Hepatopancreas and ovary are sites of vitellogenin synthesis as determined from partial cDNA encoding of vitellogenin in the marine shrimp,Penaeus vannamei

Summary

The site of yolk protein synthesis in crustaceans has long been a subject of controversy. A portion of the vitellogenin gene structure was reported recently in a freshwater giant prawn (Macrobrachium rosenbergii) and black tiger shrimp (Penaeus monodori), in which the hepatopancreas was confirmed to be the extraovarian site of vitellogenin synthesis. The ovary is also frequently reported to be the site of yolk protein synthesis in penaeid shrimp. The same PCR product was obtained using cDNA from the hepatopancreas or the ovary as a template. The deduced amino acid sequence of Vg in P. vannamei showed high identities of 57% and 78% with those from M. rosenbergii and P. monodon, respectively. The same location of the intron in the sequenced region of genomic DNA was also found between these three species. We therefore concluded that the hepatopancreas and ovary are sites of vitellogenin synthesis in P. vannamei. The partial structure of the vitellogenin gene is further presented.     

Hepatopancreas is the extraovarian site of vitellogenin synthesis in black tiger shrimp, Penaeus monodon.

The site of yolk protein synthesis in crustaceans has long been a subject of controversy. The vitellogenin gene structure was partially reported only very recently in Macrobrachium rosenbergii, after which the hepatopancreas was confirmed as the extraovarian site of vitellogenin synthesis in that species. Ovaries are the most frequently reported as the site of yolk protein synthesis in penaeid shrimp. Using cDNA reversed-transcribed from mRNA isolated from the hepatopancreas of vitellogenic female shrimp, Penaeus monodon, we found that its deduced amino acid sequence had high identity of 48% with that from M. rosenbergii vitellogenin. A similar location of the intron in the sequenced region of genomic DNA was also found between these two species. We therefore concluded that the hepatopancreas the extraovarian site of vitellogenin synthesis in P. monodon in vivo. The partial structure of vitellogenin gene is presented in this study.     

Cloning and characterization of the tiger shrimp lysozyme

Lysozymes are key proteins to invertebrates in the innate immune responses against bacterial infections. A lysozyme gene isolated from tiger shrimp, Penaeus monodon, was cloned, sequenced and characterized. The cDNA consists of a signal peptide of 18 amino acids and a mature peptide of 140 amino acids. The lysozyme is presumed to be a chicken-type lysozyme for it possesses two catalytic sites and eight cysteine residues which are highly conserved across species of chicken-type lysozymes. The lysozyme cDNAs of Penaeus semisulcatus, Litopenaeus vannamei, Macrobrachium nipponense and Macrobrachium rosenbergii were also cloned. High similarities existed among shrimp and prawn lysozymes but phylogenetic relationship of shrimps and prawns based on lysozyme molecules did not quite consistent with traditional taxonomic classification. High mRNA expression was detected in hepatopancreas, haemocytes and gill of tiger shrimp. Recombinant lysozyme exhibited potent lytic activities against fish pathogens providing evidence of the involvement of lysozyme in shrimp immunity.     

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.     

Vitellogenocytes in the Hepatopancreas of Carcinus maenas and Libinia emarginata (Decapoda brachyura)

Summary

In addition to the ovary, the hepatopanocreas of female decapod crustaceans, Carcinus maenas, and Libinia emarginata is a source of yolk protein. The specific cells in the hepatopancreas that localize vitellogenins on tissue sections are revealed with lipovitellin-specific antiserum. These cells, designated vitellogenocytes, are believed to be responsible for vitellogenin synthesis in the hepatopanocreas. This conclusion is based upon immunolocalization which demonstrates a temporal relationship with vitellogenin synthesis in the hepatopanocreas. Specifically, when the oocytes are most active in vitellogenin uptake, the hepatopanocreas is producing vitellogenins most abundantly. Vitellogenocytes are relatively large and polymorphic, similar to the reserve-inclusion cells that were described by others. Yolk protein was not detected in other cells of the hepatopancreas, male reserve-inclusion cells, or pre-vitellogenic oocytes by the same method of staining. Vitellogenocytes resemble cyanocytes, the source of hemocyanin. Whether the vitellogenocytes and their precursors are related to other populations of hepatocytes, such as cyanocytes, is not known and has not yet been studied.     

