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. To extend these observations, we have explored whether cofilin influences Ca(2+) signalling in the oocytes. Here we show that microinjection of the functionally active cofilin alters the Ca(2+) signalling mediated by the three major second messengers, InsP(3), NAADP, and cADPr. Cofilin intensifies the Ca(2+) signals induced by InsP(3) and NAADP, and delays those induced by cADPr. Furthermore, the injection of cofilin increases the Ca(2+) 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 Ca(2+) signalling.     

Increased sensitivity and clustering of elementary Ca2+ release events during oocyte maturation

The universal signal for egg activation at fertilization is a rise in cytoplasmic Ca(2+) with defined spatial and temporal kinetics. Mammalian and amphibian eggs acquire the ability to produce such Ca(2+) signals during a maturation period that precedes fertilization and encompasses resumption of meiosis and progression to metaphase II. In Xenopus, immature oocytes produce fast, saltatory Ca(2+) waves that can be oscillatory in nature in response to IP(3). In contrast, mature eggs produce a single continuous, sweeping Ca(2+) wave in response to IP(3) or sperm fusion. The mechanisms mediating the differentiation of Ca(2+) signaling during oocyte maturation are not well understood. Here, I characterized elementary Ca(2+) release events (Ca(2+) puffs) in oocytes and eggs and show that the sensitivity of IP(3)-dependent Ca(2+) release is greatly enhanced during oocyte maturation. Furthermore, Ca(2+) puffs in eggs have a larger spatial fingerprint, yet are short lived compared to oocyte puffs. Most interestingly, Ca(2+) puffs cluster during oocyte maturation resulting in a continuum of Ca(2+) release sites over space in eggs. These changes in the spatial distribution of elementary Ca(2+) release events during oocyte maturation explain the continuous nature and slower speed of the fertilization Ca(2+) wave.     

Sperm deliver a new second messenger: NAADP

NAADP is a highly potent mobilizer of Ca(2+), which in turn triggers Ca(2+)-induced Ca(2+) release pathways in a wide range of species. Nevertheless, NAADP is not presently classified as a second messenger because it has not been shown to increase in response to a physiological stimulus. We now report a dramatic increase in NAADP during sea urchin egg fertilization that was largely due to production in sperm upon contacting egg jelly. The NAADP bolus plays a physiological role upon delivery to the egg based on its ability to induce a cortical flash, a depolarization-induced activation of L-type Ca(2+) channels. Moreover, the sperm-induced cortical flash was eliminated in eggs desensitized to NAADP. We conclude that an NAADP increase plays a physiologically relevant role during fertilization and provides the first conclusive demonstration that NAADP is a genuine second messenger.     

Effects of ionomycin on egg activation and early development in starfish

Ionomycin is a Ca(2+)-selective ionophore that is widely used to increase intracellular Ca(2+) levels in cell biology laboratories. It is also occasionally used to activate eggs in the clinics practicing in vitro fertilization. However, neither the precise molecular action of ionomycin nor its secondary effects on the eggs' structure and function is well known. In this communication we have studied the effects of ionomycin on starfish oocytes and zygotes. By use of confocal microscopy, calcium imaging, as well as light and transmission electron microscopy, we have demonstrated that immature oocytes exposed to ionomycin instantly increase intracellular Ca(2+) levels and undergo structural changes in the cortex. Surprisingly, when microinjected into the cells, ionomycin produced no Ca(2+) increase. The ionomycin-induced Ca(2+) rise was followed by fast alteration of the actin cytoskeleton displaying conspicuous depolymerization at the oocyte surface and in microvilli with concomitant polymerization in the cytoplasm. In addition, cortical granules were disrupted or fused with white vesicles few minutes after the addition of ionomycin. These structural changes prevented cortical maturation of the eggs despite the normal progression of nuclear envelope breakdown. At fertilization, the ionomycin-pretreated eggs displayed reduced Ca(2+) response, no elevation of the fertilization envelope, and the lack of orderly centripetal translocation of actin fibers. These alterations led to difficulties in cell cleavage in the monospermic zygotes and eventually to a higher rate of abnormal development. In conclusion, ionomycin has various deleterious impacts on egg activation and the subsequent embryonic development in starfish. Although direct comparison is difficult to make between our findings and the use of the ionophore in the in vitro fertilization clinics, our results call for more defining investigations on the issue of a potential risk in artificial egg activation.     

