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(++).     

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.     

Characterization of molt-inhibiting hormone (MIH) action on crustacean Y-organ segments and dispersed cells in culture and a bioassay for MIH activity

Ecdysteroid secretion in vitro by gland quarters and dispersed cells of ecdysial glands (Y-organs) of the crab, Cancer antennarius Stimpson, was characterized. Optimum culture conditions are reported for maximum, sustained (72 hr) secretion and maintenance of cell viability in activated Y-organs obtained from de-eyestalked donors. Addition in vitro of eyestalk ganglia extracts containing the putative molt-inhibiting hormone (MIH) inhibited ecdysteroid production dose-dependently in the range of 0.1-4.0 and 0.01-4.0 eyestalk equivalents of MIH for gland quarters and dispersed cells, respectively. Inhibition by MIH was reversible, tissue specific as to source of MIH activity, and did not affect cell viability relative to controls. The results of replicate incubations of gland quarters with MIH were analyzed with formal statistics of parallel-line assay. The inhibitory action on ecdysteroid secretion is shown to be reproducibly linear and parallel in the dosage range, 0.1-4.0 eyestalk equivalents, amenable to calculation of relative potency among successive extracts, and of sufficiently high precision to serve as an MIH bioassay. Also, the results of these studies support the hypothesis that control of Y-organs by the eyestalks is physiologically direct.     

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.     

三疣梭子蟹蜕皮周期中MIH基因mRNA水平与蜕皮激素浓度变化

甲壳动物的蜕皮过程被认为是由位于眼柄的X器-窦腺复合体(XO-SG)分泌蜕皮抑制激素(MIH)通过调节Y器(YO)合成蜕皮激素而调控的。通过实时荧光定量PCR(qRT-PCR)发现MIH基因在三疣梭子蟹眼柄X器-窦腺复合体中表达最强。采用qRT-PCR分析了MIH基因在三疣梭子蟹蜕皮周期中的表达变化, 结果表明; A期为(0.42±0.08)倍, B期为(1.09±0.09)倍, C期为(1.35±0.16)倍, D₀亚期为(1.00±0.10)倍, D₁亚期(0.78±0.07)倍, D₂亚期为(0.27±0.08)倍, D_3/4亚期为(0.20±0.04)倍。采用高效液相色谱-电喷雾串联质谱(LC-MS/MS)法完成了三疣梭子蟹蜕皮周期中蜕皮激素(20E)浓度变化的测定。A/B期蜕皮激素的浓度较低, 低于仪器检测限0.33 pg, C期为(1.666±0.762) ng/mL, D₀亚期为(4.047±1.5133) ng/mL, D₁亚期为(6.756±4.928) ng/mL, D₂亚期为(8.609±3.827) ng/mL, D₃亚期为(19.534±4.799) ng/mL, D₄亚期为11.616 ng/mL。在三疣梭子蟹蜕皮周期中, MIH基因表达量与血淋巴中蜕皮激素浓度呈现一定拮抗性, 揭示MIH抑制Y器合成蜕皮激素而调控着三疣梭子蟹蜕皮的发生和进行。     

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.     

Stress reduces hemolymph ecdysteroid levels in the crab: mediation by the eyestalks

In decapod crustaceans, molt hormone (ecdysone) production by Y-organs is suppressed by an eyestalk neurosecretory product, molt-inhibiting hormone (MIH). Environmental stressors are known to delay or prevent molting in crabs. The present study assesses the function of the MIH-Y-organ neuroendocrine system in the crab Cancer antennarius under conditions of daily handling stress. After three days, stressed crabs showed significant suppression of hemolymph ecdysteroid levels, which continued to fall to 20% of controls by day 14. Ecdysteroid titers of stressed crabs returned to prestress levels seven days after stress termination. Ecdysteroid levels in de-eyestalked (DES) crabs rose 160% within 48 hr post-DES. Stressing DES crabs over 16 subsequent days did not significantly alter ecdysteroid levels compared with unstressed DES controls. Handling stress thus depresses hemolymph ecdysteroid levels in the crab, a response that is mediated by eyestalks and appears to result from stress-induced MIH release.     

