Peroxidase and tyrosinase are present in secondary lysosomes that degrade photosensory membranes of the crayfish photoreceptor: possible role in pigment granule formation.

Three enzymes (acid phosphatase, peroxidase, and tyrosinase) were localized by electron microscopy within the retina of crayfish Orconectes limosus. Peroxidase activity was observed only in lamellar bodies, which are secondary lysosomes and degrade photosensory membrane. After H2O2 was omitted from the reaction medium, peroxidase activity in lamellar bodies was partly inhibited but was not missing completely. After addition of sodium pyruvate, which inhibits endogenous generation of H2O2, staining of lamellar bodies was absent. Tyrosinase activity was found in lamellar bodies and in small vesicles within the rhabdoms similar to those found positive for acid phosphatase. Granules (500-700 nm in diameter) with an electron opaque matrix and mature screening pigment granules showed tyrosinase activity. Moreover, lamellar structures within membrane-bound organelles that additionally contained screening pigment-like granules were electron dense because of tyrosinase activity. After addition of phenylthiourea (PTU) to the incubation medium, lamellar bodies did not generally contain electron dense deposits, although weak staining of single membranes still was sometimes observed. After addition of sodium pyruvate in combination with PTU, no staining was detected. The possible role of tyrosinase in ommochrome synthesis within secondary lysosomes that degrade photosensory membrane is discussed.     

Atypical Granules in the Eyes of the White Mutant of Drosophila melanogaster Are Lysosome-Related Organelles

In the pigment cells of the white mutant of Drosophila melanogaster, as described earlier, two types of abnormal granules are found by conventional electron microscopy. However, both types of abnormal granules, in addition to those in pigment cell invaginations, are also present in the cytoplasm of the photoreceptor cells. Three enzymes (acid phosphatase, peroxidase, and tyrosinase) are localized within the eyes of wild type and white mutant Drosophila melanogaster by electron microscopy. Peroxidase activity is present in lamellar bodies close to the rhabdomeral microvilli of both fly types. However the organelles containing peroxidase activity are 6-fold more frequent in the wild type than in the mutant. Acid phosphatase is present in lamellar bodies between and at the bases of the rhabdomeral microvilli of the wild type, as well as in ommochrome granules of the photoreceptor cells. In the white mutant, however, acid phosphatase was located in electron lucent vacuoles in the cytoplasm of the receptor cells. These acid phosphatase-positive vacuoles also contained both types of abnormal granules. The latter result indicates that abnormal granules in the receptor cells originate from lysosomal degradation and that targeting of lysosomal enzymes is altered in the white mutant. Due to the tyrosinase activity in the hemolymph of flies, the extracellular spaces are electron dense after DOPA incubation. Since some abnormal granules within the photoreceptor cells are not surrounded by an extracellular space, they can be assumed to originate within the photoreceptor cells.     

Herpetomonas samuelpessoai: electron microscopy and cytochemistry of electron-dense granules.

Herpetomonas samuelpessoai has membrane-bound electrondense granules in its cytoplasm. The electron density independs on postfixation with osmium tetroxide and is enhanced by uranyl acetate staining. The granules contain iron, have basic proteins cytochemically detected by the silver ammoniacal method, and have a peroxidase activity as detected by the diaminobenzidine method. Some of the granules also have acid phosphatase activity. It is suggested that the granules may represent either lysosomes or a storage form of tetrapyrrole derivatives which are essential for the growth and metabolism of most Trypanosomatidae.     

Further evidence for synthesis of screening pigment granules involved in the photosensory membrane turnover of the crayfish photoreceptor

