Summary
By use of antisera raised against purified moultinhibiting (MIH) and crustacean hyperglycemic hormone (CHH) from Carcinus maenas, complete and distinct neurosecretory pathways for both hormones were demonstrated with the PAP and immunofluorescence technique. By double staining, employing a combination of silver-enhanced immunogold labelling and PAP, both antigens could be visualized in the same section. Immunoreactive structures were studied in Carcinus maenas, Liocarcinus puber, Cancer pagurus, Uca pugilator and Maja squinado. They were only observed in the X-organ sinus gland (SG) system of the eyestalks and consisted of MIH-positive perikarya, which were dispersed among the more numerous CHH-positive perikarya of the medulla terminalis X-organ (XO). The MIH-positive neurons form branching collateral plexuses adjacent to the XO and axons that are arranged around the CHH-positive central axon bundle of the principal XO-SG tract. In the SG, MIH-positive axon profiles and terminals, clustered around hemolymph lacunae, are distributed between the more abundant CHH-positive axon profiles and terminals. Colocalisation of MIH and CHH was never observed. The gross morphology of both neurosecretory systems was similar in all species examined, however, in U. pugilator and M. squinado immunostaining for MIH was relatively faint unless higher concentrations of antiserum were used. Possible reasons for this phenomenon as well as observed moult cycle-related differences in immunostaining are discussed.
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Boer HH, Schot LPC, Roubos EW, TerMaat A, Lodder JC, Reichelt D, Swaab DF (1979) ACTH-like immunoreactivity in two electronically coupled giant neurons in the pond snail Lymnaea stagnalis. Cell Tissue Res 202:231–240
Bruce MJ, Chang ES (1984) Demonstration of a molt-inhibiting hormone from the sinus gland of the American lobster Homarus americanus. Comp Biochem Physiol 79:421–424
Carlisle DB (1957) On the hormonal inhibition of moulting in decapod Crustacea. J Mar Biol Ass UK 36:291–307
Dircksen H, Zahnow CA, Gaus G, Keller R, Rao KR, Riehm JP (1987) The ultrastructure of nerve endings containing pigment-dispersing hormone (PDH) in crustacean sinus glands: Identification by an antiserum against synthetic PDH. Cell Tissue Res 250:377–387
Durand JB (1956) Neurosecretory cell types and their secretory activity in the crayfish. Biol Bull 111:62–76
Gorgels-Kallen JL, Van Herp F, Leuven RSEW (1982) A comparative immunocytochemical investigation of the crustacean hyperglycemic hormone (CHH) in the eyestalk of some decapod Crustacea. J Morphol 174:161–168
Gu J, de Mey J, Moeremans M, Polak JM (1981) Sequential use of the PAP and immunogold staining of tissue antigens. Its application to the study of regulatory peptides on the gut. Regul Pept 1:365–374
Hartnoll RG (1982) Growth. In: Bliss DE, Mantel LH (eds) The Biology of Crustacea, vol. 2. Academic Press, New York, pp 11–194
Jaros PP, Keller R (1979a) Immunocytochemical identification of hyperglycemic hormone-producing cells in the eyestalks of Carcinus maenas. Cell Tissue Res 204:379–385
Jaros PP, Keller R (1979b) Radioimmunoassay of an invertebrate peptide hormone — the crustacean hyperglycemic hormone. Experientia 35:1252–1253
Jaros PP, Dircksen H, Keller R (1985) Occurrence of immunoreactive enkephalins in a neurohemal organ and other nervous structures in the eyestalk of the shore crab, Carcinus maenas L. (Crustacea, Decapoda). Cell Tissue Res 241:111–117
Keller R (1977) Comparative electrophoretic studies of crustacean hyperglycemic and melanophore-dispersing hormones from isolated sinus glands. J Comp Physiol 122:359–373
Keller R (1981) Purification and amino acid composition of the hyperglycemic neurohormone from the sinus gland of Orconectes limosus and comparison with the hormone from Carcinus maenas. J Comp Physiol 141:445–450
Keller R (1987) RIAs and ELISAs: Peptides. In: Gilbert L, Miller TA (eds): Immunological Techniques: Arthropods. Springer Series in Experimental Entomology. Springer, Berlin Heidelberg New York (in press)
