LYSOSOMES AND GERL IN NORMAL AND CHROMATOLYTIC NEURONS OF THE RAT GANGLION NODOSUM

The rat ganglion nodosum was used to study chromatolysis following axon section. After fixation by aldehyde perfusion, frozen sections were incubated for enzyme activities used as markers for cytoplasmic organelles as follows: acid phosphatase for lysosomes and GERL (a Golgi-related region of smooth endoplasmic reticulum from which lysosomes appear to develop) (31–33); inosine diphosphatase for endoplasmic reticulum and Golgi apparatus; thiamine pyrophosphatase for Golgi apparatus; acetycholinesterase for Nissl substance (endoplasmic reticulum); NADH-tetra-Nitro BT reductase for mitochondria. All but the mitochondrial enzyme were studied by electron microscopy as well as light microscopy. In chromatolytic perikarya there occur disruption of the rough endoplasmic reticulum in the center of the cell and segregation of the remainder to the cell periphery. Golgi apparatus, GERL, mitochondria and lysosomes accumulate in the central region of the cell. GERL is prominent in both normal and operated perikarya. Electron microscopic images suggest that its smooth endoplasmic reticulum produces a variety of lysosomes in several ways: (a) coated vesicles that separate from the reticulum; (b) dense bodies that arise from focal areas dilated with granular or membranous material; (c) "multivesicular bodies" in which vesicles and other material are sequestered; (d) autophagic vacuoles containing endoplasmic reticulum and ribosomes, presumably derived from the Nissl material, and mitochondria. The number of autophagic vacuoles increases following operation.     

Cytochemical characterization of the endomembranous system during the oocyte primary growth in Serrasalmus spilopleura (Teleostei, Characiformes, Characidae)

The morphophysiological changes that occur during oocyte primary growth in Serrasalmus spilopleura were studied using ultrastructural cytochemical techniques. In the previtellogenic oocytes endoplasmic reticulum components, Golgi complex cisternae and vesicles, lysosomes, multivesicular bodies and some electron-dense vesicles react to acid phosphatase (AcPase) detection. The endoplasmic reticulum components, Golgi complex cisternae and vesicles also react to osmium tetroxide and potassium iodide impregnation (KI). These structures, except for the Golgi complex cisternae, are strongly contrasted by osmium tetroxide and zinc iodide impregnation (ZIO). Some electron-dense vesicles are ZIO-stained, while microvesicles in the multivesicular bodies and other large isolated cytoplasmic vesicles are contrasted by KI. At primary oocyte growth, the activity of the endomembranous system and the proliferation of membranous organelles are intense. The biosynthetic pathway of the lysosomal proteins such as acid phosphatase, involves the endoplasmic reticulum, Golgi complex, vesicles with inactive hydrolytic enzymes and, finally, the lysosomes. The oocyte endomembranous system have reduction capacity and are involved in the metabolism of rich in SH groups.     

Distribution of Acid Phosphatase in Paramecium caudatum: Its Relations with the Process of Digestion

SYNOPSIS. The distribution of acid phosphatase was investigated at the ultrastructural level in Paramecium caudatum. Acid phosphatase occurs in endoplasmic reticulum, Golgi apparatus, food vacuoles, autophagic vesicles, vacuolar and dense bodies. Some slight deposits are also seen in the mitochondria.
These observations point out that this hydrolase activity is related to digestive processes. The enzyme, originating from the endoplasmic reticulum and Golgi apparatus reaches the food vacuole or autophagic vesicle likely via the reticulum. The digestion of the bacteria or of the enclosed organelle gives rise to electronopaque material which is later found in dense bodies. These dense bodies are likely secondary lysosomes and it is possible that they may fuse with the young food vacuole or with autophagic vesicles.     

