Confocal laser scanning microscopy of calcium dynamics in living cells.

Confocal laser scanning microscopy (CLSM) is widely used to monitor intracellular calcium levels in living cells loaded with calcium-sensitive fluorophores. This review examines the basic advantages and limitations of CLSM in in vivo imaging analyses of calcium dynamics. The benefits of utilizing ratioed images and dextran-conjugated fluorophores are addressed, and practical aspects of handling confocal datasets are outlined. After considering some relatively new microscopical methods that can be used in conjunction with conventional CLSM, possible future applications of confocal techniques in analyses of intracellular calcium dynamics are discussed.

The ontogeny of shell secretion in Terebratalia transversa (Brachiopoda, Articulata). I. Development of the mantle.

The morphology of the mantle in free-swimming and metamorphosing larvae of the articulate brachiopod Terebratalia transversa has been examined by scanning and transmission electron microscopy. The mantle begins to form approximately 2 days after fertilization and subsequently develops into a skirtlike lobe that encircles the middle region of the larval body. A simple epithelium covers both the outer surface of the mantle lobe and the inner side situated next to the pedicle lobe of the larva. During metamorphosis, the mantle lobe is everted over the anterior end of the larva. Thus, the epithelium covering the outer part of the mantle lobe in the larva subsequently becomes the inner epithelium of the juvenile mantle. Similarly, the inner epithelium of the larval mantle lobe represents the future outer epithelium of the juvenile mantle. In free-swimming larvae, the prospective outer mantle epithelium contains two types of cells, called "lobate" and "vesicular" cells. Lobate cells initially deposit a thin layer of amorphous material, and vesicular cells produce ovoid multigranular bodies. Following settlement at about 5 days postfertilization, the vesicular cells secrete an electron-dense sheet that constitutes the basal layer of the developing periostracum. Within several hours to a day thereafter, reversal of the mantle lobe is rapidly effected, apparently by contractions of the pedicle adjustor muscles.

The ontogeny of shell secretion in Terebratalia transversa (Brachiopoda, Articulata). II. Formation of the protegulum and juvenile shell.

The fine structure of the shell and underlying mantle in young juveniles of the articulate brachiopod Terebratalia transversa has been examined by electron microscopy. The first shell produced by the mantle consists of a nonhinged protegulum that lacks concentric growth lines. The protegulum is secreted within a day after larval metamorphosis and typically measures 140-150 micron long. A thin organic periostracum constitutes the outer layer of the protegulum, and finely granular shell material occurs beneath the periostracum. Protegula resist digestion in sodium hypochlorite and are refractory to sectioning, suggesting that the subperiostracal portion of the primordial shell is mineralized. The juvenile shell at 4 days postmetamorphosis possesses incomplete sockets and rudimentary teeth that consist of nonfibrous material. The secondary layer occuring in the inner part of the juvenile shell contains imbricated fibers, whereas the outer portion of the shell comprises a bipartite periostracum and an underlying primary layer of nonfibrous shell. Deposition of the periostracum takes place within a slot that is situated between the so-called lobate and vesicular cells of the outer mantle lobe. Vesicular cells deposit the basal layer of the periostracum, while lobate cells contribute materials to the overlying periostracal superstructure. Cells with numerous tonofibrils and hemidesmosomes differentiate in the outer mantle epithelium at sites of muscle attachments, and unbranched punctae that surround mantle caeca develop throughout the subperiostracal portion of the shell. Three weeks after metamorphosis, the juvenile shell averages about 320 micron in length and is similar in ultrastructure to the shells secreted by adult articulates.

A time-lapse study of interactions between Echinostoma paraensei intramolluscan larval stages and adherent hemocytes from Biomphalaria glabrata and Helix aspersa.

In vitro interactions between intramolluscan stages (sporocyst, daughter rediae, and metacercariae) of the trematode parasite Echinostoma paraensei and adherent hemocytes from the gastropods Biomphalaria glabrata (intermediate host) and Helix aspersa (non-host) were visualized by time-lapse videomicroscopy. Hemocytes of either species not exposed to E. paraensei displayed extensive mobility and activity of cellular extensions. Image analysis disclosed no significant change in the total surface area occupied by hemocytes in a selected field of view over 2 hr. Echinostoma paraensei exerted life stage-specific effects on the behavior of B. glabrata hemocytes; the cells moved away from sporocysts and daughter rediae but not encysted metacercariae. In the presence of sporocysts, hemocytes rounded up, whereas hemocytes adjacent to rediae assumed a stringy, beady appearance. Hemocytes close to the parasite were affected more rapidly than more distant cells. In 2 hr, a hemocyte-free "halo" formed around the parasite larvae, significantly reducing the hemocyte-occupied surface area (to 43% by sporocysts and to 70% by rediae). The changes induced by sporocysts and rediae are similar to those noted in both in vivo and in vitro studies of the B. glabrata-E. paraensei model system and are interpreted as manifestations of parasite-mediated interference with host hemocyte function. Helix aspersa (non-host) hemocytes were not affected, suggesting that E. paraensei-mediated effects on hemocytes exhibit a degree of specificity.

