syngamy and cell cycle control - wiley-vchwiley-vch.de/books/sample/352730651x_c01.pdf · 2005. 10....

1

Syngamy and Cell Cycle Control

Michael WhitakerUniversity of Newcastle upon Tyne, Newcastle upon Tyne, UK

1 Formation of Gametes 61.1 Meiosis and Genetic Recombination 61.2 Male Gametes 71.3 Female Gametes 8

2 Interaction of Sperm and Egg 92.1 Sperm Activation and Chemotaxis 92.2 The Acrosome Reaction 102.3 Sperm–Egg Interaction and Speciation 112.4 Sperm–Egg Fusion and Sperm Incorporation 112.5 Syngamy 13

3 Fertilization Calcium Signals 143.1 Calcium Ions are a Universal Egg Activator at Fertilization 143.2 Calcium Signaling 143.3 Fertilization Calcium Waves 153.4 Repetitive Fertilization Calcium Transients 163.5 Signal Transduction during the Fertilization Calcium Wave 173.5.1 Reaction Diffusion Waves 173.5.2 Initiation of the Calcium Wave by the Sperm 18

4 Fertilization and the Cell Division Cycle 194.1 Fertilization Occurs at Different Stages of the Cell Division Cycle

in Different Species 194.2 Maturation Promoting Factor Plays a Part in Meiosis

and in Meiotic Arrest 194.3 Calcium Signals and Cell Cycle Progression at Fertilization 204.3.1 Metaphase Arrest 204.3.2 Interphase Arrest 214.4 The Cell Cycle and Syngamy 22

Encyclopedia of Molecular Cell Biology and Molecular Medicine, 2nd Edition. Volume 14Edited by Robert A. Meyers.Copyright 2005 Wiley-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 3-527-30651-X

2 Syngamy and Cell Cycle Control

5 Calcium and Cell Cycle Control 235.1 Cell Cycle Checkpoints 235.2 The DNA Damage Checkpoint 245.3 The Mitosis Exit Checkpoint 245.4 The Checkpoint at Cell Cycle Onset 255.5 Calcium and Cell Cycle Checkpoints 25

Bibliography 25Books and reviews 25Primary Literature 26

Keywords

ActinA small protein monomer that concatenates to produce thin filaments when itpolymerizes.

AllostericModifications to protein structure occur when the binding of a small molecule oranother protein to one site in a protein leads to a conformational change in anotherpart of the protein distant from the site of interaction.

ApoptosisA process of programmed cell death in which cellular components are dismantled inorderly fashion without leakage of cell contents.

CentromeresStretches of DNA sequence generally toward the middle of a chromosome that attractaccessory proteins that allow mechanical attachment of microtubules in the mitoticspindle and permit the movement of chromosomes into the daughter cells.

ChaperonesProteins that bind to other proteins to enable them to fold correctly or to remainsoluble.

ChromatinDNA and its accessory proteins, found condensed in chromosomes during mitosis anduncondensed in the interphase nucleus, where it is surrounded by a double nuclearmembrane.

Syngamy and Cell Cycle Control 3

Cyclic AMPA messenger molecule within cells that activates a protein kinase (protein kinase A); itis made in a single step from ATP by adenylyl cyclase under the control oftransmembrane receptors of hormones or growth factors.

CytokinesisThe process by which the daughter cells separate following mitosis; it involves theactin-based contraction of a purse stringlike contractile ring that generates the cleavagefurrow between the daughter cells.

DepolarizationThe usually transient loss of the electrical potential that cells maintain across theirplasma membranes by setting up asymmetric ionic concentration gradients; it acts as asignal, above all in nerve and muscle.

DisjunctionThe separation of the chromosomes at the start of anaphase in mitosis and meiosis;disjunction allows the chromosomes to be pulled into the daughter cells.

EchinodermsA phylum that sits at the base of the vertebrate lineage to which sea urchins and seastars belong.

EnzymesProteins that act as catalysts in chemical reactions.

GlycoproteinProtein that has been modified after translation by the addition of sugars, oftenbranching chains of mannose and glucosamine derivatives.

HistonesVery basic (positively charged) proteins that bind to and order negatively charged DNA.

HomologsThe name given to the complementary chromosome pairs that result fromrecombination.

HydrophobicMolecules prefer nonaqueous environments.

ImprintingAn epigenetic modification of key genes that occurs in placental mammals; it regulatesembryonic growth.


InterphaseComprises all stages of the cell division cycle except mitosis and cytokinesis.

IntronsStretches of DNA within a gene that do not encode amino acids and are cut out beforeprotein sequence is decoded from messenger RNA.

KinasesEnzymes that attach phosphate groups to other molecules. Protein kinases turn otherproteins on and off in this way.

Lipid bilayersSeparate the cell from the extracellular milieu and also separate the cell’s internalcompartments; they consist of cylindrical lipid molecules with a long hydrophobic tailand a water-loving head group that assemble into planar sheets, lipid tails inwards.

MicrotubulesLong cylindrical tubes that assemble from tubulin protein dimers; the dimers pack in ahelical array.

MitosisThe process that precisely segregates the genes between daughter cells after geneduplication; DNA becomes very tightly coiled and packed into chromosomes that areassembled into a disc and then pulled apart into the daughter cells by microtubules.

NADPHNicotine-adenine dinucleotide phosphate in its reduced form; it is used by cells as acofactor to build molecules as this requires a reducing agent.

PhospholipasesEnzymes that cleave phospholipids and remove some or all of the headgroup.

PolyaminesLarge linear molecules with many positively charged amine groups.

PolymorphismThe term given to small sequence differences between the same gene in differentindividuals within a species; many polymorphisms show no obvious phenotype, whileat the other end of the spectrum some polymorphisms give rise to debilitating diseasessuch as cystic fibrosis and muscular dystrophy; the sum of all polymorphisms accountfor the genetic differences between individuals.


ProteoglycansVery large proteins found in the extracellular matrix that have an amino acid backboneand very long carbohydrate side chains.

ProteolysisThe process of protein destruction; all proteins eventually undergo proteolysis, but insome cases, proteins are rapidly destroyed as part of a regulatory mechanism rapidlyinactivate their activity; proteins can be cleaved into large fragments by proteases andinto very short peptides by a cytoplasmic machine known as the proteasome.

S-phaseThe phase in the cell division cycle during which DNA synthesis occurs and thegenome is duplicated.

SpindleThe mitotic spindle is the array of microtubules that attaches to the chromosomesduring mitosis and drags the chromosomes into their respective daughter cells; in itsshape, this structure resembles spun thread wrapped around its spindle.

SympatricSpecies are found in the same or overlapping geographical areas.

VesiclesSpherical compartments bounded by a lipid bilayer membrane; their contents are oftenreleased into the extracellular milieu; this is achieved by fusion of the limiting bilayermembrane with the plasma membrane.

� The key aspect of sexual reproduction is the generation of genetic variationthrough the swapping of variable lengths of chromosome arms between pairs ofhomologous chromosomes during meiotic recombination in the germline. Followingrecombination, it is the general rule that a single (haploid) set of chromosomes ispackaged into each male and female gamete (sperm and egg). These two sets ofchromosomes, one from each parent, then come together to form a single nucleus:the process of syngamy. This simple outcome is achieved in a complex way throughthe process of fertilization. Fertilization comprises the delivery of the male set ofchromosomes, tightly packaged in a small and motile sperm, to the female set ofchromosomes, contained within a much larger nutrient laden and relatively sessileegg or oocyte; at fertilization, fusion of the two gametes occurs as a prelude tosyngamy. In addition, fertilization triggers the onset of the very rapid rounds ofcell division that quickly produce a very large number of embryonic cells. The celldivision cycle must be coordinated with the events of syngamy. Large intracellularcalcium signals occur at fertilization and ensure that the events of the cell cycle arecontrolled to permit successful melding of the parents’ genomes.


1Formation of Gametes

1.1Meiosis and Genetic Recombination

Each round of sexual reproduction shuf-fles the genetic pack giving rise to a uniquecombination of genetic alleles and thusto a unique individual. The shuffling isbrought about by recombination, a formof genetic cutting and pasting. Human ge-netic information is packaged into 22 pairsof fully recombining chromosomes (theautosomes); the X and Y sex chromosomesrecombine only in a limited (pseudoauto-somal) region and are ignored in this ac-count. Each of the 22 autosomes differs inthe genes it carries and in its shape becausechromosomes have two arms, each ofwhich varies in length between autosomepairs. The autosome pairs look identical toone another, like twins. But unlike identi-cal (monozygotic) twins, though they carrythe same genes, they are not geneticallyidentical: each member of the pair carriesdifferent alleles of the same genes. Recom-bination cuts one or more of the autosomearms in each member of the autosome pairat the same place (in fact almost always atexactly the same nucleotide position in theDNA on the two chromosomes) and thenswaps the fragments, pasting them backon the opposite chromosome (Fig. 1a).

Recombination occurs during meiosis,the specialized form of chromosome sep-aration that is believed to have evolvedfrom the more normal mitosis seen eachtime a somatic cell divides (It has recentlybeen suggested by Krylov, Nasmyth, andKoonin that meiosis may be the phyloge-netically primitive form of chromosomeseparation.). Meiosis is at the center of themechanism of sexual reproduction and oc-curs only once as each gamete is formed.

