UConn School of Medicine
263 Farmington Avenue
Farmington, CT 06030
Research in the Jaffe lab concerns the physiological mechanisms that regulate the oocyte cell cycle and fertilization. Currently, our studies are focused on regulation of meiosis in mammalian ovarian follicles by cyclic nucleotides.
Meiosis prepares the oocyte for fertilization, by reducing the number of copies of each gene from two to one, such that the female and male genomes from oocytes (eggs) and sperm can combine to make a new individual. In mammalian oocytes, meiosis begins during embryonic development of the mother, and then arrests in prophase for a prolonged period.
Meiosis prepares the oocyte for fertilization, by reducing the number of copies of each gene from two to one, such that the female and male genomes from oocytes (eggs) and sperm can combine to make a new individual. In mammalian oocytes, meiosis begins during embryonic development of the mother, and then arrests in prophase for a prolonged period.Much of the process of meiosis occurs within a spherical complex of somatic cells, called granulosa cells, that make up a follicle; the outer granulosa cells are called mural granulosa, and the granulosa cells closest to the oocyte are called cumulus (see photo below). During each reproductive cycle, a group of follicles grow to a stage at which luteinizing hormone from the pituitary can act on the mural granulosa cells, to cause resumption of meiosis and ovulation of a mature fertilizable egg.
The follicle functions as a coordinated system in which processes in the granulosa cells, as well as processes in the oocyte itself, regulate meiotic progression in the oocyte.
Work in the Jaffe lab has established that a Gs G-protein and an associated receptor, GPR3, both located in the oocyte, contribute to maintaining meiotic prophase arrest in mouse oocytes (Mehlmann et al., 2002; 2004). The activity of the Gs G-protein leads to production of cyclic AMP in the oocyte, and high cAMP keeps the cell cycle arrested during storage in the ovary .
We have also determined that the granulosa cells of the follicle contribute to maintaining meiotic prophase arrest by regulating the
hydrolysis of cAMP in the oocyte. This regulation involves another cyclic nucleotide, cyclic GMP, which diffuses from the granulosa cells into the oocyte, where it inhibits a cAMP phosphodiesterase,
PDE3A, and thus maintains the high cAMP that maintains meiotic arrest (Norris et al., 2009). Then in response to luteinizing hormone (LH), which acts on a G-protein coupled receptor in the mural granulosa cells, the signaling system in the follicle switches, such that cAMP in the oocyte decreases and meiosis proceeds. This occurs
primmarily because LH signaling lowers cGMP in the granulosa cells (Norris et al., 2009).
The cGMP decrease in the granulosa cells begins within less than a minute after LH application (Shuhaibar et al., 2015). We can monitor this change using confocal microscopy of follicles from mice expressing a FRET sensor for cGMP, cGi500. The cGMP sensor mice were made by the lab of our collaborator, Robert Feil (University of Tubingen). The LH-induced cGMP decrease occurs in a wave progressing inwards. In the outer granulosa cells, where the LH receptors are located, cGMP decreases to half of its plateau value within ~3 minutes. cGMP in the oocyte falls to half its plateau value within ~10 minutes, as a result of diffusion into the large volume of the surrounding granulosa cells. Supporting this diffusion-based mechanism for LH control of oocyte cGMP, we have previously shown that a 326-Da tracer, comparable in size to cGMP, diffuses from the oocyte through gap junctions to the granulosa cells with similar kinetics (Norris et al., 2008).
Imaging of cGMP dynamics in live mouse follicles. A. cGi500 sensor for cGMP. B. Fluorescence of a follicle expressing cGi500. C. Time course of the LH-induced cGMP decrease, moving inwards from the outer mural granulosa cells to the oocyte.
Cyclic GMP is produced in the mural granulosa and cumulus cells by the guanylyl cyclase natriuretic peptide receptor 2 (NPR2), which is activated by C-type natriuretic peptide (CNP); CNP is produced by the mural granulosa cells (work from the lab of John Eppig ; see Zhang et al., 2010, Science 330, 366-369). Our work,
in collaboration with the lab of Lincoln Potter (University of
Minnesota), has shown that LH signaling reduces cGMP in
part by reducing its production, through rapid dephosphorylation and inactivation of the guanylyl cyclase NPR2 and a slower reduction of its agonist CNP (Robinson et al., 2012; Egbert et al., 2014;
Shuhaibar et al, 2016). LH exposure
also results in rapid phosphorylation of the phosphodiesterase PDE5.
This is accompanied by a rapid increase in PDE5 activity, indicating that in parallel with decreasing cGMP
production, LH signaling increases cGMP hydrolysis (Egbert et., 2014;
Egbert et al., 2016).
LH exposure also results in rapid phosphorylation of the phosphodiesterase PDE5. This is accompanied by a rapid increase in PDE5 activity, indicating that in parallel with decreasing cGMP production, LH signaling increases cGMP hydrolysis (Egbert et., 2014; Egbert et al., 2016).
