An autoradiographic analysis of vitellogenin synthesis and secretion in the fat body of the stick insect Bacillus rossius

The fat body of the stick insect Bacillus rossius was studied with a view to clarifying the metabolic pathway leading to secretion of vitellogenin (VG). Electrophoretic analysis of ovarian follicles and hemolymph from egg-laying females showed that the two tissues shared a common polypeptide composition consisting of five major polypeptides with molecular weights ranging from 60-180 Kd. Following in vivo exposure to [(35)S]-methionine for up to 24 h, these polypeptides were labeled in a stage- and time-dependent manner, suggesting that they were transferred from the hemolymph to the oocyte during vitellogenesis. Fat body pulse-labeled with [(35)S]-methionine for up to 240 min and immunoprecipitated with an anti-yolk serum was labeled only in a fraction containing high molecular weight polypeptides. We presume these polypeptides to be VG precursors bearing a precursor-product relationship with the five major polypeptides of the hemolymph and developing ovarian follicles. Fat body exposed in vivo to [(3)H]-leucine for time intervals ranging from 20-240 min were processed for EM autoradiography. The results of this analysis showed that incorporated radioactivity was progressively transferred from the endoplasmic reticulum to the Golgi apparatus and from there to the composite granules. The data provided in this study are consonant with previous findings by which composite granules were shown to contain two compartments differing both in content and origin. In addition, the autoradiographical data of in vivo labeled fat body demonstrate that only the material partitioned into the electron-dense compartment of these granules is exocytosed.     

A fine structural survey of the development of the adult fat body of Leptinotarsa decemlineata

The fat body of a female Colorado potato beetle, Leptinotarsa decemlineata, at adult ecdysis, contains a large number of protein granules which are composed of light and dark zones. Part of the light zone in some of these granules is believed to be urate. During the first two days after adult ecdysis, fat body development is not essentially different in females reared either under long- or short-day conditions. Protein granules and large vacuoles disappear and the first cell organelles are regenerated. The effect of the photoperiod on the histological structure of the fat is expressed after these events. In females reared under long-day conditions, the fat body becomes specialized for vitellogenin synthesis. Under short-day conditions, the fat body stores massive amounts of lipid until day 6 after adult ecdysis. Then the first electron-dense protein granules develop near the nucleus, and on day 10 the first autophagic vacuoles are seen. These structure changes are discussed in connection with the known biochemical properties of the adult faty body of Leptinotarsa.     

Fine Structure of the Ovarian Development in the Orb-web Spider, Nephila clavata

ABSTRACT Fine structural changes of the ovary and cellular composition of oocyte with respect to ovarian development in the orb-web spider, Nephila clavata were examined by scanning and transmission electron microscopy. Unlike the other arthropods, the ovary of this spider has only two kinds of cells-follicle cells and oocytes. During the ovarian maturation, each oocyte bulges into the body cavity and attaches to surface of the elongated ovarian epithelium through its peculiar short stalk attachments. In the cytoplasm of the developing oocyte two main types of yolk granules, electron-dense proteid yolk and electron-lucent lipid yolk granules, are compactly aggregated with numerous glycogen particles. The cytoplasm of the developing oocyte contains a lot of ribosomes, poorly developed rough endoplasmic reticulum, mitochondria and lipid droplets. These cell organelles, however, gradually degenerate by the later stage of vitellogenesis. During the active vitellogenesis stage, the proteid yolk is very rapidly formed and the oocyte increases in size. However, the micropinocytosis invagination or pinocytotic vesicles can scarcely be recognized, although the microvilli can be found in some space between the oocyte and ovarian epithelium. During the vitellogenesis, the lipid droplets in the cytoplasm of oocytes increase in number, and become abundant in the peripheral cytoplasm close to the stalks. On completion of the yolk formation the vitelline membrane, which is composed of an inner homogeneous electron-lucent component and an outer layer of electron-dense component is formed around the oocyte.     

