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     

Immunocytochemical demonstration of the neurosecretory X-organ complex in the eyestalk of the crab Carcinus maenas

Summary An antiserum was obtained by immunizing rabbits with sinus gland extracts from Carcinus maenas. The antiserum is almost exclusively directed against neurosecretory material in the medulla terminalis X-organ (MTGXO), as demonstrated by the peroxidase—antiperoxidase (PAP) staining method in light and electron microscopic studies. Radioimmunological binding studies indicate the presence of antibodies against the crustacean hyperglycemic hormone (CHH) or the black pigment dispersing hormone (BPDH) in the antiserum. The results suggest that the neurosecretory perikarya of the MTGXO are the sites of production of CHH and/or BPDH.Supported by the Deutsche Forschungsgemeinschaft (Ke 206/2)     

Immunocytochemical demonstration of the neurosecretory systems containing putative moult-inhibiting hormone and hyperglycemic hormone in the eyestalk of brachyuran crustaceans

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.     

cDNA cloning of a mandibular organ inhibiting hormone from the spider crabLibinia emarginata

Mandibular organs (MO) produce a crustacean juvenile hormone, methyl farnesoate (MF). MO activity is negatively regulated by factors, called mandibular organ inhibiting hormones (MOIHs), from the crustacean sinus gland X-organ complex in the eyestalks. Three MOIHs have been isolated previously from the spider crabLibinia emarginata and are characterized as members of the crustacean hyperglycemic hormone (CHH) neuropeptide family. In the research reported here, a full length cDNA sequence of 972 bp of a MOIH was isolated by screening a cDNA library constructed from the eyestalks ofLibinia emarginata. This cDNA sequence encodes a preprohormone peptide with 137 amino acid residues, including a 26-amino acid long signal peptide, a 34-amino acid long precursor peptide, a dibasic peptide, the full length of 72-amino acid long MOIH, and a tri-peptide Gly-Lys-Lys which designates the potential amidation site at the C-terminus of the mature peptide.     

The spatiotemporal segregation of GAD forms defines distinct GABA signaling functions in the developing mouse olfactory system and provides novel insights into the origin and migration of GnRH neurons

Gamma‐aminobutyric acid (GABA) has a dual role as an inhibitory neurotransmitter in the adult central nervous system (CNS) and as a signaling molecule exerting largely excitatory actions during development. The rate‐limiting step of GABA synthesis is catalyzed by two glutamic acid decarboxylase isoforms GAD65 and GAD67 coexpressed in the GABAergic neurons of the CNS. Here we report that the two GADs show virtually nonoverlapping expression patterns consistent with distinct roles in the developing peripheral olfactory system. GAD65 is expressed exclusively in undifferentiated neuronal progenitors confined to the proliferative zones of the sensory vomeronasal and olfactory epithelia In contrast GAD67 is expressed in a subregion of the nonsensory epithelium/vomeronasal organ epithelium containing the putative Gonadotropin‐releasing hormone (GnRH) progenitors and GnRH neurons migrating from this region through the frontonasal mesenchyme into the basal forebrain. Only GAD67+, but not GAD65+ cells accumulate detectable GABA. We further demonstrate that GAD67 and its embryonic splice variant embryonic GAD (EGAD) concomitant with GnRH are dynamically regulated during GnRH neuronal migration in vivo and in two immortalized cell lines representing migratory (GN11) and postmigratory (GT1–7) stage GnRH neurons, respectively. Analysis of GAD65/67 single and double knock‐out embryos revealed that the two GADs play complementary (inhibitory) roles in GnRH migration ultimately modulating the speed and/or direction of GnRH migration. Our results also suggest that GAD65 and GAD67/EGAD characterized by distinct subcellular localization and kinetics have disparate functions during olfactory system development mediating proliferative and migratory responses putatively through specific subcellular GABA pools. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 249–270, 2015     

