Characterization of vitellogenin genes in European sea bass (Dicentrarchus labrax)

ABSTRACTCHARACTERIZATION OF VITELLOGENIN GENES in EUROPEAN SEA BASS (Dicentrarchus labrax)Özlem YILMAZPhD in Fisheries SciencesSupervisor: Assoc. Prof. Dr. Sadi KÖKSOYMarch 2013, 136 pagesComplete cDNA sequences encoding three different vitellogenin genes were obtained for European sea bass (Dicentrarchus labrax). Two cDNAs encoding vitellogenins with a full suite of yolk protein domains were identified as sea bass VtgAa and VtgAb using the current nomenclature for multiple teleost vitellogenins. A shorter cDNA encoded an ?incomplete? vitellogenin lacking a phosvitin domain and having an abbreviated C-terminus that was identified as sea bass VtgC. Alignments of the deduced vitellogenin peptide sequences with those from other teleosts, and similar comparisons of primary vitellogenin structures, allowed definitive identification of sea bass VtgAa, VtgAb and VtgC. The existence of each form of vitellogenins in the blood plasma, and of their product lipovitellin-derived yolk proteins in oocytes or eggs, was verified via Western blotting performed using vitellogenin type-specific antisera raised against grey mullet (Mugil cephalus) lipovitellins, which also detected all three forms of vitellogenins in plasma from males injected with estradiol-17ß. Comparisons of ovary extracts from postvitellogenic and ovulated females via Western blotting revealed that the lipovitellins derived from all three forms of vitellogenins undergo partial proteolysis during oocyte maturation. The peptide abundance and the proportional ratio of the three vitellogenins or their yolk protein products present in liver, plasma and ovary was evaluated in postvitellogenic females just completing oocyte growth via label free quantitative mass spectrometry. Based on normalized spectral counts, VtgAa and VtgAb are the dominant types of vitellogenin in sea bass, with VtgAb spectra being from 2- to 5-fold more abundant than VtgAa spectra depending on the sample type. The VtgC spectra were generally very limited, except in ovary, where they were only 0.5- to 3.0-fold lower in abundance than for VtgAa and VtgAb, respectively. This first report on the multiple vitellogenin system of D. labrax shows that all three forms of vitellogenin described for acanthomorph teleosts are produced by this species and that each type of vitellogenin contributes significantly to the store of yolk proteins deposited in growing oocytes and eggs. These findings set the stage for functional studies of the molecular details of vitellogenesis underlying oogenesis and egg quality in this important aquaculture species.KEYWORDS: Sea bass Dicentrarchus labrax, Ovary, Oocyte, Teleost, Oogenesis, Vitellogenin, Yolk