Germinal vesicle breakdown is not fully dependent on MAPK activation in maturing oocytes of marine nemertean worms.

Previously, it has been shown that oocytes of marine nemertean worms resume meiosis and undergo germinal vesicle breakdown (GVBD) following treatment with either natural seawater (NSW), or the neurohormone serotonin (5-hydroxytryptamine or 5-HT). In this investigation of the nemerteans Cerebratulus lacteus and Cerebratulus sp., immunoblots and kinase assays were used to compare the roles of two regulatory kinases: mitogen-activated protein kinase (MAPK) and Cdc2/cyclin B (referred to as maturation promoting factor or MPF). Based on such analyses, an ERK (extracellular signal regulated kinase) type of MAPK was found to be activated concurrently with Cdc2/cyclin B during NSW- and 5-HT-induced maturation. MAPK activation occurred prior to GVBD and seemed to be controlled primarily by phosphorylation rather than de novo protein synthesis. Inhibition of MAPK signaling by U0126 was capable of delaying but not permanently blocking Cdc2/cyclin B activation and GVBD in 5-HT treated oocytes and subsets of NSW-treated oocytes. Collectively such data indicated that GVBD is not fully dependent on MAPK activation, since Cdc2/cyclin B can apparently be activated by MAPK-independent mechanism(s) in maturing nemertean oocytes.

Repetitive calcium waves induced by fertilization in the nemertean worm Cerebratulus lacteus.

To analyze fertilization-induced calcium dynamics in a protostome worm, unfertilized oocytes of the nemertean Cerebratulus lacteus were co-injected with calcium green (CG) and rhodamine (Rh) dextrans for dual-channel confocal imaging of early development. Based on CG/Rh ratioed images collected every 800 msec, fertilization elicits a "cortical flash" of elevated free calcium that spreads rapidly around the oocyte without propagating as a point-source wave. A similar calcium transient occurs in unfertilized oocytes treated with KCl to depolarize the oolemma, and the fertilization-induced cortical flash is eliminated if cobalt is used to block calcium channels, collectively indicating that fertilization initially triggers an influx of calcium ions through voltage-gated calcium channels in the oolemma. However, within minutes after producing a cortical flash, C. lacteus oocytes begin to display a series of point-source, oscillating waves of elevated free calcium that are propagated at about 15 micron/sec. The first two calcium waves arise at the site of sperm fusion and typically fail to reach the antipode, but after sperm incorporation, the waves spread globally throughout the ooplasm and typically shift their origin to a pacemaker region in the vegetal cortex. About 10 oscillations with an average duration of 3.3 +/- 1.2 min are generated for approximately 60-100 min postfertilization as meiotic maturation is completed, and such waves continue to occur in cobalt-containing seawater or calcium-free seawater. Thus, wavelike calcium oscillations induced by fertilization are apparently dependent upon internal calcium stores, which in turn may contain IP3-insensitive and/or IP3-sensitive receptors based on experiments using ryanodine, caged IP3, and heparin. Unfertilized oocytes also display repetitive calcium waves following intracytoplasmic injections of whole sperm, and such oscillations are eliminated if the sperm suspensions are boiled prior to injection, suggesting the possible presence of a heat-labile sperm component that can elicit wavelike oscillations during fertilization.

Intracellular injections of a soluble sperm factor trigger calcium oscillations and meiotic maturation in unfertilized oocytes of a marine worm.

