Assistant Professor (In Residence)
I am interested in many aspects of ovarian biology, especially the development and maturation of the ovarian follicle and its enclosed oocyte. My postdoctoral research has focused on the mechanisms by which luteinizing hormone (LH) signaling triggers the resumption of oocyte meiosis. In mammals, high levels of cGMP maintain the meiotic arrest of oocytes within preovulatory follicles. LH signaling reduces cGMP levels within the follicle and oocyte, which reinitiates meiosis. These levels can be regulated in at least three different ways: cGMP synthesis, hydrolysis, and efflux from the cell. In the Jaffe lab, I have primarily studied how LH signaling regulates the synthesis and hydrolysis of cGMP.
cGMP is produced in granulosa cells by the membrane guanylyl cyclase NPR2, and diffuses through gap junctions into the oocyte. With our collaborators in the lab of Dr. Lincoln Potter at the University of Minnesota, we have shown that LH signaling rapidly decreases the cGMP-producing activity of NPR2 in both mouse and rat follicles. Dr. Potter’s group has previously shown that phosphorylation of up to 7 juxtamembrane serines and threonines is required for full NPR2 activity in vitro. To test whether LH signaling induces NPR2 dephosphorylation, I used Phos-tag gel electrophoresis, which retards the migration of phosphorylated proteins relative to their non-phosphorylated or less-phosphorylated forms. Using this technique followed by Western blotting, I showed that NPR2 in rat follicles is largely dephosphorylated after just 10 minutes of LH treatment, and is maximally dephosphorylated 30 minutes after LH (Egbert et al, 2014). If the dephosphorylation of NPR2 is prevented by using a phosphatase inhibitor, then there is no decrease in guanylyl cyclase activity in response to LH. However, the LH-induced reduction in cGMP levels is only attenuated, not prevented, suggesting that NPR2 inactivation accounts for only part of the decrease in cGMP levels after LH treatment.
The remainder of the LH-induced cGMP decrease could result from increased activity of a cGMP-hydrolyzing phosphodiesterase (PDE). In rat follicles, I showed that PDE1 and PDE5 account for most of the basal cGMP-hydrolytic activity (Egbert et al., 2016). Furthermore, PDE5 activity increases by ~70% in response to LH. Using Phos-tag gel electrophoresis, I demonstrated that PDE5 is phosphorylated on serine 92 by PKA, which has been shown to increase PDE5 activity in vitro. Thus, LH signaling both decreases the synthesis and increases the hydrolysis of cGMP. The phosphorylation and activation of PDE5 likely contribute to the LH-induced decrease in cGMP that causes meiotic resumption. I am currently testing this hypothesis using mice expressing a mutant form of PDE5 that cannot be phosphorylated. We predict that follicles from these mice will have higher post-LH cGMP levels than controls, which may inhibit or delay meiotic resumption. Together, the inactivation of the NPR2 guanylyl cyclase and the activation of the PDE5 cGMP phosphodiesterase may lower cGMP levels below a threshold that triggers meiotic resumption of the oocyte.
As I transition to research independence, I would like to examine how LH signaling regulates other events and processes in the follicle, especially ovulation, cytoskeletal rearrangements, and steroidogenesis. LH signaling primarily acts through protein kinase A (PKA), which is often integrated through one or more A-kinase anchoring proteins (AKAPs). Since several proteins involved in lipid metabolism and steroidogenesis are known to be PKA targets, disruption of signaling through AKAPs may impair steroidogenesis. AKAP regulation could be important for ovulation, since this process requires the steroid hormone progesterone. Furthermore, cytoskeletal rearrangements, which can occur through PKA signaling, are hypothesized to play a role in steroid production. Thus, I would like to pursue ultrastructural studies of the cytoskeleton and its associated organelles (e.g., mitochondria and lipid droplets) in granulosa cells during LH stimulation.
