Dephosphorylation of juxtamembrane serines and threonines of the NPR2 guanylyl cyclase regulates oocyte meiotic resumption
BMC Pharmacology and Toxicology volume 16, Article number: A30 (2015)
The meiotic cell cycle of mammalian oocytes starts during embryogenesis and then pauses until luteinizing hormone (LH) restarts the cycle. This meiotic arrest is maintained by cGMP, which is produced in the granulosa cells by C-type natriuretic peptide (CNP) activation of NPR2 . LH decreases cGMP in the granulosa cells, and via equilibration through gap junctions, cyclic GMP also decreases in the oocyte, thus releasing the meiotic arrest . LH causes dephosphorylation and inactivation of NPR2 [3, 4], but whether NPR2 dephosphorylation is required for meiotic resumption is not known. Seven regulatory NPR2 phosphorylation sites have been identified (Fig. 1) [5, 6]. Here, we generated a knock-in mouse where each site was mutated to glutamate (Npr2-7E), resulting in a “constitutively phosphorylated” enzyme that we used to investigate the role of NPR2 dephosphorylation in the rapid resumption of meiosis in response to LH.
Membranes from isolated antral follicles treated with or without LH for 20 min were assayed for guanylyl cyclase activity under physiological conditions with CNP, ATP and Mg2+GTP (Fig. 2). In wild-type follicles treated with LH, the CNP-dependent guanylyl cyclase activity decreased to ~47% of initial values (Fig. 2, left). In contrast, LH caused no significant change in CNP-dependent guanylyl cyclase activity in Npr2-7E/7E follicle membranes (Fig. 2, right). Protein loss did not explain the reduction in CNP-dependent activity because activities measured with Mn2+GTP/Triton X-100 were not reduced by LH. These findings indicate that dephosphorylation of NPR2 is necessary for the hormonal regulation of guanylyl cyclase activity in the ovarian follicle.
To investigate whether the LH-induced decrease in guanylyl cyclase activity is required for meiotic resumption, we isolated Npr2-7E/7E and wild-type follicles and observed them in culture before and after addition of LH. In wild-type follicles, LH-induced nuclear envelope breakdown (NEBD) began at 2 hours and reached ~80% by 6 hours. In contrast, in Npr2-7E/7E follicles, no evidence of meiotic resumption was observed in the first 6 hours following treatment with LH. However, by 8 hours after LH application, NEBD had occurred in ~40% of Npr2-7E/7E follicle-enclosed oocytes, and by 12 hours NEBD was seen in ~80% of oocytes. During cumulus expansion preceding ovulation, gap junction communication between the oocyte and cumulus cells is disrupted . The LH-induced cumulus expansion occurred similarly in Npr2-7E/7E and wild-type follicles, suggesting that gap junction disruption during cumulus expansion might explain why meiosis eventually resumes in the NPR2-7E/7E follicles.
Dephosphorylation of NPR2 is required for the rapid resumption of meiosis in response to LH.
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This work was supported by NIH grants R37HD014939, R01GM098309, T32AR050938 and the Fund for Science.
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Shuhaibar, L.C., Edmund, A.B., Egbert, J.R. et al. Dephosphorylation of juxtamembrane serines and threonines of the NPR2 guanylyl cyclase regulates oocyte meiotic resumption. BMC Pharmacol Toxicol 16 (Suppl 1), A30 (2015). https://doi.org/10.1186/2050-6511-16-S1-A30