Lysosomes in the cessation of vitellogenin secretion by the mosquito fat body; selective degradation of Golgi complexes and secretory granules

A massive and selective degradation of Golgi complexes, secretory granules, and RER is the mechanism responsible for the rapid termination of Vg secretion by trophocytes of the mosquito fat body. These cells are involved in an intensive synthesis of a glycoprotein, vitellogenin (Vg), which is accumulated by developing oocytes as yolk protein. Previously, assays for lysosomal enzymes have demonstrated that the cessation of Vg synthesis is characterized by a sharp increase in lysosomal activity; and fluorescent microscopy has shown that, during this intense lysosomal activity, Vg concentrates in lysosomes. In this report, electron microscopy combined with cytochemistry for lysosomal enzymes and localization of Vg with colloidal gold immunocytochemistry has shown that this lysosomal activity is directed towards selective degradation of Vg and organelles associated with its synthesis and secretion. Three organelles undergo lysosomal breakdown: the Golgi complex, Vg-containing secretory granules, and RER. The degradation of Golgi complexes occurs in two steps similar to that for RER: first, the organelle is sequestered by double isolation membranes, and the resulting pre-lysosome then fuses with a primary or secondary lysosome. In contrast, mature Vg-containing secretory granules fuse with lysosomes directly. This combination of crino- and autophagy is a specific, highly intense, and precisely timed event.     

The secretory pathway of vitellogenin in the fat body of the stick insect bacillus rossius: An ultrastructural and immunocytochemical study

The fat body of the adult female stick insect Bacillus rossius was examined ultrastructurally with a view to clarifying the secretory pathway. The absence of lipid storage in the tissue allowed visualization of a polarized distribution of all organelles in the cell cytoplasm. Composite granules were distributed along the baso-apical axis of the cell according to progressive stages of maturation. At their final stage of maturation, these granules possess two distinct compartments, an electron-translucent compartment and a more electron-dense one. The origin of each of the two compartments was traced back to other organelles in the basal cytoplasm of the fat body cell. The differential origin of the two compartments contributing to the composite granules was further investigated by cytochemical analyses. Vitellogenin was detected both in the electrondense compartment of the composite granules and in the Golgi apparatus. The electron-translucent compartment of the composite granules appeared to consist mainly of urate crystals. Such enzyme activities as acid phosphatase, peroxidase and catalase were also detected in this latter compartment. The observations support the interpretation that secretion in the fat body of B. rossius entails fusion of Golgi-derived vesicles with a specialized kind of multivesicular body. While Golgiderived vesicles convey their load of newly synthesized vitellogenin to the electron-dense compartment, the multivesicular body develops the urate crystals of the electron-translucent compartment.     

Rhipicephalus sanguinius: localization of vitellogenin synthesis by immunological methods and electron microscopy

In ovipositing Rhipicephalus sanguinius (Latrelle), complete immunological identity existed between vitellogenin from the midgut, fat body, and hemolymph and vitellin from eggs. This supported the hypothesis that the same vitellogenin was synthesized by both the midgut and fat body, then released into the hemolymph and transported to the ovary. Vitellogenin was taken up unaltered by the oocytes during vitellogenesis to become vitellin. Antivitellogenin did not react with host (dog) hemoglobin. Transmission electron microscopy showed specialized cells with large amounts of rough endoplasmic reticulum, Golgi complexes, and secretory granules in the midgut and fat body of ovipositing females that were absent in the midgut and fat body of fed males. It is suggested that these cells synthesize vitellogenin.     

Termination of vitellogenin synthesis by mosquito fat body, a programmed response to ecdysterone

ABSTRACT. In the blood-fed mosquito, peak vitellogenin synthesis occurs 24–32 h after the meal, dropping to resting levels by 40 h. Challenging fat body with ecdysterone in vitro at various times after a blood meal demonstrated a refractory period at about 50 h, when there was also a drastic decrease in mitochondria, rough endoplasmic reticulum, ribosomes, and glycogen in fat body cells. When fat bodies from sugar-fed females were incubated with continuous ecdysterone in vitro , vitellogenin synthesis reached a peak at 30 h, but then declined even in the presence of ecdysteroné. This was not due to the in vitro conditions since fat bodies were responsive, even if first exposed to ecdysterone, after 80 h in vitro. If ecdysterone was removed, vitellogenin synthesis ceased. If it was replaced, the fat body responded again only if the initial removal was done during the first 30 h. It is proposed that the falling ecdysterone titre is the major cause of cessation of vitellogenin synthesis, but that synthesis is programmed to decline even if exposure to ecdysterone is abnormally prolonged.     

