Hemolymph ecdysteroids during the last three molt cycles of the blue crab,Callinectes sapidus: quantitative and qualitative analyses and regulation

The profiles of circulating ecdysteroids during the three molt cycles prior to adulthood were monitored from the juvenile blue crab, Callinectes sapidus. Ecdysteroid patterns are remarkably similar in terms of peak concentrations ranging between 210–330 ng/ml hemolymph. Analysis of hemolymph at late premolt stage revealed six different types of ecdysteroids with ponasterone A (PoA) and 20‐OH ecdysone (20‐OH E) as the major forms. This ecdysteroid profile was consistent in all three molt cycles. Bilateral eyestalk ablation (EA) is a procedure that removes inhibitory neurohormones including crustacean hyperglycemic hormone (CHH) and molt‐inhibiting hormone (MIH) and often results in precocious molting in crustaceans. However, the inhibitory roles of these neuropeptides in vivo have not yet been tested in C. sapidus. We determined the regulatory roles of CHH and MIH in the circulating ecdysteroid from ablated animals through daily injection. A daily administration of purified native CHH and MIH at physiological concentration maintained intermolt levels of ecdysteroids in the EA animals. This suggests that Y organs (YO) require a brief exposure to CHH and MIH in order to maintain the low level of ecdysteroids. Compared to intact animals, the EA crabs did not exhibit the level of peak ecdysteroids, and the major ecdysteroid turned out to be 20‐OH E, not PoA. These results further underscore the important actions of MIH and CHH in ecdysteroidogenesis, as they not only inhibit, but also control the composition of output of the YO activity. © 2009 Wiley Periodicals, Inc.     

Proteomics and signal transduction in the crustacean molting gland

Regulation of the molting cycle in decapod crustaceans involves2 endocrine organs: the X-organ/sinus gland (XO/SG) complexlocated in the eyestalk ganglia and the Y-organ (YO) locatedin the cephalothorax. Two neuropeptides [molt-inhibiting hormone(MIH) and crustacean hyperglycemic hormone (CHH)] are producedin the XO/SG complex and inhibit ecdysteroidogenesis in theYO. Thus, YO activation is induced by eyestalk ablation (ESA),which removes the primary source of MIH and CHH. Cyclic nucleotides(cAMP and cGMP) and nitric oxide (NO) appear to mediate neuropeptidesuppression of the YO. Proteomics was used to identify potentialcomponents of signal transduction pathways ("targeted" or cell-mapproteomics) as well as assess the magnitude of protein changesin response to activation ("global" or expression proteomics)in the tropical land crab, Gecarcinus lateralis. Total proteinsin YOs from intact and ES-ablated animals were separated bytwo-dimensional gel electrophoresis and expression profileswere assessed by image analysis and gene clustering software.ESA caused a >3-fold increase in the levels of 170 proteinsand >3-fold decrease in the levels of 89 proteins; a totalof 543 proteins were quantified in total YO extracts. ESA inducedsignificant changes in the levels of 3 groups of proteins elutingfrom a phosphoprotein column and detected with phosphoproteinstaining of two-dimensional gels;     

