Analysis of Signal Transduction of Growth and Differentiation of Avian Granulosa cells

鸡、鹌鹑等鸟类卵泡颗粒细胞。在卵泡生长过程中增殖，在黄体生成素（LH）的作用下分化分泌黄体酮，此时卵泡最大，达到排卵期。为了研究鹌鹑颗粒细胞生长分化的机制和信号转导途径，本实验采用多种促性腺激素或生长因子（如表皮生长因子（EGF））培养鹌鹑颗粒细胞。培养基为McCoy’s 5a和Ham’s F12（1:1）的混合物，并添加谷氨酰胺、HEPES、青霉素和链霉素。在培养前6小时，胎牛血清浓度为10%，随后降至1%。为了评估细胞在培养后对LH的反应，将细胞在添加牛血清白蛋白的克雷布斯-林格溶液中与LH或不含LH孵育3小时，并通过特异性放射免疫法测定培养液中黄体酮的产生。在促卵泡激素（FSH）作用下，颗粒细胞对LH的反应增强，但细胞数量没有显著增加。当颗粒细胞与EGF一起培养72小时时，细胞数量显著增加，但通过孕酮的产生来评估细胞的LH反应减弱。这些结果表明卵泡成熟过程中颗粒细胞的生长和分化受FSH和EGF的共同控制。

Granulosa cells of follicles in avian species, such as chicken and Japanese quail, . proliferate during follicular growth, and differentiate to secrete progesterone in response to luteinizing hormone (LH), when the follicle becomes the largest and reaches to the time of ovulation. In order to study the mechanism and signal transduction pathway of growth and differentiation, quail granulosa cells were cultured with various gonadotrophins or growth factors, such as epidermal growth factor (EGF).The culture medium was a mixture of McCoy's 5a and Ham's F12 (1 : 1) supplemented with glutamine, HEPES,Penicilline and Streptomycin. The concentration of fetal calf serum was 10% during the initial 6 hr of the culture, and then it was reduced to 1%. In order to evaluate the LH response of the cells after the culture, the cells were incubated with or without LH for 3 hr in Krebs-Ringer solution supplemented with bovine serum albumin, and progesterone production was measured in the medium during this 3 hr of incubation by the specific radio immunoassay.When the granulosa cells were cultured with follicle stimulating hormone (FSH) for 72 hr, the response to LH was increased while the cell numbers were not significantly increased. When the granulosa cells were cultured with EGF for 72 hr, the cell numbers increased dramatically, but the LH response of the cells, which was evaluated by the production of progesterone, was diminished. These results suggested that the growth and differentiation of the granulosa cells during follicular maturation was controlled by both FSH and EGF.