Number and size of intracisternal granules in the adipokinetic hormone-secreting cells of locusts: a phase-dependent characteristic

Abstract.The intracisternal (= ergastoplasmic) granules in the adipokinetic hormone-secreting cells of the glandular lobes of the corpora cardiaca in Locusta migratoria migratorioides represent accumulations of adipokinetic prohormones within cisternae of the rough endoplasmic reticulum. Solitary locusts have more and larger intracisternal granules than gregarious locusts. This coincides with the general locomotor activity and thus the energy metabolism in solitary locusts being quite different from that of gregarious locusts, which apparently has consequences for the amounts of adipokinetic hormones synthesized and/or released and, consequently, for the storage of these hormones in the intracisternal granules. These granules apparently function as supplementary stores of secretory material.     

Differential location of peptide hormones in the secretory pathway of insect adipokinetic cells

Immunoreactivity of granules containing secretory material in the adipokinetic cells of the insect Locusta migratoria was studied using antisera specific for the adipokinetic hormone-associated peptides (AAP) I, II and III. Immunocytochemical detection of these associated peptides represents a new strategy for studying the intracellular location of the adipokinetic hormones and their prohormones. Fixation with 2% glutaraldehyde and 2% formaldehyde with low-temperature embedding in Lowicryl HM20 allowed highly selective immunogold labelling of both secretory and intracisternal granules. All three associated peptides were co-localized in secretory granules. This indicates that also all three adipokinetic hormones can be co-localized in these granules, which was confirmed by experiments in which, after secretory stimulation, adipokinetic hormone III was released from the adipokinetic cells together with adipokinetic hormones I and II. The immunopositivity of the intracisternal granules was similar to that of the secretory granules, although with the exception that the intracisternal granules did not show any specific reaction with anti-AAP III. The presence of AAP I and AAP II in intracisternal granules indicates that these granules only function as stores of adipokinetic prohormones I and II and not of adipokinetic prohormone III. The observed differences in storage in intracisternal granules among the three adipokinetic prohormones suggest differences in physiological significance of the three adipokinetic hormones in L. migratoria.     

Ageing adipokinetic cells in Locusta migratoria: an ultrastructural morphometric study

Summary A morphometric study was made of the ultrastructure of adipokinetic cells in resting adults of Locusta migratoria at 3, 23, and 43 days after imaginal ecdysis. The nucleus, rough endoplasmic reticulum, and Golgi apparatus enlarge with age, which indicates that the synthesis and packaging of secretory substances increases during ageing. The size of the storage compartment, consisting of secretory and ergastoplasmic granules, does not increase earlier than 23–43 days after imaginal ecdysis. The lysosomal compartment markedly enlarges between 3 and 23 days; later on, the growth of this compartment, especially of autophagosomes, is less prominent. This suggests that lysosomal destruction initially compensates for the production of new secretory granules, assuming that exocytosis of secretory granules by adipokinetic cells is insignificant in resting locusts. Afterwards, lysosomal destruction may no longer be sufficient to prevent over-production of secretory granules, as is suggested by the increase in the number of these granules between 23 and 43 days. This coincides with the appearance of a considerable number of large ergastoplasmic granules, which represent a spatially more efficient form of storage of secretory material than the much smaller secretory granules. The increase with age in the amount of secretory products indicates that the biosynthetic activity of the adipokinetic cells is not (finely) tuned to their releasing activity.     

Insect adipokinetic hormones: release and integration of flight energy metabolism