A germ cell destined to become a sperm oregg contains 22 autosomes from the fatherand 22 from the mother (the 22 auto-some pairs). Just before meiosis, the germcell undergoes a round of DNA synthe-sis. This generates 44 chromosome pairs,consisting of 44 autosomes with the fa-ther’s alleles (the paternal homologs) and44 autosomes with the mother’s alleles (thematernal homologs). Each homolog pair isjoined at the centromere. The outcome ofmeiosis is a haploid gamete with 22 non-identical autosomes, so the 88 autosomesat the outset of meiosis must be packagedinto four cells. This requires two roundsof cell division (Fig. 1b). The first meioticdivision (meiosis I) is very unusual. Asa consequence of recombination, pairs ofrecombined maternal/paternal homologsare formed, attached at the points on thearms at which recombination has occurred(the chiasmata). Chromosomes are pulledapart during meiosis I by microtubule-based forces, just as in mitosis. As thechromosomes line up in the central spin-dle under the tension generated by themicrotubules of the meiotic spindle at-tached to the centromeres, they are seento be arranged as pairs of pairs (tetrads).The first pairing reflects the attachmentsbetween sister chromatids of both ma-ternal and paternal homologs establishedduring DNA synthesis at the centromereand along the chromosomal arms; thesecond pairing is due to the chiasmatathat are a consequence of recombina-tion. It is a unique feature of meiosis Ithat when the spindle microtubules pullthe chromosomes apart during anaphase,the chromosomes separate at the arms(synaptonemes), not at the centromeres.This results in detachment of homologsat the chiasmata (a process known as res-olution) and disjunction of the homologpairs, leaving the sister chomatid pairs


(a) (b) (c) (d)

(e) (f)

Centrosome

Spindle microtubule

Kinetochore

Cohesin complex

Meiosis I

Meiosis II

Fig. 1 Recombination and chromatid separation during meiosis. Before meiosisbegins, the diploid germ cell contains pairs of homologous chromosomes; forsimplicity, only one chromosome pair is illustrated (a). Both chromosomes ineach pair undergo DNA synthesis (b); the newly replicated chromosomes are heldtogether along their length by the protein cohesin. Then a complex series of eventsoccurs during recombination that results in the swapping of chromosome armsbetween the replicated pairs (not shown). The outcome of these recombinationevents is revealed during the first meiotic division (c): chromosomes aligned onthe meiotic spindle can be seen as tetrads, linked by the chiasm that is formed atthe site of recombination. At anaphase cohesin falls off the chromosome arms asthe chromosomes are pulled toward the centrosomes that organize the meioticspindle (d); cohesin persists around the kinetochore, maintaining the associationbetween sister chromatids. The meiotic spindle reforms during secondmeiosis (e). This time cohesin is lost at the kinetochore during anaphase (f). Theresult is four haploid cells, two of which have swapped chromosome arms in thisillustration. With thanks to Neil Hunter for the building blocks of this illustration.

attached at the centrosome. The secondmeiotic division takes place without anyfurther genome duplication through DNAsynthesis. It involves a conventional chro-mosomal separation at the centromeres,the outcome being four haploid cells.

1.2Male Gametes

In males, each of the four haploidoutcomes of meiosis becomes a gamete.

Driven presumably by a strong selectionpressure (an unsuccessful gamete hasa wasted life and an individual whoproduces unsuccessful gametes will haveno offspring), male gametes have evolvedas small, higher energetic cells. As theymature, they lose most of their cytoplasm,develop very highly condensed chromatindue to the replacement of histones withpolyamines and grow a very motile tail(flagellum). They also retain a secretoryvesicle (the acrosome) whose purpose is


MitochondrionNucleus Head

Fertilization envelope

Tail

Acrosomal process

Fig. 2 The spermatozoon as it fertilizes the egg. Light microscope image of a spermas it fertilizes the sea urchin egg. The sperm has fused with the egg through itsacrosomal process and started the calcium wave. The local increase in calcium hascaused the local elevation of the fertilization envelope that will exclude other sperm.The sperm head contains the DNA-containing nucleus and the mitochondrion thatprovides energy to the tail. Once the sperm has fused with the egg, the tail stopsbeating and stands straight as the sperm enters the egg. The sperm is only2–3 microns across; the egg is 100 microns in diameter. The disparity in the sizes ofthe gametes is very obvious in this image.

to secrete digestive enzymes at the timethat the sperm is passing through theextracellular matrix surrounding the eggand a mitochondrion or mitochondria toprovide the ATP required for motility(Fig. 2).

1.3Female Gametes

All embryos undergo a period of au-tonomous development and during thisperiod they require a source of metabo-lites. As we have seen, sperm offer slimpickings; it is the egg that has specializedto contain a nutrient store (yolk) on whichthe embryo will live until it can itself feedor undergo uterine implantation. As a con-sequence, the embryo can grow only a littlebigger than the egg itself (by, for example,

developing fluid-filled internal cavities).The size of the egg thus determines thesize of the autonomous embryo. Chicksundergo autonomous development withinan eggshell to a very large size and thushave a very large yolk store. Frog eggs arearound 1 mm in diameter and give rise toa macroscopic tadpole. Many eggs, includ-ing mammalian eggs are in the 0.1 mmrange, with correspondingly smaller mi-croscopic autonomous embryos, but stillweigh in at ten times the size and onethousand times the volume of most so-matic cells.

Size matters in two ways. First, it setsa constraint when the male and femalegametes meet, as the much smaller spermhas had to evolve alongside mechanismsthat signal its presence to the relativelyenormous egg. We shall return to this.


Fig. 3 The female oocyte during meioticmaturation. The immature oocyte is readilyidentified by its large nucleus (germinal vesicle,GV); by this point, DNA synthesis andrecombination have taken place and the oocytecontains four copies of the genome (4n). The first

GV

PB

2n4nDNAcontent

n

M I M Il PN

meiotic division (meiosis I, MI) results in the extrusion of a polar body (PB); the oocyte now containsa 2n genomic complement. After second meiosis, a second polar body is formed and the oocytereforms a haploid (n) pronucleus (PN).

Second, it has made its mark on the waythe egg undergoes meiosis.

Strictly speaking, it is an oocyte thatundergoes meiosis, the oocyte becomingan egg only once meiosis is complete. Inmost cases, the oocyte is already a verylarge cell by the time meiosis occurs,pumped up with yolk by the surroundingnurse cells in the ovary. In the femalegermline, both meiotic divisions are veryasymmetric, both generating a very smallcell, budded as it were on the surfaceof the large oocyte. After meiosis I,the small cell (polar body) contains 46chromosomes and does not divide further.The polar body extruded during meiosis IIcontains 23 chromosomes and again hasan inconsequential fate (Fig. 3). Thus, ofthe four possible haploid cells that can begenerated by meiosis, only the large oocytesurvives in the female germline.

2Interaction of Sperm and Egg

2.1Sperm Activation and Chemotaxis

Active sperm have a very limited shelflife. Sperm of external spawners shedinto the water column have no readyaccess to respiratory substrates beyondtheir own limited stores and sperm ofthis sort cease to swim within minutes.Mammalian sperm within the uterus are

supplied with external substrates; they alsohave many more mitochondria and cansurvive as motile and fecund sperm forseveral days. Mechanisms therefore existto suppress sperm motility until sperm aredelivered to the vicinity of the egg.

A widespread mechanism for suppres-sion of sperm activation appears to be theacidic environment in testis and seminalfluid, combined with a high partial pres-sure of carbon dioxide. Once the seminalfluid is diluted, pH rapidly increases andactivates motility.

Sperm activation may also be mediatedor enhanced by signaling molecules re-leased by the egg or present in the uterus.In echinoderm sperm, small peptides re-leased from the egg coat interact withtheir receptor, a transmembrane guany-late cyclase located in the sperm tail.Stimulation of the receptor increases bothpH and calcium concentrations withinthe sperm, leading to enhanced motility(Fig. 4). Similarly, progesterone stimulatescalcium signals in mammalian sperm andenhances motility.

These agents may also be inducingchemotaxis, the directed movement ofsperm toward the egg along a chemicalgradient, though this is not yet proven.In other animals, for example, ascidi-ans and siphonophores, sperm chemotaxishas been well demonstrated. Chemotaxisbetween gametes of the fern can bedemonstrated using simple kits designedfor high school students. It has recently


Fig. 4 Calcium signaling and sperm chemotaxis. Calcium ion concentrations can be detectedinside cells by using fluorescent dyes whose intensity depends on calcium concentration;sperm head is marked with an asterisk. Sperm have low resting calcium concentrations (left).When the chemotactic peptide speract is added, calcium concentration increases in the tail(arrow); this alters swimming behavior.

been found that human sperm possessreceptors equivalent to those found inthe olfactory epithelium and that ap-propriate odorants can induce changesin swimming behavior consistent withchemotaxis.

2.2The Acrosome Reaction

The naked surface of the egg or oocyte isalways well protected, sometimes by thickproteoglycan coats and often in additionwith a cloud of accessory cells embeddedin this extracellular matrix. Sperm musttraverse these barriers in order to reach theplasma membrane and enter the egg andto do so they must dissolve the extracellularmatrix. The release of the contents of theacrosomal vesicle is a process akin to the re-lease of neurotransmitters and circulatinghormones: a large increase in intracellu-lar calcium concentration triggers fusionof the acrosomal vesicle with the spermplasma membrane. The acrosomal vesi-cle contains glycanases and proteases in

many cases, capable of hydrolyzing theextracellular matrix.

Fittingly, the release of acrosomal con-tents is triggered by the proximity of theextracellular coats themselves. In echin-oderms, the acrosome reaction can beexperimentally triggered by a componentof egg jelly in the complete absence ofthe egg itself. In mammalian sperm, thetrigger to the acrosome reaction is a gly-coprotein component of the extracellularmatrix immediately surrounding the egg,the zona pellucida.

In abalone, the contents of the acrosomalvesicle consist of a protein, lysin, whichacts not as an enzyme, but stoichiometri-cally as a structurally specific chaotropicagent (chaperone) that dissolves the eggvitelline envelope.

