Search

The crustacean hyperglycemic hormone (CHH) is involved in the regulation of endogenous blood glucose metabolism. In this paper we describe the daily rhythmicity in the blood glucose and the blood CHH content of the crayfish Orconectes limosus. Both blood CHH and blood glucose levels increase during the first hours after the beginning of darkness. The bioactivity of released CHH is far higher than that of CHH stored in the sinus gland. Moreover, the released hyperglycemic material shows an affinity for high molecular weight proteins in the hemolymph. Preliminary results suggest that subunits of hemocyanin may act as potential carrier-proteins for bioactive CHH.

The toad Xenopus laevis is able to adapt its skin color to background light intensity. In this neuroendocrine reflex, the proopiomelanocortin (POMC)-derived peptide alpha-melanophore-stimulating hormone (alphaMSH) is a key regulatory factor. In animals adapting to a black background, release of alphaMSH from the pituitary pars intermedia causes dispersal of melanin in skin melanophores. To investigate the long-term in vivo dynamics of alphaMSH production during black background adaptation, the biosynthetic rate of POMC and the contents of POMC, alphaMSH and the POMC processing enzyme precursor convertase 2 (PC2) have been studied in the pars intermedia using pulse-labeling, Western blot and radioimmunoassay. In control animals, adapted to a white background, the rate of POMC biosynthesis and the POMC content were low, while high alphaMSH and PC2 contents were found. After 1 week of adaptation to a black background, the rate of POMC biosynthesis and the POMC protein content had increased 19- and 3.7-fold respectively. These parameters attained a maximum level (28- and 5. 8-fold higher than control) after 3 weeks and remained at these elevated levels for at least 12 weeks. After 1 week, the pars intermedia content of alphaMSH was only 30% of the control level, but after 6 and 12 weeks, the alphaMSH level had increased to the control level. The PC2 content decreased to 52% of control after 1 week and stabilized after 3 weeks at a level slightly lower than the control value. The results show that during long-term background adaptation a steady-state situation is reached, with a balance between the biosynthesis, enzymatic processing and release of alphaMSH. The in vivo dynamics of the processing enzyme PC2 suggest a parallel storage and release of alphaMSH and mature PC2 in the Xenopus pituitary pars intermedia.

Seven different types of neurosecretory granules have been observed in the axon terminals of the sinus gland of the adult female lobster Homarus gammarus. They were discriminated through morphometrical and morphological criteria such as size, shape, frequency of occurrence, electron density, and axon-matrix density. Each size distribution followed a single Gaussian-type distribution. Each axon terminal contained I type of granule. The mean granule diameter ranged from 50 nm (type 1) to 161 nm (type 7). Type 6 and type I axon terminals were, respectively, the most and least frequent. An immunopositive reaction against an anti-Homarus americanus CHH was detected in the 2 largest types of granules: type 6 (mean diameter, 128 nm) and type 7 (mean diameter, 161 nm).

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 h...

Osmoregulatory capacity (OC) decreased by approximately 50% after eyestalk ablation in adult Homarus americanus when in dilute media. OC was used to assay sinus gland extracts. Injection of total extracts and of some HPLC-separated fractions of sinus glands into destalked lobsters increased OC. One of the described crustacean hyperglycemic hormone isoforms influences osmoregulation. Another fraction of the sinus gland extracts modifies osmoregulation but its nature remains unknown. Variations in OC were examined in response to ecdysterone, Phe-Met-Arg-Phe-NH2, and atrial natriuretic factor but effects were minimal.

Summary Studies on the control of reproduction in crustaceans have, for the most part, dealt with the regulation of female reproduction. However, not a great deal of attention has been paid to cellular interactions of the neurohormones involved in this regulation. In this contribution recent data on inhibitory and stimulatory neuropeptides are discussed and reflections are made on their importance for further application-directed research. Attention is paid to: histophysiological events in the oocytes during vitellogenesis; biochemical aspects of the regulating neuropeptides and cytological localization of these neuropeptides in the neuroendocrine system. Opportunities for further research are identified and appraised in light of the knowledge reviewed.

