Metabolic signalling to the ovary

NSERC支持的一项已建立的、富有成效的研究计划的长期目标是确定向卵巢传递代谢信号的机制，因此需要资金来继续这一计划。加拿大奶牛群在遗传改良产奶量方面取得了令人难以置信的成功，这也与非自愿淘汰率的戏剧性增加有关。淘汰哺乳奶牛的主要原因是繁殖问题，在过去的五年中，17%-18%的奶牛被淘汰。哺乳期奶牛的不孕症归因于产后经历的巨大新陈代谢变化。因此，有必要开发新的策略来优化哺乳期奶牛的生育能力，这可以通过加强对其不孕症发病机制的了解来实现。我的研究计划的长期目标是了解卵巢功能的代谢调节机制。资助期间的目标是研究：1)哺乳奶牛颗粒细胞转录组的差异调控；2)β-羟基丁酸(BHBA)对体外培养的牛颗粒细胞直接作用的机制；以及3)利用条件基因敲除小鼠模型研究Pappa在卵巢中的作用。对于目标1，我建议使用RNA测序技术和生物信息学工具来比较具有代谢应激的哺乳期奶牛和没有代谢应激的未分娩小母牛的卵巢颗粒细胞的转录组。在目标2中，用小母牛和哺乳期奶牛体内的BHBA刺激颗粒细胞的原代培养，以研究BHBA在颗粒细胞基因表达和功能的表观遗传调控中的作用。对于目标2，我们将建立缺乏Pappa基因的颗粒特异性小鼠系，Pappa基因是一种调节胰岛素样生长因子(IGF)生物利用度的蛋白酶。广泛的表型和分子研究将深入了解由于缺乏PappA活性而导致的IGF1信号降低对卵巢功能和女性生育能力的影响。这些研究将产生适用于加拿大奶牛的有效数据，包括哺乳期奶牛颗粒细胞的全球基因表达、代谢物BHBA的直接靶标以及Pappa蛋白酶的调控机制。此外，通过这些研究得出的见解也适用于哺乳动物卵巢和雌性生育能力的代谢调节。

Funds are requested for continuation of an established and productive NSERC-supported research program with the long-term objective of determining the mechanisms of metabolic signaling to the ovary. Incredible success in genetic improvement of milk yield in Canadian dairy herds is also associated with a dramatic increase in involuntary culling rates. The major reason for culling of lactating cows is reproductive problems, with 17-18% of the animals eliminated from herds over last five years. Infertility in lactating cows is attributed to the tremendous metabolic alterations they go through after calving. Thus, there is a need to develop new strategies to optimize fertility in lactating cows, which can be accomplished with enhanced understanding of the pathogenesis of their infertility. The long-term goal of my research program is to understand the mechanisms of metabolic regulation of ovarian function. The objectives for the funding period are to investigate: 1) the differentially regulated granulosa cell transcriptome in lactating dairy cows; 2) the mechanisms of the direct effects of beta-hydroxybutyric acid (BHBA) on bovine granulosa cells in vitro; and 3) the role of Pappa in the ovary using a conditional knockout mouse model. For Objective 1, I propose to use RNA-sequencing technology and bioinformatic tools to compare the transcriptome of ovarian granulosa cells in lactating cows that have metabolic stress and nulliparous heifers that do not have metabolic stress. For Objective 2, primary cultures of granulosa cells will be challenged with BHBA at doses found in heifers and lactating cows to investigate the role of BHBA in epigenetic regulation of granulosa cells gene expression and functions. For Objective 2, we will develop granulosa-specific mouse line lacking Pappa gene, the protease that regulates the insulin-like growth factor (IGF) bioavailability. Extensive phenotyping and molecular studies will provide insights into effects of reduced IGF1 signaling due to lack of Pappa activity on ovarian functions and female fertility. These studies will produce impactful data that are applicable to Canadian dairy cows including global gene expression in granulosa cells of lactating cows, direct targets of the metabolite BHBA and mechanisms of regulation by Pappa protease. In addition, the insights developed through these studies are also applicable to metabolic regulation of the ovary and female fertility in mammals.