Photosensory membrane degradation in crayfish occurs at first in multi-vesicular bodies (MVBs) and then, with the aid of lysosomal enzymes, in lysosome related lamellar bodies. In organ culture experiments with the isolated crayfish retina (Orconectes limosus) small screening pigment-like granules became visible under the electron microscope in such lamellar bodies and suggested a possible relation of photosensory membrane degradation and screening pigment granule synthesis. Chloroquine, an inhibitor of lysosomal activity, when added to the culture medium reduced the appearance of screening pigment-like granules in lamellar bodies, but led to the appearance of these granules in mature MVB's, indicating the involvement of lysosomal enzymes in the formation of pigmented lamellar bodies. In a second set of experiments the effect of bright light on the screening pigment granule ultrastructure of crayfish phoreceptors was investigated. It was found that after bright light exposure large numbers of little screening pigment granules (0.15-0.3 microns) were located between or close to rhabdomeral microvilli that were not at these sites in crayfish kept under natural light. MVB's were also reduced in size, and among the little screening pigmentary organelles granules of different electron density and morphology appeared. Additionally, vesicle flux to little screening pigment granules was detected. The screening pigment granules of the little type did not seem to be transported close to or between the microvilli, but appeared to be synthesized at these sites within little MVBs.     

Cytochemical demonstration of amine-storing vacuoles and lysosomes in the chicken thrombocytes

Summary A combined electron microscopic and cytochemical study of the thrombocytes of the chicken has clearly identified the amine-storing organelles and lysosomes. A chormaffin positive-reaction product was observed on the inner surface and the granules of the large electronlucent vacuoles. No acid phosphatase activity was localized in these amine-storing vacuoles. However, the acid phosphatase activity was observed in the small vesicles, the primary lysosomes, and in the large electron dense inclusions with myelin which may be secondary lysosomes. The results of this study suggest that the large empty vacuoles, with one or two very dense osmiophilic peripherally-situated granules, in the chicken thrombocytes are comparable to the vesicles with electron dense materials called dense bodies in mammalian thrombocytes.To whom offprint requests should be sent     

Lysosome formation and surface changes in stimulated peritoneal cells

Summary The cells of the peritoneum of the mouse have been studied with the electron microscope after stimulation in vitro and in vivo with glyceryl trioleate and glucan. Stimulation has two main morphological effects. There is an increase in the length of cytoplasmic processes, both finger-like and flap-like; this is apparent within an hour and lasts for several days. Several days after stimulation there is an increase in the number of lysosomes, accompanied by an increase, demonstrated cytochemically and biochemically, in acid phosphatase. The lysosomes fall into two groups, a group of small homogeneous bodies, the characteristic macrophage granules, and larger heterogeneous bodies.The small macrophage granules have a constant fine structural pattern. The morphological appearances suggest that they are in the main primary lysosomes, largely synthesized in the endoplasmic reticulum. The larger heterogeneous dense bodies probably contain varying amounts of ingested material, and can be considered as residual bodies.     

Cytochemical characterization of leucocytes from the seawater teleost,gilthead seabream (Sparus aurata L.)

The cytochemical characterization of head-kidney and peripheral blood leucocytes of gilthead seabream (Sparus aurata L.) was studied by light and electron microscopy. Neutrophilic granulocytes show some cytoplasmic granules, which are positive for alkaline phosphatase and peroxidase but acid phosphatase negative. The scarce granules found in the cytoplasm of the circulating neutrophils and their cytochemical features seem to be indicative of an immature stage. Acidophils are also alkaline phosphatase and peroxidase positive at pH 11.0. They are strongly positive for acid phosphatase and acid phosphatase activity may thus be considered a cytochemical marker to characterize and differentiate neutrophilic from acidophilic granulocytes in this fish species. Three granule populations are characterized in the cytoplasm of the gilthead seabream acidophils: the first is positive only for peroxidase and the second contains a dense core with acid and alkaline phosphatase activities, surrounded by a thin peroxidase positive electron-dense halo. The third granule type contains an eccentric core, which is strongly positive for acid and alkaline phosphatase and peroxidase. As regards their cytochemical features, the first and second granule types seem to correspond respectively to the azurophilic and specific granules found in acidophils of mammals and could be involved in phagocytic processes, thus playing an important microbicidal role in this species. The monocytes, monocyte-macrophages and macrophages show different cytochemical features. The first have scarce acid phosphatase-positive lysosomes, while blood monocyte-macrophages and macrophages are positive for acid and alkaline phosphatases and for peroxidase; the monocyte-macrophages show scarce lysosomes.     

