Insulin-like Androgenic Gland Hormone Research Articles

Genomic cloning, expression, and single nucleotide polymorphism association analysis of the insulin-like androgenic gland hormone gene in the oriental river prawn (Macrobrachium nipponense).

Increasing evidence suggests that the insulin-like androgenic gland hormone (IAG) gene plays an important role in male sexual differentiation, metabolism, and growth in crustaceans. In the present study, we isolated the full-length genome sequence of IAG by genome walking based on the cDNA sequence in Macrobrachium nipponense. Four novel single nucleotide polymorphisms (SNPs) were studied, including 509G>T, 529G>T, 590A>T in intron 1, and 2226A>G in intron 2. The association of genetic variation with growth traits [body length (BL) and body weight (BW)] was analyzed. Individuals with GG geno- type at locus 2226A>G maintained higher mean BL (P < 0.01) and BW (P < 0.05) than AA and GA individuals. These results suggest that IAG SNPs may be useful molecular markers for selecting growth traits in M. nipponense.

Molecular characterization of insulin-like androgenic gland hormone-binding protein gene from the oriental river prawn Macrobrachium nipponense and investigation of its transcriptional relationship with the insulin-like androgenic gland hormone gene

Insulin-like androgenic gland hormone-binding protein (IAGBP) has been investigated in crustaceans in vitro. However, the relationship between IAGBP and its putative binding protein partner insulin-like androgenic gland hormone (IAG) has not been studied at the transcriptional level in vivo. In the current study, we cloned the full-length cDNA of IAGBP from the oriental river prawn Macrobrachium nipponense (Mn-IAGBP) and investigated the transcriptional patterns of Mn-IAGBP and the M. nipponense IAG gene (Mn-IAG) at different developmental stages and in different tissues. Mn-IAGBP mRNA was detected in all examined tissues from adult male prawns, with the highest transcriptional levels in the testis. Mn-IAG mRNA was detected in the androgenic gland and hepatopancreas. The genomic sequences of Mn-IAGBP and Mn-IAG were isolated by genome walking and two gene copies were found in both Mn-IAGBP and Mn-IAG. The relationship between Mn-IAGBP and Mn-IAG at the transcriptional level was studied by RNA interference. Injection of Mn-IAGBP double-stranded RNA (dsRNA) significantly reduced the transcription of Mn-IAG, while injection of Mn-IAG dsRNA significantly reduced the transcription of Mn-IAGBP in testis, muscle, androgenic gland, and hepatopancreas. These results demonstrate the involvement of the IAGBP gene in IAG signaling in M. nipponense.

On the safety of RNAi usage in aquaculture: The case of all-male prawn stocks generated through manipulation of the insulin-like androgenic gland hormone

Abstract The recent introduction of RNA interference (RNAi) based biotechnology in aquaculture has aroused public awareness regarding safety aspects related to the use of such temporal transcript manipulation. In the present study, we demonstrate use of this biotechnology in large-scale production of all-male freshwater prawn Macrobrachium rosenbergii, relying on a two-phased approach. First, the insulin-like androgenic gland hormone (Mr-IAG) was silenced by dsRNA injections, causing sex-reversal of males into ‘neo-females’ (genetic males with female phenotype, with 86% success in this study). The next step involved mating the neo-females with normal males to produce all-male progeny. The administered exogenous dsRNA was fully cleared from the prawn's tissues after 7 days. Gene silencing through dsMr-IAG appeared to be temporary, with decay seen over time and nearly full expression of Mr-IAG (~ 82%) being restored after 28 days. Assessment of reproduction and fecundity in normal females versus neo-females revealed that in both groups, ~ 80% of the prawns were in a reproductive state and no significant difference was found in their reproductive output, either in terms of brood mass or number. Population structure, as indicated by the normal occurrence of male morphotypes, in both mixed-sex and all-male populations was examined in terms of weight and morphotype frequency distribution. The final harvest and marketing of the all-male prawn offspring occur about 19 months, i.e., one generation after the actual RNAi procedure. Thus, on top of the aquaculture yield advantage, the use of such RNAi procedure in crustacean aquaculture seems to be safe due to its temporary nature.

