doi: 10.1073/pnas.1423598112. Epub 2015 Mar 16.
Intercellular signaling via cyclic GMP diffusion through gap junctions restarts meiosis in mouse ovarian follicles
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- PMID:25775542
- PMCID: PMC4418852
- DOI: 10.1073/pnas.1423598112
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Intercellular signaling via cyclic GMP diffusion through gap junctions restarts meiosis in mouse ovarian follicles
Leia C Shuhaibar et al. Proc Natl Acad Sci U S A..
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Abstract
Meiosis in mammalian oocytes is paused until luteinizing hormone (LH) activates receptors in the mural granulosa cells of the ovarian follicle. Prior work has established the central role of cyclic GMP (cGMP) from the granulosa cells in maintaining meiotic arrest, but it is not clear how binding of LH to receptors that are located up to 10 cell layers away from the oocyte lowers oocyte cGMP and restarts meiosis. Here, by visualizing intercellular trafficking of cGMP in real-time in live follicles from mice expressing a FRET sensor, we show that diffusion of cGMP through gap junctions is responsible not only for maintaining meiotic arrest, but also for rapid transmission of the signal that reinitiates meiosis from the follicle surface to the oocyte. Before LH exposure, the cGMP concentration throughout the follicle is at a uniformly high level of ∼2-4 μM. Then, within 1 min of LH application, cGMP begins to decrease in the peripheral granulosa cells. As a consequence, cGMP from the oocyte diffuses into the sink provided by the large granulosa cell volume, such that by 20 min the cGMP concentration in the follicle is uniformly low, ∼100 nM. The decrease in cGMP in the oocyte relieves the inhibition of the meiotic cell cycle. This direct demonstration that a physiological signal initiated by a stimulus in one region of an intact tissue can travel across many layers of cells via cyclic nucleotide diffusion through gap junctions could provide a general mechanism for diverse cellular processes.
Keywords: cyclic GMP; gap junctions; luteinizing hormone; oocyte meiosis; ovarian follicle.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
LH receptor activation initiates an inwardly propagating cGMP decrease in the mouse ovarian follicle. (A) Isolated follicle expressing the cGi500 sensor for cGMP, showing a scanning transmission image (Left), CFP fluorescence (Center), and YFP fluorescence with the regions of measurement indicated (Right). (B) Images of the CFP/YFP ratio before LH perfusion, and at 5 and 20 min afterward, for the follicle shown inA. Before LH application, cGMP is at a uniformly high level throughout the follicle. cGMP in the surrounding theca cells is lower; the theca cells are not connected by gap junctions to the granulosa cells (17). After LH application, cGMP decreases first in the mural granulosa cells, then in the cumulus cells and oocyte, reaching a plateau at the same value in all regions. Movie S1 shows this time series. Based on ELISA measurements in wild-type follicles, the cGMP concentration before LH application is ∼4 μM, and the plateau value after LH is ∼100 nM (Fig. S1). No change in cGMP occurs in the theca cells.
Kinetics of the LH-induced cGMP decrease in mural granulosa, cumulus, and oocyte. (A) Time courses of the CFP/YFP ratios for the follicle shown in Fig. 1. For this and other graphs, ratios were calculated by dividing the mean CFP intensity in each region of interest, as shown in Fig. 1A, by the mean YFP intensity. (B) Recording from a follicle perfused with control medium without LH (representative of four experiments). (C andD) CFP/YFP ratios for mural granulosa cells, cumulus cells, and oocyte, before and at 20 min (C) or 2 h (D) after treatment with LH (16–20 follicles for each condition forC; 3–6 follicles for each condition forD). (E andF) Time to 10% or 50% of the decrease in CFP/YFP ratio in each region; results from 15 sets of measurements. Values that are indicated by an asterisk are significantly different from the control, and values not indicated by the same letter are significantly different from each other (P < 0.05); values indicate mean ± SEM.
