NAADP+ initiates the Ca2+ response during fertilization of starfish oocytes.

We have explored the role of the recently discovered second messenger nicotinic acid adenine nucleotide phosphate (NAADP+) in Ca2+ swings that accompany the fertilization process in starfish oocytes. The injection of NAADP+ deep into the cytoplasm of oocytes matured by the hormone 1-methyladenine (1-MA), mobilized Ca2+ exclusively in the cortical layer, showing that the NAADP+-sensitive Ca2+ pool is restricted to the subplasma membrane region of the cell. At variance with this, InsP3 initiated the liberation of Ca2+ next to the point of injection in the center of the cell. The initial cortical Ca2+ liberation induced by NAADP+ was followed by a spreading of the Ca2+ wave to the remainder of the cell and by a massive cortical granule exocytosis similar to that routinely observed on injection of InsP3. A striking difference in the responses to NAADP+ and InsP3 was revealed by the removal of the nucleus from immature oocytes, i.e., from oocytes not treated with 1-MA. Whereas the Ca2+ response and the cortical granule exocytosis induced by NAADP+ were unaffected by the removal of the nucleus, the Ca2+ response promoted by InsP3 was significantly slowed. In addition, the cortical granule exocytosis was completely abolished. When enucleated oocytes were fertilized, the spermatozoon still promoted the Ca2+ wave and normal cortical exocytosis, strongly suggesting that the Ca2+ response was mediated by NAADP+ and not by InsP3. InsP3-sensitive Ca2+ stores may mediate the propagation of the wave initiated by NAADP+ since its spreading was strongly affected by removal of the nucleus.

Breakdown of cytoskeletal proteins during meiosis of starfish oocytes and proteolysis induced by calpain.

Meiosis reinitiation in starfish oocytes is characterized by Ca(2+) transients in the cytosol and in the nucleus and is accompanied by the disassembly of the nuclear envelope, a process which is likely to be mediated by the cleavage of selected proteins. We have used mass spectrometry analysis (mass profile fingerprinting) on 2D polyacrylamide gels of extracts of oocytes in which meiosis resumption was induced by 1-methyladenine and have identified five proteins that were specifically degraded: alpha-tubulin, lamin B, dynamin, and two kinds of actin. They are all components of the cytoskeleton or associated with it. We then investigated whether calpain, which is activated by the increase in cell Ca(2+), could cleave the same proteins that became degraded under the influence of 1-methyladenine and thus be involved in nuclear membrane breakdown. The investigation was prompted by the finding that microinjection of calpain into the nuclei of prophase arrested oocytes induced meiosis in the absence of 1-methyladenine. Incubation of prophase arrested (disrupted) oocytes with calpain produced a 2D gel protein pattern in which some of the degradation products coincided with those seen in oocytes challenged with 1-methyladenine.

Nicotinic acid adenine dinucleotide phosphate-induced Ca(2+) release. Interactions among distinct Ca(2+) mobilizing mechanisms in starfish oocytes.

An intracellular mechanism activated by nicotinic acid adenine dinucleotide phosphate (NAADP(+)) contributes to intracellular Ca(2+) release alongside inositol 1,4,5-trisphosphate (Ins-P(3)) and ryanodine receptors. The NAADP(+)-sensitive mechanism has been shown to be operative in sea urchin eggs, ascidian eggs, and pancreatic acinar cells. Furthermore, most mammalian cell types can synthesize NAADP(+), with nicotinic acid and NADP(+) as precursors. In this contribution, NAADP(+)-induced Ca(2+) release has been investigated in starfish oocytes. Uncaging of injected NAADP(+) induced Ca(2+) mobilization in both immature oocytes and in oocytes matured by the hormone 1-methyladenine (1-MA). The role of extracellular Ca(2+) in NAADP(+)-induced Ca(2+) mobilization, which was minor in immature oocytes, was instead essential in mature oocytes. Thus, the NAADP(+)-sensitive Ca(2+) pool, which is known to be distinct from those sensitive to inositol 1,4,5-trisphosphate or cyclic ADPribose, apparently migrated closer to (or became part of) the plasma membrane during the maturation process. Inhibition of both Ins-P(3) and ryanodine receptors, but not of either alone, substantially inhibited NAADP(+)-induced Ca(2+) mobilization in both immature and mature oocytes. The data also suggest that NAADP(+)-induced Ca(2+) mobilization acted as a trigger for Ca(2+) release via Ins-P(3) and ryanodine receptors.