Electron microscopic study of intracellular digestion in intestinal cells of the ixodid tick Hyalomma asiaticum. I. Formation of primary and secondary lysosomes]

The formation of primary and secondary lysosomes in digestive cells of midgut of the tick H. asiaticum was investigated using ultracytochemical methods for acid phosphatase. This enzyme is synthesized in the rough endoplasmic reticulum cisternae to be concentrated in the Golgi complex. Vesicles 0.1-0.15 mum in diameter filled with the enzyme are propagated from the distal Golgi cisternae which are primary lysosomes. Secondary lysosomes are produced in result of fusion of primary lysosomes with heterophagosomes that appear during endocytosis. Another type of structures responsible for transport of lysosomal enzymes into heterophagosomes is represented by dense bodies 0.3-0.5 mum in size. These are rich in acid phosphatase being different stages of heterophagolysosomes and telolysosomes.     

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.     

Electron cytochemical demonstration of four hydrolytic enzymes in the rat corpora lutea at second half of gestation

Corpora lutea from rat ovaries at mid pregnancy were fixed by perfusion and studied by electron cytochemistry for localisation of four hydrolytic enzymes. Using the metal-salt methods for acid phosphatase and aryl sulphatases activity was localised in small and large lysosomes, multivesicular bodies, Golgi complex and within cisternae of endoplasmic reticulum. The azo-dye coupling method for Beta-glucuronidase was less satisfactory and gave positive results in lysosomes, lipids and in the globules within the mitochondrial matrix. The latter two localisation were probably associated with affinity of the naphthol AS-BI for lipid material. In addition to plasma membranes, the reaction product for alkaline phosphatase with the lead-salt method was seen in lysosomelike bodies, in smooth endoplasmic reticulum and in occasional Golgi elements of granulosa lutein and endothelial cells. Increased activity of lysosomal acid hydrolases occurs when regressive changes of lutein cells start at the end of gestation and this might probably reflect the initiation of lytic processes.     

Electron cytochemical demonstration of four hydrolytic enzymes in the rat corpora lutea at second half of gestation

Summary Corpora lutea from rat ovaries at mid pregnancy were fixed by perfusion and studied by electron cytochemistry for localisation of four hydrolytic enzymes. Using the metal-salt methods for acid phosphatase and aryl sulphatases activity was localised in small and large lysosomes, multivesicular bodies, Golgi complex and within cisternae of endoplasmic reticulum. The azo-dye coupling method for Beta-glucuronidase was less satisfactory and gave positive results in lysosomes, lipids and in the globules within the mitochondrial matrix. The latter two localisation were probably associated with affinity of the naphthol AS-BI for lipid material. In addition to plasma membranes, the reaction product for alkaline phosphatase with the lead-salt method was seen in lysosomelike bodies, in smooth endoplasmic reticulum and in occasional Golgi elements of granulosa lutein and endothelial cells.Increased activity of lysosomal acid hydrolases occurs when regressive changes of lutein cells start at the end of gestation and this might probably reflect the initiation of lytic processes.     

An ultrastructural study of phosphatases and aryl sulphatase in BHK and CHO cells grown in culture

Summary The ultrastructural distribution of a number of phosphatases and aryl sulphatase has been studied in BHK 21/C 13, BHK21/J 1 and CHO cells grown in culture. In all three cell lines acid -glycerophosphatase and aryl sulphatase appear to be confined to lysosomes and elements of the Golgi apparatus and glucose-6-phosphatase to the endoplasmic reticulum. With thiamine pyrophosphate at pH 7.0 in CHO cells reaction product is present in lysosomes, the Golgi apparatus, the endoplasmic reticulum and on the cell surface. Preincubation at acid pH reduces the reactions in the endoplasmic reticulum but enhances the surface activity. At pH 5.0 and pH 7.0 in CHO cells p-nitrophenylphosphatase is present in lysosomes, the Golgi apparatus and the endoplasmic reticulum and this activity is inhibited by sodium fluoride. p-nitrophenylphosphatase activity is also present on the cell surface of CHO cells and this activity is not inhibited by sodium fluoride. No activity could be demonstrated in any cells at pH 9.O. The significance of these results is discussed with respect to the possible role of surface acid phosphatase in the process of transformation.     

