Ultrastructural study of the neurosecretory granules in the sinus gland of the blue crab,Callinectes sapidus

Summary Seven morphologically different types of neurosecretory granules have been found in the axon terminals of the sinus gland of the blue crab, Callinectes sapidus. They differ from each other in size, shape, staining characteristics, solubility characteristics, core matrix characteristics, axon terminal matrix characteristics, presence or absence of space between the granule membrane and granule core matrix, and frequency of occurrence. Five of the types are segregated in different axon terminals and are believed to represent different hormone-protein complexes. Two of the types, which have lost part or all of their granular contents, are thought to be variants of the other five types. The differences in granular morphology are better revealed by some fixation procedures than others. Palade's acetate-veronal buffered osmium tetroxide, in particular, reveals striking differences. The following observations suggest that different hormone-protein complexes are segregated in different axon terminals and that these complexes may be morphologically distinguished at the level of the electron microscope.Supported by USPHS-NIH Training Grant GM-00669 and Grant GB-7595X from the National Science Foundation.     

Fine structure of the neurohemal sinus gland of the shore crab,Carcinus maenas,and immuno-electron-microscopic identification of neurosecretory endings according to their neuropeptide contents

Summary The sinus gland of the shore crab, Carcinus maenas, is a compact assembly of interdigitating neurosecretory axon endings abutting upon the thin basal lamina of a central hemolymph lacuna. Four types of axon endings are distinguishable by the size distribution, shape, electron density and core structure of their neurosecretory granules. One additional type of axon ending is characterized by electron-lucent vacuoles and vesicles. The axon profiles are surrounded by astrocyte-like glial cells. Various fixations followed by epoxy- or Lowicrylembedding were compared in order to optimize the preservation of the fine structure of the granule types and the antigenicity of their peptide hormone contents. By use of specific rabbit antisera, the crustacean hyperglycemic, molt-inhibiting, pigment-dispersing, and red-pigment-concentrating hormones were assigned to the four distinct granule types which showed no overlap of immunostaining. Epi-polarization microscopy and ultrathin section analysis of immunogold-stained Lowicrylembedded specimens revealed that immunoreactivity to Leu-enkephalin and proctolin is co-localized with moltinhibiting hormone immunoreactivity in the same type of granule. The size and core structure of the immunocytochemically identified granule types vary little with the different pretreatments but, in some cases, to a statistically significant extent. The present results are compared with those from earlier studies of sinus glands in different crustaceans. The methods of granule identification used in this study supplement the classical approach in granule typing; they are easier to perform and more reliable for the analysis of release phenomena in identified secretory neurons supplying the neurohemal sinus gland.     

The ultrastructure of the sinus gland of Carcinus maenas (Crustacea: Decapoda)

Summary The sinus gland of Carcinus maenas consists of the swollen axonal endings of the neurosecretory cells of the major ganglia and acts as a storage release centre for the membrane bound neurosecretory material. These neurosecretory granules fall into five different types based on size and electron density. Their contents are released by exocytosis of the primary granules or smaller units budded from the primary granules.I thank Professor E. Naylor for his constant advice and Professor E. W. Knight-Jones, Department of Zoology, University College, Swansea, for the provision of laboratory facilities. I am grateful to the Science Research Council for the financial support. Finally, I thank the Electron Microscope Unit, Southampton General Hospital, where the work was completed.     

Hydrolysis of G6P by a microsomal aspecific phosphatase and glucose phosphorylation by a low K m hexokinase in the digestive gland of the crab Carcinus maenas: variations during the moult cycle

