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Intercellular dynamics of cyclic nucleotides in ovarian follicles

卵巢卵泡中环核苷酸的细胞间动力学

基本信息

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
项目类别：
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

项目摘要

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.

项目总结/文摘