The initiation and propagation of the fertilization wave in sea urchin eggs

Calcium waves sweep across most eggs of the deuterostome lineage at fertilization. The precise timing of the initiation and propagation of a fertilization calcium wave has been best studied in sea urchin embryos, since the rapid depolarization caused by sperm egg fusion can be detected as a calcium influx using confocal imaging of calcium indicator dyes. The time between sperm egg fusion and the first sign of the calcium increase that constitutes the calcium wave is comparable to the time it takes for the wave to sweep across the egg, once initiated. The latency and rise time of the calcium response is sensitive to inhibitors of the InsP3 signalling pathway, as reported previously. Using calcium green dextran and confocal microscopy, we confirm that the propagation time of the calcium wave is lengthened and that initiation of the calcium wave involves activation of calcium release at hot spots that may represent clusters of calcium release channels, as has been seen in other cell types.     

Regulation of Trpm activation and calcium wave initiation during Drosophila egg activation

The transition from a developmentally arrested mature oocyte to a developing embryo requires a series of highly conserved events, collectively known as egg activation. All of these events are preceded by a ubiquitous rise of intracellular calcium, which results from influx of external calcium and/or calcium release from internal storage. In Drosophila, this calcium rise initiates from the pole(s) of the oocyte by influx of external calcium in response to mechanical triggers. It is thought to trigger calcium responsive kinases and/or phosphatases, which in turn alter the oocyte phospho‐proteome to initiate downstream events. Recent studies revealed that external calcium enters the activating Drosophila oocyte through Trpm channels, a feature conserved in mouse. The local entry of calcium raises the question of whether Trpm channels are found locally at the poles of the oocyte or are localized around the oocyte periphery, but activated only at the poles. Here, we show that Trpm is distributed all around the oocyte. This requires that it thus be specially regulated at the poles to allow calcium wave initiation. We show that neither egg shape nor local pressure is sufficient to explain this local activation of Trpm channels.     

Intracellular calcium redistribution during mating in Chlamydomonas reinhardii

Gametes of the unicellular green alga Chlamydomonas reinhardii recognize and adhere to cells of the opposite mating type by flagellar contact. Adhesion between these specialized organelles signals a rapid series of mating events which result in gamete fusion. The sequence of morphological changes (flagellar tip activation, cell wall loss, and mating structure elongation), which occur as a consequence of the sexual signalling, have been characterized. The signalling mechanisms have, however, not been defined. Calcium is known to be involved during fertilization of animal species. Increased intracellular free calcium, which can be achieved either by calcium influx or by mobilization of ions from intracellular stores, has been observed during activation of both eggs and sperm. A recent report by Bloodgood & Levin that gametes of C. reinhardii preloaded with 45Ca showed a transient increase in Ca efflux following mating, suggests that intracellular Ca redistribution may also accompany mating in this algal species. We have used X-ray microanalysis to analyze the subcellular distribution of bound calcium during mating in Chlamydomonas reinhardii. X-ray maps reveal that calcium is sequestered in discrete granules within the gamete cell body prior to mating and that during activation and cell fusion, calcium is diffuse throughout the cell. This suggests the possibility that calcium serves as a second messenger in this species.     

Egg activation in flowering plants

Compared to animals and algae, egg activation in flowering plants is still poorly understood because of the inaccessibility and complexity of the fertilization process which is double and internal. However, the development of in vitro fertilization (IVF) systems in maize and a few other plants, despite some limitations, offers new possibilities for the study of early post- fusional events and signals leading to egg activation under defined conditions. This review reports recent data on calcium events induced by gamete fusion during maize IVF and presents perspectives on the role of calcium in egg activation and in early development. Received: 2 December 2000 / Accepted: 7 June 2001     

Sperm incorporation,the pronuclear migrations,and their relation to the establishment of the first embryonic axis: Time-lapse video microscopy of the movements during fertilization of the sea urchin Lytechinus variegatus

