Relationship between peroxisomes and endoplasmic reticulum investigated by combined catalase and glucose-6-phosphatase cytochemistry

The theoretical advantages of electron microscopic cytochemistry were utilized to look for evidence of possible connections between peroxisomes and the endoplasmic reticulum in rat liver. Established cytochemical procedures for catalase (peroxisomes) and glucose-6-phosphatase (endoplasmic reticulum) were carried out, and evidence was sought of diffusion of reaction products between the organelles. No such diffusion was observed: lead phosphate was found in the endoplasmic reticulum and in the nuclear envelope but not in peroxisomes; oxidized diaminobenzidine (DAB) was seen only in peroxisomes. In addition, both types of cytochemistry were carried out on the same tissue. The two kinds of reaction product could be distinguished by virtue of their different electron opacities. No mixing of the two reaction products was observed. These results do not support the hypothesis that peroxisomes and endoplasmic reticulum may be connected; rather, they support the idea that the two organelles exist as separate cellular compartments.     

Characterization of peroxisomes in glucose-grown Hansenula polymorpha and their development after the transfer of cells into methanol-containing media

Cells of Hansenula polymorpha growing exponentially on glucose generally contained a single peroxisome of small dimension, irregular in shape and located in close proximity to the cell wall. Crystalline inclusions in the peroxisomal matrix were not observed. Associations of the organelles with one or more strands of endoplasmic reticulum were evident. In stationary phase cells the size of the peroxisomes had increased considerably. They were more cubical in form and showed a partly or completely crystalline matrix.After the transfer of cells growing exponentially on glucose into media containing methanol, large peroxisomes with a partly crystalline matrix developed in the cells within 6 h. These organelles originated from the small peroxisomes in the glucose-grown cells. De novo synthesis of peroxisomes was not observed. Prolonged cultivation in the presence of methanol resulted in a gradual increase in the number of peroxisomes by means of separation of small peroxisomes from mature organelles. During growth of peroxisomes associations with the endoplasmic reticulum remained evident.The increase in volume density of peroxisomes in stationary phase cells grown on glucose and in methanol-grown cells was accompanied by the synthesis of the peroxisomal enzymes alcohol oxidase and catalase. Cytochemical staining techniques revealed that alcohol oxidase activity was only detected when the peroxisomes contained a crystalloid inclusion. Since in peroxisomes of an alcohol oxidase-negative mutant of Hansenula polymorpha crystalline inclusions were never detected, it is concluded that the development of crystalloids inside peroxisomes is due to the accumulation of alcohol oxidase in these organelles.     

Zinc iodide-osmium tetroxide (ZIO) reactive sites in the extrafloral nectary of Citharexylum mirianthum Cham. (Verbenaceae)

This paper reports on a study of the zinc iodide-osmium tetroxide method (ZIO) applicability to formaldehyde-glutaraldehyde prefixed extrafloral nectary tissues of Citharexylum mirianthum Cham. (Verbenaceae). The ZIO solution impregnates the dictyosome stacks and adjacent vesicles, smooth endoplasmic reticulum, nuclear envelope, multivesicular bodies, and peroxisomes. The use of this method greatly facilitates the observation and recognition of organelles in each nectary region. It also allows the correlation between structure and function in nectariferous cells.     

Destructive and restorative processes of the liver in rats under intensive physical loading

The ultrastructure of rat liver cells after running exercise was investigated. When rats were trained for a month and sacrificed immediately after the last exercise it was revealed that the number of liver cells mitochondria increased, but many of them had alterations: mitochondria became swollen, had lucid matrix. There were some variations in degree of alterations between different mitochondria: a) in the same hepatocyte, b) in different hepatocytes of the same animal, that was connected with individual sensitivity of organelles on the levels of the cell and of the organ. Rough endoplasmic reticulum bore few ribosomes. Glycogen was absent. There were abundant vesicles of smooth endoplasmic reticulum, autophagic vacuoles and peroxisomes in the liver cell cytoplasm. Adaptation of rat liver to the exercise programme becomes evident by 1.5 month of exercise. Mitochondria and rough endoplasmic reticulum were numerous and of normal structure. There were many peroxisomes and glycogen granules in the cytoplasm of hepatocyte. The presence of large autophagic vacuoles in the cytoplasm of some hepatocytes were obviously connected with more rapid destruction of some organelles, than in control.     