Fertilization causes a single Ca2+ increase that fully depends on Ca2+ influx in oocytes of limpets (Phylum Mollusca, Class Gastropoda)

Mature limpet oocytes arrested at the first metaphase (MI) of meiosis are activated by the stimulation of fertilizing sperm. The aim of the present study was to clarify the spatiotemporal property and mechanism of intracellular Ca2+ increase in limpet oocytes, which is a prerequisite signal for initiation of development at fertilization. In all of the five limpet species tested, the initial Ca2+ rising phase just after fertilization took the form of a centripetal Ca2+ wave spreading from the whole cortex to the center (cortical flash), yielding a homogeneous Ca2+ elevation throughout the oocyte. The Ca2+ level remained high during the subsequent plateau phase lasting for several minutes and then returned nearly to the original value. No additional Ca2+ increase followed the plateau phase at least by the time of first cleavage. Both rising and plateau phases of Ca2+ increase at fertilization were inhibited by removal of external Ca2+, suggesting that continuous Ca2+ entry occurs throughout the Ca2+ increase. Injection of inositol 1,4,5-trisphosphate (IP3) was effective in generating a Ca2+ increase in mature limpet oocytes arrested at MI; however, their ability to show an IP3-induced Ca2+ increase was extremely low, as compared with other animals. Responsiveness to IP3 injection in immature oocytes arrested at the first prophase (PI) was similar to that in the mature oocytes, suggesting that the IP3-induced Ca2+ release system does not develop during the process of meiotic maturation in limpet oocytes. Caffeine, cyclic adenosine diphosphate ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP), the agents known to stimulate internal Ca2+ release mechanisms distinct from an IP3-dependent pathway, had no effect on intracellular Ca2+ changes in mature limpet oocytes. Labeling of the endoplasmic reticulum (ER) with DiI revealed that cortical ER clusters are only present in the localized region around meiotic chromosomes in mature oocytes. These data strongly suggest that Ca2+ release and its propagating mechanisms are undeveloped in limpet oocytes and that Ca2+ influx is the only Ca2+-mobilizing system available and functioning at fertilization.     

Maturation and fertilization in Lottia gigantea oocytes: intracellular pH, Ca(2+), and electrophysiology

Intracellular pH and Ca(2+) were measured with BCECF- and Calcium Green-dextran during maturation and fertilization of oocytes of the limpet Lottia gigantea. Maturation of oocytes from prophase to metaphase I of meiosis was induced in seawater adjusted to pH 9 with NH(4)OH. Intracellular pH rose during maturation induction, and maturation was also induced by microinjecting pH 8, but not pH 7, HEPES buffer. Intracellular Ca(2+) rose during NH(4)OH-induced maturation, but maturation was not inhibited when the increase was blocked by microinjection of BAPTA. When the metaphase I oocytes were fertilized(), there was an abrupt increase in intracellular Ca(2+), and activation (polar body formation) failed to occur in BAPTA-injected oocytes. Intracellular pH did not rise during fertilization. These observations show that maturation from prophase to metaphase I of meiosis is pH-dependent and activation of the metaphase I oocytes is Ca(2+)-dependent. A Ca(2+) action potential was present in both immature and mature oocytes but was more prominent in mature oocytes whose input resistance was higher. Fertilization produced a long-lasting (17-20 min) Na(+)-dependent fertilization potential with superimposed oscillations resembling Ca(2+) action potentials.     

Meiosis, egg activation, and nuclear envelope breakdown are differentially reliant on Ca2+, whereas germinal vesicle breakdown is Ca2+ independent in the mouse oocyte

During early development, intracellular Ca2+ mobilization is not only essential for fertilization, but has also been implicated during other meiotic and mitotic events, such as germinal vesicle breakdown (GVBD) and nuclear envelope breakdown (NEBD). In this study, the roles of intracellular and extracellular Ca2+ were examined during meiotic maturation and reinitiation at parthenogenetic activation and during first mitosis in a single species using the same methodologies. Cumulus-free metaphase II mouse oocytes immediately resumed anaphase upon the induction of a large, transient Ca2+ elevation. This resumption of meiosis and associated events, such as cortical granule discharge, were not sensitive to extracellular Ca2+ removal, but were blocked by intracellular Ca2+ chelators. In contrast, meiosis I was dependent on external Ca2+; in its absence, the formation and function of the first meiotic spindle was delayed, the first polar body did not form and an interphase-like state was induced. GVBD was not dependent on external Ca2+ and showed no associated Ca2+ changes. NEBD at first mitosis in fertilized eggs, on the other hand, was frequently, but not always associated with a brief Ca2+ transient and was dependent on Ca2+ mobilization. We conclude that GVBD is Ca2+ independent, but that the dependence of NEBD on Ca2+ suggests regulation by more than one pathway. As cells develop from Ca(2+)-independent germinal vesicle oocytes to internal Ca(2+)-dependent pronuclear eggs, internal Ca2+ pools increase by approximately fourfold.     

