Mandibular Organ Research Articles

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.

Endocrine and neuroendocrine regulations in embryos and larvae of crustaceans

Summary This review deals with the studies which have been conducted for the past 30 years on the endocrine and neuroendocrine regulations in embryos and larvae of crustaceans, mostly in decapods. Y-organs, mandibular organs and the X-organ sinus gland complex of the eyestalks are present in the first post-embryonic instar of most investigated species. Y-organs, the X-organs and the sinus glands have also been located in embryos of a few species. Larval molting appears to be regulated in a way similar to that in adults involving a MIH-ecdysteroid system. Evidence points to a control of metamorphosis through the eyestalks. Experimental evidence points to a neuroendocrine control of changes in pigmentation and of osmoregulation. Progress in the isolation and characterization of the hormones and neurohormones controlling these metabolic changes in adults should help and promote further research on regulation during the embryonic and early postembryonic development.

Isolation of Amelogenin-Positive Ameloblasts from Rat Mandibular Incisor Enamel Organs by Flow Cytometry and Fluorescence Activated Cell Sorting

The purpose of this study was to use amelogenin as a marker to examine the feasibility of isolating ameloblasts from enamel organ cell populations by fluorescence activated cell sorting. After treating dissected rat enamel organs with proteolytic enzymes to loosen cell attachments and labial connective tissues, dissociated cell suspensions were fixed, then immunostained with rabbit anti-rM179 recombinant amelogenin antibody and FITC-conjugated goat anti-rabbit Ig G antibody. Flow cytometry indicated that about 70% of the total cell sample and virtually all the larger cells therein were amelogenin-positive. Fluorescence activated cell sorting yielded a sample of amelogenin-positive cells at 97% purity. Immunofluorescence microscopy indicated that these isolated amelogenin-positive cells varied widely in size and morphology. This was attributed to loss of intercellular support for ameloblasts once they were dissociated from each other, and to some fragmentation caused when the cells were initially physically removed from the teeth. The results demonstrate that viable ameloblast cell fractions, especially representing cells at the secretory stage, can be purified from enzymic digests of rat enamel organ by sorting on the basis of cell size alone. From these fractions, subpopulations of ameloblasts may be identified when differentiation specific cell surface markers become available.

A mouse mandibular culture model permits the study of neural crest cell migration and tooth development.

A major issue in developmental biology is to determine how time and position-restricted instructions are signaled and received during morphogenesis of different phenotypes, of which tooth, Meckel's cartilage and tongue formation are classical examples. It is now evident that a hierarchy of growth factors and their downstream transcription factors regulate the timing, sequence and position of cells and tissues in forming different phenotypes during embryogenesis. Here we report the development of an early mandibular organ culture model. Explants of E8 and E9 first branchial arch were cultured and produced mandibular processes with cap stage tooth formation, Meckel's cartilage and tongue development. In tandem, vital dye (Dil) labeling studies confirmed that rhombomeres 1-4 give rise to craneal neural crest (CNC) cells which emigrate from the neural fold to the forming maxillary and mandibular arches. Furthermore, we have tested the feasibility of investigating the regulation of different phenotypes within the first branchial arch by a transcription factor using this early mandibular organ culture model. Lymphoid enhancing factor 1 (Lef1), a transcription factor, has been implicated to regulate tooth formation in vivo. We have analyzed the expression of Lef1 and studied the biological effects of Lef1 on E8 embryonic mouse first branchial arch explants in organ culture. Collectively, these results demonstrate that first branchial arch explant model is suitable for studies of rhombencephalic crest cell fate during mandibular morphogenesis and can be used as a model with direct access to investigate the molecular mechanism in regulating first branchial arch morphogenesis.

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.