Jaffe LA and Egbert JR, (2017) Regulation of Mammalian Oocyte Meiosis by Intercellular Communication Within the Ovarian Follicle. Annu. Rev. Physiol. 79: 237-260. pdf
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. pdf - supplement
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. pdf
Egbert, JR, Shuhaibar, LC, Yee, S-P, Jaffe, LA. Phosphorylation of the cGMP phosphodiesterase PDE5 is required for the LH-induced increase in cGMP-hydrolytic activity in mouse ovarian follicles. Society for the Study of Reproduction Annual Meeting; 2016 July 16-20; San Diego, California.
As a PhD student and postdoctoral fellow in the Jaffe lab, I investigated the regulation of oocyte meiosis in mammals, in particular the role of cyclic nucleotides in transmitting hormonal signals into the oocyte. In preovulatory ovarian follicles, meiotic arrest in the oocyte is maintained by cyclic GMP. Luteinizing hormone (LH) causes a decrease in cGMP, which initiates a cascade of signaling pathways inducing oocyte maturation in preparation for fertilization. How cGMP is lowered within the follicle is not completely understood.
Our research has contributed to our understanding on how luteinizing hormone acts in the follicle to restart oocyte meiosis in preparation for ovulation and fertilization. My research included developing new methods for live tissue microscopy, which I used to show for the first time that a physiological signal initiated by a stimulus in one region of an intact tissue can travel across many layers of cells via cyclic GMP diffusion through gap junctions (Shuhaibar et al., 2015). During the course of these studies, and in collaboration with Dr. Siu-Pok Yee (UCHC) and Dr. Lincoln Potter (University of Minnesota), we developed a novel mouse model in which it was shown that a constitutively phosphorylated form of the guanylyl cyclase (NPR20 responsible for cGMP production in the ovary delays the normal progression of oocyte maturation (Shuhaibar et al., 2016).
These discoveries established a new physiological mechanism that has major implications for understanding of other physiological processes as well, including the growth of bones. Bones in mice with constitutively active NPR2 are longer than bones in wildtype mice. Our experiments indicate that, just as LH signaling dephosphorylates + inactivates NPR2 in the ovary, fibroblast growth factor signaling dephosphorylates and inactivates NPR2 in the bone growth plate. These findings may have implications for development of new therapeutics for bone growth disorders such as achondroplasia.
Shuhaibar, L.C., Egbert, J.R., Edmund, A.B., Uliasz, T.F., Dickey, D.M., Yee, S.P., Potter, L.R., and Jaffe, L.A. (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. 1;409(1):194-201 pdf - supplement
Shuhaibar, L.C., Egbert, J.R., Norris, R.P., Lampe, P.D., Nikolaev, V.O., Thunemann, M., Wen, L., Feil, R., and Jaffe, L.A. (2015). Intercellular signaling via cyclic GMP diffusion through gap junctions restarts meiosis in mouse ovarian follicles. Proc. Natl. Acad. Sci. USA 112:5527-5532. pdf
Robinson, J.W., Shuhaibar, L.C., Yee, S.P., Jaffe, L.A., Potter, L.R. Changes in the phosphorylation of guanylyl cyclase-B (GC-B) regulates bone growth in a mouse model. ASBMR (American Society for Bone and Mineral Research) Annual Meeting. Atlanta, Georgia. September 2016.
As a Postdoctoral fellow in the Jaffe lab, I’m participating in a project regarding the signaling mechanisms by which luteinizing hormone causes meiotic resumption in the ovarian follicle. Prior to joining this lab, I did my Master of Science and PhD research in the laboratory of Developmental Biology at the Univ. of Pavia (Italy), with Dr. Garagna and Dr. Zuccotti, where I specialized on the biology of the ovarian follicle. My research projects were mainly focused on the identification of murine follicle cell markers of the oocyte developmental competence. I’m now interested in more functional aspects of the female gamete growth and maturation. In particular, I’m interested in investigating the role of A-kinase anchoring proteins (AKAPs) in LH signal transduction pathways within granulosa cells in the preovulatory follicle.
