Jong T. Chun

Alteration of the Cortical Actin Cytoskeleton Deregulates Ca2+ Signaling, Monospermic Fertilization, and Sperm Entry

Background When preparing for fertilization, oocytes undergo meiotic maturation during which structural changes occur in the endoplasmic reticulum (ER) that lead to a more efficient calcium response. During meiotic maturation and subsequent fertilization, the actin cytoskeleton also undergoes dramatic restructuring. We have recently observed that rearrangements of the actin cytoskeleton induced by actin-depolymerizing agents, or by actin-binding proteins, strongly modulate intracellular calcium (Ca2+) signals during the maturation process. However, the significance of the dynamic changes in F-actin within the fertilized egg has been largely unclear. Methodology/Principal Findings We have measured changes in intracellular Ca2+ signals and F-actin structures during fertilization. We also report the unexpected observation that the conventional antagonist of the InsP3 receptor, heparin, hyperpolymerizes the cortical actin cytoskeleton in postmeiotic eggs. Using heparin and other pharmacological agents that either hypo- or hyperpolymerize the cortical actin, we demonstrate that nearly all aspects of the fertilization process are profoundly affected by the dynamic restructuring of the egg cortical actin cytoskeleton. Conclusions/Significance Our findings identify important roles for subplasmalemmal actin fibers in the process of sperm-egg interaction and in the subsequent events related to fertilization: the generation of Ca2+ signals, sperm penetration, cortical granule exocytosis, and the block to polyspermy.

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. Here, we show that this Ca2+ 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 Ca2+ waves triggered by 1-MA. This indicates that the cortical actin cytoskeleton modulates Ca2+ release during meiotic maturation. The Ca2+ wave was inhibited by the classical antagonists of the InsP(3)-linked Ca2+ 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 Ca2+ 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(3), despite the massive release of Ca2+, implying that exocytosis of the cortical granules requires not only a Ca2+ 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 Ca2+ signals and in regulating cortical granule exocytosis.

Fertilization in echinoderms

For more than 150 years, echinoderm eggs have served as overly favored experimental model systems in which to study fertilization. Sea urchin and starfish belong to the same phylum and thus share many similarities in their fertilization patterns. However, several subtle but fundamental differences do exist in the fertilization of sea urchin and starfish, reflecting their phylogenetic bifurcation approximately 500 million years ago. In this article we review some of the seminal and recent findings that feature similarities and differences in sea urchin and starfish at fertilization.

The biphasic increase of PIP2 in the fertilized eggs of starfish: new roles in actin polymerization and Ca2+ signaling.

Background Fertilization of echinoderm eggs is accompanied by dynamic changes of the actin cytoskeleton and by a drastic increase of cytosolic Ca2+. Since the plasma membrane-enriched phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) serves as the precursor of inositol 1,4,5 trisphosphate (InsP3) and also regulates actin-binding proteins, PIP2 might be involved in these two processes. Methodology/Principal Findings In this report, we have studied the roles of PIP2 at fertilization of starfish eggs by using fluorescently tagged pleckstrin homology (PH) domain of PLC-δ1, which has specific binding affinity to PIP2, in combination with Ca2+ and F-actin imaging techniques and transmission electron microscopy. During fertilization, PIP2 increased at the plasma membrane in two phases rather than continually decreasing. The first increase was quickly followed by a decrease about 40 seconds after sperm-egg contact. However, these changes took place only after the Ca2+ wave had already initiated and propagated. The fertilized eggs then displayed a prolonged increase of PIP2 that was accompanied by the appearance of numerous spikes in the perivitelline space during the elevation of the fertilization envelope (FE). These spikes, protruding from the plasma membrane, were filled with microfilaments. Sequestration of PIP2 by RFP-PH at higher doses resulted in changes of subplasmalemmal actin networks which significantly delayed the intracellular Ca2+ signaling, impaired elevation of FE, and increased occurrences of polyspermic fertilization. Conclusions/Significance Our results suggest that PIP2 plays comprehensive roles in shaping Ca2+ waves and guiding structural and functional changes required for successful fertilization. We propose that the PIP2 increase and the subsequent formation of actin spikes not only provide the mechanical supports for the elevating FE, but also accommodate increased membrane surfaces during cortical granule exocytosis.

