SELCER, Kyle W.; NESPOLI, Lisa; MANGUS, Lisa; Duquesne University: Estrogen Induction of Vitellogenin Protein and mRNA in Tiger Salamanders

In oviparous vertebrates, estrogen stimulates the liver to produce the egg-yolk precursor protein vitellogenin. Knowledge of serum vitellogenin levels is valuable in studies of vertebrate reproductive cycles; furthermore, vitellogenin induction is now widely used as a biomarker for exposure of animals to environmental estrogens. Vitellogenin bioassays have been developed for numerous fish species but not for many amphibians, particularly salamanders. In the present study, we provide information on vitellogenin induction by estrogens in the tiger salamander (Ambystoma tigrinum). Adult male tiger salamanders were injected i.p. with ethinyl estradiol and blood was collected at weekly intervals. Male salamanders exposed to estrogen produced substantial quantities of a 200 kDa protein, the approximate size of most vertebrate vitellogenins. The estrogen-induced protein was purified using DEAE chromatography and subjected to N-terminal amino acid analysis. The sequence obtained showed substantial homology to known vertebrate vitellogenins. Antibodies raised against a peptide representing the A. tigrinum vitellogenin N-terminus crossreacted in ELISA assays with sera from estrogen-treated salamanders but not from control animals. RT-PCR was used to evaluate vitellogenin mRNA in livers from estrogen-treated and untreated male A. tigrinum. Degenerate vitellogenin primers were designed from known sequences of amphibian and fish species. The cDNA obtained from estrogen-treated males was sequenced and found to have strong homology to other vertebrate vitellogenins. New primers, based on the A. tigrinum sequence, are currently being tested in real-time RT-PCR. Vitellogenin assays for tiger salamander could be used to study annual reproductive cycles, to test waters for the presence of estrogenic agents or to assess exposure of wild populations to environmental estrogens.