This article describes the development and evaluation of a computer-aided-learning (CAL) program called ZOOPHYL. The CAL program was used in a first-year university level course in invertebrate zoology. It deals with the classification, biology and morphology of ten major invertebrate phyla. For each phylum, students must complete 16 ‘fill-in’ screens which are identified as themes. Each screen specifies important characteristics of each phylum, which can be compared and contrasted with the others. Approximately ten hours of instructional materials were produced to operate on PCs running under DOS. The program is described from a technical, educational and structural viewpoint. Usage and evaluation procedures and outcomes are summarized. Plans for the future are enumerated.

Summary Cryo field emission scanning electron microscopy (cryo-FE-SEM) is a versatile technique that allows the investigation of the three-dimensional organization of cells at the ultrastructural level over a wide range of magnifications. Unfortunately, cryopreparation of the specimens for this technique remains cumbersome, in particular because ice crystal formation must be prevented during freezing. Here we report that a light prefixation with glutaraldehyde and incubation in glycerol as cryoprotectant or a high-pressure freezing approach are both excellent procedures for cryopreparation of animal cells to be used in combination with cryo-FE-SEM. Using the proopiomelanocortin-producing intermediate pituitary melanotrope cells of Xenopus laevis as a physiologically inducible neuroendocrine system, we compared the ultrastructural characteristics of inactive and hyperactive neuroendocrine cells. The overall quality of the ultrastructural images was comparable for the two cryopreparation procedures, although some fine structures were better conserved using high-pressure freezing. Melanotrope cells in a secretory inactive state contained numerous storage granules and a poorly developed endoplasmic reticulum (ER), while large amounts of rough ER were present in hyperactive cells. Thus, the cryo-FE-SEM approach described here allows a fast ultrastructural study on the secretory activity of neuroendocrine cells.

A combined molecular biological, (bio)chemical and immunocytochemical study was carried out on the role of neuropeptides in the control of the biannual reproductive cycle in the female lobster.

The present study investigated changes in crustacean hyperglycaemic hormone (CHH) and serotonin (5-hydroxytryptamine, 5-HT) immunoreactivity in the retina and the X-organ/sinus gland complex (XO-SG) of the crayfish Procambarus clarkii at two developmental stages, post-embryonic stage two (PO2) and the juvenile stage, at three different times of day, under a photoperiod cycle of 12 h:12 h L:D, using qualitative and quantitative immunohistochemical methods. In the retina, CHH immunoreactivity is located in the tapetal cells, while 5-HT immunoreactivity is found in the retinular cells. In the XO-SG, CHH-immunoreactivity is localized to the CHH-producing cell perikarya and in their axons and endings in the sinus gland, while 5-HT immunoreactivity is restricted to axon endings branching into the perikarya of the CHH-producing cells. A stereological analysis demonstrates that the PO2 and juvenile stages show significant differences in the amount of the immunoreactive CHH and 5-HT material at the three selected time points, indicating daily and related changes in the levels of CHH and 5-HT in the XO-SG and the retina. Our findings therefore support the idea that daily rhythms in the secretory activity of the XO-SG complex affect the circadian sensitivity of the eye. Furthermore, the differences found between the PO2 and juvenile stages suggest that both CHH and 5-HT are key factors in the development of the circadian rhythm of retinal sensitivity.