One consequence of the acrosomereaction in many species is the formationof the acrosomal process, a fine protrusionat the tip of the sperm head that isthe locus of interaction with the eggmembrane. This fact is exemplified by thebehavior of Thyone sperm: they undergo


an acrosome reaction at the very peripheryof the egg jelly, sending a 100-µm processthrough the jelly coat to reach the eggplasma membrane. In Thyone, as in otherspecies, the process grows due to actinpolymerization. The growing plus end ofthe actin filaments is at the tip of theacrosomal process while the reservoir ofmonomeric actin lies at the base of thefilament; the rate of growth decreasesexponentially, limited by the increasingdiffusion time for actin monomers alongthe length of the growing process. Actinpolymerization is triggered by the increasein pH that accompanies the calcium risethat triggers the acrosome reaction.

2.3Sperm–Egg Interaction and Speciation

Reproductive isolation is the major mecha-nism driving the formation of new species.This often occurs as a result of geographicisolation of a population, but can also occursympatrically. Closely related sympatricspecies may remain speciated by separa-tion of habitat or by asynchronous spawn-ing, but reproductive isolation is very oftendue to rapid evolution of the molecules thatare the gatekeepers for syngamy.

A striking example is the abalone lysinreleased during the acrosome reaction.There are five species of abalone livingsympatrically on the Pacific coast of NorthAmerica. Their reproductive isolation isdue in large part to the species specificity ofthe lysin reaction: lysins from other relatedabalone species are much less potent at dis-solving the vitelline envelope. Lysins showvery little polymorphism within species.Nonconservative substitutions within theprotein are very high relative both to house-keeping proteins and to lysin introns,implying very strong positive selection.Lysin interacts with a protein designated

VERL in the vitelline envelope. VERL is avery large (>1 m kDa) protein that consistsof well conserved ∼100 amino acid repeats.Analysis indicates that it is not understrong positive selection. The hypothesisput forward is that VERL undergoes a pro-cess of concerted evolution that puts verystrong evolutionary pressure on lysin thatcan lead to speciation. Concerted evolu-tion is a phenomenon found in proteinswith multiple repeats. A mutation in onerepeat can propagate through all repeatsas a result of preferential recombinationevents. The finding that lysin shows a verylow polymorphism within a population in-dicates that positive selection produced anoptimal lysin in response to concerted evo-lution in VERL that then swept through apopulation, creating a new species.

Lysin/VERL is the only known exam-ple of how sperm/egg receptor interactionmay govern speciation. Very little is knownabout other sperm/egg receptors. It hasbeen suggested that an 18-kDa protein thatcoats the acrosomal process in abalone(a close relative of lysin, but with littlesequence similarity) may be the spermreceptor for the egg plasma membrane.Similarly, the very hydrophobic proteinbindin, which coats the echinoderm acro-somal process, may interact with a re-ceptor on the egg plasma membrane. Inmammalian eggs, it has been suggestedthat integrin/disintegrin ligand receptorpairs that are known to be involved incell–matrix interaction and signaling maybe the sperm/egg ligand/receptor at fertil-ization, but the evidence is contradictory.

2.4Sperm–Egg Fusion and SpermIncorporation

Once the sperm and egg plasma mem-branes are in close proximity, they fuse


to establish continuity of egg and spermcytoplasm. Cell–cell fusion in general isnot well understood and gamete fusion isno exception. The protein bindin that coatsthe acrosomal process in echinoderms isvery hydrophobic, as are the cores of lipidbilayers: it is thought that bindin may pro-mote membrane fusion, not least becauseit has been found to promote fusion oflipid vesicles in vitro. The 18-kDa proteinof abalone has been ascribed a similarfunction. In mammalian fertilization, theintegrin/disintegrin interaction may be fu-sogenic, but there is no sound evidencefor this.

It is humbling that we do not yetunderstand the mechanism of sperm–eggfusion: it represents the moment offertilization. A variety of observationsisolate the event of sperm–egg fusion asthe key event at fertilization:

• In echinoderms, sperm–egg fusioncauses depolarization of the egg, the firstresponse observed at fertilization that,

significantly, much reduces the proba-bility of other sperm fusing with the egg(an electrical block to polyspermy).

• In echinoderms, under certain condi-tions, sperm–egg fusion is reversible;when it is reversed, eggs may or maynot undergo activation and the longerthe sperm cytoplasm is in continuitywith the egg, the greater the chances ofegg activation.

• Sperm–egg fusion is the earliest de-tectable event at fertilization, as mea-sured by dye transfer between egg andsperm.

• Mammalian sperm carry an enzyme intheir cytoplasm that can activate the eggand is delivered to the egg when thesperm fuses with it.

Once the sperm fuses with the egg, ithas fulfilled its purpose of finding theegg and fusing with in local competitionwith tens (mammals) or millions (externalspawners) of other sperm. Incorporation

Intensity:128

33_s

2550

60_s 100_s

36_s

Intensity

2560

Fig. 5 Sperm–egg fusion in sea urchin andmouse eggs. The eggs contain a fluorescent dyesensitive to calcium concentration. When thesperm fuses with the egg, dye enters the sperm.

Sperm–egg fusion triggers an increase ofcalcium within the egg. The calcium increaseoccurs many seconds after sperm–egg fusion.Left: sea urchin. Right: mouse.


of the sperm into the egg relies on the eggitself (Fig. 5).

Immediately after sperm–egg fusion,the connection between egg and sperm istenuous. In many species, fusion occurs atthe tip of an acrosomal process little morethan 100 nm in diameter. Even duringmammalian sperm–egg fusion where it isthe side of the sperm head that fuses withthe egg plasma membrane, the area ofcontact is no more than 1 µm2. Movementof the sperm head (and eventually spermtail) into the egg is due to actin-basedtraction. Actin is a force generating proteinfound in all cells whose acme is rapid andcoordinated force generation in skeletalmuscle. Actin filaments surround thesperm head, forming an extrusion fromthe egg surface known as a fertilizationcone.

Actin filaments drag the sperm nucleusinto the egg. Mitochondria also enterthe egg cytoplasm. These degenerate; itis clear by observation and from thegenetics of mitochondrial inheritance thatall mitochondria in offspring are inheritedfrom the egg and so from the mother. Thesperm tail is incorporated. Its componentsdissolve in the egg cytoplasm, save forthe centriole, an organelle that in thesperm organizes the tail microtubules butthat, incorporated into the egg, takes overthe role of organizing the mitotic spindlemicrotubules.

Once inside the egg, the behavior ofthe sperm nucleus depends upon the cellcycle stage of the egg that the sperm hasfertilized.

2.5Syngamy

The blind aim of sexual reproductionis to ensure that one sperm pronucleuscoalesces with one female pronucleus.

Coalescence of more than the comple-mentary pair of pronuclei leads to geneticchaos. The penetration of more than onesperm nucleus into an egg is known aspolyspermy and its consequences are usu-ally fatal to the embryo.

Mechanisms exist to exclude super-numerary sperm. The electrical blockto polyspermy (depolarization), found inechinoderms and amphibians, has alreadybeen mentioned: the fertilizing sperm de-polarizes the egg, making it (for unknownreasons) much harder for subsequently ar-riving sperm to fuse with the egg. Theelectrical block is very fast and can comeinto play within 10 ms of sperm–eggfusion. Other polyspermy blocks are effec-tive, but slower, requiring tens of secondsto take effect. A common mechanism com-prises the secretion of the contents ofsecretory vesicles from the egg after fer-tilization. In many cases, this leads to theelaboration of a shell (vitelline envelope)around the fertilized egg that sperm can-not penetrate. In mammalian eggs, thesecretions lead to inactivation of the glyco-protein that triggers the acrosome reactionin incoming sperm; sperm that have notundergone the acrosome reaction cannotfuse with the egg. In fish eggs, sperm mustenter in single file down a narrow canal inthe egg investments (the micropyle). Oncethe egg is activated by the fertilizing sperm,the secretions plug the canal, obstructingthe progress of further sperm.

Not all strategies to ensure one-to-onefusion of the sperm and egg nucleus relyon sperm exclusion. Bird eggs are usuallypolyspermic. Once a lucky sperm nucleushas fused with the egg nucleus, all theother sperm nuclei present in the eggdegenerate. In ctenophores, more thanluck seems to be in play. The ctenophoreegg is naturally polyspermic. The eggnucleus roams from one sperm nucleus


to the next, eventually fusing with one ofthem; the basis on which this choice ismade is unknown.

Once the sperm nucleus has entered theegg it becomes surrounded by a nuclearenvelope membrane and is known as themale pronucleus; its counterpart in the eggis the female pronucleus.

As the male pronucleus forms, it swells.The increase in pronuclear volume is dueto chromatin decondensation consequenton the replacement of the polyamines sur-rounding sperm DNA with egg histones.

Ultimately, male and female pronucleicongress and fuse to realize syngamy. Con-gression of the pronuclei occurs becausethe male pronucleus is closely associatedwith the sperm centrosome: it organizes amicrotubule array (the aster) that spreadsthrough the egg cytoplasm and capturesthe female pronucleus, drawing it towardthe male pronucleus, while the growingaster pushes against the egg cortex to moveboth male and female pronucleus towardthe center of the egg.

3Fertilization Calcium Signals

3.1Calcium Ions are a Universal Egg Activatorat Fertilization

In the early part of the twentieth cen-tury, Jacques Loeb experimented withvarious ways of stimulating the egg toundergo development in the absence ofsperm – parthenogenesis. Treatment ofechinoderm eggs with butyric acid fol-lowed by hypertonic seawater was effective,proving Loeb’s antivitalist point that lifewas chemistry but offering no insight intothe underlying mechanisms that governedthe onset of development.