Inositol 1,4,5-triphosphate microinjection triggers activation, but not meiotic maturation in amphibian and starfish oocytes

Inositol 3,4,5-triphosphate (InsP3) brought about cortical granule exocytosis and elevation of a fertilization membrane, due to a rapid increase of free calcium in cytoplasm, when injected into oocytes of the amphibian Xenopus laevis arrested at second meiotic metaphase. The same result was observed when injection was performed into oocytes of the starfish Marthasterias glacialis arrested either at the first meiotic prophase or after completion of meiosis. Although meiotic maturation was induced in both animals by specific hormones which have been previously shown to release Ca2+ within cytoplasm, InsP3 microinjection into prophase-arrested oocytes did not release them from prophase block.     

The role of the actin cytoskeleton in calcium signaling in starfish oocytes

Ca(2+) is the most universal second messenger in cells from the very first moment of fertilization. In all animal species, fertilized eggs exhibit massive mobilization of intracellular Ca(2+) to orchestrate the initial events of development. Echinoderm eggs have been an excellent model system for studying fertilization and the cell cycle due to their large size and abundance. In preparation for fertilization, the cell cycle-arrested oocytes must undergo meiotic maturation. Studies of starfish oocytes have shown that Ca(2+) signaling is intimately involved in this process. Our knowledge of the molecular mechanism of meiotic maturation and fertilization has expanded greatly in the past two decades due to the discovery of cell cycle-related kinases and Ca(2+)-mobilizing second messengers. However, the molecular details of their actions await elucidation of other cellular elements that assist in the creation and transduction of Ca(2+) signals. In this regard, the actin cytoskeleton, the receptors for second messengers and the Ca(2+)-binding proteins also require more attention. This article reviews the physiological significance and the mechanism of intracellular Ca2+ mobilization in starfish oocytes during maturation and fertilization.     

Development of calcium releasing activity induced by inositol trisphosphate and cyclic ADP-ribose during in vitro maturation of sea urchin oocytes

During fertilization of sea urchin eggs, the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) transiently increases (Ca(2+) transient). Increased [Ca(2+)](i) results from a rapid release from intracellular stores, mediated by one or both of two signaling pathways; inositol 1,4,5-trisphosphate (IP(3)) and IP(3) receptor (IP(3)R) or cyclic GMP (cGMP), cyclic ADP-ribose (cADPR) and ryanodine receptor (RyR). During fertilization, cGMP and cADPR increase preceding the Ca(2+) transient, suggesting their contribution to this. If the RyR pathway contributed to the Ca(2+) transient, its Ca(2+) releasing activity would develop in parallel with that of the IP(3) system during maturation of oocytes. Sea urchin oocytes were cultivated in vitro and Ca(2+) transients induced by photolysis of caged IP(3) or caged cADPR were measured during maturation. Oocytes spontaneously began to maturate in seawater. More than 50% of oocytes underwent germinal vesicle breakdown within 25 h and the second meiosis within 35 h, but it took more than 24 h until they became functionally identical to in vivo-matured eggs. Both IP(3) and cADPR induced Ca(2+) transients comparable to those of in vivo-matured eggs later than 24 h from the second meiosis. However, cADPR induced a small Ca(2+) transient even before meiosis, whereas IP(3) and sperm almost did not.     

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.     