Molecular characterization and mRNA transcript profile of vitellogenin in Chinese shrimp, Fenneropenaeus chinensis

A full-length cDNA encoding vitellogenin (Vg) was cloned from Chinese shrimp, Fenneropenaeus chinensis using RACE method. The full-length cDNA consist of 7,942 nucleotides including a 7,761 bp open reading frame, which encodes 2,587 amino acid residues. The deduced amino acid sequence showed high (from 94% to 37%) identity with other known crustacean Vgs. In addition, a consensus cleavage site (R-X-K/R-R) recognized by an endopeptidase and a member of subtilisin family of serine protease were identified in the deduced Vg precursor. RT-PCR analysis shown that Vg mRNA can be detected in both ovary and hepatopancreas of vitellogenic females but not in other experimental tissues including muscle, heart, lymph organ, gill, haemocytes and intestine. These results suggest that the Vg gene may be expressed exclusively in mature females, and both ovary and hepatopancreas are the possible tissues for Vg synthesis in F. chinensis. In addition, Vg gene is detected in genomic DNA of both females and males.     

Site of vitellogenin synthesis determined from a cDNA encoding a vitellogenin fragment in the freshwater giant prawn, Macrobrachium rosenbergii.

Vitellogenesis is an important part of reproductive process in crustaceans, and the process is characterized by the synthesis and accumulation of yolk protein in the developing oocytes. The yolk proteins in crustaceans mainly consist of vitellogenin (Vg) and vitellin (Vn), which are respectively present in extra-oocyte tissues and intra-oocytes. The site and the process of yolk protein synthesis in crustaceans are still controversial. The synthesis site of Vg in a crustacean species, Macrobrachium rosenbergii, is determined by immunological and immunohistochemical techniques, and molecular cloning of a cDNA encoding the primary structure of Vn in this study. The hepatopancrease is clearly shown to be the synthesis site of Vg in this species. The length of Vg mRNA was estimated as about 6 kb from Northern blotting analysis. The partial primary structure of Vg gene is presented, and the post-translational processing are further discussed. For the first time, the partial primary structure of Vg gene and the synthesis site of Vg approached by molecular cloning in crustaceans are presented.     

Identification of the yolk receptor protein in oocytes of Nereis virens (Annelida,Polychaeta) and comparison with the locust vitellogenin receptor

Summary In oviparous animals large amounts of yolk proteins of extraovarian origin are accumulated by developing oocytes during vitellogenesis. The yolk protein precursors, the vitellogenins (VTG), are transported into the oocytes by receptor-mediated endocytosis. In oocytes of the polychaetous annelid, Nereis virens, the receptor protein for VTG was visualized by ligand blotting studies as a protein with an apparent molecular mass of 190 kDa under non-reducing conditions. Anti-Locusta VTG receptor antibodies recognize the Nereis VTG receptor protein. The Nereis VTG receptor protein binds Locusta and Schistocerca VTG; the VTG receptor proteins of both locust species bind the Nereis vitellin. These results indicate the conservation of structural elements important for internalization of VTG.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane-sulphonic acid - HBS HEPES-buffered saline - PAP peroxidase-anti-peroxidase - SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis - TRIS, TBS TRIS-buffered saline - VT vitellin - VTG vitellogenin     