Fine Structures of Oocytes and Accessory Cells of the Ovary in the Starfish Patiria (Asterina) pectinifera at Different Stages of Oogenesis and After 1-Methyladenine-Induced Maturation

Morphological changes in the growing and maturing oocytes of Patiria ( Asterina ) pectinifero were studied by electron microscopy. Oogenesis is of the solitary type. An extensive system of rough endoplasmic reticulum (ER) and Golgi complex (GC) develops in the ooplasm forming the cortical, yolk and secretory granules in its peripheral regions. The contents of the latter granules are released from the oocyte and form the vitelline membrane. At early stages of oogenesis, extensive multiplication of mitochondria results in formation of a large aggregate of these organelles in the perinuclear cytoplasm ("yolk nucleus"). After maturation of full grown oocytes has been induced by 1-methyladenine, the membranous cell structures are rapidly rearranged: vast aggregates of ER cisternae in the surface cytoplasm layer and single ER cisternae among yolk granules are disintegrated to small vesicles; the GC is reduced. These processes are suggested to be somehow related to changes in hydration of the cytoplasm and in rigidity of its surface layer. In maturing oocytes, the yolk granules form characteristic linear rows, trabeculae, traversing the cytoplasm and their boundary membranes fuse in zones of contact. Some granules are converted to multivesicular bodies, thus suggesting the activation of hydrolytic enzymes that form part of the yolk in echinoderms.     

Receptor-mediated endocytosis of storage proteins by the fat body of Helicoverpa zea

The selective uptake of storage proteins by the fat body of the corn earworm Helicoverpa zea is mediated by a membrane-bound receptor protein. In this study, the major storage proteins of this insect species, arylphorin and very high density lipoprotein, were directly labeled with colloidal gold-particles of different size. After the fat body had been incubated with the labeled storage proteins, the distribution of these proteins was examined by electron microscopy. Both storage proteins were found at the extracellular side of coated pits and within coated vesicles. Moreover, fusion products of several coated vesicles such as endosomes and multivesicular bodies contained both proteins in their lumen. Ultimately, the proteins accumulated in electron-dense storage granules. Equal numbers of either storage protein were present in each organelle, supporting the notion that a single receptor mediates the uptake of both proteins. In contrast, only small numbers of gold-labeled immunoglobulin G molecules were found in the organelles, indicating that the protein uptake is specific for storage proteins. The results show that storage protein uptake in this lepidoteran species occurs in a process of receptor-mediated endocytosis that is similar to the well-established uptake of specific proteins into mammalian tissues.     

Electron microscopic and pharmacological studies on the rat median eminence

Summary The rat median eminence contains at least three kinds of granules or vesicles: 1. large electron-dense granules (perhaps carriers of neurohypophysial hormones), 2. small electron-dense granules with or without haloes (perhaps carriers of catecholamines) and 3. synaptic vesicle-like structures (perhaps carriers of acetylcholine). The former two electrondense granules exist in separate axons but they coexist with the latter vesicles in the same axons.The pars nervosa shows basically a similar structure to the median eminence. However, the axons containing the small electron-dense granules are very few. In the pars tuberalis, there are at least two types of cells: the cells of one type contain much cytoplasm with large round nuclei and those of the other type contain a small amount of cytoplasm with polymorphic nuclei. The cells of the former include multivesicular bodies and secretory granules, but those of the latter do not. Some of capillaries of the primary plexus are surrounded by the cells of the pars tuberalis on one side and by neurosecretory axon endings on the other side.The median eminence contains high concentration of acetylcholine or an acetylcholine-like substance and shows neurohypophysial hormone activity.Aided by Grant A-3678 from the United States National Institute of Arthritis and Metabolic Diseases. The authors are indebted to Dr. Welsh, Harvard University, for the kind gift of Mytolon.     

