R. Douglas Watson

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.

Evidence that Y-organs of the crab Cancer antennarius secrete 3-dehydroecdysone

Y-organs are paired glands in crustaceans that secrete a class of steroid hormones (ecdysteroids) that regulate growth, molting and development. The glandular secretion has been assumed to be solely the ecdysteroid, ecdysone, a polyhydroxylated derivative of cholesterol. We previously reported that Y-organs of a crab (Cancer antennarius) additionally secreted an ecdysteroid that is less polar than ecdysone. Evidence is presented here that the other secretion product is 3-dehydroecdysone (3-dhE). The compound co-chromatographed with authentic 3-dhE in both normal-phase, and reversed-phase, high-performance liquid chromatography. Mass spectrometry of the ecdysteroid gave results consistent with its identity as 3-dhE. The putative 3-dhE was radiolabeled by injecting crabs with [3H]cholesterol and then incubating the Y-organs. The putative [3H]3-dhE secretion was then subjected to chemical reduction. The reaction yielded labeled products that co-chromatographed with authentic ecdysone and 3-epiecdysone. Results of other experiments gave the following results: (1) Putative 3-dhE was not altered (chromatographic criteria) by incubations with snail hydrolases. (2) Putative [3H]3-dhE, added to incubations of Y-organ halves or homogenates, was not significantly converted to ecdysone; also, no conversion was evident after incubation in medium alone in which the hemolymph serum supplement was raised to 50% of the volume. (3) [3H]Ecdysone was not converted to putative 3-dhE in vitro by Y-organ halves or homogenates.(ABSTRACT TRUNCATED AT 250 WORDS)

Molt-inhibiting hormone-mediated regulation of ecdysteroid synthesis in Y-organs of the crayfish (Procambarus clarkii): involvement of cyclic GMP and cyclic nucleotide phosphodiesterase.

Crustacean molt-inhibiting hormone (MIH), a polypeptide secreted by the X-organ/sinus gland complex of the eyestalks, regulates molting by inhibiting the synthesis of ecdysteroids by Y-organs. Previous results indicate the biosynthetic activity of Y-organs is likely controlled not only by the level of hemolymphatic MIH, but also by the responsiveness of Y-organs to MIH. The present studies were conducted to (a) identify the second messenger that mediates MIH-induced suppression of ecdysteroidogenesis, and (b) assess the possible involvement of cyclic nucleotide phosphodiesterase (PDE) in determining the responsiveness of Y-organs to MIH. Adding 8-bromo cAMP or 8-bromo cGMP to incubation medium significantly suppressed ecdysteroid production by Y-organs of the crayfish (Procambarus clarkii). Incubating Y-organs with MIH produced a significant increase in glandular cGMP, but MIH had no effect on glandular cAMP. The composite data indicate that MIH-induced suppression of ecdysteroidogenesis in Y-organs of P. clarkii is mediated by cGMP. Subsequently, Y-organs from various stages of the molt cycle were incubated with MIH, 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of PDE), or both. Y-Organs from middle and late premolt stages were poorly responsive to MIH alone. Including IBMX in the incubation medium enhanced the responsiveness of the Y-organs to MIH at these stages. Moreover, glandular PDE activity in the Y-organs at these stages was significantly higher than other stages. The combined results suggest that molt cycle-associated changes in PDE activity affect the ability of MIH to stimulate cGMP accumulation and suppress ecdysteroidogenesis in Y-organs of P. clarkii.

Molecular cloning of a cDNA encoding molt-inhibiting hormone of the crab, Cancer magister

A neuropeptide molt-inhibiting hormone (MIH) negatively regulates crustacean molting glands (Y-organs). We report here the molecular cloning of a cDNA encoding putative MIH of the Dungeness crab, Cancer magister. A cDNA library was commercially prepared using poly (A+) RNA isolated from C. magister eyestalk neural ganglia. The library was screened using as probe a previously cloned portion of a cDNA encoding MIH of the blue crab, Callinectes sapidus. DNA sequence analysis of one positive clone revealed a 339 base pair open reading frame encoding a 78 amino acid putative MIH and a 35 amino acid signal peptide. The deduced amino acid sequence of C. magister MIH shows high sequence identity (80-98%) with MIH of three other brachyuran crabs, but lower identity (26-45%) with MIH and MIH-like peptides from astacurans and shrimp. Studies using reverse transcription-polymerase chain reaction (RT-PCR) indicate the MIH gene is expressed in eyestalk but not control (muscle, gill, gonad, hepatopancreas) tissue.