Crustacean reproduction is regulated by a complex chain of hormonal interactions in which the crustacean hyperglycaemic hormones A and B (CHH-A and CHH-B) and the gonad-inhibiting hormone (GIH) play a primary role. These neurohormones are produced in the same neuroendocrine cells of the X-organ sinus gland complex, situated in the eyestalks of the American lobster, Homarus americanus. In order to obtain more information on the synthesis, storage, release and function of these three neuropeptides during the reproductive cycle, we studied the levels of their mRNAs in the X-organ, their peptide storage in the sinus gland and their concentration in the haemolymph at different stages of the female reproductive cycle. A high CHH-A mRNA level was found only in the previtellogenic stage, while elevated mRNA levels were determined for CHH-B in the mature as well as the previtellogenic stage. High CHH storage levels in the sinus gland were found during previtellogenesis. The total amount of CHH (CHH-A plus -B) in the haemolymph was significantly higher during maturation. A low level of GIH mRNA in the X-organ and a low amount of the GIH I isoform in the sinus gland were found only in the immature stage. In contrast, GIH haemolymph levels were high during the immature and previtellogenic stages. We conclude that CHH-A and -B are involved in triggering the onset of vitellogenesis and that CHH-B in particular is responsible for stimulating oocyte maturation before spawning, while GIH prevents the start of vitellogenesis in the ovary. Moreover, our results show that the balance between the haemolymph levels of the CHHs and GIH may tune the synchronization of reproduction and molting during the biannual reproductive cycle of the American lobster.

Neurohemal organs of the lobster Homarus americanus contain isoforms of the crustacean hyperglycemic hormone, which differ by the third amino acid (phenylalanyl) residue that is either in the L- or in the D-configuration. Polyclonal antisera have been raised in rabbit against synthetic octapeptides with the sequence corresponding to the N-terminal part of the L- or D-phenylalanine-containing isoforms. Their specificity was shown by immunoassays, indicating that they discriminate the isoforms of the lobster hyperglycemic neuropeptides. It was demonstrated that the two major forms of the crayfish Orconectes limosus hyperglycemic hormone also correspond to peptide isomers containing the L- or D-phenylalanyl residue. The cellular distribution of the isoforms among the neurosecreting cells of the major neuroendocrine complex in lobster and crayfish has been studied by immunohistochemistry. Every hyperglycemic hormone-containing cell was labelled with the anti-L antisera while only some of them were visualized with the anti-D antisera. These results constitute the first observation of peptide isomerization at the cellular level and suggest that the isomerization process occurs in specialized neuroendocrine cells.

Isolated posterior gills of the hyper-hyporegulating crab Pachygrapsus marmoratus were perfused with extracts of homologous sinus glands. Sinus gland extracts stimulated the influx of Na+ ions and increased the transepithelial potential difference in the gills in a dose-dependent and reversible fashion. The bioactivity of extracts prepared from crabs that had been acclimated to 10/1000 salinity for at least 1 week was not significantly different from that of extracts prepared from seawater (36/1000 salinity) crabs. The perfusion experiments with both extracts containing two sinus glands significantly increased Na+ influx by about 150% and transepithelial potential difference by about 45%. Sinus gland extracts also increased the Na+/K(+)-ATPase activity by 54% in incubated posterior gills. The bioactivity of extracts was reduced by pronase and trypsin, but not by heating for 10 min at 100 degrees. The molecular weight of the responsible factor(s) was5000 Da. Thus, the sinus gland of P. marmoratus is concluded to be involved in the neuroendocrine control of osmoregulation and to contain a peptide(s) that directly influences brachial function.

Several large peptidic neurohormones have been isolated in crustaceans. In lobster and other related species, each of these neurohormones, and particularly the crustacean hyperglycemic hormone, occurs as two isoforms having the same peptidic sequence and molecular mass. We report here that these isoforms differ by the configuration of a single amino acid residue. The third residue (Phe3) of the lobster hyperglycemic hormones is in either the L- or D-configuration. In addition, we have shown that the biological activity of the two isoforms differs when considering the kinetics of their hyperglycemic effect.

