Katsu Yoshinao, Hinago Megumi, Sone Kiyoaki, Urushitani Hiroshi, Guillette Louis J, Iguchi Taisen
Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan.
Mol Cell Endocrinol. 2007 Sep 30;276(1-2):10-7. doi: 10.1016/j.mce.2007.06.004. Epub 2007 Jun 29.
Sex-steroid hormones are essential for normal reproductive activity in both sexes. Estrogens are necessary for ovarian differentiation during a critical developmental stage in many vertebrates and promote the growth and differentiation of the female reproductive system. Androgens play essential roles in the development and functioning of the vertebrate male reproductive system as well as actively supporting spermatogenesis. Importantly, recent studies suggest that androgens and estrogens have important reproductive roles in both males and females. To understand the molecular mechanisms of estrogen and androgen actions and to evaluate estrogen and androgen receptor-ligand interactions in the mosquitofish, Gambusia affinis affinis, we used degenerate primer sets and PCR techniques to isolated DNA fragments encoding estrogen receptor alpha (ERalpha; ESR1), ERbeta1 (ERbeta1) and ERbeta2 from the ovary. Full-length mosquitofish ER (mfER) cDNAs were obtained using cDNA library screening and RACE techniques. Amino acid sequences of mfERs showed over-all homology of 46% (alpha versus beta1), 43% (alpha versus beta2), and 52% (beta1 versus beta2). We applied the ERE-luciferase reporter assay system to characterize these receptors. In this transient transfection assay system using mammalian cells, the mfER proteins displayed estrogen-dependent activation of transcription. In addition to ERs, the transactivation of mosquitofish ARs (mfARs) previously isolated by our group, were examined using an androgen-responsive MMTV-luciferase assay system. Mosquitofish ARs showed androgen-dependent activation of transcription from the MMTV promoter. These data provide a basic tool allowing future studies examining the receptor-ligand interactions and endocrine disrupting mechanisms in mosquitofish and also expands our knowledge of estrogen and androgen receptor evolution.
性类固醇激素对两性的正常生殖活动至关重要。在许多脊椎动物的关键发育阶段，雌激素对于卵巢分化是必需的，并促进雌性生殖系统的生长和分化。雄激素在脊椎动物雄性生殖系统的发育和功能中发挥着重要作用，同时积极支持精子发生。重要的是，最近的研究表明雄激素和雌激素在雄性和雌性中都具有重要的生殖作用。为了了解雌激素和雄激素作用的分子机制，并评估食蚊鱼（Gambusia affinis affinis）中雌激素和雄激素受体 - 配体相互作用，我们使用简并引物组和PCR技术从卵巢中分离出编码雌激素受体α（ERα；ESR1）、ERβ1和ERβ2的DNA片段。使用cDNA文库筛选和RACE技术获得了食蚊鱼ER（mfER）全长cDNA。mfER的氨基酸序列显示α与β1的总体同源性为46%，α与β2为43%，β1与β2为52%。我们应用ERE - 荧光素酶报告基因检测系统来表征这些受体。在这个使用哺乳动物细胞的瞬时转染检测系统中，mfER蛋白表现出雌激素依赖性的转录激活。除了ERs，我们还使用雄激素反应性MMTV - 荧光素酶检测系统检测了我们小组之前分离的食蚊鱼ARs（mfARs）的反式激活。食蚊鱼ARs显示出雄激素依赖性的MMTV启动子转录激活。这些数据提供了一个基础工具，有助于未来研究食蚊鱼中的受体 - 配体相互作用和内分泌干扰机制，也扩展了我们对雌激素和雄激素受体进化的认识。
