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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.05.549 Byline: Keiichiro Kyozuka (a), Jong T. Chun (b), Agostina Puppo (b), Gianni Gragnaniello (b), Ezio Garante (b), Luigia Santella (b) Keywords: Starfish; Oocyte; 1-methyladenine; Actin; Cytoskeleton; InsP.sub.3; Heparin; U73122; Cortical granule exocytosis; Meiosis Abstract: Before successful fertilization can occur, oocytes must undergo meiotic maturation. In starfish, this can be achieved in vitro by applying 1-methyladenine (1-MA). The immediate response to 1-MA is the fast Ca.sup.2+ release in the cell cortex. Here, we show that this Ca.sup.2+ wave always initiates in the vegetal hemisphere and propagates through the cortex, which is the space immediately under the plasma membrane. We have observed that alteration of the cortical actin cytoskeleton by latrunculin-A and jasplakinolide can potently affect the Ca.sup.2+ waves triggered by 1-MA. This indicates that the cortical actin cytoskeleton modulates Ca.sup.2+ release during meiotic maturation. The Ca.sup.2+ wave was inhibited by the classical antagonists of the InsP.sub.3-linked Ca.sup.2+ signaling pathway, U73122 and heparin. To our surprise, however, these two inhibitors induced remarkable actin hyper-polymerization in the cell cortex, suggesting that their inhibitory effect on Ca.sup.2+ release may be attributed to the perturbation of the cortical actin cytoskeleton. In post-meiotic eggs, U73122 and jasplakinolide blocked the elevation of the vitelline layer by uncaged InsP.sub.3, despite the massive release of Ca.sup.2+, implying that exocytosis of the cortical granules requires not only a Ca.sup.2+ rise, but also regulation of the cortical actin cytoskeleton. Our results suggest that the cortical actin cytoskeleton of starfish oocytes plays critical roles both in generating Ca.sup.2+ signals and in regulating cortical granule exocytosis. Author Affiliation: (a) Research Center for Marine Biology, Asamushi, Tohoku University, 039-3501 Japan (b) Laboratory of Cell Signaling, Stazione Zoologica Anton Dohrn, Villa Comunale, Napoli, I-80121, Italy Article History: Received 17 August 2007; Revised 23 May 2008; Accepted 27 May 2008

It has been known that the intracellular Ca 2+ level transiently rises at the specific stages of mitosis such as the moment of nuclear envelope breakdown and at the metaphase–anaphase transition. Comparable intracellular Ca 2+ increases may also take place during meiosis, as was intermittently reported in mouse, Xenopus , and starfish oocytes. In a majority of starfish species, the maturing oocytes display an intracellular Ca 2+ increase within few minutes after the addition of the maturation hormone, 1-methyladenine (1-MA). Although starfish oocytes at meiosis also manifest a Ca 2+ increase at the time of polar body extrusion, a similar Ca 2+ increase has never been observed during the envelope breakdown of the nucleus (germinal vesicle, GV). Here, we report, for the first time, the existence of an additional Ca 2+ response in the maturing oocytes of Asterina pectinifera at the time of GV breakdown. In contrast to the immediate early Ca 2+ response to 1-MA, which is independent of external Ca 2+ and takes a form of intracellular Ca 2+ wave traveling three times as fast as that in the fertilized eggs, this late stage Ca 2+ response comprised a train of numerous spikes representing Ca 2+ influx. These Ca 2+ spikes coinciding with GV breakdown were mostly eliminated when the GV was removed from the oocytes prior to the addition of 1-MA, suggesting that the Ca 2+ spikes are rather a consequence of the GV breakdown. In support of the idea that these Ca 2+ spikes play a physiological role, the oocytes matured in calcium-free seawater had a higher rate of cleavage failure 2 h after the fertilization in natural seawater. Specific inhibitors of L-type Ca 2+ channels, verapamil and diltiazem, severely suppressed the amplitude of the individual Ca 2+ spikes, but not their frequencies. On the other hand, latrunculin-A (LAT-A), which promotes net depolymerization of the actin cytoskeleton, had a dual effect on this late Ca 2+ response. When added immediately after the hormone-dependent period, LAT-A inhibited the occurrence (frequency) of the spikes in a dose-dependent manner, but the amplitude of the prevailing Ca 2+ spikes itself was rather significantly increased. These results suggest that the cortical actin cytoskeleton and some nuclear factors may play a role in regulating ion channel activities during this stage of meiotic progression. [ABSTRACT FROM AUTHOR]

