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.     

Some Observations on the Fine Structure of the Sinus Gland of a Land Crab, Gecarcinus lateralis

The dilated axon endings of the sinus glands of the brachyuran crab, Gecarcinus lateralis, are filled with homogeneously dense granules, each granule being bounded by a delicate membrane. The granules are of two orders of magnitude: 0.05 to 0.1 µ and 0.15 to 0.2 µ in diameter. Each axon ending contains granules of a nearly uniform size. Endings with granules of the larger size range predominate. Non-nervous cells endogenous to the sinus gland are scattered among the nerve endings. The cell contours are irregular, and cytoplasmic processes ramify between endings. The axons are unmyelinated, having only thin limiting membranes, and they possess many neurofibrils. Granules in preterminal portions of the axons tend to lie at the periphery of the fiber, and in some cases in chains at the core of the fiber. The granules appear to be storage and release centers for neurosecretory substances or their precursors.     

Some observations on the fine structure of the sinus gland of a land crab,Gecarcinus lateralis

The dilated axon endings of the sinus glands of the brachyuran crab, Gecarcinus lateralis, are filled with homogeneously dense granules, each granule being bounded by a delicate membrane. The granules are of two orders of magnitude: 0.05 to 0.1 micro and 0.15 to 0.2 micro in diameter. Each axon ending contains granules of a nearly uniform size. Endings with granules of the larger size range predominate. Non-nervous cells endogenous to the sinus gland are scattered among the nerve endings. The cell contours are irregular, and cytoplasmic processes ramify between endings. The axons are unmyelinated, having only thin limiting membranes, and they possess many neurofibrils. Granules in preterminal portions of the axons tend to lie at the periphery of the fiber, and in some cases in chains at the core of the fiber. The granules appear to be storage and release centers for neurosecretory substances or their precursors.     

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     

Localization of crustacean hyperglycemic hormone (CHH) in the X-Organ sinus gland complex in the eyestalk of the crayfish,Astacus leptodactylus (Nordmann, 1842)

The eyestalk of Astacus leptodactylus is investigated immunocytochemically by light, fluorescence, and electron microscopy, using an antiserum raised against purified crustacean hyperglycemic hormone (CHH). CHH can be visualized in a group of neurosecretory perikarya on the medualla terminalis (medulla terminalis ganglionic X-organ: MTGX), in fibers forming part of the MTGX-sinus gland tractus, and in a considerable part of the axon terminals composing the sinus gland. Immunocytochemical combined with ultrastructural investigations led to the identification of the CHH-producing cells and the CHH-containing neurosecretory granule type.     

锯缘青蟹窦腺显微和超微结构研究

借助光学和电子显微镜观察据缘青蟹（Ｓｃｙｌｌａ ｓｅｒｒａｔａ）窦腺的形态结构。窦腺位于眼柄视神经节内髓背侧近外髓处。窦腺呈羹状;腺体壁山神经分泌细胞的末梢和神经胶质细胞组成。神经末梢内充满了电子致密的神经分泌颗粒。根据颗粒的大小、形态及电子致密度等特征,可以区分出４种类型的神经末梢。一些末梢中的多形性颗粒可能是由Ⅱ型末梢中的颗粒转变而成的。一些现象表明,神经分泌物质可以通过胞吐作用或一种类似“顶浆分泌”的方式释放,从而说明神经激素的释放可能是多途径的．     

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.     

Exocytosis of neurosecretory granules from the crustacean sinus gland in freeze-fracture

Exocytosis is clearly shown in freeze-fracture preparations to be the mechanism for neurosecretion granule release from axon endings in the crayfish sinus gland. The cytoplasmic leaflet (A-face) of axon ending membrane is characterized by randomly situated depressions representing invaginations of the axolemma, which are in contact with limiting membranes of neurohormone granules in the subjacent cytoplasm. The extracellular leaflet (B-face) of the axolemma at release sites exhibits complementary volcano-shaped protrusions which are cross-fractures through necks of channels formed by invaginating plasma membrane in contact with underlying neurosecretion granules. Structural variation in B-face protrusions is consistent with a spectrum of exocytotic profiles in various stages of formation, and with granules at different stages of passage out of the endings. Evidence in this study suggests that formation of exocytotic structures may begin by alteration of axon membrane structure at the neurosecretory ending-hemolymph interface prior to contact of the neurohormone granules with the axolemma. Limiting membranes of neurosecretory granules exhibit protrusions which appear to interconnect granules adjacent to release sites and to attach granules to the axolemma. Freeze-fracture is clearly shown to be an invaluable tool for monitoring the degree of exocytosis exhibited by sinus glands under normal conditions and under experimental acceleration of hormone release. This technique is capable therefore, of detecting slight increases in numbers of exocytotic profiles much more quickly and accurately than the examination of random thin sections.     

