Dynamics of vitellogenin mRNA expression during vitellogenesis in the banana shrimp Penaeus (Fenneropenaeus) merguiensis using real-time PCR (original) (raw)
Related papers
Journal of Crustacean Biology, 2013
A complete cDNA sequence of vitellogenin (Vg-H) was cloned from the hepatopancreas of Fenneropenaeus merguiensis (De Man, 1888). The full-length Vg-H gene consists of 7958 bp and contains an ORF of 7761 bp. It encodes a polypeptide of 2587 amino acids, comprising a predicted molecular mass of 283 kD and the theoretical pI is 6.5. The amino acid sequences of the mature vitellogenin (Vg) composed of a signal peptide, three possible O-glycosylation sites, three phosphorylation sites and putative possessing sites (R-X-K/R-R) recognized by subtilisin-like endoproteases. Cleavage at residues 725 to 728 will produce two Vg subunits (78 and 200 kD), which the N-terminal amino acid sequence of 78 kD subunit is identical to that of vitellin purified from the ovary. These properties suggest that Vg protein encoding by Vg-H undergoes processing at its synthetic sites prior to being transported to developing oöcytes. The deduced primary structure of Vg-H showed the highest identity (98.8%) to the Vg-O previously cloned from the ovary of the same species. It is more related to the penaeid Vg sequences than that of nonpenaeids. In situ hybridization revealed that mRNA encoding Vg was expressed both in follicle cells in the ovary and parenchyma cells in the hepatopancreas. In the developing ovary, highest levels were detected during the early vitellogenic stage 2, declining thereafter. In the hepatopancreas, Vg mRNA levels reached a maximum at stage 3 of ovarian development. Profiles of Vg mRNA expression in the ovary and hepatopancreas suggest that the contribution of these tissues to vitellogenesis is inversely related during gonad maturation. Thus, Vg gene sequences expressed in the ovary and hepatopancreas are most likely identical and both tissues are responded for yolk protein synthesis in F. merguiensis.
Zoological Science, 2000
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.
Aquaculture, 2006
Although the gene fragment and partial cDNA for the vitellogenin (Vg) of Penaeus monodon have been reported for several years, the complete gene sequence and full-length cDNA of this Vg has not been reported. Here we report the gene organization and cloning of the full-length cDNA of P. monodon Vg. Similar to the MeVg1 of M. ensis, the Vg gene of P. monodon (PmVg1) is confirmed to consist of 14 introns and 15 exons. Additionally, genomic DNAs that show high sequence identity to PmVg1 were isolated from a single shrimp. The result confirmed the existence of multiple Vg genes in P. monodon. PmVg cDNA is 7.8 kb in size and the deduced precursor is most similar to the Vg of Penaeus merguenesis (86% identity). Contrary to the previous report, similar levels of PmVg1 transcripts can be detected from the hepatopancreas and ovary suggesting that the ovary also plays a major role in the contribution of PmVg1 transcript during vitellogenesis. In shrimp at early vitellogenesis (i.e., GSI b 3%), the expression of PmVg1 in the ovary and hepatopancreas is low; the expression reached the maximum level in shrimp with GSI 4-8% and decreased in shrimp with GSI N 9%. Farnesoic acid and 20-hydroecdysone can cause a significant stimulation of PmVg1 expression in an in vitro hepatopancreas explant culture. In conclusion, vitellogenesis in P. monodon is most likely the result of the expression of multiple vitellogenin genes and the hepatopancreas and ovary contribute equally to the production of PmVg1 transcripts.