Summary The hydrolysis of glucose-6-phospate in the digestive gland of the crab Carcinus maenas is carried out by an aspecific phosphatase. This enzyme possesses the following features: (1) insensitivity to acid treatment; (2) absence of inhibition when exposed to citrate at low pH; (3) similar affinity for G6P as the acid phosphatase for Na--glycerophosphate (K _m 2.3 and 2.0 mM, respectively). Glucose-6-phosphate and Na--glycerophate hydrolysis reactions seem to be catalysed by the same enzyme, since both activities exhibit the same distribution in a subcellular fractionation of the gland. Furthermore, as these activities are principally recovered in the subcellular fraction enriched in calcospherites (or calcium phosphate granules), it is proposed that the aspecific G6P-phosphohydrolase could play a major role in the formation of these granules. The phosphorylation of glucose is made by two low K _m hexokinases (230 and 64 M, respectively). As their level of activity shows significant changes over the moult cycle, these enzymes could be considered as having a regulatory role in the storage of glucose in the digestive gland.Abbreviations Acid Pase aspecific acid phosphatase - ATP adenosine triphosphate - DTT dithiothreitol - EDTA ethylenediaminetetra-acetate - G calcium phosphate granules fraction - G6P glucose-6-phosphate - G6Pase hepatic glucose-6-phosphatase - G6PDH glucose-6-phosphate dehydrogenase - K _m Michaelis-Menten constant - MI mitochondria and intermediate postmitochondrial particles - N nuclei fraction - NADH nicotineamide adenine dinucleotide - P microsome fraction - Pi inorganic phosphate - PMSF phenylmethylsulphonylfluoride - STI soybean trypsin inhibitor - glyP Na--glycerophosphate - T_1,2,3 transport protein 1,2,3 - TCA trichloroacetic acid     

Morphometric classification of neurosecretory granules in the neurohypophysis of the hagfish,Eptatretus burgeri

Summary Neurosecretory axons in the neurohypophysis of the hagfish, Eptatretus burgeri, were statistically classified into six types according to the size of secretory granules. These types are comparable with those in higher vertebrates. The concentration of each axon type is different in three regions: anterior dorsal wall, posterior dorsal wall, and ventral wall. The regional differences of the hagfish neurohypophysis are discussed in relation to the regional differentiation of the tetrapod neurohypophysis into the median eminence and the pars nervosa.We wish to express our appreciation to Professor Hideshi Kobayashi for the guidance of this study. It was aided by grants from the Ford Foundation and the Ministry of Education to Professor H. Kobayashi, and also by grants from the Population Council, New York and the Ministry of Education to Professor S. Ishii.     

Neurosecretion in the sinus gland of the fiddler crab,Uca pugnax

The ultrastructure of the sinus gland of the fiddler crab, Uca pugnax, was investigated and found to be similar to that in other crustaceans. Five types of neurosecretory axon terminals were tentatively identified on the basis of the size, shape, and electron density of granules within the axons. Release of neuro-secretory material appears to be by exocytosis.     

Ultrastructural changes in isolated peptidergic nerve terminals induced by digitonin permeabilization and K+ stimulation in the sinus gland of the crab,Cardisoma carnifex

Summary The preparation of isolated peptidergic nerve terminals from the sinus gland (a neurohemal organ) of the crab (Cardisoma carnifex) is described. In this species the nerve endings can have diameters up to 30 m. They release neurosecretory material as judged by the decrease in the volumetric density of granules upon depolarization with potassium. Similar results were obtained after permeabilization of the nerve terminals with digitonin, but only in the presence of micromolar concentrations of calcium. This preparation should prove useful in correlating electrical events with other cellular processes involved in stimulus-secretion coupling.     

Corrective note about R* cells of the digestive gland of the shore crab Carcinus maenas

In a previous paper, we described and discussed the possible functions of calcospherite-rich cells (R^* cells) in the digestive gland of the shore crab, Carcinus maenas. We recently realised that electron micrographs in this publication presented neither typical R^* cells nor their calcium phosphate granules. Indeed, our pictures showed spermatophores (filled with typical spermatozoa) that had contamined hepatopancreatic cell suspensions. As the present study indicates, this contamination is difficult to detect by optical microscopy because unstained R^* cells closely resemble spermatophores. However, morphological differences between these cell types appear clearly when observed by electron microscopy. The present paper describes a comparative study of cell populations isolated from female digestive glands; it validates our previous results obtained with male hepatopancreas and suggests a low containation of those male cell fractions by spermatophores.     