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.     

Immuno-electron microscopy of formation,degradation and exocytosis of the ovulation neurohormone in Lymnaea stagnalis

Summary The cerebral caudodorsal cells (CDC) of the pulmonate snail Lymnaea stagnalis are involved in the control of egg laying and associated behaviour by releasing various peptides. One of these is the ovulation hormone (CDCH). The cellular dynamics of this peptide have been studied using an antiserum raised to a synthetic portion of CDCH comprising the 20–36 amino acid sequence. With the secondary antibody-immunogold technique, specific immunoreactivity was found in all CDC. Rough endoplasmic reticulum and Golgi apparatus showed very little reactivity as did secretory granules that were in the process of being budded off from the Golgi apparatus. However, secretory granules that were being discharged from the Golgi apparatus, were strongly reactive. Secretory granules within lysosomal structures revealed various degrees of immunoreactivity, indicating their graded breakdown. Large electrondense granules, formed by the Golgi apparatus and thought to be involved in intracellular degradation of secretory material, were only slightly reactive. In the axon terminals secretory granules released their contents into the haemolymph by the process of exocytosis. The exteriorized contents were in most cases clearly immunopositive.The possibility has been discussed that CDCH is cleaved from its polypeptide precursor within secretory granules during granule discharge from the Golgi apparatus; subsequently, the mature secretory granules would be transported towards the neurohaemal axon terminals where they release CDCH into the haemolymph. Superfluous secretory material would be degraded by the lysosomal system including the large electron-dense granules.     

Ultrastructure and histochemistry of the fat body of Anthonomus grandis (Coleoptera: Curculionidae)

Abstract

Metabolic demands such as growth and reproduction of insects relate directly to the fat body. This organ is responsible for the biosynthesis and storage of nutrients, and participates in the neutralization and detoxification of xenobiotics. Studies show a relationship between its ability to accumulate nutrients and activities, such as diapause and reproduction in the beetle Anthonomus grandis, an important pest of cotton plants. However, there are no detailed studies on the structure and histochemistry of the fat body in this insect. We describe the ultrastructure and histochemistry of the fat body in A. grandis and discuss some of its characteristic features. The fat body is composed of a single cell type, the trophocyte, which is a voluminous cell with a spherical or bilobed nucleus, and cytoplasm containing several lipid droplets, glycogen granules (electron-dense), and protein granules. These trophocytes have few areas of contact with each other and form lobes, enclosed by connective tissue. A high metabolic activity of the fat body is deduced by the intense histochemical staining for protein and polysaccharide compounds, demonstrating that specific processes, such as the synthesis and secretion of vitellogenin, could be a target for more specific studies on methods to control this insect.     

Stereological study of gonadotropes in the frog,Rana pipiens,after GnRH stimulation in vitro

Summary Previous physiological results have indicated the existence of two releasable pools of gonadotropins in amphibian pituitaries: an acute releasable pool that appears independent of protein synthesis, and a storage pool involved in chronic release that depends on protein synthesis. To elucidate the ultrastructural localization of these pools and the morphological changes induced in gonadotrope cells after treatment with gonadotropin-releasing hormone, we carried out a morphometric study of immuno-identified gonadotrope cells using an in vitro superfusion system. Treatment with gonadotropin-releasing hormone induced a degranulation of small (110–255 nm) and medium (236–360 nm) secretory granules as well as hypertrophy of the endoplasmic reticulum and Golgi complex. Simultaneous incubation with gonadotropin-releasing hormone and cycloheximide inhibited the release of secretory granules although the endoplasmic reticulum and Golgi complex were hypertrophied. These morphological results strongly suggest: (1) that gonadotropin-releasing hormone induces degranulation and hypertrophy of the biosynthetic machinery in gonadotrope cells; and (2) that the activation of the endoplasmic reticulum and Golgi complex by stimulation with gonadotropin-releasing hormone is independent of protein synthesis, while the release of secretory granules is protein synthesis-dependent. In addition, the second or storage pool of gonadotropin is associated mainly with the small and medium secretory granules.     