Keller R, Kegel G (1984) Studies on crustacean eyestalk neuropeptides by use of high performance liquid chromatography. In: Hoffmann J, Porchet M (eds) Biosynthesis, metabolism and mode of action of invertebrate hormones. Springer, Berlin Heidelberg New York, pp 145–154
Keller R, O'Connor JD (1982) Neuroendocrine regulation of ecdysteroid production in the crab Pachygrapsus crassipes. Gen Comp Endocrinol 46:348
Keller R, Wunderer G (1978) Purification and amino-acid composition of the neurosecretory hyperglycemic hormone from the sinus gland of the shore crab, Carcinus maenas. Gen Comp Endocrinol 34:328–335
Keller R, Jaros PP, Kegel G (1985) Crustacean hyperglycemic neuropeptides. Am Zool 25:207–221
Mangerich S, Keller R, Dircksen H (1986) Immunocytochemical identification of structures containing putative red pigmentconcentrating hormone in two species of decapod crustaceans. Cell Tissue Res 245:377–386
Mangerich S, Keller R, Dircksen H, Rao KR, Riehm JP (1987) Localization of pigment-dispersing hormone (PDH) and coexistence with FMRFamide immunoreactivity in the eyestalks of two decapod crustaceans. Cell Tissue Res 250:365–375
Mattson MP, Spaziani E (1985) Characterization of molt-inhibiting hormone (MIH) action on crustacean Y-organ segments and dispersed cells in culture and a bioassay for MIH activity. J Exp Zool 236:93–101
Newcomb RW, Stuenkel EL, Cooke IM (1985) Characterization, biosynthesis and release of neuropeptides from the X-organ sinus gland system of the crab Cardisoma carnifex. Am Zool 25:157–171
Nordmann JJ, Morris JF (1980) Depletion of neurosecretory granules and membrane retrieval in the sinus gland of the crab. Cell Tissue Res 205:31–42
Passano LM (1953) Neurosecretory control of molting in crabs by the X-organ-sinus gland complex. Physiol Comp Oecol 3:155–189
Rehm M (1959) Observations on the localization and chemical constitution of neurosecretory material in nerve terminals in Carcinus maenas. Acta Histochem 7:88–106
Skinner DM (1985a) Molting and regeneration. In: Bliss DE, Mantel (eds) The Biology of Crustacea. Vol. 9, Academic Press, New York, pp 43–146
Skinner DM (1985b) Interacting factors in the control of the crustacean molt cycle. Am Zool 25:275–284
Smith G, Naylor E (1972) The neurosecretory system of the eyestalk of Carcinus maenas (Crustacea, Decapoda). J Zool Lond 166:313–321
Snyder MD, Chang ES (1986) Effects of sinus gland extracts on larval molting and ecdysteroid titers of the American lobster Homarus americanus. Biol Bull 170:244–254
Soumoff C, O'Connor JD (1982) Repression of Y-organ secretory activity by molt-inhibiting hormone in the crab Pachygrapsus crassipes. Gen Comp Endocrinol 48:432–439
Stefanini M, DeMartino C, Zamboni L (1967) Fixation of ejaculated spermatozoa for electron microscopy. Nature 216:173–174
Sternberger LA (1979) Immunocytochemistry, 2nd ed. John Wiley and Sons, New York Chichester Brisbane Toronto
Stuenkel EL (1986) A common precursor to two major crab neurosecretory peptides. Peptides 7:397–406
Towbin H, Staehlin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354
Webster SG (1986) Neurohormonal control of ecdysteroid biosynthesis by Carcinus maenas Y-organs in vitro, and preliminary characterisation of the putative molt-inhibiting hormone (MIH). Gen Comp Endocrinol 61:237–247
Webster SG, Keller R (1986) Purification, characterisation and amino acid composition of the putative moult-inhibiting hormone (MIH) of Carcinus maenas (Crustacea, Decapoda). J Comp Physiol 156:617–624
Webster SG, Keller R (1987) Physiology and biochemistry of crustacean neurohormonal peptides. In: Thorndyke M, Goldsworthy GJ (eds) Peptides and amines in invertebrates. Cambridge University Press, Cambridge (in press)
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Dircksen, H., Webster, S.G. & Keller, R. Immunocytochemical demonstration of the neurosecretory systems containing putative moult-inhibiting hormone and hyperglycemic hormone in the eyestalk of brachyuran crustaceans. Cell Tissue Res. 251, 3–12 (1988). https://doi.org/10.1007/BF00215441
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DOI: https://doi.org/10.1007/BF00215441