Mandibular Organ-inhibiting Hormone Research Articles

Eyestalk transcriptome and methyl farnesoate titers provide insight into the physiological changes in the male snow crab, Chionoecetes opilio, after its terminal molt

The snow crab, Chionoecetes opilio, is a giant deep-sea brachyuran. While several decapod crustaceans generally continue to molt and grow throughout their lifetime, the snow crab has a fixed number of molts. Adolescent males continue to molt proportionately to their previous size until the terminal molt at which time an allometric increase in chela size occurs and an alteration of behavioral activities occurs, ensuring breeding success. In this study, we investigated the circulating concentrations of methyl farnesoate (an innate juvenile hormone in decapods) (MF) before or after the terminal molt in males. We then conducted eyestalk RNAseq to obtain molecular insight into the regulation of physiological changes after the terminal molt. Our analyses revealed an increase in MF titers after the terminal molt. This MF surge may be caused by suppression of the genes that encode MF-degrading enzymes and mandibular organ-inhibiting hormone that negatively regulates MF biosynthesis. Moreover, our data suggests that behavioral changes after the terminal molt may be driven by the activation of biogenic amine-related pathways. These results are important not only for elucidating the physiological functions of MFs in decapod crustaceans, which are still largely unknown, but also for understanding the reproductive biology of the snow crab.

RNAi-mediated knockdown of the mandibular organ-inhibiting hormone (MOIH) gene stimulates vitellogenesis in the Chinese mitten crab Eriocheir sinensis

RNAi-mediated knockdown of the mandibular organ-inhibiting hormone (MOIH) gene stimulates vitellogenesis in the Chinese mitten crab Eriocheir sinensis

Eyestalk neuropeptide identification in the female red deep-sea crab, Chaceon quinquedens

Eyestalk-derived neuropeptides, primarily the crustacean hyperglycemic hormone (CHH) neuropeptide family, regulate vitellogenesis in decapod crustaceans. The red deep-sea crab, Chaceon quinquedens, a cold-water species inhabiting depths between 200 and 1800 m, has supported a small fishery, mainly harvesting adult males in the eastern US for over 40 years. This study aimed to understand the role of eyestalk-neuropeptides in vitellogenesis in C. quinquedens with an extended intermolt stage. Chromatography shows two CHH and one MIH peak in the sinus gland, with a CHH2 peak area four times larger than CHH1. The cDNA sequence of MIH and CHH of C. quinquedens is isolated from the eyestalk ganglia, and the qPCR assay shows MIH is significantly higher only at ovarian stages 3 than 4 and 5. However, MIH transcript and its neuropeptides do differ between stages 1 and 3. While CHH transcripts remain constant, its neuropeptide levels are higher at stages 3 than 1. Additionally, transcriptomic analysis of the de novo eyestalk ganglia assembly at ovarian stages 1 and 3 found 28 eyestalk neuropeptides. A GIH/VIH or GSH/VSH belonging to the CHH family is absent in the transcriptome. Transcripts per million (TPM) values of ten neuropeptides increase by 1.3 to 2.0-fold at stage 3 compared to stage 1: twofold for Bursicon α, followed by CHH, AKH/corazonin-like, Pyrokinin, CCAP, Glycoprotein B, PDH1, and IDLSRF-like peptide, and 1.3-fold of allatostatin A and short NP-F. WXXXRamide, the only downregulated neuropeptide, decreases TPM by ∼ 2-fold at stage 3, compared to stage 1. Interestingly, neuroparsin with the highest TPM values remains the same in stages 1 and 3. The mandibular organ-inhibiting hormone is not found in de novo assembly. We report that CHH, MIH, and eight other neuropeptides may play a role in vitellogenesis in this species.