Cytochemical demonstration of amine-storing vacuoles and lysosomes in the chicken thrombocytes

A combined electron microscopic and cytochemical study of the thrombocytes of the chicken has clearly identified the amine-storing organelles and lysosomes. A chromaffin positive-reaction product was observed on the inner surface and the granules of the large electron-lucent vacuoles. No acid phosphatase activity was localized in these amine-storing vacuoles. However, the acid phosphatase activity was observed in the small vesicles, the primary lysosomes, and in the large electron dense inclusions with myelin which may be secondary lysosomes. The results of this study suggest that the large empty vacuoles, with one or two very dense osmiophilic peripherally-situated granules, in the chicken thrombocytes are comparable to the vesicles with electron dense materials called "dense bodies" in mammalian thrombocytes.     

Density gradient separation of two populations of lysosomes from rat parotid acinar cells

Exocrine acinar cells possess two cytochemically distinct populations of secondary lysosomes. One population is Golgi associated and has demonstrable acid phosphatase (AcPase) activity, whereas the second is basally located and lacks AcPase activity but has trimetaphosphatase (TMPase) activity. The basal lysosomes are tubular in shape and rapidly label with horseradish peroxidase (HRP) after intravenous injection. In the present study using isolated rat parotid acinar cells, the two lysosomal populations were separated by cell fractionation on Percoll density gradients and were analyzed biochemically and by EM cytochemistry. On 35% Percoll gradients, two peaks of AcPase and beta-hexosaminidase, both lysosomal marker enzymes, and succinic dehydrogenase, an enzyme marker for mitochondria, could be resolved. The major peaks of beta-hexosaminidase and succinic dehydrogenase and the minor peak of AcPase corresponded with the dense lysosome fraction. The major peak of AcPase and the minor peaks for beta-hexosaminidase and succinic dehydrogenase coincided with the light membrane fraction. Galactosyl transferase (a marker enzyme for Golgi saccules) and 5'-nucleotidase (a plasma membrane marker) were also associated with this fraction. By electron microscopy, the light membrane fraction was seen to contain tubular elements, multivesicular bodies (MVB), Golgi saccules, GERL, immature secretory granules, and some mitochondria. Electron microscopic cytochemical examination showed that these tubular structures were lysosomes. The dense lysosome fraction contained lysosomes positive for both AcPase and TMPase. After continuous incubation of isolated acinar cells with HRP, reaction product was rapidly localized to the light membrane fraction (greater than 2 min), where it was found in vesicles and tubular lysosomes. By 10 min it was present in MVB and tubular lysosomes, but by 60 min no HRP reaction product had appeared in the dense lysosomes. These results demonstrate that the tubular lysosomes are separable from dense lysosomes, typical secondary lysosomes, and are involved in the initial stages of endocytosis.     

CYTOCHEMICAL LOCALIZATION OF PEROXIDATIC ACTIVITY OF CATALASE IN RAT HEPATIC MICROBODIES (PEROXISOMES)

Prominent staining of rat hepatic microbodies was obtained by incubating sections of aldehyde-fixed rat liver in a modified Graham and Karnovsky's medium for ultrastructural demonstration of peroxidase activity. The electron-opaque reaction product was deposited uniformly over the matrix of the microbodies. The microbodies were identified by their size, shape, presence of tubular nucleoids, and other morphologic characteristics, and by their relative numerical counts. The staining reaction was inhibited by the catalase inhibitor, aminotriazole, and by KCN, azide, high concentrations of H₂O₂, and by boiling of sections. These inhibition studies suggest that the peroxidatic activity of microbody catalase is responsible for the staining reaction. In the absence of exogenous H₂O₂ appreciable staining of microbodies was noted only after prolonged incubation. Addition of sodium pyruvate, which inhibits endogenous generation of H₂O₂ by tissue oxidases, or of crystalline catalase, which decomposes such tissue-generated H₂O₂, completely abolished microbody staining in the absence of H₂O₂. Neither diaminobenzidine nor the product of its oxidation had any affinity to bind nonenzymatically to microbody catalase and thus stain these organelles. The staining of microbodies was optimal at alkaline pH of 8.5. The biological significance of this alkaline pH in relation to the similar pH optima of several microbody oxidases is discussed. In addition to staining of microbodies, a heat-resistant peroxidase activity is seen in some of the peribiliary dense bodies. The relation of this reaction to the peroxidase activity of lipofuscin pigment granules is discussed.     

