Starfish oocytes of A. pectinifera reveal marked differences in sperm-induced electrical and intracellular calcium changes during oocyte maturation and at fertilization.

Although changes in membrane potential and intracellular Ca (Ca ) during fertilization in starfish oocytes have been known for long time, little is known precisely about how and what kind of channels are involved during oocyte maturation and in fertilization, and how the mechanisms of changes in Ca in oocytes develop during oocyte maturation. Since in starfish, oocyte maturation-inducing hormone, 1-methyladenine (1MA) is well known, we took advantage of it to investigate the developmental process of channel-function and changes in Ca in three different developmental stages using 1MA. Sperm-induced membrane current at voltage clamp and changes in Ca in starfish oocytes, Asterina pectinifera, were examined in stages of immature, partly mature (a state in 15-20 min after sufficient concentration, 1 µM of 1MA addition, or 30-40 min exposure to subthreshold concentration of 1MA), and mature oocytes (MO).

Development of Ca2+-release mechanisms during oocyte maturation of the starfish Asterina pectinifera.

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.

Novel Ca2+ increases in the maturing oocytes of starfish during the germinal vesicle breakdown.

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).

Soluble sperm extract specifically recapitulates the initial phase of the Ca2+ response in the fertilized oocyte of P. occelata following a G-protein/ PLCβ signaling pathway.

Matured oocytes of the annelidan worm Pseudopotamilla occelata are fertilized at the first metaphase of the meiotic division. During the activation by fertilizing spermatozoa, the mature oocyte shows a two-step intracellular Ca2+ increase. Whereas the first Ca2+ increase is localized and appears to utilize the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores, the second Ca2+ increase is global and involves Ca2+ influx via voltage-gated Ca2+ channels on the entire surface of the oocyte.

Intracellular calcium signaling in the fertilized eggs of Annelida.

Fertilization is such a universal and indispensable step in sexual reproduction, but a high degree of variability exists in the way it takes place in the animal kingdom. As discussed in other reviews in this issue, recent works on this subject clarified many points. However, important results on the mechanisms of fertilization are obtained mainly from a few restricted model organisms.

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

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.

Nitric oxide (NO) increase at fertilization in sea urchin eggs upregulates fertilization envelope hardening.

Previous studies indicate that the nitric oxide (NO) increase at fertilization in sea urchin eggs is Ca(2+)-dependent and attributed to the late Ca(2+) rise. However, its role in fertilization still remains unclear. Simultaneous measurements of the activation current, by a single electrode voltage clamp, and NO, using the NO indicator DAF-FM, showed that the NO increase occurred at the time of peak current (t(p)) which corresponds to peak [Ca(2+)](i), suggesting that NO is not related to any other ionic changes besides [Ca(2+)](i).

Actin cytoskeleton modulates calcium signaling during maturation of starfish oocytes.

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 Ca2+ release in the cell cortex.

Novel two-step Ca2+ increase and its mechanisms and functions at fertilization in oocytes of the annelidan worm Pseudopotamilla occelata.

Mature oocytes of the annelidan worm Pseudopotamilla occelata have a wide perivitelline space between the oocyte surface and the vitelline envelope and are arrested at the first metaphase (MI). We found a novel two-step Ca2+ increase in normally fertilized oocytes. The first Ca2+ increase originated at a cortex situated underneath a fertilizing sperm on the vitelline envelope, but failed to propagate beyond the center of the oocyte.

Two phases of calcium requirement during starfish meiotic maturation.

During meiosis in oocytes of the starfish, Asterina pectinifera, a Ca(2+) transient has been observed. To clarify the role of Ca(2+) during oocyte maturation in starfish, an intracellular Ca(2+) blocker, TMB-8, was applied. The oocyte maturation induced by 1-methyladenine (1-MA) was blocked by 100 microM TMB-8.

Serotonin stimulates [Ca2+]i elevation in ciliary ectodermal cells of echinoplutei through a serotonin receptor cell network in the blastocoel.

A full-length serotonin receptor mRNA from the 5Hthpr gene was sequenced from larvae of the sea urchin, Hemicentrotus pulcherrimus. The DNA sequence was most similar to 5HT-1A of the sea urchin Strongylocentrotus purpuratus found by The Sea Urchin Genome Project, and the protein sequence predicted the presence of seven transmembrane domains. Immunohistochemistry with anti-5HThpr antibodies indicated that the protein was expressed on blastocoelar cells that comprised the major blastocoelar network (serotonin receptor cell network).

Increase in intracellular cAMP is a prerequisite signal for initiation of physiological oocyte meiotic maturation in the hydrozoan Cytaeis uchidae.

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.

Modulation of calcium signalling by the actin-binding protein cofilin.

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 Ca(2+) signalling in starfish oocytes.

A novel oocyte maturation arresting factor in the central nervous system of scallops inhibits serotonin-induced oocyte maturation and spawning of bivalve mollusks.

Serotonin (5-hydroxytriptamine; 5-HT) is a major neurotransmitter that triggers oocyte maturation and sequential spawning in bivalve mollusks. A proteinous and heat-labile substance that proved to be a novel inhibitor of 5-HT-induced egg release from ovarian tissue was found in the cerebral and pedal ganglia (CPG) of the scallop Patinopecten yessoensis. The same inhibitory activity was also observed in the proteinous fraction from the supernatant of hemolymph.

NAADP and InsP3 play distinct roles at fertilization in starfish oocytes.

NAADP participates in the response of starfish oocytes to sperm by triggering the fertilization potential (FP) through the activation of a Ca2+ current which depolarizes the membrane to the threshold of activation of the voltage-gated Ca2+ channels. The aim of this study was to investigate whether this Ca2+ influx is linked to the onset of the concomitant InsP3-mediated Ca2+ wave by simultaneously employing Ca2+ 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 Ca2+ wave.

NAADP triggers the fertilization potential in starfish oocytes.

In invertebrates oocytes or eggs, the fertilization or activation potential establishes the fast electrical block to polyspermy and, in some species, provides the Ca2+ influx which contributes to the following intracellular Ca2+ wave. In echinoderms, the molecule triggering the activation potential is still unknown. The aim of this study was to assess whether nicotinic acid-adenine dinucleotide phosphate (NAADP) elicited the fertilization potential in starfish oocytes.

The M-phase-promoting factor modulates the sensitivity of the Ca2+ stores to inositol 1,4,5-trisphosphate via the actin cytoskeleton.

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 Ca2+ stores to inositol 1,4,5-trisphosphate (InsP3) to InsP3 starting at the animal hemisphere (where the germinal vesicle was originally located) and propagating along the animal/vegetal axis of the oocyte. This initiates Ca2+ 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.

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.

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.

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.

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.

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.

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.

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.

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.