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.     

Immunocytochemical identification of hyperglycemic hormone-producing cells in the eyestalk of Carcinus maenas

Summary Antiserum raised in rabbits against extracts of sinus glands from Carcinus and shown by several criteria to contain antibodies directed against the neurosecretory hyperglycemic hormone was used to locate the hormone-producing perikarya in the optic ganglia. By means of the double antibody fluorescence technique, selective staining of the large neurosecretory perikarya of the medulla terminalis ganglionic X-organ (MTGXO) and their axons is obtained. The axon endings of the sinus gland are also stained. None of the other groups of neurosecretory cells in the eyestalk shows fluorescence. Preabsorption of the antiserum with pure hyperglycemic hormone abolishes the fluorescence.Supported by the Deutsche Forschungsgemeinschaft (SFB 87, A 3; Ke 206/2). Thanks are due to E. Schmid (Ulm) for excellent technical assistance and to Prof. R. Martin and E. Weber for help and suggestions. A short version of parts of the results has been presented at theXth Conference of European Comparative Endocrinologists, Sorrento, May 1979     

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.     

The neurosecretory system of the eyestalk of Carcinus maenas (Crustacea: Decapoda)

The optic ganglia neurosecretory cells of male and female Carcinus maenas during intermoult are distinguishable into six types based on size, location, appearance and method of secretory material release from the perikaryon. Release occurs via the sinus gland and also, in one case, directly into blood capillaries among the neurosecretory cells themselves. The sinus gland consists of axonal extensions of the neurosecretory cells; no secretory granules are produced there and nuclei observed between the axonal endings are those of ill-defined glial cells.     

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.     

Neurosecretory centers from the eyestalk of Siriella armata M. Edw. (Crustacea: Mysidacea): ultrastructural variations during normal and experimentally inhibited molt cycle

Summary The ultrastructure of the medulla interna-medulla externa X-organ (MI-ME Xo)-sinus gland (SG) complex in the eyestalk of Siriella armata is described during the normal and the experimentally inhibited molt cycle. In the normal SG, four types of neurosecretory axon terminals, each containing distinguishable neurosecretory granules, can be described. Thus, type-2 granules are synthesized by G1 neurons forming the MI-ME Xo. The cell bodies and axonal endings of these cells in the sinus gland have been examined at the following molt stages: intermolt (stage C4), premolt (D0 and D2), and postmolt (A1, A2 and B). Changes in ultrastructure of the G1 cells have been monitored and correlated to inhibitions of the molt-and reproductive cycle produced by electrocauterization of the MI-ME Xo. The results obtained suggest that the neurosecretion from the G1 cells exerts a positive influence on molt and brood preparation. The occurrence of a distal group of G1 cells whose axons terminate at a different site from the SG suggests that the neural factors of the MI-ME Xo are diverse and control different physiological activities.     

Do the neurohormones VIH (vitellogenesis inhibiting hormone) and CHH (crustacean hyperglycemic hormone) of crustaceans have a common precursor? Immunolocalization of VIH and CHH in the X-organ sinus gland complex of the lobster,Homarus americanus

Summary

The present study deals with the location of the vitellogenesis inhibiting hormone (VIH)-producing cells in the eyestalk of the lobster Homarus americanus. In the present study, the neurosecretory pathways of VIH in Homarus, have been described immunocytochemically by use of a mouse serum against Homarus VIH. The location of the VIH cells was compared with the location of the crustacean hyperglycemic hormone (CHH) cells visualized by a rabbit serum raised against CHH of the crayfish Astacus leptodactylus. Immunocytochemical detection procedures, both at the light and electron microscopic level, revealed frequent but not complete co-localization of VIH and CHH in a variable number of the same group of perikarya. In the sinus gland, both neuropeptides were mostly demonstrated in distinct axonal endings characterized by different granule types. Postulations on the biosynthesis of these factors and suggestions concerning the processing of both neurohormones have been made.     

