Characterization of vitellogenin in the shrimp Metapenaeus ensis: expression studies and hormonal regulation of MeVg1 transcription in vitro.

During gonad maturation, female shrimp accumulate the major egg yolk protein vitellin (Vn) in premolt stage, and the process of molting and reproduction is synchronized. Using a polyclonal anti-Vn antibody, immunopositive signals could be detected in the ovary and among the proteins secreted by the hepatopancreas by Western blot. In the ovary, Vn immunoreactivity was located in the posterior lobe. Hepatopancreas proteins with sizes identical to ovary vitellogenin (Vg) subunits (i.e., 78 and 157 kDa) were immunoreactive to the Vn antibody and these proteins included amino acid sequences identical to parts of the MeVg1 precursor. A major 7.8 kb MeVg1 transcript, was detected in the ovary. In the hepatopancreas, the transcripts were primarily small (<2.3 kb) and while the 7.8 kb transcript which constitutes <50% of the total Vg mRNA. MeVg1 transcript could be detected in the hepatopancreas of juvenile females with a maximum level during late intermolt and early premolt. To study the effect of different hormones on expression of MeVg1, explant cultures of hepatopancreas and ovary were developed. Although several hormones (20-hydroxyecdysone, estradiol (ES), farnesoic acid (FA), juvenile hormone (JH) III, methyl farnesoate, and progesterone (PG)) apparently stimulated MeVg1 gene expression, only FA consistently stimulated MeVg1 expression by the hepatopancreas explants, while both FA and 20-hydroxyecdysone were stimulated ovarian explants. In summary, (i) Vg transcripts can be detected in both reproductive and nonreproductive females; (ii) the presence of large quantities of smaller Vg transcripts and the absence of a large Vg precursor from the hepatopancreas suggests that smaller MeVg1 transcripts provide an important contribution to Vg synthesis in shrimp. Our results suggest that there is differential processing of the MeVg1 precursor in the ovary and hepatopancreas of shrimp.

Tiu SH ，Hui JH ，He JG ，Tobe SS ，Chan SM ... -  《MOLECULAR REPRODUCTION AND DEVELOPMENT》

Vitellogenesis in the sand shrimp, metapenaeus ensis: the contribution from the hepatopancreas-specific vitellogenin gene (MeVg2).

Kung SY ，Chan SM ，Hui JH ，Tsang WS ，Mak A ，He JG ... -  《BIOLOGY OF REPRODUCTION》

Vitellogenesis in the red crab Charybdis feriatus: Hepatopancreas-specific expression and farnesoic acid stimulation of vitellogenin gene expression.

Vitellogenesis in the mature female crab Charybdis feriatus occurs all year round during which active synthesis of the vitellogenin (Vg) precursor occurs. Several polypeptides from the ovaries were shown to be immuno-reactive to the shrimp vitellin (Vn) antibody. N-terminal amino acid sequence determination revealed that several ovarian polypeptides and one polypeptide secreted by the hepatopancreas were identical to part of the C. feriatus Vg (CfVg) precursor. The full-length cDNA sequence encoding a protein with high amino acid sequence similarity to the Vg of the shrimp Metapenaeus ensis was cloned. In common with the shrimp M. ensis MeVg2, the crab vitellogenin gene is expressed only in the hepatopancreas. The expression level of CfVg is undetectable in the non-reproductive females, increases to maximum at the middle stages of vitellogenesis and drops to a lower level in late vitellogenesis. Expression of CfVg also extended to females that are undergoing brooding of developing larvae. Although the 8 kb transcript for the full-length cDNA was detected, smaller transcripts specific to CfVg mRNA were also detected, suggesting the occurrence of alternative splicing/expression of the CgVg gene to produce the smaller transcripts. Using a short term in vitro hepatopancreas explant culture assay, we have demonstrated that low concentrations of farnesoic acid (FA) stimulate CfVg gene expression in the hepatopancreas. Although both methyl farnesoate (MF) and juvenile hormone III also caused up-regulation of the CfVg gene, their effects are only significant at much higher concentrations.

Mak AS ，Choi CL ，Tiu SH ，Hui JH ，He JG ，Tobe SS ，Chan SM ... -  《MOLECULAR REPRODUCTION AND DEVELOPMENT》

Organization of the shrimp vitellogenin gene: evidence of multiple genes and tissue specific expression by the ovary and hepatopancreas.

Tsang WS ，Quackenbush LS ，Chow BK ，Tiu SH ，He JG ，Chan SM ... -  《GENE》

Molecular characterization of a cDNA encoding vitellogenin in the banana shrimp, Penaeus (Litopenaeus) merguiensis and sites of vitellogenin mRNA expression.

In order to determine the primary structure of banana shrimp, Penaeus merguiensis, vitellogenin (Vg), we previously purified vitellin (Vt) from the ovaries of vitellogenic females, and chemically analyzed the N-terminal amino acid sequence of its 78 kDa subunit. In this study, a cDNA from this species encoding Vg was cloned based on the N-terminal amino acid sequence of the major 78 kDa subunit of Vt and conserved sequences of Vg/Vt from other crustacean species. The complete nucleotide sequence of Vg cDNA was achieved by RT-PCR and 5' and 3' rapid amplification of cDNA ends (RACE) approaches. The full-length Vg cDNA consisted of 7,961 nucleotides. The open reading frame of this cDNA encoding a precursor peptide was comprised of 2,586 amino acid residues, with a putative processing site, R-X-K/R-R, recognized by subtilisin-like endoproteases. The deduced amino acid sequence was obtained from the Vg cDNA and its amino acid composition showed a high similarity to that of purified Vt. The deduced primary structure, of P. merguiensis Vg was 91.4% identical to the Vg of Penaeus semisulcatus and was also related to the Vg sequences of six other crustacean species with identities that ranged from 86.9% to 36.6%. In addition, the amino acid sequences corresponding to the signal peptide, N-terminal region and C-terminal region of P. merguiensis Vg were almost identical to the same sequences of the seven other reported crustacean species. Results from RT-PCR analysis showed that Vg mRNA expression was present in both the ovary and hepatopancreas of vitellogenic females but was not detected in other tissues including muscle, heart, and intestine of females or in the hepatopancreas of mature males. These results indicate that the Vg gene may be expressed only by mature P. merguiensis females and that both the ovary and hepatopancreas are possible sites for Vg synthesis in this species of shrimp.

Phiriyangkul P ，Utarabhand P 《MOLECULAR REPRODUCTION AND DEVELOPMENT》