Demonstration of the cellular expression of genes encoding molt-inhibiting hormone and crustacean hyperglycemic hormone in the eyestalk of the shore crab Carcinus maenas

Based on the amino acid sequence of the molt-inhibiting hormone of Carcinus maenas, two degenerated oligonucleotide primers were synthesized and used in the polymerase chain reaction. By use of complementary DNA of a library constructed from medulla terminalis-X-organ RNA of C. maenas as template, the specific complementary DNA between the primers was amplified, cloned and sequenced. This strategy revealed a DNA sequence for which the deduced amino acid sequence is identical to the recently published C. maenas molt-inhibiting hormone sequence as determined by Edman degradation. Visualization of messenger RNAs encoding molt-inhibiting hormone and crustacean hyperglycemic hormone in different perikarya of the X-organ was obtained using digoxigenin-labelled complementary RNA probes. Combination of immunocytochemical staining using polyclonal antisera against the native C. maenas neuropeptides and in situ hybridization performed on alternating sections confirmed the specificity of the reaction. The results show that there is no co-localization of molt-inhibiting hormone and crustacean hyperglycemic hormone at the messenger RNA and the protein level.     

甲壳动物高血糖激素家族生理功能研究进展

甲壳动物高血糖激素家族是甲壳动物特有的神经多肽激素家族,主要由眼柄的X-器窦腺复合体(XO-SG)合成与分泌,包括高血糖激素(CHH)、蜕皮抑制激素(MIH)、性腺抑制激素(GIH)和大颚器抑制激素(MOIH),协同调控着甲壳动物的生长、繁殖与蜕皮等生理生化过程.本文就目前CHH家族神经肽的功能研究,包括功能研究的方法、各个激素的功能以及分泌调控等研究进展作一综述.     

The crustacean hyperglycemic hormone precursors a and b of the Norway lobster differ in the preprohormone but not in the mature peptide

The neuro-endocrine X-organ sinus-gland complex of crustaceans produces and releases the neuropeptides of the crustacean hyperglycemic hormone (cHH)/molt-inhibiting hormone (MIH)/gonad-inhibiting hormone (GIH) family that regulate important physiological processes, such as growth, reproduction and molting. We cloned two full-length cDNAs encoding the preprocHH-A and preprocHH-B of the Norway lobster Nephrops norvegicus of 132 and 131 amino acid residues. The two cHHs differ in the preprohormone but not in the mature peptide sequence. The mature cHH was expressed in bacteria as GST fusion protein that, in bioassay, shows a hyperglycemic activity similar to that of native cHH present in an eyestalk extract.     

Electrophysiological effects of GABA on cat pancreatic neurons

In mammalian peripheral sympathetic ganglia GABA acts presynaptically to facilitate cholinergic transmission and postsynaptically to depolarize membrane potential. The GABA effect on parasympathetic pancreatic ganglia is unknown. We aimed to determine the effect of locally applied GABA on cat pancreatic ganglion neurons. Ganglia with attached nerve trunks were isolated from cat pancreata. Conventional intracellular recording techniques were used to record electrical responses from ganglion neurons. GABA pressure microejection depolarized membrane potential with an amplitude of 17.4 +/- 0.7 mV. Electrically evoked fast excitatory postsynaptic potentials were significantly inhibited (5.4 +/- 0.3 to 2.9 +/- 0.2 mV) after GABA application. GABA-evoked depolarizations were mimicked by the GABA(A) receptor agonist muscimol and abolished by the GABA(A) receptor antagonist bicuculline and the Cl(-) channel blocker picrotoxin. GABA was taken up and stored in ganglia during preincubation with 1 mM GABA; beta-aminobutyric acid application after GABA loading significantly (P < 0.05) increased depolarizing response to GABA (15.6 +/- 1.0 vs. 7.8 +/- 0.8 mV without GABA preincubation). Immunolabeling with antibodies to GABA, glial cell fibrillary acidic protein, protein gene product 9.5, and glutamic acid decarboxylase (GAD) immunoreactivity showed that GABA was present in glial cells, but not in neurons, and that glial cells did not contain GAD, whereas islet cells did. The data suggest that endogenous GABA released from ganglionic glial cells acts on pancreatic ganglion neurons through GABA(A) receptors.     

