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.     

Morphological Modifications of Oocyte Vitelline Envelope from Bufo arenarum during Different Functional States

The submicroscopic morphology of the vitelline envelope of Bufo arenarum's oocyte change significantly during the maturation and fertilization processes. The morphological changes are related to physiological activity in vivo and can be triggered in vitro by experimental procedures. It is our scope to present the ultrastructure differences of fascicular components of the vitelline envelope in coelomic, "pars recta" conditioned, oviductal, oviposited and fertilized oocytes. Our experimental results indicate that artificial "pars recta" treatment of coelomic oocytes arrange the fascicular components as those of oviposited oocyte, although differences still remain indicating that additional maturation processes take place while the egg pass througth the oviduct. Fertilized or activated oocytes which are refractary to sperm penetration, change the vitelline envelope fascicular components orientation giving a submicroscopical image quite different to those of none fertilized oocytes. These ultrastructural changes define in a clear cut manner the functional states of Bufo arenarum's oocyte.     

Behaviour of the vitelline envelope in Bufo arenarum oocytes matured in vitro in blockade to polyspermy

During activation of amphibian eggs, cortical granule exocytosis causes elaborate ultrastructural changes in the vitelline envelope. These changes involve modifications in the structure of the vitelline envelope and formation of a fertilization envelope (FE) that can no longer be penetrated by sperm. In Bufo arenarum, as the egg traverses the oviduct, the vitelline envelope is altered by a trypsin-like protease secreted by the oviduct, which induces an increased susceptibility of the vitelline envelope to sperm lysins. Full-grown oocytes of B. arenarum, matured in vitro by progesterone, are polyspermic, although cortical granule exocytosis seems to occur within a normal chronological sequence. These oocytes can be fertilized with or without trypsin treatment, suggesting that the vitelline envelope is totally sperm-permeable. Vitelline envelopes without trypsin treatment cannot retain either gp90 or gp96. This suggests that these glycoproteins are involved in the block to polyspermy and that trypsin treatment of matured in vitro oocytes before insemination is necessary to enable vitelline envelopes to block polyspermy. The loss of the binding capacity in vitelline envelopes isolated from B. arenarum oocytes matured in vitro with trypsin treatment and activated by electric shock suggests that previous trypsin treatment is a necessary step for sperm block to occur. When in vitro matured oocytes were incubated with the product of cortical granules obtained from in vitro matured oocytes (vCGP), vitelline envelopes with trypsin treatment were able to block sperm entry. These oocytes exhibited the characteristic signs of activation. These results support the idea that B. arenarum oocytes can be activated by external stimuli and suggest the presence of unknown oocyte surface receptors linked to the activation machinery in response to fertilization. Electrophoretic profiles obtained by SDS-PAGE of solubilized vitelline envelopes from oocytes matured in vitro revealed the conversion of gp40 (in vitro matured oocytes, without trypsin treatment) to gp38 (ascribable to trypsin activity or cortical granule product activity, CGP) and the conversion of gp70 to gp68 (ascribable to trypsin activity plus CGP activity). Taking into account that only the vitelline envelopes of in vitro matured oocytes with trypsin treatment and activated can block sperm entry, we may suggest that the conversion of gp70 to gp68 is related to the changes associated with sperm binding.     

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.     

Extracellular Mg2+ induces an intracellular Ca2+ wave during oocyte activation in the marine shrimp Sicyonia ingentis.