Working model of how LH signaling rapidly decreases cGMP in the mural granulosa cells, and then via cGMP diffusion through gap junctions, decreases cGMP in the oocyte, leading to meiotic resumption. Before LH exposure, cGMP concentrations are elevated throughout the follicle, due to a high rate of production of cGMP by the NPR2 guanylyl cyclase in the mural granulosa and cumulus cells. cGMP phosphodiesterases, including PDE1 and PDE5, hydrolyze cGMP at a rate equal to its production, thus keeping the cGMP concentration at a constant level. Through gap junctions that connect all cells of the follicle, cGMP diffuses into the oocyte, where it inhibits the activity of PDE3A, maintaining cAMP at a level that inhibits meiotic resumption. The cAMP in the oocyte is produced by an adenylyl cyclase in the oocyte that is kept active by the constitutive activity of the Gs-coupled receptor GPR3. When LH binds to its receptor in the mural granulosa cells, the activation of Gs and possibly other G-proteins results in dephosphorylation of NPR2, which decreases its rate of production of cGMP. Activation of the LH receptor also increases phosphorylation of PDE5, which increases its rate of hydrolysis of cGMP. Due to reduced NPR2 activity and increased cGMP phosphodiesterase activity, the concentration of cGMP in the mural granulosa cells decreases. Through the series of gap junctions that connects the oocyte to the large volume of the mural granulosa cells, cGMP in the oocyte diffuses down its concentration gradient, and the resulting decrease in oocyte cGMP relieves the inhibition of PDE3A in the oocyte, such that cAMP decreases. This model depicts only events occuring in the first 20 minutes after LH exposure. Subsequent events, including a decrease in gap junction permeability, an increase in EGF receptor ligands, and a decrease in C-type natriuretic peptide, also contribute to maintaining cGMP at the low level that triggers meiotic resumption. Modified from Shuhaibar et al., 2015.
Lab Member's page: See posters of current work!
Movie: Microinjection of an antral follicle-enclosed mouse oocyte
Jaffe LA and Egbert JR, (2017) Regulation of Mammalian Oocyte Meiosis by Intercellular Communication Within the Ovarian Follicle. Annu. Rev. Physiol. 79: 10.1-10.24. pdf
Egbert J.R., Uliasz T.F., Shuhaibar, L.C., Geerts A., Wunder F., Kleiman R.J., Humphrey J.M., Lampe P.D., Artemyev, N.O., Rybalkin, S.D., Beavo, J.A., Movsesian, M.A., and Jaffe, L.A. (2016) Luteinizing hormone causes phosphorylation and activation of the cyclic GMP phosphodiesterase PDE5 in rat ovarian follicles, contributing, together with PDE1 activity, to the resumption of meiosis. Biol. Reprod. 94(5):110. pdf - supplement
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. 409:194-201. 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
Egbert, J.R., Shuhaibar, L.C., Edmund, A.B., Van Helden, D.A., Robinson, J.W., Uliasz, T.F., Baena, V., Geerts, A., Wunder, F., Potter, L.R., and Jaffe, L.A. (2014). Dephosphorylation and inactivation of the NPR2 guanylyl cyclase in the granulosa cells contributes to the LH-induced decrease in cGMP that causes meiotic resumption in rat oocytes. Development. 141:3594-3604. pdf
Robinson, J.W., Zhang, M., Shuhaibar, L.C., Norris, R.P., Geerts, A., Wunder, F., Eppig, J.J., Potter, L.R., and Jaffe, L.A. (2012). Luteinizing hormone reduces the activity of the NPR2 guanylyl cyclase in mouse ovarian follicles, contributing to the cyclic GMP decrease that promotes resumption of meiosis in oocytes. Dev. Biol. 366: 308–316. pdf - supplement
Norris, R.P., Freudzon, M., Nikolaev, V.O., and Jaffe, L.A. 2010. Epidermal growth factor receptor kinase activity is required for gap junction closure and for part of the decrease in ovarian follicle cGMP in response to LH. Reproduction 140: 655-662. pdf
Norris, R.P., Ratzan, W.J., Freudzon, M., Mehlmann, L.M., Krall, J., Movsesian, M.A., Wang, H., Ke, H., Nikolaev, V.O., and Jaffe, L.A. (2009). Cyclic GMP from the surrounding somatic cells regulates cyclic AMP and meiosis in the mouse oocyte. Development 136: 1869-1878. pdf - supplement
Jaffe, L.A., Norris, R.P., Freudzon, M., Ratzan, W.J., and Mehlmann, L.M. (2009). Microinjection of follicle-enclosed mouse oocytes. Methods Mol. Biol. 518:157-173. pdf
Norris, R.P., Freudzon, M., Mehlmann, L.M., Cowan, A.E., Simon, A.M., Paul, D.L., Lampe, P.D., and Jaffe, L.A. (2008). Luteinizing hormone causes MAPK-dependent phosphorylation and closure of Cx43 gap junctions in mouse ovarian follicles: one of two paths to meiotic resumption. Development 135:3229-3238. pdf - supplement
Norris, R.P., Freudzon, L., Freudzon, M., Hand, A.R., Mehlmann, L.M., and Jaffe, L.A. (2007). A Gs linked receptor maintains meiotic arrest in mouse oocytes, but luteinizing hormone does not cause meiotic resumption by terminating receptor Gs signaling. Develop. Biol. 310: 240-249.