Insect flight involves mobilization, transport and utilization of endogenous energy reserves at extremely high rates. Peptide adipokinetic hormones (AKHs), synthesized and stored in neuroendocrine cells, integrate flight energy metabolism. The complex multifactorial control mechanism for AKH release in the locust includes both stimulatory and inhibitory factors. The AKHs are synthesized continuously, resulting in an accumulation of AKH-containing secretory granules. Additionally, secretory material is stored in large intracisternal granules. Although only a limited part of these large reserves appears to be readily releasable, this strategy allows the adipokinetic cells to comply with large variations in secretory demands; changes in secretory activity do not affect the rate of hormone biosynthesis. AKH-induced lipid release from fat body target cells has revealed a novel concept for lipid transport during exercise. Similar to sustained locomotion of mammals, insect flight activity is powered by oxidation of free fatty acids derived from endogenous reserves of triacylglycerol. However, the transport form of the lipid in the circulatory system is diacylglycerol (DAG) that is delivered to the flight muscles associated with lipoproteins. While DAG is loaded onto the multifunctional insect lipoprotein, high-density lipophorin (HDLp) and multiple copies of the exchangeable apolipoprotein III (apoLp-III) associate reversibly with the expanding particle. The resulting low-density lipophorin (LDLp) specifically shuttles DAG to the working muscles. Following DAG hydrolysis by a lipophorin lipase, apoLp-III dissociates from the particle, regenerating HDLp that is re-utilized for lipid uptake at the fat body cells, thus functioning as an efficient lipid shuttle mechanism. Many structural elements of the lipoprotein system of insects appear to be similar to their counterparts in mammals; however, the functioning of the insect lipoprotein in energy transport during flight activity is intriguingly different.     

Ultrastructural enzyme-cytochemical study of the intrinsic glandular cells in the corpus cardiacum of Locusta migratoria: relation to the secretory and endocytotic pathways,and to the lysosomal system

Summary Ultrastructural aspects of the secretory and the endocytotic pathways and the lysosomal system of corpus cardiacum glandular cells (CCG cells) of migratory locusts were studied using morphological, marker enzyme, immunocytochemical and tracer techniques. It is concluded that (1) the distribution of marker enzymes of trans Golgi cisternae and trans Golgi network (TGN) in locust CCG cells corresponds to that in most non-stimulated vertebrate secretory cell types; (2) the acid phosphatase-positive TGN in CCG cells is involved in sorting and packaging of secretory material and lysosomal enzymes; (3) these latter substances are produced continuously; (4) at the same time, superfluous secretory granules and other old cell organelles are degraded; (5) the remarkable endocytotic activity in the cell bodies and the minor endocytotic activity in cell processes are coupled mainly to constitutive uptake of nutritional and/or regulatory (macro)molecules, rather than to exocytosis; (6) plasma membrane recycling occurs mainly by direct fusion of tubular endosomal structures with the plasma membrane and little traffic passes the Golgi/TGN; and (7) so-called cytosomes arise mainly from autophagocytotic vacuoles and represent a special kind of complex secondary lysosomes involved in the final degradation of endogenous (cell organelles) and exogenous material.     

Coherence between biosynthesis and secretion of insect adipokinetic hormones

The importance of the process of continuous biosynthesis of locust adipokinetic hormones (AKHs) for the availability of these peptide hormones for release was assessed in vitro by inhibiting this biosynthesis followed by secretory stimulation. Inhibition of the biosynthetic activity for AKHs by brefeldin A caused a considerable inhibition of the AKH release induced by the endogenous crustacean cardioactive peptide (CCAP). After brefeldin A treatment followed by potassium depolarization, CCAP-induced AKH release was completely abolished. In vitro pulse-chase labeling experiments indicated that constitutive secretion from the AKH-producing cells does not occur. It is concluded that AKH secretion involves a regulated release from a relatively small pool of newly formed secretory granules, while older AKH-containing granules appear to be unavailable for release.     

Demonstration of acid phosphatase activity induced by 20-hydroxyecdysone in the fat body of Calliphora

In the larval fat body of Calliphora erythrocephala, protein accumulation and autophagic activity occur prior to the onset of puparium formation. The involvement of the lysosomal system in the degradation of sequestered protein and cell organelles can be demonstrated by the electron-microscopical cytochemical localization of the lysosomal marker enzyme acid phosphatase in so-called protein granules. These granules contain not only newly synthetized or absorbed protein but also remnants of cell organelles such as mitochondria and endoplasmic reticulum. Ligation of the larvae behind the brain-ring gland complex prevents the appearance of these acid phosphatase-positive granules. They can be induced in ligated larvae by the injection of 20-hydroxyecdysone into the abdomen. These findings are briefly discussed in relation to the role of moulting hormones in normal development, especially with regard to the induction of autophagic activity.     

