Integrative physiology of ovarian follicular development and ovulation

在过去的几十年里，牛产业的繁殖效率在全球范围内下降，这给生产者带来了重大的经济损失。这种生育能力下降的原因是多方面的;女性方面的核心问题是卵巢生理学。该研究计划的长期目标是通过研究卵泡生长，排卵和早期黄体化的最后阶段颗粒细胞（GC）的生理学来更好地了解卵泡的发育。在以前的研究中，我们已经建立了一个差异表达的基因指数GC在卵泡发育的关键阶段。该提案的目的是确定这些先前鉴定的新型差异表达基因的生物学功能，包括低密度脂蛋白受体相关蛋白8（LRP 8）和两个长链非编码RNA（lncRNA）。我们的具体目标将测试这一假设，即从卵泡膜层释放的Reelin（LRP 8的配体）和GC表达的膜受体LRP 8之间的旁分泌相互作用调节卵泡生长的最后阶段。我们将：1）定义结合由GC表达的LRP 8的细胞内和细胞外结构域的蛋白质; 2）理解LRP 8基因表达如何被激活;和3）证明在膜受体LRP 8激活后细胞外配体对GC生理学的生理作用。对于这两种新的lncRNA，我们假设它们代表控制特定基因转录的RNA调节因子。短期目标是通过定义：1）它们的蛋白质结合伴侣; 2）它们在牛基因组中的结合位点，以确定哪些基因受它们的控制，从而揭示它们在GC中的生物学功能。这项研究计划将大大提高我们对GC增殖和分化调节最终卵泡生长的关键生化过程的理解，这是卵泡对影响生育能力的代谢应激最敏感的发育阶段。了解哪些配体激活LRP 8膜受体以及LRP 8如何在GC内转导其细胞外信号将为开发激动剂以刺激LRP 8活性提供见解，这反过来可能有助于对抗代谢应激的影响。这项研究计划将进一步推进我们的理解：1）提高针对牛卵泡同步化和卵巢刺激计划的治疗效果; 2）增加用于体外受精，核移植和基因组编辑的合格卵母细胞的产量; 3）鉴定与育性相关的候选基因，其可用作遗传辅助育种的分子标记。选择参与商业化和分配雄性和雌性配子的行业的育种计划;和4）促进高素质科学人才的培训。

The last several decades have born witness to a global decline in reproductive efficiency within the bovine industry which has translated into significant economic losses for producers. The causes of this reduced fertility are multifactorial; central to the problem on the female side is ovarian physiology. The long-term goal of this research program is to better comprehend the development of ovarian follicles by studying the physiology of granulosa cells (GC) during the final stages of follicular growth, ovulation and early luteinisation. In previous studies, we have established a differentially expressed gene index for GC at critical stages of follicular development. The objectives of this proposal are to determine the biological functions of these novel and differentially expressed genes previously identified, including the low-density lipoprotein receptor-related protein 8 (LRP8) and two long non-coding RNAs (lncRNA). Our specific objectives will test the hypothesis that a paracrine interaction between Reelin (a ligand of LRP8) released from the theca layer and the membrane receptor LRP8 expressed by GC regulate the final stages of follicular growth. We will: 1) define proteins that bind the intracellular and extracellular domains of LRP8 expressed by GC; 2) comprehend how LRP8 gene expression is activated; and 3) demonstrate the physiological effects of extracellular ligands on GC physiology following activation of the membrane receptor LRP8. For the two novel lncRNAs, we hypothesize that they represent RNA regulators controlling transcription of specific genes. The short-term objective is to uncover their biological function in GC by defining: 1) their protein binding partners; and 2) their binding sites within the bovine genome to determine which genes are under their control. This research program will significantly advance our understanding of key biochemical processes involved in GC proliferation and differentiation regulating final follicular growth, a stage of development at which follicles are most sensitive to metabolic stresses that impact fertility. Understanding which ligands activate the LRP8 membrane receptor and how LRP8 transduces its extracellular signals within GC will provide insights into the development of agonists to stimulate the LRP8 activity that may in turn help contravene the effects of metabolic stresses. This research program will further advance our understanding: 1) To enhance the efficacy of treatments that aim at follicle synchronization and ovarian stimulation programs in cattle; 2) To increase the production of competent oocytes used for in vitro fertilization, nuclear transfer and genome editing; 3) To identify candidate genes associated with fertility that can be used as molecular markers for genetic assisted-selection in breeding programs of industries involved in commercializing and distributing male and female gametes; and 4) To promote the training of highly qualified personnel in the science.