Laurinda A. Jaffe

University of Connecticut, Сторс, Connecticut, United States

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Publications (81)478.63 Total impact

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    ABSTRACT: The meiotic cell cycle of mammalian oocytes starts during embryogenesis and then pauses until luteinizing hormone (LH) acts on the granulosa cells of the follicle surrounding the oocyte to restart the cell cycle. An essential event in this process is a decrease in cyclic GMP in the granulosa cells, and part of the cGMP decrease results from dephosphorylation and inactivation of the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase, also known as guanylyl cyclase B. However, it is unknown whether NPR2 dephosphorylation is essential for LH-induced meiotic resumption. Here, we prevented NPR2 dephosphorylation by generating a mouse line in which the seven regulatory serines and threonines of NPR2 were changed to the phosphomimetic amino acid glutamate (Npr2-7E). Npr2-7E/7E follicles failed to show a decrease in enzyme activity in response to LH, and the cGMP decrease was attenuated; correspondingly, LH-induced meiotic resumption was delayed. Meiotic resumption in response to EGF receptor activation was likewise delayed, indicating that NPR2 dephosphorylation is a component of the pathway by which EGF receptor activation mediates LH signaling. We also found that most of the NPR2 protein in the follicle was present in the mural granulosa cells. These findings indicate that NPR2 dephosphorylation in the mural granulosa cells is essential for the normal progression of meiosis in response to LH and EGF receptor activation. In addition, these studies provide the first demonstration that a change in phosphorylation of a transmembrane guanylyl cyclase regulates a physiological process, a mechanism that may also control other developmental events.
    No preview · Article · Nov 2015 · Developmental Biology
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    Preview · Article · Sep 2015 · BMC pharmacology & toxicology
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    ABSTRACT: Meiosis in mammalian oocytes is paused until luteinizing hormone (LH) activates receptors in the mural granulosa cells of the ovarian follicle. Prior work has established the central role of cyclic GMP (cGMP) from the granulosa cells in maintaining meiotic arrest, but it is not clear how binding of LH to receptors that are located up to 10 cell layers away from the oocyte lowers oocyte cGMP and restarts meiosis. Here, by visualizing intercellular trafficking of cGMP in real-time in live follicles from mice expressing a FRET sensor, we show that diffusion of cGMP through gap junctions is responsible not only for maintaining meiotic arrest, but also for rapid transmission of the signal that reinitiates meiosis from the follicle surface to the oocyte. Before LH exposure, the cGMP concentration throughout the follicle is at a uniformly high level of ∼2-4 μM. Then, within 1 min of LH application, cGMP begins to decrease in the peripheral granulosa cells. As a consequence, cGMP from the oocyte diffuses into the sink provided by the large granulosa cell volume, such that by 20 min the cGMP concentration in the follicle is uniformly low, ∼100 nM. The decrease in cGMP in the oocyte relieves the inhibition of the meiotic cell cycle. This direct demonstration that a physiological signal initiated by a stimulus in one region of an intact tissue can travel across many layers of cells via cyclic nucleotide diffusion through gap junctions could provide a general mechanism for diverse cellular processes.
    Full-text · Article · Mar 2015 · Proceedings of the National Academy of Sciences
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    ABSTRACT: In mammals, the meiotic cell cycle of oocytes starts during embryogenesis and then pauses. Much later, in preparation for fertilization, oocytes within preovulatory follicles resume meiosis in response to luteinizing hormone (LH). Before LH stimulation, the arrest is maintained by diffusion of cyclic (c)GMP into the oocyte from the surrounding granulosa cells, where it is produced by the guanylyl cyclase natriuretic peptide receptor 2 (NPR2). LH rapidly reduces the production of cGMP, but how this occurs is unknown. Here, using rat follicles, we show that within 10 min, LH signaling causes dephosphorylation and inactivation of NPR2 through a process that requires the activity of phosphoprotein phosphatase (PPP)-family members. The rapid dephosphorylation of NPR2 is accompanied by a rapid phosphorylation of the cGMP phosphodiesterase PDE5, an enzyme whose activity is increased upon phosphorylation. Later, levels of the NPR2 agonist C-type natriuretic peptide decrease in the follicle, and these sequential events contribute to the decrease in cGMP that causes meiosis to resume in the oocyte.