An insulin-like androgenic gland hormone gene in the mud crab, Scylla paramamosain, extensively expressed and involved in the processes of growth and female reproduction

Insulin-like androgenic gland hormone (IAG) produced by androgenic gland (AG) in male crustaceans is regarded as a key regulator of sex differentiation. As a member of the insulin/insulin-like growth factor family, IAG is also likely involved in regulating somatic growth. In this study, a full-length cDNA of IAG (termed Sp-IAG) was isolated from the mud crab, Scylla paramamosain. Genomic DNA of Sp-IAG was also cloned, analysis of which reveals that Sp-IAG gene is organized in a 4 exon/3 intron manner. RNA in situ hybridization analysis detected positive signals in both type I and type II AG cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that Sp-IAG was expressed not only in AG, but also in many other tissues. Sp-IAG expression levels in ovaries were examined at different stages of ovarian development (stages I to V); it was found that the expression was maintained at low levels during undeveloped stage (stage I) to late vitellogenic stage (stage IV) and then increased significantly at mature stage (stage V), suggesting that Sp-IAG may participate in inhibiting oocyte growth and vitellogenesis. The expression pattern of Sp-IAG during the molting cycle of the first stage crabs (C1) was also determined. Sp-IAG expression level continuously decreased from 0h C1 (postmolt) crabs to 96h C1 (premolt) crabs, and then increased significantly in the newly molted second stage crabs (C2, postmolt). The combined results suggested for the first time that IAG is involved in regulating ovarian development and somatic growth in crustaceans.

Histology of the androgenic gland and expression of the insulin-like androgenic gland hormone precursor gene in the genital organ of Pacific white shrimp Litopenaeus vannamei

The location of the androgenic gland in the genital organ in male Pacific white shrimp Litopenaeus vannamei (Boone, 1931) was analyzed by histology and expression of the insulin-like androgenic gland hormone precursor gene (Lv-IAG). The androgenic gland in L. vannamei is a group of three conspicuous semicircular cellular masses interconnected by cord-like androgenic gland cells, in parallel and attached to the distal vas deferens and terminal ampoule, which form a hook-like morphology. Lv-IAG expression occurs mainly at the terminal ampoule, but also at the distal part of the descending medium vas deferens and the narrow distal vas deferens. These specific regions of Lv-IAG expression basically match the anatomical localization of the androgenic gland observed by histology. The complete cDNA nucleotide sequence of Lv-IAG precursor had 87% to Fenneropenaeus chinensis (Osbeck, 1765), 84% identity to Penaeus monodon Fabricius, 1798 (Pm-IAG), and 82% to Marsupenaeus japonicus (Bate, 1888) (Maj-IAG). The deduced encoding sequence from an open reading frame of 507 bp contained a signal peptide, a B chain, a C peptide, and an A chain that correspond to the androgenic gland insulin-like hormone polypeptide in decapods, where six highly conserved cysteine residues of the A and B chains fold the mature peptide through disulfide bridges. The amino-acid sequence showed high identity to P. monodon (80%), F. chinensis (80%), and M. japonicus (72%), whereas the match to other decapod families Palaemonidae, Parastacidae, and Portunidae was low (24-32%). This study extends our knowledge of the molecular structure of insulin-like peptides related to sex differentiation in malacostraca.

Gene Silencing in Crustaceans: From Basic Research to Biotechnologies

Gene silencing through RNA interference (RNAi) is gaining momentum for crustaceans, both in basic research and for commercial development. RNAi has proven instrumental in a growing number of crustacean species, revealing the functionality of novel crustacean genes essential among others to development, growth, metabolism and reproduction. Extensive studies have also been done on silencing of viral transcripts in crustaceans, contributing to the understanding of the defense mechanisms of crustaceans and strategies employed by viruses to overcome these. The first practical use of gene silencing in aquaculture industry has been recently achieved, through manipulation of a crustacean insulin-like androgenic gland hormone. This review summarizes the advancements in the use of RNAi in crustaceans, and assesses the advantages of this method, as well as the current hurdles that hinder its large-scale practice.

The effects of serotonin, dopamine, gonadotropin-releasing hormones, and corazonin, on the androgenic gland of the giant freshwater prawn, Macrobrachium rosenbergii

Neurotransmitters and neurohormones are agents that control gonad maturation in decapod crustaceans. Of these, serotonin (5-HT) and dopamine (DA) are neurotransmitters with known antagonist roles in female reproduction, whilst gonadotropin-releasing hormones (GnRHs) and corazonin (Crz) are neurohormones that exercise both positive and negative controls in some invertebrates. However, the effects of these agents on the androgenic gland (AG), which controls testicular maturation and male sex development in decapods, via insulin-like androgenic gland hormone (IAG), are unknown. Therefore, we set out to assay the effects of 5-HT, DA, l-GnRH-III, oct-GnRH and Crz, on the AG of small male Macrobrachium rosenbergii (Mr), using histological studies, a BrdU proliferative cell assay, immunofluorescence of Mr-IAG, and ELISA of Mr-IAG. The results showed stimulatory effects by 5-HT and l-GnRH-III through significant increases in AG size, proliferation of AG cells, and Mr-IAG production (P<0.05). In contrast, DA and Crz caused inhibitory effects on the AG through significant decreases in AG size, proliferation of AG cells, and Mr-IAG production (P<0.05). Moreover, the prawns treated with Crz died before day 16 of the experimental period. We propose that 5-HT and certain GnRHs can be now used to stimulate reproduction in male M. rosenbergii, as they induce increases in AG and testicular size, IAG production, and spermatogenesis. The mechanisms by which these occur are part of our on-going research.