Effects of 4-aminopyrazolo (3,4-d) pyrimidine on rat hepatocytes: an ultrastructural morphometric study

The prolonged administration of the hypolipidemic drug 4-aminopyrazolo (3,4-d) pyrimidine (4APP) induced conspicuous morphological changes in rat hepatocytes, which are clearly demonstrated by stereology. We observed a significant decrease in the rough endoplasmic reticulum, and an accumulation of lipid droplets, which was coupled with a comparable rise in the hepatic concentrations of total cholesterol and triglycerides. These changes were interpreted as the expression of the 4APP-provoked impairment of the synthesis of the polypeptide chains of lipoproteins and of the consequent suppression of the assembly of lipid molecules in exportable lipoproteins. We also noted a neto lowering in smooth endoplasmic reticulum and peroxisomes, a decrease which was hypothesized to be the morphological counterpart of a reduced de novo cholesterol synthesis, due to the 4APP-induced rise in the intracellular cholesterol concentration.     

Structural and cytochemical characterization of three specialized peroxisome types in soybean

Structural and cytochemical comparisons were made between three peroxisome types in soybean [ Glycine max (L.) Merr. cv. Centennial]. Leaf peroxisomes were densely granular organelles with an amorphous nucleoid and were generally located in close proximity to the chloroplasts. Catalase (EC 1.11.1.6) and glycolate oxidase (EC 1.1.3.1) were localized in these peroxisomes although glycolate oxidase was absent from the nucleoid region. Glyoxysomes, present in the etiolated cotyledons, were coarsely granular organelles that were generally in close proximity to lipid bodies. Malate synthase (EC 4.1.3.2), catalase, and glycolate oxidase were present throughout the matrix. Although peroxisomes were found in both infected and uninfected nodule tissue, uninfected interstitial cell peroxisomes were the most developed. These organelles contained a core surrounded by a less electron-opaque periphery that frequently was in close association with (but distinct from) a network of smooth endoplasmic reticulum. Of the enzymes studied, only catalase and urate oxidase (EC 1.7.3.3) were detected in the nodule peroxisomes. Neither enzyme was detected in the peripheral area of the peroxisome. These data indicate that peroxisomes in the three tissue types have organelle associations, internal structures, enzyme constitutions and packaging that reflect their metabolic differences.     

Peroxisomes in the absorptive cells of normal,rachitic, and vitamin-D replete chick intestine: Ultrastructure and histochemistry

Using routine transmission electron microscopy and light and electron microscopic techniques for the histologic demonstration (localization) of catalase (a peroxisomal enzyme), peroxisomes in chick duodenal epithelial cells were identified and studied. In these cells, peroxisomes were seen to be small, ovoid structures, delimited by a single unit membrane. They were concentrated in the supranuclear cytoplasm in initimate association with the smooth endoplasmic reticulum. As demonstrated histochemically, the heterogeneous matrix of these organelles was catalase positive. In addition, most of the larger peroxisomes revealed central nucleoids; however, the smaller peroxisomes were generally anucleoid. It thus appears that two classes of peroxisomes exist in chick intestinal absorptive cells: (1) small, anucleoid microperoxisomes, and (2) larger, nucleoid-containing peroxisomes. In addition to the above morphological characteristics, both peroxisome types were numerous in normal and vitamin-D-replete tissues, but were conspicuously decreased or absent from the apical cytoplasm of rachitic epithelial cells. From these observations it is hypothesized that these organelles may be involved in the overall vitamin-D response of the small intestine.     

Distribution and transport of apo- and holocytochrome b5 in the endoplasmic reticulum of rat liver

The transport and distribution of apo- and holocytochrome $\text{b}{}_5$ was investigated with the aid of specific antibodies. The holoenzyme was found to be localized mainly in the rough and smooth endoplasmic reticulum and in the Golgi system but some precipitation could also be obtained in the outer mitochondrial membranes and in the peroxisomes. The apoenzyme, however, could only be detected in the endoplasmic reticulum-Golgi system, which also was shown to be the sole site for incorporation of the prosthetic heme moiety. Time-course studies revealed that the labeled enzyme appeared both as apoenzyme and as holoenzyme in the rough endoplasmic reticulum 10 min after in vivo injection of radioactive leucine and that further transport to the smooth endoplasmic reticulum occurred within 10 min. The subsequent transport to other organelles, however, required a somewhat longer time and peak radioactivity in outer mitochondrial membranes was not attained until after 40 min.     