    Full-text · Article · Sep 2014 · Development
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    ABSTRACT: The 2nd messenger cyclic guanosine monophosphate (cGMP) is well-known to regulate important functions of the cardiovascular and nervous system. cGMP is generated by NO-stimulated guanylyl cyclases in the cytosol or by particulate guanylyl cyclases at the plasma membrane. This subcellular compartmentalization may have important impacts on its physiological functions. However, it is not known when, where, and how much cGMP is generated in a mammalian organism in vivo. Genetically-encoded cGMP indicator proteins (cGi's) allow for cGMP imaging in live cells with excellent spatial and temporal resolution. We generated mouse lines expressing the most sensitive cGi variant, cGi500 (with an EC50 of ~500 nM) in the cytosol [1], or a newly-generated, membrane-targeted version of this sensor, mcGi500. In the SM22-cGi500 mouse line, the SM22 promoter drives cGi500 expression selectively in smooth muscle cells of adult mice. Other transgenic lines were generated by targeted insertion of CAG promoter-driven cGi500 or mcGi500 transgenes into the Rosa26 (R26) locus. R26-(m)cGi500-L1 lines show strong sensor expression in every cell type tested so far. R26R-(m)cGi500-L2 lines carry conditional sensor constructs, expressed only after Cre-mediated excision of a STOP cassette. Therefore, cGi500 or mcGi500 expression can be directed to any tissue of choice by mating the respective R26R-(m)cGi500-L2 line to tissue-specific Cre mice. Initially, cGMP imaging studies were performed with cells in primary culture, or tissues freshly isolated from SM22-cGi500 and R26-(m)cGi500-L1 mice. In retina from SM22-cGi500 and aorta from R26-cGi500-L1 mice, we were able to detect cGMP transients induced with the NO-donor DEA/NO. We also visualized cGMP decreases in response to luteinizing hormone in ovarian follicles from R26-cGi500-L1 mice. Moreover, cGMP imaging was also feasible in vivo by intravital microscopy of live R26-cGi500-L1 mice. Using epifluorescence microscopy, we monitored DEA/NO-induced cGMP transients in resistance-type vessels of the cremaster muscle. Using multiphoton microscopy, DEA/NO-induced cGMP transients were detected in the walls of subcutaneous blood vessels accessed through a chronic skinfold chamber. Here, the cGMP transients correlated with the occurrence and extent of NO-induced vasodilation in vivo. All in all, transgenic mice expressing cGi500 or mcGi500 allow cGMP imaging with high temporal and spatial resolution in intact cells, isolated tissues, and live mice. We believe that cGMP imaging in vivo, particularly when correlated with physiological responses, should provide deeper insights into the physiology and pathophysiology of cGMP signaling. [1] Thunemann, M., Wen, L. et al., 2013. Transgenic Mice for cGMP Imaging. Circulation research, 113(4), pp.365–71.
    No preview · Conference Paper · Apr 2014
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    Full-text · Conference Paper · Aug 2013
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    ABSTRACT: Natriuretic peptide type C (NPPC) and its receptor natriuretic peptide receptor 2 (NPR2) regulate cGMP in ovarian follicles and participate in maintaining oocyte meiotic arrest. We investigated the regulation of Nppc expression in mouse granulosa cells in vivo and in vitro. In mural granulosa cells (MGCs) in vivo, eCG caused an increase in Nppc mRNA and subsequent hCG-treatment caused a decrease. A culture system was established for MGCs isolated from follicles not stimulated with eCG to further define mechanisms controlling Nppc expression. In this system, expression of Nppc mRNA was increased by E2, with augmentation by FSH, but FSH or LH alone had no effect. Thus estrogens are important for regulating Nppc expression, probably by feedback mechanisms enhancing the action of gonadotropins. In MGCs treated with E2 plus FSH in vitro, subsequent treatment with EGF, but not LH, decreased Nppc mRNA. MGCs express higher levels of both Nppc and Lhcgr mRNAs than cumulus cells. Oocyte-derived paracrine factors suppressed cumulus cell Lhcgr but not Nppc expression. Thus, higher Nppc expression by MGCs is not the result of oocyte suppression of expression in cumulus cells. Another possible regulator of the LH-induced NPPC decrease is NPR3, an NPPC clearance receptor. hCG increased Npr3 expression in vivo and LH increased Npr3 mRNA in cultured MGCs, independently of EGF-receptor activation. Interestingly, despite the increase in Npr3 mRNA, the hCG-induced decrease in ovarian NPPC occurred normally in an Npr3 mutant (lgj), thus NPR3 probably does not participate in regulation of ovarian NPPC levels or oocyte development.