Methyl Farnesoate: Crustacean Juvenile Hormone in Search of Functions

The sesquiterpenoid methyl farnesoate (MF) is synthesized by crustacean mandibular organs and is present in the hemolymph. The chemical structure of MF is nearly identical to that of insect juvenile hormone III, differing only by the absence of an epoxide group. The functions of MF in crustaceans have been elusive, in part because the mandibular organs of most species are difficult to ablate. However, three roles of MF have emerged from a growing body of literature. They are (a) stimulation of general protein synthesis; (b) promotion of the molt cycle; and (c) reproduction in both males and females. These functions are not universal; rather, they reflect the diversity of the crustaceans that have been studied to date. This review will synthesize aspects of what is known about MF in crustaceans and outline some current avenues of research.

Distribution and Regulation of Esterases That Hydrolyze Methyl Farnesoate inHomarus americanusand Other Crustaceans

Ester hydrolysis of methyl farnesoate (MF) by crustacean tissue homogenates was measured using the substrate [3H]MF in a radiochemical partition assay. Tissues were obtained from the lobsterHomarus americanus,penaeid shrimpSicyonia ingentis,thalanassid shrimpCallianassa californiensis,sand crabEmerita analoga,and spider crabPugettia producta.The greatest specific activities were recovered from the hepatopancreas (239 to 11,500 pmol MF/min-mg total protein). Hepatopancreatic homogenates ofC. californiensiswere significantly more active than homogenates from the other species. In the lobster, esterases that hydrolyze MF were associated with lipid storage (R) cells of the hepatopancreas. Enzyme activity of lobster larval homogenates increased 1.5-fold during the second stage of development. The rate of MF hydrolysis by esterases extracted from the juvenile lobster hepatopancreas could not be correlated with molt stage or sex and was not significantly influenced by eyestalk ablation, mandibular organ ablation, or MF injection.

Mammalian craniofacial embryology in vitro.

Our review demonstrates that the whole embryo culture system established by New and his colleagues, in combination with beneficial fluorescent dye cell-tracing techniques, has greatly contributed to many advancements in the field of mammalian craniofacial embryology, especially with regard to elucidating the developmental behavior of cephalic crest cells. In addition, based on recent results, further combining whole embryo culture with mandibular organ culture methods has allowed us to trace cranial crest cells for a much longer developmental period, i.e., presently up to the cap stage in odontogenesis.

Seasonal differences in methyl farnesoate esterase activity in tissues of the spider crab Libinia emarginata

Summary Methyl farnesoate (MF), an unepoxidated form of insect JH III, is present in Crustacea. MF is synthesized by the mandibular organs and is degraded to fomesoic acid (FA) by peripheral tissues. In this study we investigated MF degradation by esterases in hepatopancreas, ovary, testes and hemolymph of the spider crab Libinia emarginata collected at different times of the year to determine seasonal differences. The conversion of MF to FA varied among the tissues. In the summer, the hepatopancreas showed the greatest esterase activity (52.8% conversion in females and 59.16% in males), and it was twice as high (28.86%) in ovaries than in the testes (12.16%), but was low in the hemolymph of both sexes (10.84% in males, and 6.97% in females). In the fall, the conversion of MF to FA was significantly reduced in all tissues (ovary 8.55%, testes 6.21%, hepatopancreas 10.22%, hemolymph 3.96%). Eyestalk ablation of animals in the fall restored MF esterase activity to summer levels. When tissues from these animals were incubated with OTFP, a specific inhibitor of JH esterase, MF metabolism was significantly reduced. These results suggest that MF esterase activity depends on direct induction by MF, and its degradation is by a specific esterase(s).

Mass Spectrometric Determination of Methyl Farnesoate Profiles and Correlation with Ovarian Development in the Edible Crab,Cancer pagurus

Evidence indicates that methyl farnesoate (MF), produced by the mandibular organs (MOs), is the crustacean equivalent of the juvenile hormones of insects. Gas chromatography–mass spectrometry with selected ion monitoring (GC–MS-SIM) was used to measure the titers of MF in the MOs of the crab, Cancer pagurus. Comparison of the amounts of MF detected in groups of left and right MOs from crabs at different stages of ovarian development showed no statistically significant variation. The amounts of MF detected in MOs were in the range of 230 ng/MO in crabs with immature ovaries to 400 ng/MO in crabs with fully developed ovaries. Application of GC–MS-SIM to the measurement of MF in hemolymph was unsuccessful, leading to the development of a high-performance liquid chromatography–MS-SIM (HPLC–MS-SIM) method for analysis of such titers. MF concentrations in hemolymph varied throughout ovarian development, exhibiting a peak at the beginning of secondary vitellogenesis (140 ng/ml), falling to basal levels thereafter.

