Direct evidence for the function of crustacean insulin-like androgenic gland factor (IAG): Total chemical synthesis of IAG

Insulin-like androgenic gland factor (IAG) is presumed to be a sex differentiation factor so-called androgenic gland hormone (AGH) in decapod crustacean, although the function of IAG peptide has not yet been reported. In this study, we synthesized IAG from the prawn, Marsupenaeus japonicus, and its function was assessed by an in vitro bioassay. As a result, IAG with the insulin-type disulfide bond arrangement showed biological activity, whereas its disulfide isomer did not. These results strongly suggest that the native IAG peptide has an insulin-type disulfide, and it is the decapod AGH.     

Molecular evolution of the androgenic hormone in terrestrial isopods

In crustaceans, the androgenic gland (AG), thanks to the synthesis of the androgenic gland hormone (AGH), controls the differentiation of the primary and secondary male sexual characters. In this study, we amplified 12 new AGH cDNAs in species belonging to five different families of the infra-order Ligiamorpha of terrestrial isopods. Putative essential amino acids for the production of a functional AGH protein exhibit signatures of negative selection and are strictly conserved including typical proteolytic cleavage motifs, a putative N-linked glycosylation motif on the A chains and the eight Cys positions. An insulin-like growth factor motif was also identified in Armadillidium AGH sequences. The phylogenetic relationships of AGH sequences allowed one to distinguish two main clades, corresponding to members of the Armadillidiidae and the Porcellionidae families which are congruent with the narrow specificity of AG heterospecific grafting. An in-depth understanding of the regulation of AGH expression would help deciphering the interaction between Wolbachia, widespread feminizing endosymbiotic bacteria in isopods, and the sex differentiation of their hosts.     

Endocrine and genetic control of sex differentiation in the Malacostracan Crustacea

Summary

Sex differentiation in Malacostraca is controlled by hormone secreted from the androgenic glands. Experimentally induced sex inversions in isopods and amphipods proved that the genetic female and male possess primordia of the androgenic glands, gonads, and gonoducts, along with sexual characteristics of both sexes. During the sensitive period, the presence or absence of androgenic gland hormone (AGH) affects the differentiation of these primordia.

Genetic control of the development of androgenic gland primordium seems to be brought about assuming of the following: 1. Both genetic female and male possess gene(s) (AGH-G) responsible for the AGH-synthesis situated on the homologous loci of the sex chromosomes and/or on the autosomes. 2. The gene(s) are activated spontaneously with the lack of inhibition of the major sex factor carried by the W or X chromosome. The W and X factors are hypostatic to major sex factor carried by the Y chromosome. The Z factor does not seem to influence sex differentiation. Sufficient allochthonous AGH seems to render the W and X factors ineffective.     

Inhibitory effects of the androgenic gland on ovarian development in the mud crab Scylla paramamosain

Isolation and characterization of androgenic hormone in decapod crustaceans depend on an effective bioassay of its action. In the present study, the effect of androgenic gland on ovarian development in the mud crab Scylla paramamosain was investigated with a view to develop a bioassay for androgenic hormone. Ovarian regression with degeneration of oocytes occurred in some female crabs implanted with androgenic gland in vivo. In vitro incubation of ovarian tissues at secondary vitellogenesis in extract of androgenic gland resulted in a significant decrease in amino acid uptake by the tissues. We propose that this inhibitory effect could be established as an effective bioassay for the isolation of androgenic hormone in the mud crab.     

The structure of a glycosylated protein hormone responsible for sex determination in the isopod, Armadillidium vulgare.

Two glycoforms (AH1 and AH2) of androgenic hormone, and its corresponding hormone precursor derived from HPLC-purified androgenic gland extract from the woodlouse Armadillidium vulgare were fully characterized by microsequencing and mass spectrometry. The amino-acid sequences of the two glycoforms were identical; they consist of two peptide chains, A and B, of 29 and 44 amino acids, respectively, with chain A carrying one N-glycosylated moiety on Asn18. The two chains are linked by two disulfide bridges. Glycoforms were only differentiated by the size and heterogeneity of the glycan chain. The androgenic hormone precursor (16.5 kDa) was shown to contain the sequence of chains A and B from the androgenic hormone, connected by a C-peptide (50 amino acids). These results were confirmed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis performed on a single hypertrophied androgenic gland. When injected into young females, both glycoforms of the androgenic hormone were able to override genetic sex-determination. In invertebrates, there is no other example where sex-differentiation is controlled by a protein hormone that is not synthesized by the gonads but by a special gland. A functional comparison with two other hormones which are believed to play a role in sex determination, i.e. ecdysone in insects and anti-Müllerian hormone in mammals, is presented. Work is in progress to clone and characterize the gene encoding androgenic hormone, moreover special attention is devoted to its regulatory regions, putative targets for the Wolbachia action.     