Cortical granule translocation during maturation of starfish oocytes requires cytoskeletal rearrangement triggered by InsP3-mediated Ca2+ release.

Cortical granules (secretory vesicles located under the cortex of mature oocytes) release their contents to the medium at fertilization. Their exocytosis modifies the extracellular environment, blocking the penetration of additional sperm. The granules translocate to the surface during the maturation process, and it has been suggested that they move to the cortex via cytoskeletal elements. In this paper we show that the increase in intracellular Ca2+, which the maturing hormone 1-methyladenine (1-MA) induces in starfish through the activation of inositol 1,4, 5-trisphosphate (InsP3) receptors, triggers changes in filamentous actin, which then direct the correct movement and reorientation of the cortical granules and the elevation of the fertilization envelope.

Cytoplasmic Ca2+ oscillation coordinates the formation of actin filaments in the sea urchin eggs activated with phorbol ester.

Changes in the intracellular Ca2+ concentration ([Ca2+]i) and the formation of actin filaments were investigated in unfertilized eggs of the sea urchin Hemicentrotus pulcherrimus after activation with a phorbol ester, 12-O-tetradecanoyl phorbol13-acetate (TPA). Intracellular Ca2+ oscillation was observed using a fluorescent Ca2+ indicator dye, calcium green dextran. From about 20 to 80 min after the addition of TPA to 100 microM, there was a rise in [Ca2+]i, which was followed by Ca2+ oscillation. A change in [Ca2+]i in response to TPA was not observed in eggs that had been injected with heparin, an inositol 1,4,5-triphosphate (IP3) receptor antagonist. Therefore, long-term exposure to a high concentration of TPA seems to induce Ca2+ release via the IP3 pathway, as well as causing the release of diacylglycerol from membrane lipids. Moreover, the elongation of actin filaments occurred in the cytoplasm during the rise in [Ca2+]i. Actin filaments also formed when TPA-induced cytoplasmic alkalization was inhibited by exposure to Na(+)-free sea water. These results suggest that the observed cytoplasmic formation of actin filaments may be related to change in the cytoplasmic [Ca2(+)]i, and not intracellular pH, induced by TPA. These phenomena may be similar to the changes in actin construction that occur during cell cycle events.

Separate activation of the cytoplasmic and nuclear calcium pools in maturing starfish oocytes.

The dynamics of the cytoplasmic and nuclear Ca2+ pools in starfish oocytes arrested at the prophase of the first meiotic division or after induction of meiosis by 1-methyladenine (1-MA) have been studied by confocal microscopy. A 70 kDa fluorescent Ca2+ indicator has been injected in either the cytoplasm or the nucleus, and shown to remain restricted to the compartment of injection. 1-MA induced a first Ca2+ transient in the cytosol, followed by a nuclear transient, and eventually by a second cytosolic transient. The latter failed to occur if the nuclear peak was suppressed. This required the nuclear injection of antagonists of the inositol 1,4,5-trisphosphate (InsP3) and cyclic-ADPribose (cADPr) Ca2+ channels, showing that both channel types were active in the inner envelope membrane. The nuclear injection of the Ca2+ channel antagonists affected the process of meiosis reinitiation: in about one third of the injected oocytes no breakdown of the nuclear envelope (GVBD) was observed. In the others, even if GVBD eventually occurred, the intermixing of the nucleoplasm and cytoplasm was inhibited.

Injection of sperm extract mimics spatiotemporal dynamics of Ca2+ responses and progression of meiosis at fertilization of ascidian oocytes.

Sperm extract (SE) of the ascidian, Ciona savignyi, injected into oocytes induced repetitive intracellular Ca2+ increases with kinetics consistent with those at fertilization and caused reinitiation and progression of meiosis as in fertilized oocytes with the formation of polar bodies. The Ca2+ response comprised two sets of Ca2+ oscillations separated by 5 minutes and correlated with the first and second meiotic metaphase. The effects of SE were dose dependent and the critical dose corresponded roughly to a single spermatozoon. In the first Ca2+ transient observed by confocal microscopy, a Ca2+ wave started from the SE injection site at the peripheral region of the oocyte and propagated across the ooplasm. The similar wave was produced by injection at the central region, starting from an arbitrary cortical area after 30 seconds, probably after SE had diffused to the cortex. The sensitivity to SE is thought to be preferentially higher in the cortex. The effective component of SE was heat-unstable, and its molecular weight was estimated as in the range between 10x10(4 )and 3x10(4) using membrane filters. These results suggest that, in ascidian fertilization, a cytosolic sperm protein factor is introduced to the oocyte cortex and induces Ca2+ waves and thereby meiotic resumption, leading to cell-cycle-correlated Ca2+ oscillations.