Comparative Genomic and Transcriptomic Analyses of CHHs and Their Putative Receptors in Scylla paramamosain, Portunus trituberculatus, and Eriocheir sinensis

Crustacean hyperglycemic hormone superfamily neuropeptides (CHHs) are typical crustacean eyestalk hormones that include the crustacean hyperglycemic hormone (CHH), moult-inhibiting hormone (MIH), vitellogenesis/gonad-inhibiting hormone (VIH/GIH) and mandibular organ-inhibiting hormone (MOIH), which are divided into two subfamilies: type I CHH (included CHH) and type II CHH (consisting of MIH, VIH/GIH, and MOIH). They are involved in various biological activities, such as metabolism, molting, reproduction, and osmotic regulation. Discovery of the ion transport peptide (ITP) in insects expanded the members of CHHs and revealed that CHHs are not restricted to crustaceans. In this study, we focused on three economically important crabs: the mud crab, Scylla paramamosain, the swimming crab, Portunus trituberculatus, and the Chinese mitten crab, Eriocheir sinensis. Their genomes, Pacbio full-length transcriptomic data as well as comparative RNA-seq data were obtained and used to analyze the genomic structures and expression patterns of CHHs and their putative receptors through bioinformatic methods. Two type I CHH members (CHH1 and CHH2) were identified, of which CHH1 had two splice variants, CHH1-v1 and CHH1-v2. One copy of type II CHH (MIH) was found in P. trituberculatus and E. sinensis. While most decapods, including S. paramamosain, have two copies of type II CHHs (MIH/VIH), these MIH/VIHs are adjacent to each other on the same chromosome. Besides type I and II CHH, ITP-like peptides have also been found in the three crabs, and they are mainly expressed in the eyestalk. Four, five, and three G protein-coupled receptors (GPCRs) were identified in S. paramamosain, P. trituberculatus, and E. sinensis, respectively, which might be putative CHH receptors. These GPCRs were divided into three groups. One group was composed of two contiguous genomic position GPCRs, and they were mainly expressed in the hepatopancreas. These findings provide a basis for further studies on CHHs receptor binding tests and on CHHs/GPCRs signaling pathways.

The Crustacean Hyperglycemic Hormone Superfamily: Progress Made in the Past Decade.

Early studies recognizing the importance of the decapod eyestalk in the endocrine regulation of crustacean physiology—molting, metabolism, reproduction, osmotic balance, etc.—helped found the field of crustacean endocrinology. Characterization of putative factors in the eyestalk using distinct functional bioassays ultimately led to the discovery of a group of structurally related and functionally diverse neuropeptides, crustacean hyperglycemic hormone (CHH), molt-inhibiting hormone (MIH), gonad-inhibiting hormone (GIH) or vitellogenesis-inhibiting hormone (VIH), and mandibular organ-inhibiting hormone (MOIH). These peptides, along with the first insect member (ion transport peptide, ITP), constitute the original arthropod members of the crustacean hyperglycemic hormone (CHH) superfamily. The presence of genes encoding the CHH-superfamily peptides across representative ecdysozoan taxa has been established. The objective of this review is to, aside from providing a general framework, highlight the progress made during the past decade or so. The progress includes the widespread identification of the CHH-superfamily peptides, in particular in non-crustaceans, which has reshaped the phylogenetic profile of the superfamily. Novel functions have been attributed to some of the newly identified members, providing exceptional opportunities for understanding the structure-function relationships of these peptides. Functional studies are challenging, especially for the peptides of crustacean and insect species, where they are widely expressed in various tissues and usually pleiotropic. Progress has been made in deciphering the roles of CHH, ITP, and their alternatively spliced counterparts (CHH-L, ITP-L) in the regulation of metabolism and ionic/osmotic hemostasis under (eco)physiological, developmental, or pathological contexts, and of MIH in the stimulation of ovarian maturation, which implicates it as a regulator for coordinating growth (molt) and reproduction. In addition, experimental elucidation of the steric structure and structure-function relationships have given better understanding of the structural basis of the functional diversification and overlapping among these peptides. Finally, an important finding was the first-ever identification of the receptors for this superfamily of peptides, specifically the receptors for ITPs of the silkworm, which will surely give great impetus to the functional study of these peptides for years to come. Studies regarding recent progress are presented and synthesized, and prospective developments remarked upon.