Characterization of Methyl Transferase Activity in the Mandibular Organ of the American Lobster, Homarus americanus.

Previous articleNext article No AccessCOMPARATIVE BIOCHEMISTRY AND PHYSIOLOGYCharacterization of Methyl Transferase Activity in the Mandibular Organ of the American Lobster, Homarus americanusT. Claerhout, W. Bendena, S. S. Tobe, and D. W. BorstT. Claerhout Search for more articles by this author , W. Bendena Search for more articles by this author , S. S. Tobe Search for more articles by this author , and D. W. Borst Search for more articles by this author PDFPDF PLUS Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by The Biological Bulletin Volume 191, Number 2October 1996 Published in association with the Marine Biological Laboratory Article DOIhttps://doi.org/10.1086/BBLv191n2p304 Views: 9Total views on this site Citations: 1Citations are reported from Crossref Copyright © 1996 by Marine Biological LaboratoryPDF download Crossref reports the following articles citing this article:J. Ogan, A. Shaub, D. L. Lovett, and D. W. Borst Relationship of Methyl Transferase Activity and Methyl Farnesoate Levels in the Spider Crab, Libinia emarginata, The Biological Bulletin 193, no.22 (Sep 2016): 267–268.https://doi.org/10.1086/BBLv193n2p267

Contribution of Early-Emigrating Midbrain Crest Cells to the Dental Mesenchyme of Mandibular Molar Teeth in Rat Embryos

Teeth are formed by reciprocal interactions between the epithelium and mesenchyme in the first pharyngeal arch. Although the contribution of midbrain and hindbrain crest cells to the first pharyngeal arch has been previously examined in rodent embryos, no direct evidence exists that these cells are actually involved in the dental mesenchyme. In order to elucidate the contribution of the cranial neural crest cells in tooth formation, we first identified the emigration sites and stages providing the crest cells that migrate to the presumed tooth-forming region of the mandibular prominence. Focal labeling with DiI was performed at the midbrain and anterior hindbrain crests in rat embryos, and the labeled embryos were cultured for 30 or 60 hr. The resultant migration patterns indicated that posterior midbrain crest cells emigrating by the end of the 4-somite stage predominantly migrated to the region where tooth buds normally develop. Second, we established a new type of long-term culture system in which whole embryo culture is followed by a mandibular organ culture. Using this system, rat embryos were maintained from the early-somite stage and the molars in the explants were able to reach the bud stage within 8 days. Finally, to ascertain if posterior midbrain crest cells emigrating by the end of the 4-somite stage were involved in the dental mesenchyme, these cells were labeled with DiI and processed for the long-term culture. Labeled crest cells were clearly detectable in the dental mesenchyme. These findings indicate that the early-emigrating posterior midbrain crest cells contribute to mandibular molar tooth development in rat embryos.

Structure and Significance of Mandibular Organ-inhibiting Hormone in the Crab, Cancer pagurus: INVOLVEMENT IN MULTIHORMONAL REGULATION OF GROWTH AND REPRODUCTION

