Shirley Hiu Kwan Tiu

The use of recombinant protein and RNA interference approaches to study the reproductive functions of a gonad-stimulating hormone from the shrimp Metapenaeus ensis

Although the crustacean crustacean hyperglycemic hormone/molt‐inhibiting hormone/gonad‐inhibiting hormone neuropeptides have been studied extensively in the last two decades and several neuropeptides from the shrimp Metapenaeus ensis have been cloned, the functions of most of these neuropeptides remained putative. In this article, we describe the use of recombinant protein and an RNA interference approach to study the reproductive function of the previously reported molt‐inhibiting hormone (MeMIH‐B) in M. ensis. When hepatopancreas and ovary explants were cultured in medium containing recombinant MeMIH‐B, the vitellogenin gene (MeVg1) expression level was upregulated in a dose‐dependent manner, reaching a maximum in explants treated with 0.3 nm recombinant MeMIH‐B. Shrimp injected with recombinant MeMIH‐B showed an increase in vitellogenin gene expression in the hepatopancreas. Moreover, a corresponding increase in the vitellogenin‐like immunoreactive protein was detected in the hemolymph and ovary of these females. Injection of MeMIH‐B dsRNA into the female shrimp caused a decrease in MeMIH‐B transcript level in thoracic ganglion and eyestalk. These shrimp also showed reduction of vitellogenin gene expression in the hepatopancreas and ovary. Furthermore, the hemolymph vitellogenin level was also reduced in these animals. In summary, the results from recombinant protein and RNA interference experiments have demonstrated the gonad‐stimulatory function of MeMIH‐B in shrimp.

From Hepatopancreas to Ovary: Molecular Characterization of a Shrimp Vitellogenin Receptor Involved in the Processing of Vitellogenin

Abstract We report the first cloning and characterization of cDNA encoding a putative vitellogenin (Vg) receptor (VgR) from the shrimp, Penaeus monodon. The shrimp VgR cDNA is 6.8 kb; the deduced protein has 1943 amino acids with a molecular weight of 211 kDa. VgR is ovary specific and consists of conserved cysteine-rich domains, epidermal growth factor-like domains, and YWTD motifs similar to the low-density lipoprotein, very low-density lipoprotein, and VgR of insects and vertebrates. VgR expression level in the ovary is low during early vitellogenesis and increases to maximum levels in females with a gonadosomatic index of 3–4, presumably when needed for receptor-mediated endocytosis during the rapid phase of extraovarian Vg production by the hepatopancreas. A peptide from the C-terminal end of VgR was synthesized for antibody production. Anti-VgR antibody recognized an ovarian membrane protein, and the level of this protein was high when extraovarian production of Vg reached peak levels. By immunohistochemical analysis, VgR was detected strongly in the membranes of larger oocytes. VgR expression was knocked down after the shrimp were injected with VgR double-stranded RNA, leading to a decrease in VgR protein content in the ovary, but an increase in the hemolymph level of Vg. This study represents the first report of the functional analysis of a putative VgR in a crustacean.

Cloning and expression study of the lobster (Homarus americanus) vitellogenin: Conservation in gene structure among decapods

This study reports the molecular characterization of the vitellogenin (Vg) of the lobster, Homarus americanus. Based on the annual collection of female lobsters, vitellogenesis commences in early March and continues through to September of each year. Using an antibody to vitellin of the lobster, H. americanus, several immunoreactive ovarian proteins were initially identified by Western blot analysis. The 80kDa protein contained the amino acid sequence APWGGNTPRC, identified subsequently by cDNA cloning to be identical to the lobster Vg. In common with the shrimp Metapenaeus ensis and crab Charybdis feriatus, the lobster HaVg1 gene comprises 14 introns and 15 exons. The deduced HaVg1 precursor is most similar to the Vg of the crayfish Cherax quadricarinatus (57%), followed by M. ensis (40-43% identity) and C. feriatus (38%). The results from genomic and RT-PCR cloning also confirmed the presence of multiple Vg genes in lobster. At early reproductive stages, the hepatopancreas HaVg1 transcript levels are low but increased to a maximum in animals with mature oocytes. The ovary, however, also expressed low levels of HaVg1. Using in vitro explant culture, treatment of hepatopancreas fragments with farnesoic acid or 20-hydroxyecdysone resulted in a significant stimulation in HaVg1 expression. From this study, it appears that Vg gene organization and expression pattern in decapods is highly conserved. Similar endocrine mechanisms may govern the process of vitellogenesis across the decapods.

Vitellogenesis in the Red Crab, Charybdis feriatus: Contributions from Small Vitellogenin Transcripts (CfVg) and Farnesoic Acid Stimulation of CfVg Expression

Abstract: During reproductive maturation of the female red crab, Charybdis feriatus, the oocytes rapidly accumulate 110‐ and 78‐kDa major polypeptides. Although the hepatopancreas expresses a high level of vitellogenin (CfVg) mRNA, tissue proteins and secreted proteins of the hepatopancreas consist of only small polypeptides. In addition to the 8.0‐kb transcripts, many smaller mRNAs specific to the CfVg gene can be detected. These results suggest that the hepatopancreas also produces smaller CfVg transcripts for small CfVg subunits. Using an RT‐PCR cloning approach, a population of the small cDNA clones were isolated. Determining the DNA sequence of these clones revealed that these transcripts were most likely the result of alternative splicing and/or alternative expression of the CfVg gene. In vitro treatment of the hepatopancreas fragments with low levels of farnesoic acid stimulated the expression of CfVg.