Ultrastructural studies on the formation of yolk granules in the statoblast of a fresh-water bryozoan,Pectinatella gelatinosa

The formation of protein-carbohydrate yolk in the statoblast of a fresh-water bryozoan, Pectinatella gelatinosa, was studied by electron microscopy. Two types (I and II) of yolk cells were distinguished. The type I yolk cells are mononucleate and comprise a large majority of the yolk cells. The type II yolk cells are small in number; they become multinucleate by fusion of cells at an early stage of vitellogenesis. In both types of yolk cells, electron-dense granules (dense bodies) are formed in Golgi or condensing vacuoles, which are then called yolk granules. For the formation of yolk granules, the following processes are considered: 1. Yolk protein is synthesized in the rough-surfaced endoplasmic reticulum (RER) of the yolk cells. 2. The synthesized protein condenses in the cisternal space of the RER and is packaged into small oval swellings, which are then released from the RER as small vesicles (Golgi vesicles, 300-600 A in diameter). 3. The small vesicles fuse with one another to form condensing vacuoles, or with pre-existing growing yolk granules. 4. In the matrix of the condensing vacuoles or growing yolk granules, electron-dense fibers are fabricated and then arranged in a paracrystalline pattern to form the dense body. 5. After the dense body reaches its full size, excess membrane is removed and eventually the yolk granules come to mature. Toward the end of vitellogenesis of the yolk cells, the cytoplasmic organelles are ingested by autophagosomes derived from multivesicular bodies and disappear.     

In Vivo Study of Vitellogenin-Gold Transport in the Ovarian Follicle and Oocyte of Xenopus laevis

The transport of injected vitellogenin (VTG)-gold in the ovarian follicle and developing oocyte in Xenopus is described. The gold particles reached the extracellular spaces of the theca and interfollicular spaces within 1 and 2 hr, respectively, after a tracer injection at 20°C. The tracers moved through channels between the constitutive cells of both the capillary endothelium and the follicle cell layer.
Compartments in the peripheral cytoplasm of vitellogenic oocytes at stage IV, which relate to yolk formation, seemed to be segregated as follows: (a) internalization compartment consisting of coated pits and vesicles of the oolemma covering the oocyte "macrovilli", (b) transport compartment of endosomes and multivesicular endosomes in the oocyte cortex, and (c) crystallization compartment of primordial yolk platelets (PYP) in the sub-cortical region. The gold particles appeared in the internalization and transport compartments at 3–6 hr after the tracer injection and in the cystallization compartment at 12–18 hr. The VTG, internalized by receptor-mediated endocytosis, was transferred from coated vesicles to multivesicular endosomes by vesicle-to-vesicle fusion. VTG crystallization took place in globular-shaped PYPs of about 1 μm. At 24 hr after the tracer injection, the gold particles appeared in completely crystallized yolk platelets, most of them clustered in the superficial layer and some integrated into the crystals.     

On vesicles and membrane compartments

Summary Two different mechanisms have been proposed to explain transport along the endocytic and biosynthetic transport routes in cells. The first involves stable compartments connected by vesicular traffic while the second argues that the key organelles (early endosomes or the cis Golgi) form de novo by fusion of vesicles and subsequently mature into later forms. In the first part of this article, I propose a classification that distinguishes between stable, preexisting membrane compartments and vesicles that are, by definition, transient organelles. In this scheme, compartments, but not vesicles, are capable of homotypic fusion while vesicles, but not compartments, are able to mature, a process defined as an irreversible set of biochemical events which lead to a physiologically distinct end-state of the vesicle prior to its vectorial fusion with a target compartment. In the second part, I summarize my current ideas about the ultrastructural organization of the ER-Golgi region. Finally, I review the cell biology of selected examples of different vesicle types in order to exemplify the fascinating diversity of functions that this class of membrane organelles has evolved.Abbreviations COP coatomer - ECV endosome carrier vesicle - ER endoplasmic reticulum - HRP horseradish peroxidase - IC intermediate compartment between ER and Golgi - MVB multivesicular body - NSF N-ethyl maleimide sensitive factor - SNAPS soluble NSF associated proteins - TGN trans Golgi network Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Dynamics of the redistribution of pigment granules in the dermal melanophores of anuran amphibians. 1. Dispersion

The dispersion of melanosomes in the dermal melanophores of the Xenopus laevis larvae has been studied by time--lapse cinematography. The process began with the appearance of distally directed melanosome flows in the cell cytoplasm. During the subsequent migration of pigment granules, the flows branched forming branches of the 2nd and higher orders. The whole cytoplasm became filled with a layer of melanosomes. During the dispersion, the movement of melanosomes in a flow is replaced by their dispersion all over the cytoplasm; these processes alternated. In the peripheral part of the cell devoid of melanosomes, membrane vesicles appeared and the cytoplasm was distinctly divided into ecto- and endoplasm. The ectoplasm contained numerous microfilaments and single microtubules, the endoplasm did not contain any cell organelles, except single electron-dense melanosomes. The active role of plasma membrane in the intracellular movement of melanin granules is suggested.     