In contrast to most systems in which oocyte activation is triggered by the fertilizing sperm, Sicyonia ingentis oocytes are activated by seawater Mg2+ during spawning. S. ingentis oocytes were spawned into Mg(2+)-free seawater and microinjected with the fluorescent Ca2+ indicator Fluo-3 to study the effects of added Mg2+ on intracellular Ca2+ levels. The Mg2+ induced a wave of fluorescence across the oocyte that traveled at a speed of 13 +/- 3 microns/sec. Extracellular Ca2+ was not required for induction of the wave. Treatment with Ca2+ ionophore in Mg(2+)-free medium or a localized injection (0.3% oocyte volume) of 3-5 microM Ca2+ also initiated the wave; injection of 250 mM Mg2+ (up to 1.5% oocyte volume) had no effect. Microinjection of 750 microM EGTA (final) suppressed the Mg(2+)-induced wave, while an identical concentration of EDTA had no inhibitory effect. Subsequent to the initial Mg(2+)-induced intracellular Ca2+ increase, a second Ca2+ increase was observed at approximately 15 min postspawning; the timing of this second increase appeared to be independent of when the Mg(2+)-induced wave was initiated, thus an event associated with spawning may be involved. While oocytes in normal seawater were monospermic, those in Mg(2+)-free seawater were polyspermic, suggesting a role for the Mg(2+)-induced Ca2+ wave in regulating sperm entry into the oocyte.     

Activation of amphibian oocytes by sperm extracts

In the fertilization of most animals, egg activation is accompanied by an increase in cytoplasmatic Ca2+; however, the mechanism through which the fertilizing sperm induce this phenomenon is still controversial. An increase in intracellular free Ca2+ is required to trigger egg activation events, a process that includes cortical granule exocytosis, resumption and completion of meiosis and DNA replication, and culminates in the first mitotic cleavage. In this work, we investigated the effect of microinjection and incubation of different fractions of homologous sperm extract on the activation of Bufo arenarum oocytes matured in vitro. Two heat treatment-sensitive fractions obtained by chromatography were able to induce oocyte activation. The sperm fraction, which contained a 24 kDa protein, induced 90% activation when it was microinjected into the oocytes. Whilst the sperm fraction, which contained a 36 kDa protein, was able to induce about 70% activation only when it was applied on the oocyte surface.     

A wave of IP3 production accompanies the fertilization Ca2+ wave in the egg of the frog, Xenopus laevis: theoretical and experimental support

The fertilization Ca2+ wave in Xenopus laevis is a single, large wave of elevated free Ca2+ that is initiated at the point of sperm-egg fusion and traverses the entire width of the egg. This Ca2+ wave involves an increase in inositol-1,4,5-trisphosphate (IP3) resulting from the interaction of the sperm and egg, which then results in the activation of the endoplasmic reticulum Ca2+ release machinery. The extraordinarily large size of this cell (1.2 mm diameter) together with the small surface region of sperm-receptor activation makes special demands on the IP3-dependent Ca2+ mobilizing machinery. We propose a detailed model of the fertilization Ca2+ wave in Xenopus eggs that requires an accompanying wave of IP3 production. While the Ca2+ wave is initiated by a localized increase of IP3 near the site of sperm-egg fusion, the Ca2+ wave propagates via IP3 production correlated with the Ca2+ wave-possibly via Ca(2+)-mediated PLC activation. Such a Ca(2+)-mediated IP(3) production wave has not been required previously to explain the fertilization Ca2+ wave in eggs; we argue this is necessary to explain the observed IP3 dynamics in Xenopus eggs. To test our hypothesis, we have measured the IP3 levels from 20 nl "sips" of the egg cortex during wave propagation. We were unable to detect the low IP3 levels in unfertilized eggs, but after fertilization, [IP3] ranged from 175 to 430 nM at the sperm entry point and from 120 to 700 nM 90 degrees away once the Ca2+ wave passed that region about 2 min after fertilization. Prior to the Ca2+ wave reaching that region the IP3 levels were undetectable. Since significant IP3 could not diffuse to this region from the sperm entry point within 2 min, this observation is consistent with a regenerative wave of IP3 production.     

