Katsu Yoshinao, Bermudez Dieldrich S, Braun Edward L, Helbing Caren, Miyagawa Shinichi, Gunderson Mark P, Kohno Satomi, Bryan Teresa A, Guillette Louis J, Iguchi Taisen
Center for Integrative Bioscience, National Institute for Basic Biology, Okazaki National Research Institutes, Higashiyama, Myodaiji, Okazaki, Japan.
Gen Comp Endocrinol. 2004 Mar;136(1):122-33. doi: 10.1016/j.ygcen.2003.11.008.
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 (ERalpha and ERbeta) were obtained and the full-length alligator ERalpha cDNA was obtained using 5' and 3' RACE. The inferred amino acid sequence of alligator ERalpha (aERalpha) was very similar to the chicken ERalpha (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 ERbeta and the progesterone receptor (PR) of the alligator, both of which show strong sequence similarities to avian ERbeta and PR. We examined the expression levels of these three steroid receptors (ERalpha, ERbeta, and PR) in the ovary of juvenile alligators and observed detectable levels of all three receptors. Quantitative RT-PCR showed that gonadal ERalpha transcript levels in juvenile alligators decreased after E2 treatment whereas ERbeta 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.
类固醇激素在脊椎动物的胚胎发育、繁殖、生长、水平衡及应激反应过程中发挥着许多重要作用。雌激素对于雌性和雄性脊椎动物的正常生殖活动至关重要，并且在某些脊椎动物的性别决定过程中似乎具有直接作用。为了开始了解雌激素在短吻鳄中发挥作用的分子机制，我们从卵巢中分离出了编码雌激素受体（ER）的cDNA。设计了特异于ER的简并PCR引物，并用于扩增短吻鳄卵巢RNA。获得了两个不同的DNA片段（ERα和ERβ），并使用5'和3' RACE获得了全长短吻鳄ERα cDNA。短吻鳄ERα（aERα）的推导氨基酸序列与鸡ERα非常相似（同一性为91%），尽管系统发育分析表明脊椎动物ER序列的序列进化速率存在显著差异。我们还分离出了编码短吻鳄ERβ和孕激素受体（PR）的部分DNA片段，它们与禽类ERβ和PR均表现出很强的序列相似性。我们检测了幼年短吻鳄卵巢中这三种类固醇受体（ERα、ERβ和PR）的表达水平，并观察到所有三种受体均有可检测水平。定量RT-PCR显示，E2处理后幼年短吻鳄性腺中ERα转录本水平下降，而ERβ和PR转录本未发生变化。这些结果提供了工具，将有助于未来研究短吻鳄类固醇受体的调控和个体发育表达，并扩展我们对脊椎动物类固醇受体进化的认识。
