Date of Completion

5-7-2015

Embargo Period

12-1-2015

Keywords

Meiosis, cyclic GMP, guanylyl cyclase, NPR2, ovarian follicle, gap junctions, bone growth

Major Advisor

Dr. Laurinda Jaffe

Associate Advisor

Dr. Ann Cowan

Associate Advisor

Dr. Bruce White

Associate Advisor

Dr. Lisa Mehlmann

Associate Advisor

Dr. Mark Terasaki and Dr. Siu-Pok Yee

Field of Study

Biomedical Science

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Meiosis in mammalian oocytes starts during embryogenesis and pauses until luteinizing hormone (LH) restarts the cycle. Within the ovary, granulosa cells surrounding each oocyte maintain meiotic arrest, by providing cGMP, which diffuses through gap-junctions into the oocyte.

LH acts on receptors located on granulosa cells up to 10 cell layers away from the oocyte, but it is unknown how this signal initiated in a distant tissue is transmitted to the oocyte. By expressing a FRET sensor for cGMP in all cells of the ovarian follicle, we visualized the spatio-temporal dynamics of cGMP before and after treatment with LH. After stimulation with LH, cGMP first decreases in the peripheral granulosa cells, and later in the cumulus cells and the oocyte, as a consequence of diffusion through gap-junctions. These findings show that diffusion of cGMP transmits the signal that reinitiates meiosis from the follicle surface to the oocyte.

cGMP is produced in granulosa cells by the guanylyl cyclase NPR2. We found that LH decreases NPR2 activity by 20 minutes, as a result of dephosphorylation of seven regulatory serine and threonine sites in NPR2. We mutated these regulatory sites to glutamates, such that NPR2 behaves as if it is constitutively phosphorylated and active, and produced knockin-mice expressing the mutated protein. The mutations inhibited the rapid LH-induced decrease in NPR2 activity and delayed meiotic resumption in follicle-enclosed oocytes. Mice with these mutations also had longer vertebrae. Our findings demonstrate that dephosphorylation of NPR2 is important for regulation of oocyte meiosis and bone growth.

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