Kyozuka K et al. (2009), Guanine nucleotides in the meiotic maturation o...

ECB-ART-41222

PLoS One 2009 Jul 20;47:e6296. doi: 10.1371/journal.pone.0006296.

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Guanine nucleotides in the meiotic maturation of starfish oocytes: regulation of the actin cytoskeleton and of Ca(2+) signaling.

Kyozuka K , Chun JT , Puppo A , Gragnaniello G , Garante E , Santella L .

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BACKGROUND: Starfish oocytes are arrested at the first prophase of meiosis until they are stimulated by 1-methyladenine (1-MA). The two most immediate responses to the maturation-inducing hormone are the quick release of intracellular Ca(2+) and the accelerated changes of the actin cytoskeleton in the cortex. Compared with the later events of oocyte maturation such as germinal vesicle breakdown, the molecular mechanisms underlying the early events involving Ca(2+) signaling and actin changes are poorly understood. Herein, we have studied the roles of G-proteins in the early stage of meiotic maturation. METHODOLOGY/PRINCIPAL FINDINGS: By microinjecting starfish oocytes with nonhydrolyzable nucleotides that stabilize either active (GTPgammaS) or inactive (GDPbetaS) forms of G-proteins, we have demonstrated that: i) GTPgammaS induces Ca(2+) release that mimics the effect of 1-MA; ii) GDPbetaS completely blocks 1-MA-induced Ca(2+); iii) GDPbetaS has little effect on the amplitude of the Ca(2+) peak, but significantly expedites the initial Ca(2+) waves induced by InsP(3) photoactivation, iv) GDPbetaS induces unexpectedly striking modification of the cortical actin networks, suggesting a link between the cytoskeletal change and the modulation of the Ca(2+) release kinetics; v) alteration of cortical actin networks with jasplakinolide, GDPbetaS, or actinase E, all led to significant changes of 1-MA-induced Ca(2+) signaling. CONCLUSIONS/SIGNIFICANCE: Taken together, these results indicate that G-proteins are implicated in the early events of meiotic maturation and support our previous proposal that the dynamic change of the actin cytoskeleton may play a regulatory role in modulating intracellular Ca(2+) release.

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Genes referenced: LOC115919910 LOC583082 LOC590297 pole