Molecular cloning of crustacean hyperglycemic hormone (CHH) family members (CHH, molt-inhibiting hormone and mandibular organ-inhibiting hormone) and their expression levels in the Jonah crab, Cancer borealis

The crustacean hyperglycemic hormone (CHH) neuropeptide family has multiple functions in the regulation of hemolymph glucose levels, molting, ion, and water balance and reproduction. In crab species, three neuroendocrine tissues: the eyestalk ganglia (medulla terminalis X-organ and -sinus gland = ES), the pericardial organ (PO), and guts synthesize a tissue-specific isoforms of CHH neuropeptides. Recently the presence of the mandibular organ-inhibiting hormone (MOIH) was reported in the stomatogastric nervous system (STNS) that regulates the rhythmic muscle movements in esophagus, cardiac sac, gastric and pyloric ports of the foregut. In this study, we aimed to determine the presence of a tissue-specific CHH isoform in the Jonah crab, Cancer borealis using PCR with degenerate primers and 5′, 3′ rapid amplification of cDNA ends (RACE) in the ES. PO, and STNS. The analysis of CHH sequences shows that C. borealis has one type of CHH isoform, unlike other crab species. We also isolated the cDNA sequence of molt-inhibiting hormone (MIH) in the ES and MOIH in the ES and STNS. The presence of CHH, MOIH and MIH in the sinus gland of adult females and males is confirmed by using a dot-blot assay with the putative peaks collected from RP-HPLC and anti-Cancer sera for CHH, MIH, and MOIH. The present of crustacean female sex hormone (CFSH) in the sinus gland of adult females was examined with a dot-blot assay with anti-Callinectes CFSH serum. Levels of CHH, MOIH, and MIH in the sinus gland and their expressions in the eyestalk ganglia are estimated in the adult males, where CHH is the predominant form among these neuropeptides.

CRUSTACEAN HYPERGLYCEMIC HORMONE: STRUCTURAL VARIANTS, PHYSIOLOGICAL FUNCTION, AND CELLULAR MECHANISM OF ACTION

Crustacean hyperglycemic hormone (CHH) is a peptide hormone originally identified in the X-organ/sinus gland (XO/ SG) complex of the eyestalks. It belongs to the CHH family which also includes molt-inhibiting hormone (MIH), vitellogenesis-inhibiting hormone (VIH), and mandibular organ-inhibiting hormone (MOIH), and ion transport peptide (ITP). Multiple molecular variants of CHH are generated by both post-transcriptional and post-translational mechanisms. In addition to the XO/SG complex, CHH gene is widely expressed in many extra-eyestalk tissues. Functionally, available data indicate that CHH is involved with the regulation of carbohydrate metabolism and stress-induced hyperglycemia. Several other physiological processes, including molting, ion and water balance, reproduction, and immunity, are likely also regulated by CHH. The functional role(s) of the alternatively splice form CHH-like peptide (CHH-L) are different from those of CHH and remain unknown. Combined results showed that cyclic guanosine 3',5'-monophosphate (cGMP) plays important roles in mediating the effects of CHH on carbohydrate metabolism. Recent studies of receptor guanylyl cyclase (rGC) may lead to characterization and identification of CHH receptor(s). Future efforts are needed to fully understand the functional roles of the different CHH variants; identification of CHH receptor(s) hold the promise of revealing in greater depth the structure/functional relationships of the various structural variants.

