Metabolic signalling to the ovary

资金要求继续建立和生产NSERC支持的研究计划，长期目标是确定卵巢代谢信号的机制。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急剧增加有关。淘汰泌乳奶牛的主要原因是生殖问题，在过去五年中，有17-18%的动物从牛群中淘汰。泌乳奶牛的不孕症归因于产犊后它们经历的巨大代谢变化。因此，需要开发新的策略来优化泌乳奶牛的生育力，这可以通过增强对其不孕症发病机制的理解来实现。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。资助期的目标是研究：1）泌乳奶牛中差异调节的颗粒细胞转录组; 2）β-羟基丁酸（BHBA）对体外牛颗粒细胞直接影响的机制; 3）使用条件性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有代谢应激的泌乳奶牛和没有代谢应激的未产小母牛的卵巢颗粒细胞的转录组。对于目标2，将用小母牛和泌乳奶牛中发现的剂量的BHBA挑战颗粒细胞的原代培养物，以研究BHBA在颗粒细胞基因表达和功能的表观遗传调节中的作用。对于目标2，我们将开发缺乏Pappa基因（调节胰岛素样生长因子（IGF）生物利用度的蛋白酶）的颗粒细胞特异性小鼠系。广泛的表型和分子研究将提供深入了解由于缺乏Pappa活性而导致的IGF 1信号传导减少对卵巢功能和女性生育力的影响。这些研究将产生适用于加拿大奶牛的有影响力的数据，包括泌乳奶牛颗粒细胞中的全球基因表达，代谢物BHBA的直接靶点以及Pappa蛋白酶的调节机制。此外，通过这些研究开发的见解也适用于哺乳动物卵巢和雌性生育力的代谢调节。

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.