Regulation of circulating levels of the crustacean hyperglycemic hormone: evidence for a dual feedback control system

The effects of glutamate, aspartate, glycine, proline, alanine, taurine, glycerol, glucose and lactate injections on the haemolymph levels of the crustancean hyperglycemic hormone and/or glucose and lactate in the shore crab, Carcinus maenas, were investigated. Only glucose and lactate caused significant changes of hyperglycaemic hormone levels. Glucose injections resulted in a drop of both hormone and lactate, while lactate had an opposite effect, i.e. it raised both crustacean hormone and glucose levels. The results suggest that during increases in glycolytic flux, lactate may cause a release of hormone by a positive feedback mechanism. The hormone would then stimulate glycogenolysis, thus increasing glucose availability. If more glucose is released than is metabolized, excess glucose may leak from the cells and suppress crustancean hyperglycemic hormone release from the X-organ/sinus gland complex by negative feedback.Abbreviations ABTS 2,2-azino-bis (3-ethylbenzthiazoline sulphonic acid) - ANOVA one-way analysis of variance - BSA bovine serum albumin - BW body weight - CHH crustacean hyperglycemic hormone - ELISA cnzyme-liked immunosorbent assay - GIH gonadinhibiting hormone - IgG immunoglobin G - MIH moult-inhibiting hormone - MTGXO medulla terminalis X-organ - PB sodium phosphate buffer - PBS phosphate buffered saline - Pi inorganic phosphate - XO-SG X-organ-sinus gland complex     

Synaptic junctions in the sinus gland of the freshwater prawn Palaemon paucidens

Summary Two types of neurosecretory fibers, designated as Type 5 and Type 6 axons, in the sinus gland of the freshwater prawn, Palaemon, establish contact with other neurosecretory axons by means of synaptic junctions. This finding strongly supports the view that release of some neurohormones from the eyestalk may be regulated by neurosecretory neurons through synaptic transmission.The author wishes to express his sincere appreciation to Prof. T. Aoto for his invaluable advice during the course of this study, to Mr. S. Kubota for collecting the material, and to Mr. Y. Takakuwa for his excellent assistance in electron microscopy     

Ultrastructural location of calcium and magnesium during mineralisation of the cuticle of the shore crab,as determined by the K-pyroantimonate method and X-ray microanalysis

We have investigated the distribution of Ca²⁺ and Mg²⁺ in the new cuticle of moulting shore crabs (Carcinus maenas), using the K-pyroantimonate method in combination with X-ray microanalysis in order to identify antimony precipitates. During the premoult period, Ca²⁺ and Mg²⁺ accumulate in well-defined sites of the new pigmented layer. After moulting, mineralisation appears to begin preferntially at these sites. These form a honeycomb-like structure that quickly increases the rigidity of the new cuticle, with a small recruitment of material from extraneous sources. Mineralisation of the principal layer, on the other hand, immediately follows deposition of the organic matrix. Our experiments also provide evidence that the epidermal cell extensions associated with the pore canals are the means by which Ca²⁺ and Mg²⁺ are transferred from the epidermis into the mineralising cuticular layers. The plasma membrane of these cell extensions appears densely lined by particles of antimony precipitate that probably mark the location of the transporting sites. Shortly after moulting, the distribution of mineral deposits is such that the cell extensions cross the mineralised lamellae of the principal layer and constitute preferential access routes to the pigmented layer, where mineralisation is still in progress.     

The ultrastructure of the sinus gland of the crayfish Astacus leptodactylus (Nordmann)

Summary An ultrastructural study of the sinus gland of the crayfish Astacus leptodactylus demonstrates that this gland is mainly composed of glial cells, axons and axon terminals. On the basis of the size, shape and electron density of the neurosecretory granules, we could distinguish five different types of axon terminals.     

