Intercellular dynamics of cyclic nucleotides in ovarian follicles

项目摘要/摘要 在哺乳动物的卵巢中，黄体生成素的周期中期激增作用于卵巢颗粒细胞。 排卵前卵泡，以触发卵母细胞成熟、排卵和黄体化。通过黄体生成素受体传递信号 发生主要通过Gs介导的腺酰环化酶的顺序激活，产生第二个 信使cAMP和蛋白激酶A依赖的磷酸化。然而，夏令营是否以及如何 信号从表达促黄体生成素受体(LHR)的细胞亚群传播，以实现许多结果 黄体生成素激增是一个长期存在的问题，仍有待解决。原封不动地测试这个问题和其他问题 将从全球表达新开发的荧光传感器的小鼠身上分离出排卵前卵泡 CAMP被称为R-FlincA，与cAMP结合后，亮度最高可增加600%。使用R-FlincA和 最先进的光片显微镜，当单个细胞升高cAMP时，可以直接在 对黄体生成素的反应，或通过细胞间的扩散。如果检测到扩散，则假定是通过缝隙连接， 它将所有颗粒细胞相互连接，并连接到卵母细胞。为了测试这一点，卵泡将在一个 缝隙连接抑制剂，甘草酮，在黄体生成素治疗之前。据推测，只有部分细胞会升高 CAMP，与表达LHR的细胞相对应，细胞间无扩散。CAMP在GAP中的扩散 交界处会出现一个悖论，因为黄体生成素激增也会导致卵母细胞内cAMP的减少，这是 是恢复减数分裂所必需的。推测促黄体生成素通过表皮生长因子传递信号 受体(EGFR)激酶关闭颗粒细胞和卵母细胞之间的缝隙连接，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 EGFR激酶活性。在ag1478的存在下，预测cAMP将继续通过GAP扩散。 从颗粒细胞到卵母细胞的连接，这将阻止卵母细胞成熟。黄体生成素激增也 导致颗粒细胞和卵母细胞中另一种环核苷酸cGMP的迅速减少。这 卵母细胞cGMP的减少允许卵母细胞cAMP的减少，然后触发卵母细胞成熟为 可受精卵。然而，颗粒细胞cAMP升高和cGMP降低的机制尚不清楚。 相关的。这将通过用另一个表达A基因的小鼠品系来培育R-FlincA小鼠来进行研究 CGMP荧光传感器。假设最初升高cAMP以回应黄体生成素的细胞将 CGMP水平迅速下降，提示cAMP升高是降低cGMP水平所必需的。 这个项目将有助于理解黄体生成素信号如何导致受精卵，并可能导致 人类体外成熟(IVM)的改善，这取决于 卵母细胞。因此，该项目可能导致生殖健康方面的临床进展。

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.