GROWTH FACTOR CONTROL OF OVARIAN ANDROGEN BIOSYNTHESIS

卵巢卵泡膜间质细胞（TIC）是卵巢上皮细胞的主要来源。 卵泡雌激素生物合成的雄激素底物。 证据 文献清楚地表明，TIC的及时进展 分化是正常卵巢功能所必需的，然而， 激素调节TIC分化的机制很差， 明白 为了研究TIC的分子机制， 在健康和疾病状态的差异，我们已经制定了一个 分离高度纯化的TIC群体的独特方法， 没有颗粒细胞污染。 重要的是 纯化的TIC对无血清中的激素调节有反应， 培养基，并提供最佳可用模型来研究激素 TIC分化过程中基因表达的调节。 我们有 表明LH是调节TIC分化重要激素， 但LH本身不能解释基因表达的模式， 在发育卵泡的TIC中观察到。 在第一阶段 TIC表达LH受体和胆固醇侧 链裂解（P450 SCC），但不含17 α-羟化酶/C17-20裂解酶 （P45017 α），尽管P450 SCC和P45017 α的mRNA 都被转录了 我们已经证明胰岛素样生长因子I （IGF-I）单独刺激P450 SCC和P45017 α mRNA， 选择性表达P450 SCC蛋白。 的含义 这一发现表明IGF-I可能在脑缺血再灌注损伤中发挥重要的生理作用。 在调节TIC分化的早期阶段。 整体 本申请的目的是了解的生理作用 IGF-I在调节TIC分化中的作用，并阐明 IGF-I单独和与LH协同作用的分子机制 调节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发育过程中基因表达 发育和闭锁。 IGF-I在卵巢颗粒细胞中的表达 细胞与TIC中LH和IGF-I的表达相关 受体，cAMP依赖性蛋白激酶调节和催化 亚基，和P450 SCC，3 β-羟基类固醇脱氢酶， P45017阿尔法 此外，我们还试图确定 基因表达发生在卵泡膜发育的第一阶段， 当TIC从成纤维细胞样前体分化时的分化 细胞转化为特征性内膜细胞。 研究将 进行比较和对比的转录和 使用核径流测定的IGF-I与LH的翻译作用， RT-PCR和免疫沉淀技术。 最后，研究将 确定第二信使系统的细节 相互作用负责协同作用之间 IGF-I和LH。 从这些研究中，我们希望了解基本的新 关于早期鞘分化的生理学的信息， 卵泡发育和闭锁，以及这些过程是如何 由当地生产的荷尔蒙调节。 这些结果将帮助我们 了解高雄激素血症在疾病中的机制， 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.