presented on micrographs: similarities and differences are compared to coelomocytes of related species.     

Estrogen receptor in rabbit endocervical cells isolated by velocity sedimentation

Three nonciliated rabbit endocervical epithelial cell types were isolated by velocity sedimentation at unit gravity. Cell Types I and II contained heterogeneous secretory granules, whereas Type III cells were characterized by empty cytoplasmic vacuoles. Previous studies had confirmed the ultrastructural and functional integrity of each cell type and suggested hormone dependence of cell population sizes. Since the concentration of total cytosol estrogen receptor in endocervical epithelial cells from progesterone-dominated, 5-day pseudopregnant rabbits was reduced to approximately 23% of the value for estrous rabbits, receptor concentrations were measured in the three nonciliated epithelial cell types from animals in both hormonal states. The results indicate that the reduction in the estrogen receptor content in progesterone-dominated animals can be attributed to a reduction in the number of Type I and Type II cells, and to a significant (P less than 0.05) reduction in their estrogen receptor concentrations.     

Granulation staining and cytochemistry of peripheral blood leucocytes in healthy carp (Cyprinus carpio L.) II: Monocytes

Granulation staining and cytochemistry of peripheral blood monocytes in healthy carp ( Cyprinus carpio L.) are described. Blood smears were stained for periodic acid-Schiff (PAS), peroxidase, oxidase, alkaline and acid phosphatase, α-naphthyl acetate esterase, α-naphthyl-butyrate esterase, naphthol-AS-chloroacetate esterase (AS-D), naphthol-AS-acetate esterase and β-glucuronidase. For representation of different granulations triazide-staining for eosinophil and neutrophil granules and aqueous methylere-blue staining for basophil granules were used. Lipids were shown by sudan-black-reaction. Monocytes showed only basophil granulation and weak lipid reaction. All tested enzymes were detected, with the exception of peroxidase. The PAS reaction for glycogen proof was negative.     

Intravascular granuloma induced by intravenous inoculation ofCryptococcus neoformans

In rodents an intravenous administration of viableCryptococcus (C.) neoformans cells frequently resulted in attachment of intravascular cryptococcal granulomas to inner walls of the large to medium-sized veins of various organs, including the lungs, liver and spleen. In order to elucidate the pathogenesis of granulomatous changes, the cells composing the intravascular granulomas were observed by electron microscopic peroxidase (PO) cytochemistry. The granuloma composing cells could be divided into the following four types according to the pattern of endogenous peroxidase activity: exudate macrophage (Mφ, type I), PO-negative Mφ (type II), resident Mφ (type III) and other inflammatory cells (type IV). In the intravenous granulomas of the lung, the percentages of composed cells were 39.0% for type I, 57.9% for type II, 0% for type III and 3.1% for type IV. By contrast, in the interstitial granulomas in the lung, type III Mφs, possibly derived from alveolar Mφs, played a significant role in granuloma formation. This may indicate that the intravascular granuloma is almost composed of macrophages derived from monocytes rather than alveolar macrophages. The expression of ICAM-1 on endothelia of the pulmonary veins was examined by immunoelectron microscopy. An immunogold labeling index was significantly augmented on the surface of endothelia in response to intravenous challenge ofC. neoformans. The intravascular granuloma demonstrates that the monocytes develop into the granuloma-composing macrophages and suppress the cryptococcal activities even hi the peripheral blood resulting in an assistance of endothelial functions.     

Composition and ultrastructure of elasmobranch granulocytes. II. Rays (Rajiformes)

The blood granulocyte composition of seven species of ray is given together with ultrastructural observations made on the epigonal organ and blood of Pavoraja spinifera and the spleen of a deepwater rajid skate. Under the light microscope three granulocyte types, eosinophils, eosinophilic granulocytes and neutrophilic granulocytes could be distinguished. At the EM level two granulocyte types were apparent, one with elongated granules containing longitudinal fibrils that consolidated to form an axial rod-like inclusion, and the other with large, spherical, uniformly electron-dense granules. Correlation of light and electron microscope observations indicated that the neutrophilic granulocytes with weakly basophilic, elongated granules become weakly eosinophilic, as eosinophilic granulocytes, and these in turn develop to eosinophils with granules containing axial rods. The other granulocyte type forms another population of eosinophils with spherical granules.
The inter-relationship of these granulocytes, the identification of eosinophilic granulocytes, or heterophils, as immature eosinophils, and the co-existence of two morphologically distinct eosinophil forms are discussed.     

