Characterization of heat shock protein 90 in the shrimp Metapenaeus ensis: Evidence for its role in the regulation of vitellogenin synthesis.

Estrogen hormones play a vital role in the regulation of female reproductive maturation. In oviparous vertebrates, the synthesis of vitellogenin (VTG) is tightly controlled by estrogen hormone signal transduction pathway, which is mediated by estrogen receptor and heat shock protein 90 (Hsp90). In order to investigate whether a similar mechanism exists in crustaceans, the Hsp90 gene was cloned and isolated from the shrimp Metapenaeus ensis by homology cloning strategy. The Hsp90 is 2,524 bp in length, containing an open reading frame of 2,163 bp that encodes a 720 amino acid polypeptide (83 kD). The Hsp90-coding region is interrupted by four introns. MeHsp90 is differentially expressed in eyestalk, ovary, and hepatopancreas at different ovarian maturation stages, and consistently expressed in other tissues including heart, gill, gut, muscle, and central nervous system. In vitro ovary explant assay reveals that MeHsp90 expression in immature ovary can be induced by the addition of exogenous estradiol-17beta, but expression in fully mature ovary exhibits no response to estradiol-17beta treatment. In situ hybridization shows that MeHsp90 is highly expressed in previtellogenic oocytes and its expression decreases with the progress of maturation, and finally stops in late-vitellogenic oocytes. Our results indicate a strong correlation between estrogen hormones and Hsp90 expression in shrimp, suggesting that the expression of VTG may be under the regulation of estrogen hormones through a mechanism similar to that in vertebrates. The result provides insights on the control of vitellogenesis in invertebrates.

Wu LT ，Chu KH 《-》

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》

[Molecular identification and expression of heat shock cognate 70 (HSC70) in the Pacific white shrimp Litopenaeus vannamei].

Wu R ，Sun L ，Lei M ，Xie ST ... -  《-》

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》