Characterization and cDNA cloning of androgenic gland hormone of the terrestrial isopod Armadillidium vulgare.

The sex differentiation in crustaceans is known to be controlled by a peptide hormone called androgenic gland hormone (AGH). AGH was extracted and purified from the androgenic glands (AGs) of the male isopod Armadillidium vulgare by high-performance liquid chromatography. AGH consisted of two peptide chains and their N-terminal amino acid sequences were determined. A cDNA encoding AGH was cloned by PCR and sequenced. The cDNA had an open reading frame of 432 bp, which encoded a preproAGH consisting of a signal peptide (21 residues), B chain (44 residues), C peptide (46 residues), and A chain (29 residues). Through processing, the A and B chains might form a heterodimer interlinked by disulfide bonds. The A chain possessed a putative N-linked glycosylation site. A Northern blot analysis using the cDNA as a probe detected a hybridization signal with 0.8 kb in the RNA preparation only from the AGs.     

Hormonal Control of Sexual Differentiation and Reproduction in Crustacea

SYNOPSIS. Sexual differentiation in malacostracan Crustaceais controlled by the androgenic gland hormone (AGH). In males,the primordial androgenic glands (AG) develop and AGH inducesmale morphogenesis. In females, the primordial AG does not developand the ovaries differentiate spontaneously. Implantation ofthe AG into females yields various results, showing that thesensitivity to AGH differs with the species and the receptiveorgans. Purified AGH of the isopod Armadillidium vulgare consistsof at least two molecular forms, which exist as monomeric proteinswith molecular weights of 17,000 ± 800 and 18,300 ±1,000 Da and with isoelectric points of about 4.5 and 4.3, respectively.The antiserum raised against purified AGH makes it possibleto measure AGH activity by immunoassay. Neurohormones control male and female reproduction. In males,they are involved in the maintenance of the male germinativezone and the control of AG activity. In females, the secondaryvitellogenesis is controlled by the vitellogenesis-inhibitinghormone (VIH) and the vitellogenesis- stimulating hormone (VSH).VIH isolated from the lobster Homarus americanus is a peptidewith a molecular weight of 9,135 Da and shows homology to thecrustacean hyperglycemic hormone and moltinhibiting hormone.Involvement of the molting hormone and the juvenile hormone-likecompound in the secondary vitellogenesis have also been suggested.In the amphipod Orchestia gammarella, the vitellogenesis- stimulatingovarian hormone (VSOH) seems to control vitellogenin synthesis     

Mechanisms of parasite-induced sex reversal in Gammarus duebeni

The amphipod Gammarus duebeni is host to the feminising microsporidian parasite Nosema granulosis that converts males into functional females. To test the hypothesis that the parasite acts through endocrine disruption we compared the morphology of the gonad and activity of the androgenic gland, which coordinates male sexual differentiation, in infected and uninfected animals. Male gonad consisted of testis, seminal vesicle and vas deferens that was anchored to the genital papilla on segment 7. The androgenic gland was associated with the distal end of the vas deferens. In female and intersex animals the bi-lobed ovary opened into the oviduct at segment 5, vestigial vas deferens and vestigial androgenic gland were retained. The majority of parasitised individuals (38/39) were either phenotypic females or intersexes with fully developed ovaries and an undifferentiated androgenic gland. Our data suggest that the parasite prevents differentiation of the androgenic gland. In further support of this hypothesis, mass spectrometry of a single androgenic gland from males revealed a dominant molecular ion with a mass/charge ratio of 4818.4+H, corresponding to a peptide of androgenic gland hormone from Armadillidium vulgare. In contrast the vestigial androgenic gland from parasitised and unparasitised females showed only low intensity peaks. Our observations demonstrate that the parasite manipulates host sex by preventing androgenic gland differentiation, androgenic gland hormone production and consequently male differentiation. This is in agreement with observations of A. vulgare with inherited Wolbachia infection, suggesting that phylogenetically distant feminisers manipulate hosts through a common mechanism. The high frequency of infection in intersexes (89.3%) suggests that this phenotype results from incomplete feminisation by the parasite.     