Cytoplasmic activation of starfish oocytes by sperm and divalent ionophore A-23187

The relationship between onset of the early cytoplasmic stages of oocyte activation (vitelline membrane separation and elevation) and nuclear meiotic maturation was investigated in starfish oocytes after their exposure to divalent ionophore (A-23187) or sperm. Meiotically mature oocytes, isolated in calcium-free seawater, underwent activation in response to sperm or ionophore as previously reported. Large, immature starfish oocytes, arrested in prophase I of meiosis (germinal vesicle stage), underwent vitelline membrane elevation when treated with divalent ionophore A-23187 or starfish sperm. Histological studies demonstrated that cortical granule breakdown in the oocyte cortex was associated with vitelline membrane elevation after these treatments. Activation of oocytes by sperm occurred only in response to starfish sperm. Sea urchin, sand dollar, surf clam, or marine worm sperm did not induce vitelline membrane elevation of either immature or mature starfish oocytes. Sperm- or ionophore-activated immature oocytes underwent nuclear maturation after addition of the meiosis-inducing hormone, l-methyladenine; however, parthenogenetic development did not occur and embryonic development was markedly inhibited. In contrast to previous studies, the present results indicate that cytoplasmic activation can be initiated before and without hormone induction of the nuclear maturation process. Differentiation of the oocyte cell surface or cortex reactivity therefore appears to occur during oogenesis rather than as a consequence of maturation. The data further support the view that divalent ions mediate certain of the early activation responses initiated by sperm at the time of fertilization and that synchronization of fertilization to the meiotic process in the oocyte is important for the occurrence of normal development.     

Measurement of intracellular IP3 during Ca2+ oscillations in mouse eggs with GFP-based FRET probe

Intracellular Ca2+ oscillations in fertilized mammalian eggs, the key signal that stimulates egg activation and early embryonic development, are regulated by inositol 1,4,5-trisphosphate (IP3) signaling pathway. We investigated temporal changes in intracellular IP3 concentration ([IP3]i) in mouse eggs, using a fluorescent probe based on fluorescence resonance energy transfer between two green fluorescent protein variants, during Ca2+ oscillations induced by fertilization or expression of phospholipase Czeta (PLCzeta), an egg-activating sperm factor candidate. Fluorescence measurements suggested the elevation of [IP3]i in fertilized eggs, and the enhancement of PLCzeta-mediated IP3 production by cytoplasmic Ca2+ was observed during Ca2+ oscillations or in response to CaCl2 microinjection. The results supported the view that PLCzeta is the sperm factor to stimulate IP3 pathway, and suggested that high Ca2+ sensitivity of PLCzeta activity and positive feedback from released Ca2+ are important for triggering and maintaining Ca2+ oscillations.     

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. Strikingly, the self-desensitization of NAADP receptors either abolished the Ca2+ response or resulted in abnormal oocyte activation, i.e., the membrane depolarization followed the Ca2+ wave and the oocyte was polyspermic. The inhibition of InsP3 signaling only impaired the propagation of the Ca2+ wave and shortened the FP. The duration of FP was also reduced in low-Na+ sea water. Finally, uncaged InsP3 produced a Ca2+ increase, which depolarized the membrane upon the activation of a Ca2+-sensitive cation current. These results support the hypothesis that Ca2+ entry during the NAADP-triggered FP is required for the onset of the Ca2+ wave at fertilization. The InsP3-mediated Ca2+ wave, in turn, may interact with the NAADP-evoked depolarization by activating a Ca2+-dependent Na+ entry.     

Ultrastructural studies on the nematode Xiphinema diversicaudatum: Oogenesis and fertilization

Oogenesis and fertilization in longidorid nematodes has been examined for the first time at electron microscope level in Xiphinema diversicaudatum. Oogonia in the germinative zone of the ovary are irregularly shaped and lie adjacent to each other or separated by processes of the epithelial cells of the ovary. Developing oocytes pass in single file up to the growth zone and fibrogranular formation occurs around their nucleus. The perinuclear deposits remain until the oocyte is fully grown. Oocytes increase rapidly in volume because of the production of secretory granules. Three types of granules are recognizable. Type 1 granules are spherical, amorphous in structure and delimited by a lighter area, probably consisting of lipoprotein. Type 2 granules, electron lucent, arranged in groups, are lipid inclusions. Type 3 are dense spheres and may represent yolk bodies. The two last are then utilized by the developing embryo. Mature oocytes assume a smooth, cylindrical configuration as they traverse the oviduct. A cone of fertilization seems to be formed at the distal pole of the oocyte, where the sperm penetrates. The sperm totally penetrates the oocyte, through an invagination formed at the oocyte surface. The oocyte continues to undergo two unequal cytoplasmic divisions, resulting in the formation of a female pronucleus and two polar bodies. Under the stimulus of fertilization, a new egg cell membrane is produced, the first one becoming the vitelline envelope.     