How sperm trigger activating calcium transients in eggs remains a central, unresolved question in fertilization biology. To determine if a soluble sperm factor can generate a fertilization-like calcium response in the absence of sperm-egg binding, aqueous extracts of sperm from the nemertean worm Cerebratulus lacteus were mixed with Ca2+-sensitive fluorescent dyes and injected into unfertilized, metaphase-I-arrested oocytes. Based on confocal imaging analyses, unfertilized oocytes that had been injected with sperm extract routinely produced oscillating Ca2+ waves and resumed meiotic maturation in a manner that closely resembled normal fertilization. Calcium oscillations and maturation were typically lacking in control oocytes that had been (i) injected with buffer alone or with buffer containing added calcium, (ii) given external treatments of the sperm factor, or (iii) injected with extracts made from cells other than sperm. Boiling or protease treatment essentially abolished the potency of the sperm extract, and nonboiled extracts retained full activity in >10-kDa fractions, but not in <10-kDa fractions. Collectively, such findings suggest that the sperm of C. lacteus possess a soluble protein that can bypass oolemmal surface receptors to act within the ooplasm as a trigger of repetitive Ca2+ waves and meiotic maturation. Results obtained in this study are discussed with respect to the minimum amount of extract needed for egg activation and whether the oscillogenic substance is sufficiently concentrated in a single sperm to play a biological role during fertilization.

Time-lapse confocal imaging of calcium dynamics in starfish embryos.

During fertilization and cleavage, embryos undergo transient rises in their intracellular free calcium levels that are postulated to provide essential signals enabling normal development to proceed. In order to analyze the spatiotemporal patterns and possible biological significance of these calcium transients, time-lapse confocal microscopy was used to monitor starfish embryos during normal development and following experimental manipulations that disrupted cleavage and/or the release of calcium ions from internal stores in the embryo. For such analyses, oocytes were co-injected with dextran-conjugated forms of the calcium-sensitive fluorochrome calcium green (CG) and the calcium-insensitive dye rhodamine (Rh) for dual-channel confocal ratioing. Based on CG/Rh ratioed images obtained every 15 sec for the first few hours of development, no prominent calcium spikes were typically evident at the onset of the first cell cycle as hormone-treated oocytes resumed maturation and underwent germinal vesicle breakdown (GVBD). Subsequently, fertilizations of post-GVBD oocytes caused a single prolonged calcium wave that reached relatively uniform amplitudes throughout the ooplasm. Within 90 min after fertilization, most starfish zygotes began to display clusters of repetitive calcium oscillations that typically-but not invariably-preceded nuclear envelope breakdown, anaphase onset, and the formation of the first cleavage furrow. Rapidly decaying calcium oscillations continued through at least the first five cleavages in specimens that developed into normal blastulae, and unlike fertilization-induced calcium waves, such spikes tended to be more pronounced in the cortical cytoplasm during early cleavages. Thus, three different types of calcium dynamics--no marked transients, a single nonoscillating wave, and repetitive oscillations--were observed as normally developing starfish underwent prefertilization maturation, fertilization, and cleavage, respectively, suggesting that the spatiotemporal patterns of calcium spiking can change during starfish early development. In specimens microinjected with colchicine, calcium transients were also visible in the absence of cell divisions, indicating that calcium spiking can be uncoupled from cytokinesis. To assess whether calcium fluxes are required for normal development, oocytes were also treated with heparin to block calcium release mediated by inositol 1,4,5-trisphosphate (IP3). Injections of heparin, but not the control molecule de-N-sulfated heparin, caused abnormal fertilization-induced calcium dynamics in a dose-dependent fashion and typically abolished marked postfertilization calcium oscillations and normal cleavage. Based on correlative studies using caged IP3, heparin interfered with IP3-mediated calcium release, suggesting that such release is involved in the production of the free calcium elevations that occur in normally developing starfish embryos.

Comparative biology of calcium signaling during fertilization and egg activation in animals.

During animal fertilizations, each oocyte or egg must produce a proper intracellular calcium signal for development to proceed normally. As a supplement to recent synopses of fertilization-induced calcium responses in mammals, this paper reviews the spatiotemporal properties of calcium signaling during fertilization and egg activation in marine invertebrates and compares these patterns with what has been reported for other animals. Based on the current database, fertilization causes most oocytes or eggs to generate multiple wavelike calcium oscillations that arise at least in part from the release of internal calcium stores sensitive to inositol 1,4,5-trisphosphate (IP3). Such calcium waves are modulated by upstream pathways involving oolemmal receptors and/or soluble sperm factors and in turn regulate calcium-sensitive targets required for subsequent development. Both "protostome" animals (e.g., mollusks, annelids, and arthropods) and "deuterostomes" (e.g., echinoderms and chordates) display fertilization-induced calcium waves, IP3-mediated calcium signaling, and the ability to use a combination of external calcium influx and internal calcium release. Such findings fail to support the dichotomy in calcium signaling modes that had previously been proposed for protostomes vs deuterostomes and instead suggest that various features of fertilization-induced calcium signals are widely shared throughout the animal kingdom.