Androgenic gland cell structure and spermatogenesis during the molt cycle and correlation to morphotypic differentiation in the giant freshwater prawn, Macrobrachium rosenbergii

The freshwater prawn Macrobrachium rosenbergii shows three male morphotypes: blue-claw males (final stage having high mating activity), orange-claw males (transitional stage showing rapid somatic growth), and small males (primary stage showing sneak copulation). This morphotypic differentiation is considered to be controlled by androgenic gland hormone, which is probably a peptide hormone. However, its physiological roles are not fully understood. In the present study, we examined the correlation of androgenic gland cell structure to spermatogenic activity and morphotypic differentiation histologically in M. rosenbergii. spermatogenic activity showed close correlation to the molt cycle in orange-claw males and small males. spermatogonia increased in number in the late premolt stage, becoming spermatocytes in the postmolt stage, and spermatocytes differentiated into spermatozoa in the intermolt and early premolt stages. Ultrastructure of the androgenic gland was additionally compared among the molt stages, but, distinct histological changes were not observed in relation to spermatogenesis during the molt cycle. On the other hand, among the three morphotypes, the androgenic gland was largest in the blue-claw males, containing developed rough endoplasmic reticulum in the cytoplasm. These results suggest that, during spermatogenesis which is related to the molt cycle, the androgenic gland hormone is at rather constant levels and plays a role in maintaining spermatogenesis rather than directly regulating the onset of a specific spermatogenesis stage and that, during the morphotypic differentiation, the androgenic gland is most active in the blue-claw males and plays a role in regulating the observed high mating activity in M. rosenbergii.     

甲壳动物胰岛素样促雄腺激素功能及 作用机制的研究进展

甲壳动物的雄性性别分化主要由其促雄腺（AG）分泌的胰岛素样促雄腺激素（IAG）负责调控。在罗氏沼虾（Macrobrachium rosenbergii）中,通过单个IAG的操作可以成功性反转,进而实现全雄养殖。因此,基于IAG的性别调控技术具有良好的应用潜力。目前,IAG在许多经济甲壳动物中得到研究报道,发现其表达不仅局限于促雄腺,功能也更加广泛。此外,随着RNA干扰技术在水产动物中的广泛运用,基因功能的研究更易实现,IAG如何执行其生理作用的信号机制及上游的调控网络逐渐成为学者们探究的热点。本文综述了近年来有关IAG研究的进展,从IAG的分子特征、生理功能、作用机制及上游调控机理等方面展开探讨,为深入阐明IAG的生理功能及作用机制提供基础。     

Bilateral eyestalk ablation of the blue swimmer crab, Portunus pelagicus, produces hypertrophy of the androgenic gland and an increase of cells producing insulin-like androgenic gland hormone

The androgenic glands (AG) of male decapod crustaceans produce insulin-like androgenic gland (IAG) hormone that controls male sex differentiation, growth and behavior. Functions of the AG are inhibited by gonad-inhibiting hormone originating from X-organ-sinus gland complex in the eyestalk. The AG, and its interaction with the eyestalk, had not been studied in the blue swimmer crab, Portunus pelagicus, so we investigated the AG structure, and then changes of the AG and IAG-producing cells following eyestalk ablation. The AG of P. pelagicus is a small endrocrine organ ensheathed in a connective tissue and attached to the distal part of spermatic duct and ejaculatory bulb. The gland is composed of several lobules, each containing two major cell types. Type I cells are located near the periphery of each lobule, and distinguished as small globular cells of 5-7 μm in diameter, with nuclei containing mostly heterochromatin. Type II cells are 13-15 μm in diameter, with nuclei containing mostly euchromatin and prominent nucleoli. Both cell types were immunoreactive with anti-IAG. Following bilateral eyestalk ablation, the AG underwent hypertrophy, and at day 8 had increased approximately 3-fold in size. The percentage of type I cells had increased more than twice compared with controls, while type II cells showed a corresponding decrease.     