In the 1970s, a sharp tool becameavailable that enabled these mechanismsto be defined: a calcium ionophore with thedesignation A23187. Calcium ionophoresare small molecules that can carry calciumions across lipid membranes. It was shownthat treatment of unfertilized eggs ofboth vertebrates and invertebrates withA23187 led to many of the sequelae offertilization: secretion of cortical secretorygranules, DNA synthesis, and migrationof the female pronucleus. The eggs hadbeen activated, but did not divide. Whatwas missing? It turned out to be thecentrosomes that are required to formthe mitotic spindle and that are donatedto the egg by the sperm at fertilization.When centrosomes were induced to formin the echinoderm egg using hypertonicseawater, full development ensued inmany cases and these parthenotes couldbe raised to adulthood.

These investigations set calcium ionscenter stage at fertilization.

3.2Calcium Signaling

Calcium ions are the bearers of messagesdelivered by universal signaling systemsfound in all higher eukaryotes. Calciumion concentrations are very low in the cyto-plasm of all cells and are below micromolarconcentrations at rest. It is postulated thatcalcium ion concentrations must of neces-sity be very low in the cytoplasm becauseof the existence of high concentrations ofinorganic phosphate, itself a key compo-nent of intermediary metabolism. Highconcentrations of calcium and phosphatecannot coexist because of the low solu-bility product of calcium phosphate salt.On the other hand, calcium concentra-tions are in the millimolar range in theseas and in the body fluids of metazoans,


leading to very large gradients of calciumconcentration across cell membranes. Theexistence of these gradients has two con-sequences. One is that calcium ion pumpsdriven by the cell’s metabolism must con-stantly eject calcium from the cell sinceno lipid membrane is completely imper-meant to calcium. The second is thatrelatively small calcium fluxes across theplasma membrane can give rise to largeand rapid changes in the intracellularcalcium concentrations. The latter con-sequence makes the cytoplasmic calciumconcentration very suitable as an energy-efficient cell signal.

Calcium fluxes across plasma mem-branes control neurotransmitter releaseand are the basis of all neuronal activ-ity. In skeletal muscle, calcium causescontraction but is released into the cyto-plasm not from the extracellular fluid butfrom a pervasive network of reticular inter-nal membranes that form a compartmentknown as the sarcoplasmic reticulum. Thisarrangement allows calcium ion concen-trations to increase rapidly throughout thecytoplasm without the diffusion delays thatarise when calcium enters through theplasma membrane. The reticular networkis present in other cell types, where it isknown as endoplasmic reticulum (ER). Itexists not solely as a source of calciumsignals; in fact, its primary purpose isto permit the folding and export of pro-teins destined for the plasma membraneeither as integral membrane proteins oras secreted products. The concentration ofcalcium ions in ER is closer to the con-centrations in extracellular fluid than tothat of the cytoplasm and is maintainedby a calcium pump. We know that foldingand export of ER proteins is compromisedwhen the calcium concentration in the ERfalls, so it is reasonable to suppose that

the compartment’s high calcium concen-tration exists to provide an environmentfor proteins comparable to that they willexperience once they leave the cell. But, aswith the plasma membrane calcium gra-dient, the ER calcium gradient has beenexploited in evolution as a calcium signal-ing system.

3.3Fertilization Calcium Waves

When the calcium ionophore is used toactivate an unfertilized egg, it is causingthe release of calcium from the internalcalcium stores of the ER. This is easilydemonstrated by removal of all calciumexternal to the egg: this has no effect onegg activation. It is also possible, thoughrather more complicated, to demonstratethat calcium ion concentrations increaseat fertilization.

The first demonstrations of fertilizationcalcium increases employed a protein (ae-quorin) that emits light in response tocalcium in a graded way. The increasein calcium at fertilization could be seenas a ring of light travelling through theperipheral cytoplasm of a large medakafish egg. This established the basic fea-tures of the fertilization calcium signal as alarge increase in cytoplasmic calcium thatarises at the point of sperm–egg fusionand traverses the egg as a constant veloc-ity reaction-diffusion wave. Similar waveswere observed in frog, echinoderm, ne-matode, and mammalian eggs using bothaequorin and more convenient fluorescentcalcium indicators developed by Dr RogerTsien (Fig. 6).

Fertilization calcium waves are thelargest and longest calcium signals mea-sured. They rise to around 5–10 µM andlast for many minutes. Preparation forthese large explosive signals takes place


4

3

2

1

4

3

2

1

4

3

2

1

4

3

2

1

4

3

2

1

4

3

2

1

4

3

2

1

4

3

2

1

36.3 s26.4 s

9.9 s

3.3 s 6.6 s

0.0 s−3.3 s

16.5 s

Fig. 6 Fertilization calcium waves in sea urchinand frog eggs. Calcium sensitive dyes are usedto detect calcium waves. Warm colors reflecthigh calcium concentrations. The calcium wavecrosses the sea urchin egg in around 15 s with avelocity of around 5 µm s−1. The wave has asimilar velocity in the frog egg, but here it takes

5 min to traverse the egg as it is over 1 mm indiameter. Left: frog egg. Right: sea urchin egg. Asmall early increase in calcium concentration canbe seen at the egg periphery, a consequence of acalcium action potential that is triggered bysperm–egg fusion.

during meiosis. As meiosis progresses,the oocyte becomes competent to gener-ate a large calcium wave; after fertilization,this competence disappears. The calciumionophore experiments tell us that thewaves’ purpose is to initiate develop-ment and it is reasonable to supposethat their extent, magnitude, and dura-tion are necessary to ensure that each partof the egg cytoplasm receives the mes-sage that fertilization has occurred. Theyensure that the small and local interac-tion that occurs between egg and spermis amplified and transmitted throughoutthe egg.

3.4Repetitive Fertilization Calcium Transients

Both ascidian and mammalian oocytesmanifest multiple large calcium transientsat fertilization. In ascidian oocytes, thelocus of initiation of the calcium wavemigrates with the male pronucleus as itmoves toward the oocyte’s vegetal pole.In mammalian oocytes, the first few cal-cium transients originate at the point ofsperm – egg fusion, but thereafter, eachcalcium transient appears to rise homoge-neously within the oocyte cytoplasm. Thisbehavior can be explained by proposing


that each transient is generated by a factorassociated with the sperm and male pronu-cleus. In ascidian oocytes, it is supposedthat this factor remains closely associatedwith the male pronucleus, while in mam-malian oocytes, the factor slowly diffusesaway from the male pronucleus to becomeuniformly distributed in the cytoplasm.We shall consider the likely purpose of therepetitive transients when we come to dis-cuss the link between fertilization and thecell division cycle.

3.5Signal Transduction during theFertilization Calcium Wave

The question before us is how sperm–eggfusion, an event that initially involves acytoplasmic bridge as little as 100 nm wide,is able to trigger the large fertilizationcalcium wave. An analogy is that thefertilization calcium wave is an explosionand that the sperm lights a fuse to set itoff. This leads to two further questions:

what gives the calcium wave its explosiveproperties and how is the fuse lit? We shallanswer them in turn.

3.5.1 Reaction Diffusion WavesExplosions occur because the products ofa chemical reaction further increase therate of the reaction itself in a processof positive feedback. The fuel for thecalcium explosion at fertilization is thecalcium stored within the ER. Positivefeedback occurs because of the particularproperties of calcium channels that existin the ER membrane: the probability oftheir opening increases when cytoplasmiccalcium increases. Once calcium rises ata point within the cytoplasm, a wave ofcalcium release will propagate from thatpoint (Fig. 7). There is good evidence forthis mechanism in heart cells made topropagate artificial calcium waves.

The identity of the calcium channelin the ER responsible for the fertiliza-tion calcium wave was determined by

ER lumen

Ca CaInsP3 InsP3

Cytoplasm

PtdlnsP2PtdlnsP2

Fig. 7 Mechanism of propagation of thefertilization calcium wave. The calcium wavepropagates via calcium-dependent production ofInsP3 and diffusion of calcium from one release

channel to the next. Both InsP3 and calcium arerequired in the vicinity of the channel to triggerchannel opening and movement of calcium fromthe ER into the cytoplasm.


investigation of the phosphoinositide mes-senger pathway, a ubiquitous signalingpathway in cells of higher eukaryotes.The substrate of this pathway is phos-phatidylinositol bisphosphate (PtdInsP2).A signaling event, for example, the bind-ing of a hormone or neurotransmitterto its receptor, leads to activation of aspecific phospholipase C that hydrolyzesPtdInsP2 to produce two products, inositoltrisphosphate (InsP3) and diacylglycerol.InsP3 is a small sugar that causes calciumrelease from ER. It achieves this by bind-ing to and allosterically activating an ERcalcium channel known as the InsP3 recep-tor (InsP3R), causing it to open. Calciumin the cytoplasm is a coactivator of theInsP3R. The sequence in which InsP3 andcalcium are presented to the channel is cru-cial: if calcium binds after InsP3, then thechannel is activated, but if it binds before,InsP3 is unable to open the channel.

Calcium waves can be triggered ineggs by injecting very small amountsof InsP3 into unfertilized eggs and canbe blocked by agents that prevent InsP3

from interacting fruitfully with the InsP3R.These observations demonstrate that theInsP3R is the calcium channel responsiblefor propagation of the fertilization calciumwave, but they also suggest that themechanism by which the wave propagatesis more complex than that established forcalcium waves in heart cells.

The properties of the InsP3R are notimmediately consistent with the observa-tion that very small quantities of InsP3

are sufficient to trigger a calcium wavesince once the locally injected InsP3 is di-luted by diffusion into the large volumeof the egg, its concentration is insufficientto activate the InsP3R which, as we haveseen, requires coactivation by both InsP3

and calcium. There is evidence, in fact, that

increases in calcium induce further hydrol-ysis of PtdInsP2, generating InsP3 locallyas the wave propagates. The mechanismof wave propagation thus relies on twointertwined positive feedback pathways,calcium-induced production of InsP3 andcoactivation of the InsP3R by InsP3 andcalcium (Fig. 7).

