Dephosphorylation and inactivation of NPR2 guanylyl cyclase in granulosa cells contributes to the LH-induced decrease in cGMP that causes resumption of meiosis in rat oocytes

Abstract

In mammals, the meiotic cell cycle of oocytes starts during embryogenesis and then pauses. Much later, in preparation for fertilization, oocytes within preovulatory follicles resume meiosis in response to luteinizing hormone (LH). Before LH stimulation, the arrest is maintained by diffusion of cyclic (c)GMP into the oocyte from the surrounding granulosa cells, where it is produced by the guanylyl cyclase natriuretic peptide receptor 2 (NPR2). LH rapidly reduces the production of cGMP, but how this occurs is unknown. Here, using rat follicles, we show that within 10 min, LH signaling causes dephosphorylation and inactivation of NPR2 through a process that requires the activity of phosphoprotein phosphatase (PPP)-family members. The rapid dephosphorylation of NPR2 is accompanied by a rapid phosphorylation of the cGMP phosphodiesterase PDE5, an enzyme whose activity is increased upon phosphorylation. Later, levels of the NPR2 agonist C-type natriuretic peptide decrease in the follicle, and these sequential events contribute to the decrease in cGMP that causes meiosis to resume in the oocyte.

Dephosphorylation is the mechanism of fibroblast growth factor inhibition of guanylyl cyclase-B

“…Because inhibition of GC-B by multiple hormones and signaling factors is correlated with receptor dephosphorylation [23, 24, 27, 35, 39, 40], we investigated whether dephosphorylation contributes to the FGF2-dependent inhibition of GC-B in RCS cells. To determine GC-B phosphate levels, we employed two different phosphate detection methods, Phos-tag and ProQ Diamond.…”

“…For analysis of phosphorylation by Phos-tag, GC-B was immunoprecipitated as previously described [23]. Briefly, ~200–500 μg crude membrane protein was diluted to 0.5 or 1 ml in 50 mM Tris-HCl pH 7.5, 50 mM NaF, 10 mM NaH 2 PO 4 , 2 mM EDTA, 0.5% deoxycholate, 0.1% SDS, 1% NP-40, 100 mM NaCl, 10 mM NaH 2 PO 4 , 1X Roche Protease Inhibitor Cocktail, and 1 μM microcystin.…”

“…Recently, luteinizing hormone (LH) was shown to stimulate GC-B dephosphorylation and inactivation in ovarian follicles by a process that requires a PPP family serine and threonine protein phosphatase [23]. Importantly, a knock-in mouse (GC-B 7E/7E ) expressing GC-B-7E at the normal GC-B genetic locus was immune to LH-dependent GC-B inactivation and displayed a 5-h delay in the resumption of meiosis in the oocyte [24].…”

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

“…Because cGMP phosphodiesterases are actively hydrolyzing cGMP in the granulosa cells (5), the decrease in the rate of cGMP production results in a lower equilibrium level of cGMP. In addition, there could be an increase in the activity of the cGMP phosphodiesterase PDE5, as indicated by evidence that PDE5 is phosphorylated (11) and evidence from studies of other cells that phosphorylation of PDE5 is associated with increased activity (30-32). G s -mediated elevation of cAMP in the mural granulosa cells is very likely a step in this process, because in response to adenylyl cyclase activation by forskolin, cGMP in these cells decreases rapidly, reaching levels comparable to those seen with LH (Fig.…”

“…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)(5)(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)(8)(9)(10)(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).…”