A comparative ultrastructural study of ‘glue’ production and secretion of the salivary glands in different species of theDrosophila melanogaster group

Summary Salivary gland cells of members of theDrosophila melanogaster group (from four different subgroups) were examined electron microscopically and histochemically during the late larval period of development. The secretory product, which is supposed to be utilized as glue at the time of puparium formation, appears, by analogy to Palade and Jamieson's results, to be synthesized partially in the rough endoplasmic reticulum (RER) and partially in the Golgi complex. The latter is also the usual site of the packaging of the product into secretory granules, except in the case of one of the secretory granule components ofD. lucipennis. The phylogenetic relationships among the subgroups, implied by the morphological appearance of the secretory granules, fit well with the existing phylogenetic relationships within the group. The secretory granules of each species have their own morphological features; granules of species of the same subgroup share some of these features. Secretion occurs from the cells via exocytosis during which the morphology of the secretory granules changes. Light microscope examination of PAS (Periodic Acid-Schiff reaction) stained glands shows a strong positive reaction in most species, with the exception of the species of thesuzukii subgroup which show a weak, or a negative reaction (D. rajasekari). Electron histochemical localization of polysaccharides in the secretory granules was possible inD. melanogaster and the species of theananassae subgroup.     

Sex determining genes and vitellogenin synthesis in Drosophila melanogaster

Summary This study investigates the relationship between sexual phenotype and ability to synthesize vitellogenin (yolk proteins, YPs) in Drosophila. Various mutations were used to transform XX and XY animals into intersexes or pseudomales (Table 1). The presence or absence of YPs in the haemolymph and in the fat body was determined by SDS gel electrophoresis, fluorography, and precipitation of YPs with anit-YP antibody (see Fig. 1). YPs were synthesized whenever the flies displayed at least some female morphological characteristics, regardless of their sex chromosome constitution (Table 1; Fig. 2). Pseudomales (definition see p. 1) did not produce detectable amounts of YPs despite their female XX-karyotype. Immature ovaries, transplanted into adult males or pseudomales, developed normally and synthesized YPs, but the fat bodies of the host males or pseudomales were not induced to synthesize YPs. Vitellogenesis was, however, induced in the fat bodies of males and pseudomales by injection of 20-hydroxyecdysone (ecdysterone) (Fig. 3). The results are interpreted to mean that the sexual pathways are controlled by a small number of key genes that regulate the synthetic activities of many sex-specific genes. However, the female-specific YP genes can be activated with ecdysterone although the genetic signals are set for male differentiation.     

The role of the fat body, midgut and ovary in vitellogenin production and vitellogenesis in the female tick, Dermacentor variabilis.

Polyclonal antibodies directed against D. variabilis vitellin were utilized for immunocytochemistry at the ultrastructural level. We localized vitellogenin (Vg) in rough endoplasmic reticulum cisternae, secretory granules and secreted products of fat body trophocytes and midgut vitellogenic cells from feeding and ovipositing females. Vg was localized in the oocyte Golgi bodies and in the yolk bodies of both feeding and ovipositing females. Uptake of exogenous Vg was indicated by the presence of immunospecific gold probe in coated pits and coated vesicles at the apical plasma membrane of oocytes from females in rapid engorgement and oviposition. In unmated females little detectable evidence of Vg uptake by developing oocytes suggests that mating and host detachment signal the beginning of vitellogenesis. We conclude that fat body trophocytes, midgut vitellogenic cells and oocytes are involved in the synthesis and/or processing of Vg and that feeding is the signal associated with the initiation of Vg synthesis and/or processing.     

