Intercellular dynamics of cyclic nucleotides in ovarian follicles

项目总结/摘要 在哺乳动物卵巢中，黄体生成素（LH）的中期激增作用于卵巢颗粒细胞， 排卵前卵泡触发卵母细胞成熟、排卵和黄体化。通过LH受体的信号传导 发生主要是通过连续的GS介导的腺苷酸环化酶激活，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）的细胞亚群传播，以实现许多结果， LH激增是一个长期存在的问题，仍有待解决。为了测试这个和其他问题， 排卵前卵泡将从小鼠中分离出来，这些小鼠全面表达新开发的荧光传感器， 称为R-FlincA的cAMP在cAMP结合后可以增加亮度高达600%。使用R-FlincA和 最先进的光片显微镜，单个细胞可以成像，因为它们直接在细胞中升高cAMP。 对LH的反应，或通过细胞间的扩散。如果检测到扩散，则假设是通过间隙连接， 连接所有颗粒细胞和卵母细胞。为了测试这一点，卵泡将在 间隙连接抑制剂甘珀酸，LH治疗前。据推测，只有一些细胞会升高 cAMP，对应于LHR表达细胞，细胞间无扩散。cAMP通过间隙的扩散 由于LH峰也会导致卵母细胞中cAMP的减少， 是减数分裂恢复所必需的。假设LH信号通过表皮生长因子 EGFR受体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AG 1478进行测试，AG 1478是一种 EGFR激酶活性。在AG 1478的存在下，预测cAMP将继续通过间隙扩散 连接从颗粒细胞进入卵母细胞，这将阻止卵母细胞成熟。LH激增也 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的减少允许卵母细胞cAMP的减少，其然后触发卵母细胞成熟为一个成熟的卵母细胞。 受精卵然而，颗粒细胞cAMP增加和cGMP减少是如何发生的尚不清楚。 相关.这将通过将R-FlincA小鼠与另一种表达一种表达的小鼠品系进行繁殖来研究。 cGMP的荧光传感器。假设最初响应LH升高cAMP的细胞将 快速表现出降低的cGMP水平，表明cAMP增加是降低cGMP水平所必需的。 该项目将有助于了解LH信号如何导致受精卵，并可能导致 人体外成熟（IVM）的改善，这取决于细胞中的最佳环核苷酸水平。 卵母细胞因此，该项目可能导致生殖健康的临床进展。

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.