Calcium, protease action, and the regulation of the cell cycle.

Proteolysis is a key event in the control of the cell cycle. Most of the proteins which are degraded at specific cycle points, e.g. cyclins A, B, and E, are substrates of the ubiquitin/proteasome pathway. The Ca2+ dependent neutral protease calpain also cleaves cell cycle proteins, among them cyclin D1 and the c-mos proto-oncogene product which is a component of the CSF. The proteasome itself, however, may be under Ca2+ control through the binding of Ca2+ to its 29 kDa regulatory subunit. Calpain undergoes relocation among cell compartments during the various steps of the mitotic and meitotic cycles. It promotes the initiation and the progression of mitosis when injected into the perinuclear space of synchronized PtK1 cells, and the resumption of meiosis when directly injected into the nuclei of prophase-arrested starfish oocytes. Apart from the proteins mentioned above, most of the substrates of calpain which become cleaved during mitosis and meiosis are still unknown. Microtubule-associated proteins are likely candidates.

Effects of 1-methyladenine on nuclear Ca2+ transients and meiosis resumption in starfish oocytes are mimicked by the nuclear injection of inositol 1,4,5-trisphosphate and cADP-ribose.

The treatment of prophase-arrested starfish oocytes with the hormone 1-methyladenine (1-MA) induces the elevation of Ca2+ in both the cytoplasm and the germinal vesicle (nucleus), and is followed by the resumption of meiosis. The injection of the modulators of the intracellular Ca2+ channels inositol 1,4,5-trisphosphate (InsP3) or cyclic adenosine diphosphate ribose (cADPr) into the germinal vesicle promoted meiosis resumption in the absence of 1-MA in about 50% of the oocytes. Caged InsP3 or caged cADPr were injected into the nuclei of oocytes together with the Ca2+ indicator calcium green dextran; their photoreleasing elicited nuclear calcium spikes which, in the case of cADPr, had repetitive behaviour. The spikes were abolished by the nuclear injection of antagonists or antibodies to the InsP3 or cADPr-sensitive Ca2+ channels. cADPr-modulated channels were localized on the membranes of the nuclear envelope using specific antibodies conjugated with IgG-gold complexes.

The proteasome is an essential mediator of the activation of pre-MPF during starfish oocyte maturation.

Starfish oocyte maturation was blocked by the addition of 100 microM MG115, a potent proteasome inhibitor, whereas no inhibition was observed by membrane permeable cysteine protease inhibitor, E-64-d. The inhibition by MG115 was diminished by adding at a time corresponding to the half time required for germinal vesicle breakdown. Potent inhibition of germinal vesicle breakdown was also observed by microinjection of anti-proteasome-a-subunit antibodies. The antibody-injected oocytes failed to activate pre-maturation promoting factor (pre-MPF), since the dephosphorylation of phospho-Tyr15 in cdc2 kinase was not observed even in the presence of 1-methyadenine, a maturation-inducing hormone. These results indicate that the proteasome triggers the activation of pre-MPF via the dephosphorylation of cdc2 kinase in the signal transduction pathway in response to the hormonal stimulus during starfish oocyte maturation.

A soluble extract from human spermatozoa activates ascidian oocytes.

A soluble extract from human spermatozoa induced calcium oscillations and extrusion of the first polar body when injected into oocytes of the ascidian Ciona intestinalis. The properties of calcium oscillations and time of polar body extrusion precisely mimic oocyte activation induced by C. intestinalis sperm or sperm extracts. The data suggest that human sperm extracts can activate oocytes of different phyla by the same mechanism as homologous spermatozoa. Injection of inositol 1,4,5-trisphosphate (IP3) into C. intestinalis oocytes mimicked to some extent the initial stages of oocyte activation, but the results demonstrate that ascidian oocyte activation by human sperm extract cannot be explained solely in terms of IP3-induced calcium release. Injection of other calcium releasing second messengers, cyclic adenosine diphosphate ribose, or calcium ions, does not lead to oocyte activation or release intracellular calcium in ascidian oocyte. It was concluded that human spermatozoa contain one or more molecules than can trigger intracellular calcium release in oocytes from different phyla.

Association of calmodulin with nuclear structures in starfish oocytes and its role in the resumption of meiosis.