Cloning and molecular structure analysis of crustacean hyperglycemic hormone(Ers-CHH)in Eriocheir sinensis

Crustacean hyperglycemic hormone(CHH)is a peptide hormone originally identified in a crustacean neurosecretory complex,the X-organ/sinus gland complex,and located within the eyestalks.CHH is placed in the crustacean hyperglycemic hormone family,which also includes molt-inhibiting hormone(MIH),vitellogenesis-inhibiting hormone(VIH)or gonad-inhibiting hormone(GIH),mandibular organ-inhibiting hormone(MOIH)and insect ion transport peptide(ITP).CHH plays a significant role in the growth,molting and reproduction of crustaceans.By using degenerate primers which were designed according to the N-terminal amino acid sequences of CHH isolated by our lab and the sequences of homologous species CHH,a novel cDNA of crustacean hyperglycemic hormone gene(Ers-CHH,GenBank Accession No:JX485664)was cloned from Chinese mitten crab(Eriocheir sinensis).The full length cDNA consists of 585 bp with a 453 bp open reading frame,encoding 150 amino acids,containing a 30 amino acids signal peptide,and a 42 amino acids CHH precursor-related peptide,a 78 amino acids mature peptide.The mature peptide contains 6 conserved cysteines which formed three disulfide bonds C7-C43、C23-C39、C26-C52.There was an arthropod CHH/MIH/GIH neurohormones family signature in this domain.The three-dimensional structure prediction showed that Ers-CHH mature peptide consisted of 4 α-helixes,and not with the β-sheet structure.5 Prosite patterns,which were analyzed by a Swiss-PdbViewer(v4.0.4),are P5:Protein kinase C phosphorylation site(Ser6,Lys8);P6:Casein kinase Ⅱ phosphorylation site(Ser17,Glu20);P8:N-myristoylation site(Gly38,Cys39,Asn42,Cys43);PS00342:Microbodies C-terminal targeting signal(Ser17,Lys18,Leu19);PS01250:Arthropod CHH/MIH/GIH neurohormones family(Val35,Cys39,Arg40,Asn42,Cys43,Tyr44,Asn46,Phe49,Cys52).The results of the structural model comparison show that the structure of Ers-CHH has high similarity with other CHHs and Ers-CHH.They have no β-sheet structure,except that CHH has 4α-helixes,MIH has 5α-helixes.Although α1 helix of MIH could not be found in CHHs,the position and the amino acid number of α2-α5 helixes in MIH molecular were same as the α1-α4 helixes in CHH.It might be able to interpret the function similarity between CHH and MIH.Multiple alignment results showed that Ers-CHH has the highest identity with Ptychognathus pusillus CHH(92%),and it also shared high identities with Neohelice granulata(86%)and Pachygrapsus marmoratus(72%).However,it showed lower identity with CHH from crayfishes,such as Nephrops norvegicus(49%)and Homarus gammarus(46%).Multiple alignments of Ers-CHH and MIH from other crustaceans showed that only six amino acid residues(Arg13,Asp25,Asn28,Arg31,Arg40,Phe49)in Ers-CHH sequence were same with MIH sequences.It may be helpful for further research on regulation mechanism of CHH in the process of crab growth and carbohydrate metabolism.

Crustacean hyperglycemic hormone (CHH) neuropeptides family: Functions, titer, and binding to target tissues

The removal of the eyestalk (s) induces molting and reproduction promoted the presence of regulatory substances in the eyestalk (ES), particularly medulla terminalis X-organ and the sinus gland (MTXO–SG). The PCR-based cloning strategies have allowed for isolating a great number of cDNAs sequences of crustacean hyperglycemic hormone (CHH) neuropeptides family from the eyestalk and non-eyestalk tissues, e.g., pericardial organs and fore- and hindguts. However, the translated corresponding neuropeptides in these tissues, their circulating concentrations, the mode of actions, and specific physiological functions have not been well described. The profiles of CHH neuropeptides present in the MTXO–SG may differ among decapod crustacean species, but they can be largely divided into two sub-groups on the basis of structural homology: (1) CHH and (2) molt-inhibiting hormone (MIH)/mandibular organ-inhibiting hormone (MOIH)/vitellogenesis/gonad-inhibiting hormone (V/GIH). CHH typically elevating the level of circulating glucose from animals under stressful conditions (hyper- and hypothermia, hypoxia, and low salinity) has multiple target tissues and functions such as ecdysteroidogenesis, osmoregulation, and vitellogenesis. Recently, MIH, known for exclusively suppressing ecdysteroidogenesis in Y-organs, is also reported to have an additional role in vitellogenesis of adult female crustacean species, suggesting that some CHH neuropeptides may acquire an extra regulatory role in reproduction at adult stage. This paper reviews the regulatory roles of CHH and MIH at the levels of specific functions, temporal and spatial expression, titers, their binding sites on the target tissues, and second messengers from two crab species: the blue crab, Callinectes sapidus, and the European green crab, Carcinus maenas. It further discusses the diverse regulatory roles of these neuropeptides and the functional plasticity of these neuropeptides in regard to life stage and species-specific physiology.