Gene, 2003
Vitellogenin is the major egg yolk protein synthesized in female shrimp during gonad maturation. Although there are several reports for the cloning of vitellogenin complementary DNA (cDNA) in different crustaceans, little is known of the gene organization of this protein. This study reports the first cloning and characterization of a full-length gene encoding the vitellogenin precursor from the shrimp Metapenaeus ensis. By genomic DNA library screening, six different lambda clones were isolated using shrimp partial gene sequence as probe. Initial DNA sequence determination revealed that these clones are derived from different genes with coding sequence similar to other crustacean vitellogenins. Two of these clones were used for further analysis. One of the lambda clones (l3.3) carries most of the coding sequence that correspond to the M. ensis vitellogenin gene (MeVg1) and the other clone (l8.3) carries a smaller portion of the coding sequence of a different vitellogenin gene (MeVg2). The l3.3 clone was chosen for further characterization. To clone the remaining 5 0 end upstream promoter region, 5 0 untranslated region and the remaining coding sequence of MeVg1, a polymerase chain reaction (PCR)-based gene walking approach was used. Subsequently, a PCR clone with overlapping sequence identical to the genomic clone was obtained and the organization of MeVg1 gene was constructed. The MeVg1 gene consists of 15 exons and 14 introns spanning approximately 10 kb. Several potential cleavage sites were identified from the deduced vitellogenin precursor. Cleaving of the precursor in these sites would result in the production of several vitellogenin subunits. To clone the cDNA for the vitellogenin, 5 0 and 3 0 rapid amplification of cDNA ends was performed using ovary cDNA of the shrimp. A 4.4 kb 5 0 cDNA clone and a 4 kb 3 0 end cDNA clone were isolated. The size of the reconstructed cDNA for M. ensis Vg is 7.97 kb and consists of the longest open reading frame of 7776 bp. Unlike the vitellogenin precursor of most insects and vertebrates, the deduced vitellogenin precursor lacks the polyserine domain important for receptor-mediated endocytosis. Phylogenetic analysis revealed a closer relationship of the MeVg1 with other crustacean vitellogenins but distantly related to other invertebrate and vertebrate vitellogenins. By reverse transcription-PCR, we have demonstrated that the shrimp MeVg1 gene is expressed only in the ovary and hepatopancreas while the MeVg2 gene is expressed exclusively in the hepatopancreas. In conclusion, the shrimp ovary also contribute significantly in the production of vitellogenin at transcription level and the gene organization of the shrimp protein may provide an insight in the evolution of this group of important proteins. q
Biology of …, 2004
An additional vitellogenin gene (MeVg2) that is structurally different from MeVg1 was cloned and characterized from the shrimp Metapenaeus ensis. The MeVg2 gene consists of fewer exons-introns and is most likely evolved from the MeVg1 gene. The cDNA for MeVg2 is 8.0 kilobases (kb) in size, and the deduced MeVg2 precursor shared an overall 54% amino sequence identity to the MeVg1 gene of the same shrimp. As compared to the MeVg1 precursor, MeVg2 precursor consists of more potential subunit cleavage sites, suggesting that the precursor may be processed into many smaller subunits. The MeVg2 is expressed only in the hepatopancreas, and the expression level of MeVg2 in adult female increases from the early vitellogenic stage, reaching a maximum at the middle vitellogenic stage, and remains high toward the end of vitellogenic cycle. In addition to the 8-kb mRNA, smaller transcripts of 1.5-2.5 kb for MeVg2 were identified, and the 8-kb transcript only constitutes less than 10% of the overall MeVg2-derived transcripts. To confirm the presence of the small transcripts, we screened the shrimp hepatopancreas cDNA library and isolated two smaller MeVg2specific cDNA clones. These clones shared greater than 99% overall identity to the corresponding C-terminal region of the MeVg2 precursor, suggesting that an alternative expression/ splicing of the MeVg2 gene occurred. By immunohistochemical analysis, vitellin-immunopositive signals were localized in the lumen and extracellular fraction of the hepatopancreas. Amino acid sequence determination of the tissue protein and secreted protein from the hepatopancreas revealed that the 76-kDa vitellogenin subunit is most likely processed into smaller-sized subunits. Taken together, these results suggest that the hepatopancreas is an important organ for the synthesis of vitellogenin and may contribute to vitellogenesis by producing a large quantity of smaller MeVg2 subunit for ovarian uptake.
Crustacean Vitellogenesis: Its Role in Oocyte Development1
American Zoologist, 2001
SYNOPSIS. One of the major changes that occurs during the maturation of oocytes is the accumulation of yolk protein, or vitellin (Vn). To better understand how this process is regulated, we characterized the Vn of the ridgeback shrimp, Sicyonia ingentis (Penaeoidea). This Vn is a 322 kDa molecule composed of three subunits. Using purified Vn, we developed an anti-Vn antiserum and used it to characterize vitellogenin by Western blot analysis. The antiserum was also used in an ELISA to measure hemolymph levels of vitellogenin. Previous studies suggested the presence of vertebrate-type steroids might stimulate reproductive processes in decapod crustaceans. Treatment of sexually quiescent female shrimp with progesterone, hydroxyprogesterone, and estradiol did not increase hemolymph levels of yolk protein precursor. The absence of a response to 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 screened using the anti-Vn antiserum. A 2.9 kilobase clone was isolated from both cDNA libraries suggesting that both tissues are sites of vitellogenin synthesis. These molecular tools should be useful for in vitro studies of vitellogenin synthesis.