The resumption of meiosis in prophase-arrested starfish oocytes is induced by the hormone 1-methyladenine, which has been shown previously to induce a calcium transient in the nucleus which at this stage is called the germinal vesicle. This transient precedes the breakdown of the germinal vesicle (GVBD). Experiments were performed to establish whether nuclear calmodulin (CaM) was involved in the progression of the meiotic cycle. CaM antagonists, antibodies, and an inhibitory peptide corresponding to the CaM-binding domain of myosin-light-chain kinase have been injected into the nucleus of prophase-arrested starfish oocytes. The antagonists failed to affect the final response to 1-methyladenine, i.e. GVBD, although two antagonists delayed it, whereas the peptide inhibitor and the antibodies completely inhibited it. The antibodies suppressed the nuclear Ca2+ spikes that were shown by previous work to be induced by the photoreleasing of caged adenosine 3',5'-(cyclic)diphosphate ribose in the germinal vesicle. Immunofluorescence staining of isolated starfish oocyte nuclei with CaM antibodies showed CaM in the envelope and in the nucleolus. Immunogold labelling of oocytes revealed aggregates of CaM and of a 36-kDa protein, of the heterogeneous ribonucleoprotein particles (hnRNP), in electron-dense hnRNP in the nuclear matrix. 1-Methyladenine induced the disappearance of these hnRNP from the nucleoplasm and the translocation of CaM and the 36-kDa protein previously associated with them to the cytoplasm, prior to the breakdown of the nuclear envelope.

Change in intracellular Ca2+ is not involved in serotonin-induced meiosis reinitiation from the first prophase in oocytes of the marine bivalve Crassostrea gigas.

In response to the neurohormone serotonin (5-hydroxytryptamine, 5-HT), prophase-arrested oocytes of the marine bivalve Crassostrea gigas (oyster) reinitiate meiosis, undergo germinal vesicle breakdown (GVBD), and are arrested again at metaphase I. We examined the pharmacological characteristics of 5-HT receptors and the signal transduction pathway following 5-HT stimulation in oyster oocytes. Among 5-HT agonists tested, only alpha-methyl 5-HT, a 5-HT2 agonist, induced GVBD although it was 1000 times less sensitive than 5-HT. The rank order of the potency of 5-HT antagonists to inhibit GVBD was propranolol, cyproheptadine > metoclopramide > mianserin. These results are quite different from those reported for other mollusks, suggesting the presence of unique 5-HT receptors on oyster oocytes. Using the fluorescent Ca2+ dyes fura 2 and calcium green and the pH indicator 1-hydroxypyrene-3,6,8-trisulfonic acid, we examined changes in intracellular Ca2+ ([Ca2+]i) and intracellular pH (pHi) during 5-HT-induced meiosis reinitiation. 5-HT did not trigger any changes in [Ca2+]i. However, an increase in pHi was observed during the 5-HT-induced meiosis reinitiation. The increased pHi level was rather small before GVBD and not necessary for GVBD, because lowering pHi by sodium acetate seawater (pH 7.0) did not prevent 5-HT-induced GVBD. Measurement of the kinase activity toward a peptide substrate specific to cdc2 demonstrated that maturation-promoting factor (MPF) was activated in accordance with the occurrence of GVBD in response to 5-HT. Therefore, it is likely that in oyster oocytes the signal transduction pathways and intracellular effectors participating in 5-HT-induced meiosis reinitiation via the activation of MPF are insensitive to [Ca2+]i and pHi.

Maturation promoting factor in ascidian oocytes is regulated by different intracellular signals at meiosis I and II.