3.5.2 Initiation of the Calcium Wave bythe SpermThe simplest idea is that the spermgenerates a small amount of InsP3, sosetting off the calcium wave in muchthe same way as happens when InsP3 ismicroinjected. This is almost certainly thecase, though there is no direct evidence foran increase in InsP3 in the tiny volumeclose to the point of sperm–egg fusion.

The evidence that an increase in InsP3triggers the fertilization calcium wave ismore subtle. A significant amount of timeelapses after sperm–egg fusion beforethe increase in cytoplasmic calcium thatmarks the onset of the wave is seen.In echinoderms, around 10–20 s elapses,comparable to the time it then takes for thewave to cross the egg; in mammalian eggs,several minutes pass. What is found is thatan agent that interferes with the activationof the InsP3R increases this latency.

InsP3 is generated by activation ofPtdInsP2-specific phospholipases. Therehas been a debate about which of thesephospholipases is responsible for trig-gering the calcium wave. It was initiallythought that an interaction of the spermwith an egg receptor might activate theβ-form of phospholipase C (PLCβ); PLCβ

is known to be coupled to transmembranereceptors in all cell types examined. How-ever, as it became clearer that sperm–eggfusion was the key activating event at fertil-ization, attention shifted to another formof phospholipase, PLCγ , that is activated


by phosphorylation on tyrosine. Proteinfragments that were able to compete withand block either PLCγ or tyrosine (Srcfamily) kinases were found to increase thelatency of the fertilization response andat higher concentrations, to block it com-pletely. Mammalian eggs were, however,an exception, being completely insensitiveto these inhibitors. On the assumptionthat the activating phospholipase would bepresent in sperm, the mammalian testiswas screened for the existence of appropri-ate phospholipases. A novel phospholipasefound only in testis (PLCζ ) was shown toinduce repetitive calcium transients whenexpressed in unfertilized mouse eggs.

The picture we have of signal trans-duction at fertilization is that a protein(Src kinase, PLCγ , PLCζ , depending onspecies) passes into the egg when spermand egg fuse. The presence of this proteinin the egg leads to the very local productionof InsP3. As the latent period progresses,a concentration of InsP3 builds up suffi-ciently to trigger the onset of the explosivefertilization calcium wave (Fig. 7). Obser-vation of the repetitive fertilization calciumtransients in ascidians and mammals sug-gests that in these species, the activatingprotein is bound to a component of thesperm head. In ascidian oocytes, the as-sociation persists as the male pronucleusforms, while in mammalian eggs, the pro-tein (PLCζ ) is thought to dissociate fromthe male pronucleus (see Sects. 3.4 and4.3.1).

4Fertilization and the Cell Division Cycle

4.1Fertilization Occurs at Different Stages ofthe Cell Division Cycle in Different Species

It is striking given the ubiquity of calciumsignals at fertilization that there are signif-icant differences between eggs of differentspecies in the cell cycle stage in meio-sis at which fertilization occurs. Meiosiscomprises two cell divisions without an in-tervening S-phase. Fertilization can occurbefore the first meiotic division begins, atmetaphase of first meiosis, at metaphaseof second meiosis, and in interphase oncemeiosis is complete (Fig. 8). With very fewexceptions, this is because at the time thatthe egg is most receptive to sperm, itscell cycle has arrested at one of the abovestages, a stage specific to the species inquestion.

4.2Maturation Promoting Factor Plays a Partin Meiosis and in Meiotic Arrest

Classic experiments in the field of cellcycle control showed that cytoplasm fromoocytes arrested and awaiting fertilizationin meiosis I or meiosis II would inducethe maturation of immature oocytes.Similarly, classical experiments in thegenetics of yeast cell cycle control genesdetermined that two key components of

Fig. 8 Cell cycle arrest before fertilization. Oocyteof different species arrest before fertilization atdifferent points of the meiotic division cycle.Some are fertilized as immature oocytes (i: clam),some during first meiotic metaphase (ii: othermolluscs, ascidian), some during second meioticmetaphase (iii: frog, starfish, mammals) andsome after meiosis at the start of the first mitoticcell division cycle (iv: sea urchin).

DNA content 4n 2n n

GV M I M II PN

PB

i ii iviii


maturation promoting factor activity werecdk1, a protein kinase, and its activatingpartner, cyclin B.

Cdk1/cyclin B activation linked to accu-mulation of cyclin B drives the cell intomeiosis or mitosis, causing condensationof chromatin and formation of the mi-totic or meiotic spindle. Oocytes (and othercells) arrested at metaphase show highlevels of cdk1/cyclin B activity, but whatmarks them out is the persistence of thisnormally transient activity: the persistenceleads to cell cycle arrest.

Stabilization of cdk1/cyclin B activityduring metaphase cell cycle arrest isdue to what appear to be the two othercomponents of maturation promotingfactor, a protein termed mos and its target,MAP kinase. Mos is a protein uniqueto meiotic maturation. Its transcriptionearly during maturation leads to increasinglevels of MAP kinase activity. MAP kinaseactivity remains high throughout meiosisuntil fertilization occurs. It is responsiblefor the persistence of cdk1/cyclin Bactivity (Fig. 9) and, incidentally, for thesuppression of S-phase between meiosis Iand meiosis II.

Persistent activation of cdk1/cyclin Baccounts for the two metaphase cell cyclearrest points during meiosis observed invarious species, but other explanations arenecessary to explain cell cycle arrest beforemeiosis begins or after it is completed.

Cell cycle arrest before the onset ofmeiosis is seen in most organisms. In most

species, premeiotic arrest is a distinct stagepreceding the prefertilization arrest thatoccurs later in meiosis and that has alreadybeen discussed earlier. Ovaries containmany immature oocytes, a proportionof which are recruited by hormonalstimulation to begin maturation. It isthought that hormonal stimulation leadsto a fall in the second messenger cyclicadenosine monophosphate (cAMP). Inspecies whose oocytes are fertilized whenimmature, it is the fertilization calciumsignal that triggers the onset of maturationand reinitiation of meiosis (In somemolluscs fertilized at this stage, calciuminflux across the plasma membrane takesthe place of a calcium wave.).

Cell cycle arrest in interphase of thefirst mitotic cell cycle occurs in someechinoderms. This arrest can be explainedby the suppression of protein synthesis bya relatively acidic cytoplasmic pH and themaintenance of a redox state that lead tolow levels of the synthetic nicotine-adeninedinucleotide phosphate (NADPH).

4.3Calcium Signals and Cell CycleProgression at Fertilization

4.3.1 Metaphase ArrestPersistent cdk1/cyclin B activity drivenby mos/MAP kinase is the cause of thecell cycle arrest at metaphase in oocytes.Inactivation of these kinases by removalof both cyclin B and mos is how the

MAPK

p34cdc2

Fig. 9 MAP kinase and cdk1during meiotic maturation.MAP kinase activity remainshigh throughout meiosis, whilecdk1 activity dips betweenmeiosis I and meiosis II.


fertilization calcium signal causes meiosisto resume.

Frog oocytes are arrested during meio-sis II. The fertilization calcium wavetriggers cyclin proteolysis by the protea-some through activating calmodulin andso calmodulin-dependent protein kinaseII (CamKII). CamKII in turn activates an-other molecular machine known as theanaphase promoting complex (APC) thattargets cyclin for destruction by the pro-teasome. A parallel mechanism, activationof the calcium-dependent protease calpain,leads to proteolysis of mos. In frog oocytes,the proximal cause of anaphase onset isthe destruction of cyclin; destruction ofmos occurs later than cyclin destruction.Mouse oocytes are also arrested at meiosisII awaiting fertilization. In mammals, 10 to12 h elapses between fertilization and for-mation of the nuclear membrane aroundmale and female pronuclei, the lengthyperiod coinciding with the process of im-printing, found only in mammals. Duringthis period, calcium transients occur ev-ery few minutes and cyclin B is slowlydegraded, the rate extent of cyclin degra-dation determined by the frequency ofcalcium signals. Formation of the pronu-clear membranes marks the end of meiosisand coincides with the final disappearanceof cyclin B. Mos, on the other hand, isdegraded within a few hours. Ascidianoocytes are arrested before fertilization atmetaphase of meiosis I. A first set of cal-cium transients occurs immediately afterfertilization, leading to degradation of cy-clin B and exit from meiosis I. Mos/MAPkinase activity is not affected by this firstset of transients (remember that a role ofmos/MAP kinase is to suppress S-phasein the interval between the two meioticdivisions). The repetitive transients beginagain as the oocyte enters meiosis II, againleading to degradation of cyclin B and,

on this occasion, degradation of mos too.The events described in this paragraph aresummarized in Fig. 10.

The reader will have noted a correla-tion between the state of the nucleus andthe occurrence of repetitive calcium tran-sients: the transients are present duringmeiotic divisions when chromatin is con-densed and the nuclear envelope is absent,and are absent during interphase whenchromatin in decondensed and a nuclearenvelope surrounds the membrane. Thetransients may indeed be triggered by afactor that is sequestered in the nucleusand released when the nuclear envelopebreaks down as the oocyte enters meioticdivision. The evidence for this is that nu-clei isolated from first cell cycle interphasein mouse embryos will break down underthe influence of cdk1/cyclin B when trans-ferred to unfertilized mouse oocytes andinduce repetitive calcium transients. Thesimplest explanation is that PLCζ , the eggactivator from the sperm, is sequesteredinto the nucleus as the nuclear envelopereforms. This conjecture is consistent withobservations that repetitive calcium tran-sients continue indefinitely when importof proteins into the nucleus though thenuclear pores are blocked.

4.3.2 Interphase ArrestLittle more is known about how calciumlifts the cell cycle blockade after fertiliza-tion of immature oocytes than that calciumsignals are known to be capable of antago-nizing signaling by cyclic AMP.