Conserved role of cyclic nucleotides in the regulation of ecdysteroidogenesis by the crustacean molting gland

Molting processes in crustaceans are regulated by ecdysteroids produced in the molting gland (Y-organ), and molting is indirectly controlled by circulating factors that inhibit the production of these polyhydroxylated steroids. Two of these regulatory factors are the neuropeptides molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH). CHH appears to inhibit ecdysteroidogenesis in the Y-organ through the activation of a receptor guanylyl cyclase. The signaling pathway activated by MIH, however, remains a subject of controversy. It is clear that neuropeptides inhibit ecdysteroidogenesis by simultaneously suppressing ecdysteroid biosynthetic processes, protein synthesis, and uptake of high density lipoproteins. Data demonstrate that cAMP is the primary regulator of critical catabolic, anabolic, and transport processes, which ultimately support the capacity for ecdysteroid production by the Y-organ. While cAMP also regulates acute ecdysteroidogenesis to some extent, data indicate that cGMP is the primary signaling molecule responsible for acute inhibition by neuropeptides. It is clear that the regulatory roles filled by cAMP and cGMP are conserved among decapod crustaceans. It is unknown if these complementary second messengers are linked in a single signaling pathway or are components of independent pathways activated by different factors present in extracts of eyestalk ganglia.     

Expression of crustacean (Callinectes sapidus) molt-inhibiting hormone in insect cells using recombinant baculovirus.

Molt-inhibiting hormone (MIH) negatively regulates the synthesis of ecdysteroid molting hormones by crustacean Y-organs. We report here the expression of blue crab (Callinectes sapidus) MIH in insect cells using recombinant baculovirus. Insect Sf9 cells were transfected with recombinant baculovirus containing a DNA insert encoding the C. sapidus MIH prohormone (signal sequence plus mature hormone). The construct was designed to yield a mature, fully processed recombinant MIH (recMIH). Several baculovirus recombinants showing no contamination with wild-type viral DNA were subsequently analyzed for their ability to direct expression of recMIH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from infected cells revealed time-dependent expression of two proteins of approximately the predicted size for the C. sapidus MIH prohormone and mature hormone. Western blot results (using antiserum against MIH of Carcinus maenas) indicated that the proteins were MIH-immunoreactive. N-Terminal amino acid sequence data and mass spectral analysis indicated the expressed proteins were of the correct sequence and molecular mass. Cell lysates containing the recombinant protein dose-dependently suppressed the synthesis of ecdysteroids by Y-organs in vitro. We anticipate the recombinant peptide will prove useful for studies of the structure and function of MIH.     

The role of the Y-organ and cephalic gland in ecdysteroid production and the control of molting in the crayfish,Orconectes limosus

Summary The production of ecdysteroids (monitored by RIA) by Y-organs and cephalic glands in vitro was measured and hemolymph ecdysteroid levels were determined in the crayfish,Orconectes limosus, both after eyestalk ablation and as a function of time during natural premolt. Y-organ synthesis of ecdysteroid increased in parallel with a rise in hemolymph ecdysteroid concentrations under both conditions, peaking in substage D₂ of premolt. Y-organ ecdysteroid output after eyestalk ablation was 3–4 times higher. Thus, removal of the inhibiting system of the eyestalk effectively removes not only the principal control but also any modulation of ecdysteroid secretion by the Y-organs. Ecdysteroid levels remained low in Y-organ-ectomized crayfish, although premolt was initiated in some animals. The cephalic gland does not appear to contribute to the regulation of molting inOrconectes limosus. The Y-organs, on the other hand, are a principal source of ecdysteroids which regulate the major synthetic activities of premolt.     

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.     

Uptake of high-density lipoprotein by Y-organs of the crab,cancer antennarius. II. formal characterization of receptor-mediation with isolated membranes

Y-organs are the ecdysial glands of crustaceans, responsible for synthesis and secretion of ecdysteroid hormones. For this purpose, the glands acquire cholesterol as obligate precursor entirely from circulating high-density lipoprotein (HDL). A preceding study provided evidence for the mechanism of acquisition: Y-organs take up cholesterol bound to HDL by an energy-requiring process, receptor-mediated absorptive endocytosis. The present study characterized the receptors involved utilizing isolated Y-organ membranes. HDL binding was saturable and specific; a dissociation constant (K_d) of 1.08 × 10^?7 M and a binding maximum at equilibrium (B_max) of 70 μg HDL protein/mg membrane protein, were obtained. Binding was decreased by protease and was dependent upon calcium. Y-organs are regulated negatively by a peptide hormone from the eystalks, molt-inhibiting hormone (MIH). Y-organ membranes from de-eyestalked crabs (MIH absent) exhibited the same K_d value as membranes from intact crabs, but a B_max 17% higher. Thus, MIH activity apparently does not change the binding affinity of HDL, but decreases the number of binding sites. These results agree with our previous findings that MIH depresses ecdysteroid synthesis in part by inhibiting cholesterol uptake. Generally, Y-organ cells appear to contain receptors for HDL that are of high affinity and high binding capacity, similar to the characteristics reported for the binding of insect HDL (vitellogenin) to fat bodies and oocytes. © 1995 Wiley-Liss, Inc.     

