Assistant Professor (in Residence)
Department of Cell Biology
263 Farmington Avenue
Farmington, CT 06030
My research interests are focused on physiological mechanisms of hormone signaling in the ovary, both within and between cells. During my postdoctoral research in the laboratory of Laurinda Jaffe, and now as an assistant professor-in-residence, I am studying the mechanisms by which luteinizing hormone (LH) acts on the outermost layer of granulosa cells to reduce cGMP levels within the follicle and cause oocyte meiotic resumption (Jaffe and Egbert, 2017). I am currently working on two projects that make use of novel mouse models:
Identifying the phosphatase(s) that mediate NPR2 dephosphorylation in response to LH
We have shown that LH triggers the rapid dephosphorylation and inactivation of the guanylyl cyclase, natriuretic peptide receptor 2 (NPR2), which reduces cGMP production in the granulosa cells (Egbert et al., 2014). This lowers cGMP throughout the follicle and oocyte within 20 min, leading to resumption of oocyte meiosis. NPR2 dephosphorylation is required for the normal time course of oocyte meiotic resumption (Shuhaibar et al., 2016), but the LH-activated phosphatase(s) that is responsible for this process remains unknown. A previous study of LH signaling in rat granulosa cells identified PPP2 and its regulatory subunit, PPP2R5D, as a strong candidate (Flynn et al., 2008, Mol Endo 22:1695-1710). PPP2R5D, also called PP2A-B56delta, increases the activity of the catalytic subunit of PPP2 (also called PP2A) when phosphorylated by PKA on serine 566 (Ahn et al., 2007, PNAS 104:2979-84). This proposed model is summarized in Fig. 1.
Fig. 1: Proposed model of initial signaling events by which luteinizing hormone (LH) acts on mural granulosa cells of mouse ovarian follicles to cause oocyte meiotic resumption. LHR, luteinizing hormone receptor; AC, adenylyl cyclase; PKA, protein kinase A.
Because a phosphospecific antibody is not currently available, we have established an assay for monitoring changes in PPP2R5D phosphorylation in isolated mouse follicles using Phos-tag gel electrophoresis, which separates differentially-phosphorylated protein species. We have confirmed the LH-induced phosphorylation of PPP2R5D-S566 in isolated rat follicles using mass spectrometry in collaboration with lab of Henning Urlaub (Göttingen, Germany). Importantly, although this analysis also identified the expression of 21 other phosphatase proteins that contain phosphorylated sites, only four proteins exhibited changes in phosphorylation in response to LH (PPP1R2, PPP1R3G, PPP1R12A, and PPP6R2). For these four, there are no definitive links between the observed changes in phosphorylation and altered activity of the catalytic phosphatase. Thus, the LH-induced phosphorylation of PPP2R5D-S566, which is well established to increase PPP2 catalytic activity, is the leading candidate for mediating the LH-induced dephosphorylation of NPR2 that causes oocyte meiotic resumption. We are in the process of making a mouse in which S566 is mutated to alanine such that it cannot be phosphorylated. With this Ppp2r5d-S566A mouse, we will test the hypothesis that LH signaling phosphorylates PPP2R5D-S566, leading to NPR2 dephosphorylation, cGMP decrease in the follicle, and oocyte meiotic resumption. We expect that these processes will be attenuated or blocked in follicles from Ppp2r5d-S566A mice.
LH-induced cAMP dynamics within and between granulosa cells
Using a mouse with a hemagglutinin (HA) epitope tag attached to the LH receptor (HA-LHR), it was recently revealed that LHR is expressed heterogeneously throughout the outer mural granulosa cells (Baena et al., 2020). In fact, only 10-50% of these cells are estimated to express LHR, which raises the question of how LH signaling can rapidly induce changes in cells throughout the follicle and oocyte. Although the spatiotemporal dynamics of cGMP in ovarian follicles have been investigated extensively, using a mouse line expressing the fluorescent sensor cGi500 (Shuhaibar et al., 2015), there is a gap in corresponding information for cAMP, due to lack of a mouse line with an optimal cAMP sensor. To study cAMP dynamics in intact follicles at cellular resolution, we are making a mouse that expresses R-FlincA, a newly described high-affinity fluorescent sensor for cAMP (Ohta et al., 2018, Sci Rep 8:1866; Fig. 2).
Fig. 2: (A) Generation and activation of the R-FlincA cAMP sensor. (B) R-FlincA expressed in 293 T cells increases brightness by 600% in response to the adenylyl cyclase activator forskolin (50 µM). Modified from Ohta et al., 2018, Sci Rep 8:1866.
Combined with lightsheet microscopy, R-FlincA should allow us to understand how LH-induced cAMP signaling is initiated and propagated throughout the granulosa cells. Specifically, we will test whether LH exposure initially causes only a subset of outer mural granulosa cells to elevate cAMP, as suggested by the heterogenous expression of LHR. In addition, we will test whether cAMP diffuses from these LHR-expressing cells through gap junctions into adjacent cells that lack LHR. Because NPR2 is inactivated by cAMP signaling, possibly by way of PPP2R5D-S566 phosphorylation, such a mechanism could explain the rapid, widespread cGMP decrease that is necessary for meiotic resumption of the oocyte.
These projects will investigate long-standing questions in membrane guanylyl cyclase regulation and cyclic nucleotide signaling, increasing our understanding of how the mid-cycle LH surge rapidly alters cyclic nucleotide levels in the follicle and oocyte to trigger timely oocyte meiotic resumption and release a fertilizable egg.
Cyclic AMP links luteinizing hormone signaling to dephosphorylation and inactivation of the NPR2 guanylyl cyclase in ovarian follicles. Biol Reprod. 104 (5):939-941.
Baena, V., Owen, C.M., Uliasz, T.F., Lowther, K.M., Yee, S.-P., Terasaski, M., Egbert, J.E., and Jaffe, L.A. (2020). Cellular heterogeneity of the LH receptor and its significance for cyclic GMP signaling in mouse preovulatory follicles. Endocrinology 161 (7):bqaa074.
Egbert, J.R., Fahey, P.G., Reimer, J., Owen, C.M, Evsikov, A.V., Nikolaev, V.O., Griesbeck, O., Ray, R.S., Tolias, A.S., and Jaffe, L.A. (2019) Follicle-stimulating hormone and luteinizing hormone increase Ca2+ in the granulosa cells of mouse ovarian follicles. Biol. Reprod., 101(2), 433–444.
Egbert, J.R., Yee, S.-P., and Jaffe, L.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. Develop. Biol. 435:6-14.
Jaffe LA and Egbert JR. (2017) Regulation of Mammalian Oocyte Meiosis by Intercellular Communication Within the Ovarian Follicle. Annu. Rev. Physiol. 79: 237-260.
Egbert JR, Uliasz TF, Shuhaibar LC, Geerts A, Wunder F, Kleiman RJ, Humphrey JM, Lampe PD, Artemyev NO, Rybalkin SD, Beavo JA, Movsesian MA, Jaffe LA. (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(5) :110. Supplement.pdf.
Egbert JR, Shuhaibar LC, Edmund AB, Van Helden DA, Robinson JW, Uliasz TF, Baena V, Geerts A, Wunder F, Potter LR, Jaffe LA. (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(18):3594-604.