Fertilization and development of quail oocytes after intracytoplasmic sperm injection

The present study was conducted to establish the intracytoplasmic sperm injection (ICSI) method for in vitro fertilization and development in quail. The efficiency of fertilization of oocytes was compared 1) between spontaneous and premature ovulation and 2) among testicular round spermatids, elongated spermatids, and immature and mature spermatozoa. The oocytes were injected with a single spermatozoon or spermatid and cultured for 24 h. Cell division was histologically observed with hematoxylin-eosin (HE) and a nucleus-specific fluorescent dye (DAPI). Five of 30 (16.6%) and 4 of 30 (13.3%) oocytes injected with mature sperm were fertilized in the spontaneous and induced ovulation group, respectively. Those embryos showed development at stages II-VII. Half the number (three of six) of the oocytes injected with testicular spermatozoa were fertilized and developed to stages IV-VII, and two of five oocytes injected with elongated spermatids were fertilized and developed to stage VI. All ooocytes injected with round spermatids were unfertilized. The results demonstrate that intracytoplasmic injection of a single sperm into quail oocyte can activate the oocyte and lead to fertilization. Oocytes prematurely ovulated are capable of fertilizing with mature sperm as are those spontaneously ovulated. In addition, the results suggest that the testicular round spermatids may not possess sufficient oocyte-activating potency but that the elongated spermatids and immature spermatozoa are competent to participate in fertilization and early embryonic development in quail.     

The Chaetopterus vitelline envelope is not necessary for the gamete interactions that lead to fertilization

It has been recently shown that, in several genera of annelids, including Chaetopterus, fertilizing sperm attach to and fuse with egg microvilli which penetrate the vitelline envelope. This suggests that the annelid vitelline envelope may have no direct or obligatory role in normal fertilization. The present study was undertaken to investigate the involvement of the vitelline envelope in fertilization in Chaetopterus experimentally, by examining the fertilization of vitelline envelope-free eggs quantitatively and qualitatively. Brief exposure of the eggs to isotonic sucrose-EDTA removed the vitelline envelope as determined by both phase-contrast and electron microscopy, rendered the eggs more sensitive to polyspermy and substantially reduced the binding of supernumerary sperm to eggs but did not decrease fertilizability as determined by sperm dilution assay and did not make the eggs more sensitive to cross-fertilization. The events of fertilization were examined by electron microscopy and found to be very similar in vitelline envelope-free eggs to those in intact eggs. We conclude that the vitelline envelope in Chaetopterus has binding sites for sperm but that it has no obligatory role in fertilization and is primarily involved in the prevention of polyspermy.     

STIM1 is required for Ca2+ signaling during mammalian fertilization

During fertilization in mammals, a series of oscillations in the oocyte's intracellular free Ca(2+) concentration is responsible for oocyte activation and stimulation of embryonic development. The oscillations are associated with influx of Ca(2+) across the plasma membrane that is probably triggered by the depletion of the intracellular stores, a mechanism known as store-operated Ca(2+) entry. Recently, STIM1 has been identified in oocytes as a key component of the machinery that generates the Ca(2+) influx after store depletion. In this study, the involvement of STIM1 in the sperm-induced Ca(2+) oscillations and its significance in supporting subsequent embryo development were investigated. Downregulation of STIM1 levels in pig oocytes by siRNA completely inhibited the repetitive Ca(2+) signal triggered by the fertilizing sperm. In addition, a significantly lower percentage of oocytes cleaved or formed blastocysts when STIM1 was downregulated prior to fertilization compared to the control groups. Restoring STIM1 levels after fertilization in such oocytes by means of mRNA injection could not rescue embryonic development that in most cases was arrested at the 2-cell stage. On the other hand, STIM1 overexpression prior to fertilization did not alter the pattern of sperm-induced Ca(2+) oscillations and development of these fertilized oocytes up to the blastocyst stage was also similar to that registered in the control group. Finally, downregulation of STIM1 had no effect on oocyte activation when activation was stimulated artificially by inducing a single large elevation in the oocyte's intracellular free Ca(2+) concentration. These findings suggest that STIM1 is essential for normal fertilization as it is involved in the maintenance of the long-lasting repetitive Ca(2+) signal.     

