Bronson D D, Stumpf W E
Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill 27599-7090.
Prog Histochem Cytochem. 1991;22(4):1-59. doi: 10.1016/s0079-6336(11)80050-1.
Xenopus laevis oocytes resume meiosis in response to progesterone. The initial interaction involves surface binding to numerous low-affinity receptor proteins. The mechanism of entry and functions of intracellular steroid are unknown. Because the latter are important for understanding progesterone-induced maturation, a dry-mount autoradiographic technique for analyzing entry and intracellular distribution of radiolabeled steroids was developed and tested. The distinguishing feature of this cryo-technique is sample preparation directly in incubation media using uncross-linked polyacrylamide for inert support. The external ligand functions as an internal standard, so quantitation is by simple ratio (bound/free). The entry kinetics and subcellular binding patterns in large oocytes were studied using this method at nM levels of radiolabeled steroids and model compounds. Progesterone, estradiol, corticosterone, and 1,25-dihydroxycholecalciferol all showed rapid entry (P approximately 10(-6) cm/sec). Entry rates were not saturable with unlabeled steroid. Intracellular patterns of these steroids were highly specific and negatively associated with yolk protein and lipid. Intracellular binding in animal hemisphere ooplasm was 10x that of the yolk-rich vegetative ooplasm. In contrast, dexamethasone, ponasterone-A, and ecdysone displayed entry rates 20-60x slower than progesterone with little compartmentalization. Glycerol, glucose, and leucine entered over 10x slower than progesterone. Cholesterol and Ca++ had entry rates below detection. Evidence for mediated entry of progesterone included the rapid saturation of a cortical compartment equivalent in magnitude to reported receptor numbers. The kinetics and specificity of cortical uptake were consistent with low-affinity, high capacity protein binding. Intracellular binding was seen to correlate with rhodamine 123 patterns, suggesting involvement of mitochondrial or other microtubule-associated structures in steroid responses. Mitochondrial binding is consistent with the limited steroid metabolism seen in oocytes. Since several maturation events are consistent with respiratory uncoupling, reported by others for steroids and isolated organelles, and since mitochondria contain nearly all of the oocyte DNA, a role for these organelles in steroid-induced oocyte maturation was proposed.
非洲爪蟾卵母细胞会因孕酮而恢复减数分裂。最初的相互作用涉及与众多低亲和力受体蛋白的表面结合。细胞内类固醇的进入机制和功能尚不清楚。由于后者对于理解孕酮诱导的成熟很重要，因此开发并测试了一种用于分析放射性标记类固醇的进入和细胞内分布的干装放射自显影技术。这种低温技术的显著特点是直接在使用未交联聚丙烯酰胺作为惰性支持物的孵育介质中制备样品。外部配体用作内标，因此通过简单的比率（结合/游离）进行定量。使用该方法在纳摩尔水平的放射性标记类固醇和模型化合物下研究了大卵母细胞中的进入动力学和亚细胞结合模式。孕酮、雌二醇、皮质酮和1,25-二羟基胆钙化醇均显示快速进入（P约为10^(-6)厘米/秒）。未标记的类固醇不会使进入速率饱和。这些类固醇的细胞内模式具有高度特异性，并且与卵黄蛋白和脂质呈负相关。动物半球卵质中的细胞内结合是富含卵黄的营养卵质的10倍。相比之下，地塞米松、蜕皮甾酮A和蜕皮激素的进入速率比孕酮慢20-60倍，且几乎没有区室化。甘油、葡萄糖和亮氨酸的进入速度比孕酮慢10倍以上。胆固醇和Ca++的进入速率低于检测水平。孕酮介导进入的证据包括一个皮质区室的快速饱和，其大小与报道的受体数量相当。皮质摄取的动力学和特异性与低亲和力、高容量的蛋白质结合一致。细胞内结合与罗丹明123模式相关，表明线粒体或其他微管相关结构参与了类固醇反应。线粒体结合与卵母细胞中有限的类固醇代谢一致。由于其他人报道的类固醇和分离细胞器的几种成熟事件与呼吸解偶联一致，并且由于线粒体几乎包含所有的卵母细胞DNA，因此提出了这些细胞器在类固醇诱导的卵母细胞成熟中的作用。
