The objective of this project is to understand the signaling mechanisms by which eggs are activated to begin development. The current renewal application, for years 30-34, addresses the question of how luteinizing hormone (LH) from the pituitary acts on the ovary to cause oocytes to progress to the stage at which they can be fertilized. Mammalian oocytes are stored in the ovary, arrested at meiotic prophase, for decades in women. Then in response to luteinizing hormone signaling in the surrounding follicle, meiosis resumes, and the mature egg is ovulated. Studies in mice have shown that meiotic arrest in antral follicles is maintained by cyclic GMP that is produced by the granulosa cells and diffuses into the oocyte through gap junctions;LH signaling decreases gap junction permeability and cGMP production by the granulosa cells, thus lowering cGMP in the oocyte. The cGMP decrease leads to the release of inhibition of the meiotic cell cycle. Despite knowledge of this cascade, many important questions still remain. This proposal focuses on the key event of the reduction in cGMP by investigating how LH signaling reduces the guanylyl cyclase activity of natriuretic peptide receptor 2 (NPR2) (aims 1 and 2), and how LH signaling lowers cGMP through activity of cGMP phosphodiesterases (aim 3).
Aim 1 will test whether LH signaling decreases NPR2 guanylyl cyclase activity by dephosphorylating NPR2 regulatory sites.
Aim 2 will investigate how LH activation of G-proteins and the EGF receptor initiates the decrease in the guanylyl cyclase activity of NPR2.
Aim 3 will investigate which cGMP phosphodiesterases function to lower cGMP in the follicle, and whether they are stimulated by LH. The methods to be used include isolation and culture of ovarian follicles and granulosa cells from mice and rats, confocal microscopy, immunoprecipitation, Western blotting, use of transgenic mice and specific enzyme inhibitors, use of optical sensors to measure cGMP and calcium in live ovarian follicles, and assays of enzyme activities in vitro. The conclusions reached from these studies will also be applicable to understanding the regulation of meiosis in women. In vitro oocyte maturation, in which LH receptor stimulation is performed in vitro, is an emerging component of methods for human in vitro fertilization, particularly for patients with polycystic ovary syndrome and in combination with cryopreservation of ovarian tissue from cancer patients. The proposed studies will contribute to understanding of the biochemistry and cell biology underlying clinical advances in in vitro maturation.

Public Health Relevance

The proposed research concerns the mechanisms by which hormonal signals cause oocytes, which are stored in the ovary, to develop to the stage at which they can be ovulated and fertilized. This research will advance knowledge of a crucial physiological process, and establish a basis for future clinical developments, especially in treatment of infertility.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Method to Extend Research in Time (MERIT) Award (R37)
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Cellular, Molecular and Integrative Reproduction Study Section (CMIR)
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Taymans, Susan
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University of Connecticut
Anatomy/Cell Biology
Schools of Medicine
United States
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Jaffe, Laurinda A (2018) The fast block to polyspermy: New insight into a century-old problem. J Gen Physiol 150:1233-1234
Vigone, Giulia; Shuhaibar, Leia C; Egbert, Jeremy R et al. (2018) Multiple cAMP Phosphodiesterases Act Together to Prevent Premature Oocyte Meiosis and Ovulation. Endocrinology 159:2142-2152
Egbert, Jeremy R; Yee, Siu-Pok; Jaffe, Laurinda A (2018) Luteinizing hormone signaling phosphorylates and activates the cyclic GMP phosphodiesterase PDE5 in mouse ovarian follicles, contributing an additional component to the hormonally induced decrease in cyclic GMP that reinitiates meiosis. Dev Biol 435:6-14
Jaffe, Laurinda A; Egbert, Jeremy R (2017) Regulation of Mammalian Oocyte Meiosis by Intercellular Communication Within the Ovarian Follicle. Annu Rev Physiol 79:237-260
Robinson, Jerid W; Egbert, Jeremy R; Davydova, Julia et al. (2017) Dephosphorylation is the mechanism of fibroblast growth factor inhibition of guanylyl cyclase-B. Cell Signal 40:222-229
Shuhaibar, Leia C; Robinson, Jerid W; Vigone, Giulia et al. (2017) Dephosphorylation of the NPR2 guanylyl cyclase contributes to inhibition of bone growth by fibroblast growth factor. Elife 6:
Shuhaibar, Leia C; Egbert, Jeremy R; Edmund, Aaron B et al. (2016) Dephosphorylation of juxtamembrane serines and threonines of the NPR2 guanylyl cyclase is required for rapid resumption of oocyte meiosis in response to luteinizing hormone. Dev Biol 409:194-201
Egbert, Jeremy R; Uliasz, Tracy F; Shuhaibar, Leia C et al. (2016) Luteinizing Hormone Causes Phosphorylation and Activation of the cGMP Phosphodiesterase PDE5 in Rat Ovarian Follicles, Contributing, Together with PDE1 Activity, to the Resumption of Meiosis. Biol Reprod 94:110
Shuhaibar, Leia C; Egbert, Jeremy R; Norris, Rachael P et al. (2015) Intercellular signaling via cyclic GMP diffusion through gap junctions restarts meiosis in mouse ovarian follicles. Proc Natl Acad Sci U S A 112:5527-32
Egbert, Jeremy R; Shuhaibar, Leia C; Edmund, Aaron B et al. (2014) 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. Development 141:3594-604

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