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

鸡和日本鹌鹑等鸟类卵泡的颗粒细胞。在卵泡生长期间增殖，并在卵泡变得最大并达到排卵时间时分化以响应促黄体生成素（LH）分泌孕酮。为了研究鹌鹑颗粒细胞生长分化的机制和信号转导途径，本研究采用麦考伊's 5a和Ham's F12（1：1）混合培养液，添加谷氨酰胺、HEPES、青霉素和链霉素，并加入不同的促性腺激素或生长因子，如表皮生长因子（EGF）等，对鹌鹑颗粒细胞进行培养。在培养的最初6小时期间胎牛血清的浓度为10%，然后将其降低至1%。为了评价培养后细胞的LH反应，将细胞在添加有牛血清白蛋白的Krebs-Ringer溶液中与LH或不与LH一起孵育3小时，并通过特异性放射免疫测定法测量在孵育3小时期间培养基中孕酮的产生。对LH的反应增加，而细胞数量没有显著增加。当颗粒细胞与EGF培养72小时时，细胞数量显著增加，但细胞的LH反应，这是通过孕酮的产生来评估的，被减弱。提示卵泡成熟过程中颗粒细胞的生长和分化受FSH和EGF共同调控。

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.