Ultrastructural Localization of Acid Phosphatase in Starved Tokophrya infusionum*

SYNOPSIS. Young organisms of Tokophrya infusionum starved for several hr, are best suited for a study of the fine structure of this organism including the distribution of its organelles. Acid phosphatase was localized by a combined electron microscopy and cytochemical approach using modified Gomori methods. The enzyme was found in small dense bodies, spheroid vesicles, missile-like bodies, rough-surfaced endoplasmic reticulum, residue and autophagic vacuoles. The small dense bodies are thought to be primary lysosomes since electron micrographs show a) a continuity between the membrane of the rough-surfaced endoplasmic reticulum and that of the dense bodies and b) a connection between the contents of both structures when the dense bodies form from the endoplasmic reticulum.     

Woronin bodies fromPenicillium chrysogenum: Isolation and characterization by analytical subcellular fractionation

Differential centrifugation of whole homogenates ofPenicillium chrysogenum, disrupted by a modified Ballotini bead method, resulted in the enrichment of Woronin bodies between 800g (5 minutes) and 6000g (10 minutes). Isolated Woronin bodies are membrane-bounded, electron-opaque, approximately spherical organelles, 0.11 to 0.29 μm in diameter. Woronin bodies have a buoyant density (ϱ) of 1.21 g cm⁻³ and S_20,w values of 6300 to 37,600 in sucrose gradients. Analytical subcellular fractionation of whole homogenates in a zonal rotor showed that Woronin bodies did not cosediment with marker enzymes for lysosomes (acid phosphatase), peroxisomes (catalase), mitochondria (cytochrome c oxidase), or endoplasmic reticulum (NADPH cytochrome c reductase).     

Acid phosphatase in the guinea-pig oocytes

In guinea-pig oocytes, at every developmental stage, acid phosphatase is found histochemically in cytoplasmic granules. Ultracytochemically the reaction product is located in lysosomes and is some cisternae of the rough endoplasmic reticulum, but not in cortical granules or in vesicles with a rough endoplasmic reticulum membrane which are filled with a moderately dense homogeneous substance. It is discussed whether the acid phosphatase transforms reserve material into a storable form as has been proposed for the deposition of vitellogenin in the oocytes of lower vertebrates.     

CYTOLOGICAL STUDIES ON TWO FUNCTIONAL HEPATOMAS : Interrelations of Endoplasmic Reticulum, Golgi Apparatus, and Lysosomes

The Reuber hepatoma H-35 and Morris hepatoma 5123 have been studied by electron microscopy and by cytochemical staining methods for a number of phosphatases. These studies emphasize the resemblances of the two tumors to rat liver, but they also indicate distinctive features in each of the three tissues. Secretory product accumulates within the cisternae of the Golgi apparatus that dilate to form the Golgi vacuoles. The vacuoles apparently separate, and secretory material undergoes further condensation within them. These "secretory vacuoles" possess acid phosphatase activity and may thus be considered lysosomes. The membranes of the Golgi apparatus are without acid phosphatase activity but show high levels of thiaminepyrophosphatase activity. The endoplasmic reticulum also hydrolyzes thiaminepyrophosphate but at a lower rate; it hydrolyzes the diphosphates of uridine, guanosine, and inosine rapidly. These observations and the electron microscopic images are consistent with the view that the cytomembranes are in a dynamic state of flux, movement, and transformation in the living cell, and that smooth surfaced derivatives of the endoplasmic reticulum become refashioned into the Golgi membranes as the Golgi membranes are being refashioned into those that delimit secretory vacuoles. The variations encountered in the two hepatomas are described. The electron microscope literature dealing with the relations of the Golgi apparatus to secretory granules, on the one hand, and the endoplasmic reticulum, on the other, is reviewed briefly.     