Uptake of high-density lipoprotein by Y-organs of the crab,Cancer antennarius. I. characterization in vitro and effects of stimulators and inhibitors

Cholesterol is the obligate precursor for ecdysteroid hormone synthesis by the ecdysial glands (Y-organs) in crustaceans, and all cholesterol in the hemolymph is bound to high-density lipoprotein (HDL). The mechanism was studied of how Y-organ cells acquire cholesterol. Y-organ segments were incubated with HDL isolated from hemolymph and labeled with ¹²⁵I. After incubation, tissue was homogenized in acid to determine radioactivity in acid-precipitable (cell associated, intact) HDL and in acid-soluble (degraded) HDL. Both HDL uptake and degradation showed saturation kinetics. At saturation most of the total counts represented degraded HDL; by 3 h, degradation was 80%. Rates of HDL uptake and breakdown were higher in Y-organs from de-eyestalked crabs (deprived thereby of molt-inhibiting hormone, MIH) than in glands from intact crabs. Both parameters were depressed by inhibitors of glycolysis and oxidative phosphorylation dose dependently and by low temperature. HDL uptake also was depressed by cAMP added to the medium experimentally or through efflux from the tissue during incubation. These results indicate a mechanism for HDL uptake that entails receptor-mediated, energy-dependent endocytosis of the entire HDL-cholesterol complex. Also the results suggest that HDL uptake and degradation are mediated by cAMP and depressed by an eyestalk factor, presumably MIH. © 1995 Wiley-Liss, Inc.     

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.     

Further Studies on Signaling Pathways for Ecdysteroidogenesis in Crustacean Y-Organs

The Y-organs of crustaceans secrete ecdysteroids (molting hormones)and are regulated (negatively) by a neurosecretory peptide,molt-inhibiting hormone (MIH). Signaling path(s) in Y-organswere explored that connect MIH receptors ultimately with suppressionof receptor number for the uptake of cholesterol (ecdysteroidprecursor) and of gene expression of steroidogenic enzymes.Experiments were conducted in vitro with Y-organs of crabs (Cancerantennarius, Menippe mercenaria) and crayfishes (Orconectessp.). It was confirmed in all species that steroidogenesis occursin the absence of external calcium (Ca⁺⁺), but increases toa maximum as Ca⁺⁺ is increased to 1 to 10 mM and is substantiallyinhibited at higher Ca⁺⁺ concentrations. MIH does not requireexternal Ca⁺⁺ for inhibitory action, but inhibition is eliminatedby high Ca⁺⁺concentrations. Several experimental approachesfailed to find evidence of phospholipase C activation, turnoverof inositol triphosphate or diacylglycerol generation connectedwith steroidogenesis. Unbinding or chelation of intracellularCa⁺⁺ with thapsigargin or TMB-8, respectively both caused dose-dependentinhibition of ecdysteroid output. Blockade of Ca⁺⁺ channelswith verapamil, nifedipine or nicardipine also inhibited steroidogenesis;highest doses inhibited profoundly to below Ca⁺⁺-free basallevels. Inhibition also was obtained with all doses of the Ca⁺⁺channel agonist/antagonist (–) BAY K 8644 in crabs, butin crayfishes lower doses were stimulatory. However, if thecrayfish cells were depolarized, allowing greater Ca⁺⁺ influx,the previously stimulatory doses of BAY K 8644 became inhibitory.Y-organ protein kinase C (PKC) is Ca⁺⁺-sensitive. Activationof PKC was uniformly stimulatory in crabs, but inhibitory incrayfishes. Cytochalasin D, which disrupts the actin cytoskeleton,and which causes moderate Ca⁺⁺ influx, stimulated hormone formation.These results are interpreted to indicate a regulatory rolefor Ca⁺⁺ in ecdysteroidogenesis, involving a local, submembranecirculation of Ca⁺⁺ through ion channels and Ca⁺⁺ pumps andinteraction with PKC in phosphorylating key proteins. An optimallocal Ca⁺⁺ environment fostering hormone synthesis is evidentsince too little or too much Ca⁺⁺ is inhibitory. Methyl farnesoate (MF) had no effect on ecdysone productionin crab or crayfish Y-organs in 24-hr incubations with MF at100 pM to 10 µM.     

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.