Sea urchin eggs are arrested after com-pletion of meiosis, in interphase of the firstcell cycle. The fertilization calcium wavestimulates a calcium-dependent NAD+ ki-nase to generate NADP, which is rapidlyreduced in the cell to NADPH, provid-ing fuel for protein synthesis. Activationof phospholipase C is known to generate


Mos

MosCyclin

Cyclin

10 min 200 minCalcium transients in ascidian oocytes Calcium transients in mouse oocytes

Fig. 10 Destruction of mos and cyclin B at fertilization. Two calcium-dependent pathways forproteolysis at fertilization. One degrades mos by activating calpain. The other activatesCaMKinase II to stimulate the APC and cyclin degradation by the proteasome. Calciumdependent proteolysis is rapid in ascidians; in mouse and other mammals mos is rapidlydegraded, but cyclin degradation takes several hours.

a diacylglycerol from PtdInsP2 in addi-tion to InsP3 (Fig. 7). At fertilization inthe sea urchin egg, the diacylglycerol sogenerated activates a protein kinase C (norelation of phospholipase C) in the eggplasma membrane in turn stimulating asodium–hydrogen antiporter that ejectshydrogen ions from the egg, returningthe cytoplasm to its normal pH of 7.2. Thesuppression of the protein synthetic ma-chinery is lifted, cyclin synthesis begins,and the cell cycle proceeds.

4.4The Cell Cycle and Syngamy

The behavior of the male pronucleus onceinside the oocyte or egg after fertilization

depends on the point at which fertil-ization occurs: during or after meiosis.Oocytes fertilized during meiosis mustcomplete meiosis to generate a hap-loid female pronucleus with which themale pronucleus can undergo syngamy.In this case, the male pronucleus re-mains separate with its chromatin in acondensed state. Once meiosis II is com-pleted, cyclin B/cdk1 activity falls andthe male pronucleus develops a nuclearenvelope, as does the female pronu-cleus. Syngamy comprises the mixingof male and female genomes and oc-curs through fusion of the pronuclei’snuclear envelopes. S-phase of the firstmitotic cell cycle often begins before syn-gamy. In an extreme case, in mouse


zygotes, S-phase is completed and thenuclear envelope breaks down as thezygote enters the first mitotic division be-fore syngamy occurs. Syngamy occurs onthe metaphase plate as male and femalechromosomes are captured by spindlemicrotubules.

5Calcium and Cell Cycle Control

5.1Cell Cycle Checkpoints

It is noteworthy that the same cytoplasmiccalcium signal, the wave generated by thesperm, operates to trigger resumption ofthe cell cycle at three distinct points in thecell cycle in different organisms: G2/M,metaphase of MI and MII (which areformally equivalent) and G1. The questionarises as to whether these fertilizationmechanisms are specific examples of amore general requirement for calciumduring cell cycle progression of all celltypes.

More general study of the regulation ofthe cell cycle in yeast and in mammaliansomatic cells as well as in embryos has ledto the concept of cell cycle checkpoints.The concept is straightforward: before acell makes a crucial step through the cellcycle, a checkpoint mechanism is invokedthat is designed to ensure that everythingis correctly in place, a preflight checklistas it were. Two examples of this are theDNA damage checkpoint and the mitosisexit checkpoint.

DNA replication is not a faultlessprocess. Errors are introduced duringcopying of the DNA strands that are thenrepaired during late S-phase. The pointhere is that if mitosis were to occur beforerepair had taken place, then one or both

daughter cells would inherit mutationsthat resulted from errors introduced atS-phase. The DNA damage checkpointmechanism is invoked by damaged DNA;while it is active, the cell cannot proceedinto mitosis.

The mitosis exit checkpoint is invokedduring mitosis to ensure that all chro-mosome pairs are captured and properlyaligned on the metaphase plate beforethey are separated by the processes ofanaphase. A single misaligned chromo-some will prevent mitosis. The checkpointmachinery monitors attachment of twosets of microtubules to the chromosomeand also verifies by measuring tension inthe microtubules that they are correctlyattached to the spindle poles. In this in-stance, the checkpoint mechanism aimsto avoid an unequal distribution of chro-mosomes into the daughter cells. Thiscondition known as aneuploidy leads tocell death or on occasion to uncontrolledcell proliferation. A third checkpoint ex-ists at the beginning of the cell cycle inG1. Here, a cell decides, on the basis ofsignals from its environment, whether itwill differentiate, commit suicide (apop-tosis), or progress into the cell cycle anddivide. The cell cycle stage at which oocytesare found to arrest in the cell cycle cor-respond to the stages at which thesecheckpoints act.

Oocytes arrested in G2 are stoppedat the stage at which the DNA dam-age checkpoint acts, those arrested atmetaphase are stopped at the mitosis exitcheckpoint stage, and those arrested inG1 resemble those cells making choicesabout whether to divide, die, or differ-entiate. It is thus very likely that cellcycle arrest before fertilization makesuse of underlying cell cycle checkpointmechanisms.


5.2The DNA Damage Checkpoint

The DNA damage checkpoint operatesby preventing activation of the mitotickinase cdk1/cyclin B. It does this bypreventing a phosphatase (cdc25) fromremoving a phosphate group on cdk1that inhibits kinase function. It has beenshown in mouse oocytes that activationof cdc25 is necessary and sufficientto bypass the G2/M block and triggerresumption of meiosis. In most oocytes, asmentioned earlier, resumption of meiosisis triggered by a fall in cyclic AMP(Fig. 11). In mouse oocytes, it has beenshown that cdc25 is downstream of thereduction in cyclic AMP levels. Thecalcium transient in oocytes fertilized atG2/M may activate cdc25 via calmodulin-dependent protein kinase II, as it hasbeen shown that progression through

the G2/M checkpoint in mammaliansomatic cells involves activation of thiskinase.

5.3The Mitosis Exit Checkpoint

The mitosis exit checkpoint operates bypreventing activation of the APC that tar-gets key proteins for destruction includingsecurin and cyclin B. Loss of securinleads to loss of adhesion between pairedchromosomes on the metaphase plate, al-lowing them to separate and spring to thepoles; loss of cyclin B leads to inactivationof the cdk1/cyclin B kinase, permittingdecondensation of chromatin and refor-mation of the nuclear envelope. Theseevents are triggered by the fertilizationcalcium transient in oocytes arrested atmetaphase meiosis I and meiosis II beforefertilization.

cdc25

cdc25

cdc2

Activemitotic kinase

cdc2cdc2

cak

Cyclin BCyclin B

wee1

Increased calciumDecreased cyclic AMP

Cyclin B

p

pp

pp

Fig. 11 Activation of mitotic kinase by the cdc25 phosphatase. Cdk activating kinase (cak)and wee1 kinase phosphorylate the cdk1/cyclin complex. Cak phosphorylation activates themitotic kinase and wee1 phosphorylation is inhibitory. Cdc25 phosphatase removes theinhibitory phosphates. Cdc25 is controlled by cAMP and calcium.


5.4The Checkpoint at Cell Cycle Onset

There are analogies between fertilizationin species such as echinoderms that arefertilized in G1 and the G1 decision pointin mammalian somatic cells. One strikingexample is starfish oocytes: if they do notsense a fertilization calcium transient, theyproceed to apoptosis. Other similarities arethe activation of an MAP kinase signal-ing cascade, stimulation of the sodium-hydrogen antiporter by protein kinase C,and stimulation of the protein syntheticmachinery. Nonetheless, the comparisonis less precise at the molecular level.

5.5Calcium and Cell Cycle Checkpoints

The existence of identical molecular mech-anisms in arrested oocytes and in thecheckpoint mechanisms of somatic cellsstrongly suggests that the arrest before fer-tilization uses the checkpoint machineryand raises a question: is calcium signalinga component of general cell cycle regula-tion or is control of the checkpoint ma-chinery by calcium unique to fertilization?

That calcium is a cell cycle regulatorysignal is clear in some cases, and lessclear in others. In some embryos, calciumsignals can be identified at the timesat which the checkpoint mechanismsoperate; the amplitude of the signals issmall, less than one-tenth of the sizeof the fertilization calcium signal andsignals are often local to the nucleus. Thecalcium-calmodulin-calmodulin kinase IIpathway has been shown to control entryinto mitosis and calcium and calmodulincontrol separation of the chromosomes atmitosis. Embryos do not possess a G1decision point mechanism.

In somatic cells, it has been shown,for example, that calmodulin-dependentprotein kinase II must be activated atmitosis entry and that it phosphorylatescdc25, the activator of cdk1/cyclin B. Ithas also been shown that rapidly raisingintracellular calcium can induce anaphase.Checkpoint-associated calcium signals canbe detected, but they are not present inall circumstances. The jury is out on theuniversality of cell cycle calcium signaling,but if the hypothesis turns out to betrue, then the calcium signals central tofertilization will have pointed the way.

See also Calcium Biochemistry; Fe-male Reproduction System, Molec-ular Biology of; In vitro Fertiliza-tion; Male Reproductive System:Testis Development and Spermato-genesis.

Bibliography

Books and reviews

Berridge, M.J., Lipp, P., Bootman, M.D. (2000)The versatility and universality of calciumsignalling, Nat. Rev. Mol. Cell Biol. 1, 11–21.

Darszon, A., Labarca, P., Nishigaki, T., Espinosa,F. (1999) Ion channels in sperm physiology,Physiol. Rev. 79, 481–510.

Eisenbach, M. (1999) Sperm chemotaxis, Rev.Reprod. 4, 56–66.

Heilbrunn, L.V. (1928) The Colloid Chemistryof Protoplasm, Verlag von GebruederBorntraeger, Berlin, Germany.

Hepler, P.K. (1992) Calcium and mitosis, Int.Rev. Cytol. 138, 239–268.

Jaffe, L.F. (1980) Calcium explosions as triggersof development, Ann. N.Y. Acad. Sci. 339,86–101.

Jaffe, L.A., Cross, N.L. (1986) Electrical regulationof sperm-egg fusion, Annu. Rev. Physiol. 48,191–200.