Defective calcium release during in vitro fertilization of maturing oocytes of LT/Sv mice

Oocytes of LT/Sv mice have anomalous cytoplasmic and nuclear maturation. Here, we show that in contrast to the oocytes of wild-type mice, a significant fraction of LT/Sv oocytes remains arrested at the metaphase of the first meiotic division and is unable to undergo sperm-induced activation when fertilized 15 hours after the resumption of meiosis. We also show that LT/Sv oocytes experimentally induced to resume meiosis and to reach metaphase II are unable to undergo activation in response to sperm penetration. However, the ability for sperm-induced activation developed during prolonged in vitro culture. Both types of LT/Sv oocytes, i.e. metaphase I and those that were experimentally induced to reach metaphase II, underwent activation when they were fertilized 21 hours after germinal vesicle breakdown (GVBD). Thus, the ability of LT/Sv oocytes to become activated by sperm depends on cytoplasmic maturation rather than on nuclear maturation i.e. on the progression of meiotic division. We also show that sperm penetration induces fewer Ca(2+) transients in LT/Sv oocytes than in control wild-type oocytes. In addition, we found that the levels of mRNA encoding different isoforms of protein kinase C (alpha, delta and zeta), that are involved in meiotic maturation and signal transduction during fertilization, differed between metaphase I LT/Sv oocytes which cannot be activated by sperm, and those which are able to undergo activation after fertilization. However, no significant differences between these oocytes were found at the level of mRNA encoding IP(3) receptors which participate in calcium release during oocyte fertilization.     

Intracellular Ca2+ increase induces post-fertilization events via MAP kinase dephosphorylation in eggs of the hydrozoan jellyfish Cladonema pacificum

Naturally spawned eggs of the hydrozoan jellyfish Cladonema pacificum are arrested at G1-like pronuclear stage until fertilization. Fertilized eggs of Cladonema undergo a series of post-fertilization events, including loss of sperm-attracting ability, expression of adhesive materials on the egg surface, and initiation of cell cycle leading to DNA synthesis and cleavage. Here, we investigate whether these events are regulated by changes in intracellular Ca2+ concentration and mitogen-activated protein kinase (MAP kinase) activity in Cladonema eggs. We found that MAP kinase is maintained in the phosphorylated form in unfertilized eggs. Initiation of sperm-induced Ca2+ increase, which is the first sign of fertilization, was immediately followed by MAP kinase dephosphorylation within a few minutes of fertilization. The fertilized eggs typically stopped sperm attraction by an additional 5 min and became sticky around this time. They further underwent cytokinesis yielding 2-cell embryos at approximately 1 h post-fertilization, which was preceded by DNA synthesis evidenced by BrdU incorporation into the nuclei. Injection of inositol 1,4,5-trisphosphate (IP3) into unfertilized eggs, which produced a Ca2+ increase similar to that seen at fertilization, triggered MAP kinase dephosphorylation and the above post-fertilization events without insemination. Conversely, injection of BAPTA/Ca2+ into fertilized eggs at approximately 10 s after the initiation of Ca2+ increase immediately lowered the elevating Ca2+ level and inhibited the subsequent post-fertilization events. Treatment with U0126, an inhibitor of MAP kinase kinase (MEK), triggered the post-fertilization events in unfertilized eggs, where MAP kinase dephosphorylation but not Ca2+ increase was generated. Conversely, preinjection of the glutathione S-transferase (GST) fusion protein of MAP kinase kinase kinase (Mos), which maintained the phosphorylated state of MAP kinase, blocked the post-fertilization events in fertilized eggs without preventing a Ca2+ increase. These results strongly suggest that all of the three post-fertilization events, cessation of sperm attraction, expression of surface adhesion, and progression of cell cycle, lie downstream of MAP kinase dephosphorylation that is triggered by a Ca2+ increase.     