    No preview · Article · Dec 2012 · Biology of Reproduction
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    ABSTRACT: In preovulatory ovarian follicles of mice, meiotic prophase arrest in the oocyte is maintained by cyclic GMP from the surrounding granulosa cells that diffuses into the oocyte through gap junctions. The cGMP is synthesized in the granulosa cells by the transmembrane guanylyl cyclase natriuretic peptide receptor 2 (NPR2) in response to the agonist C-type natriuretic peptide (CNP). In response to luteinizing hormone (LH), cGMP in the granulosa cells decreases, and as a consequence, oocyte cGMP decreases and meiosis resumes. Here we report that within 20 min, LH treatment results in decreased guanylyl cyclase activity of NPR2, as determined in the presence of a maximally activating concentration of CNP. This occurs by a process that does not reduce the amount of NPR2 protein. We also show that by a slower process, first detected at 2h, LH decreases the amount of CNP available to bind to the receptor. Both of these LH actions contribute to decreasing cGMP in the follicle, thus signaling meiotic resumption in the oocyte.
    Full-text · Article · Apr 2012 · Developmental Biology
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    ABSTRACT: Voltage-sensitive phosphatases (VSPs) are unique proteins in which membrane potential controls enzyme activity. They are comprised of the voltage sensor domain of an ion channel coupled to a lipid phosphatase specific for phosphoinositides, and for ascidian and zebrafish VSPs, the phosphatase activity has been found to be activated by membrane depolarization. The physiological functions of these proteins are unknown, but their expression in testis and embryos suggests a role in fertilization or development. Here we investigate the expression pattern and voltage dependence of VSPs in two frog species, Xenopus laevis and Xenopus tropicalis, that are well suited for experimental studies of these possible functions. X. laevis has two VSP genes (Xl-VSP1 and Xl-VSP2), whereas X. tropicalis has only one gene (Xt-VSP). The highest expression of these genes was observed in testis, ovary, liver, and kidney. Our results show that while Xl-VSP2 activates only at positive membrane potentials outside of the physiological range, Xl-VSP1 and Xt-VSP phosphatase activity is regulated in the voltage range that regulates sperm-egg fusion at fertilization.
    Full-text · Article · Nov 2011 · Journal of Cellular Physiology
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    ABSTRACT: The meiotic cell cycle in mouse oocytes is arrested in prophase, and then restarted when LH acts on the surrounding granulosa cells. The granulosa cells keep meiosis arrested by providing a source of cGMP that diffuses into the oocyte through gap junctions, and LH restarts the cell cycle by closing the junctions and by decreasing granulosa cell cGMP, thus lowering oocyte cGMP. Epidermal growth factor receptor (EGFR) activation is an essential step in triggering LH-induced meiotic resumption, but its relationship to the cGMP decrease in the follicle is incompletely understood, and its possible function in causing gap junction closure has not been investigated. Here, we use EGFR agonists (epiregulin and amphiregulin) and an EGFR kinase inhibitor (AG1478) to study the function of the EGFR in the signaling pathways leading to the release of oocytes from prophase arrest. Our results indicate that the EGFR kinase contributes to LH-induced meiotic resumption in two different ways. First, it is required for gap junction closure. Second, it is required for an essential component of the decrease in follicle cGMP. Our data show that the EGFR kinase-dependent component of the cGMP decrease is required for LH-induced meiotic resumption, but they also indicate that an as yet unidentified pathway accounts for a large part of the cGMP decrease.