Ferromagnetic isolation of endosomes: a novel method for subcellular fractionation of Xenopus oocytes

A novel method has been developed using ferric particles to label endosomes, and to achieve magnetic sorting of the various endocytic compartments involved in lipoprotein uptake into cells. Ferric particles conjugated to a receptor-recognized ligand are bound to coated membrane pits and become internalized into the cytoplasm inside coated vesicles. After apparent fusion of the vesicles to tubular endosomes, the conjugates accumulate and finally discharge into multivesicular endosomes. Pulse-chase experiments elucidate the pathway of internalized conjugates and allow both early compartments (pinosomes and tubular endosomes) and late compartments (multivesicular endosomes and storage organelles) to be selectively labelled. After ferroloading of the various transport compartments, the cells are homogenized and subcellularly fractionated. Sorting of labelled endosomes is performed by a specially designed "free-flow" magnetic chamber. Prophase I-arrested oocytes of the toad Xenopus laevis are used as a model system for studying the transport pathway and the conversion of the yolk precursor vitellogenin. It is possible to follow the route of internalization of vitellogenin-iron conjugates via coated pits, coated vesicles, uncoated vesicles, tubular endosomes, multivesicular endosomes, and light primordial yolk platelets. These endosomes shuttle the ferric particles together with the vitellogenin from oolemma to performed heavy yolk organelles which are still growing. In addition, these various compartments can be isolated according to their function and subjected to electron microscopy and to gel electrophoresis for detailed characterization of their limiting membranes as well as their contents.     

Cysteine proprotease colocalizes with vitellogenin in compound granules of the cockroach fat body

A cysteine proprotease has been identified in developing embryos of the cockroach Blattella germanica and found to be a maternally encoded gene product that is transferred endocytically to the oocyte. The present study aims at establishing how this maternally derived proprotease is synthesized, packaged, and secreted during vitellogenesis. To this end, proprotease was localized immunocytochemically in the fat body of postmating females and its localization compared with that of vitellogenin over the same developmental periods. Fat bodies in cockroaches are comprised of two different cell types: trophocytes and bacteriocytes. Data show that proprotease and vitellogenin come to colocalize in compound granules of the fat body trophocytes. While synthesis of vitellogenin can be traced back to granules resulting from the coalescence of Golgi-derived vesicles in the trophocyte cytoplasm, proprotease appears to be localized predominantly on the cytolysosomes of both trophocytes and bacteriocytes. When probed with an anti-proprotease antiserum, bacteria are also positively labeled, regardless of whether they are segregated inside the cytolysosomes or free in the bacteriocyte cytoplasm. Since vitellogenin and proprotease colocalize within the same cell organelle, it is assumed that Golgi-derived vesicles, which contain vitellogenin, may fuse with cytolysosomes bearing proprotease to yield compound secretory granules. To account for the present observations, the origin and role of proprotease are discussed in relation to the turnover of bacteria in the fat body and to the requirements of endosymbiosis.     

Precursors of monoamine-storage organelles in developing megakaryocytes of the rat

Summary Identification and distribution of the precursors of aminestorage organelles in rat megakaryocytes during cell maturation were studied, using the uranaffin reaction for adenine nucleotide. The precursors of the amine-storage organelles appeared as 200–300 nm vesicles having an uranaffin electron dense granule, whereas they appeared as empty vesicles by conventional glutaraldehyde-OsO₄ fixation. X-ray probe microanalysis confirmed the existence of U and P in the uranaffin reaction positive vesicles. The precursors appeared in the immature megakaryocytes, especially at the trans(mature) face of the Golgi apparatus, and rapidly increased in number in the maturing cells. The size of the uranaffin granules in the precursory organelles increased gradually during cell maturation and became almost equivalent to the dense body of blood platelets in the final stage of cell maturation.     