Actin, more than just a housekeeping protein at the scene of fertilization

Since the first demonstration of sperm entry into the fertilized eggs of Mediterranean sea urchin Paracentrotus lividus by Hertwig (1876), enormous progress and insights have been made on this topic. However, the precise molecular mechanisms underlying fertilization are largely unknown. The two most dramatic changes taking place in the zygote immediately after fertilization are: (i) a sharp increase of intracellular Ca2+ that initiates at the sperm interaction site and traverses the egg cytoplasm as a wave, and (ii) the concomitant dynamic rearrangement of the actin cytoskeleton. Traditionally, this has been studied most extensively in the sea urchin eggs, but another echinoderm, starfish, whose eggs are much bigger and transparent, has facilitated experimental approaches using microinjection and fluorescent imaging methodologies. Thus in starfish, it has been shown that the sperm-induced Ca2+ increase in the fertilized egg can be recapitulated by several Ca2+-evoking second messengers, namely inositol 1,4,5-trisphosphate (InsP3), cyclic ADP-ribose (cADPr) and nicotinic acid adenine dinucleotide phosphate (NAADP), which may play distinct roles in the generation and propagation of the Ca2+ waves. Interestingly, it has also been found that the dynamic rearrangement of the actin cytoskeleton in the fertilized eggs plays pivotal roles in guiding monospermic sperm entry and in the fine modulation of the intracellular Ca2+ signaling. As it is well known that Ca2+ regulates the structure of the actin cytoskeleton, our finding that Ca2+ signaling can be reciprocally affected by the state of the actin cytoskeleton raises an intriguing possibility that actin and Ca2+ signaling may form a ‘positive feedback loop’ that accelerates the downstream events of fertilization. Perturbation of the cortical actin networks also inhibits cortical granules exocytosis. Polymerizing actin bundles also compose the ‘acrosome process,’ a tubular structure protruding from the head of fertilizing sperm. Hence, actin, which is one of the most strictly conserved proteins in eukaryotes, modulates almost all major aspects of fertilization.

Guanine Nucleotides in the Meiotic Maturation of Starfish Oocytes: Regulation of the Actin Cytoskeleton and of Ca2+ 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 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.

Early events of fertilization in sea urchin eggs are sensitive to actin-binding organic molecules.

We previously demonstrated that many aspects of the intracellular Ca(2+) increase in fertilized eggs of starfish are significantly influenced by the state of the actin cytoskeleton. In addition, the actin cytoskeleton appeared to play comprehensive roles in modulating cortical granules exocytosis and sperm entry during the early phase of fertilization. In the present communication, we have extended our work to sea urchin which is believed to have bifurcated from the common ancestor in the phylogenetic tree some 500 million years ago. To corroborate our earlier findings in starfish, we have tested how the early events of fertilization in sea urchin eggs are influenced by four different actin-binding drugs that promote either depolymerization or stabilization of actin filaments. We found that all the actin drugs commonly blocked sperm entry in high doses and significantly reduced the speed of the Ca(2+) wave. At low doses, however, cytochalasin B and phalloidin increased the rate of polyspermy. Overall, certain aspects of Ca(2+) signaling in these eggs were in line with the morphological changes induced by the actin drugs. That is, the time interval between the cortical flash and the first Ca(2+) spot at the sperm interaction site (the latent period) was significantly prolonged in the eggs pretreated with cytochalasin B or latrunculin A, whereas the Ca(2+) decay kinetics after the peak was specifically attenuated in the eggs pretreated with jasplakinolide or phalloidin. In addition, the sperm interacting with the eggs pretreated with actin drugs often generated multiple Ca(2+) waves, but tended to fail to enter the egg. Thus, our results indicated that generation of massive Ca(2+) waves is neither indicative of sperm entry nor sufficient for cortical granules exocytosis in the inseminated sea urchin eggs, whereas the structure and functionality of the actin cytoskeleton are the major determining factors in the two processes.