Residual bodies resulting from photosensory membrane degradation are taken up by pigment cells in the eyes of the flyLucilia sp.

Retinae of blowflies (Lucilia sp.) were exposed to light for 12 h and then investigated by routine electron microscopy. Residual bodies and multi-vesicular bodies containing electron-dense structures were found in the photoreceptor cells. These structures appeared indistinguishable from material inside the pigment granules of secondary pigment cells. The residual bodies were found in interdigitations between photoreceptor and pigment cells and were often in close contact with mitochondria. Lamellar bodies and pigment granules were also found in the extracellular space between photoreceptor and pigment cells. In a second set of experiments, a membrane-impermeable reagent [sulfosuccinimidyl-6-(biotinamido) hexanoate] that should covalently biotinylate the surface of the photosensory membrane was introduced into the ommatidial cavity. The marker was detected, 4 h after application, inside the ommatidial cavity, on the rhabdomeric microvilli, and on residual bodies inside the photoreceptor cells, by streptavidin-gold binding on ultrathin sections. After 6 h of exposure to the reagent, pigment granules of the adjacent pigment cells were also labeled. The results suggest that the photosensory membrane is taken up and degraded together with the marker. Residual bodies resulting from this degradative process may thus be transported into the pigment cells; eventually material originating from photosensory membrane degradation may then be involved in pigment granule synthesis.     

Localization of acid phosphatase activity in collagen-secreting and collagen-resorbing fibroblasts

The ultrastructural localization of acid phosphatase (ACPase) activity was examined in cultured human gingival fibroblasts in the formative and resorptive phases. In the collagen-secreting fibroblasts, weak ACPase activity was demonstrated in the lysosomes, inner Golgi cisternae, and condensing vacuoles, and none was found in the Golgi-associated endoplasmic reticulum-lysosome system (GERL), presecretory granules, or secretory granules. On the contrary, collagen phagocytosis induced strong ACPase activity in the GERL, which was in addition to the weaker activity found in the same sites as those in the collagen-secreting cells. At the same time, collagen secretion was suppressed, and dense elongated secretory bodies associated with ACPase activity accumulated within the cells. When collagen fibrils had been interiorized in whole or in part within the phagosomes, primary lysosomes derived from the Golgi-GERL complex then fused with them to form phagolysosomes. Collagen degradation occurred within these bodies. The observations indicate significant differences in ACPase activity used as a marker for lysosomal enzyme activities in the different functional phases of fibroblasts. These results suggest that fibroblasts work only one way at a given time, viz., collagen synthesis or collagen degradation.     

Basophilic Lamellar Systems in the Crayfish Spermatocyte

Histochemical procedures for the demonstration of RNA have shown the presence of intensely basophilic bodies in the cytoplasm of spermatocytes of the crayfish, Cambarus virilis. The staining of thick sections, cut alternately with thin sections for electron microscopy, has permitted identification of the basophilic bodies with two types of lamellar systems. One of these, a set of straight annulate lamellae, is restricted to meiotic prophase. The second type of lamellar systems has been found from late prophase to early spermatid stages. It consists of an ellipsoidal lamellar set which intersects a number of straight lamellae. Within the region of intersection, the ellipsoidal lamellae break up into an array of small tubules of about 150 A diameter. The term tubulate lamellar system was chosen to designate this type of lamellar complex. Small RNA-containing granules could not be detected in annulate lamellar systems. While there are a few granules in the marginal regions of the tubulate lamellar system, their distribution cannot be responsible for the basophilia which is intense within all regions of the lamellar body.     