Signaling Pathways for Ecdysteroid Hormone Synthesis in Crustacean Y-organs

The Y-organs of crustaceans secrete steroid hormones (ecdysteroids)which are responsible for molting and regeneration. The Y-organsin turn are controlled (negatively) by the eyestalk peptide,molt-inhibiting hormone (MIH). We are exploring the signalingpaths in Y-organ cells that lead to ecdysteroid generation whenactivated by the absence of MIH. The objective is to understandthe connections between MIH-receptor occupancy and the depressionof genes that express ecdysteroidogenic enzymes. MIH actionis mediated by a rise in cyclic 5' adenosine monophosphate (cAMP);cGMP also is involved in some species. That a cyclic nucleotideis a central regulatory component is indicated by the followingselection of results: dibutyryl cAMP, activators of adenylylcyclase or inhibitors of cyclic nucleotide phosphodiesteraseeach mimic the inhibitory action of MIH. Cyclic AMP inhibitsthe receptor-mediated uptake of cholesterol (the obligate ecdysteroidprecursor), by decreasing the number of receptor sites for thelipoprotein carrier of cholesterol. MIH via cAMP also depressesde novo protein synthesis upon which ecdysteroidogenesis dependsin part. A role for cellular free calcium (Ca⁺⁺) is indicatedby the ability of Ca⁺⁺ (or a Ca⁺⁺ionophore) to stimulate ecdysteroidproduction,thereby antagonizing MIH action. The mechanism involvesloweringcAMP levels by enhancing phosphodiesterase activity via calmodulin,not by affecting adenylate cyclase activity. Ca⁺⁺ counters thesuppressive action of MIH or cAMP on protein synthesis. Consistentwith the MIH-Ca⁺⁺ mutual antagonism, MIH increases Ca⁺⁺ effluxfrom ⁴⁵Ca-preloaded cells. Y-Organ cells contain protein kinaseC (PKC), the activation of which increases ecdysteroid production.PKC activity is not affected by MIH, but is stimulated by Ca⁺⁺.These and related experiments indicate that the PKC-activatedincrease in ecdysteroidogenesis involves events downstream fromthe production of cAMP and the degradation of cAMP by Ca⁺⁺.In relation to the latter, specific and non-specific inhibitorsof protein tyrosine kinases (PTK) inhibit ecdysteroid synthesisdose-dependently. The relationship of PTK with MIH-cAMP andCa⁺⁺-PKC systems is under study.     

Crustacean molt-inhibiting hormone: Structure,function, and cellular mode of action

In Crustacea, secretion of ecdysteroid molting hormones by Y-organs is regulated, at least in part, by molt-inhibiting hormone (MIH), a polypeptide neurohormone produced by neurosecretory cells of the eyestalks. This article reviews current knowledge of MIH, with particular emphasis on recent findings regarding the (a) structure of the MIH peptide and gene, (b) levels of MIH in eyestalks and hemolymph, (c) cellular mechanism of action of MIH, and (d) responsiveness of Y-organs to MIH. At least 26 MIH/MIH-like sequences have been directly determined by protein sequencing or deduced from cloned cDNA. Recent studies reveal the existence of multiple forms of MIH/MIH-like molecules among penaeids and raise the possibility that molecular polymorphism may exist more generally among MIH (type II) peptides. The hemolymphatic MIH titer has been determined for two species, a crayfish (Procambarus clarkii) and a crab (Carcinus maenas). The data are dissimilar and additional studies are needed. Composite data indicate cellular signaling pathways involving cGMP, cAMP, or both may play a role in MIH-induced suppression of ecdysteroidogenesis. Data from the two species studied in our laboratories (P. clarkii and Callinectes sapidus) strongly favor cGMP as the physiologically relevant second messenger. Ligand-binding studies show an MIH receptor exists in Y-organ plasma membranes, but the MIH receptor has not been isolated or fully characterized for any species. Such studies are critical to understanding the cellular mechanism by which MIH regulates ecdysteroidogenesis. Rates of ecdysteroid synthesis appear also to be influenced by stage-specific changes in the responsiveness of Y-organs to MIH. The changes in responsiveness result, at least in part, from changes in glandular phosphodiesterase (PDE) activity. The PDE isotype (PDE1) present in Y-organs of C. sapidus is calcium/calmodulin dependent. Thus, calcium may regulate ecdysteroidogenesis through activation of glandular PDE.     

Expression of recombinant eyestalk crustacean hyperglycemic hormone from the tropical land crab, <Emphasis Type="Italic">Gecarcinus lateralis</Emphasis>, that inhibits Y-organ ecdysteroidogenesis in vitro

