MANGUS, Lisa; NESPOLI, Lisa; HUANG, Yue-Wern; SELCER, Kyle W; Duquesne University; Duquesne University; University of Missouri, Rolla; Duquesne University: Isolation and Characterization of Vitellogenin in Two North American Salamanders, Ambystoma tigrinum and Cryptobranchus alleganiensis

Vitellogenin is the egg yolk precursor protein produced by the liver of female oviparous vertebrates under estrogen stimulation. Previous work has shown that vitellogenin can be used as a biomarker for exposure of animals to environmental estrogens, which are chemical agents capable of disrupting endocrine pathways. These compounds may have deleterious effects on humans and wildlife. Our lab has been developing assays to detect the vitellogenin protein and hepatic mRNA in various aquatic vertebrates after stimulation by estrogen and estrogenic agents. In this study, we have successfully isolated and characterized the vitellogenins of two North American salamanders, the tiger salamander (Ambystoma tigrinum), and the hellbender (Cryptobranchus alleganiensis). The protein was purified from serum of estrogen-treated females using DEAE chromatography and SDS-PAGE, followed by N-terminal amino acid sequencing. The resulting sequences showed significant homology with the known vitellogenin sequence of Xenopus laevis (African clawed frog). An RT-PCR procedure was conducted on RNA extracted from livers of female and estrogen-treated male salamanders using degenerate primers designed from known vitellogenin cDNA sequences of various amphibian and fish species. The cDNAs obtained from these reactions was sequenced and new species-specific primers were developed. Sequencing of cDNAs revealed strong homology to other vertebrate vitellogenins. Based on the protein and cDNA sequences found in this study, we are developing specific salamander vitellogenin antibodies and PCR primers that may be useful in field studies of endocrine disruption in salamander populations.