The initial cGMP decrease in the cumulus cells requires gap junction communication. (A) A cGi500-expressing follicle was pretreated for 2 h with 200 μM carbenoxolone (CBX), and then perfused with LH. (B) CFP/YFP ratios for mural granulosa cells, cumulus, and oocyte, without LH exposure, for 10 follicles with CBX treatment; these are compared with the 20 control follicles from Fig. 2C. (C andD) CFP/YFP ratios for mural granulosa, cumulus cells, and oocytes, before and at 20 min (C) or 2 h (D) after treatment with LH, for follicles in the presence of CBX (7–15 follicles for each condition). Because CBX treatment lowers cGMP in the oocyte to approximately the same level attained after LH (B), LH treatment of follicles in the presence of CBX caused little further change in cGMP in the oocyte (A,C, andD). Values that are indicated by an asterisk are significantly different from the control (P < 0.05); values indicate mean ± SEM.
Kinetics of the LH-induced decrease in gap junction permeability. (A andB) Fluorescence redistribution after photobleaching of Alexa-488 in the mural granulosa cells. (A) shows images of a follicle before photobleaching and at 5 and 60 s afterward, before (Top) or 10 min after LH perfusion (Middle). The bottom row shows a separate follicle that was photobleached 60 min after LH treatment. The graphs show the time courses of fluorescence recovery. (B) The percent recovery in the first minute after bleaching, for eight follicles before and after a 10-min LH treatment, and three follicles after a 60-min LH treatment. (C) Kinetics of Cx43 phosphorylation on serines 279 and 282 after applying LH to follicles. Similar results were obtained in another identical experiment. The value indicated by an asterisk is significantly different from the control (P < 0.05); values indicate mean ± SEM.
Working model of how LH signaling rapidly decreases cGMP in the mural granulosa cells, and then via cGMP diffusion through gap junctions, decreases cGMP in the oocyte, leading to meiotic resumption. (A) Before LH exposure, cGMP concentrations are elevated throughout the follicle, because of a high rate of production of cGMP by the NPR2 guanylyl cyclase in the mural granulosa and cumulus cells. cGMP phosphodiesterases, including PDE5, degrade cGMP at a rate equal to its production, thus keeping the cGMP concentration at a constant level. Through gap junctions that connect all cells of the follicle, cGMP diffuses into the oocyte, where it inhibits the activity of PDE3A, maintaining cAMP at a level that inhibits meiotic resumption. The cAMP in the oocyte is produced by adenylyl cyclase 3 in the oocyte (39), and AC3 is kept active by the constitutive activity of the Gs-coupled receptor GPR3 (40). (B) When LH binds to its receptor in the mural granulosa cells, the activation of Gs and possibly other G proteins results in dephosphorylation of NPR2, which decreases its rate of production of cGMP. Activation of the LH receptor also increases phosphorylation of PDE5, and from studies of other cells, this should increase its rate of degradation of cGMP. Because of reduced NPR2 activity and increased cGMP phosphodiesterase activity, the concentration of cGMP in the mural granulosa cells decreases. Through the series of gap junctions that connects the oocyte to the large volume of the mural granulosa cells, cGMP in the oocyte diffuses down its concentration gradient, and the resulting decrease in oocyte cGMP relieves the inhibition of PDE3A in the oocyte, such that cAMP decreases. This model depicts only events occurring in the first 20 min after LH exposure. Subsequent events, including a decrease in gap junction permeability, an increase in EGF receptor ligands, and a decrease in C-type natriuretic peptide, also contribute to maintaining cGMP at the low level that triggers meiotic resumption. EGF receptor activation may also contribute to the early decrease in cGMP, although findings about this question are variable (Fig. S4). References and further discussion of this model are included in the text.
References
- Holt JE, Lane SIR, Jones KT. The control of meiotic maturation in mammalian oocytes. Curr Top Dev Biol. 2013;102:207–226. - PubMed
- Hunzicker-Dunn M, Mayo K. Gonadotropin signaling in the ovary. In: Plant TM, Zeleznik AJ, editors. Knobil and Neill’s Physiology of Reproduction. 4th Ed. Academic; San Diego: 2015. pp. 895–945.
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