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

禽类中滤泡的颗粒细胞，如鸡和日本鹌鹑。在卵泡生长过程中增殖，并在卵泡最大和排卵时分化为分泌孕酮以响应黄体生成素(L H)。为了研究颗粒细胞生长分化的机制和信号转导途径，用不同的促性腺激素或生长因子，如表皮生长因子(EGF)，在添加谷氨酰胺、HEPES、青霉素和链霉素1：1的McCoy‘s 5a和Ham’s F12(1：1)混合液中对颗粒细胞进行培养。在培养的最初6小时内，胎牛血清浓度为10%，然后降至1%。为评价卵泡刺激素(FSH)对卵泡颗粒细胞的促黄体生成素(LH)反应，将培养的颗粒细胞分别加入或不加入促黄体生成素(LH3h)，在添加牛血清白蛋白的Krebs-Ringer液中孵育3h，用放射免疫法测定培养液中孕酮的产生。颗粒细胞与EGF共同培养72小时后，细胞数显著增加，但黄体生成素反应减弱。这些结果提示卵泡成熟过程中颗粒细胞的生长和分化受FSH和EGF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.