Can sieve-element plastids in Panicum maximum (Poaceae) leaves act in the blockage of injured sieve-tube elements?

The ultrastructural features and the plastid changes caused by sample preparation were studied in sieve elements of Panicum maximum leaves. Samples of expanded leaves, taken near the ligule region, were fixed and processed by common light and transmission electron microscopy methods. In mature sieve-tube elements, the protoplast is electron-translucent and plastids are the most frequent organelles. Mitochondria and smooth endoplasmic reticulum segments are also visible and occupy a parietal position within the cell. The plastids are globular and show electron-dense proteinaceous inclusions in the stroma. The protein crystals are predominantly cuneate, but thin crystalloids and amorphous and/or filamentous proteins also occur. The presence of intact plastids plus others in different phases of plastid envelope rupture were interpreted as evidence that this rupture is a normal event in response to injury. This plastid envelope rupture is possibly activated by the release of pressure in the sieve-tube element. After plastid membrane vesiculation, the stroma and the protein crystals are dispersed within the sieve-element ground cytoplasm. The vesicles originating from the plastid envelope move to one cell pole, while protein crystalloids move to the opposite pole and agglomerate in the sieve-plate region. Our findings indicate that these protein crystalloids, which deposit in the sieve plate, may act in sieve-plate pores occlusion, preventing the release of phloem sap, similar to the role of P-protein in dicotyledons.     

Dorsal lingual epithelium of Platemys pallidipectoris (Pleurodira,Chelidae)

Scanning electron microscopy reveals that the flat tongue of Platemys pallidipectoris has shallow grooves and no lingual papillae. The surface of the tongue is covered with dome-shaped bulges, each corresponding to a single cell. Short microvilli are distributed over the cell surface. Light microscopy shows a stratified cuboidal epithelium with an underlying strong connective tissue. Transmission electron microscopy indicates four layers. The basal cells of the epithelium are electron-translucent and have a large central nucleus and a cytoplasm with keratin tonofilaments. Plasma cells with abundant rough endoplasmic reticulum and mitochondria occur in the basal layer. Production of secretory granules begins in the more electron-dense intermediate layers and increases as the cells move toward the surface. The membranes of the cells of the deep intermediate layer form processes that project into relatively wide intercellular spaces. In the superficial intermediate layer, the cytoplasm of the cells contains numerous fine granules; these increase in number but not in size in more distal layers. The cells of the surface layer are electron-translucent with a round nucleus. Contents of their fine granules are secreted into the oral cavity. © 1995 Wiley-Liss, Inc.     

The cytology of the testaceous rhizopod Lesquereusia spiralis (Ehrenberg) Penard. I. Ultrastructure and shell formation

Ultrastructure and shell formation in the testaceous ameba, Lesquereusia spiralis, were investigated with both scanning and transmission electron microscopy and X-ray microanalysis. The nucleus, surrounded by a fibrous lamina, contains multiple nucleoli. The cytoplasm, containing a well developed granular endoplasmic reticulum, also contains remnants of starch granules in stages of digestion. Spherical aggregates of ribosome-like particles may be seen. Golgi complexes seem to produce both a nonordered fibrous material and an electron dense vesicle. Only the latter appears to bleb off from the Golgi complex. X-ray microanalysis demonstration of silicon in Golgi vesicles and in some dense vesicles suggests that the fibrous component of the cisternae may take up and concentrate silica to form the electron-dense component of the vesicles. Membrane-bound siliceous crystals are often seen adjacent to the Golgi, suggesting either a Golgi origin or platelet formation in vesicles after release from the Golgi complex. Both electron-dense bodies and siliceous platelets are released from the cell by a process similar to apocrine secretion and may be seen outside the cell in route to the shell during shell morphogenesis. Shell development involves fusion of electron-dense bodies to form a matrix, positioning of siliceous platelets in this matrix parallel to the shell surface, and development of a system of matrix chambers. A particulate glycoconjugate is released to the shell surface upon rupture of the matrix chamber.     

