Molt-inhibiting Hormone Activity Research Articles

Molt-inhibiting hormone (MIH) gene from the green mud crabScylla paramamosainand its expression during the molting and ovarian cycle

Mud crab is a group of commercially important species that are found throughout the Indo-Pacific region. In this study, the full-length cDNA of molt-inhibiting hormone (Scp-MIH) was cloned from the green mud crab, Scylla paramamosain. The conceptually translated peptide precursor consists of a 78-residue mature peptide, preceded by a 35-residue signal peptide. The mature peptide shares a high identity with the other known MIHs, and contains six conserved cysteine residues that have been proposed to be functionally critical for MIH activity. Analysis of genomic sequence revealed that Scp-MIH gene was organized in a 3 exons/2 introns manner. Quantitative real-time PCR showed that Scp-MIH transcripts were detected mainly in the eyestalk ganglion and brain. During the molting cycle, Scp-MIH mRNA increased significantly from postmolt stage to intermolt stage, then decreased sharply at premolt stage. It implies that MIH is closely related to ecdysteroid production in S. paramamosain. In the ovarian cycle, Scp-MIH transcripts in some neural tissues (eyestalk ganglion and brain) increased from stage I to stage II, and reached the peak value at stage III, then declined at later stage. It suggests that MIH might be involved in the ovarian maturation in S. paramamosain.

Characterization and sequence elucidation of a novel peptide with molt-inhibiting activity from the South African spiny lobster, Jasus lalandii

We have isolated a peptide from extracts of sinus glands from a South African spiny lobster species, Jasus lalandii, by high-performance liquid chromatography (HPLC) and identified it as a putative molt-inhibiting hormone (MIH) by (i) an in vitro assay with J. lalandii Y-organs to measure the inhibition of ecdysteroid synthesis and (ii) an immunoassay using antiserum raised against MIH of the edible crab. The MIH of J. lalandii has 74 amino acid residues, a molecular mass of 9006 Da, a free N-terminus and an amidated C-terminus. The full primary sequence has been obtained from sequencing various digest fragments (tryptic, endoproteinase Asp-N, cyanogen bromide) of the unreduced (native) peptide: RFTFDCPGMMGQRYLYEQVEQVCDDCYNLYREEKIAVNCRENCFLNSWFTVCLQATMREHETPRFDIWRSIILKA-NH 2. Structural comparisons with other peptides show that the J. lalandii MIH belongs to the peptide family which includes the crustacean hyperglycemic hormone, molt-inhibiting hormone and vitellogenesis-inhibiting hormone (cHH/MIH/VIH). This novel peptide has 36–43% sequence identity to putative MIHs from other decapod crustaceans and 32–34% identity to the two cHH peptides previously identified in this spiny lobster species. This is the first report of a peptide with MIH activity in the Palinuridae infraorder.

A Novel Neuropeptide with Molt-inhibiting Activity from the Sinus Gland of the Crayfish, Procamharus clarkii

Abstract Molt-inhibiting hormone (MIH) activity was tested in terms of the inhibitory activity of ecdysteroid secretion by cultured Y-organs of the crayfish, Procamharus clarkii. Several neuropeptides were separated from a crude extract of sinus glands of P. clarkii by means of reverse-phase high-performance liquid chromatography (HPLC) and tested for MIH activity. An extremely high level of activity was found in one fraction in reverse-phase HPLC, suggesting that the novel neuropeptide in this fraction may be a P. clarkii MIH.

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

AbstractY‐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 (Kd) of 1.08 × 10−7 M and a binding maximum at equilibrium (Bmax) 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 Kd value as membranes from intact crabs, but a Bmax 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.

Biosynthesis of ecdysteroid hormones by crustacean Y-organs: conversion of cholesterol to 7-dehydrocholesterol is suppressed by a steroid 5α-reductase inhibitor

A pair of glands (Y-organs) in crustaceans synthesize and secrete ecdysteroid hormones; the obligate precursor for synthesis is circulating cholesterol. Ecdysteroid output by the Y-organs is regulated negatively by an eyestalk neurosecretory peptide, molt-inhibiting hormone (MIH). The question was addressed, does MIH suppress ecdysteroid synthesis by decreasing cholesterol supply (uptake) or its utilization or both? Experiments were conducted with Y-organs in vitro from the crab, Menippe mercenaria, in the presence of labeled cholesterol, with or without the steroid 5α-reductase inhibitor, L-645390 (Merck). Other experiments superimposed the presence or absence of eyestalk extract containing MIH activity. L-645390 greatly depressed incorporation of cholesterol into an early intermediate, 7-dehydrocholesterol and into secreted ecdysteroids. At the same time, cholesterol accumulated in the Y-organs, to levels significantly higher than in untreated controls. MIH alone depressed both cholesterol uptake and incorporation. MIH together with L-645390 produced the greatest depression of cholesterol incorporation while also preventing the cholesterol accumulation seen with L-645390 alone. These results indicate that cholesterol uptake and its metabolic utilization in Y-organs are separable events representing separate sites of regulation by MIH.