The movements during fertilization have been investigated with differential interference optics and recorded by time-lapse video microscopy of the clear egg of the sea urchin Lytechinus variegatus. Sperm-egg binding occurs rapidly, and following a time when the sperm gyrates on the egg surface, gamete fusion occurs. A rapid cortical contraction radiates from the fusion site and is succeeded by the elevation of the fertilization coat. Sperm incorporation occurs in two stages: the fertilization cone enlarges around and above the erect and immotile sperm and then the sperm head, midpiece, and tail are displaced along the subsurface region of the egg at an average rate of 3.5 μm/min. The formation of the sperm aster moves the male pronucleus from the subsurface region of the egg toward the egg center at a rate of 4.9 μm/min. When the rays of the radial sperm aster appear to contact the female pronucleus, the female pronucleus migrates at a rate of 14.6 μm/min to the center of the sperm aster. The now adjacent pronuclei are moved to the egg center by the continuing enlargement of the sperm aster at a rate of 2.6 μm/min. Syngamy is usually preceded by the disassembly of the sperm aster. The centripetal migration of the pronuclei appears involved in the establishment of the first embryonic axis; cleavage occurs within 8° of the direction of this centering motion.     

Role of the Fyn kinase in calcium release during fertilization of the sea urchin egg

Protein tyrosine kinase activity has been implicated as part of the signaling mechanism leading to the sperm-induced calcium transient following fertilization. In the present study, we have tested the role of the Fyn kinase in triggering the calcium transient by microinjecting domain-specific fusion proteins encoding regions of Fyn sequence as inhibitors of Fyn function in vivo. A fusion protein encoding the SH2 domain of Fyn caused an increase in the latent period between sperm-egg fusion and the beginning of the calcium transient and reduced the amplitude of the calcium signal. A fusion protein encoding the U + SH3 domains also caused a small increase in the latent period. Microscopic examination revealed that a large percentage of eggs injected with the U+SH3 or SH2 domains became polyspermic as a result of the delayed block to polyspermy. Affinity experiments demonstrated that the U+SH3 and SH2 domains of Fyn were capable of forming a stable complex with phospholipase Cgamma from the sea urchin egg. The results suggest that the Fyn kinase participates in the signaling events leading up to the calcium transient and may directly regulate phospholipase Cgamma activity at fertilization.     

The influence of ultrasound and constant magnetic field on gametes, zygotes, and embryos of the sea urchin

The influence of constant magnetic field, power 7 T, and ultrasound, frequency 2, 4 and 8 MHz, on gametes, fertization, embryos and larvae of the sea urchin was studied. It was shown that magnetic field breaks the process of the gamete fusion but does not influence gametes, embryos, and larvae. Ultrasound impairs the motility of spermatozoa and larvae, prevents the fertilization, and breaks the embryonic development. It is assumed that the effect of the magnetic field is connected with the response of the cortical cytoskeleton, which consists of bundles of actin microfilaments. The rearrangement of the cortical cytoskeleton occurs during the first 20 minutes after the contact of sperm with the egg. Also there is effect of magnetic fields on calcium ions, which are liberated during the first seconds after gamete contact. The effect of the ultrasound is explained by a small increase in water temperature and cavitation process, which break celluar structures.     

Signal transduction at fertilization: the Ca2+ release pathway in echinoderms and other invertebrate deuterostomes

Gamete interaction and fusion triggers a number of events that lead to egg activation and development of a new organism. A key event at fertilization is the rise in intracellular calcium. In deuterostomes, this calcium is released from the egg's endoplasmic reticulum and is necessary for proper activation. This article reviews recent data regarding how gamete interaction triggers the initial calcium release, focusing on the echinoderms (invertebrate deuterostomes) as model systems. In eggs of these animals, Src-type kinases and phospholipase C-gamma are required components of the initial calcium trigger pathway in eggs.     

Studies on the participation of epididymal sperm protein DE/CRISP-1 in egg activation.

