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: 3330470
• 负责人: Denis A Magoffin
• 金额: $ 16.96万
• 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/3330470
• 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.

卵巢的主要功能是周期性的生产和排出 一个可受精的卵母细胞。 这一进程的核心是及时和 卵泡膜细胞和颗粒细胞的协调分化。 两个细胞， 卵泡雌激素生物合成的双促性腺激素概念是 卵泡细胞分化的基本机制。 那里 然而，卵巢生理学的许多方面无法解释 促性腺激素的作用 最近，自分泌和 卵巢中的旁分泌调节受到了相当大的关注， 发现许多最初被描述为生长的肽 这些因子由卵巢中的卵泡膜和颗粒细胞局部产生。 这个概念特别有吸引力，因为它有助于解释 卵泡膜和颗粒细胞可以相互沟通， 来自大脑的主要信号 在初步研究中， 获得的证据表明，转化生长因子-β（TGF-β），产生 可能是卵泡膜细胞的重要调节因子， 雄激素的产生，并可能赋予FSH对颗粒细胞的反应性 在小卵泡中。 本提案的总体目标是审查 TGF-β在卵泡发育、选择和闭锁中的作用。 到 为实现这一目标，我们将实施四个具体目标。 一是 TGF-β控制卵泡膜类固醇生成的机制将是 研究了 将对LH进行时程和剂量反应研究 和TGF-β单独和组合以确定TGF-β 对P450、3 β-HSD和P45017 α mRNA水平的影响 转录酶复制cDNA，然后进行聚合酶链反应， 放大信号。 然后我们将确定mRNA水平的变化是否 是使用核径流分析的转录效应。 其次，由于 关于TGF-β分泌的调节是未知的， 通过卵泡膜细胞激活，我们将测试LH，IGF-I，雄激素 和雌激素对卵泡膜TGF-β分泌和激活的影响， TGF-β RIA和生物测定。 第三，我们的初步数据显示， 来自非雌激素环境（例如小卵泡中）的细胞则不会 对FSH刺激有反应。 TGF-β治疗使颗粒细胞 对FSH有反应。 我们将阐明TGF-β 通过检测TGF-β对FSH受体的影响， FSH/cAMP信号通路的含量和亲和力，以及 P450 SCC、3 β-HSD和P450 arom在颗粒细胞中表达。 最后利用 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TGF-β的表达与 卵泡膜和颗粒细胞中类固醇生成酶的表达 健康和闭锁的卵泡，试图了解TGF-β在 卵泡选择和闭锁。 我们建议的研究结果将 进一步了解卵泡的自分泌和旁分泌调节 发展，选择和闭锁，并可能导致了解 多囊卵巢综合征