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.     

A Possible Role of the Non-GAT1 GABA Transporters in Transfer of GABA From GABAergic to Glutamatergic Neurons in Mouse Cerebellar Neuronal Cultures

Cultures of dissociated cerebellum from 7-day-old mice were used to investigate the mechanism involved in synthesis and cellular redistribution of GABA in these cultures consisting primarily of glutamatergic granule neurons and a smaller population of GABAergic Golgi and stellate neurons. The distribution of GAD, GABA and the vesicular glutamate transporter VGlut-1 was assessed using specific antibodies combined with immunofluorescence microscopy. Additionally, tiagabine, SKF 89976-A, betaine, β-alanine, nipecotic acid and guvacine were used to inhibit the GAT1, betaine/GABA (BGT1), GAT2 and GAT3 transporters. Only a small population of cells were immuno-stained for GAD while many cells exhibited VGlut-1 like immuno-reactivity which, however, never co-localized with GAD positive neurons. This likely reflects the small number of GABAergic neurons compared to the glutamatergic granule neurons constituting the majority of the cells. GABA uptake exhibited the kinetics of high affinity transport and could be partly (20%) inhibited by betaine (IC₅₀ 142 μM), β-alanine (30%) and almost fully (90%) inhibited by SKF 89976-A (IC₅₀ 0.8 μM) or nipecotic acid and guvacine at 1 mM concentrations (95%). Essentially all neurons showed GABA like immunostaining albeit with differences in intensity. The results indicate that GABA which is synthesized in a small population of GAD-positive neurons is redistributed to essentially all neurons including the glutamatergic granule cells. GAT1 is not likely involved in this redistribution since addition of 15 μM tiagabine (GAT1 inhibitor) to the culture medium had no effect on the overall GABA content of the cells. Likewise the BGT1 transporter cannot alone account for the redistribution since inclusion of 3 mM betaine in the culture medium had no effect on the overall GABA content. The inhibitory action of β-alanine and high concentrations of nipecotic acid and guvacine on GABA transport strongly suggests that also GAT2 or GAT3 (HUGO nomenclature) could play a role.     

Immunocytochemistry of GABA and glutamic acid decarboxylase in the thoracic ganglion of the crab Eriphia spinifrons

We have used specific antisera against protein-conjugated -aminobutyric acid (GABA) and rat-brain glutamic acid decarboxylase (GAD) in immunocytochemical preparations to study the distribution of putatively GABAergic neurons in the fused thoracic ganglion of the crab Eriphia spinifrons. In the thoracic neuromeres, about 2000 neurons with somata arranged in clusters or located singly in the cell cortex exhibited both GABA-like and GAD-like immunoreactivity. In addition, more than a hundred cells showed only GABA-like immunoreactivity. Fibrous immunoreactive staining to GAD and GABA was distributed throughout the neuropil of the thoracic ganglion, and several fiber tracts contained immunoreactive processes. Sets of serially homologous neurons exhibited GABA-like and GAD-like immunoreactivity in the thoracic neuromeres. Especially prominent were one medial and four ventro-lateral clusters of somata, together with thirteen individually recognized cells in each neuromere. Six of these cells in the ventro-medial cell cortex may be the somata of inhibitory motoneurons. The leg nerves contained three immunoreactive fibers, corresponding to the previously described common inhibitory motoneuron and the two specific inhibitors. The results present further evidence for GABA being the neurotransmitter of all inhibitory leg motorneurons, and suggest its presence and role as a neurotransmitter in a considerable number of interneurons in the thoracic ganglion of the crab.     