    Preview · Article · Nov 2010 · Reproduction
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    Laurinda A. Jaffe · Rachael P. Norris
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    ABSTRACT: Mammalian oocytes are arrested in meiotic prophase for prolonged periods, and then resume meiosis in response to luteinizing hormone from the pituitary, which binds to the somatic cells surrounding the oocyte. This chapter reviews current knowledge and questions about the molecular mechanisms that mediate communication between the somatic cells and oocyte to control the prophase-to-metaphase transition.
    Preview · Article · Apr 2010
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    Preview · Article · Jan 2010
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    ABSTRACT: Mammalian oocytes are arrested in meiotic prophase by an inhibitory signal from the surrounding somatic cells in the ovarian follicle. In response to luteinizing hormone (LH), which binds to receptors on the somatic cells, the oocyte proceeds to second metaphase, where it can be fertilized. Here we investigate how the somatic cells regulate the prophase-to-metaphase transition in the oocyte, and show that the inhibitory signal from the somatic cells is cGMP. Using FRET-based cyclic nucleotide sensors in follicle-enclosed mouse oocytes, we find that cGMP passes through gap junctions into the oocyte, where it inhibits the hydrolysis of cAMP by the phosphodiesterase PDE3A. This inhibition maintains a high concentration of cAMP and thus blocks meiotic progression. LH reverses the inhibitory signal by lowering cGMP levels in the somatic cells (from approximately 2 microM to approximately 80 nM at 1 hour after LH stimulation) and by closing gap junctions between the somatic cells. The resulting decrease in oocyte cGMP (from approximately 1 microM to approximately 40 nM) relieves the inhibition of PDE3A, increasing its activity by approximately 5-fold. This causes a decrease in oocyte cAMP (from approximately 700 nM to approximately 140 nM), leading to the resumption of meiosis.
    No preview · Article · Jul 2009 · Development
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    Full-text · Article · Jul 2009 · Developmental Biology
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    ABSTRACT: The mammalian oocyte develops within a complex of somatic cells known as a follicle, within which signals from the somatic cells regulate the oocyte, and signals from the oocyte regulate the somatic cells. Because isolation of the oocyte from the follicle disrupts these communication pathways, oocyte physiology is best studied within an intact follicle. Here we describe methods for quantitative microinjection of follicle-enclosed mouse oocytes, thus allowing the introduction of signaling molecules as well as optical probes into the oocyte within its physiological environment.
    Preview · Article · Feb 2009 · Methods in Molecular Biology
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    William J. Ratzan · Yasushi Okamura · Laurinda A. Jaffe

    Preview · Article · Feb 2009 · Biophysical Journal
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    ABSTRACT: Luteinizing hormone (LH) acts on ovarian follicles to reinitiate meiosis in prophase-arrested mammalian oocytes, and this has been proposed to occur by interruption of a meioisis-inhibitory signal that is transmitted through gap junctions into the oocyte from the somatic cells that surround it. To investigate this idea, we microinjected fluorescent tracers into live antral follicle-enclosed mouse oocytes, and we demonstrate for the first time that LH causes a decrease in the gap junction permeability between the somatic cells, prior to nuclear envelope breakdown (NEBD). The decreased permeability results from the MAP kinase-dependent phosphorylation of connexin 43 on serines 255, 262 and 279/282. We then tested whether the inhibition of gap junction communication was sufficient and necessary for the reinitiation of meiosis. Inhibitors that reduced gap junction permeability caused NEBD, but an inhibitor of MAP kinase activation that blocked gap junction closure in response to LH did not prevent NEBD. Thus, both MAP kinase-dependent gap junction closure and another redundant pathway function in parallel to ensure that meiosis resumes in response to LH.