Methyl farnesoate synthesis in the lobster mandibular organ: The roles of HMG-CoA reductase and farnesoic acid O-methyltransferase

Eyestalk ablation (ESA) increases crustacean production of methyl farnesoate (MF), a juvenile hormone-like compound, but the biochemical steps involved are not completely understood. We measured the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and farnesoic acid O -methyl transferase (FAOMeT), an early step and the last step in MF synthesis. ESA elevated hemolymph levels of MF in male lobsters. Enzyme activity suggested that increased MF production on day one was due largely to elevated HMGR activity while changes in FAOMeT activity closely paralleled changes in MF levels on day 14. Transcript levels for HMGR and FAOMeT changed little on day one, but both increased substantially on day 14. We treated ESA males with a partially purified mandibular organ-inhibiting hormone (MOIH) and observed a significant decline in MF levels, FAOMeT activity, and FAOMeT–mRNA levels after 5 h. However, no effect was observed on HMGR activity or its mRNA indicating that they must be regulated by a separate sinus gland peptide. We confirmed that lobster HMGR was not a phosphoprotein and was not regulated by reversible phosphorylation, an important mechanism for regulating other HMGRs. Nevertheless, molecular modeling indicated that the catalytic mechanisms of lobster and mammalian HMGR were similar.

Members of the crustacean hyperglycemic hormone (CHH) peptide family are differentially distributed both between and within the neuroendocrine organs ofCancercrabs: implications for differential release and pleiotropic function

The crustacean hyperglycemic hormone (CHH) family of peptides includes CHH, moult-inhibiting hormone (MIH) and mandibular organ-inhibiting hormone (MOIH). In the crab Cancer pagurus, isoforms of these peptides, as well as CHH precursor-related peptide (CPRP), have been identified in the X-organ-sinus gland (XO-SG) system. Using peptides isolated from the C. pagurus SG, antibodies to each family member and CPRP were generated. These sera were then used to map the distributions and co-localization patterns of these peptides in the neuroendocrine organs of seven Cancer species: Cancer antennarius, Cancer anthonyi, Cancer borealis, Cancer gracilis, Cancer irroratus, Cancer magister and Cancer productus. In addition to the XO-SG, the pericardial organ (PO) and two other neuroendocrine sites contained within the stomatogastric nervous system, the anterior cardiac plexus (ACP) and the anterior commissural organ (ACO), were studied. In all species, the peptides were found to be differentially distributed between the neuroendocrine sites in conserved patterns: i.e. CHH, CPRP, MIH and MOIH in the XO-SG, CHH, CPRP and MOIH in the PO, and MOIH in the ACP (no immunolabeling was found in the ACO). Moreover, in C. productus (and probably in all species), the peptides present in the XO-SG and PO were differentially distributed between the neurons within each of these neuroendocrine organs (e.g. CHH and CPRP in one set of XO somata with MIH and MOIH co-localized in a different set of cell bodies). Taken collectively, the differential distributions of CHH family members and CPRP both between and within the neuroendocrine organs of crabs of the genus Cancer suggests that each of these peptides may be released into the circulatory system in response to varied, tissue-specific cues and that the PO- and/or ACP-derived isoforms may possess functions distinct from those classically ascribed to their release from the SG.

