Characterization and processing of superoxide dismutase-fused vitellogenin in the diapause embryo formation: a special developmental pathway in the brine shrimp, Artemia parthenogenetica.

To withstand environmental stress, Artemia release diapause cysts via an oviparous pathway instead of producing swimming nauplius larvae by the ovoviviparous pathway. Encased in such a cyst, the embryos at diapause can survive for many years. Vitellogenin (Vtg), the precursor of vitellins, the main yolk proteins, is crucial for embryonic development. This study compares vitellogenesis between oviparity and ovoviviparity, the two reproductive modes occurring in A. parthenogenetica. A Vtg gene was cloned, based on N-terminal amino acid sequence analysis, PCR amplification, and cDNA library construction and screening, and was found to consist of 6778 bp with a 6657 bp open reading frame encoding 2219 amino acids. From the deduced primary structure, Artemia vitellogenin (ArVtg) was found to possess six copies of the consensus cleavage site, R-X-X-R, and to contain a superoxide dismutase (SOD)-like domain at the N-terminus. This is an unusual finding for crustacean Vtg proteins, having been reported only in one previous crustacean, Daphnia magna. Using Northern blot analysis and in situ hybridization, ArVtg gene expression was observed at early stages of vitellogenesis in the connective tissue located in the cephalothorax, with trace expression in the ovary. Western blot analysis and several N-terminal sequences revealed that ArVtg was cleaved at each consensus cleavage site and that more than 10 subunits were formed during posttranslational processing in ovarian maturation. Of these, only the SOD-containing subunits (∼90 and 60 kDa) showed different profiles between the oviparous and ovoviviparous pathways. This suggests that these high concentration components have an important function for the encysted diapaused embryos during long-term cell-cycle arrest, which has remained unknown up until now.