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, I studied signaling by luteinizing hormone (LH) in the mammalian preovulatory ovarian follicle, which consists of a prophase-arrested oocyte that is connected by gap junctions to ~10 layers of surrounding granulosa cells. The granulosa cells produce cGMP, which diffuses through gap junctions into the oocyte and maintains meiotic arrest. Using mice and rats, I studied the mechanisms by which (LH) acts on the outermost layer of granulosa cells to reduce cGMP levels within the follicle and cause oocyte meiotic resumption. This work showed that LH induces the dephosphorylation and inactivation of the NPR2 guanylyl cyclase, which reduces cGMP production in the granulosa cells (Egbert et al., 2014). LH signaling also phosphorylates the cGMP phosphodiesterase PDE5, which increases its cGMP hydrolytic activity (Egbert et al., 2016; 2018). These mechanisms are important, but preventing them does not completely prevent the LH-induced cGMP decrease, suggesting that LH activates other mechanisms to lower cGMP. One candidate is the calcium/calmodulin-activated cGMP phosphodiesterase PDE1, which is highly expressed in granulosa cells. If LH increases granulosa cell calcium, it would increase PDE1 activity and contribute to the cGMP decrease. However, the role of calcium in gonadotropin signaling has been a long-standing unanswered question.
Currently, I am investigating this question using mice that express one of two high-affinity calcium sensors, allowing us to measure hormone-induced calcium changes in intact follicles for the first time.
Figure 1: High-affinity calcium sensors have been designed and genetically encoded into mice in the last few years. We are using mice that ubiquitously express one of these two different types of calcium sensor to study hormone-induced calcium changes in granulosa cells of ovarian follicles.
I am using these mice to study the dynamics of calcium changes within granulosa cells in response to both LH and follicle-stimulating hormone (FSH), which is responsible for granulosa cell proliferation, follicle growth, and LH receptor expression. These studies involve live imaging of calcium within the cells of intact follicles using both confocal and light sheet microscopy.
Figure 2: A preantral ovarian follicle expressing the Twitch-2B FRET sensor for calcium, visualized with a 40X/1.2 NA objective on a Zeiss LSM Pascal confocal microscope. A thin layer of theca cells surrounds the follicle and is separated from the granulosa cells by a basement membrane. Twitch-2B expression is fairly uniform throughout the granulosa cells, but is often higher in the theca and always very low in the oocyte.
I have found that FSH induces a single, transient calcium increase lasting several minutes that is uniform across all granulosa cells and is dependent on extracellular calcium.
Figure 3: FSH-induced calcium changes within the outer granulosa cells of a preantral follicle expressing Twitch-2B. Top: YFP/CFP FRET ratio (which reflects relative intracellular calcium levels) does not change in response to perfusions of control medium (MEMa), but increases in response to FSH before decreasing to near baseline. Bottom: YFP and CFP channels shown separately, revealing the inverse changes that are diagnostic for FRET only in response to FSH, not MEMa, perfusion.
In contrast, LH treatment immediately causes a subset of granulosa cells to undergo repeated calcium oscillations that persist for at least 6 hours and are dependent on Gq-family G-protein signaling (Egbert et al., 2019).
Movie 1: Section of the outer region of a preovulatory follicle expressing GCaMP6s, showing calcium oscillations within several outer granulosa cells in response to LH. GCaMP6s brightness increases with increasing intracellular calcium levels. The movie contains two 5-minute perfusions of control medium, followed by LH perfusion for 10 minutes. In this example, the single layer of flat theca cells surrounding the follicle undergoes a brief spike in calcium immediately after LH, followed by the outer granulosa cell oscillations.
In ongoing work, I am investigating the functional significance of these LH- and FSH-induced calcium increases. My overall goal is to develop an independent research program directed at understanding hormone signaling in the ovary and other three-dimensional tissues.
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.