Evidence that ecdysteroids and methyl farnesoate control allometric growth and differentiation in a crustacean

Allometric, or disproportionate, growth of body parts is a basic problem in morphogenesis. Male spider crabs, Libinia emarginata, have several forms or morphotypes. During the terminal molt, the propodus enlarges disproportionately, exceeding the carapace length by as much as 35%. Even though shorter clawed males are reproductive, the large-clawed males become primary reproductives. We stimulated penultimate stage males to molt by eyestalk ablation, which removes molt inhibiting hormone (MIH) and mandibular organ inhibiting hormone (MIOH), and measured ecdysones by radioimmunoassay and methyl farnesoate (MF) in hemolymph by high-performance liquid chromatography (HPLC) using an internal standard. Eyestalk ablation accelerated molting and increased ecdysteroids to peak at 150 ng/ml before the molt. In control animals the ecdysteroids peaked at 90 ng/ml 3 days before the molt, with MF remaining less than 0.5 ng/ml. These became large males with large allometric claws. In contrast, the ablated ones, with increased MF (1 to 1.5 ng/ml), increased carapace size, but retained shorter non-allometric claws, with length shorter than the carapace. The results are consistent with experiments that we have performed with MF administration (Abdu et al., Biol. Bull., Woods Hole, MA 195 (1998) 112; Laufer et al., Gen. Comp. Endocrinol. 111 (1998) 113; Laufer et al., in: IV Symp. Aquaculture in Central America: Focusing on Shrimp and Tilapia, (1997a), p. 161; Laufer et al., Invert. Reprod. Dev. 31 (1997b) 63) which led to the interpretation that ecdysteroids and low MF concentrations promote allometric growth, while ecdysones with relatively higher concentrations of MF inhibited allometric growth. These results indicate and support the conclusion that MF and ecdysteroids determine the control of morphogenesis in allometric growth of Crustacea.

Crustacean Hyperglycemic Hormone Family: Old Paradigms and New Perspectives

I present an overview of recent research on the isolation and characterization of members of the crustacean hyperglycemic hormone (CHH) neuropeptide family. Members of this arthropod-specific family include CHH, molt-inhibiting hormone (MIH), vitellogenesis-inhibiting hormone (VIH), and mandibular organ-inhibiting hormone (MOIH). There are two subfamilies of this neuropeptide group, based upon the presence or absence of a C-terminal CHH precursor-related peptide. There are also sequence motif differences between these subfamilies. Most of the peptides comprising this neuropeptide family are synthesized and released by the eyestalk X-organ/sinus gland complex. Recent experiments have demonstrated the presence of extra-eyestalk cells that produce CHH and the assignment of additional functions to this hormone family.

Crustacean Hyperglycemic Hormone Family: Old Paradigms and New Perspectives1

I present an overview of recent research on the isolation and characterization of members of the crustacean hyperglycemic hormone (CHH) neuropeptide family. Members of this arthropod-specific family include CHH, molt-inhibiting hormone (MIH), vitellogenesis-inhibiting hormone (VIH), and mandibular organ-inhibiting hormone (MOIH). There are two subfamilies of this neuropeptide group, based upon the presence or absence of a C-terminal CHH precursor-related peptide. There are also sequence motif differences between these subfamilies. Most of the peptides comprising this neuropeptide family are synthesized and released by the eyestalk X-organ/sinus gland complex. Recent experiments have demonstrated the presence of extra-eyestalk cells that produce CHH and the assignment of additional functions to this hormone family.