Crustacean hyperglycemic hormone (CHH) is a pleiotropic neuropeptide that regulates carbohydrate and lipid metabolism, molting, reproduction, and osmoregulation in decapod crustaceans. CHH elevates glucose levels in the hemolymph by stimulating glycogenolysis in target tissues. It also inhibits ecdysteroidogenesis in the molting gland, or Y-organ (YO), possibly as a response to environmental stress. CHH acts via binding to a membrane receptor guanylyl cyclase, which is expressed in most tissues, including the YO. Large amounts of biologically active neuropeptide are required to investigate the mechanism of CHH signaling in the YO. Consequently, the eyestalk ganglia CHH (EG-CHH) isoform was cloned into a yeast (Pichia pastoris) expression vector to express recombinant mature peptide (rEG-CHH) with or without a C-terminal c-Myc/polyhistidine tag. Yeast cultures with untagged or tagged rEG-CHH inhibited ecdysteroidogenesis in YOs from European green crab (Carcinus maenas) 36% (P < 0.002) and 51% (P < 0.006), respectively. Purified tagged EG-CHH inhibited YO ecdysteroidogenesis 32% (P < 0.002), but lacked hyperglycemic activity in vivo. This is the first report of recombinant EG-CHH inhibiting YO ecdysteroidogenesis. The data suggest that the tagged recombinant peptide can be used to elucidate the CHH signaling pathway in the crustacean molting gland.     

Trimeric G proteins in crustacean (Callinectes sapidus) Y-organs: occurrence and functional link to protein synthesis

Crustacean Y-organs produce ecdysteroid molting hormones. Regulation of ecdysteroidogenesis appears to be complex, involving regulatory ligands (including but not limited to molt-inhibiting hormone, an eyestalk neurohormone) and the capacity of the Y-organs to respond to those ligands. Available data indicate cell signaling pathways involving cAMP, cGMP, or both may be involved in regulation of Y-organ function. Trimeric G proteins link receptor occupancy to regulation of intracellular cAMP levels. In studies reported here, we have assessed the occurrence of G proteins in blue crab (Callinectes sapidus) Y-organs, and the link of G proteins to Y-organ function. Bacterial toxin-catalyzed ADP-ribosylation revealed a PTX-sensitive (alpha i-like) protein in Y-organ membranes, but failed to reveal a CTX-sensitive (alpha s-like) protein in Y-organ membranes. Western blotting with primary antibodies raised against conserved regions of mammalian G proteins detected an alpha i-immunoreactive protein (approximately 40 kDa) and two alpha s-immunoreactive proteins (approximately 50 and approximately 57 kDa) in Y-organ membrane preparations. Incubation of Y-organ membrane fractions with cholera toxin significantly suppressed incorporation of [35S]-methionine into TCA-precipitable Y-organ proteins, but had no detectable effect on ecdysteroidogenesis in short-term (6 h) incubations. The combined results indicate that C. sapidus Y-organs possess both Gi and Gs proteins, and that alpha s is functionally linked to regulation of glandular protein synthesis.     

Regulation of protein synthesis in Y-organs of the blue crab (Callinectes sapidus): involvement of cyclic AMP

Paired Y-organs secrete ecdysteroid hormones that control cycles of growth and molting in crustaceans. Y-Organs are regulated, at least in part, by molt-inhibiting hormone (MIH), a polypeptide produced and released by the X-organ/sinus gland complex of the eyestalks. In the present studies, crab (Callinectes sapidus) Y-organs were incubated in vitro in the presence of [(35)S]methionine, and cyclic nucleotide analogs or experimental agents that influence the cAMP signaling pathway. In 4-hr incubations, 8-Br-cAMP and db-cAMP (but not 8-Br-cGMP) suppressed incorporation of [(35)S]methionine into Y-organ proteins; the effect of 8-Br-cAMP was concentration-dependent. Autoradiograms of radiolabeled Y-organ proteins separated on SDS-PAGE gels indicated the effect of 8-Br-cAMP was general (as opposed to selective) suppression of protein synthesis. Addition of both forskolin (an adenylyl cyclase activator) and 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor) likewise suppressed incorporation of [(35)S]methionine into Y-organ proteins. Cycloheximide (a protein synthesis inhibitor) suppressed incorporation of [(35)S]methionine into Y-organ proteins and secretion of ecdysteroids. The combined results suggest that cAMP is involved in regulation of protein synthesis in C. sapidus Y-organs. We are currently investigating the link of protein synthesis to ecdysteroid production, and the possibility of cross-talk between cAMP and other cellular signaling pathways in Y-organs.     