Using the fluorescent dye Calcium Green-dextran, we measured intracellular Ca2+ in oocytes of the ascidian Ciona intestinalis at fertilization and during progression through meiosis. The relative fluorescence intensity increased shortly after insemination in a single transient, the activation peak, and this was followed by several smaller oscillations that lasted for approximately 5 minutes (phase 1). The first polar body was extruded after the completion of the phase 1 transients, about 9 minutes after insemination, and then the intracellular calcium level remained at baseline for a period of 5 minutes (phase 2). At 14 minutes postinsemination a second series of oscillations was initiated that lasted 11 minutes (phase 3) and terminated at the time of second polar body extrusion. Phases 1 and 3 were inhibited by preloading oocytes with 5 mM heparin. Simultaneous measurements of membrane currents, in the whole-cell clamp configuration, showed that the 1-2 nA inward fertilization current correlated temporally with the activation peak, while a series of smaller oscillations of 0.1-0.3 nA amplitude were generated at the time of the phase 3 oscillations. Biochemical characterization of Maturation Promoting Factor (MPF) in ascidian oocytes led to the identification of a Cdc2-like kinase activity. Using p13suc1-sepharose as a reagent to precipitate the MPF complex, a 67 kDa (67 x 10(3) Mr) protein was identified as cyclin B. Histone H1 kinase activity was high at metaphase I and decreased within 5 minutes of insemination reaching a minimum level during phase 2, corresponding to telophase I. During phase 3, H1 kinase activity increased and then decayed again during telophase II. Oocytes preloaded with BAPTA and subsequently inseminated did not generate any calcium transients, nonetheless H1 kinase activity decreased 5 minutes after insemination, as in the controls, and remained low for at least 30 minutes. Injection of BAPTA during phase 2 suppressed the phase 3 calcium transients, and inhibited both the increase in H1 kinase activity normally encountered at metaphase II and second polar body extrusion.

Serotonergic Ligands Induce Spawning but not Oocyte Maturation in the Bivalve Mactra chinensis from Central Japan.

We examined the spawning sensitivity to serotonin and serotonergic ligands in the Japanese bivalve Mactra chinensis. Spawning was induced by both injected and externally applied scrotonin (5-hydroxytryptamine. 5-HT). The vertebrate 5-HT2 receptor agonist alpha-methyl 5-HT and the selective 5HT1A agonist 8-OH-DPAT were also effective at inducing spawning. However TFMPP (m-trifluoromethylphenylpiperazine, a verterbrate 5-HT1 receptor agonist) and 1-methyl-chlorophenyl biguanide (a vertebrate 5-HT3 agonist) were not effective spawning inducers. The 5-HT-induced spawning was blocked by mianserin (a vertebrate 5-HT2 antagonist). The rank order of potency of the agonists was: 5-HT > alpha-methyl 5-HT > 8-OH-DPAT > TFMPP > 1-methyl-chlorophenyl biguanide; these data support a growing body of literature invoking a mixed 5-HT1/5-HT2 pharmacological profile for serotonin receptors mediating reproductive processes in bivalves. However, neither 5-HT nor 8-OH-DPAT induced germinal vesicle breakdown (GVBD) in Mactra oocytes. Sperm induced GVBD in a high percentage of oocytes. This is the first report of a bivalve in which spawning, but not GVBD, can be induced by 5-HT. This result might be expected because Mactra spawns germinal vesicle oocytes that normally undergo GVBD upon fertilization, but is in contrast to the case of the closely related Spisula spp. in which serotonin induces both processes. The ability of 5-HT to induce spawning but not GVBD makes Mactra chinensis a model organism for studying spawning and meiotic mechanisms in bivalves.

Characterization of zebra mussel (Dreissena polymorpha) sperm morphology and their motility prior to and after spawning.

In order to understand more about the reproductive physiology of the zebra mussel (Dreissena polymorpha), we have examined the effect of spawning on the kinematics and fertilization ability of their spermatozoa. Sperm morphology was characterized using scanning electron microscopy, differences in sperm kinematics were examined using CASA following manual collection of sperm from the gonad or spawning induced by 5-hydroxytryptamine (5-HT), and the fertilization ability of spawned and manually removed spermatozoa were compared. D. polymorpha sperm have a tail 50 microns long, a midpiece consisting of four spherical mitochondria-rich regions approximately 0.6 micron in diameter, a conical nucleus 2.52 microns in length, and a cylindrical acrosome 0.72 micron in height and width. Kinematic analysis revealed that manually removed sperm exhibit significantly lower motion characteristics as compared to spawned sperm. Treatment with 5-HT had no significant effect on the sperm kinematics of either manually removed or spawned spermatozoa. These data suggest that D. polymorpha spermatozoa have a similar morphology to other bivalve sperm, spawning results in a significant enhancement in sperm kinematics, and 5-HT does not directly alter sperm motion.

The Effect of Salinity and Temperature on Spawning and Fertilization in the Zebra Mussel Dreissena polymorpha (Pallas) from North America.