Ultrastructural localization of some phosphatases in the prothoracic gland of the insect Leucophaea Maderae

Summary The cytochemical localization of acid phosphatase and thiamine pyrophosphatase activity was studied by light and electron microscopy in prothoracic gland cells of the cockroach Leucophaea moderae. Nymphal and young adult animals were used.Prominent sites of acid phosphatase activity included large membrane-bounded dense bodies or lysosomes, and certain cisternae of the Golgi apparatus. The results suggest a possible difference in the enzymatic activity toward glycerophosphate and aromatic phosphates as substrates.Thiamine pyrophosphatase activity was localized in elements of the Golgi apparatus and endoplasmic reticulum, and in lysosome-like dense bodies. This latter activity was abolished by sodium fluoride treatment, whereas the phosphatase activity in the Golgi apparatus and endoplasmic reticulum is unaffected by such inhibition.The cytochemical results confirm through direct evidence the suggestions of Scharrer (1964), that the large dense bodies present in the prothoracic gland cells are lysosomes, and that their activity may be related to stages in the life history of the glands. Furthermore, the lysosomes or their derivative structures may play an essential role in the autolysis of the prothoracic glands toward the end of their active period.The enzymatic activity of the endoplasmic reticulum may indicate the involvement of this organelle in the metabolism of steroid-like precursor materials necessary for the synthesis of ecdysone.This study was supported by U.S.P.H.S. grants 5 T1-MH-6418 and NB-05219, and grant RO 1-AM-3984 to Dr. Berta Scharrer. I would like to express my appreciation to Dr. Scharrer for her encouragement and assistance during this study. I also wish to thank Mrs. Sarah Wurzelmann for her competent technical aid.     

Acid phosphatase and peroxidase in ‘resting’ acinar cells of the major salivary glands of cats and their possible movement into secretory granules

Synopsis After fixation by perarterial perfusion using an aldehyde mixture, salivary tissues were prepared for ultrastructural cytochemistry of acid phosphatase or peroxidase. Great variations in the distributions of the reaction products occurred, often within the same cell. Acid phosphatase staining occurred not only in lysosomes and sometimes in a GERL system, but a diffuse cytoplasmic component was also found in submandibular central acinar cells and to a lesser extent in parotid acini and variable staining occurred in the secretory granules of these cells. Peroxidase was variably associated with rough endoplasmic reticulum in submandibular demilunar cells, parotid acini, and more strongly in some sublingual cells. The secretory granules of the latter were darkly stained, but in parotid granules there was varibale staining and least staining occurred in the granules of submandibular demilunes.These results are thought to indicate that not all enzymes present in secretory granules have reached there by an elective secretory process. Sometimes they appear to have entered the granules haphazardly, possibly having been enzymes associated with intracellular cisternal channels for transport or metabolism of other secretory substances and ultimately to have passed into the cisternal channels by chance or as part of a natural removal of redundant material.     

Fonction catabolique de l'épithélium mammaire

Summary The histochemical (iron, lipopigments, acid phosphatase, leucine aminopeptidase) and cytologic (lysosomes) changes occuring during pregnancy, lactation and involution of mouse, rat, rabbit, guinea-pig mammary glands are studied by light microscopy and electron microscopy.In all the animals examined, the mammary epithelium has an intracellular digestive system which is adapted to subserve two functions. The first one is the segregation of cytoplasmic components which often precedes cellular involution. The second one is the regulation of secretory processes in the non lactating glands. This digestion of endogenous materials results in the formation of various lytic bodies: dense bodies sometimes containing ferritin, vacuolated dense bodies with membranous residues, autophagic vacuoles. The lysosomes can give large complex dense bodies like lipofuscin pigments with or without ferritin.Leucine aminopeptidase which always disappears in the mouse mammary epithelium during lactation is not present in rat, rabbit, guinea-pig mammary epithelium. In these species only the vascular tissue contains the enzyme. This observation indicates that leucine aminopeptidase does not take care of the overproduction of secretory products in the non-lactating glands.Acid phosphatase is concentrated in secretory granules and in lytic bodies: multivesicular bodies, dense bodies with ferritin, vacuolated dense bodies, lipopigments. This enzyme constitutes probably a mechanism for controlling and triggering the destruction of the secretory material with no active elimination.The iron of the mammary epithelium appears in virgin mice older than 30 weeks and in mice, rats, rabbits, guinea-pigs during glandular cells involution. This is a catabolic iron located in lysosomes. Its amount depends upon the iron content of the milk and upon the competitive secretory and catabolic activities of the glandular cells. An explanation of iron disappearance during a second pregnancy and lactation is discussed.     