Nuclear envelope disassembly and nuclear lamina depolymerization during germinal vesicle breakdown in starfish.

During germinal vesicle breakdown (GVBD) in starfish, the nuclear envelope disassembles before the nuclear lamina completely depolymerizes, judging from correlative ultrastructural, immunolabeling, and light microscopic analyses. At 13 degrees C, prophase-arrested oocytes of Pisaster ochraceus begin GVBD and rapidly undergo nuclear envelope disassembly about 50 min after addition of the maturation-inducing hormone 1-methyladenine (1-MA). The nuclear lamina of these oocytes, however, remains present for 10-20 min following the vesiculation of the nuclear envelope. Completion of GVBD, as evidenced by a blending of the nuclear contents with the surrounding cytoplasm, occurs within about 15 min after the nuclear lamina has fully depolymerized. Immunofluorescence studies also indicate that a marked increase in the phosphorylations of nuclear proteins precedes the structural reorganizations of the nuclear envelope and nuclear lamina during GVBD.

Multiple triggers of oocyte maturation in nemertean worms: the roles of calcium and serotonin.

To analyze the process of oocyte maturation in nemertean worms, oocytes with a large nucleus (=germinal vesicle, or GV) were removed from gravid ovaries of Cerebratulus lacteus and Micrura alaskensis. Following transfer to natural seawater (NSW), fully grown oocytes spontaneously matured as indicated by their completion of germinal vesicle breakdown (GVBD), whereas GVBD was reversibly blocked if the oocytes were initially placed in calcium-free seawater (CaFSW). Similarly, calcium ionophore treatments triggered GVBD in calcium-containing artificial seawater (ASW) but not in CaFSW, suggesting that external calcium influx may facilitate maturation. However, compared to the overall levels of maturation elicited by ASW, significantly higher percentages of GVBD were achieved with NSW or with ASW that had been conditioned with marine sediment. Moreover, calcium channel blockers decreased GVBD rates in ASW but not in NSW, which is consistent with the view that substances other than external calcium ions can trigger maturation. Accordingly, oocytes underwent equally high levels of GVBD when treated with serotonin (=5-hydroxytryptamine, or 5-HT) in ASW or CaFSW. The 5-HT-induced maturation was blocked by inhibitors of 5-HT receptors but continued to occur in the presence of calcium channel blockers or the calcium chelator BAPTA. In addition, oocytes microinjected with fluorescent calcium indicators underwent GVBD in response to 5-HT without displaying marked calcium transients during confocal imaging runs. Collectively, such findings suggest that nemertean oocytes can mature via multiple pathways that may include external calcium influx or a 5-HT-induced signaling cascade that lacks prominent calcium fluctuations. J. Exp. Zool. 287:243-261, 2000.

Morphological changes during maturation of starfish oocytes: surface ultrastructure and cortical actin.

The cell surface and extracellular investments of oocytes of the starfish Pisaster ochraceus are analyzed by Nomarski differential interference contrast microscopy and by scanning electron microscopy. The investing coats include a thin sheet of follicle cells, a jelly coat, and a vitelline layer; their morphologies are described. Methods are outlined for systematically removing them without altering the behavior of the oocyte so that the cell surface can be examined directly. The topography of denuded oocytes changes dramatically when they are treated with the maturation-inducing hormone, 1-methyladenine. The major topographical change is the early and transient formation of prominent surface spikes. These structures arise due to the rapid, reversible polymerization of actin into stout bundles. Polymerization and subsequent depolymerization of cortical actin is monitored by epifluorescence microscopy of oocytes stained with NBD-phallacidin, a stain which is specific for polymerized actin. Based on scanning electron microscopy, spikes apparently utilize preexisting plasma membrane of microvilli, and plasma membrane is apparently lost when spikes collapse. Long after microvilli are eliminated due to spike formation, the number of microvilli is somewhat restored, especially around the animal pole where the polar body forms. A chronology of events observed during oocyte maturation is discussed with reference to the possible mechanisms and implications of polymerization and depolymerization of cortical actin.

Oocyte Maturation in the Brachiopod Terebratalia transversa: Role of Follicle Cell-Oocyte Attachments During Ovulation and Germinal Vesicle Breakdown.