Mastoparan induces Ca2+-independent cortical granule exocytosis in sea urchin eggs

In most species, cortical granule exocytosis is characteristic of egg activation by sperm. It is a Ca(2+)-mediated event which results in elevation of the vitelline coat to block permanently the polyspermy at fertilization. We examined the effect of mastoparan, an activator of G-proteins, on the sea urchin egg activation. Mastoparan was able to induce, in a concentration-dependent manner, the egg cortical granule exocytosis; mastoparan-17, an inactive analogue of mastoparan, had no effect. Mastoparan, but not sperm, induced cortical granule exocytosis in eggs preloaded with BAPTA, a Ca(2+) chelator. In isolated egg cortical lawns, which are vitelline layers and membrane fragments with endogenously docked cortical granules, mastoparan induced cortical granule fusion in a Ca(2+)-independent manner. By contrast, mastoparan-17 did not trigger fusion. We conclude that in sea urchin eggs mastoparan stimulates exocytosis at a Ca(2+)-independent late site of the signaling pathway that culminates in cortical granule discharge.     

Calcium and other ion dynamics during gamete maturation and fertilization

Ion currents and cytosolic free calcium ([Ca(2+)](i)) elevations are crucial events in triggering the complex machinery involved in both gamete maturation and fertilization. Oocyte maturation is triggered by hormone signaling which causes ion currents and [Ca(2+)](i) increase. Extracellular calcium seems to be required for meiosis progression since: (i) calcium depletion in the maturation medium severely affects oocyte developmental competence; (ii) the activity of plasma membrane L-type Ca(2+) currents decreases during maturation; (iii) the exposure to verapamil, a specific Ca(2+) channel blocker, decreases in vitro maturation efficiency. In spermatozoa, maturation initiates inside the epididymis and ends in the female genital tract. During their journey through the female reproductive tract, sperm undergo a dramatic selection and capacitation achieving fertilization competence. Adhesion to the tubal epithelium extends sperm life through depression of [Ca(2+)](i) until capacitation signals trigger an [Ca(2+)](i) elevation followed by sperm release. At fertilization, egg-sperm interaction evokes well-described transient and almost simultaneous events: i.e., fertilization current, a change in resting potential, and an increase in free [Ca(2+)](i) concentration. These events, termed oocyte activation, are the direct consequence of sperm interaction via either activation of a receptor or entry of a sperm factor. The latter hypothesis has been recently supported by the discovery of PCLzeta, a sperm-specific isozyme triggering a dramatic [Ca(2+)](i) increase via inositol 1,4,5-trisphosphate (IP(3)) production. The course of ion currents and [Ca(2+)](i) transients during maturation and fertilization plays a pivotal role in correct embryo development.     

Calcium entry through L-type calcium channels causes mitochondrial disruption and chromaffin cell death

Sustained, mild K+ depolarization caused bovine chromaffin cell death through a Ca(2+)-dependent mechanism. During depolarization, Ca(2+) entered preferentially through L-channels to induce necrotic or apoptotic cell death, depending on the duration of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) signal, as proven by the following. (i) The L-type Ca(2+) channel activators Bay K 8644 and FPL64176, more than doubled the cytotoxic effects of 30 mm K+; (ii) the L-type Ca(2+) channel blocker nimodipine suppressed the cytotoxic effects of K+ alone or K+ plus FPL64176; (iii) the potentiation by FPL64176 of the K+ -evoked [Ca(2+)](c) elevation was totally suppressed by nimodipine. Cell exposure to K+ plus the L-type calcium channel agonist FPL64176 caused an initial peak rise followed by a sustained elevation of the [Ca(2+)](c) that, in turn, increased [Ca(2+)](m) and caused mitochondrial membrane depolarization. Cyclosporin A, a blocker of the mitochondrial transition pore, and superoxide dismutase prevented the apoptotic cell death induced by Ca(2+) overload through L-channels. These results suggest that Ca(2+) entry through L-channels causes both calcium overload and mitochondrial disruption that will lead to the release of mediators responsible for the activation of the apoptotic cascade and cell death. This predominant role of L-type Ca(2+) channels is not shared by other subtypes of high threshold voltage-dependent neuronal Ca(2+) channels (i.e. N, P/Q) expressed by bovine chromaffin cells.     