A neuropeptide with molt-inhibiting hormone activity from the sinus gland of the mexican crayfish Procambarus bouvieri (Ortmann)

1. 1. By means of reverse-phase high-performance liquid chromatography (RP-HPLC) on a μBondapak-Phenyl column, a neuropeptide with molt-inhibiting hormone (MIH) activity was isolated from a crude extract of 2000 sinus glands from the crayfish Procambarus bouvieri (Ortmann) with a yield of 25 ng/SG. 2. 2. The activity was tested in a heterology in vitro assay as the depression of ecdysteroid biosynthesis by cultured Orconectes limosus' Y-organs. 3. 3. On SDS-PAGE, the peptide migrated as a single band of approximately 6000 Da, and on IEF, it migrated as a single band to a pI of 5.50. 4. 4. The N-terminus was shown to be blocked by means of the dansyl-Cl method, while the C-terminus was shown to be isoleucine by means of carboxypeptidase-Y digestion. 5. 5. The following amino acid average composition of MIH was obtained: (Asx) 7, (Val) 6, (Glx, Leu, Arg) 5, (Cys, Lys) 4, (Ala, Ile, Tyr, Phe) 3, (Ser) 2, (Thr, Gly) 1–2, (Pro) 1. MIH has 53–55 amino acid residues and its minimal molecular mass is calculated as 6243–6402 Da. It does not contain Trp, His nor Met. 6. 6. 37.7 μg (6.3 nmol) of the molt-inhibiting hormone (MIH) and 70.7 μg (11.8 nmol) of the major peak of the crustacean hyperglycemic hormone (CHH-B) were reduced, carboxymethylated and digested with trypsin. The tryptic peptides of each of the hormones were purified on an Ultrasphere-ODS column and the peptide maps compared with respect to retention times and the composition of each peak. 7. 7. 57.4% of the total amount of amino acid residues were accounted or in five peptides which coincided in elution time and composition. 8. 8. As we have found a clear-cut difference of only two residues between MIH and CHH-B (without taking into account the amides of aspartic and glutamic acids), the present results confirm that the two hormones belong to a family of neuropeptides comprising at least one molt-inhibiting hormone and two hyperglycemic hormones. 9. 9. Evidence is presented for the existence of microheterogeneity in the structure of each of these hormones.

Regulation of Y-organ ecdysteroidogenesis by molt-inhibiting hormone in crabs: Involvement of cyclic AMP-mediated protein synthesis

The crustacean neuropeptide, molt-inhibiting hormone (MIH), directly inhibits Y-organ ecdysteroidogenesis, an effect mediated by cyclic AMP (cAMP) and antagonized by calcium-calmodulin. We investigated regulation of Y-organ protein, RNA, and DNA syntheses by MIH, cAMP, and calcium in relation to steroidogenesis in vitro. Ecdysteroid production and [ 3H]leucine incorporation into protein were inhibited 50–60 and 80–90%, respectively, by MIH activity in eyestalk extracts (4 eyestalk equivalents), 10 −6 M forskolin, or a combination of 10 −2 M dibutyryl cAMP and 10 −4 M 3-isobutyl-1-methylxanthine (dbcAMP-IBMX). Calcium ionophore A23187 (10 −4 M) stimulated ecdysteroidogenesis two-fold, did not affect the relatively high basal (control) rate of protein synthesis, and reduced the inhibitory effects of forskolin on steroidogenesis and protein synthesis. Incorporation of [ 3H]uridine into RNA was unaffected by MIH, forskolin, or A23187 but was reduced 50% by dbcAMP-IBMX. Basal rates of [ 3H]thymidine incorporation into DNA were low and were not affected by treatments. The effects of MIH were specific; extracts of brain or muscle did not alter Y-organ steroidogenesis or protein synthesis, while muscle extract increased precursor incorporation into RNA. Eyestalk extract did not affect [ 3H]leucine incorporation into protein of brain, muscle, or gill. Cycloheximide (5 μg/ml) depressed protein synthesis 90% and steroidogenesis 60%, enhanced the inhibition induced by MIH, and blocked the stimulation of steroidogenesis induced by A23187; effects on basal steroidogenesis were evident after 1 hr. Actinomycin D (1 μg/ml) depressed RNA synthesis 86% but did not alter basal, MIH-inhibited, or A23187-stimulated ecdysteroidogenesis during incubations. These results indicate that MIH suppresses Y-organ steroidogenesis in part by inhibiting protein synthesis at the translational level; the effect is mediated by cAMP. The stimulation of steroidogenesis by calcium, mediated by lowering cAMP, also appears to depend in part upon protein synthesis.

