Metabolic signalling to the ovary

卵巢的代谢信号

• 批准号: RGPIN-2014-03956
• 负责人: Duggavathi, Raj
• 金额: $ 2.19万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=610746
• 关键词: Metabolic; signalling; ovary

Financial support is sought for the continuation of my NSERC research program with a long-term goal of understanding the mechanisms metabolic signaling to the ovary. The short-term goal for the present period of funding is to understand the role of insulin like growth factor 1 (IGF1) system in the regulation of ovarian function. Specific objectives are: 1) To explore the importance of the IGF1 of liver and granulosa cell origin; 2) to determine the mechanisms of IGF1 receptor in FSH signalling; and 3) to decipher molecular mechanisms by which gonadotropins regulate the expression of PAPPA in bovine granulosa cells RATIONALE: Infertility in lactating dairy cattle has been attributed to severe negative energy balance due to high metabolic demands of milk production. Abnormal levels of metabolic hormone such as insulin, leptin and IGF1 are the major feature negative energy balance. Our recent data demonstrate that circulating concentrations of IGF1 are dramatically reduced in lactating cows as compared to heifers. This abnormal endocrine milieu is reflected in the follicular fluid of the dominant follicles of lactating cows as compared to those at similar developmental stage in heifers. Reduced follicular fluid IGF1 concentration is associated with decreased expression of PAPPA in granulosa cells of the dominant follicle in lactating cows. As PAPPA is a protease of IGF1 binding proteins, these data indicate that the follicular microenvironment of lactating cows is severely deficient of bioactive IGF1. Thus, a better understanding of the molecular mechanisms of IGF1 in ovarian follicles will contribute toward development of novel treatment and/or management strategies to improve fertility in lactating dairy cows. For objective one, we propose to develop liver- and granulosa-specific Igf1 knockout mice using our expertise in cre-recombinase directed deletion of genes. For this we will breed mice carrying conditional allele of Igf1 with mice expressing Cre-recombinase specifically in the liver (Alb1-Cre) and granulosa cells (Cyp19-Cre), respectively. We will undertake extensive phenotyping approaches to determine consequences of loss of Igf1 production in the liver or granulosa cells on ovarian follicular dynamics, reproductive hormonal profile and fertility. For the second objective, we will use a well-established culture system of bovine primary granulosa cells to investigate the signaling pathways of IGF1R in the regulation of granulosa cell proliferation and steroidogenesis. We will employ gene knockdown and pharmacologic inhibition strategies to test signaling pathways of IGF1R in bovine granulosa cells. For the third objective, we will first employ an in vivo ultrasound guided follicular aspiration strategy to synchronize follicular wave and collect samples. We will systematically analyze expression pattern of PAPPA, its abundance in follicular fluid and its protease activity in follicles at specific developmental stages. We will then investigate the molecular mechanisms by which FSH regulates the expression of PAPPA in bovine granulosa cells using cutting edge molecular biology techniques. Our aim will be to determine signaling pathways and transcription factors involved in FSH-regulated expression of PAPPA. Overall, the results of the proposed research program should significantly enhance our understanding how IGF1 system regulates ovarian functions and thus female fertility.

我的NSERC研究项目的长期目标是了解卵巢代谢信号的机制，因此我寻求资金支持。目前资金的短期目标是了解胰岛素样生长因子1(IGF1)系统在卵巢功能调节中的作用。具体目标是：1)探讨IGF1在肝脏和颗粒细胞起源中的重要性；2)确定IGF1受体在FSH信号转导中的机制；以及3)破译促性腺激素调节牛颗粒细胞PappA表达的分子机制。 基本原理：泌乳奶牛的不孕不育是由于产奶量的高代谢需求造成的严重的能量负平衡。胰岛素、瘦素、IGF1等代谢激素水平异常是能量负平衡的主要特征。我们最近的数据表明，与小母牛相比，哺乳期奶牛体内IGF1的循环浓度显著降低。与处于类似发育阶段的小母牛相比，这种异常的内分泌环境反映在哺乳期奶牛优势卵泡的卵泡液中。在哺乳期奶牛，卵泡液IGF1浓度降低与优势卵泡颗粒细胞中Pappa表达减少有关。由于PappA是IGF1结合蛋白的一种蛋白酶，这些数据表明奶牛卵泡微环境中严重缺乏具有生物活性的IGF1。因此，更好地了解IGF1在卵泡中的分子机制将有助于开发新的治疗和/或管理策略来提高奶牛的繁殖力。 对于第一个目标，我们建议利用我们在重组酶引导的基因缺失方面的专业知识来开发肝脏和颗粒特异性的Igf1基因敲除小鼠。为此，我们将培育携带条件等位基因Igf1的小鼠和分别在肝脏(Alb1-Cre)和颗粒细胞(Cyp19-Cre)特异表达Cre-重组酶的小鼠。我们将采用广泛的表型方法来确定肝脏或颗粒细胞中Igf1产生缺失对卵巢卵泡动力学、生殖激素谱和生育能力的影响。对于第二个目标，我们将使用成熟的牛原代颗粒细胞培养系统来研究IGF1R在颗粒细胞增殖和类固醇生成调节中的信号通路。我们将使用基因敲除和药物抑制策略来测试牛颗粒细胞中IGF1R的信号通路。对于第三个目标，我们将首先使用体内超声引导的卵泡抽吸策略来同步卵泡波并收集样本。我们将系统分析Pappa在卵泡液中的表达模式、在卵泡液中的丰度以及在特定发育阶段的卵泡中的蛋白酶活性。然后，我们将利用前沿的分子生物学技术研究FSH调节牛颗粒细胞中Pappa表达的分子机制。我们的目标将是确定参与FSH调控的Pappa表达的信号通路和转录因子。 总体而言，拟议的研究计划的结果将显著增强我们对IGF1系统如何调节卵巢功能从而调节女性生育能力的理解。