DIFFERENCES IN ENZYME CONTENT OF AZUROPHIL AND SPECIFIC GRANULES OF POLYMORPHONUCLEAR LEUKOCYTES : II. Cytochemistry and Electron Microscopy of Bone Marrow Cells

In the previous paper we presented findings which indicated that enzyme heterogeneity exists among PMN leukocyte granules. From histochemical staining of bone marrow smears, we obtained evidence that azurophil and specific granules differ in their enzyme content. Moreover, a given enzyme appeared to be restricted to one of the two types. Clear results were obtained with alkaline phosphatase, but those with a number of other enzymes were suggestive rather than conclusive. Since the approach used previously was indirect, it was of interest to localize the enzymes directly in the granules. Toward this end, we carried out cytochemical procedures for five enzymes on normal rabbit bone marrow cells which had been fixed and incubated in suspension. The localization of reaction product in the granules was determined by electron microscopy. In accordance with the results obtained on smears, azurophil granules were found to contain peroxidase and three lysosomal enzymes: acid phosphatase, arylsulfatase, and 5'-nucleotidase; specific granules were found to contain alkaline phosphate. Specific granules also contained small amounts of phosphatasic activity at acid pH. Another finding was that enzyme activity could not be demonstrated in mature granules with metal salt methods (all except peroxidase); reaction product was seen only in immature granules. The findings confirm and extend those obtained previously, indicating that azurophil granules correspond to lysosomes whereas specific granules represent a different secretory product.     

ELECTRON MICROSCOPY OF LYSOSOME-RICH FRACTIONS FROM RAT LIVER

A preliminary electron microscope study has revealed the presence in lysosome-rich fractions, isolated from rat liver, of hitherto undescribed cytoplasmic particles, called "dense bodies." Approximately 0.37 µ in length, the dense bodies often possess an internal cavity and external membrane. They contain many electron-dense granules 55 to 77 A, or less, in diameter. Such dense bodies are also visible in electron micrographs of parenchymatous cells in liver sections. The correlations between dense bodies and lysosomes are listed, but until pure preparations are available it is not possible to assert that dense bodies and lysosomes are identical.     

SECRETION AND ENDOCYTOSIS IN INSULIN-STIMULATED RAT ADRENAL MEDULLA CELLS

Insulin was used to deplete the adrenalin stores of rat adrenal medulla cells. Release of secretion was observed to occur by exocytosis. In addition, during the stages of massive release of secretory granules, the insulin-treated preparations showed greatly enhanced endocytic uptake of horseradish peroxidase. The tracer was taken up within vesicles, tubules, multivesicular bodies, and dense bodies. From acid phosphatase studies and from previous work it appears that many of the structures in which peroxidase accumulates are lysosomes or are destined to fuse with lysosomes. Subsequent to the period of intense exocytosis and endocytosis, there is a transient accumulation of lipid droplets in the adrenalin cells. The cells then regranulate, with new granules forming near the Golgi region. These results suggest that under the conditions used, much of the membrane that initially surrounds secretory granules is degraded after release of the granules.     

Pyridoxal 5'-phosphate: a possible physiological substrate for alkaline phosphatase in human neutrophils

The intracellular localization of pyridoxal phosphatase activity was demonstrated in human neutrophils by electron microscope cytochemistry. Under alkaline conditions, an enzyme active against pyridoxal phosphate was localized to a cytoplasmic granule population, the phosphasome. These granules have previously been shown by electron microscope cytochemical techniques and by subcellular fractionation to be rich in alkaline phosphatase. Under acidic conditions, a phosphatase activity against pyridoxal phosphate was localized to intracellular multilamellar bodies resembling secondary lysosomes. These were quite distinct from the primary, secondary and phosphasome granules and this unique localization corresponds to that previously demonstrated (tertiary granules) by subcellular fractionation studies of these cells. The similarity in the enzyme reaction requirements of alkaline pyridoxal phosphatase and alkaline phosphatase, and their localization to the same subcellular organelle, suggests that pyridoxal phosphate may be a physiological substrate for human neutrophil alkaline phosphatase.