Metabolic signalling to the ovary

我的长期研究目标是了解卵巢代谢信号的机制，为我的NSERC研究项目的继续寻求资金支持。目前资助的短期目标是了解胰岛素样生长因子1 （IGF1）系统在调节卵巢功能中的作用。具体目的是：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信号传导中的作用机制；3)揭示促性腺激素调节牛颗粒细胞中PAPPA表达的分子机制**理论依据：泌乳奶牛不育是由于产奶代谢需求高导致的严重负能量平衡所致。代谢激素如胰岛素、瘦素和IGF1水平异常是负能量平衡的主要特征。我们最近的数据表明，与小母牛相比，泌乳母牛体内IGF1的循环浓度显著降低。与处于相似发育阶段的母牛相比，泌乳母牛的优势卵泡的卵泡液反映了这种异常的内分泌环境。乳牛卵泡液IGF1浓度降低与显性卵泡颗粒细胞中PAPPA表达降低有关。由于PAPPA是IGF1结合蛋白的蛋白酶，这些数据表明，泌乳奶牛的卵泡微环境严重缺乏生物活性的IGF1。因此，更好地了解卵巢卵泡中IGF1的分子机制将有助于开发新的治疗和/或管理策略，以提高泌乳奶牛的生育能力。**对于目标一，我们建议利用我们在cree -recombinase定向基因删除方面的专业知识，开发肝脏和颗粒特异性Igf1敲除小鼠。为此，我们将携带Igf1条件等位基因的小鼠与分别在肝脏（Alb1-Cre）和颗粒细胞（Cyp19-Cre）中特异性表达cre -重组酶的小鼠杂交。我们将采用广泛的表型方法来确定肝脏或颗粒细胞中Igf1产生的减少对卵巢卵泡动力学、生殖激素谱和生育能力的影响。第二个目的，我们将使用一个完善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控中的信号通路。我们将采用基因敲低和药物抑制策略来检测牛颗粒细胞中IGF1R的信号通路。对于第三个目标，我们将首先采用一种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的表达模式、其在卵泡液中的丰度及其在卵泡特定发育阶段的蛋白酶活性。然后，我们将利用尖端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。我们的目标是确定参与fsh调控的PAPPA表达的信号通路和转录因子。**总的来说，本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。

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.