PARACRINE ROLE OF OVARIAN TRANSFORMING GROWTH FACTOR-B

卵巢转化生长因子-B 的旁分泌作用

• 批准号: 3330469
• 负责人: Denis A Magoffin
• 金额: $ 17.28万
• 依托单位: CEDARS-SINAI MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3330469
• 关键词: RNA directed DNA polymerase; androgens; autoradiography; bioassay; cell growth regulation; cyclic AMP; cytochrome P450; enzyme activity; estrogens; follicle stimulating hormone; gene expression; granulosa cell; hormone regulation /control mechanism; hydroxysteroid dehydrogenases; hypophysectomy; immunocytochemistry; immunofluorescence technique; in situ hybridization; insulinlike growth factor; laboratory rat; luteinizing hormone; messenger RNA; nuclear runoff assay; ovary; paracrine; polymerase chain reaction; radioimmunoassay; steroid hormone biosynthesis; steroid hormone receptor; tissue /cell culture; transforming growth factors

The principal purpose of the ovary is the periodic production and expulsion of a fertilizable oocyte. Central to this process is the timely and coordinate-differentiation of the theca and granulosa cells. The two-cell, two-gonadotropin concept of follicle estrogen biosynthesis is the fundamental mechanism by which follicle cell differentiation occurs. There are, however many aspects of ovarian physiology which cannot be explained by the gonadotropins alone. Recently, the concepts of autocrine and paracrine regulation in the ovary have received considerable attention with the discovery that a number of peptides originally described as growth factors are produced locally by theca and granulosa cells in the ovary. This concept is particularly attractive because it helps to explain how the theca and granulosa cells can communicate with each other and fine tune the primary signals coming from the brain. In preliminary studies, we have obtained evidence that transforming growth factor-beta (TGF-beta), produced primarily by the theca cells, may be an important regulator of thecal androgen production and may confer FSH responsiveness to granulosa cells in-small follicles. The overall goal of this proposal is to examine the role of TGF-beta in follicle development, selection and atresia. To accomplish this goal we will execute four specific aims. First, the mechanism by which TGF-beta controls thecal steroidogenesis will be studied. Time course and dose-response studies will be performed with LH and TGF-beta alone and in combination to determine the effects of TGF-beta on thecal P450 , 3beta-HSD and P45017alpha mRNA levels using reverse transcriptase to copy cDNA and then the polymerase chain reaction to amplify the signal. We will then determine if the changes in mRNA levels are transcriptional effects using nuclear runoff assays. Second, since nothing is known regarding the regulation of TGF-beta secretion and activation by theca cells, we will test the effects of LH, IGF-I, androgens and estrogens on thecal TGF-beta secretion and activation using a specific TGF-beta RIA and bioassay. Third, our preliminary data show that granulosa cells from a non-estrogenic environment such as in small follicles do not respond to FSH stimulation. TGF-beta treatment renders the granulosa cells responsive to FSH. We will elucidate the mechanism by which TGF-beta exerts this effect by examining the effects of TGF-beta on FSH receptor content and affinity the FSH/cAMP signaling pathway, and the expression of P450scc, 3beta-HSD and P450arom in granulosa cells. Finally, using the techniques of in situ hybridization, immuncytochemistry, autoradiography and immunofluorescence we will correlate the expression of TGF-beta with the expression of steroidogenic enzymes in the theca and granulosa cells in healthy and atretic follicles to try to understand the role of TGF-beta in follicle selection and atresia. The results of our proposed studies will further our understanding of autocrine and paracrine regulation in follicle development, selection and atresia and may lead to an understanding of polycystic ovarian disease.

卵巢的主要目的是周期性地产生和排出。 指可受精的卵母细胞。这一进程的核心是及时和 膜细胞和颗粒细胞的协调分化。两个牢房， 双促性腺激素概念的卵泡雌激素的生物合成是 毛囊细胞分化发生的基本机制。那里 然而，卵巢生理的许多方面是无法解释的 仅靠促性腺激素。最近，自分泌的概念和 卵巢中的旁分泌调节受到了相当大的关注 一些最初被描述为生长的多肽的发现 因子由卵巢中的卵泡膜和颗粒细胞局部产生。 这个概念特别吸引人，因为它有助于解释 卵泡膜细胞和颗粒细胞可以相互通信，并微调 主要信号来自大脑。在初步研究中，我们有 获得的证据表明转化生长因子-β(转化生长因子-β)，产生 主要由膜细胞调节，可能是膜细胞的重要调节因子 雄激素的产生和FSH对颗粒细胞的反应性 小卵泡。这项提案的总体目标是研究 转化生长因子-β在卵泡发育、选择和闭锁中的作用至 实现这一目标，我们将落实四个具体目标。首先， 转化生长因子-β调控鞘类固醇合成的机制将是 学习。将对黄体生成素进行时间进程和剂量-反应研究 和转化生长因子-β单独及联合应用以确定转化生长因子-β的作用 用逆转录法检测卵泡膜P450、3β-HSD和P45017α基因的表达 转录酶复制cDNA，然后进行聚合酶链式反应 放大信号。然后我们将确定mRNA水平的变化 是使用核径流分析的转录效应。第二，因为 关于转化生长因子-β的分泌和调节尚不清楚 被膜细胞激活后，我们将测试促黄体生成素、胰岛素样生长因子-I、雄激素的作用 和雌激素对卵泡膜转化生长因子-β分泌和激活的影响 转化生长因子-β放射免疫分析和生物测定。第三，我们的初步数据显示，颗粒 来自非雌激素环境的细胞，如小卵泡，不会 对促卵泡激素刺激的反应。转化生长因子-β处理使颗粒细胞 对卵泡刺激素有反应。我们将阐明转化生长因子-β的作用机制 通过检测转化生长因子-β对卵泡刺激素受体的影响来发挥这种作用 FSH/cAMP信号通路的含量和亲和力，以及 颗粒细胞表达P450scc、3β-HSD和P450arom。最后，使用 原位杂交、免疫细胞化学、放射自显影技术 和免疫荧光我们将转化生长因子-β的表达与 类固醇产生酶在幼鼠卵泡膜细胞和颗粒细胞中的表达 了解转化生长因子-β在卵泡发育中的作用 卵泡选择和闭锁。我们建议的研究结果将 加深对卵泡自分泌和旁分泌调节的认识 发展、选择和闭锁，并可能导致对 多囊性卵巢疾病。