GRANULOSA CELL FUNCTION DURING FOLLICULOGENESIS

卵泡发生过程中颗粒细胞的功能

• 批准号: 3325100
• 负责人: VIRENDRA B MAHESH
• 金额: $ 14.12万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 1993-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3325100
• 关键词: androgens; aromatase; cell differentiation; cell growth regulation; cyclic AMP; density gradient ultracentrifugation; diethylstilbestrol; electron microscopy; estradiol; flow cytometry; follicle stimulating hormone; gonadotropins; graafian follicles; granulosa cell; hormone receptor; laboratory rat; luteinizing hormone; progesterone; somatotropin; tissue /cell culture; transfection

The growth and development of ovarian follicles is dependent upon the population of granulosa cells present in the follicle. A systematic study of the growth and differentiation of granulosa cells has been difficult because of the differences in granulosa cells within the same follicle. Functionally different populations of granulosa cells can be separated using Percoll gradients from the ovaries of immature rats treated with diethyl stilbestrol (DES) to induce follicular growth and pregnant mare's serum gonadotropins (PMSG) to induce follicular growth and differentiation. Two populations of granulosa cells gave been shown to exist by flow cytometry and EM studies. Granulosa cells from DES and PMSG treated rats will be separated by the use of an EPICS 5 flow cytometer and Percoll gradients. The cells will be characterized by light and electron microscopy, presence of FSH and LH receptors and FSH induced estradiol and progesterone production. A combination of the enrichment of ovarian follicles by size and granulosa cell separation is expected to yield populations of granulosa cells in various stages of growth and differentiation. The use of the DES and PMSG primed immature rats is being made to work out experimental procedures in a cost effective manner with minimal animal use and the results obtained will be verified in the adult cycling rat. In order to obtain sufficient numbers of granulosa cells for further studies in different stages of growth and differentiation with use of a minimal number of animals, immortalization by transfection with the large T antigen of the SV40 virus will be attempted to generate stable cell lines. Such transfection studies have been successful in our hands using bovine adrenal cells in which the cells retained their pretransfection characteristics. With purified granulosa cell population with different growth and differentiation characteristics, detailed studies will then be done on the regulation of growth and differentiation by agents such as growth factors, estradiol, cAMP analogues, and FSH singly or in combination in a serum free defined medium. The effect of androgens on the stage dependent stimulation or inhibition of aromatase will also be studied. These studies are expected to provide new insights into the factors that lead to healthy ovulatory follicles or atretic follicles during the process of folliculogenesis.

卵泡的生长发育取决于 卵泡中存在的颗粒细胞群。 系统研究 颗粒细胞的生长和分化一直很困难 因为同一卵泡内的颗粒细胞存在差异。 可以分离功能不同的颗粒细胞群 使用来自经以下处理的未成熟大鼠卵巢的 Percoll 梯度 己烯雌酚 (DES) 诱导卵泡生长和怀孕母马 血清促性腺激素（PMSG）诱导卵泡生长和分化。 流动表明存在两个颗粒细胞群 细胞计数和电镜研究。 来自 DES 和 PMSG 处理的大鼠的颗粒细胞 将使用 EPICS 5 流式细胞仪和 Percoll 进行分离 梯度。 细胞将通过光和电子来表征 显微镜下观察，FSH 和 LH 受体的存在以及 FSH 诱导的雌二醇和 黄体酮的产生。 丰富卵巢的组合 按大小和颗粒细胞分离的卵泡预计产量 不同生长阶段的颗粒细胞群 差异化。 DES 和 PMSG 引发的未成熟大鼠的使用正在 以具有成本效益的方式制定实验程序 尽量减少动物的使用，所获得的结果将在成人中得到验证 骑自行车的老鼠。 为了获得足够数量的颗粒细胞 进一步研究不同生长阶段和分化的使用 最少数量的动物，通过转染永生化 SV40病毒大T抗原将尝试产生稳定细胞 线。 此类转染研究已在我们手中取得成功，使用 牛肾上腺细胞，其中细胞保留转染前的状态 特征。 纯化的颗粒细胞群具有不同的 生长和分化特征，随后将进行详细研究 对生长和分化的调节进行了研究，例如 生长因子、雌二醇、cAMP 类似物和 FSH 单独或组合 在无血清成分确定的培养基中。 雄激素对舞台的影响 还将研究芳香酶的依赖性刺激或抑制。 这些研究有望为影响因素提供新的见解 在此过程中产生健康的排卵卵泡或闭锁卵泡 卵泡发生。