Gutiérrez C G, Campbell B K, Webb R
Roslin Institute, Roslin, Midlothian, United Kingdom.
Biol Reprod. 1997 Mar;56(3):608-16. doi: 10.1095/biolreprod56.3.608.
The objectives of this study were to develop a serum-free bovine granulosa cell culture system in which FSH-responsive estradiol production could be induced and maintained, and to use this system to evaluate the effects of FSH, insulin, and IGF-I on steroidogenesis and proliferation of bovine granulosa cells from different follicle size categories (< 4-, 4-8, and > 8-mm diameter). In the presence of FSH, granulosa cells from small follicles differentiated in vitro, and estradiol secretion increased with time (p < 0.01) so that by the end of the culture period it was similar to that of cells from large follicles. Granulosa cells from medium and large follicles secreted estradiol throughout the culture period. Cells cultured in plasma-coated culture wells had an increased proliferative response but had lower estradiol production compared to cells cultured under serum-free conditions (p < 0.01). Insulin promoted proliferation and estradiol production by granulosa cells from the three follicle-size categories (p < 0.01). Physiological concentrations of FSH induced proliferation and estradiol secretion (p < 0.01) by granulosa cells in a dose-responsive manner. The inclusion of IGF-I in the culture system enhanced proliferation and estradiol production (p < 0.01), even in the absence of gonadotropic support, demonstrating the gonadotropic characteristics of this growth factor. These results demonstrate the development of a relevant physiological culture system for bovine granulosa cells. This system will permit the detailed study of the key factors controlling the differentiation and proliferation of bovine granulosa cells.
本研究的目的是建立一种无血清牛颗粒细胞培养系统，在该系统中可诱导并维持促卵泡素（FSH）反应性雌二醇的产生，并利用该系统评估FSH、胰岛素和胰岛素样生长因子-I（IGF-I）对来自不同卵泡大小类别（直径<4mm、4 - 8mm和>8mm）的牛颗粒细胞类固醇生成和增殖的影响。在FSH存在的情况下，来自小卵泡的颗粒细胞在体外发生分化，雌二醇分泌随时间增加（p<0.01），因此在培养期结束时，其分泌水平与来自大卵泡的细胞相似。来自中卵泡和大卵泡的颗粒细胞在整个培养期都分泌雌二醇。与在无血清条件下培养的细胞相比，在血浆包被培养孔中培养的细胞增殖反应增强，但雌二醇产生量较低（p<0.01）。胰岛素促进了来自三种卵泡大小类别的颗粒细胞的增殖和雌二醇产生（p<0.01）。生理浓度的FSH以剂量反应方式诱导颗粒细胞增殖和雌二醇分泌（p<0.01）。即使在没有促性腺激素支持的情况下，在培养系统中加入IGF-I也能增强增殖和雌二醇产生（p<0.01），这表明该生长因子具有促性腺激素特性。这些结果证明了一种适用于牛颗粒细胞的相关生理培养系统的建立。该系统将有助于详细研究控制牛颗粒细胞分化和增殖的关键因素。