QUANTITATIVE CHARACTERIZATION OF DENSE BODY, AUTOPHAGIC VACUOLE, AND ACID PHOSPHATASE-BEARING PARTICLE POPULATIONS DURING THE EARLY PHASES OF GLUCAGON-INDUCED AUTOPHAGY IN RAT LIVER

Quantitative characterization of dense body, autophagic vacuole and acid phosphatase-bearing particle populations of rat liver have been made at 10 min intervals during the first 50 min following the intraperitoneal administration of glucagon. Beginning 10 to 20 min postinjection, increases in the number of autophagic vacuoles and in the osmotic sensitivity of acid phosphatase-bearing particles were observed, associated with a progressive disappearance of dense bodies. These changes appeared to reach a maximum 50 min after treatment. The average volume of autophagic vacuoles was found to be 440–870% greater than that of normal dense bodies during this time period. No consistent change in total acid phosphatase activity was noted. A detailed study of autophagic vacuole profile populations revealed the presence of five different types of profiles, two of which, types I and II, accounted for 76.3–94.4% of the profiles examined. Type I profiles primarily contained elements of the endoplasmic reticulum, free ribosomes, and ground cytoplasm. Type II profiles had mitochondrial profiles as their principal constituent, but endoplasmic reticulum and free ribosomes were also seen. At all time points type I profiles predominated, comprising 55–69% of the profiles found. Both profile types were bounded by single and double limiting membranes, the former being predominate. A time-dependent change in the ratio of single to double membrane-limited profiles could not be demonstrated. Morphometric parameters derived from profile size distributions indicated that the number of types I and II autophagic vacuoles increased with time, the rate being greater for the type II particle, except between 40 and 50 min. The average volume of the type II autophagic vacuole was consistently greater than that of the type I.     

Ontogeny of the endocrine pancreas in sea bass (Dicentrarchus labrax): an ultrastructural study. II. The big and secondary islets

The big and secondary islets of sea bass larvae were characterized ultrastructurally from, 25 to 60 days after hatching. From the 25th day, big islets consisted of inner type II and III, external type I and peripheral type IV cells. From the 55th day, type V cells appeared in limited peripheral areas. Secondary islets, first found in 32-day-old larvae, were made up of inner type II and III, external type I, and peripheral either type IV and V cells (type I islets), or only type V cells (type II islets). Type I cells contained secretory granules with a fine granular, low-medium electron-dense material, whereas the secretory granules of type II cells were smaller and had a high electron-dense core with diffused limits; needle and rod-like crystalloid contents were occasionally found. Type III secretory granules posessed a homogeneous, high or medium electron-dense material with or without a clear halo. Type IV cells had secretory granules with a polygonal dense core embedded in a granular matrix and granules containing a high or medium electron-dense material. Type V cells had secretory granules with a fine granular, high or medium electron-dense content. These cell-types correlated with cells previously identified immuno-cytochemically, as regards to their distribution in the islets, and related to those characterized ultrastructurally in adult specimens. Thus, types I, II, III, IV and V correspond to D₁, B, D₂, A and PP cells, respectively. From the 32nd day onwards, endocrine cells of all the different types were found grouped, type V cells also being observed in isolation close to pancreatic ducts and/or blood vessels. Small groups consisting of type I and II cells were found in 40-day-old larvae. A mitotic centroacinar ductular cell containing some secretory granules similar to those of type I cells, was seen adjacent to a type I cell. As the larvae grew older, the endoplasmic reticulum developed, the number of free ribosomes decreased, and the number and size of the secretory granules increased. Dark type I, II, III, IV and V cells were found in the islets and cell clusters from the 55th day onwards.