Intercellular signaling via cyclic GMP diffusion through gap junctions restarts meiosis in mouse ovarian follicles

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.cyclic GMP | gap junctions | ovarian follicle | oocyte meiosis | luteinizing hormone M eiosis in mammalian oocytes begins during embryonic development and then arrests in late prophase, for up to 50 y in women and for many months in mice. At the time of ovulation, luteinizing hormone (LH) acts on the granulosa cells of the follicle surrounding the oocyte to release the arrest and restart meiosis in preparation for fertilization (1-3). In the mouse preovulatory follicle, inhibition of meiotic progression is dependent upon the cyclic nucleotide cyclic GMP (cGMP), which diffuses from the granulosa cells into the oocyte through gap junctions that connect all cells of the follicle (4-6). The cGMP is produced by the transmembrane guanylyl cyclase natriuretic peptide receptor 2 (NPR2, also known as guanylyl cyclase B), which is present in all of the granulosa cells, but not in the oocyte (7-11). In the oocyte, cGMP inhibits the degradation of another cyclic nucleotide, cAMP, which depends primarily on the phosphodiesterase PDE3A, an enzyme whose activity is antagonized by cGMP (4, 5). The resulting high level of cAMP, through a series of intermediate steps, inhibits meiotic progression (2, 3, 12).LH signaling is initiated in the outer (mural) layers of granulosa cells; receptors for LH are absent in the oocyte and in the granulosa cells that directly surround it (the cumulus cells) (13-15). Ensuing events cause meiosis to resume by reducing cGMP in the oocyte (4, 5), but how LH receptor activation up to 10 cell layers away lowers oocyt...

Oocyte maturation and quality: role of cyclic nucleotides

“…Whether this pre-ovulatory loss of CC-oocyte GJC causes meiotic resumption due to the termination of cAMP transfer from CCs to oocyte, as originally hypothesised (Dekel & Beers 1978), has remained the subject of much debate for decades. There is strong evidence supporting the hypothesis that diffusion of cGMP from the oocyte to the somatic compartment through functional GJs during GVBD has a crucial role in the re-initiation of meiosis (Norris et al 2009, Vaccari et al 2009, Shuhaibar et al 2015.…”

“…The involvement of cGMP in the process of meiotic resumption was recently strengthened by the work of Shuhaibar et al (Shuhaibar et al 2015). Using follicles from mice expressing a FRET sensor, real-time monitoring of cGMP showed that within 1 min of LH exposure, cGMP concentrations start to decrease from the peripheral granulosa cells and by 20 min the concentration of cGMP decreased by more than 20-fold and was uniformly low across the follicle (Shuhaibar et al 2015). Consequently, it is likely that oocyte cAMP concentration decreases because of the relief of the inhibitory actions of cGMP on PDE3A in the oocyte (Norris et al 2009, Vaccari et al 2009.…”

“…LH administration leads to a fall in follicular cGMP (Hubbard 1986) and a loss of gap junctions (SelaAbramovich et al 2005). The involvement of cGMP in the process of meiotic resumption was recently strengthened by the work of Shuhaibar et al (Shuhaibar et al 2015). Using follicles from mice expressing a FRET sensor, real-time monitoring of cGMP showed that within 1 min of LH exposure, cGMP concentrations start to decrease from the peripheral granulosa cells and by 20 min the concentration of cGMP decreased by more than 20-fold and was uniformly low across the follicle (Shuhaibar et al 2015).…”

Aging-related premature luteinization of granulosa cells is avoided by early oocyte retrieval

“…Transcription in oocytes depends on the presence of attached CCs (De La Fuente & Eppig 2001). In addition, the maintenance of oocyte arrest before recruitment also relies on the contribution of autocrine and paracrine factors synthesized in GCs, including cAMP/cGMP (Webb et al 2002, Wigglesworth et al 2013, Shuhaibar et al 2015, purine (Downs 1993), kit ligand (Ye et al 2009), and NPR2 , Tsuji et al 2012, Wigglesworth et al 2013. Oocytes from antral follicles resume and complete meiosis spontaneously after removal of surrounding CCs (Buccione et al 1990, Mehlmann 2005, suggesting that CCs control oocyte nuclear maturation.…”

Follicular environment and oocyte maturation: roles of local peptides and steroids

“…Later studies demonstrated that NPPC is expressed by bovine cumulus cells, and that, like in the mouse, NPPC also inhibits germinal vesicle breakdown (GVBD) in cattle De Cesaro et al, 2015). For meiosis resumption to occur, LH inhibits NPPC production by granulosa cells and reduces the flow of cGMP from the outer layers of the cumulus to the oocyte (Kawamura et al, 2011;Shuhaibar et al, 2015). Reduced gap junction functionality after the LH surge is believed to be a consequence of the production/secretion of EGF-like peptides that bind to the EGFR to induce mitogen activated protein kinase (MAPK) dependent phosphorylation of connexins, the main components of gap junctions (Conti et al, 2012).…”