PEROXISOMES IN INNER ADRENOCORTICAL CELLS OF FETAL AND ADULT GUINEA PIGS

Abundant membrane-bounded granules, 0.1–0.45 µm in diameter, occur among the elements of the smooth-surfaced endoplasmic reticulum in zona fasciculata and zona reticularis adrenocortical cells of guinea pigs. Acid phosphatase cannot be cytochemically demonstrated in them, and they are therefore distinct from lysosomes. Incubation in medium containing 3,3'-diaminobenzidine results in dense staining of the granules, identifying them as peroxisomes. These small peroxisomes increase in number as fetal adrenocortical cells differentiate, and they appear to arise from dilated regions of endoplasmic reticulum. They maintain interconnections with the smooth endoplasmic reticulum and with one another.     

Effects of prolactin on the morphology and function of rat Leydig cells: short-term versus long-term administration

Summary The bolus administration of prolactin (PRL) to adult rats did not cause any apparent change in the basal and luteinizing hormone (LH)-stimulated blood levels of testosterone (as estimated by radioimmune assay). Prolonged PRL infusion did not affect either basal testosterone plasma concentration or the morphology of Leydig cells (as evaluated by electron microscopy and stereology). Conversely, prolonged PRL treatment notably increased the gonadotrophic effects of chronic LH administration; this mainly consisted of a rise in the blood concentration of testosterone and a conspicuous hypertrophy of Leydig cells. The LH-induced increase in the volume of Leydig cells was the result of an increase in the volumes of all the organelles involved in steroid synthesis (i.e., smooth endoplasmic reticulum, peroxisomes and mitochondria). However, the trophic effects of PRL infusion exclusively concerned smooth endoplasmic reticulum and peroxisomes. In the light of these findings, the hypothesis is advanced that the mechanism underlying the gonadotrophic action of PRL involves an enhancement of the endogenous cholesterol synthesis, which could provide an abundance of precursors for testosterone synthesis, the post-cholesterol steps of which, in turn, would be exclusively controlled by LH.     

Contribution of the endoplasmic reticulum to peroxisome formation

How peroxisomes are formed in eukaryotic cells is unknown but important for insight into a variety of diseases. Both human and yeast cells lacking peroxisomes due to mutations in PEX3 or PEX19 genes regenerate the organelles upon reintroduction of the corresponding wild-type version. To evaluate how and from where new peroxisomes are formed, we followed the trafficking route of newly made YFP-tagged Pex3 and Pex19 proteins by real-time fluorescence microscopy in Saccharomyces cerevisiae. Remarkably, Pex3 (an integral membrane protein) could first be observed in the endoplasmic reticulum (ER), where it concentrates in foci that then bud off in a Pex19-dependent manner and mature into fully functional peroxisomes. Pex19 (a farnesylated, mostly cytosolic protein) enriches first at the Pex3 foci on the ER and then on the maturing peroxisomes. This trafficking route of Pex3-YFP is the same in wild-type cells. These results demonstrate that peroxisomes are generated from domains in the ER.     

The smooth endoplasmic reticulum in neurohypophysial axons of the rat: Possible involvement in transport,storage and release of neurosecretory material

Summary The intra-axonal organization of the smooth endoplasmic reticulum was studied in the neurohypophysis of rats during and after water deprivation. Parallel to conventional electron microscopy, the material was treated with a double impregnation staining technique specifically designed to contrast the intracellular membranous system. In conventionally stained ultrathin sections from severely dehydrated rats most axons appeared to be free of membranous organelles, whereas corresponding axons treated with the double-impregnation technique generally exhibited a highly developed system of smooth endoplasmic reticulum. In axonal endings, both techniques revealed a profusion of microvesicles in intimate relationship with tubular elements of the smooth endoplasmic reticulum. In short-term (12 h) rehydrated rats, a similarly developed system of smooth endoplasmic reticulum was still observed at all axonal levels with both procedures. After 24 to 48 h of rehydration the tubules of the smooth endoplasmic reticulum exhibited, in double impregnated material, numerous dilatations which resembled the adjacent neurosecretory granules. In conventionally stained ultrathin sections, an accumulation of electron dense material occurred within tubules of the smooth endoplasmic reticulum in the more proximal axonal segments, while in the more terminal segments, which contained numerous elongated granules, membrane continuity was frequently observed between newly formed granules and the smooth endoplasmic reticulum. After 7 days of rehydration the general pattern of the axonal smooth endoplasmic reticulum was comparable to that in untreated rats. These results are discussed in the light of a suggested involvement of the axonal smooth endoplasmic reticulum in the non-granular transport of neurosecretory material in connection with (1) storage in distally formed granules, and (2) release via microvesicles. Acknowledgements: The authors wish to express their gratitude to Mrs. M. Balmefrézol for her skillful technical assistance     