Novel structures in secreting the androgenic gland hormone

The secretory granules in the androgenic gland of the terrestrial isopod Armadillidium vulgare, which have been indistinct for long time because of vulnerable structures, were revealed by using the rapid-freezing and freeze-substitution method. The fine structure of the androgenic gland is conspicuous by the distribution of numerous particular organelles in the cytoplasm consisting of the endoplasmic reticulum and the Golgi complex, and by having a number of highly organized structures developed between the androgenic gland cells. The structures connect to the intercellular space, which is seen as intercellular canaliculi for exporting the androgenic gland hormone. The plasma membranes near the particular structure of the intercellular canaliculi in the androgenic gland are often specialized to form cellular junctions. The secretory granules including the electron-dense materials, which are supposed to be peptides of androgenic gland hormone, are distributed beside the particular structure of the intercellular canaliculi. Some of the granules are seen to fuse with the plasma membranes. This observation suggests that, in the Armadillidium vulgare, the secretory granules containing androgenic gland hormone are transferred to the extracellular space through the intercellular canaliculi particularly developed for exporting the peptide hormone. This is the first evidence to show the secretory mechanism of the androgenic gland hormone in the Isopoda.     

Evolutionary transition to XY sex chromosomes associated with Y-linked duplication of a male hormone gene in a terrestrial isopod

Sex chromosomes are highly variable in some taxonomic groups, but the evolutionary mechanisms underlying this diversity are not well understood. In terrestrial isopod crustaceans, evolutionary turnovers in sex chromosomes are frequent, possibly caused by Wolbachia, a vertically-transmitted endosymbiont causing male-to-female sex reversal. Here, we use surgical manipulations and genetic crosses, plus genome sequencing, to examine sex chromosomes in the terrestrial isopod Trachelipus rathkei. Although an earlier cytogenetics study suggested a ZZ/ZW sex chromosome system in this species, we surprisingly find multiple lines of evidence that in our study population, sex is determined by an XX/XY system. Consistent with a recent evolutionary origin for this XX/XY system, the putative male-specific region of the genome is small. The genome shows evidence of Y-linked duplications of the gene encoding the androgenic gland hormone, a major component of male sexual differentiation in isopods. Our analyses also uncover sequences horizontally acquired from past Wolbachia infections, consistent with the hypothesis that Wolbachia may have interfered with the evolution of sex determination in T. rathkei. Overall, these results provide evidence for the co-occurrence of multiple sex chromosome systems within T. rathkei, further highlighting the relevance of terrestrial isopods as models for the study of sex chromosome evolution.Subject terms: Evolutionary genetics, Genome evolution     

Chemical synthesis of insulin-like peptide 1 and its potential role in vitellogenesis of the kuruma prawn Marsupenaeus japonicus

The insulin superfamily comprises a group of peptides with diverse physiological functions and is conserved across the animal kingdom. Insulin-like peptides (ILPs) of crustaceans are classified into four major types: insulin, relaxin, gonadulin, and androgenic gland hormone (AGH)/insulin-like androgenic gland factor (IAG). Of these, the physiological functions of AGH/IAG have been clarified to be the regulation of male sex differentiation, but those of the other types have not been uncovered. In this study, we chemically synthesized Maj-ILP1, an ILP identified in the ovary of the kuruma prawn Marsupenaeus japonicus, using a combination of solid-phase peptide synthesis and regioselective disulfide bond formation reactions. As the circular dichroism spectral pattern of synthetic Maj-ILP1 is typical of other ILPs reported thus far, the synthetic peptide likely possessed the proper conformation. Functional analysis using ex vivo tissue incubation revealed that Maj-ILP1 significantly increased the expression of the yolk protein genes Maj-Vg1 and Maj-Vg2 in the hepatopancreas and Maj-Vg1 in the ovary of adolescent prawns. This is the first report on the synthesis of a crustacean ILP other than IAGs and also shows the positive relationship between the reproductive process and female-dominant ILP.     

Structure–activity relationship of crustacean peptide hormones

In crustaceans, various physiological events, such as molting, vitellogenesis, and sex differentiation, are regulated by peptide hormones. To understanding the functional sites of these hormones, many structure–activity relationship (SAR) studies have been published. In this review, the author focuses the SAR of crustacean hyperglycemic hormone-family peptides and androgenic gland hormone and describes the detailed results of our and other research groups. The future perspectives will be also discussed.     