The lytic granules of natural killer cells are dual-function organelles combining secretory and pre-lysosomal compartments

Cytolytic lymphocytes contain specialized lytic granules whose secretion during cell-mediated cytolysis results in target cell death. Using serial section EM of RNK-16, a natural killer cell line, we show that there are structurally distinct types of granules. Each type is composed of varying proportions of a dense core domain and a multivesicular cortical domain. The dense core domains contain secretory proteins thought to play a role in cytolysis, including cytolysin and chondroitin sulfate proteoglycan. In contrast, the multivesicular domains contain lysosomal proteins, including acid phosphatase, alpha-glucosidase, cathepsin D, and LGP-120. In addition to their protein content, the lytic granules have other properties in common with lysosomes. The multivesicular regions of the granules have an acidic pH, comparable to that of endosomes and lysosomes. The granules take up exogenous cationized ferritin with lysosome-like kinetics, and this uptake is blocked by weak bases and low temperature. The multivesicular domains of the granules are rich in the 270-kD mannose-6-phosphate receptor, a marker which is absent from mature lysosomes but present in earlier endocytic compartments. Thus, the natural killer granules represent an unusual dual-function organelle, where a regulated secretory compartment, the dense core, is contained within a pre-lysosomal compartment, the multivesicular domain.     

Liver ultrastructure and a new cell type in the Japanese newt, Cynops pyrrhogaster.

The liver of the Japanese newt, Cynops pyrrhogaster, has been investigated using light, scanning, and transmission electron microscopy. Hepatic parenchyma was composed of clusters and cords or tubules of polyhedral cells separated by a sinusoidal net. Hepatocytes had spherical, euchromatic nuclei with one or more nucleoli and stacked mitochondria with sparse cristae and dense bodies. Rough endoplasmic reticula formed peribiliary stacks and diffusely scattered vesicles and tubules. Smooth endoplasmic reticula were more pronounced in glycogen-rich hepatocytes. Most hepatocytes contained peroxisomes, Golgi complexes and large numbers of fat droplets within the cytoplasm along with glycogen. Some cells were mainly glycogen-storing and contained few or no fat droplets. A special feature of the newt liver was biliary atresia. Bile canaliculi had short, stout microvilli which were entirely atretic in some canaliculi. Canaliculi were sealed off by junctional complexes including zonulae occludentes and maculae adherentes. The latter showed extraordinary wider desmosomal gaps in the vicinity of the atretic bile canaliculi. The sinusoid wall was non-distinctive and contained fenestrated endothelial cells connected to Kupffer cells by zonulae occludentes. A distinctive new cell type (OG cell) was observed in the newt liver. These cells were found individually or in small clusters in proximity with the sinusoidal surfaces. They had small nuclei, a paucity of cytoplasmic organelles, but numerous, unique, osmiophilic granules of two distinct types. Less numerous Type I granules contained homogeneous electron-dense material, and a predominant Type II granule contained circumferentially arranged subparticulation. Granules of both types were detected within the cytoplasm of endothelial cells and within sinusoids together with blood elements. The function of this secretory type cell remains obscure, though it may represent a stage of melanophore.     

Immunocytochemical localization of pleurocidin to the cytoplasmic granules of eosinophilic granular cells from the winter flounder gill

The teleost gill is considered to be of significant immunological importance, as it is one of the first tissues exposed to environmental or pathogenic challenge and thus should be well equipped to mount an effective immune response. This study characterizes ultrastructurally and immunocytochemically a tissue granulocyte (eosinophilic granular cell) from the winter flounder gill that was previously determined to be involved in the gene expression and synthesis of a known antimicrobial peptide (pleurocidin). The cell is irregular in shape with a cytoplasm characterized by numerous large, electron-dense, membrane-bounded granules. The nucleus is euchromatic and closely associated with a prominent rough endoplasmic reticulum. The cytoplasm typically contains two to three mitochondria and a centralized Golgi apparatus surrounded by numerous electron-lucent vesicles. Immunogold staining of the cells with an anti-pleurocidin antibody shows large number of gold particles in direct association with the electron-dense granules. These data provide the first evidence definitively showing storage of an antimicrobial peptide in the cytoplasmic granules of an eosinophilic granule cell resident in gill tissue.