Jaffe, L.F., Creton, R. (1998) On the conservationof calcium wave speeds, Cell Calcium 24, 1–8.

Jaffe, L.A., Giusti, A.F., Carroll, D.J., Foltz, K.R.(2001) Ca2+ signalling during fertilization ofechinoderm eggs, Semin. Cell Dev. Biol. 12,45–51.

Kishimoto, T. (2003) Cell-cycle control duringmeiotic maturation, Curr. Opin. Cell Biol. 15,654–663.

Lillie, F.R. (1919) Problems of Fertilization,University of Chicago Press, Chicago, IL.

Loeb, J. (1913) Artificial Parthenogenesis andFertilization, University of Chicago Press,Chicago, IL.

Maller, J.L. (1998) Recurring themes in oocytematuration, Biol. Cell 90, 453–460.

Rieder, C. (1998) Mitosis and Meiosis, AcademicPress, New York, p. 504.

Rothschild, L. (1956) Fertilization, Methuen,London, England.

Schatten, G. (1994) The centrosome and itsmode of inheritance: the reduction of thecentrosome during gametogenesis and itsrestoration during fertilization, Dev. Biol. 165,299–335.

Stricker, S.A. (1999) Comparative biology ofcalcium signaling during fertilization and eggactivation in animals, Dev. Biol. 211, 157–176.

Wassarman, P.M. (1999) Fertilization in animals,Dev. Genet. 25, 83–86.

Whitaker, M. (1996) Control of meiotic arrest,Rev. Reprod. 1, 127–135.

Whitaker, M.J. (2005) Calcium signalling in eggsand embryos, Physiol. Rev. in press.

Whitaker, M.J., Steinhardt, R.A. (1982) Ionicregulation of egg activation, Q. Rev. Biophys.15, 593–666.

Whitaker, M.J., Patel, R. (1990) Calcium and cellcycle control, Development 108, 525–542.

Whitaker, M., Swann, K. (1993) Lighting the fuseat fertilization, Development 117, 1–12.

Primary Literature

Abassi, Y.A., Carroll, D.J., Giusti, A.F., Belton,R.J., Foltz, K.R. (2000) Evidence that Src-type tyrosine kinase activity is necessary forinitiation of calcium release at fertilization insea urchin eggs, Dev. Biol. 218, 206–219.

Adkins, C.E., Taylor, C.W. (1999) Lateralinhibition of inositol 1,4,5-trisphosphatereceptors by cytosolic Ca2+, Curr. Biol.: CB9, 1115–1118.

Aizawa, H., Kawahara, H., Tanaka, K., Yokosawa,H. (1996) Activation of the proteasomeduring Xenopus egg activation implies alink between proteasome activation andintracellular calcium release, Biochem. Biophys.Res. Commun. 218, 224–228.

Albrieux, M., Sardet, C., Villaz, M. (1997)The two intracellular Ca2+ release channels,ryanodine receptor and inositol 1,4,5-trisphosphate receptor, play different rolesduring fertilization in ascidians, Dev. Biol. 189,174–185.

Albrieux, M., Lee, H.C., Villaz, M. (1998) Calciumsignaling by cyclic ADP-ribose, NAADP, andinositol trisphosphate are involved in distinctfunctions in ascidian oocytes, J. Biol. Chem.273, 14566–14574.

Allen, R.D., Griffin, J.L. (1958) The timesequence of early events in the fertilizationof sea urchin eggs, Exp. Cell Res. 15, 163–173.

Ayabe, T., Kopf, G.S., Schultz, R.M. (1995)Regulation of mouse egg activation: presenceof ryanodine receptors and effects ofmicroinjected ryanodine and cyclic ADPribose on uninseminated and inseminatedeggs, Development (Cambridge, England) 121,2233–2244.

Baitinger, C., Alderton, J., Poenie, M., Schulman,H., Steinhardt, R.A. (1990) MultifunctionalCa2+ /calmodulin-dependent protein kinaseis necessary for nuclear-envelope breakdown,J. Cell Biol. 111, 1763–1773.

Bandyopadhyay, J., Lee, J., Lee, J., Lee Jin,I., Yu, J.-R., Jee, C., Cho, J.-H., Jung,S., Lee Myon, H., Zannoni, S., Singson,A., Kim Do, H., Koo, H.-S., Ahnn, J.(2002) Calcineurin, a calcium/calmodulin-dependent protein phosphatase, is involvedin movement, fertility, egg laying, and growthin Caenorhabditis elegans, Mol. Biol. Cell 13,3281–3293.

Begg, D.A., Rebhun, L.I. (1979) pH regulates thepolymerization of actin in the sea urchin eggcortex, J. Cell Biol. 83, 241–248.

Bement, W.M., Capco, D.G. (1989) Activatorsof protein kinase C trigger cortical granuleexocytosis, cortical contraction, and cleavagefurrow formation in Xenopus laevis oocytesand eggs, J. Cell Biol. 108, 885–892.

Berridge, G., Dickinson, G., Parrington, J.,Galione, A., Patel, S. (2002) Solubilizationof receptors for the novel Ca2+-mobilizing messenger, nicotinic acid adenine


dinucleotide phosphate, J. Biol. Chem. 277,43717–43723.

Bloom, T.L., Szuts, E.Z., Eckberg, W.R. (1988)Inositol trisphosphate, inositol phospholipidmetabolism, and germinal vesicle breakdownin surf clam oocytes, Dev. Biol. 129, 532–540.

Brandriff, B., Hinegardner, R.I., Steinhardt,R. (1975) Development and life cycle ofthe parthenogenetically activated sea urchinembryo, J. Exp. Zool. 192, 13–24.

Brind, S., Swann, K., Carroll, J. (2000)Inositol 1,4,5-trisphosphate receptors aredownregulated in mouse oocytes in responseto sperm or adenophostin A but notto increases in intracellular Ca2+ or eggactivation, Dev. Biol. 223, 251–265.

Busa, W.B., Nuccitelli, R. (1985) An elevatedfree cytosolic Ca2+ wave follows fertilizationin eggs of the frog, Xenopus laevis, J. Cell Biol.100, 1325–1329.

Busa, W.B., Ferguson, J.E., Joseph, S.K.,Williamson, J.R., Nuccitelli, R. (1985)Activation of frog (Xenopus laevis) eggs byinositol trisphosphate. I. Characterization ofCa2+ release from intracellular stores, J. CellBiol. 101, 677–682.

Campbell, K.D., Reed, W.A., White, K.L.(2000) Ability of integrins to mediatefertilization, intracellular calcium release,and parthenogenetic development in bovineoocytes, Biol. Reprod. 62, 1702–1709.

Carrasco, D., Allende, C.C., Allende, J.E.(1990) The incorporation of myo-inositol intophosphatidylinositol derivatives is stimulatedduring hormone-induced meiotic maturation,Exp. Cell Res. 191, 313–318.

Carroll, J., Swann, K. (1992) Spontaneouscytosolic calcium oscillations driven by inositoltrisphosphate occur during in vitro maturationof mouse oocytes, J. Biol. Chem. 267,11196–11201.

Carroll, J., Swann, K., Whittingham, D.,Whitaker, M. (1994) Spatiotemporal dynamicsof intracellular [Ca2+]i oscillations during thegrowth and meiotic maturation of mouseoocytes, Development (Cambridge, England)120, 3507–3517.

Carroll, D.J., Albay, D.T., Terasaki, M., Jaffe,L.A., Foltz, K.R. (1999) Identification of PLCgamma-dependent and -independent eventsduring fertilization of sea urchin eggs, Dev.Biol. 206, 232–247.

Carroll, D.J., Ramarao, C.S., Mehlmann, L.M.,Roche, S., Terasaki, M., Jaffe, L.A. (1997)

Calcium release at fertilization in starfish eggsis mediated by phospholipase Cgamma, J. CellBiol. 138, 1303–1311.

Carroll, M., Levasseur, M., Wood, C., Whitaker,M.J., Jones, K.T., McDougall, A. (2003)Exploring the mechanism of action of thesperm-triggered calcium-wave pacemaker inascidian zygotes, J. Cell. Sci. 116, 4997–5004.

Cavalier-Smith, T. (2002) Origins of themachinery of recombination and sex, Heredity88, 125–141.

Centonze, V.E., White, J.G. (1995) Calcium os-cillations during early C. elegans development.http://elegans.swmed.edu/wli/[wm95e148].(cited with permission).

Chambers, E.L. (1980) Fertilization and cleavageof the eggs of the sea urchin Lytechinusvariegatus in Ca2+-free sea water, Eur. J. CellBiol. 22, 476.

Chambers, E.L., de Armendi, J. (1979)Membrane potential, action potential andactivation potential of eggs of the sea urchin,Lytechinus variegatus, Exp. Cell Res. 122,203–218.

Chambers, E.L., Pressman, B.C., Rose, B. (1974)The activation of sea urchin eggs by thedivalent ionophores A23187 and X-537A,Biochem. Biophys. Res. Commun. 60, 126–132.

Charbonneau, M., Moreau, M., Picheral, B.,Vilain, J.P., Guerrier, P. (1983) Fertilizationof amphibian eggs: a comparison of electricalresponses between anurans and urodeles, Dev.Biol. 98, 304–318.

Cheung, A., Swann, K., Carroll, J. (2000) Theability to generate normal Ca2+ transientsin response to spermatozoa develops duringthe final stages of oocyte growth andmaturation, Hum. Reprod. (Oxford, England)15, 1389–1395.

Chiba, K., Kado, R.T., Jaffe, L.A. (1990)Development of calcium release mechanismsduring starfish oocyte maturation, Dev. Biol.140, 300–306.

Chini, E.N., Beers, K.W., Chini, C.C., Dousa,T.P. (1995) Specific modulation of cyclic ADP-ribose-induced Ca2+ release by polyamines,Am. J. Physiol. 269, C1042–C1047.