Porcine oocyte activation induced by a cytosolic sperm factor

It is not known how the fertilizing sperm elicits the release of Ca(2+) from the oocyte's intracellular stores. We investigated whether a crude extract isolated from boar sperm could induce the Ca(2+) release and trigger subsequent early and late activation events upon injection into matured porcine oocytes. The sperm extract induced an immediate rise in the intracellular free Ca(2+) concentration in all oocytes tested, which was followed by repetitive Ca(2+) transients in 11 out of 14 oocytes. Heat or trypsin treatment of the extract totally abolished the Ca(2+) releasing activity of the sperm factor. The injected oocytes showed cortical granule exocytosis, they resumed meiosis and entered first interphase: pronuclei were formed in 89.2% (132/148) of the cases. Pronuclear formation was accompanied by the appearance of a new 22 kDa protein as normally seen at fertilization. Of the successfully injected oocytes 51.7% (105/203) cleaved and 2.0% (4/203) developed to the blastocyst stage after being cultured for 7 days in NCSU 23 medium. Injection of the carrier medium could not trigger these changes. The results indicate that the sperm might activate porcine oocytes by introducing a soluble factor into the oocyte's cytoplasm after gamete fusion.     

Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release

Calcium-induced calcium release (CICR) has been observed in cardiac myocytes as elementary calcium release events (calcium sparks) associated with the opening of L-type Ca(2+) channels. In heart cells, a tight coupling between the gating of single L-type Ca(2+) channels and ryanodine receptors (RYRs) underlies calcium release. Here we demonstrate that L-type Ca(2+) channels activate RYRs to produce CICR in smooth muscle cells in the form of Ca(2+) sparks and propagated Ca(2+) waves. However, unlike CICR in cardiac muscle, RYR channel opening is not tightly linked to the gating of L-type Ca(2+) channels. L-type Ca(2+) channels can open without triggering Ca(2+) sparks and triggered Ca(2+) sparks are often observed after channel closure. CICR is a function of the net flux of Ca(2+) ions into the cytosol, rather than the single channel amplitude of L-type Ca(2+) channels. Moreover, unlike CICR in striated muscle, calcium release is completely eliminated by cytosolic calcium buffering. Thus, L-type Ca(2+) channels are loosely coupled to RYR through an increase in global [Ca(2+)] due to an increase in the effective distance between L-type Ca(2+) channels and RYR, resulting in an uncoupling of the obligate relationship that exists in striated muscle between the action potential and calcium release.     

Role of cumulus cells during maturation of porcine oocytes in the rise in intracellular Ca2+ induced by inositol 1,4,5-trisphosphate

At the time of fertilization, release of inositol 1,4,5-trisphosphate (IP3) into the cytoplasm of oocytes is said to be induced by hydrolysis of phosphatidylinositol bis phosphate (PI2) via activation of phospholipase C and is responsible for the Ca2+ oscillation in oocytes immediately after sperm penetration. On the other hand, cumulus cells have been reported to play an important role in cytoplasmic maturation of mammalian oocytes and to affect embryonic development after fertilization. To obtain more information on the role of cumulus cells in cytoplasmic maturation of oocytes, the effects of cumulus cells on the rise in [Ca2+]i and the rates of activation and development of porcine mature oocytes induced by IP3 injection were investigated. Mature porcine oocytes that had been denuded of their cumulus cells in the early stage of the maturation period had a depressed rise in [Ca2+]i (4.0-6.0) and reduced rates of activation (31.4-36.8%) and development (10.0-24.4%) induced by IP3 injection compared with those of their cumulus-enclosed counterparts (7.3, 69.1% and 43.8%; P < 0.05). The [Ca2+]i rise and the rates of activation and development depressed by the removal of cumulus cells were restored by adding pyruvate to the maturation medium. Furthermore, the IP3 injection-induced depression of [Ca2+]i rise in mature oocytes derived from cumulus-denuded oocytes (DOs) was restored when they were cultured in a medium with pyruvate (3.9-6.3, P < 0.05). Also, mature oocytes from cumulus-oocyte complexes (COCs) cultured in a medium without glucose had a lower rise in [Ca2+]i than that in mature oocytes from COCs cultured with glucose (7.4-6.0, P < 0.05). Cumulus cells supported porcine oocytes during maturation in the rise in [Ca2+]i induced by IP3 and the following activation and development of porcine oocytes after injection of IP3. Moreover, we inferred that a function of cumulus cells is to produce pyruvate by metabolizing glucose and to provide oocytes with pyruvate during maturation, thereby promoting oocyte sensitivity to IP3.     