Clustering of mandibular organ-inhibiting hormone and moult-inhibiting hormone genes in the crab, Cancer pagurus, and implications for regulation of expression

Development and reproduction of crustaceans is regulated by a combination of neuropeptide hormones, ecdysteroids (moulting hormones) and the isoprenoid, methyl farnesoate (MF), the unepoxidised analogue of insect juvenile hormone-III (JH-III). MF and the ecdysteroids are respectively synthesised under the negative control of the sinus gland-derived mandibular organ-inhibiting hormones (MO-IHs) and moult-inhibiting hormone (MIH) that are produced in eyestalk neural ganglia. Previous work has demonstrated the existence of two isoforms of MO-IH, called MO-IH-1 and -2, that differ by a single amino acid in the mature peptide and one in the putative signal peptide. To study the structural organisation of the crab MIH and MO-IH genes, a genomic DNA library was constructed from DNA of an individual female crab and screened with both MO-IH and MIH probes. The results from genomic Southern blot analysis and library screening indicated that the Cancer pagurus genome contains at least two copies of the MIH gene and three copies of the MO-IH genes. Upon screening, two types of overlapping genomic clone were isolated. Each member of one type of genomic clone contains a single copy of each of the convergently transcribed MO-IH-1 and MIH genes clustered within 6.5 kb. The other type contains only the MO-IH-2 gene, which is not closely linked to an MIH gene. There are three exons and two introns in all MIH and MO-IH genes analysed. The exon–intron boundary of the crab MIH and MO-IH genes follows Chambon's rule (GT–AG) for the splice donor and acceptor sites. The first intron occurs within the signal peptide region and the second intron occurs in the coding region of the mature peptide. Sequence analysis of upstream regions of MO-IH and MIH genes showed that they contained promoter elements with characteristics similar to other eukaryotic genes. These included sequences with high degrees of similarity to the arthropod initiator, TATA box and cAMP response element binding protein. Additionally, putative CF1/USP and Broad Complex Z2 transcription factor elements were found in the upstream regions of MIH and MO-IH genes respectively. The implications of the presence of the latter two putative transcription factor binding-elements for control of expression of MIH and MO-IH genes is discussed. Phylogenetic analysis and gene organisation show that MO-IH and MIH genes are closely related. Their relationship suggests that they represent an example of evolutionary divergence of crustacean hormones.

Molecular characterization and expression of mandibular organ-inhibiting hormone, a recently discovered neuropeptide involved in the regulation of growth and reproduction in the crab Cancer pagurus

Molecular characterization and expression of mandibular organ-inhibiting hormone, a recently discovered neuropeptide involved in the regulation of growth and reproduction in the crab Cancer pagurus

Molecular characterization and expression of mandibular organ-inhibiting hormone, a recently discovered neuropeptide involved in the regulation of growth and reproduction in the crab Cancer pagurus

Methyl farnesoate, the crustacean juvenoid, is synthesized and secreted from the mandibular organs of crustaceans under the negative control of the sinus gland-derived mandibular organ-inhibiting hormone (MO-IH). Previously we isolated and sequenced two isoforms, MO-IH-1 and MO-IH-2, differing by just one amino acid, from sinus glands of the edible crab, Cancer pagurus. We now report the isolation of cDNAs encoding MO-IH-1 and MO-IH-2 by a combination of reverse-transcriptase-mediated PCR in conjunction with 5' and 3' rapid amplification of cDNA ends ('RACE'). Full-length clones of MO-IH-1 and MO-IH-2 encoded a 34-residue putative signal peptide and the mature 78-residue MO-IH sequences. Northern blot analysis of various tissues showed that MO-IH expression is confined to the X-organ (a cluster of perikarya within the eye). Southern blot analysis indicated that there are approx. 10 copies of the gene for MO-IH in C. pagurus. Additional Southern blotting experiments detected MO-IH-hybridizing bands in another Cancer species, C. antennarius. In support of this, an HPLC-radioimmunoassay analysis of sinus gland extracts of C. antennarius and C. magister also revealed MO-IH-like immunoreactivity.

