Expression of an Androgenic Gland-Specific Insulin-Like Peptide during the Course of Prawn Sexual and Morphotypic Differentiation

The crustacean male-specific androgenic gland (AG) regulates sexual differentiation. In the prawn Macrobrachium rosenbergii, silencing an AG-specific insulin-like encoding transcript (Mr-IAG) inhibited the development of male sexual characters, suggesting that Mr-IAG is a key androgenic hormone. We used recombinant pro-Mr-IAG peptide to generate antibodies that recognized the peptide in AG cells and extracts, as verified by mass spectrometry. We revealed the temporal expression pattern of Mr-IAG and studied its relevance to the timetable of sex differentiation processes in juveniles and after puberty. Mr-IAG was expressed from as early as 20 days after metamorphosis, prior to the appearance of external male sexual characters. Mr-IAG expression was lower in the less reproductively active orange-clawed males than in both the dominant blue-clawed males and the actively sneak mating small males. These results suggest a role for Mr-IAG both in the timing of male sexual differentiation and in regulating reproductive strategies

The protandric life history of the Northern spot shrimp Pandalus platyceros: molecular insights and implications for fishery management.

The Northern spot shrimp, Pandalus platyceros, a protandric hermaphrodite of commercial importance in North America, is the primary target species for shrimp fisheries within Southeast Alaska. Fishery data obtained from the Alaska Department of Fish and Game indicate that spot shrimp populations have been declining significantly over the past 25 years. We collected spot shrimps in Southeast Alaska and measured reproductive-related morphological, gonadal and molecular changes during the entire life history. The appendix masculina, a major sexual morphological indicator, is indicative of the reproductive phase of the animal, lengthening during maturation from juvenile to the male phase and then gradually shortening throughout the transitional stages until its complete disappearance upon transformation to a female. This morphological change occurs in parallel with the degeneration of testicular tissue in the ovotestis and enhanced ovarian vitellogenesis. Moreover, we obtained the entire mRNA sequence of the yolk protein precursor, vitellogenin, and monitored its transcript levels throughout the entire shrimp life-cycle. Vitellogenin transcript levels in the hepatopancreas increased in the early transitional stage until reaching a peak prior to extruding eggs. Such transcriptomic analyses, coupled with a comprehensive description of the gonad, external sex characters and timing of the reproductive life history of spot shrimps contribute to a better understanding of the hermaphroditic reproduction process in the cold Southeast Alaskan waters. This knowledge can contribute to a revision of current conservation efforts to maintain wild populations sustainable for both commercial and ecological considerations.Ye

A Sexual Shift Induced by Silencing of a Single Insulin-Like Gene in Crayfish: Ovarian Upregulation and Testicular Degeneration

In sequential hermaphrodites, intersexuality occurs naturally, usually as a transition state during sexual re-differentiation processes. In crustaceans, male sexual differentiation is controlled by the male-specific androgenic gland (AG). An AG-specific insulin-like gene, previously identified in the red-claw crayfish Cherax quadricarinatus (designated Cq-IAG), was found in this study to be the prominent transcript in an AG cDNA subtractive library. In C. quadricarinatus, sexual plasticity is exhibited by intersex individuals in the form of an active male reproductive system and male secondary sex characters, along with a constantly arrested ovary. This intersexuality was exploited to follow changes caused by single gene silencing, accomplished via dsRNA injection. Cq-IAG silencing induced dramatic sex-related alterations, including male feature feminization, a reduction in sperm production, extensive testicular degeneration, expression of the vitellogenin gene, and accumulation of yolk proteins in the developing oocytes. Upon silencing of the gene, AG cells hypertrophied, possibly to compensate for low hormone levels, as reflected in the poor production of the insulin-like hormone (and revealed by immunohistochemistry). These results demonstrate both the functionality of Cq-IAG as an androgenic hormone-encoding gene and the dependence of male gonad viability on the Cq-IAG product. This study is the first to provide evidence that silencing an insulin-like gene in intersex C. quadricarinatus feminizes male-related phenotypes. These findings, moreover, contribute to the understanding of the regulation of sexual shifts, whether naturally occurring in sequential hermaphrodites or abnormally induced by endocrine disruptors found in the environment, and offer insight into an unusual gender-related link to the evolution of insulins

