Laurinda A. Jaffe

Cyclic GMP from the surrounding somatic cells regulates cyclic AMP and meiosis in the mouse oocyte.

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 ∼2 μM to ∼80 nM at 1 hour after LH stimulation) and by closing gap junctions between the somatic cells. The resulting decrease in oocyte cGMP (from ∼1 μM to ∼40 nM) relieves the inhibition of PDE3A, increasing its activity by ∼5-fold. This causes a decrease in oocyte cAMP (from ∼700 nM to ∼140 nM), leading to the resumption of meiosis.

Luteinizing hormone causes MAP kinase-dependent phosphorylation and closure of connexin 43 gap junctions in mouse ovarian follicles: one of two paths to meiotic resumption

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.

Development of calcium release mechanisms during starfish oocyte maturation

In response to the maturation-inducing hormone 1-methyladenine, starfish oocytes acquire increased sensitivity to sperm and inositol trisphosphate (InsP3), stimuli that cause a release of calcium from intracellular stores and a rise in intracellular free calcium. In the immature oocyte, the calcium release in response to 10 sperm entries is less than that seen with a single sperm entry in the mature egg. Likewise, the sensitivity to injected InsP3 is less in the immature oocyte. Approximately 100 times as much InsP3 is required to obtain the same calcium release in an immature oocyte as in a mature egg. However, with saturating amounts of InsP3, immature oocytes and mature eggs release comparable amounts of calcium. These results indicate that although calcium stores are well-developed in the immature oocyte, mechanisms for releasing the calcium develop fully only during oocyte maturation.

Absence of an electrical polyspermy block in the mouse

To examine the possibility of an electrical polyspermy block in the mouse, we recorded the electrophysiological properties of zona-free mouse eggs during fertilization. Starting from an unfertilized value of -41 +/- 4 mV (SD), the membrane potential undergoes an oscillation (seen in 8 of 11 records) of 4 +/- 1 mV in amplitude, starting 7 +/- 5 min after insemination, and lasting about 1 min. However, except for this small oscillation, the membrane potential is constant during the 60 min following insemination; the average range (11 +/- 4 mV) is not significantly different from that which is observed in 60-min recordings from unfertilized eggs. These results indicate that the polyspermy block which is established during this period (D. P. Wolf, 1978, Dev. Biol. 64, 1-10) is not electrically mediated. Consistent with this finding, reduction of the sodium or calcium concentration in the external medium does not induce polyspermy. As a consequence of fertilization, the resistance of the egg membrane decreases from 96 +/- 34 to 44 +/- 15 M omega; this change accompanies the voltage oscillation.

Requirement of a Src Family Kinase for Initiating Calcium Release at Fertilization in Starfish Eggs

Signal transduction leading to calcium release in echinoderm eggs at fertilization requires phospholipase Cγ-mediated production of inositol trisphosphate (IP3), indicating that a tyrosine kinase is a likely upstream regulator. Because previous work has shown a fertilization-dependent association between the Src homology 2 (SH2) domains of phospholipase Cγ and a Src family kinase, we examined whether a Src family kinase was required for Ca2+ release at fertilization. To inhibit the function of kinases in this family, we injected starfish eggs with the SH2 domains of Src and Fyn kinases. This inhibited Ca2+ release in response to fertilization but not in response to injection of IP3. We further established the specificity of the inhibition by showing that the SH2 domains of several other tyrosine kinases (Abl, Syk, and ZAP-70), and the SH3 domain of Src, were not inhibitory. Also, a point-mutated Src SH2 domain, which has reduced affinity for phosphotyrosine, was a correspondingly less effective inhibitor of fertilization-induced Ca2+ release. These results indicate that a Src family kinase, by way of its SH2 domain, links sperm-egg interaction to IP3-mediated Ca2+ release at fertilization in starfish eggs.

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

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.

Membrane potential of the unfertilized sea urchin egg

Abstract The membrane potential, specific resistance, and potassium selectivity of the unfertilized Strongylocentrotus purpuratus egg were determined by two independent methods: tracer flux and microelectrode. The potassium influx was 0.50 ± 0.2 pmole/cm2· sec, which was greater than the sodium, chloride, and calcium influxes by factors of 4, 7, and 75, respectively. By means of the constant-field equations, the flux data were used to calculate membrane potential (−70 mV) and specific resistance (420 kΩ · cm2). The effect of the external potassium concentration on the sodium influx was determined and the results closely fit the result expected if the membrane behaved as a potassium electrode. Microelectrode measurements of the potential and resistance were −75 ± 3 mV and 380 ± kΩ · cm2.

The time course of cortical vesicle fusion in sea urchin eggs observed as membrane capacitance changes

Abstract The addition of cortical vesicle membrane to the plasma membrane during the first 2 min after insemination in Strongylocentrotus purpuratus eggs was monitored continuously by measuring the resulting capacitance increase with AC current (400 Hz), using intracellular electrodes. The capacitance increase was first detected at 30 to 45 sec after the rise of the activation potential, thus allowing a more precise determination of the “latent period.” The time interval from a 10 to 50% change in capacitance was always less than that from the 50 to 90% level, suggesting that the traveling wave of cortical vesicle fusion slows down as it propagates.

Regulation of meiotic prophase arrest in mouse oocytes by GPR3, a constitutive activator of the Gs G protein

The arrest of meiotic prophase in mouse oocytes within antral follicles requires the G protein Gs and an orphan member of the G protein–coupled receptor family, GPR3. To determine whether GPR3 activates Gs, the localization of Gαs in follicle-enclosed oocytes from Gpr3 +/+ and Gpr3 −/− mice was compared by using immunofluorescence and GαsGFP. GPR3 decreased the ratio of Gαs in the oocyte plasma membrane versus the cytoplasm and also decreased the amount of Gαs in the oocyte. Both of these properties indicate that GPR3 activates Gs. The follicle cells around the oocyte are also necessary to keep the oocyte in prophase, suggesting that they might activate GPR3. However, GPR3-dependent Gs 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.

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

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.