(311) Regulation of Gap Junction Communication is Required for the Normal Timing of the Oocyte cAMP Changes that Re-Initiate Meiosis

Jeremy R. Egbert¹; Iris Nakashima¹; Siu-Pok Yee¹; Laurinda A. Jaffe¹

 
1. Department of Cell Biology, UConn Health, Farmington, CT, USA

Abstract Text:

In mammalian oocytes within preovulatory follicles, re-entry into meiosis is mediated by changes in cAMP and cGMP levels. The oocyte produces cAMP to maintain meiotic arrest, but it also expresses the cAMP phosphodiesterase PDE3A, which is competitively inhibited by cGMP that diffuses through gap junctions into the oocyte from the surrounding ~10 layers of granulosa cells. During each reproductive cycle, the surge in luteinizing hormone (LH) acts on its Gs-linked receptor in the outermost granulosa cells, leading to rapid dephosphorylation and inactivation of the NPR2 guanylyl cyclase that produces the cGMP. As a result, cGMP levels fall throughout the follicle and oocyte, allowing PDE3A to hydrolyze oocyte cAMP and trigger meiotic resumption. By 1 hour after LH, gap junctions between follicle cells close, potentially contributing to these changes. However, especially because LH signaling elevates cAMP throughout the follicle, it remains unclear how these events are coordinated to achieve the reduction in oocyte cGMP and cAMP that is required for meiosis to resume. To address this question, we used fluorescent sensors for cAMP and cGMP (both genetically encoded and quantitatively microinjected into the oocyte), as well as modulators of gap junction communication. With these tools, live confocal imaging allowed us to monitor cyclic nucleotide levels in isolated follicles and their oocytes in real time, as well as to determine the timing of meiotic resumption for each oocyte. We found that several minutes after LH exposure, the wave of elevated cAMP reached the oocyte, where it remained elevated for 10 - 70 minutes before declining to a level below baseline. Preventing gap junction communication with carbenoxolone (200 µM, 2 hr) reduced the LH-induced cAMP increase in the follicle interior and blocked the oocyte cAMP increase. Application of an inhibitor (500 nM AG1478, 1 hr) that prevents the LH-induced closure of gap junctions between granulosa cells delayed both the fall in oocyte cAMP and meiotic resumption by several hours. This indicates that gap junction closure facilitates the fall in oocyte cAMP and is required for the normal time course of meiotic resumption. Simultaneously imaging changes in cAMP and cGMP in follicle-enclosed oocytes revealed that the fall in cGMP after LH is consistently rapid, while the timing of the fall in cAMP is much more variable. This variability might result from differences in the kinetics of gap junction closure, resulting in variable amounts of cAMP diffusing into the oocyte from the granulosa cells. Our results reveal that gap junction closure, rather than being primarily a “fail-safe” mechanism, is an active regulator of the decrease in oocyte cAMP that reinitiates meiosis. Supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (R03 HD104879 to J.R.E.).