Lee W M, Cheng C Y, Bardin C W, Gunsalus G L, Musto N A
Endocrinology. 1986 Nov;119(5):1914-21. doi: 10.1210/endo-119-5-1914.
Many proteins secreted by Sertoli cell-enriched cultures are maximally stimulated by a combination of FSH and testosterone. Since very few are stimulated primarily by FSH, we thought it pertinent to identify such proteins. Sertoli cell-enriched cultures were prepared from testes of 20-day-old rats and grown in serum-free medium containing insulin, transferrin, and epidermal growth factor and in such medium supplemented with FSH, testosterone, or FSH plus testosterone. Media were fractionated using HPLC, and proteins were identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. A protein designated CMB-2, with an apparent mol wt of 22,000, was shown to increase in response to FSH. Antiserum was raised using denatured protein eluted from SDS-polyacrylamide gels as the antigen, and a specific immunoassay using a combination of SDS-polyacrylamide gel electrophoresis and Western blotting was developed. The production of CMB-2 by primary Sertoli cell-enriched cultures was found to increase in a dose-dependent manner in response to FSH (30-1000 ng/ml); secretion was not significantly affected by testosterone (2 X 10(-13) M). An investigation of the tissue distribution of CMB-2 showed that the puberty, CMB-2 is secreted into the rete testis and accumulates in the epididymis in high concentration. We conclude that CMB-2 will be a useful marker to study the action of FSH on the rat testis.
富含支持细胞的培养物分泌的许多蛋白质，在促卵泡激素（FSH）和睾酮共同作用下受到最大程度的刺激。由于很少有蛋白质主要受FSH刺激，我们认为识别此类蛋白质很有必要。从20日龄大鼠的睾丸制备富含支持细胞的培养物，并在含有胰岛素、转铁蛋白和表皮生长因子的无血清培养基中培养，以及在补充有FSH、睾酮或FSH加睾酮的此类培养基中培养。使用高效液相色谱法（HPLC）对培养基进行分级分离，并用十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳鉴定蛋白质。一种命名为CMB-2的蛋白质，表观分子量为22000，显示出对FSH有反应性增加。以从SDS-聚丙烯酰胺凝胶上洗脱的变性蛋白作为抗原制备抗血清，并开发了一种结合SDS-聚丙烯酰胺凝胶电泳和蛋白质印迹法的特异性免疫测定法。发现原代富含支持细胞的培养物中CMB-2的产生对FSH（30 - 1000 ng/ml）呈剂量依赖性增加；睾酮（2×10⁻¹³ M）对其分泌无显著影响。对CMB-2组织分布的研究表明，在青春期，CMB-2分泌到睾丸网中，并在附睾中高浓度积累。我们得出结论，CMB-2将是研究FSH对大鼠睾丸作用的有用标志物。
