Braun Alyssa M, Thomas Peter
University of Texas at Austin, Marine Science Institute, Port Aransas, Texas 78373, USA.
Biol Reprod. 2004 Jul;71(1):146-55. doi: 10.1095/biolreprod.103.025825. Epub 2004 Feb 25.
Membrane androgen receptors have been biochemically characterized in only a few vertebrate species to date. Therefore, the purpose of the current study was to comprehensively investigate the binding characteristics of a putative membrane androgen receptor in the ovary of the teleost, Atlantic croaker (Micropogonias undulatus). Specific androgen binding to an ovarian plasma membrane fraction was demonstrated using a radioreceptor assay protocol consisting of a short-term incubation with [(3)H]testosterone (T) and subsequent filtration of bound steroid from free steroid. Saturation and Scatchard analyses of T binding to an ovarian plasma membrane fraction indicated the presence of a single, high-affinity (K(d) = 15.32 +/- 2.68 nM [mean +/- SEM]), low-capacity (B(max) = 2.81 +/- 0.31 pmol/mg protein), androgen-binding site. Specific androgen binding to the receptor was readily displaceable, and the association and dissociation kinetics were rapid (half-time = 3.7 +/- 1.7 and 4.7 +/- 0.2 min, respectively). Competitive binding assays showed that 5alpha-dihydrotestosterone, T, and 11-ketotestosterone had relative binding affinities (RBAs) of 193%, 100%, and 13%, respectively, whereas none of the C(18) or C(21) steroids tested bound with high affinity except for progesterone (RBA = 191%). This androgen-binding moiety with high affinity for progesterone is unlikely to mediate the physiological actions of progestins in croaker, because it has low binding affinity for fish progestin hormones. Androgen-binding sites were also detected in membrane fractions of the brain, liver, kidney, and drumming muscle, whereas little or no binding was detected in the trunk muscle, heart, gills, or intestine. Receptor levels increased 10-fold during ovarian recrudescence, reaching maximum levels in fully mature ovaries, which suggests a likely physiological role for this receptor during the reproductive cycle of female croaker. It is concluded that the androgen-binding moiety identified in the plasma membrane fraction of Atlantic croaker ovarian tissue fulfils all the criteria for its designation as a steroid receptor.
迄今为止，仅在少数脊椎动物物种中对膜雄激素受体进行了生物化学特征分析。因此，本研究的目的是全面研究硬骨鱼大西洋黄鱼（Micropogonias undulatus）卵巢中假定的膜雄激素受体的结合特性。使用放射性受体分析方法证明了特异性雄激素与卵巢质膜部分的结合，该方法包括与[（3）H]睾酮（T）进行短期孵育，随后从游离类固醇中过滤结合的类固醇。对T与卵巢质膜部分结合的饱和分析和Scatchard分析表明存在单一的高亲和力（K（d）= 15.32 +/- 2.68 nM [平均值+/-标准误]）、低容量（B（max）= 2.81 +/- 0.31 pmol/mg蛋白质）的雄激素结合位点。特异性雄激素与受体的结合易于被取代，并且结合和解离动力学很快（半衰期分别为3.7 +/- 1.7和4.7 +/- 0.2分钟）。竞争性结合试验表明，5α-二氢睾酮、T和11-酮睾酮的相对结合亲和力（RBA）分别为193%、100%和13%，而除孕酮（RBA = 191%）外，所测试的C（18）或C（21）类固醇均未以高亲和力结合。这种对孕酮具有高亲和力的雄激素结合部分不太可能介导孕酮在黄鱼中的生理作用，因为它对鱼类孕酮激素的结合亲和力较低。在脑、肝、肾和击鼓肌的膜部分也检测到了雄激素结合位点，而在躯干肌、心脏、鳃或肠道中几乎没有检测到结合。在卵巢复壮期间，受体水平增加了10倍，在完全成熟的卵巢中达到最高水平，这表明该受体在雌性黄鱼的生殖周期中可能具有生理作用。结论是，在大西洋黄鱼卵巢组织质膜部分鉴定出的雄激素结合部分符合将其指定为类固醇受体的所有标准。
