Denari Daniela, Ceballos Nora R
Laboratorio de Endocrinología Comparada and PRHOM-CONICET, Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.
Gen Comp Endocrinol. 2006 Jul;147(3):247-54. doi: 10.1016/j.ygcen.2006.01.006. Epub 2006 Feb 17.
Glucocorticoids (GC) are the hormonal mediators of stress. In mammals, high levels of GC have negative effects on reproductive physiology. For instance, GC can inhibit testicular testosterone synthesis by acting via glucocorticoid receptors (GR), the extent of the inhibition being dependent on GC levels. However, the effect of GC on testicular function and even the presence of GR in amphibians are still unclear. The purpose of this work was to characterise testicular cytosolic GR in Bufo arenarum, determining the seasonal changes in its binding parameters as well as the intratesticular localisation. The binding assays were performed in testis cytosol with [3H]dexamethasone (DEX) and [3H]corticosterone (CORT). Binding kinetics of DEX and CORT fitted to a one-site model. Results were expressed as means +/- standard error. Apparent number of binding sites (Bapp) was similar for both steroids (Bapp DEX = 352.53 +/- 72.08 fmol/mg protein; Bapp CORT = 454.24 +/- 134.97 fmol/mg protein) suggesting that both hormones bind to the same site. Competition studies with different steroids showed that the order of displacement of [3H]DEX and [3H]CORT specific binding is: DEX approximately RU486 approximately deoxycorticosterone (DOC) > CORT > aldosterone > RU28362 > progesterone >>> 11-dehydroCORT. The affinity of GR for DEX (Kd = 11.2 +/- 1.5 nM) remained constant throughout the year while circulating CORT clearly increased during the reproductive season. Therefore, testis sensitivity to GC action would depend mainly on inactivating mechanisms (11beta-hydroxysteroid dehydrogenase type 2) and CORT plasma levels. Since total and free CORT are higher in the reproductive than in the non-reproductive period, the magnitude of GC actions could be higher during the breeding season. The intratesticular localisation of the GR was determined after separation of cells by a Percoll density gradient followed by binding assays in each fraction. DEX binds to two different fractions corresponding to Leydig and Sertoli cells. In conclusion, in the testis of B. arenarum GC could regulate the function of both cellular types particularly during breeding when CORT reaches the highest plasma concentration.
糖皮质激素（GC）是应激反应的激素介质。在哺乳动物中，高水平的GC对生殖生理有负面影响。例如，GC可通过糖皮质激素受体（GR）发挥作用，抑制睾丸睾酮的合成，抑制程度取决于GC水平。然而，GC对两栖动物睾丸功能的影响以及GR在两栖动物睾丸中的存在情况仍不明确。本研究旨在表征布氏蟾蜍睾丸胞质中的GR，确定其结合参数的季节性变化以及在睾丸内的定位。结合实验在睾丸胞质中使用[3H]地塞米松（DEX）和[3H]皮质酮（CORT）进行。DEX和CORT的结合动力学符合单点模型。结果以平均值±标准误差表示。两种类固醇的表观结合位点数（Bapp）相似（Bapp DEX = 352.53 ± 72.08 fmol/mg蛋白质；Bapp CORT = 454.24 ± 134.97 fmol/mg蛋白质），表明两种激素结合到同一位点。用不同类固醇进行的竞争研究表明，[3H]DEX和[3H]CORT特异性结合的置换顺序为：DEX≈RU486≈脱氧皮质酮（DOC）> CORT>醛固酮> RU28362>孕酮>>> 11-脱氢CORT。GR对DEX的亲和力（Kd = 11.2 ± 1.5 nM）全年保持恒定，而循环中的CORT在繁殖季节明显升高。因此，睾丸对GC作用的敏感性将主要取决于失活机制（2型11β-羟基类固醇脱氢酶）和CORT血浆水平。由于繁殖期的总CORT和游离CORT均高于非繁殖期，因此繁殖季节GC作用的强度可能更高。通过Percoll密度梯度分离细胞后，在每个组分中进行结合实验，确定GR在睾丸内的定位。DEX与对应于睾丸间质细胞和支持细胞的两个不同组分结合。总之，在布氏蟾蜍的睾丸中，GC可能调节这两种细胞类型的功能，尤其是在繁殖期间，此时CORT达到最高血浆浓度。