Farnesoic acid and methyl farnesoate production during lobster reproduction: possible functional correlation with retinoid X receptor expression.

Farnesoic acid (FA) and methyl farnesoate (MF) are juvenile hormone-related compounds secreted by the mandibular organ (MO) of crustaceans and play an important role in stimulation of ovarian maturation. To better understand how the MO activity influences female reproduction by secretion of FA and MF, the biosynthesis and release of these two compounds were measured in vitro by the incorporation of l-[(3)H-methyl]methionine into MF and [2-(14)C]acetate into FA by the MO of Homarus americanus. The production of FA is 7.5 times that of MF, and most FA and MF synthesized remained within the gland, and was not released into the surrounding medium. Most FA and MF were synthesized in the anterior fan-fold region of the MO. The rates of biosynthesis of FA and MF were stage-related, with maximal production occurring during secondary vitellogenesis (i.e. stages 4 and 5). A potential juvenoid receptor, retinoid X receptor (RXR), HaRXR, was characterized using PCR cloning techniques. HaRXR belongs to the nuclear hormone receptor superfamily and its deduced amino acid sequence shares a high homology to other RXRs of crustaceans, insects, and vertebrates. Transcripts of HaRXR can be detected in many tissues, and significant high expression level was detected in the MO, especially in the anterior fan-fold region. Expression of HaRXR was also related to reproductive stage, and maximal level of expression was observed at stage 4, in which secondary vitellogenesis is occurring. Changes in transcript level of HaRXR and the rates of FA/MF biosynthesis in the female reproductive cycle indicate that HaRXR and FA/MF may play important roles in crustacean reproduction.

The effects of farnesoic acid and 20-hydroxyecdysone on vitellogenin gene expression in the lobster, Homarus americanus, and possible roles in the reproductive process

Reproduction in female lobster (Homarus americanus) is characterized by the maturation of the ovary, with a gradual increase in its size as a result of uptake of yolk protein precursor, vitellogenin (Vg) to the final product vitellin (Vn). Vn is formed by aggregation of several Vg subunits. In most decapods, the hepatopancreas is the major site of vitellogenin biosynthesis. The production of vitellogenin is controlled by endocrine factors. In this study, the effect of farnesoic acid (FA) and 20-hydroxyecdysone (20E) on production of vitellogenin by hepatopancreas (HaVg1) was investigated by in vitro organ explant HaVg1 gene expression was stimulated by FA or 20E in a dose-dependent manner. A 2-fold and 2.2-fold increase in HaVg1 gene expression was observed with 4.2 microM FA and 0.7 microM 20E, respectively. The stimulatory effect by either FA or 20E was observed principally during the first 90 min. Stimulation of HaVg1 gene expression by FA and 20E together is greater (3.3-fold increase) than that of either hormone alone. This stimulation was also observed within the first 90 min. To study the synergistic effect of these two hormones, FA and 20E were tested separately and together at low concentration (42.3 nM and 6.7 nM, respectively). Combined use of FA and 20E increased HaVg1 gene expression synergistically, but not additively. These findings should contribute to our understanding of lobster reproduction and provide insights into manipulation of lobster reproduction in aquaculture or under captive conditions.

Suppression of JH biosynthesis by JH analog treatment: Mechanism of suppression and roles of allatostatins and nervous connections in the cockroach Diploptera punctata

Juvenile hormone analogs are known to inhibit the production of juvenile hormone (JH) by the corpora allata (CA). However, the mechanism of this inhibition remains undefined. We have used two JH mimics, fenoxycarb and pyriproxyfen, to examine the mechanism of suppression in the cockroach, Diploptera punctata. Denervation experiments demonstrated the importance of nervous connections between the brain and CA for the inhibition of JH biosynthesis by fenoxycarb. Fenoxycarb treatment alters the sensitivity of CA to allatostatin treatment in vitro. Suppression of JH biosynthesis by fenoxycarb following denervation of the CA showed that innervation was in part responsible for the inhibition. Similarly, maximal inhibition by Dippu-AST7 requires intact nervous connections between the brain and CA, particularly during rapid vitellogenesis. qPCR analysis of brain, CA, ovary and midgut extracts revealed that both allatostatin and its receptor Dippu-ASTR2 show increased levels of expression following topical fenoxycarb treatment, particularly in brain tissue on days 4 and 5 of the first gonadotrophic cycle and in CA on day 4. The correlation between inhibition of JH biosynthesis and increased expression of AST and ASTR2 in brains and CA, together with increased sensitivity of CA to allatostatin in vitro, suggests that allatostatin may be one of the effectors by which fenoxycarb inhibits JH biosynthesis.