GROWTH FACTOR CONTROL OF OVARIAN ANDROGEN BIOSYNTHESIS

卵巢膜间质细胞(TIC)是卵泡膜间质细胞(TIC)的主要来源。 卵泡雌激素生物合成的雄激素底物。证据在 文献清楚地表明，TIC的及时进展 分化对于正常卵巢功能是必不可少的，然而 激素调节TIC分化的机制很差 明白了。为了研究TIC的分子机制 在健康和疾病状态下的区分，我们已经开发出一种 一种分离高纯度TIC种群的独特方法 没有颗粒细胞污染。值得注意的是， 纯化的TIC在无血清条件下对激素调节的反应 为激素研究提供了最佳的可用模型 TIC分化过程中基因表达的调控。我们有 提示黄体生成素是调节TIC分化的重要激素。 但是单凭黄体生成素不能解释基因表达的模式。 在TIC中观察到发育中的卵泡。在第一阶段 分化方面，TIC表达黄体生成素受体和胆固醇侧 链断裂(P450scc)，但不是17α-羟基酶/C17-20裂解酶 (P45017pha)即使P450scc和P45017pha的mRNA 都被转录了下来。我们已经证明胰岛素样生长因子I IGF-I单独刺激P450scc和P45017α基因的表达 P450scc蛋白在TIC中的选择性表达隐含的含义 这一发现表明，IGF-I可能起着重要的生理作用 在调节TIC分化的早期阶段。整体而言 本应用程序的目标是了解 IGF-I在调节TIC分化中的作用及其机制 胰岛素样生长因子-I单独及与黄体生成素协同作用的分子机制 目的：调控TIC中基因的表达。原位杂交 将与放射自显影、组织化学和 免疫荧光法检测卵泡中基因的表达 发育和闭锁。胰岛素样生长因子-I在颗粒细胞中的表达 细胞与TIC中促黄体生成素和胰岛素样生长因子-I的表达相关 受体，cAMP依赖的蛋白激酶调节和催化 亚基和P450scc、3β-羟基类固醇脱氢酶和 P45017α。此外，我们还试图确定 卵泡膜第一阶段的基因表达 TIC与成纤维细胞样前体细胞的分化 细胞分化成典型的内膜细胞。研究将会是 执行以比较和对比转录和 用核径流分析研究胰岛素样生长因子-I与黄体生成素的翻译效应， RT-PCR和免疫沉淀技术。最后，研究将是 执行以确定第二信使系统的细节 负责两国之间协同互动的互动 胰岛素样生长因子-I和促黄体生成素。从这些研究中，我们希望学到基本的新知识 关于早期鞘分化的生理学信息， 卵泡发育和闭锁以及这些过程是如何发生的 由当地生产的荷尔蒙调节。这些结果将有助于我们 要了解高雄激素性疾病的机制，如 高胰岛素血症和多囊卵巢疾病。

Ovarian theca-interstitial cells (TIC) are the obligatory source of androgen substrate for follicle estrogen biosynthesis. Evidence in the literature clearly shows that the timely progression of TIC differentiation is essential for normal ovarian function, however the mechanisms by which hormones regulate TIC differentiation are poorly understood. In order to study the molecular mechanisms of TIC differentiation in healthy and diseased states, we have developed a unique method for isolating a population of highly purified TIC which are free from granulosa cell contamination. Significantly, the purified TIC are responsive to hormone regulation in serum-free medium and provide the best available model to study hormone regulation of gene expression during TIC differentiation. We have shown that LH is an important hormone regulating TIC differentiation, but LH alone cannot account for the pattern of gene expression observed in TIC from developing follicles. During the first stages of differentiation, TIC express LH receptors and cholesterol side chain cleavage (P450scc) but not 17alpha-hydroxylase/C17-20 lyase (P45017alpha) even though the mRNA for both P450scc and P45017alpha are transcribed. We have shown that insulin-like growth factor I (IGF-I) alone stimulates both P450scc and P45017alpha mRNA and the selective expression of P450scc protein in TIC. The implication of this finding is that IGF-I may play an important physiological role in regulating the early stages of TIC differentiation. The overall goals of this application are to understand the physiological role of IGF-I in regulating TIC differentiation and to elucidate the molecular mechanisms by which IGF-I acts alone and in concert with LH to regulate the expression of genes in TIC. In situ hybridization will be used together with autoradiography, histochemistry and immunofluorescence to examine gene expression during follicle development and atresia. The expression of IGF-I in the granulosa cells will be correlated with the expression in TIC of LH and IGF-I receptors, cAMP-dependent protein kinase regulatory and catalytic subunits, and P450scc, 3beta-hydroxysteroid dehydrogenase and P45017alpha. In addition we attempt to determine the sequence of gene expression that occurs during the first stages of thecal differentiation when TIC differentiate from fibroblast-like precursor cells into characteristic theca interna cells. Studies will be performed to compare and contrast the transcriptional and translational effects of IGF-I with LH using nuclear runoff assays, RT-PCR and immunoprecipitation techniques. Finally, studies will be performed to determine the details of the second messenger system interactions responsible for the synergistic interactions between IGF-I and LH. From these studies we expect to learn fundamental new information regarding the physiology of early thecal differentiation, follicle development and atresia and how these processes are regulated by locally produced hormones. These results will help us to understand the mechanism of hyperandrogenism in diseases such as hyperinsulinemia and polycystic ovarian disease.