SELCER, K.W.*; DAY, A.M.; VERBANIC, J.D.: Northern Leopard Frog (Rana pipiens) Vitellogenin: Purification, Development of an Immunoassay, and Characterization of Induction Following Immersion in Diethylstilbestrol

In oviparous vertebrates, estrogen stimulates the female liver to produce the egg yolk precursor, vitellogenin. This protein circulates in the blood, is taken up by oocytes, and is cleaved into egg-yolk proteins. Vitellogenin is now being used as a biomarker for exposure of animals to environmental estrogens. Thus, there is a need for assays capable of measuring vitellogenin induction in various species. The purpose of this study was to characterize vitellogenin induction in adult male Rana pipiens after injection with estradiol-17BETA and after immersion in the potent estrogenic compound diethylstilbestrol (DES). Sera from estrogen-treated frogs had significantly elevated total protein levels, compared to control frogs. Denaturing gel electrophoresis revealed increased amounts of a large (160 kDa) protein, presumably vitellogenin. The identity of the putative vitellogenin was confirmed by its cross-reactivity in Western blots with an antibody generated against a Xenopus laevis vitellogenin peptide and by its elution from DEAE-chromatography under high-salt conditions, which is typical of vertebrate vitellogenins. An anti-Rana pipiens vitellogenin antibody was also generated from the purified vitellogenin. An enzyme-linked immunosorbent assay was developed for Rana pipiens vitellogenin and the assay was used to characterize vitellogenin induction in frogs immersed in DES for various times and at different doses. Elevations in serum and hepatic cytosol vitellogenin were detected within five days after initial immersion in 1 mg/L DES and vitellogenin levels continued to increase through 20 days. Induction of vitellogenin in Rana pipiens may be a useful model system for field studies of environmental estrogens.