Characterization of vitellogenin from white sturgeon,Acipenser transmontanus

Sturgeon are an ancient family (Acipenseridae) of fishes that lie close to the divergence of fish that eventually evolved into terrestrial animals and those that evolved into modern teleost species. Therefore, white sturgeon vitellogenin sequences fill a gap in the current understanding of the functional domains of this protein family. Vitellogenin cDNA was sequenced and used to investigate gene expression in white sturgeon, Acipenser transmontanus. Estrogen-induced vitellogenin mRNA was detected in the livers of both males and females and was also detected in undifferentiated gonads of estrogen-treated fish. Low levels of vitellogenin mRNA were also detected in the testis of both control and estrogen-treated males. The cDNA encoded a 186-kDa protein that was missing only six to seven of the amino-terminal amino acids. Comparisons to silver lamprey, Xenopus, and chicken vitellogenin sequences indicate that the overall structure of the yolk protein domains were highly conserved. There was considerable homology in three regions of the lipovitellin I domain. These conserved sequences are likely to be involved in vitellogenin receptor binding. The phosvitin domain of white sturgeon vitellogenin contained fewer and shorter serine repeats as predicted from yolk protein phosphate content of fish compared to Xenopus and chicken. However, the vitellogenin of white sturgeon had a lower serine content as compared with silver lamprey, indicating that an increased serine content is not strictly a function of evolutionary age. Correspondence to: C. A. Bidwell     

Molecular cloning and expression analysis of the interferon-γ-inducible lysosomal thiol reductase gene from the shrimp <Emphasis Type="Italic">Penaeus monodon</Emphasis>

The interferon-γ-inducible lysosomal thiol reductase enzymes (GILT) have been shown to play an important role in the processing of exogenous antigens by catalyzing disulfide bond reduction, that facilitates unfolding of the native protein antigen to simplify further cleavage by cellular proteases. In this study a Penaeus monodon GILT (PmGILT) gene was isolated from an EST library of white spot syndrome virus (WSSV)-infected P. monodon. The full-length cDNA of the PmGILT gene was 780 bp and contained an open reading frame of 657 bp that encoded 218 amino acid residues with a predicted protein molecular weight of 24 kDa. The deduced amino acid sequence of PmGILT contains an active site CXXS motif, a GILT signature sequence (CQHGX₂ECX₂NX₄C) and 10 conserved cysteines together with other signature characteristics of GILT proteins. RT-PCR analysis showed that the PmGILT mRNA expression level was clearly up-regulated in the lymphoid organ of both the LPS-induced and WSSV-infected shrimp, compared to normal shrimp. In response to WSSV infection, the penaeid shrimp JAK/STAT pathway is reported to play an important role in the lymphoid organ. We hypothesize that this activated STAT may stimulate GILT expression so that it can be involved in the shrimp immune response system.     

Dynamics of vitellogenin mRNA expression during vitellogenesis in the banana shrimp Penaeus (Fenneropenaeus) merguiensis using real-time PCR

An open reading frame (ORF) of vitellogenin (Vg) cDNA was amplified from the ovaries of the banana shrimp, Penaeus merguiensis. An examination of Vg-deduced amino acid sequence revealed the presence of cleavage sites at a consensus motif for subtilisin-like endoproteases prior to the N-terminal sequences of purified vitellin (Vt) subunits. A comparison of the primary structures of Vg molecules in decapod crustacean species revealed the existence of a common characteristic structure, and phylogenetic analysis reflected the current taxonomic classifications of crustaceans. A PCR product of 1.1 kb encoding the 3'-end of Vg cDNA was cloned from the hepatopancreas. Although its sequence was almost identical to that of the same region of the ovarian Vg, with only 18 nucleotide differences, analysis suggests that they have been subjected to natural selection, indicating that there may be two different, tissue-specific Vg genes in P. merguiensis. This is consistent with the different expression patterns of Vg mRNA, as determined by real-time PCR. Vg mRNA levels were maintained at low levels during the previtellogenic stage and they increased as vitellogenesis progressed to reach a peak at the early vitellogenic stage in the ovary or at the vitellogenic stage in the hepatopancreas, and thereafter, levels decreased. Expression of Vg mRNA was much higher in the ovary compared to the hepatopancreas at all stages of ovarian development, implying that the ovary is mainly responsible for Vt synthesis. These indicate that penaeids constitute a unique model for vitellogenesis, showing intraovarian gene expression and synthesis of yolk protein.     