Regulation of inositol 1,4,5-trisphosphate receptor function during mouse oocyte maturation

At the time of fertilization, an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) underlies egg activation and initiation of development in all species studied to date. The inositol 1,4,5-trisphosphate receptor (IP(3)R1), which is mostly located in the endoplasmic reticulum (ER) mediates the majority of this Ca(2+) release. The sensitivity of IP(3)R1, that is, its Ca(2+) releasing capability, is increased during oocyte maturation so that the optimum [Ca(2+)](i) response concurs with fertilization, which in mammals occurs at metaphase of second meiosis. Multiple IP(3)R1 modifications affect its sensitivity, including phosphorylation, sub-cellular localization, and ER Ca(2+) concentration ([Ca(2+)](ER)). Here, we evaluated using mouse oocytes how each of these factors affected IP(3)R1 sensitivity. The capacity for IP(3)-induced Ca(2+) release markedly increased at the germinal vesicle breakdown stage, although oocytes only acquire the ability to initiate fertilization-like oscillations at later stages of maturation. The increase in IP(3)R1 sensitivity was underpinned by an increase in [Ca(2+)](ER) and receptor phosphorylation(s) but not by changes in IP(3)R1 cellular distribution, as inhibition of the former factors reduced Ca(2+) release, whereas inhibition of the latter had no impact. Therefore, the results suggest that the regulation of [Ca(2+)](ER) and IP(3)R1 phosphorylation during maturation enhance IP(3)R1 sensitivity rendering oocytes competent to initiate oscillations at the expected time of fertilization. The temporal discrepancy between the initiation of changes in IP(3)R1 sensitivity and acquisition of mature oscillatory capacity suggest that other mechanisms that regulate Ca(2+) homeostasis also shape the pattern of oscillations in mammalian eggs.     

Ca2+ and Na+ current patterns during oocyte maturation, fertilization, and early developmental stages of Ciona intestinalis

Using the whole-cell voltage clamp technique, the electrical changes in oocyte and embryo plasma membrane were followed during different meiotic and developmental stages in Ciona intestinalis. We show, for the first time, an electrophysiological characterization of the plasma membrane in oocytes at the germinal vesicle (GV) stage with high L-type calcium (Ca2+) current activity that decreased through meiosis. Moreover, the absence of Ca2+ reduced germinal vesicle breakdown (GVBD), which is consistent with a role of Ca2+ currents in the prophase/metaphase transition. In mature oocytes at the metaphase I (MI) stage, Ca2+ currents decreased and then disappeared and sodium (Na+) currents first appeared remaining high up to the zygote stage. Intracellular Ca2+ release was higher in MI than in GV, indicating that Ca2+ currents in GV may contribute to fill the stores which are essential for oocyte contraction at fertilization. The fertilization current generated in Na+ free sea water was significantly lower than the control; furthermore, oocytes fertilized in the absence of Na+ showed high development of anomalous "rosette" embryos. Current amplitudes became negligible in embryos at the 2- and 4-cell stage, suggesting that signaling pathways that mediate first cleavage do not rely on ion current activities. At the 8-cell stage embryo, a resumption of Na+ current activity and conductance occurred, without a correlation with specific blastomeres. Taken together, these results imply: (i) an involvement of L-type Ca2+ currents in meiotic progression from the GV to MI stage; (ii) a role of Na+ currents during electrical events at fertilization and subsequent development; (iii) a major role of plasma membrane permeability and a minor function of specific currents during initial cell line segregation events.     

A C-type lectin associated and translocated with cortical granules during oocyte maturation and egg fertilization in fish

Oocyte maturation and egg fertilization in both vertebrates and invertebrates are marked by orchestrated cytoplasmic translocation of secretory vesicles known as cortical granules. It is thought that such redistribution of cellular content is critical for asymmetrical cell division during early development, but the mechanism and regulation of the process is poorly understood. Here we report the identification, purification and cDNA cloning of a C-type lectin from oocytes of a freshwater fish species gibel carp (Carassius auratus gibelio). The purified protein has been demonstrated to have lectin activity and to be a Ca(2+)-dependent C-type lectin by hemagglutination activity assay. Immunocytochemistry revealed that the lectin is associated with cortical granules, gradually translocated to the cell surface during oocyte maturation, and discharged to the egg envelope upon fertilization. Interestingly, the lectin becomes phosphorylated on threonine residues upon induction of exocytosis by fertilization and returns to its original state after morula stage of embryonic development, suggesting that this posttranslational modification may represent a critical molecular switch for early embryonic development.