Purification of two lipovitellins and development of immunoassays for two forms of their precursors (vitellogenins) in medaka (Oryzias latipes)

Two distinct yolk proteins (YP1 and YP2) were purified from the ovary of medaka, and specific antisera against YPs were generated to characterize YPs and reveal their relation to two vitellogenins (Vg1 and Vg2). The molecular masses of purified YP1 and YP2 on gel filtration were 270 and 380 kDa, respectively. YPs were confirmed to be lipoproteins by staining with Sudan black. Amino acid compositions of YP1 and YP2 were similar to those of Vg1 and Vg2, respectively. In double immunodiffusion using anti-Vg1, a precipitin line of YP1 formed a spur against the Vg1 line. YP2 and Vg2 were reacted with anti-Vg2, and a precipitin line of YP2 formed a fuse against the Vg2 line. These biochemical and immunological analyses of purified YPs revealed that YP1 is lipovitellin 1 (Lv1) derived from Vg1 and YP2 is lipovitellin 2 (Lv2) derived from Vg2. Using specific antibodies against Lvs and Vgs, specific, high sensitivity chemiluminescent immunoassays (CLIAs) for two Vgs were developed to reveal basal Vg levels and response to exogenous estradiol-17β (E2). The measurable range of both CLIAs was from 0.975 to 1000 ng/ml. The cross-reactivity to the alternative Vg in each CLIA was extremely low (≦0.57%). When immature fish were immersed in water containing E2 for 1 h, both Vgs were induced by 0.5 μg/L of E2 at 24 h after treatment. Vg1 increased in a concentration-dependant manner up to 100 μg/L E2, while Vg2 reached a plateau at 10 μg/L of E2. The ratio of Vg1:Vg2 in E2-treated fish changed in a concentration-dependent manner from 1.5:1 to 8.5:1. The results from E2-treatment suggest that differential regulation may control the expression of medaka Vgs.