Involvement of adenosine cyclic-3′,5′-monophosphate in the signal transduction pathway of mandibular organ-inhibiting hormone of the edible crab, Cancer pagurus

The juvenoid, methyl farnesoate (MF), is synthesized in the mandibular organs (MOs) of crustaceans, under the control of mandibular organ-inhibiting hormone (MO-IH). Using an in vitro assay to measure synthesis of MF by MOs, the effect of a variety of agents that affect signal transduction pathways was investigated. Of the compounds tested, only agents which affect cAMP (forskolin and 8-bromoadenosine cyclic-3′,5′-monophosphate) levels were found to mimic the inhibitory action of MO-IH on MF synthesis. To further support these findings, the effect of MO-IH-1 on production of cAMP was investigated. The results demonstrated that MO-IH stimulated a dose-dependent increase in cAMP levels. Furthermore, a maximal 2-fold increase in cAMP was detected after a 5-min exposure of MO membranes to 100 nM MO-IH-1, falling to basal levels thereafter. The results presented strongly support a role for cAMP in the signal transduction mechanism of MO-IH that leads to inhibition of MF synthesis in MOs.

Neuropeptide regulation of biosynthesis of the juvenoid, methyl farnesoate, in the edible crab, Cancer pagurus.

The neuropeptide mandibular organ (MO)-inhibiting hormone (MO-IH), synthesized and secreted from the X-organ-sinus-gland complex of the eyestalk, regulates the biosynthesis of the putative crustacean juvenile hormone, methyl farnesoate (MF). Using radiolabelled acetate as a precursor for isoprenoid biosynthesis, farnesoic acid (FA), farnesol, farnesal, MF and geranyl geraniol were detected in MOs cultured for 24 h. Treatment of MOs with extract of sinus gland inhibited the final step of biosynthesis of MF, catalysed by FA O-methyltransferase. Additionally, treatment of MOs with purified MO-IH exhibited a dose-dependent inhibition of this final step of MF synthesis. The extent of this inhibition was dependent on the ovary stage of the MO-donor animal, being maximal in MOs from animals in the early stages of ovarian development. Assay of FA O-methyltransferase activity, using [3H]FA in the presence of S-adenosyl-l-methionine, demonstrated that the enzyme was located in the cytosolic fraction of MOs and was inhibited by incubation of MOs with MO-IH prior to preparation of subcellular fractions. For cytosolic preparations taken from vitellogenic animals, both Vmax and Km were appreciably lower than for those taken from non-vitellogenic animals. Conversely, eyestalk ablation of early-vitellogenic animals, which removes the source of MO-IH in vivo, resulted in enhancement of the cytosolic FA O-methyltransferase activity. Although both Vmax and Km show an appreciable increase upon eyestalk ablation, the increased enzyme activity is probably reflected by the fact that Vmax/Km (an approximate indication of kcat) has increased 5-fold. The combined evidence demonstrates that MO-IH inhibits FA O-methyltransferase, the enzyme which catalyses the final step of MF biosynthesis in MOs.

CDNA cloning of a mandibular organ inhibiting hormone from the spider crab Libinia emarginata.

Mandibular organs (MO) produce a crustacean juvenile hormone, methyl farnesoate (MF). MO activity is negatively regulated by factors, called mandibular organ inhibiting hormones (MOIHs), from the crustacean sinus gland X-organ complex in the eyestalks. Three MOIHs have been isolated previously from the spider crab Libinia emarginata and are characterized as members of the crustacean hyperglycemic hormone (CHH) neuropeptide family. In the research reported here, a full length cDNA sequence of 972 bp of a MOIH was isolated by screening a cDNA library constructed from the eyestalks of Libinia emarginata. This cDNA sequence encodes a preprohormone peptide with 137 amino acid residues, including a 26-amino acid long signal peptide, a 34-amino acid long precursor peptide, a dibasic peptide, the full length of 72-amino acid long MOIH, and a tri-peptide Gly-Lys-Lys which designates the potential amidation site at the C-terminus of the mature peptide.