The localization of messenger RNAs encoding the crustacean hyperglycemic hormone, involved in regulation of carbohydrate metabolism and the gonad inhibiting hormone, which inhibits vitellogenesis, was studied in the eyestalk of the lobster Homarus americanus using complementary RNA probes for in situ hybridization. For the detection of gonad inhibiting hormone messenger RNA, we cloned and sequenced a partial complementary DNA encoding lobster gonad inhibiting hormone and for crustacean hyperglycemic hormone messenger RNA detection an available complementary DNA was used. This approach reveals that there is a frequent but inconsistent cellular co-localization of the two neurohormones. Furthermore, our data show that male lobsters contain an equal number of neuroendocrine gonad inhibiting hormone cells as female lobsters. An additional study, involving the use of in situ hybridization in combination with immunocytochemistry, shows that the synthetic activity of the crustacean hyperglycemic hormone- and gonad inhibiting hormone-producing cells can be followed at the messenger RNA as well as the protein level. This reveals that when strong immunostaining is present, the messenger RNA staining is usually weak or absent and vice versa. In conclusion, the presence of cells, containing only gonad inhibiting hormone messenger RNA or only crustacean hyperglycemic hormone messenger RNA, indicates that lobster crustacean hyperglycemic hormone and gonad inhibiting hormone originate from two different precursors. Co-localization of the two neurohormone messenger RNAs confirms the co-localization at the peptidergic level found by immunocytochemistry and thus these findings were not due to cross-reactions between the two antisera.(ABSTRACT TRUNCATED AT 250 WORDS)

The crustacean hyperglycemic hormone (CHH) is synthesized as part of a larger preprohormone in which the sequence of CHH is N-terminally flanked by a peptide for which the name CPRP (CHH precursor-related peptide) is proposed. Both CHH and CPRP are present in the sinus gland, the neurohemal organ of neurosecretory cells located in the eyestalk of decapod crustaceans. This paper describes the isolation and sequence analysis of CPRPs isolated from sinus glands of the crab Carcinus maenas , the crayfish Orconectes limosus and the lobster Homarus americanus . The published sequence of “peptide H” isolated from the land crab, Cardisoma carnifex , has now been recognized as a CPRP in this species. Sequence comparison reveals a high level of identity for the N-terminal region (residues 1–13) between all four peptides, while identity in the C-terminal domain is high between lobster and crayfish CPRP on the one hand, and between both crab species on the other. Conserved N-terminal residues include a putative monobasic processing site at position 11, which suggests that CPRP may be a biosynthetic intermediate from which a potentially bioactive decapeptide can be derived.

With the use of a two-step HPLC purification procedure, two sets of two isoforms of the crustacean hyperglycemic hormone (CHH) were isolated from sinus glands of the lobster Homarus americanus. Structural differences between the two groups of isoforms were found in their amino acid sequences, amino acid compositions and precise molecular weights. Using peptide mapping, the difference between the isoforms in each group was located within the first eight amino acids at the N-termini. The nature of this difference remained unclear as all four peptides had the same N-terminal amino acid sequence unto residue 19.

1. 1. The importance of the MTGX2 sinus gland complex in the production and the storage of CHH material has been demonstrated by bioassays using extracts of the different eyestalks structure. 2. 2. The purification of the hyperglycemic material (CHH) was carried out in Palaemon serratus by two procedures, starting from total eyestalk extracts or from extirpated sinus glands. 3. 3. There is evidence for the presence of 3 different forms of the hyperglycemic active fractions expressed by their electrophoretic mobility and molecular weight. 4. 4. The main fraction has a molecular weight of around 8000 and a r m of 0.59 in Davis electrophoresis but small peptides (around 2000 mol. wt) are also hyperglycemic if injected in eyestalkless shrimps.

The eyestalk of the astacideans Orconects limosus, Nephrops norvegicus, and Homarus gammarus, and the palinuran Palinurus vulgaris, was examined with an antiserum raised against purified crustacean hyperglycemic hormone (CHH) of the astacidean species Astacus leptodactylus. A distinct immunopositive reaction occurs in a group of neurosecretory cells in the medulla terminalis ganglionic X-organ (MTGX), in the MTGX-sinus gland tractus, and in a considerable part of the sinus gland. The immunoreactive sites in the eyestalk of the investigated species correspond to the site of production, storage, and release of the CHH. Preliminary investigations with this antiserum also indicate that a positive immunoreaction can be obtained in the eyestalk of other decapod crustaceans, for example, of the brachyuran Macropipus puber and the caridean Palaemon serratus.