Vigone G, Egbert JR, Jaffe LA. Synergistic activities of multiple cyclic AMP phosphodiesterases prevent premature meiotic progression and ovulation in mouse ovarian follicles. Cold Spring Harbor Laboratory meeting “Germ Cells”, Cold Spring Harbor, NY, USA - October 4-8, 2016.
I am currently a postdoctoral fellow in the Jaffe and Terasaki labs. In the Jaffe lab, I am investigating a phenotype in bones from mice with a mutation in the NPR2 guanylyl cyclase. Compared to the wild type mice, the substitution of the phosphomimetic amino acid glutamate for regulatory serines and threonines in this guanylyl cyclase causes increased bone length. I am contributing to this study by investigating the growth of wild type and mutant bones in culture, and comparing the structure of the wild type and mutant growth plates using histology.
Research Assistant 2
I am a research assistant in the Department of Cell Biology working jointly in the labs of Dr. Laurinda Jaffe and Dr. Lisa Mehlmann. Both labs are interested in the signaling pathways that regulate meiotic cell cycling. In the Jaffe lab, I have been involved in the study of the regulation and activity of guanylyl cyclase (NPR2) and its ligand CNP, which function to maintain high levels of cGMP in the ovarian follicle and meiotic arrest of the oocyte. We are investigating how luteinizing hormone regulates this system to cause meiosis to resume. (In particular, we are looking at signaling in cultured, primary granulosa cells.) In the Mehlmann lab the work I am involved in is focused on how the signaling pathway of the constitutively active GPR3 receptor regulates cAMP levels which is also involved in the maintenance of meiotic arrest.
Past Lab Members
- Seth Lapuk (Research Assistant, 1982-1983), Pediatric Cardiologist, Connecticut Children's Medical Center, Hartford, CT
- Ana Iglesias (Research Assistant, 1983-1984), Professor, Universidad Politechnica de Madrid, Spain
- Lyanne Schlichter (Postdoc, 1983-1984), Professor, University of Toronto, Canada
- Lavinia (Muncy) Ross (Research Assistant, 1984-1990, 2001-2003), Musician, Sweet Home, OR
- Doug Kline (Postdoc, 1985-1988), Professor, Kent State University
- Paul Turner (Ph.D., 1987), Field Applications Specialist, Affymetrics
- Kazuyoshi Chiba (Postdoc, 1988-1989), Professor, Ochanomizu University, Tokyo, Japan
- Fraser Shilling (Postdoc, 1991-1993), Research Scientist, University of California, Davis
- Chris Gallo (Postdoc, 1991-1995), Associate Research Fellow, Pfizer, Andover, MA
- David Carroll (Ph.D., 1996), Professor, Florida Institute of Technology
- David Serwanski (Research Assistant, 1995-2002), Retired
- Linda Runft (Ph.D., 2000), Senior Medical Writer, Amgen Inc.
- Lisa Mehlmann (Postdoc, 1996-2001), Assistant Professor, UConn Health Center, CT
- Andrew Giusti (Postdoc, 2000-2002), Deceased
- Rebecca Kalinowski (Ph.D., 2004), Senior Analyst at Expense Reduction Analysts, Hartford. Specializing in medical, pharmaceutical, and laboratory cost reduction.
- Leon Freudzon (Research Assistant, 2004-2005), Assistant Professor of Medicine, Department of Anesthesiology, Yale School of Medicine
- Marina Freudzon (Undergraduate Honors Student and Research Assistant, 2005-2007), resident in dermatology, Yale School of Medicine.
- Mikhail Tsesis (Research Assistant, 2009-10), Dentist, Putnam, CT.
- Rachael Norris (Ph.D., 2009), Postdoc, UConn Health Center, CT
- William Ratzan (Ph.D., 2011), Postdoc, Montana State University, MT
- Valentina Baena (Research Assistant, 2013-2015), Graduate Student, UConn Health Center.