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 Ca2+ 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 Ca2+ 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 (GTPγS) or inactive (GDPβS) forms of G-proteins, we have demonstrated that: i) GTPγS induces Ca2+ release that mimics the effect of 1-MA; ii) GDPβS completely blocks 1-MA-induced Ca2+; iii) GDPβS has little effect on the amplitude of the Ca2+ peak, but significantly expedites the initial Ca2+ waves induced by InsP3 photoactivation, iv) GDPβS induces unexpectedly striking modification of the cortical actin networks, suggesting a link between the cytoskeletal change and the modulation of the Ca2+ release kinetics; v) alteration of cortical actin networks with jasplakinolide, GDPβS, or actinase E, all led to significant changes of 1-MA-induced Ca2+ 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 Ca2+ release. [ABSTRACT FROM AUTHOR]

The resumption of the meiotic cycle (maturation) induced by 1-methyladenine in prophase-arrested starfish oocytes is indicated by the breakdown of the germinal vesicle and is characterized by the increased sensitivity of the Ca[sup 2+] stores to inositol 1,4,5-trisphosphate (InsP[sub 3]) to InsP[sub 3] starting at the animal hemisphere (where the germinal vesicle was originally located) and propagating along the animal/vegetal axis of the oocyte. This initiates Ca[sup 2+] signals around the germinal vesicle before nuclear envelope breakdown. Previous studies have suggested that the final activation of the maturation-promoting factor (MPF), a cyclin-dependent kinase, which is the major element controlling the entry of eukaryotic cells into the M phase, occurs in the nucleus. MPF is then exported to the cytoplasm where its activity is autocatalytically amplified following a similar animal/vegetal spatial pattern. We have investigated whether activated MPF was involved in the increased sensitivity of the Ca[sup 2+] response to InsP[sub 3]. We have found that the development of increased sensitivity of the Ca[sup 2+] stores to InsP[sub 3] receptors together with the Ca[sup 2+] signals in the perinuclear region was blocked in oocytes treated with the specific MPF inhibitor roscovitine. That the nuclear MPF activation is indeed required for changes of the InsP[sub 3] receptors sensitivity was shown by enucleating or by dissecting oocytes into vegetal and animal hemispheres prior to the addition of 1-MA. MPF activity 50 min after 1-methyladenine addition was much lower in the enucleated oocytes and in the vegetal hemisphere, which did not contain the germinal vesicle, as compared with the animal hemisphere, which did contain it. The Ca[sup 2+] increase induced by InsP[sub 3] under these experimental conditions correlated with the changes in actin cytoskeleton induced by MPF. [ABSTRACT FROM AUTHOR]

Abstract: Cofilin is a small protein that belongs to the family of actin-depolymerizing factors (ADF). The main cellular function of cofilin is to change cytoskeletal dynamics and thus to modulate cell motility and cytokinesis. We have recently demonstrated that the actin cytoskeleton is involved in the modulation of Ca2+ signalling in starfish oocytes. To extend these observations, we have explored whether cofilin influences Ca2+ signalling in the oocytes. Here we show that microinjection of the functionally active cofilin alters the Ca2+ signalling mediated by the three major second messengers, InsP3, NAADP, and cADPr. Cofilin intensifies the Ca2+ signals induced by InsP3 and NAADP, and delays those induced by cADPr. Furthermore, the injection of cofilin increases the Ca2+ signals during hormone-induced oocyte maturation and fertilization. The results suggest that the dynamic regulation of F-actin by its binding proteins may play an important role in the modulation of intracellular Ca2+ signalling. [Copyright &y& Elsevier]