Ontogenesis of the eyestalk structures in Palaemon serratus has been studied during the larval and postlarval life by means of scanning electron microscopy (SEM) and light microscopy (LM). At hatching, a larval sensory pore is observed in the zoea. The organ of Bellonci is present with its typical onion bodies. During larval life, this organ grows and differentiates. Vacuolization in the organ of Bellonci starts in stage 4. At the same time, the giant neuron appears, but the sinus gland cannot be recognized before stage 5.During larval life, vesicular cells are present in the optic medullae with an increase during stages 4 and 5. The differentiation of the neurosecretory cells of MEX (medulla externa X-organ) and MTGX (medulla terminalis ganglionaris X-organ) occurs with metamorphosis. The main sensory pore appears with metamorphosis and becomes clearly visible in postlarval life.The movements of the structures, which occupy different positions in larvae and adults, are described in relation to the longitudinal axis of the eyestalk. A schematic representation explains the torsion of the eyestalk and the dynamics and growth during the formation of the eyestalk structures.

Summary A two-step high performance liquid chromatography (HPLC) separation system, based on ion exchange and hydrophobic properties, was developed in order to isolate and purify several neuropeptides from the sinus gland of the lobster Homarus americanus. With this system, most of the thus far reported neurohormonal activities, such as hyperglycemia, gonad and pigment controlling factors are detectable. Special attention is given to two pairs of closely related peptides, all reacting with a polyclonal rabbit antiserum raised against the hyperglycemic hormone (CHH) of the crayfish Astacus leptodactylus. Sodium dodecyl sulphate-urea-polyacrylamide gel electrophoresis (SDS-urea-PAGE) of these substances reveals two peptides of about 6.5 kDa, and two more of about 8 kDa. A hyperglycemic response is provoked by both 6.5 kDa peptides and the second 8 kDa component. A stimulatory effect on the oocyte growth is associated with the elution time of both 8 kDa peptides. Furthermore, the presence of two factors with...

Summary After solubilization of membranes from vitellogenic oocytes of the crayfish Orconectes limosus, a component with an apparent molecular weight between 28 and 30 kDa that specifically binds vitellogenin, could be identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and by electroblotting. Because this component was pronase sensitive, we assume that we are dealing with a protein. A polyclonal rabbit antiserum was produced against this component and its tissue specificity was verified.

The criteria for determination, given by Drach (1939), have been adapted to divide the molt cycle into different stages for larvae and adults of the crayfish Astacus leptodactylus . The setal formation in the transparent appendages can be used for the definition of stages and substages in the premolt period. In order to evaluate the post-and intermolt stages in more detail, one can combine this method with changes occurring in the cuticle.

1. 1. Extracts of the hepatopancreas from sinus glandless and from control crayfish do not show any significant differences in their alkaline phosphotase activities. 2. 2. In both groups the alkaline phosphatase activity increases over a period of captivity of 21 days. 3. 3. Histochemically alkaline phosphotase appeared uniquely in the brush border of the rest cells and absorption cells, and around the vacuoles in B-cells.

Hyperglycemic hormone (HGH) from Astacus leptodactylus can be visualized by an immunocytochemical procedure using a specific antiserum against HGH and the peroxidase-anti-peroxidase (PAP) method. HGH containing cells are localized in the most distal portion of the x-organ in the medulla terminalis. Their cell diameter is about 47 +/- 7 micrometer; the nucleus measures about 22 +/- 2 micrometer. They form a subgroup of neurosecretory cell type 1, already described for the crayfish. A large amount of this HGH material is homogeneously stored in the sinus gland. A group of fibres in the x-organ - sinus gland - tractus can be followed, due to their positive reaction with the PAP-reagent.