Protein DE (32 kDa) associates with sperm during epididymal maturation and participates in sperm-egg fusion through its binding to complementary sites on the egg surface. In the present work we investigated the participation of DE in two mechanisms probably involved in egg activation: the ability of DE to trigger activation by its interaction with the binding sites on the egg surface (receptor model) and its ability to regulate intracellular calcium channels (sperm factor model). The incubation of eggs with DE did not promote activation parameters such as calcium oscillations or meiosis resumption. Secondly, microinjection of DE into eggs was ineffective in either eliciting calcium release or modifying oscillations induced by an activating sperm extract. Together, these results argue against the participation of DE in egg activation, restricting the activity of this protein and its egg binding sites to the sperm-egg fusion process.     

Osmolarity-regulated swelling initiates egg activation in Drosophila

Egg activation is a series of highly coordinated processes that prepare the mature oocyte for embryogenesis. Typically associated with fertilization, egg activation results in many downstream outcomes, including the resumption of the meiotic cell cycle, translation of maternal mRNAs and cross-linking of the vitelline membrane. While some aspects of egg activation, such as initiation factors in mammals and environmental cues in sea animals, have been well-documented, the mechanics of egg activation in insects are less well-understood. For many insects, egg activation can be triggered independently of fertilization. In Drosophila melanogaster, egg activation occurs in the oviduct resulting in a single calcium wave propagating from the posterior pole of the oocyte. Here we use physical manipulations, genetics and live imaging to demonstrate the requirement of a volume increase for calcium entry at egg activation in ex vivo mature Drosophila oocytes. The addition of water, modified with sucrose to a specific osmolarity, is sufficient to trigger the calcium wave in the mature oocyte and the downstream events associated with egg activation. We show that the swelling process is regulated by the conserved osmoregulatory channels, aquaporins and DEGenerin/Epithelial Na⁺ channels. Furthermore, through pharmacological and genetic disruption, we reveal a concentration-dependent requirement of transient receptor potential M channels to transport calcium, most probably from the perivitelline space, across the plasma membrane into the mature oocyte. Our data establish osmotic pressure as a mechanism that initiates egg activation in Drosophila and are consistent with previous work from evolutionarily distant insects, including dragonflies and mosquitos, and show remarkable similarities to the mechanism of egg activation in some plants.     

A calcium‐mediated actin redistribution at egg activation in Drosophila

Egg activation is the essential process in which mature oocytes gain the competency to proceed into embryonic development. Many events of egg activation are conserved, including an initial rise of intracellular calcium. In some species, such as echinoderms and mammals, changes in the actin cytoskeleton occur around the time of fertilization and egg activation. However, the interplay between calcium and actin during egg activation remains unclear. Here, we use imaging, genetics, pharmacological treatment, and physical manipulation to elucidate the relationship between calcium and actin in living Drosophila eggs. We show that, before egg activation, actin is smoothly distributed between ridges in the cortex of the dehydrated mature oocytes. At the onset of egg activation, we observe actin spreading out as the egg swells though the intake of fluid. We show that a relaxed actin cytoskeleton is required for the intracellular rise of calcium to initiate and propagate. Once the swelling is complete and the calcium wave is traversing the egg, it leads to a reorganization of actin in a wavelike manner. After the calcium wave, the actin cytoskeleton has an even distribution of foci at the cortex. Together, our data show that calcium resets the actin cytoskeleton at egg activation, a model that we propose to be likely conserved in other species.     

Calcium in sea urchin egg during fertilization

Calcium plays a strikingly important role in two of the major events in developmental biology: cell activation and differentiation. In this review we begin with the location and quantity of intracellular calcium in sea urchin oocytes, and then discuss the changes that occur during fertilization and egg activation, placing special emphasis on the mobilization and redistribution of intracellular calcium. We also discuss the propagation of the calcium wave and the role of the burst of calcium on the process of reorganizing the egg cortex at fertilization.     