From hepatopancreas to ovary: molecular characterization of a shrimp vitellogenin receptor involved in the processing of vitellogenin

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.     

Induction of Ovarian Maturation and Spawning in <Emphasis Type="Italic">Penaeus monodon</Emphasis> Broodstock by Double-Stranded RNA

Ovarian maturation in crustacean is under the control of gonad-inhibiting hormone (GIH); a neuropeptide secreted from X-organ sinus gland complex in eyestalks. Unilateral eyestalk ablation that partially destroys GIH source is therefore a general practice in Penaeus monodon hatchery to induce ovarian maturation and spawning. Our previous report showed that silencing of GIH expression by GIH-specific double-stranded RNA (GIH-dsRNA) resulted in an increased expression level of vitellogenin in P. monodon, thus suggesting that GIH-dsRNA could be an alternative method to induce ovarian maturation in female P. monodon broodstock. In this study, we further demonstrated that a single injection of GIH-dsRNA into previtellogenic female P. monodon at the concentration of 3 μg GIH-dsRNA per gram body weight of shrimp was able to inhibit GIH expression for a minimum of 30 days. This dsRNA-mediated GIH silencing led to ovarian maturation and eventual spawning in both domesticated and wild female broodstock, particularly with a comparable effect to eyestalk ablation in wild shrimp. This is the first report that demonstrates a potential strategy to induce ovarian maturation in female P. monodon broodstock by GIH-dsRNA and thus provides a possible substitute for the cruel and detrimental eyestalk ablation practice.     

日本沼虾卵黄蛋白原合成部位的初步研究

十足目卵巢中卵黄的来源,在过去的几十年研究中一直存在争议,内源性合成和外源性合成均有报道。以雌性日本沼虾为实验材料,根据外形观察和组织学研究确定其发育阶段,可以分为:卵原细胞增殖期;卵黄发生前期;初级卵黄发生期;次级卵黄发生期;成熟期和抱卵期(消退期)。从处于不同发育期的卵巢和肝胰腺中提取总RNA,用RT-PCR方法探讨不同发育期的日本沼虾卵巢和肝胰腺卵黄蛋白原mRNA表达,确定是否有卵黄蛋白原的合成功能。检测结果可以初步判定日本沼虾卵巢和肝胰腺都具有卵黄蛋白原mRNA表达功能,都是卵黄蛋白原的合成部位,其合成的量与沼虾卵巢的发育阶段相关。     

Molecular Characterization of a cDNA Encoding Vitellogenin and Its Expression in the Hepatopancreas and Ovary during Vitellogenesis in the Kuruma Prawn, Penaeus japonicus

In Crustacea, reproductive function and mechanisms regulating vitellogenesis have not been fully elucidated. This is due in great part to a lack of information concerning the biochemical nature of the vitellogenin molecule, the hemolymph precursor of yolk protein, vitellin, as well as the functional expression of the vitellogenin-encoding gene. We have therefore cloned a cDNA encoding vitellogenin in the kuruma prawn, Penaeus japonicus based on the N-terminal amino acid sequence of the 91 kDa subunit of vitellin. The open reading frame of this cDNA encoded 2,587 amino acid residues. This is the first investigation reporting a full-length cDNA and its corresponding amino acid sequence for vitellogenin in any crustacean species.Northern blot analysis and in situ hybridization have revealed that mRNA encoding vitellogenin was expressed in both the follicle cells in the ovary and the parenchymal cells in the hepatopancreas. In nonvitellogenic females, vitellogenin mRNA levels were negligible in both the ovary and hepatopancreas, but in vitellogenic females, levels were dramatically increased in both tissues. In the ovary, highest levels were observed during the early exogenous vitellogenic stage, and thereafter rapidly decreased, whereas in the hepatopancreas, high levels were maintained until the onset of the late vitellogenic stage. Differing profiles of vitellogenin mRNA levels in the ovary and hepatopancreas suggest that the contribution of these tissues to vitellogenin synthesis harbor separate and complementary roles during vitellogenesis.