A biochemical analysis was made of the crustacean hyperglycemic hormone (CHH) of the crayfish Astacus leptodactylus, as present in the CHH-producing perikarya, in the axonal tract and in the sinus gland, respectively. Hyperglycemic material was analyzed by polacrylamide gel electrophoresis (PAGE and SDS-PAGE) and high-pressure liquid chromatography (HPLC) in combination with a dotting immunobinding assay (DIA) and a bioassay for hyperglycemic activity. After electrophoretic analyses, the predominant biologically as well as immunologically detectable product present in all parts of the cell has an apparent molecular radius of approximately 7000 Da. In the perikarya extract, a second factor with lower electrophoretic mobility was found, which may represent the prohormone or precursor of CHH. The analyses by means of HPLC showed two predominant immunopositive peaks with an elution time of 28–29 and 52–54 min, respectively. For both HPLC peaks, electrophoretic analyses indicate a molecular weight of 7000 Da.

Following a serotonin injection, a rise of exocytosis in the sinus gland of the crayfish Astacus leptodactylus is observed together with an increase of the glucose level in the hemolymph. The phenomenon of exocytosis reaches a peak between 2 and 4 h after injection, wheras the highest glucose level in the hemolymph appears between 4 and 6 h after the same injection.

Five types secretory granules are distinguished in the sinus gland of Palaemon serratus on the basis of differences in their diameter and their electron density. Variations in the density of the acoplasm and of the granules are observed for three of these granule types.

Using the alcian blue-alcian yellow staining method, the neurosecretory system in the eyestalk of the Crayfish Astacus leptodactylus is studied during the period following the first, second, third and fourth moult after hatching. On the basis of their staining affinities, six different cell types are distinguishable, always spread over the four optic ganglia and varying in number depending upon the moment of sampling. A clearly recognizable sinus gland is present in the period following the third and fourth moult after hatching. During the successive stages, there occurs a shift in staining pattern of the sinus gland, which is also expressed by the variation in number of the various stained cell types.

By use of a specific antiserum against the molluscan cardio-excitatory tetrapeptide FMRF-amide in combination with the PAP-method it was possible to obtain positive immunocytochemical reactions in several neurosecretory regions of the eyestalk of the prawn Palaemon serratus. FMRF-amide-like material was found in perikarya and nerve fibers of the medulla terminalis and in neurons in the lamina ganglionaris. The immunoreactivity observed in the glandular tissue located at the basal insertion of the eyestalk muscles must be ascribed to a non-specific reaction. The identification of immunopositive nerve fibers, ending on a nerve bundle in the medulla terminalis, and the fact that immunoreactive material was absent in the neurohemal sinus gland seem to indicate a neurotransmitter/neuromodulator function.

The occurrence of adipokinetic hormone (AKH) and red pigment concentrating hormone (RPCH)-like neuropeptides in the central nervous system of different invertebrate species other than insects was investigated immunocytochemically with polyclonal antisera to N- and C-terminal regions of the AKH molecule. Substances reacting with the C-terminal specific antiserum code 241 were present in neurons of the pond snail, Lymnaea stagnalis, the sowbug Porcellio scaber, the centiped Lithobius forficatus and the crayfish Astacus leptodactylus. Substances revealed by the N-terminal specific antiserum code 433 were demonstrated in the latter two species. The distribution of immunoreactive substances in neuropile areas of several ganglia suggests that these substances act as neurotransmitter/neuromodulator. Their presence in neurohemal organs such as the periphery of the pedal and visceral nerves in the pond snail and in the sinus gland of the crayfish suggests a neurohormonal role in these species.

Several large peptidic neurohormones have been isolated in crustaceans. In lobster and other related species, each of these neurohormones, and particularly the crustacean hyperglycemic hormone, occurs as two isoforms having the same peptidic sequence and molecular mass. We report here that these isoforms differ by the configuration of a single amino acid residue. The third residue (Phe3) of the lobster hyperglycemic hormones is in either the L- or D-configuration. In addition, we have shown that the biological activity of the two isoforms differs when considering the kinetics of their hyperglycemic effect.