The ultrastructure of the sinus gland of Gammarus oceanicus (Crustacea: Amphipoda)

Summary The sinus gland of Gammarus oceanicus, like that of other crustaceans, is composed of three elements: neurosecretory axons, glial cells and stromal sheath. Five neurosecretory axon types are identified on the basis of granule diameter, shape, and electron density, and axon matrix density. Exocytosis appears to be the major release mechanism of neurosecretory material. The preterminal regions of neurosecretory axons contain axoplasmic reticulum and neurotubules. Their arrangement in the axon and relationship with one another suggest a transport function. Multilamellar bodies are found in the terminal regions of neurosecretory axons. They arise from mitochondria and may be involved in granulolysis.The technical assistance of G.A. Bance, statistical assistance of D. MacCharles and D.W. Hagen, and financial support provided by the University of New Brunswick Research Fund to K.H. are gratefully acknowledged     

Structure and possible functions of the calcospherite-rich cells (R* cells) in the digestive gland of the shore crab CArcinus maenas

Summary R^*-cells of the digestive gland of Carcinus maenas have been investigated functionally and morphologically. A comparison of the capacity of separated cell suspensions to synthesize glycogen gave support to the hypothesis that R and R^* cells belong to the same cell line. The unexpected observation of R^* cells in gastric juice suggests that their release could represent a mode of redistribution of carbohydrate stores when the feeding activity of the crab is lower. Under electron microscopy, the calcospherites of R^* cells appeared to be surrounded by multiple membranous layers, and displayed tubular and vesicular structures in their core. High glucose-6-phosphatase (G6Pase) activity in the subcellular fraction that is enriched in calcospherites suggests that these membranes are derived from the endoplasmic reticulum, via a process in which the enzyme plays a key role. We propose that this is the way by which the R cell differentiates into R^* cell.     

Depletion of neurosecretory granules and membrane retrieval in the sinus gland of the crab

Summary Ultrastructural aspects of hormone release from the sinus gland of the crab Carcinus maenas, have been studied by incubation of glands in vitro (i) in high potassium-containing media to induce hormone release; (ii) in a high potassium-containing calcium-free medium in which depolarisation but no hormone release would be expected; and (iii) in control saline. Uptake of horseradish peroxidase into subcellular organelles was also studied.Many neurosecretory granules could be found in the nerve terminals but, in contrast to mammalian neurosecretory systems, structures resembling microvesicles were extremely scarce. High potassium stimulation in the presence of calcium caused an 18 % loss of granules from the nerve terminals associated with images of single and multiple exocytosis. It further caused an increase in vacuoles which could have accounted for 33 % of the membrane of the granules exocytosed. After incubation in high potassium-containing, calcium-free media there was no evidence either of exocytosis of granules or of an increase in the vacuole population. The population of sparse microvesicle-like structures was not significantly altered by incubation in either high potassium medium. Horseradish peroxidase reaction product could be found only in vacuoles of tissues stimulated by high potassium concentrations in the presence of calcium. It is concluded that this depolarising stimulus produces, in the presence of calcium, the release by exocytosis of about one sixth of all the granules in the sinus gland, and that vacuoles are the organelle responsible for the recapture of membrane after the exocytosis.     

Occurrence of immunoreactive enkephalins in a neurohemal organ and other nervous structures in the eyestalk of the shore crab,Carcinus maenas L. (Crustacea,Decapoda)

Summary Light-microscopical observations with immunofluorescence and peroxidase staining procedures revealed leu-enkephalin-like immunoreactivity in axon profiles of the sinus gland (SG) and in single small neurons in the optic ganglia of the eyestalk of Carcinus maenas. Electron microscopy of the SG showed reactivity to be associated with neurosecretory granules 82±23 nm in diameter. High performance liquid chromatography of SG-extracts revealed radioimmunoreactive substances with the retention times of synthetic met- and leu-enkephalin and met-enkephalin-Arg⁶-Phe⁷, respectively.     