In the brachiopod Terebratalia transversa, each ovarian follicle consists of (i) a prophase-arrested oocyte with an enlarged nucleus (= the germinal vesicle, or GV), and (ii) a surrounding sheath of follicle cells that attach to the oocyte by means of junctional complexes. Within about 80 min after removing a follicle from the ovary, the follicular sheath undergoes a microfilament-mediated retraction, and the ovulated oocyte that emerges from the retracted sheath subsequently completes germinal vesicle breakdown (GVBD). Based on the experimental manipulations reported here, it appears that the follicle must be detached from the ovary for such ovulation and GVBD to occur. Moreover, GVBD can be prevented if the oocytes are mechanically stripped of their follicle cells up to 30 min after being isolated from the ovary. GVBD proceeds normally, however, if follicle cells are removed more than 40 min after the follicle is obtained from the ovary. The percentage of oocytes that undergo GVBD is also diminished following treatment with drugs that uncouple gap junctions. Collectively, these data suggest that removing a follicle from the ovary stimulates follicle cells to produce a maturation-inducing factor that uses the follicle cell-oocyte junctional complexes to reach the oocyte within about 30-40 min after follicular removal. The significance of these findings is discussed relative to previous reports on oocyte maturation in brachiopods and other animals.

5-HT causes an increase in cAMP that stimulates, rather than inhibits, oocyte maturation in marine nemertean worms.

In the nemertean worms Cerebratulus lacteus and Micrura alaskensis, 5-HT (=5-hydroxytryptamine, or serotonin) causes prophase-arrested oocytes to mature and complete germinal vesicle breakdown (GVBD). To identify the intracellular pathway that mediates 5-HT stimulation, follicle-free oocytes of nemerteans were assessed for GVBD rates in the presence or absence of 5-HT after being treated with various modulators of cAMP, a well known transducer of 5-HT signaling and an important regulator of hormone-induced maturation in general. Unlike in many animals where high levels of intra-oocytic cAMP block maturation, treatment of follicle-free nemertean oocytes with agents that elevate cAMP (8-bromo-cAMP, forskolin or inhibitors of phosphodiesterases) triggered GVBD in the absence of added 5-HT. Similarly, 5-HT caused a substantial cAMP increase prior to GVBD in nemertean oocytes that had been pre-injected with a cAMP fluorosensor. Such a rise in cAMP seemed to involve G-protein-mediated signaling and protein kinase A (PKA) stimulation, based on the inhibition of 5-HT-induced GVBD by specific antagonists of these transduction steps. Although the downstream targets of activated PKA remain unknown, neither the synthesis of new proteins nor the activation of MAPKs (mitogen-activated protein kinases) appeared to be required for GVBD after 5-HT stimulation. Alternatively, pre-incubation in roscovitine, an inhibitor of maturation-promoting factor (MPF), prevented GVBD, indicating that maturing oocytes eventually need to elevate their MPF levels, as has been documented for other animals. Collectively, this study demonstrates for the first time that 5-HT can cause immature oocytes to undergo an increase in cAMP that stimulates, rather than inhibits, meiotic maturation. The possible relationship between such a form of oocyte maturation and that observed in other animals is discussed.

Calcium dynamics during starfish oocyte maturation and fertilization.

Intracellular free calcium levels in starfish oocytes have been monitored during meiotic maturation and fertilization using calcium-sensitive fluorescent dyes combined with confocal laser scanning microscopy or fura ratioing techniques. In time-lapse analyses of prophase-arrested and maturing oocytes, calcium transients were elicited by inositol 1,4,5-trisphosphate (IP3), ryanodine, or caffeine, indicating that both the IP3-sensitive and IP3-insensitive receptors of the oocyte's calcium release channels could be stimulated to mobilize calcium ions. Fertilization also triggered a global calcium wave that appeared to travel faster around the cortex than through the center of the oocyte, and maturing oocytes developed normally after their fertilization-induced calcium waves had been imaged. Prophase-arrested specimens, on the other hand, did not undergo germinal vesicle breakdown or cleavage after displaying a fertilization-induced calcium transient throughout their cytoplasm and nucleus, confirming previous observations that calcium spikes are not sufficient to induce development in immature oocytes. In addition, although the calcium spikes triggered by sperm or caffeine reached similar normalized peak heights, fertilization-induced calcium waves in maturing oocytes tended to be more prolonged than the fertilization waves observed in prophase-arrested oocytes or the caffeine-triggered spikes elicited at any stage of maturation. Collectively, such findings suggest that the total amount of releasable calcium does not vary appreciably during maturation, but the patterns of the calcium transients can differ depending on the stage of maturation and/or the type of calcium-releasing agent. Possible artifacts affecting these findings are assessed, and the results are discussed relative to the functioning of calcium release pathways during starfish oocyte maturation and fertilization.