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	Figure 1. 1-MA-induced Ca2+ signaling in starfish oocyte is mimicked by microinjection of GTPγS. A. pectinifera oocytes loaded with calcium dye were either incubated with 1-MA or microinjected with 50 mM of GTPγS. (A) Relative fluorescence pseudo-colored images of the Ca2+ indicator. To compare the kinetics of Ca2+ rise, the moment of the first detectable Ca2+ signal was set to t = 0 in both cases. (B) Quantification of intracellular Ca2+ levels induced by 1-MA (green curve) or GTPγS (brown curve).
	Figure 2. Preinjection of GDPβS blocks 1-MA-induced Ca2+ signaling and GVBD in starfish oocytes. A. pectinifera oocytes were microinjected with either GDPβS or the vehicle buffer and incubated for 15 min prior to the exposure to 1-MA. (A) Quantification of intracellular Ca2+ levels induced by 1-MA in the presence (brown curves, n = 4) or absence (green curves, n = 4) of GDPβS (100 mM, pipette concentration). (B) The effect of GDPβS on GVBD. Oocytes were injected with various amount of GDPβS and exposed to 1-MA for 1 h. The concentration of GDPβS in the histogram refers to the concentration in the microinjection pipette. The amount delivered into the oocyte was 1% of the cell volume. The frequency of oocytes that had undergone successful GVBD were calculated for each concentration of GDPβS (n = 84).
	Figure 3. Effects of GDPβS on InsP3-dependent intracellular Ca2+ release and on the actin cytoskeleton in immature oocytes. A. pectinifera oocytes loaded with calcium dye and caged InsP3 were microinjected with GDPβS (100 mM, pipette concentration) or with the vehicle buffer. After 20 min incubation, the oocytes were illuminated with UV to uncage InsP3 and analyzed for intracellular Ca2+ release. To compare the kinetics of Ca2+ rise, the moment of the first detectable Ca2+ signal was set to t = 0 in both cases. (A) The relative fluorescence pseudo-colored images of the Ca2+ indicator at 7 sec. In control oocytes, InsP3-induced Ca2+ signals initiated from the cortex at the animal pole (arrow) near the nucleus (germinal vesicle, marked with n). In oocytes GDPβS-injected oocytes, this characteristic mode of Ca2+ wave initiation is lost. (B) Quantification of intracellular Ca2+ levels induced by uncaged InsP3 in the presence (brown curves, n = 8) or absence (green curves, n = 5) of GDPβS. The duration of photoactivation is marked with the violet bar labeled UV. (C) Comparison of the average Ca2+ peaks in the control (n = 5) and the GDPβS-injected (n = 8) oocytes (P<0.01). (D) Comparison of the kinetics of Ca2+ rises in the control (n = 5) and the GDPβS-injected (n = 8) oocytes. The time required for reaching 0.1 RFU was scored for each case, and the average and standard deviation of the values in the control and GDPβS-injected oocytes were presented in the histogram (P<0.001). (E) The state of the actin cytoskeleton in the control and GDPβS-injected oocytes. After 30 min incubation, actin filaments were visualized in live oocytes with Alexa Fluor 568-conjugated phalloidin (concentration in injection pipette, 50 µM). The arrowhead indicates enhancement of actin networks by GDPβS.
	Figure 4. Effects of GDPβS on InsP3-dependent intracellular Ca2+ release and on the actin cytoskeleton in mature eggs. A. pectinifera oocytes were loaded with calcium dye and caged InsP3 and exposed to 1-MA for 1 h. The eggs displaying successful GVBD were microinjected with GDPβS (100 mM, pipette concentration) or with the vehicle buffer. After 20 min incubation, the oocytes were illuminated with UV to uncage InsP3 and analyzed for intracellular Ca2+ release. To compare the kinetics of Ca2+ rise, the moment of the first detectable Ca2+ release was set to t = 0 in both cases. (A) The state of the actin cytoskeleton in the control and GDPβS-injected eggs. After 30 min incubation, actin filaments were visualized with Alexa Fluor 568-conjugated phalloidin. The arrowhead indicates enhancement of cortical actin networks by GDPβS. (B) Quantification of intracellular Ca2+ levels induced by uncaged InsP3 in the presence (brown curves, n = 6) or absence (green curves, n = 9) of GDPβS. (C) Comparison of the average amplitude of the Ca2+ peaks in the control (n = 9) and the GDPβS-injected (n = 6) oocytes (P>0.1). (D) Comparison of the kinetics of Ca2+ rises in the control (n = 9) and the GDPβS-injected (n = 6) oocytes. The time required for reaching 0.1 RFU was scored for each case, and the average and standard deviation of the values in the control and GDPβS-injected eggs were presented in the histogram (P<0.001). (E) Elevation of vitelline layers in response to InsP3-induced intracellular Ca2+ release is largely blocked in the eggs pre-injected with GDPβS (n = 4). Partial elevation of the membrane is observed only in a limited area of the egg surface (arrowhead).
	Figure 5. Effects of cortical actin networks on the Ca2+ waves generated by 1-MA and GTPγS. A. pectinifera oocytes loaded with calcium dye were incubated in the presence or absence of 12 µM JAS for 30 min and subjected to 1-MA or GTPγS treatments. (A) Alteration of cortical actin networks by JAS, as visualized by Alexa Fluor 568-conjugated phalloidin. Enhanced F-actin structures in the subplasmalemmal regions were marked with an arrowhead. (B) The Ca2+ response to 1-MA is nearly eliminated in JAS-treated oocytes. (C) The release of intracellular Ca2+ in response to GTPγS injection is either blocked or significantly delayed by JAS.
	Figure 6. Alteration of the cortical actin network and propagation of Ca2+ signals in denuded oocytes.The vitelline coats of A. pectinifera oocytes were removed as described in Experimental Procedures. The denuded and intact (control) oocytes were then loaded with calcium dye and exposed to 1-MA. (A) Alteration of cortical actin networks in denuded oocytes, as visualized by Alexa Fluor 568-conjugated phalloidin. Abolishment of F-actin structures in the subplasmalemmal regions was marked with an arrowhead. (B) The relative fluorescence pseudo-colored images of the Ca2+ indicator after the addition of 1-MA. To compare the kinetics of Ca2+ rise, the moment of the first detectable Ca2+ signal was set to t = 0 in both cases. Ca2+ signals initiate at the vegetal hemisphere in control oocytes. In contrast, Ca2+ signals aberrantly arise at the animal pole near the nucleus (n) and from multiple spots (arrowheads) in the denuded oocytes. (C) Quantification of 1-MA-induced Ca2+ signals in the control (green) and denuded (brown) oocytes.

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