Methods for quantitating sea urchin sperm-egg binding

Two simple photometric methods are described for determining the average number of bound sea urchin spermatozoa per egg between 0 and 60 sec after insemination. One method is based on stopping gamete interaction with formaldehyde and then, after the eggs settle with sperm bound to their surfaces, measuring the turbidity of sperm remaining in suspension. An alternative method involves removal of the dead, unbound sperm from the formaldehyde fixed eggs by repeated washing in sea water. The bound sperm are then released from the egg surface by pronase digestion and the turbidity of the sperm suspension measured and related to sperm concentration by direct cell counts. Two phases of gamete interaction exist (1) the binding phase, from 0 to 20 or 25 sec, during which time sperm continuously bind to the egg surfaces; (2) the unbinding phase, from 20 or 25 to 50 sec, during which time the sperm are unbound from the eggs and return to the suspension. The results of experiments in which the method is used to assess sperm binding at different pH values and at different calcium concentrations are presented. Data are presented suggesting that a definite number of sperm binding sites may exist on the vitelline layer.     

Fusion of membranes during fertilization. Increases of the sea urchin egg''s membrane capacitance and membrane conductance at the site of contact with the sperm

The early events of fertilization that precede and cause activation of an egg have not been fully elucidated. The earliest electrophysiological change in the sea urchin egg is a sperm-evoked increase of the egg's membrane conductance. The resulting depolarization facilitates entry of the fertilizing sperm and precludes the entry of supernumerary sperm. The sequence of the increase in the egg's membrane conductance, gamete membrane fusion, egg activation, and sperm entry, including causal relationships between these events, are not known. This study reports the use of whole egg voltage clamp and loose patch clamp to monitor simultaneously changes of membrane conductance and capacitance at the site of sperm-egg contact. Measurements were made during sperm-egg interactions where sperm entry readily proceeded or was precluded by maintaining the egg's membrane potential either at large, negative values or at positive values. Whenever the sperm evoked an increase of the egg's membrane conductance, that increase initiated abruptly, was localized to the site of sperm attachment, and was accompanied by a simultaneous abrupt increase of the membrane capacitance. This increase of capacitance indicated the establishment of electrical continuity between gametes (possibly fusion of the gametes' plasma membranes). If sperm entry was blocked by large negative membrane potentials, the capacitance cut off rapidly and simultaneously with a decrease of the membrane conductance, indicating that electrical continuity between gametes was disrupted. When sperm entry was precluded by positive membrane potentials, neither conductance nor capacitance increased, indicating that sperm entry was halted before the fusion of membranes. A second, smooth increase of capacitance was associated with the exocytosis of cortical granules near the sperm in eggs that were activated. Electrical continuity between the gametes always preceded activation of the egg, but transient electrical continuity between the gametes alone was not always sufficient to induce activation.     

Mammalian phospholipase Czeta induces oocyte activation from the sperm perinuclear matrix

Mammalian sperm-borne oocyte activating factor (SOAF) induces oocyte activation from a compartment that engages the oocyte cytoplasm, but it is not known how. A SOAF-containing extract (SE) was solubilized from the submembrane perinuclear matrix, a domain that enters the egg. SE initiated activation sufficient for full development. Microinjection coupled to tandem mass spectrometry enabled functional correlation profiling of fractionated SE without a priori assumptions about its chemical nature. Phospholipase C-zeta (PLCzeta) correlated absolutely with activating ability. Immunoblotting confirmed this and showed that the perinuclear matrix is the major site of 72-kDa PLCzeta. Oocyte activation was efficiently induced by 1.25 fg of sperm PLCzeta, corresponding to a fraction of one sperm equivalent (approximately 0.03). Immunofluorescence microscopy localized sperm head PLCzeta to a post-acrosomal region that becomes rapidly exposed to the ooplasm following gamete fusion. This multifaceted approach suggests a mechanism by which PLCzeta originates from an oocyte-penetrating assembly--the sperm perinuclear matrix--to induce mammalian oocyte activation at fertilization.     