Immunocytochemical and autoradiographic localization of GABA system in the vertebrate retina

Summary The localization of -aminobutyric acid (GABA) neurons in the goldfish and the rabbit retina has been studied by immunocytochemical localization of the GABA-synthesizing enzyme L-glutamate decarboxylase (GAD, L-glutamate 1-carboxy-lase, EC 4.1.1.15) and by [³H] GABA uptake autoradiography. In the goldfish retina, GAD is localized in some horizontal cells (H1 type), a few amacrine cells and sublamina b of the inner plexiform layer. Results from immunocytochemical studies of GAD-containing neurons and autoradiographic studies of GABA uptake reveals a marked similarity in the labeling pattern suggesting that in goldfish retina, the neurons which possess a high-affinity system for GABA uptake also contain significant levels of GAD. In the rabbit retina, when Triton X-100 was included in immunocytochemical incubations with a modified protein A-peroxidase-antiperoxidase method, reaction product was found in four broad, evenly spaced laminae within the inner plexiform layer. In the absence of the detergent, these laminae were seen to be composed of small, punctate deposits. When colchicine was injected intravitreally before glutamate decarboxylase staining, cell bodies with the characteristic shape and location of amacrine cells were found to be immunochemically labeled. Electron microscopic examination showed that these processes were presynaptic to ganglion cell dendrites (infrequently), amacrine cell telodendrons, and bipolar cell terminals. Often, bipolar cell terminals were found which were densely innervated by several GAD-positive processes. No definite synapses were observed in which a GAD-positive process represented the postsynaptic element. In autoradiographic studies by intravitreal injection of [³H] GABA a diffuse labeling of the inner plexiform layer and a dense labeling of certain amacrine cell bodies in the inner nuclear layer was observed. Both immunocytochemical and autoradiographic results support the notion that certain, if not all, amacrine cells use GABA as their neurotransmitter.     

Dynamics of biosynthesis and release of crustacean hyperglycemic hormone isoforms in the X-organ-sinus gland complex of the crayfish Orconectes limosus.

The crustacean hyperglycemic hormone (CHH) is the major neuropeptide produced by the X-organ-sinus gland neurosecretory system of the crayfish, Orconectes limosus. This hormone is synthesized by two different cell types, as two isomers (CHH and D-Phe3-CHH) which display different activities The aim of this report is to analyze and compare the synthetic and secretory activities of these specialized cells. In vitro pulse-chase incubations and time-course experiments were conducted on isolated X-organ-sinus gland (XO-SG) complexes, followed by analysis of the labeled peptides. The different steps of the post-translational processing of the CHH precursor, including proteolytic cleavage of the propeptide, C-terminal amidation and N-terminal pyroglutamylation were characterized and the kinetics of CHHs maturation were estimated in the different parts of the neuroendocrine complex. Furthermore, synthesis of CHHs in XO-SG complexes and release in incubation media were investigated using combined HPLC/immunoassay. Under basal conditions, i.e. without stimulation, similar dynamics for both isomers were found and results indicate that newly synthesized CHHs are preferentially released.     

Localization of crustacean hyperglycemic hormone (CHH) and gonad-inhibiting hormone (GIH) in the eyestalk ofHomarus gammarus larvae by immunocytochemistry and in situ hybridization

This study deals with the localization of crustacean hyperglycemic hormone (CHH) and gonad-inhibiting hormone (GIH) in the eyestalk of larvae and postlarvae ofHomarus gammarus, by immunocytochemistry and in situ hybridization. The CHH and GIH neuropeptides are located in the perikarya of neuroendocrine cells belonging to the X-organ of the medulla terminalis, in their tract joining the sinus gland, and in the neurohemal organ itself, at larval stages I, II and III and at the first postlarval stage (stage IV). In all the investigated stages, the mRNA encoding the aforementioned neuropeptides could only be detected in the perikarya of these neuroendocrine cells. In stage I, approximately 19 CHH-immunopositive and 20 GIH-immunopositive cells are present, both with a mean diameter of 7±1 μm. GIH cells are preferably localized at the periphery of the X-organ surrounding the CHH cells that are centrally situated. Colocalization of CHH and GIH immunoreactions can be observed in some cells. The cell system producing CHH and GIH in the larval and postlarval eyestalk is thus functional and is morphologically comparable to the corresponding neuroendocrine center in the adult lobster.     

