SELCER, KW*; VERBANIK, JL; Duquesne Univ; Duquesne Univ: Vitellogenin in the Northern Leopard Frog (Rana pipiens): Purification, Characterization and Development of a Immunoassay

Vitellogenin is the egg-yolk precursor protein for all oviparous vertebrates. It is produced by the female liver under estrogen stimulation. Recently, vitellogenin has proven useful as a biomarker for endocrine disruption in a variety of fishes. The purpose of this study was to develop an immunoassay for vitellogenin induction in the Northern leopard frog, Rana pipiens for use as a tool to study endocrine disruption in this species. Sera from estrogen-treated frogs had significantly elevated total protein levels, primarily due to substantially increased amounts of a 160-180 kDa protein, presumably vitellogenin. The identity of the putative vitellogenin was confirmed by its cross-reactivity in Western blots with an antiserum generated against a Xenopus laevis vitellogenin peptide that was based on a conserved region of vertebrate vitellogenins. The vitellogenin was purified by DEAE chromatography and used to generate a polyclonal antibody in rabbits. A competitive enzyme-linked immunosorbent assay (ELISA) was developed for Rana pipiens vitellogenin, and the assay was used to characterize vitellogenin induction in frogs immersed in the potent estrogen diethylstilbestrol for various times and at different doses. Elevations in serum vitellogenin were detected within five days after initial immersion in one mg/L DES and vitellogenin levels continued to increase through 20 days. In a 20-day dose-response experiment, increases in serum vitellogenin were detected in frogs immersed in as little as 0.01 mg/L DES. Immersion in other weak xenobiotic estrogens for 20 days resulted in vitellogenin induction in some individuals but not others. Induction of vitellogenin in Rana pipiens may be a useful model system for field studies of environmental estrogens, especially given the paucity of information available on endocrine disruption in amphibians.