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

Lethal Congenital Contractural Syndrome Type 2 (LCCS2) Is Caused by a Mutation in ERBB3 (Her3), a Modulator of the Phosphatidylinositol-3-Kinase/Akt Pathway

Lethal congenital contractural syndrome type 2 (LCCS2) is an autosomal recessive neurogenic form of arthrogryposis that is associated with atrophy of the anterior horn of the spinal cord. We previously mapped LCCS2 to 6.4 Mb on chromosome 12q13 and have now narrowed the locus to 4.6 Mb. We show that the disease is caused by aberrant splicing of ERBB3, which leads to a predicted truncated protein. ERBB3 (Her3), an activator of the phosphatidylinositol-3-kinase/Akt pathway—regulating cell survival and vesicle trafficking—is essential for the generation of precursors of Schwann cells that normally accompany peripheral axons of motor neurons. Gain-of-function mutations in members of the epidermal growth-factor tyrosine kinase–receptor family have been associated with predilection to cancer. This is the first report of a human phenotype resulting from loss of function of a member of this group

Temporal silencing of an androgenic gland-specific insulin-like gene affecting phenotypical gender differences and spermatogenesis

Androgenic glands (AGs) of the freshwater prawn Macrobrachium rosenbergii were subjected to endocrine manipulation, causing them to hypertrophy. Transcripts from these glands were used in the construction of an AG cDNA subtractive library. Screening of the library revealed an AG-specific gene, termed the M. rosenbergii insulin-like AG (Mr-IAG) gene. The cDNA of this gene was then cloned and fully sequenced. The cysteine backbone of the predicted mature Mr-IAG peptide (B and A chains) showed high similarity to that of other crustacean AG-specific insulin-like peptides. In vivo silencing of the gene, by injecting the prawns with Mr-IAG double-stranded RNA, temporarily prevented the regeneration of male secondary sexual characteristics, accompanied by a lag in molt and a reduction in growth parameters, which are typically higher in males of the species. In terms of reproductive parameters, silencing of Mr-IAG led to the arrest of testicular spermatogenesis and of spermatophore development in the terminal ampullae of the sperm duct, accompanied by hypertrophy and hyperplasia of the AGs. This study constitutes the first report of the silencing of a gene expressed specifically in the AG, which caused a transient adverse effect on male phenotypical gender differences and spermatogenesis. (Endocrinology 150: 1278 -1286, 2009) E ver since it was first proposed as the source of a hypothetical masculinizing hormone in crustaceans, the androgenic gland (AG) has been studied thoroughly in many crustacean species. The consensus emerging from these studies is that the AG plays a unifying role in the bewilderingly varied sex differentiation mechanisms in crustaceans (1-5). The AG constitutes a feature unique to male crustaceans in that it is an organ regulating sex differentiation separated from the gametogenic organ (unlike the single organ of vertebrate species). This separation enables manipulation of sex differentiation without affecting the gonads (6). In decapod male crustaceans, there are two AGs, each attached to the ejaculatory region of a vas deferens. In research spanning several decades, the functioning of the AG was investigated in a number of crustacean species by following the morphological and physiological effects of AG removal or transplantation on primary and secondary sex characteristics. In the amphipod Orchestia gamarella, for example, bilateral AG ablation decreased spermatogenesis and prevented the development of secondary male characteristics (7). In the crayfish Procambarus clarkii, injection of AG extracts accelerated the development of external male characteristics (8). In the giant freshwater prawn, Macrobrachium rosenbergii, a degree of masculinization was recorded in AG-implanted females (9). In the same species, fully functional sex reversal from males to neo-females (10) and from females to neo-males (11) was achieved by bilateral AG ablation and transplantation, respectively. The possibility of sex reversal has economical implications for the farming of this sexually dimorphic species because males grow faster than females (12). It is currently widely accepted that the AG of decapod crustaceans secretes the hormone(s) responsible for male differentiation, with a high probability of such a hormone(s) being proteinaceous in nature (13). This premise is supported by a histological study in the shore crab Phachygrapsus crassipes Multicellular organisms express various insulin-like peptides differentially. The insulin-like peptides discovered in inverte- Abbreviations: AG, Androgenic gland; CHH, crustacean hyperglycemic hormone; dsRNA, double-stranded RNA; GFP, green fluorescent protein; hAG, hypertrophy and hyperplasia of the androgenic gland; Mr-IAG, Macrobrachium rosenbergii insulin-like androgenic gland gene; RNAi, RNA interference; T7P, T7 promoter site at the 5Ј of one primer; UTR, untranslated region; XO-SG, X-organ sinus gland complex

