Skinner M K, Osteen K G
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Endocrinology. 1988 Sep;123(3):1668-75. doi: 10.1210/endo-123-3-1668.
Bovine granulosa cells were isolated from small antral, medium antral, and large Graffian follicles (i.e. small, medium, and large preovulatory follicles). Serum-free cultures of granulosa cells were established and found to be viable for 3-6 days of cell culture. Radiolabeled granulosa cell-secreted proteins were obtained and analyzed electrophoretically. No major changes were detected in the protein profiles of small, medium, and large follicle granulosa cells. FSH and insulin, however, had a dramatic effect on granulosa cell-secreted proteins and increased the apparent production of 200K, 65K, 25K, and 15K proteins. The effects of these hormones on the radiolabeled secreted proteins were similar for small, medium, and large follicle granulosa cells. Aromatase activity was high for the first day of serum-free granulosa cell culture and subsequently declined to low levels. Both FSH and insulin alone stimulated aromatase activity, while a combination of hormones resulted in an additive response similar to the stimulation observed with 10% calf serum. Although the level of aromatase activity increased slightly with the size of the follicle, the effects of hormones were independent of follicle size. Progesterone production was low on days 1 and 2 of serum-free granulosa cell culture and high on days 3 and 6 of cell culture. Interestingly, FSH and insulin suppressed progesterone production on day 1 of cell culture for small and medium follicle granulosa cells, but not for large follicle cells. In contrast, hormones stimulated progesterone production on days 3 and 6 of granulosa cell culture, and the level of progesterone production increased with the size of the follicle. The stimulatory effects of hormones on days 3 and 6 of the culture were similar for medium and large follicle granulosa cells, but were altered for small follicle cells. Results indicate that when aromatase activity is high and stimulated by hormones, progesterone levels are low and generally suppressed by the same regulatory agents. Conversely when progesterone levels are high and hormone responsive, aromatase activity is low. The inverse relationship between aromatase activity and progesterone production implies that bovine granulosa cells alter their differentiated state in culture from an estrogen-producing cell to a progesterone-producing cell. Combined observations indicate that the results obtained on day 1 of culture probably reflect the developmental and hormonal regulation of granulosa cell function in the preovulatory follicle, while data obtained at later times in culture reflect the ability of the cell to synthesize progesterone and develop a luteinization-like activity.(ABSTRACT TRUNCATED AT 400 WORDS)
牛颗粒细胞从小窦卵泡、中窦卵泡和大格拉夫卵泡（即小、中、大排卵前卵泡）中分离得到。建立了颗粒细胞的无血清培养体系，发现其在细胞培养3 - 6天内保持存活。获取了放射性标记的颗粒细胞分泌蛋白并进行了电泳分析。小、中、大卵泡颗粒细胞的蛋白质谱未检测到重大变化。然而，促卵泡激素（FSH）和胰岛素对颗粒细胞分泌蛋白有显著影响，增加了200K、65K、25K和15K蛋白的表观产量。这些激素对小、中、大卵泡颗粒细胞放射性标记分泌蛋白的影响相似。在无血清颗粒细胞培养的第一天，芳香化酶活性较高，随后降至低水平。单独的FSH和胰岛素均刺激芳香化酶活性，而激素组合产生的相加反应类似于用10%小牛血清观察到的刺激作用。尽管芳香化酶活性水平随卵泡大小略有增加，但激素的作用与卵泡大小无关。在无血清颗粒细胞培养的第1天和第2天，孕酮产量较低，而在细胞培养的第3天和第6天较高。有趣的是，对于小卵泡和中卵泡颗粒细胞，FSH和胰岛素在细胞培养第1天抑制孕酮产生，但对大卵泡颗粒细胞无此作用。相反，激素在颗粒细胞培养的第3天和第6天刺激孕酮产生，且孕酮产量水平随卵泡大小增加。激素在培养第3天和第6天对中卵泡和大卵泡颗粒细胞的刺激作用相似，但对小卵泡颗粒细胞有所改变。结果表明，当芳香化酶活性较高且受激素刺激时，孕酮水平较低且通常受到相同调节因子的抑制。相反，当孕酮水平较高且对激素有反应时，芳香化酶活性较低。芳香化酶活性与孕酮产生之间的负相关关系表明，牛颗粒细胞在培养中从产生雌激素的细胞转变为产生孕酮的细胞，改变了其分化状态。综合观察表明，培养第1天获得的结果可能反映了排卵前卵泡中颗粒细胞功能的发育和激素调节，而培养后期获得的数据反映了细胞合成孕酮和发展黄体化样活性的能力。（摘要截于400字）