Lectin-binding pattern in parotid acinar cells

Summary The localization of complex carbohydrates in the Golgi apparatus, secretory granules and plasmalemma of mouse parotid acinar cells was studied using the fracture-labelling method. The hexose residues of glycoconjugates were identified using ferritin conjugated with Wheat Germ Agglutinin (WGA-), Ricinnus Communis Agglutinin II (RCA-II-), Phaseolus Vulgaris Agglutinin (PHA-) and Limulus Polyphemus Agglutinin (LPA-). We found that the tracture-labelling method allows not only the labelling of membrane faces but also analysis of the compartment's content that is exposed during the fracturing of the tissue. Our results revealed differences in the hexose residues located in the Golgi apparatus, secretory granules and the apical and lateral plasmalemma. Numerous binding sites for WGA-, PHA-and RCA-II-ferritin were demonstrable in the Golgi apparatus. In secretory granules, the WGA-and RCA-II-ferritin binding sites were most numerous, while LPA-ferritin binding sites were very rate. The density of the binding sites for PHA-ferritin showed considerable variation in secretory granules. The apical plasmalemma exhibited a high density of binding sites for all of the lectins used. In the lateral plasmalemma, LPA-ferritin was not bound, and there were fewer binding sites for WGA-, RCA-H-and PHA-ferritin.     

Ultrastructural visualization of galactosyl residues in various alimentary epithelial cells with the peanut lectin-horseradish peroxidase procedure

Summary A conjugate of peanut lectin with horseradish peroxidase (PL-HRP) has been employed for ultrastructural localization of glycoprotein with presumed terminal galactose residues in mouse alimentary epithelial cells. The PL-HRP conjugate imparted electron opacity in sites that stain at the light microscopic level, as for example, Golgi cisternae in surface epithelial cells of the stomach and in superficial and deep crypt cells and goblet cells of the large intestine. Ultrastructural staining revealed that Golgi cisternae intermediate between the trans and cis faces stained selectively in these sites. Secretion stored in secretory granules or Golgi vesicles in the cells lacked affinity for PL-HRP conjugate, however. Selective staining of intermediate Golgi cisternae in cells with unreactive secretory product is interpreted as indicating the site of galactosyl transferase activity and a location where glactose occurs transitorily as the terminal sugar in the glycoprotein side chains. The luminal aspect of the surface epithelial cells in the stomach and columnar cells in the colon also stained, but with some variability. Staining of these surfaces was considered possibly attributable to PL affinity of some of the secretory glycoprotein which, after absorbing to the cell surface, lost terminal sialic acid through action of luminal enzyme. PL-HRP conjugate stained granules in pancreatic zymogen cells near the block surface but not in other cells, presumably because of limited penetration of reagent. Secretion on the surface of pancreatic acinar cells or in the lumen also exhibited affinity for PL-HRP complex as did the luminal surface of gastric chief cells. Staining of secretion in the pancreatic zymogen cells and gastric chief cells for galactose appeared inconsistent with lack of evidence for presence of glycoprotein in these sites which failed to stain with the periodic acid-Schiff or periodic acid-thiocarbohydrazide-silver proteinate methods for demonstrating glycoprotein at the light and electron microscopic levels. This discrepancy points to possible selective binding of PL-HRP conjugate to a moiety other than terminal galactose of glycoprotein in a few histologic sites. These results demonstrate the applicability of the PL-HRP technique at the ultrastructural level and provide information concerning the chemical structure of epithelial cell glycoproteins and their biosynthesis.     

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.     

Ultracytochemistry of glycoproteins in the eccrine nasolabial glands of the North American raccoon (Procyon lotor)

The distribution and quality of glycoproteins was studied by means of electron microscopic cytochemical methods, particularly lectin cytochemistry, in the secretory cells of the eccrine nasolabial glands of the North American raccoon (Procyon lotor). In the dark and clear glandular cells, complex glycoconjugates were demonstrable, predominantly, in secretory granules, the cisternae of the Golgi apparatus, the surface coat of the plasma membrane, and as glycogen particles. Secretory granules found in the dark cells contained a variety of saccharide residues, such as α-d-mannose, β-d-galactose, β-N-acetyl-d-glucosamine and sialic acid. Several sugars were also detectable in the surface coat of the plasma membrane and the Golgi apparatus.The results obtained may be helpful to understand the specific functions of the glandular secretions of the raccoon nasolabial glands. These could be, particularly, binding of water on the snout surface and protection against microbial hazards, to maintain the structural and functional integrity of the relatively thin snout epidermis in carnivores.     