Mosquitofish (Gambusia affinis) vitellogenin: identification, purification, and immunoassay

Vitellogenin is a phospholipoglycoprotein precursor of egg yolk. In mature female fish, vitellogenin is synthesized and secreted by the liver in response to circulating estrogens. Vitellogenin is normally undetectable in the blood of male fish, but can be induced by exposure to compounds possessing estrogenic activity. Thus, the presence of vitellogenin in blood of male fish can serve as a useful biomarker for assessing previous exposure to estrogenic compounds. In the present study, we report identification and purification of vitellogenin in the mosquitofish (Gambusia affinis). Anti-vitellogenin immune serum was generated and used to develop an immunoblot assay for detection of vitellogenin. A combination of immunoblotting and densitometric scanning was used to assess the time- and dose-dependent effects of 17alpha-ethynylestradiol on vitellogenesis in male G. affinis. The results indicate that changes in the level of vitellogenin in mosquitofish blood can be reliably detected by the immunoblot assay, and that the mosquitofish may be a useful bioindicator organism for detecting estrogenic contamination of the aquatic environment.

Expression of crustacean (Callinectes sapidus) molt-inhibiting hormone in Escherichia coli : Characterization of the recombinant peptide and assessment of its effects on cellular signaling pathways in Y-organs

A neuropeptide, 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 Escherichia coli. Bacteria were transformed with an expression plasmid containing a cDNA insert encoding MIH. After induction of protein synthesis, recombinant MIH (recMIH) was detected in the insoluble fraction of cell lysates. The insoluble recMIH was refolded and purified by reversed-phase high performance liquid chromatography (RP-HPLC). The refolded peptide was MIH-immunoreactive and comigrated with native MIH on RP-HPLC. Mass and CD spectral analyses showed the mass number and secondary structure of the recombinant peptide were as predicted for MIH. Bioassays showed recMIH dose-dependently suppresses ecdysteroid synthesis by Y-organs. The combined results suggest that recMIH is properly folded. In subsequent experiments, recMIH was used to assess cellular signaling pathways linked to MIH-mediated suppression of ecdysteroidogenesis. Incubation of Y-organs with recMIH produced an increase in intracellular cGMP content, but had no effect on intracellular cAMP. Further, a cGMP analog significantly suppressed ecdysteroid production, but neither cAMP analogs nor an activator of adenylyl cyclase had a detectable effect on ecdysteroidogenesis. The results are consistent with the hypothesis that MIH-induced suppression of ecdysteroidogenesis in Y-organs of C. sapidus is mediated by a cGMP second messenger. We anticipate recMIH will be a useful tool for additional studies of the cellular actions and physiological functions of MIH.

Structural and functional comparisons and production of recombinant crustacean hyperglycemic hormone (CHH) and CHH-like peptides from the mud crab Scylla olivacea.

Sco-CHH and Sco-CHH-L (CHH-like peptide), two structural variants of the crustacean hyperglycemic hormone family identified in the mud crab (Scylla olivacea), are presumably alternatively spliced gene products. In this study, Sco-CHH and Sco-CHH-L were isolated from the tissues using high performance liquid chromatography. Identity of the native peptides was confirmed using mass spectrometric (MS) analyses of purified materials and of trypsin-digested peptide fragments. Additionally, characterizations using circular dichroism (CD) spectrometry revealed that the 2 peptides have similar CD spectral profiles, showing they are composed mainly of alpha-helices, and are similarly thermo-stable with a melting temperature of 74-75 degrees C. Results of bioassays indicated that Sco-CHH exerted hyperglycemic and molt-inhibiting activity, whereas Sco-CHH-L did not. Further, recombinant Sco-CHH-Gly (rSco-CHH-Gly, a glycine extended Sco-CHH) and Sco-CHH-L (rSco-CHH-L) were produced using an Escherichia coli expression system, refolded, and purified. rSco-CHH-Gly was further alpha-amidated at the C-terminal end to produce rSco-CHH. MS analyses of enzyme-digested peptide fragments of rSco-CHH-Gly and rSco-CHH-L showed that the two peptides share a common disulfide bond pattern: C7-C43, C23-C39, and C26-C52. Circular dichroism analyses and hyperglycemic assay revealed that rSco-CHH and rSco-CHH-L resemble their native counterparts, in terms of CD spectral profiles, melting curve profiles, and biological activity. rSco-CHH-Gly has a lower alpha-helical content (32%) than rSco-CHH (47%), a structural deviation that may be responsible for the significant decrease in the biological activity of rSco-CHH-Gly. Finally, modeled structure of Sco-CHH and Sco-CHH-L indicated that they are similarly folded, each with an N-terminal tail region and 4 alpha-helices. Putative surface residues located in corresponding positions of Sco-CHH and Sco-CHH-L but with side chains of different properties were identified. The combined results support the notion that Sco-CHH and Sco-CHH-L are functionally different, but resemble each other at higher-level structures. Functional diversity between the 2 peptides is probably due to critical residues located in the C-terminus. The availability of large amounts of recombinant proteins will permit additional functional and structural studies of these CHH family peptides.