Current evidence indicates that methyl farnesoate is the crustacean equivalent of the juvenile hormones of insects. This putative hormone is produced by the mandibular organs and is negatively regulated by a neuropeptide produced and secreted by the X-organ-sinus gland complex of the eyestalk. To identify this neuropeptide, a bioassay was developed which measures the inhibition of methyl farnesoate synthesis by mandibular organs exposed to fractionated sinus gland extracts from the crab, Cancer pagurus. Two neuropeptides, named mandibular organ-inhibiting hormones (MOIH-1 and -2) repressed methyl farnesoate synthesis. MOIH-1 was fully sequenced by automated Edman degradation of endoproteinase-derived fragments and further characterized by mass spectrometry. This peptide consisted of 78 residues (Mr 9235.6), with unblocked termini and three intrachain disulfide bridges. MOIH-2 appeared to be almost identical to MOIH-1 with the exception of a Gln for Lys substitution at position 33. Comparison with previously sequenced crustacean neuropeptides shows that these MOIHs are members of the ever expanding crustacean hyperglycemic hormone family, with significant sequence similarity to molt-inhibiting hormones (MIHs). It is possible that these two structurally similar peptides (MIH, MOIH) may control mutually exclusive physiological phenomena (somatic and gonadal growth), suggesting a complex hormonal integration of these processes in crustaceans.

Isolation and characterization of sinus gland neuropeptides with both mandibular organ inhibiting and hyperglycemic effects from the spider crab Libinia emarginata

Using two-step reverse phase HPLC, we isolated and purified three peptides with mandibular organ inhibiting hormone (MO-IH) activity from the spider crab Libinia emarginata. One of the peptides, P22, gave a yield of 355 ng/SG. The others gave lower yields: P21, 9 ng/SG; P25, 67.5 ng/SG. The molecular weight was determined to be 8,439 for P25, 8,474 for P22, and 8,398 for P21 by mass spectrometry. All three peptides have similar amino acid compositions and contain 72–76 residues. We believe these peptides to be different isoforms of one family. The MO is more sensitive to the two minor isoforms, P21 and P25. All three isoforms can inhibit MO activity to a maximum inhibition of 70%. All three isoforms gave a significant hyperglycemic effect when injected into de-eyestalked fiddler crabs Uca pugilator. We believe the MO-IHs to be members of the crustacean hyperglycemic hormone (CHH) family, having similar amino acid compositions and both biological activities. © 1996 Wiley-Liss, Inc.

Isolation and characterization of sinus gland neuropeptides with both mandibular organ inhibiting and hyperglycemic effects from the spider crabLibinia emarginata

Using two-step reverse phase HPLC, we isolated and purified three peptides with mandibular organ inhibiting hormone (MO-IH) activity from the spider crab Libinia emarginata. One of the peptides, P22, gave a yield of 355 ng/SG. The others gave lower yields: P21, 9 ng/SG; P25, 67.5 ng/SG. The molecular weight was determined to be 8,439 for P25, 8,474 for P22, and 8,398 for P21 by mass spectrometry. All three peptides have similar amino acid compositions and contain 72–76 residues. We believe these peptides to be different isoforms of one family. The MO is more sensitive to the two minor isoforms, P21 and P25. All three isoforms can inhibit MO activity to a maximum inhibition of 70%. All three isoforms gave a significant hyperglycemic effect when injected into de-eyestalked fiddler crabs Uca pugilator. We believe the MO-IHs to be members of the crustacean hyperglycemic hormone (CHH) family, having similar amino acid compositions and both biological activities. © 1996 Wiley-Liss, Inc.

Effect of solubilization on the binding activity of a G-protein from the mandibular organ of the lobster Homarus americanus (Nephropidae, Decapoda)

The binding of [35SIGTPγS by the G-protein from the lobster mandibular organ was not affected by biogenic amines, synthetic eyestalk peptides or an extract of the X-organ-sinus gland complex from the eyestalk. This suggests that the G-protein is not part of the receptor apparatus that controls the inhibition of methyl farnesoate synthesis. The G-protein is deactivated during extraction from the membrane by the anionic detergents sodium cholate and SDS but may be solubilized using the nonionic detergents Triton X-100, Lubrol PX or Nonidet P-40.