Lethal Contractural Syndrome Type 3 (LCCS3) Is Caused by a Mutation in PIP5K1C, Which Encodes PIPKIγ of the Phophatidylinsitol Pathway

Lethal congenital contractural syndrome (LCCS) is a severe form of arthrogryposis. To date, two autosomal recessive forms of the disease (LCCS and LCCS2) have been described and mapped to chromosomes 9q34 and 12q13, respectively. We now describe a third LCCS phenotype (LCCS3)—similar to LCCS2 yet without neurogenic bladder. Using 10K single-nucleotide–polymorphism arrays, we mapped the disease-associated gene to 8.8 Mb on chromosome 19p13. Further analysis using microsatallite markers narrowed the locus to a 3.4-Mb region harboring 120 genes. Of these genes, 30 candidates were sequenced, which identified a single homozygous mutation in PIP5K1C. PIP5K1C encodes phosphatidylinositol-4-phosphate 5-kinase, type I, gamma (PIPKIγ), an enzyme that phophorylates phosphatidylinositol 4-phosphate to generate phosphatidylinositol-4,5-bisphosphate (PIP2). We demonstrate that the mutation causes substitution of aspartic acid with asparagine at amino acid 253 (D253N), abrogating the kinase activity of PIPKIγ. Thus, a defect in the phosphatidylinositol pathway leading to a decrease in synthesis of PIP2, a molecule active in endocytosis of synaptic vesicle proteins, culminates in lethal congenital arthrogryposis

The association between maternal serum first trimester free βhCG, second trimester intact hCG levels and foetal growth restriction and preeclampsia

The purpose of this study was to analyse the association between free beta hCG (fβhCG) increased levels and pregnancy complications (PC), foetal growth restriction (FGR) and preeclampsia (PE). This connection was evaluated in two stages (i) investigating the association between those PC with first trimester fβhCG and second trimester intact hCG (ihCG), and (ii) studying the association between these two analytes in the same pregnancy. This was a retrospective study in two settings: medical centre that provided data on fβhCG and ihCG levels in pregnancies with FGR and PE, and central laboratory that provided fβhCG and ihCG levels that were compared in the same pregnancy. No association was found between those PC and the hCG analytes, except for elevated ihCG levels and FGR. Elevated fβhCG (>3.00 MoM) was found in 570/16,849 (3.4%) women. However, only 14% of whom had elevated second trimester ihCG. A positive correlation was found between the magnitude of first trimester fβhCG levels and the percentage of women who had elevated second trimester ihCG. This association was determined by the magnitude of the elevation of fβhCG levels.Impact statement What is already known on this subject: The two analytes, first trimester fβhCG and second trimester ihCG, are independently produced and parameters of the biochemical screening during pregnancy. What the results of this study add: Referring to 3.00 MoM as cut-off levels, most pregnancies with elevated levels of first trimester fβhCG will have normal ihCG second trimester levels. What the implications are of these findings for clinical practice and/or further research: The risk of developing pregnancy complications, FGR and PE should be associated with second trimester ihCG levels. About 3.5% of women had high fβhCG levels during the first trimester. However, only 14% also had increased ihCG levels, defined as >3.00 MoM; additional studies are needed to explore the association between increased first trimester fβhCG levels and the risk of developing pregnancy complications, independent of ihCG levels in the second trimester