Involvement of microtubules in prothoracicotropic hormone‐stimulated ecdysteroidogenesis by insect (Manduca sexta) prothoracic glands

Secretion of ecdysteroid molting hormones by insect prothoracic glands is stimulated by neuropeptide prothoracicotropic hormones (PTTH). Studies reported here were conducted to assess the effects of microfilament and microtubule inhibitors on in vitro ecdysteroidogenesis by prothoracic glands of Manduca sexta. Microfilament inhibitors (cytochalasins B and D) had no effect on basal or big PTTH-stimulated ecdysteroidogenesis. Microtubule inhibitors (colchicine, podophyllotoxin, nocodazole) had no effect on basal ecdysteroid secretion, but suppressed PTTH-stimulated secretion in a concentration-dependent manner. The effect of nocodazole was partially reversible, suggesting it was not due to nonspecific toxicity. Colchicine had no effect on glandular ecdysteroid levels, indicating that inhibition was not due solely to blockage of secretion. The combined results are consistent with the hypothesis that microtubule-mediated transport of ecdysteroid precursors plays a critical role in stimulation of ecdysteroidogenesis by PTTH.

DEVELOPMENTAL CHANGES IN THE LEVEL OF VITELLIN- IMMUNOREACTIVE PROTEINS IN HEMOLYMPH AND TISSUES OF THE BLUE CRAB CALLINECTES SAPIDUS: RELATION TO VITELLOGENESIS

ABSTRACT Female blue crabs (Callinectes sapidus) were assigned to 1 of 6 stages based on oocyte development. An immunohistochemical study showed vitellin (Vn)-immunoreactivity was absent from ovaries in stages I and II, and was present in stages III, IV, V, and VI (postspawning). Vn-immunoreactivity was not detected in hepatopancreas, regardless of stage. Hemolymph vitellogenin (Vg) was quantified by enzyme-linked immunosorbent assay. Vg was not detected in hemolymph of stage I and II animals, but was detected in some animals in stages III, IV, V, and VI. The percentage of crabs having detectable hemolymph Vg was 17% (III), 32% (IV), 73% (V), and 100% (VI). Mean Vg levels were 0.02 ± 0.01 mg/ml (III), 0.02 ± 0.04 mg/ml (IV), 0.12 ± 0.15 mg/ml (V), and 0.14 ± 0.04 mg/ml (VI). The facts that Vg was present at low concentration in only a portion of the vitellogenic crabs, and that the level of Vg did not reach maximum until after spawning, are not consistent with the hypothesis that Vg is synthesized by extraovarian tissues and transported via hemolymph to the ovaries. It is possible that the presence of Vg in hemolymph may be a result of leakage of yolk proteins from the ovary due to oocyte reabsorption. The existence of atypical, possible atretic, oocytes in stage V and VI ovaries is consistent with this hypothesis. Considered together with the results of previous studies, these findings suggest that the ovary is the exclusive site of Vn synthesis in C. sapidus.

Heme Oxygenase-1 Protects Corexit 9500A-Induced Respiratory Epithelial Injury across Species

The effects of Corexit 9500A (CE) on respiratory epithelial surfaces of terrestrial mammals and marine animals are largely unknown. This study investigated the role of CE-induced heme oxygenase-1 (HO-1), a cytoprotective enzyme with anti-apoptotic and antioxidant activity, in human bronchial airway epithelium and the gills of exposed aquatic animals. We evaluated CE-mediated alterations in human airway epithelial cells, mice lungs and gills from zebrafish and blue crabs. Our results demonstrated that CE induced an increase in gill epithelial edema and human epithelial monolayer permeability, suggesting an acute injury caused by CE exposure. CE induced the expression of HO-1 as well as C-reactive protein (CRP) and NADPH oxidase 4 (NOX4), which are associated with ROS production. Importantly, CE induced caspase-3 activation and subsequent apoptosis of epithelial cells. The expression of the intercellular junctional proteins, such as tight junction proteins occludin, zonula occludens (ZO-1), ZO-2 and adherens junctional proteins E-cadherin and Focal Adhesion Kinase (FAK), were remarkably inhibited by CE, suggesting that these proteins are involved in CE-induced increased permeability and subsequent apoptosis. The cytoskeletal protein F-actin was also disrupted by CE. Treatment with carbon monoxide releasing molecule-2 (CORM-2) significantly inhibited CE-induced ROS production, while the addition of HO-1 inhibitor, significantly increased CE-induced ROS production and apoptosis, suggesting a protective role of HO-1 or its reaction product, CO, in CE-induced apoptosis. Using HO-1 knockout mice, we further demonstrated that HO-1 protected against CE-induced inflammation and cellular apoptosis and corrected CE-mediated inhibition of E-cadherin and FAK. These observations suggest that CE activates CRP and NOX4-mediated ROS production, alters permeability by inhibition of junctional proteins, and leads to caspase-3 dependent apoptosis of epithelial cells, while HO-1 and its reaction products protect against oxidative stress and apoptosis.