    Full-text · Article · Nov 2008 · Development
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    ABSTRACT: The maintenance of meiotic prophase arrest in fully grown vertebrate oocytes depends on the activity of a G(s) G-protein that activates adenylyl cyclase and elevates cAMP, and in the mouse oocyte, G(s) is activated by a constitutively active orphan receptor, GPR3. To determine whether the action of luteinizing hormone (LH) on the mouse ovarian follicle causes meiotic resumption by inhibiting GPR3-G(s) signaling, we examined the effect of LH on the localization of Galpha(s). G(s) activation in response to stimulation of an exogenously expressed beta(2)-adrenergic receptor causes Galpha(s) to move from the oocyte plasma membrane into the cytoplasm, whereas G(s) inactivation in response to inhibition of the beta(2)-adrenergic receptor causes Galpha(s) to move back to the plasma membrane. However, LH does not cause a change in Galpha(s) localization, indicating that LH does not act by terminating receptor-G(s) signaling.
    Preview · Article · Nov 2007 · Developmental Biology
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    ABSTRACT: The signaling pathway by which luteinizing hormone (LH) acts on the somatic cells of vertebrate ovarian follicles to stimulate meiotic resumption in the oocyte requires a decrease in cAMP in the oocyte, but how cAMP is decreased is unknown. Activation of Gi family G proteins can lower cAMP by inhibiting adenylate cyclase or stimulating a cyclic nucleotide phosphodiesterase, but we show here that inhibition of this class of G proteins by injection of pertussis toxin into follicle-enclosed mouse oocytes does not prevent meiotic resumption in response to LH. Likewise, elevation of Ca2+ can lower cAMP through its action on Ca2+-sensitive adenylate cyclases or phosphodiesterases, but inhibition of a Ca2+ rise by injection of EGTA into follicle-enclosed mouse oocytes does not inhibit the LH response. Thus, neither of these well-known mechanisms of cAMP regulation can account for LH signaling to the oocyte in the mouse ovary.
    Preview · Article · Dec 2006 · Developmental Biology
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    ABSTRACT: The arrest of meiotic prophase in mouse oocytes within antral follicles requires the G protein G(s) and an orphan member of the G protein-coupled receptor family, GPR3. To determine whether GPR3 activates G(s), the localization of Galpha(s) in follicle-enclosed oocytes from Gpr3(+/+) and Gpr3(-/-) mice was compared by using immunofluorescence and Galpha(s)GFP. GPR3 decreased the ratio of Galpha(s) in the oocyte plasma membrane versus the cytoplasm and also decreased the amount of Galpha(s) in the oocyte. Both of these properties indicate that GPR3 activates G(s). The follicle cells around the oocyte are also necessary to keep the oocyte in prophase, suggesting that they might activate GPR3. However, GPR3-dependent G(s) activity was similar in follicle-enclosed and follicle-free oocytes. Thus, the maintenance of prophase arrest depends on the constitutive activity of GPR3 in the oocyte, and the follicle cell signal acts by a means other than increasing GPR3 activity.
    Full-text · Article · Nov 2005 · The Journal of Cell Biology

Publication Stats

5k Citations
478.63 Total Impact Points


  • 2009-2014
    • University of Connecticut
      Сторс, Connecticut, United States
    • Brandeis University
      Волтам, Massachusetts, United States
  • 1997
    • French Institute of Health and Medical Research
      Lutetia Parisorum, Île-de-France, France
  • 1990-1996
    • Marine Biological Laboratory
      Falmouth, Massachusetts, United States
  • 1993
    • National Institute of Neurological Disorders and Strokes
      Chicago, Illinois, United States
  • 1991-1993
    • National Institutes of Health
      • Laboratory of Virology (LV)
      Maryland, United States
  • 1989
    • Yamaguchi University
      • Faculty of Science
      Yamaguti, Yamaguchi, Japan
  • 1986
    • University of Maine at Farmington
      Фармингтон, Maine, United States
  • 1983-1985
    • University of California, Davis
      Davis, California, United States
  • 1978
    • University of California, Los Angeles
      • Department of Physiology
      Los Ángeles, California, United States