An androgenic gland membrane-anchored gene associated with the crustacean insulin-like androgenic gland hormone

Crustacean male sexual differentiation is governed by the androgenic gland (AG) and specifically by the secreted insulin-like AG hormone (IAG), thus far identified in several decapod species including the Australian red claw crayfish Cherax quadricarinatus (termed Cq-IAG). While a few insulin-like AG genes have been identified in crustaceans, other AG-specific genes have not been documented until now. In the present study, we describe the recent identification of a non-IAG AG-specific transcript obtained from the C. quadricarinatus AG cDNA library. This transcript, termed C. quadricarinatus membrane-anchored AG-specific factor (Cq-MAG), was fully sequenced and found to encode a putative product of 189 amino acids including a signal anchoring peptide. Expression of a recombinant GFP fusion protein lacking the signal anchor encoding sequence dramatically affected recombinant protein localization pattern. While the expression of the deleterious fusion protein was observed throughout most of the cell, the native GFP::Cq-MAG fusion protein was observed mainly surrounding the periphery of the nucleus, demonstrating an endoplasmic reticulum (ER)-like localization pattern. Moreover, co-expression of the wild-type Cq-MAG (fused to GFP) and the Cq-IAG hormone revealed that these peptides indeed co-localize. This study is the first to report a protein specifically associated with the insulin-like AG hormone in addition to the finding of another AG-specific transcript in crustaceans. Previous knowledge suggests that insulin/insulin-like factor secretion involves tissue-specific transcripts and membrane-anchored proteins. In this regard, Cq-MAG's tissue specificity, anchoring properties and intracellular co-localization with Cq-IAG suggest that it may play a role in the processing and secretion of this insulin-like AG hormone.

Two spliced variants of insulin-like androgenic gland hormone gene in the Chinese shrimp, Fenneropenaeus chinensis

More and more evidence indicates that the insulin-like androgenic gland hormone (IAG) plays an important role in male sexual differentiation in crustaceans. In the present study, two IAG isoforms (Fc-IAG1 and Fc-IAG2) were identified from the penaeid shrimp Fenneropenaeus chinensis. Sequence analysis of IAG gene (Fc-IAG) showed that Fc-IAG1 and Fc-IAG2 were generated by alternative splicing of Fc-IAG pre-mRNA, and they shared almost the same deduced amino acid sequence. Both of them were composed of signal peptide, B chain, C peptide and A chain. They both contained the six conserved cysteine residues and a putative N-linked glycosylated site like IAGs reported in other crustacean species. Tissue distribution and in situ hybridization analysis revealed that they had the highest expression level in the androgenic gland. The transcripts of Fc-IAG1 and Fc-IAG2 could also be detected in hepatopancreas and nerve cord of both sexes at a low expression level. Analysis on their temporal expression profiles showed that they expressed at all embryonic and post-larvae stages. The expression of Fc-IAG1 at different developmental stages displayed a low and stable manner, while the expression of Fc-IAG2 began to increase from post-larvae stages, which suggested that Fc-IAG2 might be involved in male sexual differentiation. In the 5′ flanking sequence of Fc-IAG, putative binding sites for transcription factors regulating transcription of hormone genes and genes related to sexual development were predicted, which provided us a primary understanding on the regulation mechanism of Fc-IAG gene. This is the first time to report the gene structure of IAG gene and distinct variants of IAG transcripts in crustaceans.

Cells producing insulin-like androgenic gland hormone of the giant freshwater prawn, Macrobrachium rosenbergii, proliferate following bilateral eyestalk-ablation

We found that the androgenic gland (AG) of Macrobrachium rosenbergii possesses three cell types. Type I cells are small polygonal shaped-cells (13.4 μm in diameter), stain strongly with hematoxylin–eosin (H&E), have abundant multilayered rough endoplasmic reticulum (rER), and nuclei containing mostly heterochromatin. Type II cells are slightly larger (18.6 μm in diameter), stain lightly with H&E, have rER with dilated cisternae, and nuclei containing mostly euchromatin. Type III cells (previously undescribed) are similar in size and shape to type I cells, but the cytoplasm is unstained and they have a high amount of smooth endoplasmic reticulum (sER) and mitochondria with tubular cristae. Bilateral eyestalk-ablation resulted in AG hypertrophy with a proliferation and predominance of type I cells as determined by bromodeoxyuridine (BrdU) assays. Expression of insulin-like androgenic gland hormone (Mr-IAG), determined by immunohistochemistry, was weak in type I cells, strong in type II cells of both the intact and eyestalk-ablated, and negative in type III cells. It was also detected in spermatogonia, nurse cells, and epithelium lining of the spermatic duct. The function of Mr-IAG in these tissues is yet to be elucidated but the distribution implies a strong role in male reproduction.

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.