Fine structural localization of acid and alkaline phosphatase activities in the absorbing cells of the duodenum of rodents

Summary The morphology of the absorbing cells of the duodenal villi in the mouse, the rat, the hamster and the guinea-pig is described. The polymorphism of the dense bodies is pointed out. The fine localization of acid and alkaline phosphatase is investigated and compared. In all the species, acid phosphatase activity is observed in the dense bodies, Golgi vesicles and rare smooth endoplasmic profiles. Alkaline phosphatase is localized on the microvilli, Golgi apparatus, some smooth endoplasmic cisternae and numerous dense bodies. The presence of an alkaline phosphatase reaction in the dense bodies, probably lysosomes, of the absorbing cells is discussed. It is assumed that this enzyme follows a catabolic pathway and is finally degraded in the lysosomes.Abbreviations used AlPase alkaline phosphatase - AcPase acid phosphatase This work was done thanks to the contract C.E.N./A.I.E.A. N 347/RB and thanks to grants from the Fonds de la Recherche scientifique fondamentale collective.     

Tubular lysosomes in rat and gerbil pinealocytes

Ultracytochemical demonstration of acid phosphatase (AcPase) in pinealocytes of rat and Mongolian gerbil (Meriones unguiculatus) has revealed an existence of tubular lysosomes (30-200 nm in diameter and more than 5 micron long) in their cytoplasm. The tubular lysosomes arise by bulging from GERL cisternae (Golgi apparatus, endoplasmic reticulum, lysosomes) and spread throughout the whole cell body without forming an anastomosing network. Numerous varicosities are characteristic for the tubular lysosomes whose similarity with grumose bodies has lead to conclusion that the vermiculate variety of the latter are almost certainly tubular lysosomes. The role of these organelles is unknown. Their possible engagement in a rapid cytoplasmic remodelling of the pinealocyte body in answer to various stimuli has been discussed.     

Acid phosphatase in the guinea-pig oocytes

Summary In guinea-pig oocytes, at every developmental stage, acid phosphatase is found histochemically in cytoplasmic granules, Ultracytochemically the reaction product is located in lysosomes and in some cisternae of the rough endoplasmic reticulum, but not in cortical granules or in vesicles with a rough endoplasmic reticulum membrane which are filled with a moderately dense homogenous substance. It is discussed whether the acid phosphatase transforms reserve material into a storable form as has been proposed for the deposition of vitellogenin in the oocytes of lower vertebrates.This investigation was supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Lysosomes and yolk formation in the oocytes of Asterina gibbosa (Echinodermata,Asteroidea)

Summary

Primary lysosomes appear in the oocytes of A. gibbosa at the end of previtellogenesis. The lysosomes fuse with the cisternae of the endoplasmic reticulum and give rise to yolk globules containing acid phosphatase. The yolk globules then grow by fusion.     

The Fine-Structural Localization of Phosphatases in Neurosecretory Cells Within the Ganglia of Certain Gastropod Snails

The neurosecretory cells in the cerebral ganglia of the snail,Planorbis trivolvis, have been examined for structural detailsand distribution of phosphatase activities. A more preliminarystudy on the localization of phosphatases in the neurosecretorycells of Helix aspersa has also been made. These neurons inboth species contain typical elementary neurosecretory granuleswhich appear to be elaborated or condensed in the Golgi saccules.Immature elementary granules are identifiable as "primary lysosomes"in that they contain acid phosphatase activity as well as nucleosidephosphatases. The mature elementary granules display no phosphataseactivity. Some saccules of the Golgi are also reactive for severalnucleoside phosphatases, including thiamine pyrophosphatase(TPPase). In Planorbis, TPPase is also present in the cisternaeof the endoplasmic reticulum. Larger, membrane bound, electron-densebodies (lipochondria) are found in close spatial associationwith the Golgi region. These also possess acid phosphatase;in addition, they contain nucleoside diphosphatases and (inPlanorbis) adenosine triphosphatase. Their content of acid phosphataseand the features of their fine structure indicate that theyare lysosomes akin to the dense bodies of vertebrate neurones. The significance and implications of these results are considered,as are other details of the neuronal and glial structure inPlanorbis.     