Methyl Farnesoate Levels in Male Spider Crabs Exhibiting Active Reproductive Behavior

The concentration of methyl farnesoate (MF) in the hemolymph and its synthesis by the mandibular organs (MOs) were investigated to determine whether this compound is related to the differences in the size of the reproductive system and the mating behavior among male morphotypes of the spider crab, Libinia emarginata. Large-claw abraded males displayed mating behavior under competitive conditions. They have the largest reproductive systems, their MOs synthesize large amounts of MF in vitro, and the concentration of MF in their hemolymph is high. Small-claw abraded males displayed mating behavior with receptive females only when isolated. These smaller crabs have intermediate-sized reproductive systems, their MOs synthesize the most MF, and they have the highest circulating level of MF relative to their body size. The unabraded males did not display mating behavior; their reproductive systems are smaller; their MO activity is low, as is their circulating level of MF. The strong relationship between MF levels and the intensity of reproductive behavior suggests that MF may be one of the driving forces behind mating behavior in Crustacea.

Regional Differences in Methyl Farnesoate Production by the Lobster Mandibular Organ.

Visual examination of the mandibular organ (MO) from the lobster, Homarus americanus, disclosed two distinct morphological regions: a fan-folded region along one edge of the gland, and a smooth, unfolded region comprising the rest of the gland. Because MOs produce methyl farnesoate (MF), the MF content of both regions was measured. In freshly dissected glands, more than 95% of the MF was found in the fan-folded region of the gland. In MO sections incubated with [3H-methyl]methionine (a radiolabeled precursor of MF), more than 90% of MF synthesis was found in the fan-folded region. Eyestalk ablation, a procedure that increases MO activity, caused the MF content of MOs to increase more than 130-fold, but had little effect on the regional distribution of MF. Histological observations indicated that these two regions had different cellular compositions. The fan-folded region contained two cell types (A and B). The A cells were mitotically active and appeared to be undifferentiated. The B cells contained a large number of small vacuoles. The unfolded region was largely composed of a third cell type (C). The C cells were large and morphologically complex, containing many mitochondria and large vacuolar-like structures. They contained relatively few small vacuoles. On the basis of appearance and location, B cells appear to be the likely site of MF synthesis. The physiological importance of C cells is unknown.

Distinct Reproductive Types of Male Spider Crabs Libinia emarginata Differ in Circulating and Synthesizing Methyl Farnesoate.

Levels of methyl farnesoate in the blood and in vitro rates of methyl farnesoate synthesis by the mandibular organ were investigated to determine whether this compound is related to the differences in morphology and reproductive states of distinct types of male spider crabs described by Homola et al. (1992) in winter populations. Three male types, selected from a summer population, were investigated in detail: (1) males with relatively large propoduses (claws) and worn exoskeletons (abraded), (2) males with relatively large propoduses and exoskeletons covered with epicuticle (unabraded), and (3) males with small propoduses and unabraded exoskeletons (small). All males examined had sperm, but abraded males, identical in propodus and body size to unabraded males, had a reproductive system that weighed twice as much. Large-clawed unabraded males had relatively small reproductive systems. Small-clawed males possessed a small reproductive system. Abraded males possessed larger mandibular organs, containing almost twice the total protein, and their mandibular organs synthesized significantly more methyl farnesoate in vitro than did the other types of males. Circulating levels of methyl farnesoate, in the hemolymph of the abraded males, were more then twice as high as the levels detected in any other type of male. The strong relationship between methyl farnesoate levels, male morphology, and reproductive system development calls for further studies on the role of methyl farnesoate in the regulation of reproduction and morphogenesis in male crustaceans.

The Role of Juvenile Hormones in Crustacean Reproduction

Synopsis. Similar to juvenile hormone production in adult insects, the production of methyl farnesoate (MF) seems to be related to reproduction in both sexes in mature crustaceans. The mandibular organ (MO) synthesizes MF, and its activity corresponds to reproductive stages. High MF levels in the blood are found in vitellogenic females and reproduc? tively active males. Conversely, low levels are found in immature females, non-vitellogenic females, and males that are in reproductive diapause. The MO is under inhibitory control by neurosecretory factors from the sinus gland located in the eyestalks, and may be stimulated by factors from the brain and/or thoracic ganglion.