GABAergic Neurons in the Embryonic Olfactory Pit/Vomeronasal Organ: Maintenance of Functional GABAergic Synapses in Olfactory Explants

In previous work, we showed a robust γ-aminobutyric acid (GABAergic) synaptic input onto embryonic luteinizing hormone-releasing hormone (LHRH) neurons maintained in olfactory explants. In this study, we identify GABAergic neurons in olfactory pit (OP) of embryonic micein vivoand study, using patch-pipet whole-cell current and voltage clamp techniques, synaptic interactions of these neurons in explant cultures.In vivo,glutamate decarboxylase (GAD, the enzyme which synthesizes GABA) mRNA was first detected in nasal regions on Embryonic Day (E) 11.5. From E12.5 to E13.5, robust GAD expression was localized to cells primarily in the ventral aspect of the OP. GAD mRNA was not detected over dorsally located cells in olfactory sensory or respiratory epithelium. In addition, GAD mRNA was not observed in cells along olfactory axons. GAD mRNA was dramatically reduced in the OP/vomeronasal organ by E16.5. Using antibodies against both GABA and GAD, immunopositive axonal-like tracts were detected in the nasal septum on E12.5. GABAergic staining decreased by E13.5. To examine synaptic interactions of these GABAergic cells, embryonic olfactory explants were generated and maintained in serum-free media. As explants spread, neuron-like cells migrated into the periphery, sometimes forming ganglion-like clusters. Cells were recorded, marked intracellularly with Lucifer Yellow and post-fixation, immunocytochemically examined. Forty-six cells, typically multipolar, were GABAergic, had resting potentials around −50 mV, and exhibited spontaneous action potentials which were generated by spontaneous depolarizing GABAergic (GABA_A) synaptic activity. OP neurons depolarized in response to GABA by increasing Cl⁻conductance. The biophysical properties of OP-derived GABAergic neurons were distinct from those reported for olfactory receptor neurons but similar to embryonic LHRH neurons. However, unlike LHRH neurons, GABAergic neurons did not migrate large distances in olfactory explants or appear to leave the olfactory pitin vivo.     

Stimulation of TM3 Leydig cell proliferation via GABA<Subscript>A</Subscript> receptors: A new role for testicular GABA

The neurotransmitter gamma-aminobutyric acid (GABA) and subtypes of GABA receptors were recently identified in adult testes. Since adult Leydig cells possess both the GABA biosynthetic enzyme glutamate decarboxylase (GAD), as well as GABA_A and GABA_B receptors, it is possible that GABA may act as auto-/paracrine molecule to regulate Leydig cell function. The present study was aimed to examine effects of GABA, which may include trophic action. This assumption is based on reports pinpointing GABA as regulator of proliferation and differentiation of developing neurons via GABA_A receptors. Assuming such a role for the developing testis, we studied whether GABA synthesis and GABA receptors are already present in the postnatal testis, where fetal Leydig cells and, to a much greater extend, cells of the adult Leydig cell lineage proliferate. Immunohistochemistry, RT-PCR, Western blotting and a radioactive enzymatic GAD assay evidenced that fetal Leydig cells of five-six days old rats possess active GAD protein, and that both fetal Leydig cells and cells of the adult Leydig cell lineage possess GABA_A receptor subunits. TM3 cells, a proliferating mouse Leydig cell line, which we showed to possess GABA_A receptor subunits by RT-PCR, served to study effects of GABA on proliferation. Using a colorimetric proliferation assay and Western Blotting for proliferating cell nuclear antigen (PCNA) we demonstrated that GABA or the GABA_A agonist isoguvacine significantly increased TM3 cell number and PCNA content in TM3 cells. These effects were blocked by the GABA_A antagonist bicuculline, implying a role for GABA_A receptors. In conclusion, GABA increases proliferation of TM3 Leydig cells via GABA_A receptor activation and proliferating Leydig cells in the postnatal rodent testis bear a GABAergic system. Thus testicular GABA may play an as yet unrecognized role in the development of Leydig cells during the differentiation of the testicular interstitial compartment.     

