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.     

Chemical synthesis of N‐glycosylated insulin‐like androgenic gland factor from the freshwater prawn Macrobrachium rosenbergii

Crustacean insulin‐like androgenic gland factor (IAG) of Macrobrachium rosenbergii, a heterodimeric peptide having both four disulfide bonds and an N‐linked glycan, was synthesized by the combination of solid‐phase peptide synthesis and the regioselective disulfide formation reactions. The disulfide isomer of IAG could also be synthesized by the same manner. The conformational analysis of these peptides by circular dichroism (CD) spectral measurement indicated that the disulfide bond arrangement affected the peptide conformation in IAG. On the other hand, the N‐linked glycan attached at A chain showed no effect on CD spectra of IAG. This is the first report for the chemical synthesis of insulin‐like heterodimeric glycopeptide having three interchain disulfides, and the synthetic strategy shown here might be useful for the synthesis of other glycosylated four‐disulfide insulin‐like peptides.     

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.     

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。     

The Crustacean Androgen: A Hormone in an Isopod and Androgenic Activity in Decapods

The androgenic gland has been described in a variety of crustaceanspecies—isopods, amphipods and decapods. It has been shownto play a role in the regulation of male differentiation andin the inhibition of female differentiation. Upon its applicationfor endocrine manipulation, it inhibits female characteristics.Recently, the androgenic hormone from the isopod Armadillidiumvulgare was purified and characterized on the basis of a morphologicalbioassay. The hormone is a glycosylated protein composed oftwo peptide chains connected each to the other by two disulfidebridges. The pro-hormone consists of the same two chains connectedby a third peptide in a complex that resembles the insulin superfamily hormones. The study of the androgenic gland in decapodslags behind that in the isopods, and a decapod androgenic hormonehas yet to be identified. In this review, five decapod speciesare described as models, in which the androgenic gland exertsmorphological, anatomical, physiological and behavioral effects.These models could serve as the basis of possible bioassaysfor the study of the structure and mode of action of the androgenichormone in decapod crustaceans.     

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.     

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.     

几种甲壳动物促雄性腺素结构预测分析

利用生物信息学方法对目前已知的3种甲壳动物促雄性腺素前体(AGH precursor)和5种类胰岛素促雄性腺因子(insulin-like AG factor)进行分析,探讨了促雄性腺素前体的氨基酸理化特性、信号肽、跨膜结构域、二级结构、motif等,并利用Phyre软件对其三级进行同源性收索。结果显示:促雄性腺素前体包含信号肽,存在跨膜结构域,并和信号肽同位。PDB库中没有找到匹配的motif。3种促雄性腺素前体的二级结构有比较高的相似性,比如都包含两个中心螺旋区。Phyre搜索显示,与8种蛋白的三级结构匹配的均为胰岛素家族的蛋白,这也进一步证实了促雄性腺素前体和胰岛素原的相似性。     

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.     

Immunological identification of crustacean androgenic gland hormone, a glycopeptide

Androgenic gland hormone (AGH) is known to be responsible for sex differentiation in crustaceans. The amino acid sequence of AGH-active fraction purified from androgenic glands of the terrestrial isopod Armadillidium vulgare was determined by immunoprecipitation employing three types of antibodies raised against differing parts of the amino acid sequence deduced from the putative AGH cDNA sequence. As all antibodies adsorbed AGH activity, it was confirmed that the sequence examined was that of AGH. The affinity of AGH to certain lectins indicated that AGH possesses a carbohydrate moiety, which is in agreement with the observation that AGH possesses an N-glycosylation consensus sequence.     

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     

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.     

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.     

Determination of phosphate residues participating in the formation of the spatial structure of tRNA- Leu IAG from cow mammary glands

The phosphates of the tRNA-Leu IAG from cow mammary gland (tRNA which has a long variable loop) participating in the formation of three-dimensional structure were studied by alkylation with ethylnitrosourea and methylnitrosourea. A low degree of modification was observed for the phosphates of the following nucleotides: 7, 8, 9, 10 (at the bend site between the acceptor and D-stem); 18, 19, 20A and 21 in the D-loop; 47H and 49 at the joint of variable and T-stem; 57, 58 and 59 in the T-loop.     

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.     

A structural and functional comparison of nematode and crustacean PDH-like sequences

The elucidation of the whole genome of the nematode Caenorhabditis elegans allowed for the identification of ortholog genes belonging to the pigment dispersing hormone/factor (PDH/PDF) peptide family. Members of this peptide family are known from crustaceans, insects and nematodes and seem to exist exclusively in ecdysozoans where they play a role in different processes, ranging from the dispersion of integumental and eye (retinal) pigments in decapod crustaceans to circadian rhythms in insects and locomotion in C. elegans. Two pdf genes (pdf-1 and pdf-2) encoding three different peptides: PDF-1a, PDF-1b and PDF-2 have been identified in C. elegans. These three C. elegans PDH-like peptides are similar but not identical in primary structure to PDHs from decapod crustaceans. We investigate whether this divergence has an influence on the pigment dispersing function of the peptides in a decapod crustacean, namely the shrimp Palaemon pacificus. We show that C. elegans PDF-1a and b peptides display cross-functional activity by dispersing pigments in the epithelium of P. pacificus at physiological doses. Moreover, by means of a comparative amino acid sequence analysis of nematode and crustacean PDH-like peptides, we can pinpoint several potentially important residues for eliciting pigment dispersing activity in decapod crustaceans. Although there is no sequence information on a receptor for PDH in decapod crustaceans, we postulate that there is general conservation of the PDH/PDF signaling system based on structural similarities of precursor proteins and receptors (including those from a branchiopod crustacean and from C. elegans).     

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.