Receptor/Ligand Signals for Ovarian Follicular Angiogenesis

描述（由申请人提供）：卵巢血管在正常卵泡和卵巢癌/肿瘤的发育过程中增加，但启动卵巢血管生成的分子机制尚未明确。通过对囊性和非囊性卵泡颗粒细胞基因表达的高密度芯片分析，我们发现囊性卵泡颗粒细胞血管生成素（ANG） mRNA表达上调，成纤维细胞生长因子-9 (FGF9) mRNA表达下调，表明血管生成因子参与了囊肿的形成。我们的总体假设是，在卵泡发育过程中，颗粒细胞通过分泌ANG和FGF9向内膜发出信号，调节卵泡和血管细胞的分化和增殖。随着卵泡的生长，卵泡和颗粒细胞必须以最小的分化增殖，同时卵泡血管化增加，以便卵泡在卵母细胞完全成熟之前不会过早排卵或黄体化。因此，ANG和/或FGF9的异常产生可能导致血管功能障碍和卵巢疾病。在我们的牛颗粒和卵泡细胞模型中建立的实验范式的初步研究中，我们证明：1)囊泡和正常卵泡的牛颗粒细胞中ANG和FGF9 mRNA的丰度不同；2)颗粒细胞中胰岛素样生长因子- 1 （IGF1）增加了ANG mRNA的丰度；3) FGF9刺激卵泡膜细胞有丝分裂，抑制卵泡膜细胞分化。在Specific Aim 1中，我们将利用实时定量逆转录聚合酶链反应（qRT-PCR）研究在体内卵泡发育过程中颗粒细胞中ANG和FGF9 mRNA以及卵泡细胞中FGF9受体的发育调控，并通过ELISA检测卵泡液中ANG和FGF9的含量。特异性Aim 2将通过qRT-PCR （mRNA）和ELISA（蛋白）确定颗粒细胞产生ANG和FGF9的激素调节。在Specific Aim 3中，我们将确定细胞周期蛋白和甾体生成酶的变化是否与FGF9改变的卵泡细胞增殖和甾体生成有关，并使用微阵列技术确定FGF9调节的其他基因。这些研究将深入了解ANG和FGF9产生的生理控制，并确定ANG和FGF9调节卵泡膜细胞功能的机制。我们的研究已经发现了囊性卵泡的新标记物，也可能为改善女性不育和卵巢疾病（如多囊卵巢疾病和卵巢癌）的治疗提供新的选择。建议的研究将为学生提供宝贵的研究训练经验，并使更多的学生接触到科学发现的兴奋。

DESCRIPTION (provided by applicant): Ovarian vasculature increases during development of normal follicles as well as ovarian cancer/tumors, but molecular mechanisms that initiate ovarian angiogenesis are not well defined. Based on high-density microarray analysis comparing granulosa cell gene expression in cystic and noncystic follicles, we discovered that granulosa cell angiogenin (ANG) mRNA is up-regulated and fibroblast growth factor-9 (FGF9) mRNA is down-regulated in cystic follicles suggesting that angiogenic factors are involved in cyst formation. Our overall hypothesis is that granulosa cells signal theca interna via ANG and FGF9 secretion to regulate theca and vascular cell differentiation and proliferation during follicular development. As the follicle grows, theca and granulosa cells must proliferate with minimal differentiation while thecal vascularization increases so that follicles do not prematurely ovulate or luteinize before the oocyte is fully mature. Thus, aberrant production of ANG and/or FGF9 may lead to vascular dysfunction and ovarian disorders. In preliminary studies using experimental paradigms developed in our bovine granulosa and theca cell model, we demonstrated that: 1) abundance of ANG and FGF9 mRNA in bovine granulosa cells of cystic and normal follicles differ; 2) abundance of ANG mRNA is increased by insulin-like growth factor-I (IGF1) in granulosa cells; and 3) FGF9 stimulates theca cell mitosis while inhibiting theca cell differentiation. In Specific Aim 1, we will investigate the developmental regulation of ANG and FGF9 mRNA in granulosa cells and FGF9 receptors in theca cells during follicular development in vivo using real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) as well as measure ANG and FGF9 in follicular fluid via ELISA. Specific Aim 2 will determine the hormonal regulation of ANG and FGF9 production by granulosa cells using qRT-PCR (mRNA) and ELISA (protein). In Specific Aim 3, we will determine if changes in cell-cycle proteins and steroidogenic enzymes are associated with FGF9-altered theca cell proliferation and steroidogenesis, and identify additional genes regulated by FGF9 using microarray technology. These studies will provide insight into the physiological control of ANG and FGF9 production and determine mechanisms by which ANG and FGF9 regulate theca cell functions. Our studies have lead to newly discovered markers for cystic follicles and may also lead to new options for improved treatment of female infertility and ovarian pathologies such as polycystic ovarian disease and ovarian cancer. The proposed research will provide valuable research training experiences for students and expose many more students to the excitement of scientific discovery. PUBLIC HEALTH RELEVANCE: Increased vascularity occurs during normal ovarian follicle development, and studies show that blood vessel growth (angiogenesis) is activated in ovarian carcinomas. Our findings of increased expression of angiogenin in cystic follicles may indicate that an increased vascularity is part of the mechanism of development of cystic follicles. Along with providing advances in our understanding of female fertility and valuable research training experiences for undergraduate students, these studies should lead to new options for improved treatment of female infertility and ovarian pathologies such as polycystic ovarian disease and ovarian cancer.