Pex3p initiates the formation of a preperoxisomal compartment from a subdomain of the endoplasmic reticulum in Saccharomyces cerevisiae

Peroxisomes are dynamic organelles that often proliferate in response to compounds that they metabolize. Peroxisomes can proliferate by two apparent mechanisms, division of preexisting peroxisomes and de novo synthesis of peroxisomes. Evidence for de novo peroxisome synthesis comes from studies of cells lacking the peroxisomal integral membrane peroxin Pex3p. These cells lack peroxisomes, but peroxisomes can assemble upon reintroduction of Pex3p. The source of these peroxisomes has been the subject of debate. Here, we show that the amino-terminal 46 amino acids of Pex3p of Saccharomyces cerevisiae target to a subdomain of the endoplasmic reticulum and initiate the formation of a preperoxisomal compartment for de novo peroxisome synthesis. In vivo video microscopy showed that this preperoxisomal compartment can import both peroxisomal matrix and membrane proteins leading to the formation of bona fide peroxisomes through the continued activity of full-length Pex3p. Peroxisome formation from the preperoxisomal compartment depends on the activity of the genes PEX14 and PEX19, which are required for the targeting of peroxisomal matrix and membrane proteins, respectively. Our findings support a direct role for the endoplasmic reticulum in de novo peroxisome formation.     

Electron microscopic study of the orthograde axonal transport of horseradish peroxidase in the supraoptico-neurohypophysial tract of the rat

Summary Horseradish peroxidase (HRP) has been used as a protein tracer in order to visualize the ultrastructural sites of the orthograde transport of protein macromolecules in the hypothalamo-neurohypophysial tract of the rat. After a local injection of HRP within the supraoptic nucleus, the reaction product was observed: (1) mainly in tubules of the smooth endoplasmic reticulum in the more proximal part of the axons, and (2) in granules and microvesicles of the axon terminals. Observations on thick sections clearly showed the existence of a relationship between the smooth endoplasmic reticulum containing HRP and the labeled granules or microvesicles. These data are in good agreement with previous findings showing the existence of direct continuity between tubules of the smooth endoplasmic reticulum and a fraction of the neurosecretory granules and microvesicles. This evidence further reinforces the hypothesis that the latter organelles may possibly originate locally in the axons from the tubules of the smooth endoplasmic reticulum which may therefore be proposed as a possible vehicle for a non-granular intra-axonal transport of neurosecretory material in neurosecretory neurons.     

THE DEVELOPMENT OF MICROBODIES AND PEROXISOMAL ENZYMES IN GREENING BEAN LEAVES

The ontogeny of leaf microbodies (peroxisomes) has been followed by (a) fixing primary bean leaves at various stages of greening and examining them ultrastructurally, and (b) homogenizing leaves at the same stages and assaying them for three peroxisomal enzymes. A study employing light-grown seedlings showed that when the leaves are still below ground and achlorophyllous, microbodies are present as small organelles (e.g., 0.3 µm in diameter) associated with endoplasmic reticulum, and that after the leaves have turned green and expanded fully, the microbodies occur as much larger organelles (e.g., 1.5 µm in diameter) associated with chloroplasts. Specific activities of the peroxisomal enzymes increase 3- to 10-fold during this period. A second study showed that when etiolated seedlings are transferred to light, the microbodies do not appear to undergo any immediate morphological change, but that by 72 h they have attained approximately the size and enzymatic activity possessed by microbodies in the mature primary leaves of light-grown plants. It is concluded from the ultrastructural observations that leaf microbodies form as small particles and gradually develop into larger ones through contributions from smooth portions of endoplasmic reticulum. In certain aspects, the development of peroxisomes appears analogous to that of chloroplasts. The possibility is examined that microbodies in green leaves may be relatively long-lived organelles.     