Secretory granule formation and membrane recycling by the trans-Golgi network in adipokinetic cells of Locusta migratoria in relation to flight and rest

The influence of flight activity on the formation of secretory granules and the concomitant membrane recycling by the rans-Golgi network in the peptidergic neurosecretory adipokinetic cells of Locusta migratoria was investigated by means of ultrastructural morphometric methods. The patterns of labelling of the trans-Golgi network by the exogenous adsorptive endocytotic tracer wheat-germ agglutinin-conjugated horseradish peroxidase and by the endogenous marker enzyme acid phosphatase were used as parameters and were measured by an automatic image analysis system. The results show that endocytosed fragments of plasma membrane with bound peroxidase label were transported to the trans-Golgi network and used to build new secretory granules. The amounts of peroxidase and especially of acid phosphatase within the trans-Golgi network showed a strong tendency to be smaller in flight-stimulated cells than in non-stimulated cells. The amounts of acid phosphatase in the immature secretory granules originating from the trans-Golgi network were significantly smaller in stimulated cells. The number of immature secretory granules positive for acid phosphatase tended to be higher in stimulated cells. Thus, flight stimulation of adipokinetic cells for 1 h influences the functioning of the trans-Golgi network; this most probably results in a slight enhancement of the production of secretory granules by the trans-Golgi network.     

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.     

Internal defenses of the snail Biomphalaria glabrata.

We describe three distinct types of cells among Biomphalaria glabrata hemocytes: large cells with a tubulo-vesicular compartment, a component of the endocytic system, and with numerous mitochondria and large aggregates of glycogen particles; medium-size cells poor in organelles and glycogen; and small cells with organelles and few secretory granules. Other small hemocytes can be interpreted as juvenile cells. B. glabrata hemocytes contain few enzymes and do not show specific secretory granules, except for a subpopulation of large cells richer in acid phosphatase vesicles. Hemocytes have different aspects corresponding to different physiological states and their transitions: in quiescent hemocytes, the cell cortex is narrow and organelles are scattered in the cytoplasm, both in circulating cells characterized by thin-folded filopods and large macropinocytic vacuoles and in sedentary cells in which extended filopods connect to the extracellular matrix. In stress-activated hemocytes, the cortical region is thickened by polymerization of actin, and organelles are gathered around the nucleus. Fixed phagocytes are components of the connective tissue; the presence of numerous lysosomes and residual bodies and of acid phosphatase and peroxidase activities suggests a high phagocytic activity.     

Are rhoptries in Apicomplexan parasites secretory granules or secretory lysosomal granules?

The club-shaped rhoptries in Apicomplexan parasites are one of the most unusual secretory organelles among the eukaryotes, containing unusual lipid and protein cargo that is specialized for intracellular parasitism. Rhoptries have traditionally been viewed strictly as regulated secretory granules. We discuss in this article recent data on the cargo, function and biogenesis of rhoptries in two parasitic model systems, Toxoplasma and Plasmodium. Current findings suggest that rhoptries receive products from both biosynthetic and endocytic pathways and, therefore, they are most analogous to secretory lysosomal granules found in mammalian cells.     

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.     

Ultrastructure of neurosecretory cells in the pars intercerebralis of Rhodnius prolixus (Hemiptera).

Six neuron types are distinguished in the pars intercerebralis of the starved fifth instar of Rhodnius prolixus. All neuron types contain electron dense secretory granules derived from Golgi complexes which are of characteristic size and morphology in each type. The neuron types are not thought to represent stages in a secretory cycle. The variety of neuron types described is related to that revealed by staining sections of the same cells with paraldehyde fuchsin. Active synthesis of neurosecretory granules continues throughout starvation and the lysosomal system appears to be involved in the continual degradation of secretory granules. Some of the variations in granule morphology observed may be a consequence of granule fusion and the importance of cytoplasmic events in the development of neurosecretory granules is discussed.     

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.     