Antibody against the actin-binding protein depactin attenuates Ca2+ signaling in starfish eggs.

Being present in starfish oocytes, the cofilin/ADF (actin-depolymerizing factor) family protein depactin severs actin filaments. Previously, we reported that exogenous cofilin microinjected into starfish eggs significantly augmented the Ca(2+) release in response to inositol 1,4,5-trisphosphate (InsP3) or fertilizing sperm, raising the possibility that intracellular Ca(2+) signaling could be modulated by the actin cytoskeleton. In this communication, we have targeted the endogenous depactin by use of the specific antibody that was raised against its actin-binding domain. The anti-depactin antibody microinjected into the starfish oocytes and eggs effectively altered the structure of the actin cytoskeleton, and significantly delayed the meiotic progression induced by 1-methyladenine. When microinjected into the mature eggs, the anti-depactin antibody markedly reduced the amplitude of the Ca(2+) response in a dose-dependent manner, corroborating the results of our previous study with cofilin. In addition, the eggs microinjected with the anti-depactin antibody displayed reduced rate of successful elevation of the fertilization envelope and an elevated tendency of polyspermic interaction. Taken together, our data suggest that the actin cytoskeleton is implicated not only in meiotic maturation and intracellular Ca(2+) signaling, but also in the fine regulation of gametes interaction and cortical granules exocytosis.

The actin cytoskeleton in meiotic maturation and fertilization of starfish eggs.

In evolutionary terms, actin is a eukaryotic invention, which is employed in a variety of cell activities beyond muscle contraction. Reflecting its fundamental roles in cells, actin is expressed from multiple genes as different isoforms in a species, and its amino acid sequence is strictly conserved across the entire spectrum of phyla. Inside cells, actin plays not only a structural role as an element of cytoskeleton, but also diverse functional roles. The incorporation of actin monomers into actin filaments is highly preferred in one end (barded or plus end) than the other (pointed or minus end). Due to this phenomenon, actin filaments exhibit constant ‘treadmilling’, and the turnover rate of an average actin filament inside a cell is order of minutes [1]. Such a dynamic remodelling of the actin cytoskeleton is facilitated or directed in a certain way by the concerted action of actin-binding proteins and the signaling pathways evoked by external cues. With the aid of these accessory proteins, the selfreorganizing actin cytoskeleton plays dynamic roles in controlling cell shapes, motility, and intracellular transport [2–5]. The actin cytoskeleton can also serve as a cytoplasmic framework on which certain metabolic enzymes and the protein synthetic machinery exert their functions [6,7]. On the other hand, actin in the nucleus appears to play quite distinct roles from those of the cytoplasmic actin. Actin may serve as a component of the transcription apparatus or a chromatin-remodelling complex in the nucleus [8–12]. Hence, actin can be used for many different purposes inside the same cell, depending on the subcellular context. In animal cells, polarized actin filaments can be organized at least in three different modes: (i) antiparallel arrays of filaments in the inner cytoplasm, e.g. stress fibers, (ii) parallel arrays of filaments in the protruding cell periphery e.g. microspikes or filopodia, and (iii) isotropic arrays of filaments underneath the plasma membrane. The eggs of echinoderms, which have been widely used for studying fertilization, also display such levels of subcellular actin organization. The structure and organization of subplasmalemmal and endoplasmic actin filaments undergo dramatic changes inside eggs during meiotic maturation and fertilization. In this article, we will review some of the early and recent works regarding the roles of the actin cytoskeleton in the meiotic maturation and fertilization of starfish eggs.

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). 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 2h 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.