Comparative proteomic profiling during ovarian development of the shrimp Metapenaeus ensis.

Two-dimensional electrophoresis and mass spectrometry were used to identify proteins that are differentially expressed during ovarian maturation in Metapenaeus ensis. 87 spots with consistently significant quantitative differences (≥ 1.5-fold for vol%) among stage I, III and V ovaries were chosen for MS/MS analysis. 45 spots were significantly matched to known proteins in the database (Mascot score >40). Half of them were down-regulated, in contrast to 9 out of 45 proteins that were up-regulated as ovarian maturation proceeded. Functionally, these identified proteins could be classified into five major groups, including cytoskeleton (11 %), metabolism (18 %), signal transduction (32 %), gene expression (14 %) and immune response (7 %). Among the differentially expressed reproduction-related proteins, the mRNA expression level of cellular retinoic acid/retinol binding protein in M. ensis (MeCRABP) during ovarian maturation was further characterized by quantitative real-time PCR. It was down-regulated during ovarian maturation. In situ hybridization further revealed that MeCRABP transcript was localized in ooplasm of previtellogenic oocytes but not in vitellogenic oocytes. These results demonstrate the application of proteomic analysis for identification of proteins involved in shrimp ovarian maturation and they provide new insights into ovarian development.

Origin and evolution of yolk proteins: expansion and functional diversification of large lipid transfer protein superfamily.

Vitellogenin (VTG) and apolipoprotein (APO) play a central role in animal reproduction and lipid circulation, respectively. Although previous studies have examined the structural and functional relationships of these large lipid transfer proteins (LLTPs) from an evolutionary perspective, the mechanism in generating these different families have not been addressed in invertebrates. In this study, the most comprehensive phylogenetic and genomic analysis of the LLTP superfamily genes is carried out. We propose the expansion and diversification of LLTPs in invertebrates are mediated via retrotransposon-mediated duplications, followed by either subfunctionalization or neofunctionalization in different lineages. In agreement with a previous hypothesis, our analysis suggests that all LLTPs originate from a series of duplications of a primitive yolk protein gene similar to VTG. Two early consecutive duplications of the yolk protein genes resulted in the formation of microsomal triglyceride transfer protein (MTP) and the APO gene ancestor. Gains and losses of domains and genes occurred in each of these families in different animal lineages, with MTP becoming truncated. MTP maintained only the components stabilizing the huge lipoprotein particle. Surprisingly, for the first time, two VTG-like protein families were found to independently arise in the lineages of insects. This work consolidates the reconstruction of the evolutionary roadmap of the LLTP superfamily and provides the first mechanistic explanation on the expansion of family members via retrotransposition in invertebrates.

Characterization of an ovary-specific glutathione peroxidase from the shrimp Metapenaeus ensis and its role in crustacean reproduction.

In vertebrates, both reactive oxygen species (ROS) and the corresponding scavenging system components especially glutathione peroxidase (GPx) are indispensible for normal development of the gonads. To investigate the function of GPx in crustaceans, we cloned and characterized a full length GPx (MeGPx) transcript in the penaeid shrimp Metapenaeus ensis. Phylogenetic analysis showed that MeGPx clustered with the GPx from mollusks and nematodes and shared much higher similarity with vertebrate GPx3 and GPx5 than with GPx1 or GPx2. Multiple sequence alignment further demonstrated that MeGPx is evolutionarily conserved among invertebrates, with common functionally important motifs. MeGPx was specifically expressed in shrimp ovaries, but not in other tissues studied, including testis. In situ hybridization showed that MeGPx was highly expressed in pre-vitellogenic and mid-vitellogenic oocytes, while no expression was detected in late-vitellogenic oocytes. Moreover, real time PCR showed high level expression of MeGPx in the early ovaries. Since active protein synthesis and deposition occurred in mid-vitellogenic oocytes, MeGPx might play a pivotal role in preventing oocytes from oxidative damage and balancing ROS production. The present findings on shrimp GPx provide insights on the regulation of ROS in the ovarian maturation process and the role of GPx in crustacean reproductive biology.

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.

[Cloning and expression of tumor necrosis factor (TNFalpha) cDNA from red seabream pagrus major].

A fragment of TNFalpha cDNA sequence from red seabream was cloned by homology cloning approach with two degenerated primers which were designed based on the conserved regions of other animals' TNF sequences. The sequence was elongated by 3' and 5' RACE to get the full length CDS sequence. This sequence contained 1264 nucleotides that included a 5' UTR of 85 bp, a 3' UTR of 514 bp and an open reading frame (ORF) of 666 bp which could encode 222 amino acids propeptide. In 3' UTR, there were several mRNA instability motifs and three endotoxin-responsive sequences, but the sequence lacked the polyadenylation signal. The deduced peptide had a clear transmembrane domain, a TNFalpha family signature and a TNF2 family profile. The cell attachment sequence and the glycosaminoglycan attachment sites were also found in the sequence. The red seabream TNF sequence shared relatively high similarity with both mammalian TNFalpha and TNFbeta by multiple sequence alignments. Phylogenetic analysis showed that the piscine TNFalpha were located independently in a different branch compared with mammalian TNFalpha and TNFbeta. Based on the primary and secondary structure analysis and gene expression study, we could concluded that the red seabream TNF should be a TNFalpha, not TNFbeta. RT-PCR was used to study TNFalpha transcript expression. 24 h after the red seabream was challenged by Vibrio anguillarum, the RS TNFalpha transcript expression were detected in blood, brain, gill, heart, head kidney, kidney, liver, muscle and spleen. Results showed that TNFalpha mRNA was constitutively expressed in parts of the tissues both in stimulated and unstimulated fish and the expression could be enhanced after the pathogen infection.