Molecular mechanism of molt-inhibiting hormone (MIH) induced suppression of ecdysteroidogenesis in the Y-organ of mud crab: Scylla serrata

The present study was focused on the regulation of ecdysteroidogenesis in the Y-organ of Scylla serrata during molting cycle. A strong expression of molt-inhibiting hormone (MIH) and phosphorylation of ERK was predominantly observed in the postmolt and intermolt stages of Y-organs, whereas protein kinase C, steroidogenic acute regulatory protein (StAR) and cytochrome P450(scc) activity were exclusively seen in the premolt stages. Interestingly, inhibition of ERK phosphorylation by PD98059 in the early postmolt (A), middle postmolt (B) and intermolt (C) stages resulted in the prominent expression of PKC and StAR in the postmolt stages. This result indicates that phosphorylation of ERK is required for suppression of ecdysteroid biosynthesis with the involvement of protein kinase C, and StAR protein.     

Stage-specific changes in calcium concentration in crustacean (Callinectes sapidus) Y-organs during a natural molting cycle, and their relation to the hemolymphatic ecdysteroid titer

Secretion of ecdysteroid molting hormones by crustacean Y-organs is suppressed by molt-inhibiting hormone (MIH). The suppressive effect of MIH on ecdysteroidogenesis is mediated by one or more cyclic nucleotide second messengers. In addition, existing data indicate that ecdysteroidogenesis is positively regulated (stimulated) by intracellular Ca(++). Despite the apparent critical role of calcium in regulating ecdysteroidogenesis, the level of Ca(++) in Y-organ cells has not been previously measured during a natural molting cycle for any crustacean species. In studies reported here, a fluorescent calcium indicator (Fluo-4) was used to measure Ca(++) levels in Y-organs during a molting cycle of the blue crab, Callinectes sapidus. Mean calcium fluorescence increased 5.8-fold between intermolt (C4) and stage D3 of premolt, and then dropped abruptly, reaching a level in postmolt (A) that was not significantly different from that in intermolt (P>0.05). The level of ecdysteroids in hemolymph of Y-organ donor crabs (measured by radioimmunoassay) showed an overall pattern similar to that observed for calcium fluorescence, rising from 2.9 ng/mL in intermolt to 357.1 ng/mL in D3 (P<0.05), and then dropping to 55.3 ng/mL in D4 (P<0.05). The combined results are consistent with the hypothesis that ecdysteroidogenesis is stimulated by an increase in intracellular Ca(++).     

甲壳动物蜕皮抑制激素调控机制的研究进展

甲壳动物的蜕皮过程主要是由Y器(Y-organ)分泌的蜕皮类固醇激素与X器-窦腺复合体(X-organ-sinus gland,XO-SG)分泌的蜕皮抑制激素MIH相互拮抗而进行调控的。而MIH调控机制较为复杂,且存在争议。本文就MIH调控机制的研究进展,包括研究方法,以及目前调控机制中争议最大的3个问题:MIH受体、cAMP与cGMP功能以及Ca2+功能作一综述。     

Neurohormonal control of ecdysone production: Comparison of insects and crustaceans

Summary

Ecdysteroid synthesis is regulated in insects by prothoracicotropic hormone (PTTH) and in crustaceans by molt-inhibiting hormone (MIH). These neurohormones exert opposite effects on their respective target tissues, PTTH stimulating the prothoracic glands and MIH inhibiting the Y-organs. The present work reviews recent progress in the neurohormonal regulation of prothoracic gland and Y-organ function. The steroid products of these glands are briefly discussed, as is current information on the structures of PTTH and MIH. Focus is placed on the mechanism of action of these hormones at the cellular level, as well as developmental changes in cellular sensitivity to PTTH. Though exerting different effects on ecdysteroid secretion, both PTTH and MIH increase cyclic nucleotide second messengers, are influenced by alterations in cellular calcium, and are likely to activate protein kinases. The contrasting steroidogenic effects of PTTH and MIH probably arise from differences in the cellular kinase substrates. In insects, such substrates enhance ecdysteroid secretion, possibly by increasing the translation of glandular proteins. In crustaceans, MIH-stimulated changes lead to the inhibition of both protein synthesis and steroidogenesis.     

