Knecht M, Shinohara O, Catt K J
Endocrinology. 1986 Sep;119(3):1388-96. doi: 10.1210/endo-119-3-1388.
The synthesis of cellular and secreted proteins by differentiating granulosa cells from diethylstilbestrol-treated immature rats was studied by one- and two-dimensional polyacrylamide gel electrophoresis. In cultured granulosa cells, FSH altered the relative biosynthesis of specific cellular and secreted proteins in a concentration- and time-dependent manner. The incorporation of [35S]methionine into cellular proteins of Mr 42,000, 48,000, and 58,000 was enhanced by increasing amounts of the gonadotropin, whereas the labeling of a 44,000 Mr protein was reduced. Similarly, FSH increased the labeling of secreted proteins with relative Mr of 16,000, 17,000, 20,000, 25,000, 36,000, 41,000, 46,000, 111,000, and 153,000, and decreased that of proteins with Mr of 38,000, 48,000, 191,000, and 250,000. The expression of specific proteins was related to the degree of cellular maturation, since some proteins were newly synthesized during the early stages of granulosa cell development (less than 6 h), whereas others were more evident in the middle (24 h) or later (48 h) phases of culture. Also, the level of specific protein synthesis was variable since certain proteins were progressively produced during culture, and the biosynthesis of others fluctuated or was reduced during development. The effects of FSH on protein synthesis were mimicked by other cAMP-inducing ligands, including cholera toxin, forskolin, and 8-bromo-cAMP. Removal of FSH at 24 h of culture was followed by reversion of the protein biosynthetic pattern at 48 h to that of control cells, indicating that continued exposure to the gonadotropin is required during development. Cells cultured in the absence of ligands for 24 h synthesized proteins characteristic of differentiated cells when subsequently cultured with forskolin. These results indicate that FSH selectively alters the biosynthesis of cell-associated and secreted proteins during granulosa cell maturation. The characterization of these gene products and the mechanisms controlling their expression should ultimately clarify the sequential events involved in the hormonal regulation of granulosa cell development.
通过一维及二维聚丙烯酰胺凝胶电泳，研究了己烯雌酚处理的未成熟大鼠分化颗粒细胞中细胞蛋白和分泌蛋白的合成。在培养的颗粒细胞中，促卵泡激素（FSH）以浓度和时间依赖的方式改变特定细胞蛋白和分泌蛋白的相对生物合成。随着促性腺激素量的增加，[35S]甲硫氨酸掺入分子量为42,000、48,000和58,000的细胞蛋白中的量增加，而分子量为44,000的蛋白的标记减少。同样，FSH增加了相对分子量为16,000、17,000、20,000、25,000、36,000、41,000、46,000、111,000和153,000的分泌蛋白的标记，并减少了分子量为38,000、48,000、191,000和2,50,000的蛋白的标记。特定蛋白的表达与细胞成熟程度相关，因为一些蛋白在颗粒细胞发育的早期阶段（少于6小时）新合成，而其他蛋白在培养的中期（24小时）或后期（48小时）更明显。此外，特定蛋白合成水平是可变的，因为某些蛋白在培养过程中逐渐产生，而其他蛋白的生物合成在发育过程中波动或减少。FSH对蛋白合成的作用被其他诱导环磷酸腺苷（cAMP）的配体模拟，包括霍乱毒素、福斯可林和8-溴-cAMP。在培养24小时时去除FSH后，48小时时蛋白生物合成模式恢复到对照细胞的模式，表明在发育过程中需要持续暴露于促性腺激素。在无配体的情况下培养24小时的细胞，随后用福斯可林培养时，合成了分化细胞特有的蛋白。这些结果表明，FSH在颗粒细胞成熟过程中选择性地改变细胞相关蛋白和分泌蛋白的生物合成。这些基因产物的特征以及控制其表达的机制最终应能阐明颗粒细胞发育激素调节中涉及的一系列事件。