The trans Golgi face in rat small intestinal absorptive cells

In the small intestine cell differentiation from immature crypt cells to mature absorptive cells localized along the villi is accompanied by alterations in the organization of the trans Golgi side. In immature crypt cells the transmost Golgi cisterna is usually located closely adjacent to the other cisternae thus being a component of the stack. Concomitantly with cell differentiation the transmost cisterna of an increasing number of Golgi stacks sets off from the other cisternae being then located at various distances to the stacks. This transmost cisterna has, as in several other cell types, been interpreted as "GERL" (Golgi associated endoplasmic reticulum lysosomes [20, 28]) and thus, has been postulated to represent a specialized region of the endoplasmic reticulum. Our results, however, have shown that the cytochemical staining pattern which has been used as a basis for the differentiation of GERL from Golgi components is not present in crypt cells nor in mature absorptive cells of the proximal small intestine: identical cisternae react for thiamine pyrophosphatase, inosine diphosphatase, and acid phosphatase. Thiamine pyrophosphatase and inosine diphosphatase--enzymes characteristic for Golgi cisternae--are apparent over transmost cisternae defined as GERL, too, and in addition, acid phosphatase--postulated as GERL-marker--is demonstrable over stacked Golgi cisternae. This overlapping cytochemical reaction, as well as the alterations during cell differentiation, indicate that those structures which have been described as GERL are to be interpreted as Golgi components rather than as endoplasmic reticulum. On the other hand, endoplasmic reticulum is a constant component of the trans Golgi face in undifferentiated crypt-base cells and in maturing cells of the crypt-top region. From its localization closely adjacent to trans Golgi cisternae it may be termed "Golgi-associated endoplasmic reticulum"; however, these cisternae of endoplasmic reticulum are constantly devoid of acid phosphatase. No indications exist for continuities with the thiamine pyrophosphatase-, inosine diphosphatase-, and acid phosphatase-positive transmost Golgi cisternae, and for an engagement in production of lysosomes.     

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.     

The fine localization of acid phosphatase activity in the unvacuolated notochordal cells of the early chick embryo

Summary The electron microscopical localization of acid phosphatase activity was investigated in ultra-thin and semi-thin sections of unvacuolated notochordal cells of chick embryos from stages 9 to 14 (as defined by Hamburger & Hamilton). At stage 9, many notochordal cells show a lightly positive reaction for acid phosphatase activity. Thereafter, the acid phosphatase-positive cells of the notochord increase in number and, at stage 14, the reaction products for the enzyme are distributed throughout almost all the cisternae of the nuclear envelope and a well-differentiated endoplasmic reticulum, the parallel cisternal and reticular parts of the Golgi complex, and various lysosomes in nearly all notochordal cells. In the cisternae of the nuclear envelope and endoplasmic reticulum, the acid phosphatase reaction products are in a fine granular form. In the outermost layer of the cisternal parts of the Golgi complex, faint lead deposits similar to those in the endoplasmic reticulum are found, but in other cisternal and reticular regions which may correspond to the GERL, considerable amounts of reaction products are present. Knob-like projections are also seen protruding from the reticular parts of the Golgi complex. These results suggest that, at least up to stage 14, the notochordal cells are actively synthesizing acid phosphatase which is directly transported from the endoplasmic reticulum to the Golgi complex. The enzyme may be accumulated by the Golgi complex from which primary lysosomes are formed. Furthermore, the pattern of the ultrastructural localization of acid phosphatase activity in embryonic notochordal cells of the chick differs from that of adult cells of other animals.