Calcium and endoplasmic reticulum dynamics during oocyte maturation and fertilization in the marine worm Cerebratulus lacteus.

To monitor calcium and endoplasmic reticulum (ER) dynamics during oocyte maturation and fertilization, oocytes of the marine worm Cerebratulus lacteus were injected with the calcium-sensitive indicator calcium green dextran and/or the ER-specific probe "DiI." Based on time-lapse confocal imaging of such specimens, prophase-arrested immature oocytes failed to develop normally after insemination and typically produced non-wave-like calcium transients that were lower in amplitude and less persistent than the wave-like oscillations observed during fertilizations of mature oocytes. Accordingly, the ER of DiI-loaded immature oocytes lacked an obvious substructure, whereas ER clusters, or "microdomains," began to form in maturing specimens at about the time that these oocytes became competent to undergo normal fertilization-induced calcium dynamics and cleavage. The ER microdomains of mature oocytes typically reached widths of 1-8 micrometer and disappeared approximately 1 h after fertilization, which in turn coincided with the termination of the calcium oscillations. Collectively, these findings indicate: (i) changes in ER structure are temporally correlated with the onset and cessation of the calcium oscillations required for subsequent cleavage, and (ii) such ER reorganizations may play an important role in early development by enabling mature oocytes to generate a normal calcium response.

Injections of porcine sperm extracts trigger fertilization-like calcium oscillations in oocytes of a marine worm.

The precise mechanisms by which sperm trigger calcium transients in eggs or oocytes during fertilization remain unknown. Based on time-lapse confocal microscopy, we show that intracellular injections of porcine sperm extracts cause the oocytes of a marine nemertean worm to undergo repetitive calcium oscillations resembling those obtained during normal fertilizations. Such findings are consistent with the view that fertilization involves a soluble sperm factor (SF) which is capable of eliciting calcium transients without binding to externally situated receptors on the oocyte plasmalemma. This study also describes for the first time the wave-like propagation patterns of SF-induced calcium transients that are generated in a heterologous combination of gametes obtained from different phyla of animals. Such cross-reactivity between distantly related taxa suggests that the intracellular signaling pathways triggered by sperm factors can be well conserved.

Confocal microscopy of fertilization-induced calcium dynamics in sea urchin eggs.

Although confocal microscopy has typically been utilized in studies of fixed specimens, its potential for exploring dynamic processes in living cells is rapidly being realized. In this report, confocal laser scanning microscopy is used to analyze the calcium wave that occurs following fertilization in living sea urchin eggs microinjected with the calcium-sensitive fluorescent probes fluo-3 or calcium green. Time-lapse recordings of optical sections depicting calcium dynamics within the eggs are also subjected to volumetric reconstructions. Such analyses indicate that (1) cytoplasmic free calcium levels become elevated throughout the fertilized egg, (2) fertilization also causes the egg nucleus to undergo a transient increase in free calcium, and (3) normal cleavage can be obtained following time-lapse imaging of the calcium waves.

NO is necessary and sufficient for egg activation at fertilization.

The early steps that lead to the rise in calcium and egg activation at fertilization are unknown but of great interest--particularly with the advent of in vitro fertilization techniques for treating male infertility and whole-animal cloning by nuclear transfer. This calcium rise is required for egg activation and the subsequent events of development in eggs of all species. Injection of intact sperm or sperm extracts can activate eggs, suggesting that sperm-derived factors may be involved. Here we show that nitric oxide synthase is present at high concentration and active in sperm after activation by the acrosome reaction. An increase in nitrosation within eggs is evident seconds after insemination and precedes the calcium pulse of fertilization. Microinjection of nitric oxide donors or recombinant nitric oxide synthase recapitulates events of egg activation, whereas prior injection of oxyhaemoglobin, a physiological nitric oxide scavenger, prevents egg activation after fertilization. We conclude that nitric oxide synthase and nitric-oxide-related bioactivity satisfy the primary criteria of an egg activator: they are present in an appropriate place, active at an appropriate time, and are necessary and sufficient for successful fertilization.