Zebra mussels have dispersed from their original site of introduction in the Great Lakes into the Mississippi River, Hudson River, and other watersheds in which they will encroach upon brackish water estuaries. To investigate their likelihood of reproductive success in such estuaries, we investigated the conditions of temperature, salinity, and acclimation under which spawning and fertilization could occur. Reproductive function of mussels that were acclimated to salinities up to 7.0 parts per thousand (ppt) at 12°, 20°, and 27°C for 1 to 21 days was tested. Reproductive function of non-acclimated mussels that had been maintained in fresh-water aquaria was also tested in various salinities. Spawning was induced by serotonin, previously demonstrated to elicit spawning of fertile gametes in fresh water. Successful fertilization was indicated by oocyte cleavage after adding sperm. Nonacclimated mussels spawned in salinities of 1.75 and 3.5 ppt at 12°, 20°, and 27°C, but not at 7.0 ppt. Fertilization using gametes from non-acclimated mussels occurred only in fresh water and at 1.75 ppt. Acclimation for as little as 2 days enhanced spawning. Fertilization rate in a salinity of 3.5 ppt improved within 4 days of acclimation and continued at a high level for as long as 21 days of acclimation. Although animals acclimated for 4 days in 3.5 ppt spawned readily when tested in salinities as high as 7.0 ppt, almost no fertilization occurred in 7.0 ppt. The reduction in fertilization at increasing salinities may be due in part to reduced sperm motility. Unfertilized oocytes remain intact for hours in fresh water; however, in salinities as low as 0.7 ppt, unfertilized oocytes tended to rupture within 2 hours. These data show that although sudden increases in salinity produce an immediate decrease in the reproductive capacity of zebra mussels, acclimation to brackish water can occur, and zebra mussels may be able to reproduce in brackish water below 7.0 ppt.

Isolation, characterization, and primary structure of three major proteins obtained from Mytilus edulis sperm.

Acrosomal proteins from Mytilus edulis sperms were separated into 11 fractions by reverse phase HPLC. The three major proteins, named M3, M6, and M7, showed strong egg vitelline coat lysin and first polar body releasing activities. The amino acid sequences of these proteins were determined. M6 and M7 were composed of 180 amino acid residues and showed high sequence homology (76%), while M3 was composed of 149 residues and showed 26% homology with M6 and M7. The disulfide linkage motif of the three proteins was similar and resembled the carbohydrate recognition domain (CRD) of C-type lectin. The C-terminal half of these proteins showed sequence homology with CRD of C-type lectins, but no homology with vitelline coat lysins of other mollusks. The proteins bound to asialofetuin-Sepharose in the presence of Ca2+ and were eluted with EDTA, indicating that they are Ca(2+)-dependent carbohydrate-binding proteins.

In vivo and in vitro induction of germinal vesicle breakdown in a freshwater bivalve, the zebra mussel Dreissena polymorpha (Pallas).

Oocyte maturation and germinal vesicle breakdown (GVBD) was induced in zebra mussel (Dreissena polymorpha) oocytes by in vivo and in vitro application of serotonin (5-hydroxytryptamine, 5-HT), and in vitro application of 8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT, a 5-HT1A receptor agonist). Oocytes initiated GVBD approximately 30 minutes after exposure to 5-HT (10(-3) M) at 23 degrees C, and by 40-50 minutes after exposure, most oocytes lacked a germinal vesicle. An exposure time to 5-HT as brief as five to ten minutes was required to trigger the maturation process, which terminates in spawning of fertilizable oocytes in nearly all mussels. But, with an exposure time of less than five minutes, spawning was reduced by application of 10(-4) M methiothepin (a potent blocker of 5-HT-induced spawning in zebra mussels). Thus, the sequence of oocyte maturation events in zebra mussels was determined. Oocytes are arrested at the germinal vesicle stage (prophase I) within the ovary. 5-HT reinitiates the maturation process, including GVBD and spawning of metaphase I oocytes, which are further arrested until fertilization. To our knowledge this is the first demonstration of oocyte maturation induction by serotonergic ligands in a freshwater bivalve.

Reinitiation of meiosis in starfish oocytes requires an increase in nuclear Ca2+.

Here we report that an increase in nuclear Ca2+ is hormonally regulated and is required for reinitiation of meiosis in starfish oocytes. We have shown in Asterina pectinifera that a Ca2+ transient and a baseline increase of Ca2+ occur both in the cytoplasm and in the nucleus. These increases are in response to 1-methyladenine (1-MA), the hormone responsible for the induction of meiotic maturation. The Ca2+ transients are not necessary for the reinitiation of meiosis. However, blocking the baseline increase in nuclear Ca2+ blocks both the nuclear envelope breakdown (NEBD) and the continuation of meiosis.