Biochemical characterization of a membrane androgen receptor in the ovary of the Atlantic croaker (Micropogonias undulatus)
Biochemical characterization of a membrane androgen receptor in the ovary of the Atlantic croaker (Micropogonias undulatus)
复制标题
DOI：
10.1095/biolreprod.103.025825
复制
发表时间：
2004-07-01
影响因子：
3.6
通讯作者：
Thomas, P
中科院分区：
文献类型：
--
作者：
Braun, AM;Thomas, P
迄今为止，只有少数脊椎动物的膜雄激素受体具有生物化学特征。因此，本研究的目的是全面研究一种假定的膜雄激素受体在硬骨鱼大西洋croaker （Micropogonias undulatus）卵巢中的结合特性。使用放射受体试验方案，包括与[H-3]睾酮(T)短期孵育和随后从游离类固醇中过滤结合的类固醇，证明了特异性雄激素与卵巢质膜部分的结合。T结合卵巢质膜片段的饱和和Scatchard分析表明，存在一个单一的高亲和力(K-d 15.32 +/- 2.68 nM（平均+/- SEM%）低容量（B-max = 2.81 +/- 0.31 pmol/mg蛋白）雄激素结合位点。特异性雄激素与受体的结合很容易被取代，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 min）。竞争结合实验显示，5 α -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C-18或C-21类固醇除了孕酮（RBA = 191%）外，都没有高亲和力的结合。这种对黄体酮具有高亲和力的雄激素结合片段不太可能介导黄体酮在黄花鱼体内的生理作用，因为它对鱼类黄体酮激素的结合亲和力较低。雄激素结合位点也在脑、肝、肾和鼓膜肌的膜部分检测到，而在躯干肌、心脏、鳃或肠中检测到很少或没有结合。受体水平在卵巢复发期间增加10倍，在完全成熟的卵巢中达到最高水平，这表明该受体在雌鱼生殖周期中可能具有生理作用。结论：在大西洋花鱼卵巢组织质膜片段中发现的雄激素结合片段满足其作为类固醇受体的所有标准。
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 [H-3]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.