Changes in intracellular calcium concentration in crustacean (Callinectes sapidus) Y-organs: relation to the hemolymphatic ecdysteroid titer

Secretion of ecdysteroid molting hormones by crustacean Y-organs is negatively regulated (inhibited) by molt-inhibiting hormone (MIH), a neuropeptide produced by neurosecretory cells in eyestalk ganglia. The inhibitory effect of MIH is mediated by one or more cyclic nucleotide second messengers. In addition, available data indicate that ecdysteroidogenesis is positively regulated (stimulated) by intracellular calcium. However, despite the apparent critical role of calcium in regulating ecdysteroidogenesis, the level of Ca(2+) in Y-organs cells has not been previously determined. In studies reported here, eyestalks were ablated from blue crabs (Callinectes sapidus) to remove the endogenous source of MIH and activate Y-organs. At 0, 3, 6, and 9 days after eyestalk ablation (D0, D3, D6, and D9, respectively), the level of Ca(2+) in Y-organ cells was determined using a fluorescent calcium indicator (Fluo-4), and the hemolymphatic ecdysteroid titer was determined by radioimmunoassay. Calcium fluorescence in D6 Y-organs was 3.5-fold higher than that in D0 controls; calcium fluorescence in D9 Y-organs was 3.9-fold higher than in D0 controls (P<0.05). Measurement of fluorescence along a transect drawn through representative cells indicated that the calcium fluorescence was localized to cytoplasm and not to nuclei. Associated with the increase in intracellular Ca(2+) was a significant increase in the hemolymphatic ecdysteroid titer: The level of ecdysteroids in hemolymph rose from 5.5?ng/mL on D0 to 49.6?ng/mL on D6 and 87.2?ng/mL on D9 (P<0.05). The results are consistent with the hypothesis that ecdysteroidogenesis is stimulated by an increase in intracellular Ca(2+).     

The solution structure of molt-inhibiting hormone from the Kuruma prawn Marsupenaeus japonicus

Molting in crustaceans is controlled by molt-inhibiting hormone (MIH) and ecdysteroids. It is presumed that MIH inhibits the synthesis and the secretion of ecdysteroids by the Y-organ, resulting in molt suppression. The amino acid sequence of MIH is similar to that of crustacean hyperglycemic hormone (CHH), and therefore, they form a peptide family referred to as the CHH family. Most of the CHH family peptides show no cross-activity, whereas a few peptides show multiple hormonal activities. To reveal the structural basis of this functional specificity, we determined the solution structure of MIH from the Kuruma prawn Marsupenaeus japonicus and compared the solution structure of MIH with a homology-modeled structure of M. japonicus CHH. The solution structure of MIH consisted of five alpha-helices and no beta-structures, constituting a novel structural motif. The homology-modeled structure of M. japonicus CHH was very similar to the solution structure of MIH with the exception of the absence of the N-terminal alpha-helix and the C-terminal tail, which were sterically close to each other. The surface properties of MIH around this region were quite different from those of CHH. These results strongly suggest that this region is a functionally important site for conferring molt-inhibiting activity.     

Purification,characterisation and amino acid composition of the putative moult-inhibiting hormone (MIH) ofCarcinus maenas (Crustacea,Decapoda)

Summary Using a Y-organ in vitro assay to measure repression of ecdysteroid synthesis in the presence of putative moult-inhibiting hormone (MIH), in conjunction with HPLC separation of sinus gland neuropeptides ofCarcinus maenas, it was found that both the hyperglycemic hormone (CHH) and a novel peptide (argued to represent the MIH) inhibited ecdysteroid synthesis. The latter was purified to homogeneity, and amino acid analysis showed that it is a 61 residue peptide (minimum molecular mass 7,200 Da) with the following amino acid composition: Asx₉; Thr₂; Ser₂; Glx₇; Pro₁; Gly₄; Ala₂; 1/2 Cys₄; Val₄; Met₁; Ile₃; Leu₅; Tyr₁; Phe₃; His₃; Trp₂; Lys₂; Arg₆. The N-terminus appears to be blocked. MIH is at least 20 times more potent than CHH in repressing ecdysteroid synthesis and is active at concentrations of less than 250 pmol/l. There may be structural similarities between CHH and MIH, howeve, MIH displays no CHH radioimmunoreactivity or hyperglycemic activity. The physiological significance of CHH in controlling ecdysteroid titres is not known.Abbreviations CHH hyperglycemic hormone - MIH moult inhibiting hormone - PAGE polyacrylamide gel electrophoresis - RIA radioimmunoassay - SDS sodium dodecyl sulfate - SG smus gland(s) - SGE sinus gland equivalent - TFA trifluoroacetic acid     