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.

Cyclic AMP mediates the negative regulation of Y-organ ecdysteroid production

The putative neuropeptide, molt-inhibiting hormone (MIH), regulates crustacean growth by periodically suppressing the secretion of ecdysteriod molting hormone from peripheral glands (Y-organs). A mediating role for cyclic AMP (cAMP) in MIH action was evaluated with isolated Y-organs of the crab, Cancer antennarius. MIH activity in eyestalk extracts inhibited ecdysteroid secretion but increased cAMP levels dose-dependently in 24-h incubations. The cAMP rise preceded the onset of ecdysteroid suppression. Dibutyryl cAMP, activators of adenylate cyclase (forskolin, choleragen), and an inhibitor of phosphodiesterase (IBMX), but not AMP or cGMP, mimicked the inhibitory action of MIH.

5-HYDROXYTRYPTAMINE MEDIATES RELEASE OF MOLTINHIBITING HORMONE ACTIVITY FROM ISOLATED CRAB EYESTALK GANGLIA

Neurosecretory cells in crustacean eyestalk ganglia produce a putative molt-inhibiting hormone (MIH) which directly suppresses production of the steroid molting hormone, ecdysone, by the peripheral Y-organs. Neurotransmitter mediation of MIH release from isolated eyestalk ganglia of the crab, Cancer antennarius, was explored using an MIH bioassay based upon in vitro inhibition of Y-organ ecdysteroid production by eyestalk ganglion-conditioned saline. The conditioned saline (0.01-1.0 eyestalk equivalent) inhibited Y -organ ecdysteroid production dose-dependently and reversibly, and the effect of the saline was specific as to conditioning tissue. Isolated ganglia released a significant portion of their MIH activity in 2-h incubations, but also retained a significant portion. 5-hydroxytryptamine (5-HT) enhanced MIH release at concentrations of 10-10 M to 10-6 M. Acetylcholine, dopamine, octopamine, norepinephrine, or gamma-aminobutyric acid (10-7 M-10-6 M) did not alter basal MIH release. The 5-HT precursor,...

Some effects of the molt-inhibiting hormone and 20-hydroxyecdysone upon molting in the grass shrimp, Palaemonetes pugio

Several aspects of the chemistry and physiological effects of molt-inhibiting hormone (MIH) and 20-hydroxyecdysone and their interaction in the control of molting in Palaemonetes pugio were investigated. More MIH activity was extracted from eyestalks using phosphate buffer (pH 11) than with distilled water, saline, ethanol, or methanol-chloroform-water. A crude eyestalk extract was filtered through a Sephadex G-25 gel column. The retention constant ( R = void vol elution vol ) for MIH was 0.67. A direct relationship between dosage and response for MIH was found for crude eyestalk extracts over a range of 0.25 to 2.5 eyestalk equivalents per animal. A direct dosage-response relationship for 20-hydroxyecdysone from concentrations of 0.005 to 5.0 μg/animal was also shown. Higher doses caused abnormal setogenesis in the uropods. Precocious proecdysis was also induced in ovigerous females when injected with 20-hydroxyecdysone. When MIH was injected into the last abdominal segment and followed by an injection of 20-hydroxyecdysone into the thoraco-abdominal junction of the same shrimp, a delay in setogenesis in the uropods, as compared to the accelerated setogenesis in the antennal scale, was observed. The results of this latter experiment are consistent with the hypotheses that concentration gradients of the two hormones were established in the shrimp and that MIH acted antagonistically to 20-hydroxyecdysone at the level of the target cells of the epidermis.