Crustacean vitellogenesis: its role in oocyte development
American Zoologist, 2001
SYNOPSIS. One of the major changes that occurs during the maturation of oocytes is the accumulation of yolk protein, or vitellin (Vn). To better understand how this process is regulated, we characterized the Vn of the ridgeback shrimp, Sicyonia ingentis (Penaeoidea). This Vn is a 322 kDa molecule composed of three subunits. Using purified Vn, we developed an anti-Vn antiserum and used it to characterize vitellogenin by Western blot analysis. The antiserum was also used in an ELISA to measure hemolymph levels of vitellogenin. Previous studies suggested the presence of vertebrate-type steroids might stimulate reproductive processes in decapod crustaceans. Treatment of sexually quiescent female shrimp with progesterone, hydroxyprogesterone, and estradiol did not increase hemolymph levels of yolk protein precursor. The absence of a response to 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 screened using the anti-Vn antiserum. A 2.9 kilobase clone was isolated from both cDNA libraries suggesting that both tissues are sites of vitellogenin synthesis. These molecular tools should be useful for in vitro studies of vitellogenin synthesis.
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2001
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. ᮊ
Is There Extraovarian Synthesis of Vitellogenin in Penaeid Shrimp?
The Biological Bulletin, 1992
Extraovarian synthesis of vitellogenin (Vg), has been reported for several crustaceans, mainly in the subepidermal adipose tissue (SAT) or the hepatopancreas (HEP). The precise site(s) of Vg synthesis in penaeid shrimp is hitherto unknown and was investigated in a large local species Pcnaeus semisulcalus de Haan. Protein synthesis was determined in SAT and HEP tissue pieces incubated //; vitro. Incubations were at 25 C for eight hours in an oxygen enriched atmosphere, under sterile conditions in a physiological medium, containing 14 Cleucine. At the end of the incubation period, tissue homogenates and medium samples were analyzed for de novo protein synthesis. Total protein synthesis was determined by trichloroacetic acid precipitation. Specific vitellin (Vt) synthesis was determined by radioimmunoprecipitation with a polyclonal Vt-specific antiserum. Characterization of other de novo synthesized proteins was carried out by fluorography from polyacrylamide gels. Subepidermal adipose tissues removed from females at all stages of ovarian development did not synthesize Vtspecific proteins, in spite of the fact that total protein synthesis levels were high. The major protein synthesized de novo in the SAT of males and females is a protein with an identical electrophoretic mobility as hemocyanin in polyacrylamide gels. In vitro protein synthesis in HEP tissues was low compared to SAT or ovary systems. Vtspecific de novo synthesized protein was identified in HEP's from early vitellogenic females, but constituted less than 15% of total protein synthesis. We have previously shown that ovarian tissues from vitellogenic females incubated in vitro exhibited high levels of protein synthesis, an average of 38% of which is Vt-specific (Browdy et ai. 1990, J. Exp. Zoo/. 255: 205-2 1 5). The calculated Vt syn
Biological Bulletin, 1992
The concentration of vitellogenin (Vg) in the hemolymph of Penaeus semisulcatus was found to increase from an average of 50 pg ml-' to 439 pg ml-' in female shrimp during ovarian development. The most significant increase in Vg occurred concomitant with the increase in the vitellin (Vt) content of oocytes with an average diameter (AOD) ranging between 150 and 250 pm. The amount of Vt in the oocytes was found to increase linearly from a mean of 0.0 126 mg to 4.55 mg per gm body weight. However, the percentage of Vt in the total protein was found to decrease, from 67% in ovaries with AOD of 150-250 pm, to 39.7% in ovaries with AOD of 350 pm or larger. The volume of the hemolymph was found to be 0.4 ml per gm body weight and did not change significantly during ovarian development. Assuming that Vg in the hemolymph represents either an extraovarian origin of Vt or an active secretion from the ovary, a turnover rate of two to three times per day was calculated over one full cycle of oocyte development. However, during the most significant increase in Vt in the ovary (in ovaries with AOD of 150-250 pm), the turnover rate in the hemolymph could reach seven to eight times per day. The results lead to the conclusion that the contribution of Vg to the formation of Vt in the ovary is quantitatively insignificant.