Hormonal Control of Molting in Decapod Crustacea

The involvement of the molting hormone, 20-hydroxyecdysone,in the mediation of molting in decapod crustaceans is brieflyreviewed. Aspects of the secretion and metabolism of its precursor,ecdysone, are discussed. Experiments are described that demonstratethe presence of a molt-inhibiting hormone (MIH) in the sinusglands of juvenile lobsters (Homarus americanus). Assays forMIH include measurement of the molt interval and radioimmunoassayof circulating titers of ecdysteroids in eyestalk-ablated lobsters.This latter assay indicates that sinus gland extracts significantlydecrease the concentration of circulating ecdysteroids 24 hrafter injection. Data are also presented on the circulatingtiters of ecdysteroids during multiple molt cycles of lobstersfollowing eyestalk ablation. These data indicate that theremust be another factor that ultimately regulates the circulatinglevels of the molting hormone.     

A Crayfish Insulin-like-binding Protein: ANOTHER PIECE IN THE ANDROGENIC GLAND INSULIN-LIKE HORMONE PUZZLE IS REVEALED*

Across the animal kingdom, the involvement of insulin-like peptide (ILP) signaling in sex-related differentiation processes is attracting increasing attention. Recently, a gender-specific ILP was identified as the androgenic sex hormone in Crustacea. However, moieties modulating the actions of this androgenic insulin-like growth factor were yet to be revealed. Through molecular screening of an androgenic gland (AG) cDNA library prepared from the crayfish Cherax quadricarinatus, we have identified a novel insulin-like growth factor-binding protein (IGFBP) termed Cq-IGFBP. Based on bioinformatics analyses, the deduced Cq-IGFBP was shown to share high sequence homology with IGFBP family members from both invertebrates and vertebrates. The protein also includes a sequence determinant proven crucial for ligand binding, which according to three-dimensional modeling is assigned to the exposed outer surface of the protein. Recombinant Cq-IGFBP (rCq-IGFBP) protein was produced and, using a “pulldown” methodology, was shown to specifically interact with the insulin-like AG hormone of the crayfish (Cq-IAG). Particularly, using both mass spectral analysis and an immunological tool, rCq-IGFBP was shown to bind the Cq-IAG prohormone. Furthermore, a peptide corresponding to residues 23–38 of the Cq-IAG A-chain was found sufficient for in vitro recognition by rCq-IGFBP. Cq-IGFBP is the first IGFBP family member shown to specifically interact with a gender-specific ILP. Unlike their ILP ligands, IGFBPs are highly conserved across evolution, from ancient arthropods, like crustaceans, to humans. Such conservation places ILP signaling at the center of sex-related phenomena in early animal development.     

Crustacean Vitellogenesis: Its Role in Oocyte Development

One of the major changes that occurs during the maturation ofoocytes is the accumulation of yolk protein, or vitellin (Vn).To better understand how this process is regulated, we characterizedthe Vn of the ridgeback shrimp, Sicyonia ingentis (Penaeoidea).This Vn is a 322 kDa molecule composed of three subunits. Usingpurified Vn, we developed an anti-Vn antiserum and used it tocharacterize vitellogenin by Western blot analysis. The antiserumwas also used in an ELISA to measure hemolymph levels of vitellogenin.Previous studies suggested the presence of vertebrate-type steroidsmight stimulate reproductive processes in decapod crustaceans.Treatment of sexually quiescent female shrimp with progesterone,hydroxyprogesterone, and estradiol did not increase hemolymphlevels of yolk protein precursor. The absence of a responseto these steroids may reflect the presence of other hormones(such as the gonad-inhibiting hormone) that prevent oocyte development.To examine the molecular basis for the regulation of vitellogenesis,ovarian and hepatopancreas expression cDNA libraries were screenedusing the anti-Vn antiserum. A 2.9 kilobase clone was isolatedfrom both cDNA libraries suggesting that both tissues are sitesof vitellogenin synthesis. These molecular tools should be usefulfor in vitro studies of vitellogenin synthesis.     

Preparation of an active recombinant peptide of crustacean androgenic gland hormone

In crustaceans, male sexual characteristics are induced by a hormone referred to as androgenic gland hormone. We have recently cloned a candidate cDNA in the terrestrial isopod Armadillidium vulgare. In order to prove that this cDNA encodes the hormone, recombinant single-chain precursor molecules consisting of B chain, C peptide and A chain were produced using both baculovirus and bacterial expression systems. Neither recombinant precursors showed activity. Digestion of only the precursor carrying a glycan moiety with lysyl endopeptidase gave a heterodimeric peptide with hormonal activity by removing a part of C peptide. These results indicate that the cDNA encodes the hormone.