A novel in vitro system for gamete fusion in maize

Various systems by using electric pulse, calcium, or polyethylene glycol have been developed in the past decade for the in vitro fusion of plant gametes. These in vitro systems provide a new way to study the fertilization mechanisms of plants. In this study, we developed a bovine serum albumin （BSA）-mediated fusion system for the in vitro fusion of maize gametes. The in vitro fusion of the isolated single egg cell and sperm cell of maize was observed microscopically in the BSA solution and the fertilized egg cell showed normal cell wall regeneration and nuclear division. The effects of the BSA concentration, pH value and calcium level on the efficiency of the maize gamete fusion were also assessed. BSA concentration and pH value did significantly affect the efficiency of the gamete fusion. Calcium was not necessary for the gamete fusion when BSA was present. The optimal solution for the gamete fusion contained 0.1% BSA, pH 6.0. The fusion frequency was as high as 96.7% in that optimal solution. This new in vitro fertilization system offers an alternative tool for the in vitro study of fertilization mechanisms with much simpler manipulating procedure than PEG system, and it will be especially useful for the in vitro study of the calcium dynamics during plant fertilization.     

A C. elegans sperm TRP protein required for sperm-egg interactions during fertilization

Fertilization, a critical step in animal reproduction, is triggered by a series of specialized sperm-egg interactions. However, the molecular mechanisms underlying fertilization are not well understood. Here, we identify a sperm-enriched C. elegans TRPC homolog, TRP-3. Mutations in trp-3 lead to sterility in both hermaphrodites and males due to a defect in their sperm. trp-3 mutant sperm are motile, but fail to fertilize oocytes after gamete contact. TRP-3 is initially localized in intracellular vesicles, and then translocates to the plasma membrane during sperm activation. This translocation coincides with a marked increase in store-operated calcium entry, providing an in vivo mechanism for the regulation of TRP-3 activity. As C. elegans oocytes lack egg coats, our data suggest that some TRPC family channels might function to mediate calcium influx during sperm-egg plasma membrane interactions leading to fertilization.     

Female‐induced remote regulation of sperm physiology may provide opportunities for gamete‐level mate choice

In sedentary externally fertilizing species, direct interactions between mating partners are limited and prefertilization communication between sexes occurs largely at the gamete level. Certain combinations of eggs and sperm often have higher fertilization success than others, which may be contingent on egg‐derived chemical factors that preferentially attract sperm from compatible males. Here, we examine the mechanisms underlying such effects in the marine mussel Mytilus galloprovincialis, where differential sperm attraction has recently been shown to be associated with variation in offspring viability. Specifically, we focus on the sperm surface glycans, an individually unique layer of carbohydrates that moderate self‐recognition and other cellular‐level interactions. In many species egg‐derived factors trigger remarkable changes in the sperm's glycan layer, physiology, and swimming behavior, and thus potentially moderate mate choice at the gamete level. Here, we show that sperm glycan modifications and the strength of acrosome reaction are both dependent on specific male–female interactions (male–female combination). We also find associations between female‐induced sperm glycan changes and the Ca²⁺ influx into sperm–‐a key regulator of fertilization processes from sperm capacitation to gamete fusion. Together, our results suggest that female‐induced remote regulation of sperm physiology may constitute a novel mechanism of gamete‐level mate choice.     

Ca(2+) entry through store-operated channels in mouse sperm is initiated by egg ZP3 and drives the acrosome reaction

Fertilization occurs after the completion of the sperm acrosome reaction, a secretory event that is triggered during gamete adhesion. ZP3, an egg zona pellucida glycoprotein, produces a sustained increase of the internal Ca(2+) concentration in mouse sperm, leading to acrosome reactions. Here we show that the sustained Ca(2+) concentration increase is due to the persistent activation of a Ca(2+) influx mechanism during the late stages of ZP3 signal transduction. These cells also possess a Ca(2+) store depletion-activated Ca(2+) entry pathway that is open after treatment with thapsigargin. Thapsigargin and ZP3 activate the same Ca(2+) permeation mechanism, as demonstrated by fluorescence quenching experiments and by channel antagonists. These studies show that ZP3 generates a sustained Ca(2+) influx through a store depletion-operated pathway and that this drives the exocytotic acrosome reaction.