Intercellular dynamics of cyclic nucleotides in ovarian follicles

卵巢卵泡中环核苷酸的细胞间动力学

• 批准号: 10380740
• 负责人: Jeremy R. Egbert
• 金额: $ 8.2万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2023-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10380740
• 关键词: Address; Adenylate Cyclase; Attenuated; Basal lamina; Binding; Breeding; Carbenoxolone; Cells; Clinical; Communication; Confocal Microscopy; Crossbreeding; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic Nucleotides; Diffuse; Diffusion; Dose; Environment; Epidermal Growth Factor Receptor; Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor; Epitopes; Exhibits; Exposure to; Family; Fluorescence Resonance Energy Transfer; Future; Gap Junctions; Hormone use; Hour; Human; Image; In Vitro; Incubated; Individual; Knowledge; LH Receptors; Lead; Light; Luteinization; Luteinizing Hormone; Mammals; Measurement; Mediating; Meiosis; Microscope; Microscopy; Mus; Oocytes; Outcome; Ovarian Follicle; Ovary; Ovulation; PKA inhibitor; Pattern; Phosphorylation; Physiological; Physiological Processes; Production; Receptor Activation; Reproductive Health; Research; Resolution; Second Messenger Systems; Signal Transduction; Somatic Cell; Suggestion; Testing; Time; Tissues; Toxic effect; egg; granulosa cell; hormonal signals; infertility treatment; inhibitor; novel strategies; oocyte maturation; prevent; response; sensor; spatiotemporal

Project Summary/Abstract In mammalian ovaries, the mid-cycle surge of luteinizing hormone (LH) acts on the granulosa cells of preovulatory follicles to trigger oocyte maturation, ovulation, and luteinization. Signaling through LH receptors occurs primarily occurs through sequential Gs-mediated adenylyl cyclase activation, production of the second messenger cAMP, and protein kinase A-dependent phosphorylation. However, whether and how the cAMP signal spreads from the subset of cells that express the LH receptor (LHR) to achieve the many outcomes of the LH surge is a long-standing question that remains to be resolved. To test this and other questions, intact preovulatory follicles will be isolated from mice that globally express a newly developed fluorescent sensor for cAMP called R-FlincA that can increase brightness by up to 600% upon cAMP binding. Using R-FlincA and state-of-the-art light sheet microscopy, individual cells can be imaged as they elevate cAMP either directly in response to LH, or by diffusion between cells. If diffusion is detected, it is hypothesized to be via gap junctions, which connect all granulosa cells to each other, and to the oocyte. To test this, follicles will be incubated in a gap junction inhibitor, carbenoxolone, prior to LH treatment. It is hypothesized that only some cells will elevate cAMP, corresponding to LHR-expressing cells, with no diffusion between cells. Diffusion of cAMP through gap junctions would present a paradox because the LH surge also causes a decrease in cAMP in the oocyte, which is necessary for meiotic resumption. It is hypothesized that LH signaling through epidermal growth factor receptor (EGFR) kinase closes gap juctions between granulosa cells and the oocyte, allowing cAMP to decline in the oocyte while remaining elevated in granulosa cells. This will be tested using AG1478, an inhibitor of EGFR kinase activity. In the presence of AG1478, it is predicted that cAMP will continue to diffuse through gap junctions from granulosa cells into the oocyte, which would prevent oocyte maturation. The LH surge also causes the rapid decrease of another cyclic nucleotide, cGMP, in the granulosa cells and oocyte. This decrease in oocyte cGMP allows for the decrease in oocyte cAMP, which then triggers oocyte maturation into a fertilizable egg. However, it is not known how the granulosa cell cAMP increase and cGMP decrease are related. This will be investigated by breeding R-FlincA mice with another mouse line that expresses a fluorescent sensor for cGMP. It is hypothesized that cells that initially elevate cAMP in response to LH will rapidly exhibit decreased cGMP levels, suggesting that the cAMP increase is required to lower cGMP levels. This project will contribute to understanding of how LH signaling leads to a fertilizable egg, and could lead to improvements in human in vitro maturation (IVM), which depends on optimal cyclic nucleotide levels in the oocyte. Thus this project could lead to clinical advances in reproductive health.

项目概要/摘要 在哺乳动物卵巢中，黄体生成素 (LH) 的周期中期激增作用于颗粒细胞 排卵前卵泡触发卵母细胞成熟、排卵和黄体化。通过 LH 受体发出信号 主要通过连续 Gs 介导的腺苷酸环化酶激活发生，产生第二个 信使 cAMP 和蛋白激酶 A 依赖性磷酸化。然而，cAMP 是否以及如何 信号从表达 LH 受体 (LHR) 的细胞子集传播，以实现许多结果 LH 激增是一个长期存在的问题，有待解决。为了测试这个和其他问题，完好无损 将从小鼠体内分离出排卵前卵泡，这些卵泡在整体上表达新开发的荧光传感器 cAMP 称为 R-FlincA，在 cAMP 结合后可将亮度提高高达 600%。使用 R-FlincA 和 最先进的光片显微镜，可以在单个细胞升高 cAMP 时直接对它们进行成像 对 LH 的反应，或通过细胞之间的扩散。如果检测到扩散，则假设是通过间隙连接， 它将所有颗粒细胞相互连接并与卵母细胞连接。为了测试这一点，将卵泡培养在 LH 治疗前，间隙连接抑制剂，carbenoxolone。据推测，只有部分细胞会升高 cAMP，对应于 LHR 表达细胞，细胞间无扩散。 cAMP 通过间隙扩散 连接处会出现一个悖论，因为 LH 激增也会导致卵母细胞中 cAMP 的减少，从而 对于减数分裂恢复是必要的。据推测，LH 信号通过表皮生长因子 受体 (EGFR) 激酶关闭颗粒细胞和卵母细胞之间的间隙连接，使 cAMP 下降 在卵母细胞中，而在颗粒细胞中保持升高。这将使用 AG1478 进行测试，AG1478 是一种抑制剂 EGFR 激酶活性。在 AG1478 存在的情况下，预计 cAMP 将继续通过间隙扩散 颗粒细胞与卵母细胞的连接，这会阻止卵母细胞成熟。 LH 也激增 导致颗粒细胞和卵母细胞中另一种环核苷酸 cGMP 的快速减少。这 卵母细胞 cGMP 的减少导致卵母细胞 cAMP 的减少，从而触发卵母细胞成熟为 受精卵。然而，尚不清楚颗粒细胞cAMP增加和cGMP减少是如何发生的。 有关的。这将通过将 R-FlincA 小鼠与表达 cGMP 荧光传感器。据推测，最初响应 LH 升高 cAMP 的细胞将 迅速表现出 cGMP 水平下降，表明需要增加 cAMP 来降低 cGMP 水平。 该项目将有助于了解 LH 信号传导如何导致受精卵，并可能导致 人类体外成熟（IVM）的改善，这取决于体内的最佳环核苷酸水平 卵母细胞。因此，该项目可能会导致生殖健康方面的临床进展。