Immunohistochemical Identification of Hyperglycemic Hormone- and Molt-Inhibiting Hormone-Producing Cells in the Eyestalk of the Kuruma Prawn, Penaeus japonicus

This study deals with the localization of crustacean hyperglycemic hormone (CHH, Pej-SGPIII) and molt-inhibiting hormone (MIH, Pej-SGP-IV) in the eyestalk of the kuruma prawn Penaeus japonicus using immunohistochemistry. High-titer and highly specific antisera were raised in rabbits against synthetic Pej-SGP-III C-terminal peptide (Glu-Glu-His-Met-Ala-Ala-Met-Gln-Thr-Val-NH2) and Pej-SGP-IV C-terminal peptide (Val-Trp-Ile-Ser-Ile-Leu-Asn-Ala-Gly-Gln-OH), both of which were conjugated with bovine serum albumin by a cross linker. Eyestalks were removed from mature male prawns at the intermolt stage of the molting cycle and fixed in Bouin's solution. Serial sections stained immunohistochemically showed that neurosecretory cells of Pej-SGP-III and Pej-SGP-IV were located in the same cluster of the medulla terminalis ganglionic X-organ (MTGX), and that three kinds of neurosecretory cells, which were stained with anti-PejSGP-III antiserum and/or anti-Pej-SGP-IV antiserum were present. The number of neurosecretory cells which stained with both antisera was much fewer than that of neurosecretory cells which stained with one of the antisera only. The axon and axon terminals in the sinus gland were also stained and the staining density of the sinus gland was always deeper than that of the neurosecretory cells.     

Identification of a putative egg-laying hormone in neural and ovarian tissues of the black tiger shrimp, Penaeus monodon, using immunocytochemistry

The existence of an egg-laying hormone (ELH) was identified for the first time in the black tiger shrimp, Penaeus monodon, by means of immunoenzyme and immunofluorescence techniques. This was achieved using a polyclonal antibody produced against expressed recombinant ELH of the female Australian blacklip abalone, Haliotis rubra. The shrimp ELH reactive material was found to be localised within female neurosecretory tissues and the secretory tissue of the antennal gland, but was not identified in the X-organ sinus gland within the eyestalk. It was also present in the ovary, where the amount of ELH present was observed to be greatest in the period prior to spawning. These findings implied that the induction of P. monodon spawning might be involved with humoral regulation relating to ELH expression.     

Appearance and innervation of CHH-producing cells in the eyestalk of the crayfish Astacus leptodactylus examined after tracing with Lucifer Yellow

Summary By injection of the fluorescent dye Lucifer Yellow into individual Crustacean Hyperglycemic Hormone (CHH)-producing cells, the shape of these neurosecretory cells in the eyestalk of the crayfish Astacus leptodactylus can be traced. A highly fluorescent perikaryon gives rise to an axon that can be followed by the fluorescent label to the neurohemal region, the sinus gland. The proximal part of that axon sends out extensive branches into the neuropil of the medulla terminalis. Electron-microscopic investigations reveal synaptic input to these axonal ramifications.     

Relationship between Rous sarcoma virus production in golden hamster cells and the cell cycle]

The axon terminals of neurosecretory cells, containing elementary granules of neurosecretory materials, have been described on the basis of light and electron microscopy. No allochthonous neurosecretory elements were found in the sinus gland. The discharge of the granules from some terminals of the sinus gland occur with the changed salinity of the environment. There is a great difference in the structure of the sinus gland when salinity is changed. The structure of the sinus gland and its modification under experimental conditions indicate a possibility of neurohormonal regulation of the hydromineral balance.     

The ultrastructure of nerve endings containing pigment-dispersing hormone (PDH) in crustacean sinus glands: Identification by an antiserum against a synthetic PDH

Summary A high-liter antiserum has been obtained from two rabbits immunized with a glutaraldehyde conjugate of synthetic pigment-dispersing hormone (PDH) from Uca pugilator and bovine thyroglobulin. The antiserum blocked melanophore-dispersing activity of the peptide in vivo. In sinus glands (SG) of Carcinus maenas, Cancer pagurus, Uca pugilator and Orconectes limosus, electron-microscopic immunocytochemistry revealed sparsely distributed axon endings containing a distinct PDH-immunoreactive type of neurosecretory granules (diameter 90–130 nm). Exocytotic figures indicating release of the content of these granules into hemolymph lacunae were occasionally observed. Preservation of fine structure and antigenicity of the PDH granules were markedly dependent on the fixation procedure used. A preliminary experiment with C. maenas showed that preterminal axon dilatations near the basal lamina seemed to accumulate PDH-granules when animals were kept in complete darkness for three days. Immunodot blotting of fractions after high pressure liquid chromatography (HPLC) of extracts from SGs of C. maenas and O. limosus revealed a strongly immunoreactive substance at a retention time very similar to those of synthetic PDHs of Uca pugilator and Pandalus borealis. It is also coincident with a zone of biological activity. Thus, the antigen demonstrated by immunocytochemistry is identical or very similar to one of the known PDHs.