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Molecular Cloning of the Estrogen and Progesterone Receptors of the American Alligator

Center for Integrative Bioscience, National Institute for Basic Biology, Okazaki National Research Institutes, Okazaki, Japan (Y.K., S.K. & T.I.),  Department of Zoology, 223 Bartram Hall, University of Florida, Gainesville, FL 32611, USA (D.S.B., E.L.B., M.P.G, T.A.B. & L.J.G.),  Department of Biochemistry and Microbiology, University of Victoria, Victoria, Canada (C.H.),  Department of Molecular Biomechanics, School of Life Sciences, Graduate University of Advanced Studies, Okazaki, Japan (S.M. & T.I.),  and CREST, Japan Science and Technology Corporation, Kawasaki, Japan (T.I.)

Steroid hormones perform many essential roles in vertebrates during embryonic development, reproduction, growth, water balance and responses to stress. The estrogens are essential for normal reproductive activity in female and male vertebrates and appear to have direct actions during sex determination in some vertebrates. To begin to understand the molecular mechanisms of estrogen action in alligators, we have isolated cDNAs encoding the estrogen receptors (ER) from the ovary. Degenerate PCR primers specific to ER were designed and used to amplify alligator ovary RNA. Two different DNA fragments (ERα and ERβ) were obtained and the full-length alligator ERα cDNA was obtained using 5' and 3' RACE. The inferred amino acid sequence of alligator ERα (aERα) was very similar to the chicken ERα (91 % identity), although phylogenetic analyses suggested profound differences in the rate of sequence evolution for vertebrate ER sequences. We also isolated partial DNA fragments encoding ERβ and the progesterone receptor (PR) of the alligator, both of which show strong sequence similarities to avian ERβ and PR. We examined the expression levels of these three steroid receptors (ERα, ERβ and PR) in the ovary of juvenile alligators and observed detectable levels of all three receptors. Quantitative RT-PCR showed that gonadal ERα transcript levels in juvenile alligators decreased after E2 treatment whereas ERβ and PR transcripts were not changed. These results provide tools that will allow future studies examining the regulation and ontogenic expression of steroid receptors in alligators and expand our knowledge of vertebrate steroid receptor evolution.