Ultrastructural characterization of neurosecretory fibres immunoreactive for vasotocin,isotocin, somatostatin,LHRH and CRF in the pituitary of a teleost fish,Poecilia latipinna

Summary Using the electron-microscopic immunogold method, vasotocin, isotocin, somatostatin (SRIF), gonadotrophin-releasing hormone (LHRH) and corticotrophin-releasing factor (CRF)-like immunoreactivities were localized in separate neurosecretory fibres in the pituitary of a teleost fish Poecilia latipinna. Antigenicities were preserved in sections of conventionally fixed tissue, except in the case of LHRH and CRF-like substances which were sensitive to osmium postfixation. Under the same fixation conditions, ultrastructural differences were observed between the 5 fibre types, and morphometric analysis of their granule sizes revealed significant differences in mean diameter except between vasotocin and isotocin fibres.Terminal-like regions of each type were identified on blood vessels, glial cells or other fibres in the neurohypophysis, on the basement lamina of the adenohypophysis, or directly on adenohypophysial endocrine cells. The fibres containing the two neurohypophysial hormones, originating from separate preoptic perikarya, were intermingled with, and may form endings near all the adenohypophysial cell types except those secreting prolactin. Although both types had similar mean granule diameters, the granules in the vasotocin fibres (mean 135 nm) were markedly less electron dense than those in the isotocin fibres (mean 140 nm). SRIF-immunoreactive fibres (mean 101 nm) appeared to form synapse-like endings on the somatotrophs, and a few thyrotrophs in the proximal pars distalis, and near the pars intermedia cells. An LHRH-positive type (mean 103 nm) contacted only the gonadotrophs of the proximal pars distalis. The rarer CRF-like fibres (mean 116 nm) appeared to project mainly towards the pars intermedia, but a few appeared to terminate rostrally near the adrenocorticotrophic cells.The significance of these observations is discussed in relation to the direct neurosecretory control of adenohypophysial function in teleosts.     

Sex-specific mediation of foraging in the shore crab, Carcinus maenas

Experiments were conducted to investigate the sex-specific differences to feeding responses of the shore crab Carcinus maenas throughout the year. Results demonstrate that female shore crabs exhibit stronger feeding responses than males throughout the year with a significantly reduced feeding response in males during the summer months' reproductive season. We also studied the possible function(s) of the moulting hormone, 20-hydroxyecdysone (Crustecdysone) that has been described as a potential female-produced sex pheromone to initiate male reproductive behaviour in a number of crustaceans. We recently presented evidence that for shore crabs this is not the case and now show that the steroid is instead functioning as a sex-specific feeding deterrent protecting the moulting 'soft' female crabs. Whilst male shore crabs were deterred from prey (Mytilus edulis) and synthetic feeding stimulants glycine and taurine when these feeding stimulants were spiked with crustecdysone, intermoult female crabs were significantly less affected and rarely deterred from feeding. This sex specificity of the moulting hormone, in combination with the female sex pheromone, which has no anti-feeding properties, ensures that male crabs mate with soft-shelled, moulted females rather than engage in cannibalism, such as found frequently in cases when soft-shelled females are exposed to intermoult females.     

The hair-peg organs of the shore crab,Carcinus maenas (Crustacea,Decapoda): Ultrastructure and functional properties of sensilla sensitive to changes in seawater concentration

Summary The hair-peg organs of the shore crab, Carcinus maenas, are modified hair-sensilla. A small hair shaft (peg) is surrounded by a tuft of solid cuticular bristles (hairs). Each hair-peg organ is innervated by 6 sensory neurons, 2 of which have scolopidial (type-I) dendrites. The outer segments of all dendrites pass through a cuticular canal extending to the articulated hair base in which the 2 type-I dendrites terminate. The other 4 (type-II) dendrites reach the clavate tip of the hair shaft and have access to a terminal pore and a large sickle-shaped aperture. Three inner and 8–12 outer enveloping cells belong to a hair-peg organ. The innermost enveloping cell contains a scolopale, which has desmosomal connections to the ciliary rootlets of the type-I dendrites. An inner and an outer sensillum lymph space are present. The ultrastructural features of the dendrites and the cuticular apparatus indicate that the hair-peg organs are bimodal sensilla, comprising 2 mechano- and 4 chemosensitive sensory neurons. Extracellular recordings from the leg nerve indicate that the chemosensitive neurons of the hair-peg organs respond to changes in seawater concentration in the physiological range of Carcinus maenas.Supported by the Deutsche Forschungsgemeinschaft (SFB 45/A1; W. Gnatzy)