GABA, Glutamic Acid Decarboxylase, and GABA Transaminase Levels in the Myenteric Plexus in the Intestine of Humans and Other Mammals

Abstract: Regional distribution of endogenous γ- aminobutyric acid (GABA), its synthesizing enzyme, glutamic acid decarboxylase (GAD), and metabolic enzyme, GABA transaminase (GABA-T), were determined in the intestinal tract of guinea pigs and cats and the findings compared with the number of ganglion cells in Auerbach's plexus. There were positive correlations among the GABA contents and the numbers of neural cells of the plexus. The precise localization of GABA and GAD in individual layers (mucosa, circular and longitudinal muscles, and Auerbach's plexus) in the human and cat colon was also determined. The endogenous GABA contents and GAD activity were the highest in Auerbach's plexus in tissues of both species. These results indicate that GABA is synthesized and localized in Auerbach's plexus and probably plays a significant role in the enteric nervous system.     

Molecular cloning and localization of a cDNA encoding a crustacean hyperglycemic hormone from the South African spiny lobster,Jasus lalandii

A cDNA, encoding a crustacean hyperglycemic hormone (cHH) of the South African spiny lobster, Jasus lalandii has been cloned. The cDNA consists of 1773 bp with an open reading frame of 399 bp that encodes a preprohormone of 133 amino acid residues. The preprohormone consists of a 25 amino acid hydrophobic signal peptide, a 32 amino acid cHH precursor-related peptide (CPRP) and the cHH sequence of 72 amino acid residues. The cHH sequence is flanked N-terminally by a Lys-Arg cleavage site and C-terminally by Gly-Lys, where Gly serves as an amidation site. The deduced amino acid sequence of the CPRP is in complete agreement with a peptide previously elucidated from sinus glands of J. lalandii, code-named CPRP 2 and the sequence of the cHH peptide matches that of the minor cHH isoform of J. lalandii, i.e. crustacean hyperglycemic hormone-II (cHH-II), which was also previously obtained by peptide sequencing. In situ hybridization on eyestalks revealed strong cHH-II mRNA expression in a subset of neurosecretory cells of the X-organ.     

Ontogeny of neuroendocrine centers in the eyestalk of Homarus gammarus embryos: an anatomical and hormonal approach

Summary

The ontogeny of the eyestalk neuroendocrine centers of the European lobster, Homarus gammarus, throughout embryonic development has been studied using light and electron microscopy, and the localization of specific neuroendocrine substances has been identified by immunocytochemistry. The procephalic lobes, which are the prospective eyestalks, develop progressively during embryonic development. In the nauplius stage two neuron masses are well defined. The visual structure originates from one of them and the neuroendocrine structure from the other. The four definitive optic ganglia are present at the mid-metanauplius stage and retain their appearance and location in larvae and adults. The organ of Bellonci, an internal sensory structure, appears at the mid-metanauplius stage and is mainly characterized by onion bodies. The medulla terminalis X-organ complex, an important neuroendocrine system, is present and already functional at the beginning of the embryonic metanauplius stage. Two neurohormones have been visualized immunocytochemically: the crustacean hyperglycemic hormone (CHH) and the gonad inhibiting hormone (GIH). Both neuropeptides are localized in the perikarya of neuroendocrine cells of the X-organ as well as in their tracts joining the presumptive sinus gland. However, the sinus gland has only been observed in the early larval stages just after hatching.