Endocytosis in flight-stimulated adipokinetic cells ofLocusta migratoria

An ultrastructural morphometric study was made of the influence of flight activity on endocytosis in the adipokinetic cells ofLocusta migratoria. The endocytotic pathway was revealed by the endocytotic tracer horseradish peroxidase. Endocytosis appeared to be stimulated by flight, as indicated by an increase in the number of tracer-containing endocytotic pits and various intracellular endocytotic and lysosomal organelles. This stimulatory effect continued for at least 10 min after cessation of flight. An increase in the numbers of both tracer-labelled endocytotic pits and endosomal tubules was detected in the cell bodies of flight-stimulated adipokinetic cells. Endosomal tubules are supposed to be involved in recycling membrane material to the plasma membrane soon after it has been endocytosed. It is concluded that the increase in endocytosis in the flight-stimulated cell bodies serves to enlarge the uptake of nutritional and/or regulatory substances. An increase in number of tracer-labelled endocytotic pits was also observed in the cell processes of flight-stimulated adipokinetic cells, which was, however, not accompanied by an increase in number of labelled endosomal tubules, the latter being practically absent in the processes. This indicates that the increase in endocytotic activity in the cell processes is a form of adaptive endocytosis that compensates for membrane material added to the plasma membrane during flight-induced exocytotic release of adipokinetic hormones.     

SPECULATIONS BASED ON THE MORPHOLOGY OF THE GOLGI SYSTEMS IN SEVERAL TYPES OF PROTEIN-SECRETING CELLS

Electron microscopical observations on the relationship of the Golgi region to other intracellular organelles in certain protein-secreting cells have substantiated and extended existing hypotheses. In micrographs of several cell types, the juxtanuclear Golgi regions were observed to be closely associated with nuclear "pores." The "transition elements" of the ergastoplasmic membranes possess "blebs" which may represent a transport process facilitating the movement of intracisternal contents into the Golgi zone. A "blebbing" process of this nature may be one source of the small variety of Golgi vesicles. Zymogen granules of different densities were observed and their significance was postulated. Light Golgi vacuoles were observed. It is suggested that these vacuoles represent accumulations of relatively fluid material segregated from the secretory product in these cell types. These hypotheses from inferential evidence are discussed and extended.     

SECRETION AND ENDOCYTOSIS IN INSULIN-STIMULATED RAT ADRENAL MEDULLA CELLS

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

Co-localization of the adipokinetic hormones I and II in the same glandular cells and in the same secretory granules of corpus cardiacum of Locusta migratoria and Schistocerca gregaria

Summary The immunocytochemical reactivity of the glandular cells of the corpus cardiacum (CCG-cells) of Locusta migratoria and Schistocerca gregaria was investigated at the electron-microscopic level, using the protein A-gold method, with three antisera against fragments of the adipokinetic hormones AKH I and AKH II. This combination of antisera permitted discrimination between anti-AKH I and anti-AKH II immunoreactivity. Fixation in a mixture of 2% glutaraldehyde and 2% formaldehyde, in combination with low-temperature embedding in Lowicryl K₄M, produced the highest and most consistent selective immunogold labelling of the secretory and ergastoplasmic granules. All secretory granules in all CCG-cells investigated possessed a distinct anti-AKH I-immunopositive reaction, whereas most secretory granules showed a weaker anti-AKH II immunoreaction. Ergastoplasmic granules reacted similar to the secretory granules. The average immunolabelling of the secretory granules was higher in the processes than in the cell bodies of the CCG-cells. The results in Schistocerca gregaria were essentially similar to those in Locusta migratoria. It is concluded that (i) the individual CCG-cells synthesize AKH I as well as AKH II; (ii) these hormones coexist in the same ergastoplasmic and secretory granules; and (iii) these granules contain a higher content of AKH I than AKH II.     

Ultrastructural immunocytochemical localization of prolactin and growth hormone in the porcine pituitary

Summary The immunocytochemical peroxidase-antiperoxidase technique was used to identify prolactin- and growth hormone-producing cells in the porcine pituitary at the ultrastructural level. The growth hormone-producing cells contain round secretory granules (300 nm to 500 nm in diameter). The prolactin-producing cells can be identified by their distinct round and ovoid secretory granules which vary in size. Most of these cells contain large granules (450 nm to 750 nm in diameter), but some prolactin-producing cells display smaller secretory granules (250 nm to 500 nm). The two hormones were localized exclusively in the secretory granules. Staining for prolactin was observed in round and ovoid granules, as well as in small and polymorphic granules within the Golgi complex. This study confirmed (i) that the two hormones are located in different cells, and (ii) that under normal physiological conditions no one cell can synthesize and store both hormones simultaneously.     

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

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