Premature sperm incorporation into the primary oocyte of the polychaete Pectinaria: Male pronuclear formation and oocyte maturation

Immature oocytes of the annelid Pectinaria were prematurely fertilized while in the germinal vesicle stage. Fertilization was morphologically normal except for the formation of an enlarged fertilization cone which persisted even after sperm incorporation. However, at 30 min postinsemination, no signs of male pronuclear morphogenesis were detected. Ultrastructural data show that in the cytoplasm of a GV-stage oocyte the sperm nuclear envelope remains intact and the enclosed chromatin remains condensed. Prematurely fertilized eggs were then induced to undergo germinal vesicle breakdown (GVBD). Subsequently male pronuclear development occurred. Thus, the factors in the Pectinaria oocyte which are necessary for sperm transformation develop in the maturing cytoplasm and are dependent upon GVBD. Such prematurely fertilized oocytes fail to display the normal arrest of meiosis at Metaphase I, but instead progress directly to formation of the female pronucleus. Occurrences of normal first cleavage were observed suggesting that prematurely incorporated sperm can be recruited for participation in development.     

Lectin binding sites in the vitelline envelope of Bufo arenarum oocytes: Role in fertilization

Incubation of dejellied spawned oocytes from Bufo arenarum with different lectins results in a decrease of oocyte fertility. Concanavalin A was the most effective lectin; phytohem-agglutinin P and wheat germ lectin were less effective. Agglutinin from soybean was scarcely active. These lectin effects could be ascribed to a hindering of specific sites for some proteases, since the same treatment renders the oocyte vitelline envelope insensitive to spermatolysin (an essential requisite for fertilization) and to trypsin. Also in this case concanavalin A was the most effective lectin. Univalent concanavalin A was also effective in blocking the fertility of dejellied oocytes. These results indicate that the residues of α-D-glucose and α-D-mannose present in the vitelline envelope are involved in gamete interactions in Bufo arenarum. This idea is also supported by the finding that dejellied oocytes (fertilizables) have a number of binding sites for concanavalin A that is three or four orders of magnitude higher than coelomic or fertilized oocytes (both not penetrable by spermatozoa).     

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.     

Accumulation of fluorescently labeled actin in the cortical layer in sea urchin eggs after fertilization

Actin from sea urchin eggs was fluorescently labeled with fluorescein isothiocyanate (FITC), N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM), or 5-iodoacetamidofluorescein (IAF) and microinjected into sea urchin eggs and oocytes. It distributed evenly in the cytoplasm of unfertilized eggs. Upon fertilization, actin accumulated first around the sperm binding site and, soon afterwards, in the fertilization cone. The accumulation propagated all over the cortex after a latent period of 10-20 sec. In the case of Clypeaster japonicus eggs, propagation of the accumulation coincided with a shape change in the egg, suggesting that the accumulated actin in the cortex generates forces. FITC-actin was incorporated into microvilli and retained in the cortex after cleavage. On the other hand, DACM- or IAF-actin was not incorporated into microvilli and was dispersed from the cortex by cleavage. These differences may be attributable to differences in the properties of the actins labeled at different sites. After photobleaching by laser light irradiation, FITC- or IAF-actin redistributed in the cortex of fertilized egg as quickly as it did before fertilization. When an unfertilized egg was injected with both actin and a calcium buffer (intracellular free Ca2+ concentration 9 microM), the actin accumulation was similar to that during fertilization but without the latent period. This suggests that the accumulation depended on the increase in the intracellular free Ca2+ concentration. When the unfertilized egg was injected with 0.2 M EGTA after injection of labeled actin and then inseminated, it accumulated only in the protrusion of cytoplasm where the sperm had entered, and fertilization was not completed. In immature oocytes, the accumulation was observed in the cortical region, including the huge protrusion of the cytoplasm where the sperm had entered. These results suggest that actin accumulation in the sperm binding site plays an important role in the sperm reception mechanism of the egg.