Expression of a Recombinant Molt-Inhibiting Hormone of the Kuruma Prawn Penaeus japonicus in Escherichia coli

The crustacean molt-inhibiting hormone (MIH) suppresses ecdysteroid synthesis by the Y-organ. The MIH of the kuruma prawn Penaeus japonicus has recently been isolated and its cDNA cloned. In this study, we expressed the MIH in Escherichia coli to obtain a large quantity of this hormone with biological activity. The MIH cDNA was processed and ligated into an expression plasmid. E. coli was transformed with this plasmid, and then the recombinant MIH (r-MIH) was expressed. The r-MIH was put through the refolding reaction and was purified by reverse-phase HPLC. N-terminal amino acid sequence and time-of-flight mass spectral analyses supported the idea that the r-MIH had the entire sequence. By in vitro bioassay using the Y-organ of the crayfish, the r-MIH was found to be comparable to natural MIH in inhibiting ecdysteroid synthesis.     

Functional relations of crab molt-inhibiting hormone and neurohypophysial peptides

Biological and immunological relationships between molt-inhibiting hormone (MIH) activity in eyestalk ganglia extracts of the crab, Cancer antennarius Stimpson, and peptides of the vasopressin-oxytocin family were assessed. Lysine vasopressin (LVP), arginine vasopressin (AVP), vasotocin (VT), and oxytocin (OT) mimicked MIH action by inhibiting ecdysteroid production of Y-organ segments in vitro with the relative potencies LVP greater than AVP greater than VT much much greater than OT. The inhibitory effect was reversible and specific (6 other peptides did not alter Y-organ activity). MIH and LVP increased Y-organ cyclic adenosine 3',5' monophosphate (cAMP) levels dose-dependently and with identical time course in which the rise in cAMP preceded inhibition of ecdysteroid production. The synthetic vasopressin antidiuretic agonist 1-deamino-8-D-AVP (dDAVP) inhibited Y-organ steroidogenesis dose-dependently; the vasopressin analog ([1(B-mercapto-beta, beta-cyclopentamethylenepropionic acid), 2-(O-methyl)tyrosine[AVP) (d(CH2)5Tyr(Me)AVP), a vasopressor antagonist, had no effect on basal or MIH-suppressed steroidogenesis. AVP antiserum abolished the inhibitory action of MIH, LVP, and AVP. Competitive binding curves for MIH, LVP, AVP, VT, and OT with the AVP antiserum suggested that MIH is most closely related to LVP. MIH may be structurally related to the vasopressins and act on Y-organ cells via type V2 (cAMP-linked) receptors.     

Biochemical and functional aspects of crustacean hyperglycemic hormone in decapod crustaceans: review and update

In crustaceans, neuroendocrine centers are located in different structures of the nervous system. One of these structures, the X-organ-sinus gland complex of the eyestalk, produces several neuropeptides that belong to the two main functionally different families: firstly, the chromatophorotropins, and secondly, a large family comprising various closely related peptides, commonly named CHH/MIH/GIH family. This review updates some aspects of the structural, biochemical and functional properties of the main hyperglycemic neuropeptide of this family, the crustacean hyperglycemic hormone (CHH). The first part of this work is a survey of the neuroendocrine system that produces the neurohormones of the CHH/MIH/GIH family, focusing on recent reports that propose new possible neuroendocrine loci of CHH production, secondly we revise general aspects of the CHH biochemical, and structural characteristics and thirdly, we present a review of the role of CHH in the regulation of several physiological processes of crustaceans as well as new reports on the ontogenetic aspects of CHH. The review is centered only on one group of malacostracan crustaceans, the Decapoda.