Ultrastructural changes in the fat body of adult female Aedes aegypti in relationship to vitellogenin synthesis

Summary The ultrastructure of the fat body of Aedes aegypti was followed from emergence through a blood meal. Changes in the volume of protein granules and lipid droplets were also examined. The relationship of these events to the known endocrinology of vitellogenin synthesis in mosquitoes is discussed.Massachusetts Experiment Station Paper No. 2130. Supported by the Massachusetts Agricultural Experiment Station, Project n° 356, and by NIH grant n° AI-11909     

Developmental stages and localization of peroxidatic activity in the leucocytes of three teleost species (Cyprinus carpio L.; Tinca tinca L.; Salmo gairdneri Richardson)

Summary The ultrastructural localization of peroxidase (PO) in the leucocytes of three teleosts (Cyprinus carpio L., Tinca tinca L., Salmo gairdneri R.) has been investigated using the 3,3-diaminobenzidine method. In the heterophilic granulocytes the granules show a species specific structure and are PO-positive at pH 7.6. They can be traced back to small granules arising near the Golgi apparatus (GA) in the promyelocyte. They coalesce to form larger granules and gradually change into the mature type. Myelocytes contain small unreactive granules, and these represent a second granule population. Eosinophils contain one PO-positive granule type (at pH 9), and these granules show a varying density during cell maturation.Basophils are present only in the Cyprinid species, and contain unreactive granules originating from precursors displaying a weakly positive reaction at pH 7.6. The active secretory organelles (RER, GA) are PO-negative, except for a weakly positive reaction in the flocculent matrix of the inner G-cisternae.In promonocytes and monocytes the granules are unreactive, but in the macrophages PO-positive staining occurs in a few small to medium sized granules, and in large vacuoles. At least some of these latter are apparently derived from phagolysosomes containing digested erythrocytes. Thrombocytes and lymphocytes are unreactive.The successive development of PO-positive and negative granule populations in the heterophils, and the PO-reactivity of eosinophils and basophils, show some similarities to the corresponding cells in higher vertebrates, but an analogous PO-positive (azurophil) granule type in monocytes seems to be absent.     

The Golgi apparatus in epidermal mucoid and ellipsoid-vacuolate cells ofAeolidia papillosa andCoryphella rufibranchialis (Nudibranchia)

Summary The epidermal cell layer of the apical end of the ceras was investigated in two species of aeolid nudibranchs. Based on cellular inclusions, mostly two cell types were found: mucoid and ellipsoid-vacuolate cells. Mucoid cells ofCoryphella rufibranchialis have large heterogeneous and fibrillar secretory granules whereas inAeolidia papillosa, the granules are homogeneous, but vary in electron density from one cell to another. Ellipsoid-vacuolate cells contained large quantities of small vacuoles with an included ellipsoidal structure. Both species contained very numerous ellipsoid-vacuolate cells. Secretory granules and ellipsoid-vacuoles appear to arise from the Golgi apparatus and these contents stain with PAS, suggesting a polysaccharide composition. Mucoid cells contained both secretory granules and ellipsoid-vacuoles which may arise from the same Golgi apparatus.     

Clathrin-coated vesicular transport of secretory proteins during the formation of ACTH-containing secretory granules in AtT20 cells

We have studied by electron microscopy and immunocytochemistry the formation of secretory granules containing adrenocorticotropic hormone (ACTH) in murine pituitary cells of the AtT20 line. The first compartment in which condensed secretory protein appears is a complex reticular network at the extreme trans side of the Golgi stacks beyond the TPPase-positive cisternae. Condensed secretory protein accumulates in dilated regions of this trans Golgi network. Examination of en face and serial sections revealed that "condensing vacuoles" are in fact dilations of the trans Golgi network and not detached vacuoles. Only after presumptive secretory granules have reached an advanced stage of morphological maturation do they detach from the trans Golgi network. Frequently both the dilations of the trans Golgi network containing condensing secretory protein and the detached immature granules in the peri-Golgi region have surface coats which were identified as clathrin by immunocytochemistry. Moreover both are the site of budding (or fusion) of coated vesicles, some of which contain condensed secretory protein. The mature granules below the plasma membrane do not, however, have surface coats. Immunoperoxidase labeling with an antiserum specific for ACTH and its precursor polypeptide confirmed that many of the coated vesicles associated with the trans Golgi network contain ACTH. The involvement of the trans Golgi network and coated vesicles in the formation of secretory granules is discussed.