Calcium-Induced Ultrastructural Interactions in Adrenocorticocytes

Steroidogenesis in adrenocorticocytes is closely related to intracellular [Ca²⁺]. To detect ultrastructural changes induced by growth in cytosolic [Ca²⁺], we used a rat adrenocortical cell culture, which was examined with electron microscopy and morphometric analysis. We established that either KCl-induced membrane depolarization evoking Ca²⁺ influx into the cell via voltage-operated Ca channels and Ca²⁺ ionophore, A23187, induced remarkable ultrastructural interactions between several cytosolic organelles. Lipid droplets known as key elements for Ca²⁺-induced steroidogenesis directly contacted with organelles containing the enzymes providing steroidogenic reactions (mitochondria, smooth and rough endoplasmic reticulum, nucleus, peroxisomes, and lysosomes). In most cases, the lipid droplets formed a specialized morphological structure at the sites of contact with the partner organelles. These structures are interpreted as a specialized transporting system, which provides contacts between organelles and exchange of intermediate products of the steroidogenesis process between the droplet and organelles.     

Epoxide hydrolase is a marker for the smooth endoplasmic reticulum in rat liver.

Epoxide hydrolase (EH, EC 3.3.2.3) was chosen as a potential marker for smooth endoplasmic reticulum, because this enzyme is inducible by drugs such as phenobarbital. The hypothesis was verified in rat liver using immunochemical and immunocytochemical techniques. Antibodies were raised to the purified protein. These antibodies were affinity purified using the enzyme immobilized on Sepharose Ultrogel. The specificity of the antibodies was assayed by immunoelectrotransfer (Western blot). The labelling of rat liver thin frozen sections with protein A-gold particles demonstrated that the antibodies specifically recognised smooth endoplasmic reticulum membranes. Rough endoplasmic reticulum, other intracellular organelles and plasma membrane were unlabelled.     

Observations on the Leydig cells in the male East African spring hare (Pedetes surdaster larvalis).

The morphology of Leydig cells of the testis of sexually mature and sexually immature spring hares was studied. The cytoplasm of the Leydig of cells the sexually immature spring hares was packed with large lipid droplets leaving little space for the other organelles. Smooth endoplasmic reticulum was poorly developed and occasionally formed concentric layers of fenestrated cisterns around the large lipid droplets. The Leydig of cells the sexually mature spring hares were almost devoid of lipid droplets and their cytoplasm was occupied by abundant tubular smooth endoplasmic reticulum. Cells which shared characteristics with both immature Leydig cells and undifferentiated mesenchymal cells were observed in the limiting membrane of the seminiferous tubulus. These Leydig-like cells may play a role in the differentiation of Leydig cells in the spring hare.     

Mise en evidence d'une activité catalasique dans les peroxysomes de Micrasterias fimbriata (Ralfs)

Summary By ultrastructural methods, cytology, and cytochemistry it is shown that peroxysomes are present during all stages of the life cycle of the green unicellular alga Micrasterias fimbriata, cultivated on mineral medium. These organelles, surrounded by a single membrane, are in connection with endoplasmic reticulum. In full-grown cells, they are preferentially situated near chloroplasts and cell walls. The number of peroxisomes increase before cellular division and the organelles flow into the young bulge in front of the chloroplast.Application of a modified Graham and Karnosky's medium using DAB at pH 9 shows that an important activity of catalase is present not only at the level of peroxisomes but also at the level of the cell walls and certain Golgi vesicles.The topographic relations of peroxisomes with different cellular organelles and their possible functions in cell wall or mucus synthesis are discussed.     

The proliferative response of hepatic peroxidomes of neonatal rats to treatment with SU-13 437 (nafenopin)

The repeated administration of the hypolipidemic agent Su-13 437 (nafenopin) to neonatal rats roughly doubled the number of peroxisomes in the liver tissue and caused a sixfold volumetric expansion of the peroxisomal compartment. During the proliferative response, the size- distribution of the peroxisomes was reversibly altered, enlarged particles appearing in numbers varying according to the dose given. By means of a new method for quantitative autoradiography, it was shown that (a) the concentration of silver grains over peroxisomes was comparable to that found over the endoplasmic reticulum; (b) the peak incorporation of [3H]arginine into the peroxisomes was dealyed in comparison with that into the endoplasmic reticulum; (c) the label, once incorporated into the expanding peroxisomal compartment, displayed the same shift to large particles as did the whole population. These results are compatible with the biosynthetic pathway for peroxisomal catalase proposed earlier (cf. reference 12), and with the notion that the drug-induced size-shift might have resulted from progressive growth of a particular class of peroxisomes formed in the presence of the agent. Evidence is presented to show that during the recovery period the larger peroxisomes are removed preferentially.