Cho, C., Bunch, D.O., Faure, J.E., Goulding,E.H., Eddy, E.M., Primakoff, P., Myles,D.G. (1998) Fertilization defects in spermfrom mice lacking fertilin beta, Science 281,1857–1859.

Churchill Grant, C., Okada, Y., Thomas Justyn,M., Genazzani Armando, A., Patel, S., Galione,


A. (2002) NAADP mobilizes Ca(2+) fromreserve granules, lysosome-related organelles,in sea urchin eggs, Cell 111, 703–708.

Ciapa, B., Whitaker, M. (1986) Two phasesof inositol polyphosphate and diacylglycerolproduction at fertilisation, FEBS Lett. 195,347–351.

Ciapa, B., Epel, D. (1991) A rapid changein phosphorylation on tyrosine accompaniesfertilization of sea urchin eggs, FEBS Lett. 295,167–170.

Ciapa, B., Borg, B., Whitaker, M. (1992)Polyphosphoinositide metabolism during thefertilization wave in sea urchin eggs,Development (Cambridge, England) 115,187–195.

Ciapa, B., Pesando, D., Wilding, M., Whitaker, M.(1994) Cell-cycle calcium transients driven bycyclic changes in inositol trisphosphate levels,Nature 368, 875–878.

Cosson, M.P., Carre, D., Cosson, J. (1984)Sperm chemotaxis in siphonophores. II.Calcium-dependent asymmetrical movementof spermatozoa induced by the attractant, J.Cell Sci. 68, 163–181.

Coward, K., Campos-Mendoza, A., Larman, M.,Hibbitt, O., McAndrew, B., Bromage, N.,Parrington, J. (2003) Teleost fish spermatozoacontain a cytosolic protein factor thatinduces calcium release in sea urchin egghomogenates and triggers calcium oscillationswhen injected into mouse oocytes, Biochem.Biophys. Res. Commun. 305, 299–304.

Cox, L.J., Larman, M.G., Saunders, C.M.,Hashimoto, K., Swann, K., Lai, F.A. (2002)Sperm phospholipase Czeta from humansand cynomolgus monkeys triggers Ca2+oscillations, activation and development ofmouse oocytes, Reproduction (Cambridge,England) 124, 611–623.

Crossley, I., Swann, K., Chambers, E., Whitaker,M. (1988) Activation of sea urchin eggs byinositol phosphates is independent of externalcalcium, Biochem. J. 252, 257–262.

Dale, B., De Belice, L.J., Taglietti, V. (1978)Membrane noise and conductance increaseduring single spermatozoon-egg interactions,Nature 275, 217–219.

Day, M.L., McGuinness, O.M., Berridge,M.J., Johnson, M.H. (2000) Regulation offertilization-induced Ca2+ spiking in themouse zygote, Cell Calcium 28, 47–54.

Deguchi, R., Osanai, K. (1994) Repetitiveintracellular Ca2+ increases at fertilization

and the role of Ca2+ in meiosis reinitiationfrom the first metaphase in oocytes of marinebivalves, Dev. Biol. 163, 162–174.

Deng, M.Q., Huang, X.Y., Tang, T.S., Sun,F.Z. (1998) Spontaneous and fertilization-induced Ca2+ oscillations in mouse immaturegerminal vesicle-stage oocytes, Biol. Reprod.58, 807–813.

Dong, J.B., Tang, T.S., Sun, F.Z. (2000) Xenopusand chicken sperm contain a cytosolic solubleprotein factor which can trigger calciumoscillations in mouse eggs, Biochem. Biophys.Res. Commun. 268, 947–951.

Dube, F. (1988) The relationships between earlyionic events, the pattern of protein synthesis,and oocyte activation in the surf clam, Spisulasolidissima, Dev. Biol. 126, 233–241.

Dube, F., Guerrier, P. (1982) Activation of Barneacandida (Mollusca, Pelecypoda) oocytes bysperm of KCl, but not by NH4Cl, requiresa calcium influx, Dev. Biol. 92, 408–417.

Ducibella, T., Huneau, D., Angelichio, E., Xu,Z., Schultz Richard, M., Kopf Gregory, S.,Fissore, R., Madoux, S., Ozil, J.-P. (2002) Egg-to-embryo transition is driven by differentialresponses to Ca(2+) oscillation number, Dev.Biol. 250, 280–291.

Dumollard, R., Sardet, C. (2001) Three differentcalcium wave pacemakers in ascidian eggs, J.Cell. Sci. 114, 2471–2481.

Dumollard, R., Marangos, P., Fitzharris, G.,Swann, K., Duchen, M., Carroll, J. (2004)Sperm-triggered [Ca2+] oscillations and Ca2+homeostasis in the mouse egg have anabsolute requirement for mitochondrial ATPproduction, Development (Cambridge, England)131, 3057–3067.

Dumollard, R., Hammar, K., Porterfield, M.,Smith Peter, J., Cibert, C., Rouviere, C., Sardet,C. (2003) Mitochondrial respiration and Ca2+waves are linked during fertilization andmeiosis completion, Development (Cambridge,England) 130, 683–692.

Eisen, A., Reynolds, G.T. (1984) Calciumtransients during early development in singlestarfish (Asterias forbesi) oocytes, J. Cell Biol.99, 1878–1882.

Eisen, A., Reynolds, G.T. (1985) Source and sinksfor the calcium released during fertilizationof single sea urchin eggs, J. Cell Biol. 100,1522–1527.

Eisen, A., Kiehart, D.P., Wieland, S.J., Reynolds,G.T. (1984) Temporal sequence and spatialdistribution of early events of fertilization


in single sea urchin eggs, J. Cell Biol. 99,1647–1654.

Evans, J.P., Kopf, G.S., Schultz, R.M.(1997) Characterization of the binding ofrecombinant mouse sperm fertilin betasubunit to mouse eggs: evidence for adhesiveactivity via an egg beta1 integrin-mediatedinteraction, Dev. Biol. 187, 79–93.

Evans, T., Rosenthal, E.T., Youngblom, J., Distel,D., Hunt, T. (1983) Cyclin: a protein specifiedby maternal mRNA in sea urchin eggs thatis destroyed at each cleavage division, Cell 33,389–396.

Fechter, J., Schoneberg, A., Schatten, G. (1996)Excision and disassembly of sperm tailmicrotubules during sea urchin fertilization:requirements for microtubule dynamics, CellMotil. Cytoskeleton 35, 281–288.

FitzHarris, G., Marangos, P., Carroll, J.(2003) Cell cycle-dependent regulation ofthe structure of the endoplasmic reticulumand inositol 1,4,5-trisphosphate-induced Ca2+release in mouse oocytes and embryos, Mol.Biol. Cell 14, 288–301.

Florman, H.M., Wassarman, P.M. (1985) O-linked oligosaccharides of mouse egg ZP3account for its sperm receptor activity, Cell41, 313–324.

Fluck, R., Abraham, V., Miller, A., Galione,A. (1999) Microinjection of cyclic ADP-ribosetriggers a regenerative wave of Ca2+ releaseand exocytosis of cortical alveoli in medakaeggs, Zygote 7, 285–292.

Fontanilla, R.A., Nuccitelli, R. (1998)Characterization of the sperm-inducedcalcium wave in Xenopus eggs using confocalmicroscopy, Biophys. J. 75, 2079–2087.

Fujiwara, T., Nakada, K., Shirakawa, H.,Miyazaki, S. (1993) Development ofinositol trisphosphate-induced calcium releasemechanism during maturation of hamsteroocytes, Dev. Biol. 156, 69–79.

Galione, A., McDougall, A., Busa, W.B.,Willmott, N., Gillot, I., Whitaker, M. (1993)Redundant mechanisms of calcium-inducedcalcium release underlying calcium wavesduring fertilization of sea urchin eggs, Science261, 348–352.

Gilkey, J.C., Jaffe, L.F., Ridgway, E.B., Reynolds,G.T. (1978) A free calcium wave traverses theactivating egg of the medaka, Oryzias latipes, J.Cell Biol. 76, 448–466.

Gillot, I., Whitaker, M. (1994) Calcium signalsin and around the nucleus in sea urchin eggs,Cell Calcium 16, 269–278.

Giusti, A.F., Xu, W., Hinkle, B., Terasaki, M.,Jaffe, L.A. (2000) Evidence that fertilizationactivates starfish eggs by sequential activationof a Src-like kinase and phospholipasecgamma, J. Biol. Chem. 275, 16788–16794.

Giusti, A.F., O’Neill, F.J., Yamasu, K., Foltz, K.R.,Jaffe, L.A. (2003) Function of a sea urchin eggSrc family kinase in initiating Ca2+ release atfertilization, Dev. Biol. 256, 367–378.

Giusti, A.F., Carroll, D.J., Abassi, Y.A., Terasaki,M., Foltz, K.R., Jaffe, L.A. (1999) Requirementof a Src family kinase for initiating calciumrelease at fertilization in starfish eggs, J. Biol.Chem. 274, 29318–29322.

Glabe, C.G. (1985) Interaction of the spermadhesive protein, bindin, with phospholipidvesicles. II. Bindin induces the fusionof mixed-phase vesicles that containphosphatidylcholine and phosphatidylserinein vitro, J. Cell Biol. 100, 800–806.

Glahn, D., Mark, S.D., Behr, R.K., Nuccitelli, R.(1999) Tyrosine kinase inhibitors block sperm-induced egg activation in Xenopus laevis, Dev.Biol. 205, 171–180.

Gordo, A.C., Rodrigues, P., Kurokawa, M.,Jellerette, T., Exley, G.E., Warner, C., Fissore,R. (2002) Intracellular calcium oscillationssignal apoptosis rather than activation inin vitro aged mouse eggs, