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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.06.034 Byline: Noriyo Takeda (a)(b), Keiichiro Kyozuka (b), Ryusaku Deguchi (a) Keywords: Jellyfish; Cyclic AMP; Meiosis; GVBD; cAMP-dependent protein kinase; Umbrella-free medusa; Spawning; 8-Br-cAMP; Caged cAMP; H-89 Abstract: In medusae of the hydrozoan Cytaeis uchidae, oocyte meiotic maturation and spawning occur as a consequence of dark-light transition. In this study, we investigated the mechanism underlying the initiation of meiotic maturation using in vitro (isolated oocytes from ovaries) and in vivo (ovarian oocytes in medusae) systems. Injection of cAMP derivatives into isolated oocytes induced meiotic maturation in a dose-dependent manner. Meiotic maturation was also achieved in isolated oocytes preloaded with caged cAMP and exposed to UV irradiation. The caged cAMP/UV irradiation-induced meiotic maturation was completely inhibited by blockers of protein kinase A (PKA), H-89, KT5720, and Rp-cAMPS. The medusae from which most parts of the umbrella were removed (umbrella-free medusae) survived for at least 2 weeks, during which time oocyte meiotic maturation and spawning occurred. When H-89 and Rp-cAMPS were injected into ovarian oocytes of umbrella-free medusae within 3 min of dark-light stimulation, meiotic maturation was inhibited or delayed. An increase in intracellular cAMP was confirmed by FlCRhR, a fluorescent cAMP indicator, in ovarian oocytes exposed to dark-light transition as well as in isolated oocytes stimulated by caged cAMP/UV irradiation. These results indicate that the cAMP/PKA signaling pathway positively contributes to light-triggered physiological oocyte meiotic maturation in Cytaeis uchidae. Author Affiliation: (a) Department of Biology, Miyagi University of Education, Aoba-ku, Sendai, Miyagi 980-0845, Japan (b) Research Center for Marine Biology, Asamushi, Graduate School of Life Science, Tohoku University, Asamushi, Aomori 039-3501, Japan Article History: Received 15 April 2006; Revised 16 June 2006; Accepted 21 June 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.02.011 Byline: Francesco Moccia (a), Gilda A. Nusco (a), Dmitry Lim (a), Keiichiro Kyozuka (b), Luigia Santella (a) Keywords: NAADP; InsP.sub.3; Ca.sup.2+ wave; Fertilization; Oocytes; Sperm; Starfish Abstract: NAADP participates in the response of starfish oocytes to sperm by triggering the fertilization potential (FP) through the activation of a Ca.sup.2+ current which depolarizes the membrane to the threshold of activation of the voltage-gated Ca.sup.2+ channels. The aim of this study was to investigate whether this Ca.sup.2+ influx is linked to the onset of the concomitant InsP.sub.3-mediated Ca.sup.2+ wave by simultaneously employing Ca.sup.2+ imaging and single-electrode intracellular recording techniques. In control oocytes, the sperm-induced membrane depolarization always preceded by a few seconds the onset of the Ca.sup.2+ wave. Strikingly, the self-desensitization of NAADP receptors either abolished the Ca.sup.2+ response or resulted in abnormal oocyte activation, i.e., the membrane depolarization followed the Ca.sup.2+ wave and the oocyte was polyspermic. The inhibition of InsP.sub.3 signaling only impaired the propagation of the Ca.sup.2+ wave and shortened the FP. The duration of FP was also reduced in low-Na.sup.+ sea water. Finally, uncaged InsP.sub.3 produced a Ca.sup.2+ increase, which depolarized the membrane upon the activation of a Ca.sup.2+-sensitive cation current. These results support the hypothesis that Ca.sup.2+ entry during the NAADP-triggered FP is required for the onset of the Ca.sup.2+ wave at fertilization. The InsP.sub.3-mediated Ca.sup.2+ wave, in turn, may interact with the NAADP-evoked depolarization by activating a Ca.sup.2+-dependent Na.sup.+ entry. Author Affiliation: (a) Laboratory of Cell Signaling, Stazione Zoologica 'Anton Dohrn', Villa Comunale, 80121 Naples, Italy (b) Asamushi Marine Biological Station, Aomori 039-3501, Japan Article History: Received 16 September 2005; Revised 6 February 2006; Accepted 14 February 2006

An important step for successful fertilization and further development is the increase in intracellular Ca2+ in the activated oocyte. It has been known that starfish oocytes become increasingly sensitive to inositol 1,4,5-trisphosphate (IP3) during meiotic maturation to exhibit highly efficient IP3-induced Ca2+ release (IICR) by the time of germinal vesicle breakdown (GVBD). However, we noted that the peak level of intracellular Ca2+ increase after insemination is already high in the maturing oocytes before GVBD. Using maturing oocytes before GVBD, we investigated Ca2+ release mechanisms other than IICR. We report here that Ca2+-release mechanisms dependent on nicotinic acid adenine dinucleotide phosphate (NAADP) and nicotinamide adenine dinucleotide (NADP), the precursor of NAADP, became functional prior to the development of IICR mechanisms. As with IP3, but unlike NAADP, the Ca2+ stores responsive to NADP are sensitized during the meiotic maturation induced by 1-methyladenine (1-MA). This suggests that the process may represent a physiological response to the maturation hormone. NADP-dependent Ca2+ release in immature oocytes, however, did not induce oocyte maturation by itself, but was enhanced by the conditions mimicking the increases of intracellular Ca2+ and pH that take place in the maturing oocytes of starfish. [ABSTRACT FROM PUBLISHER]

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]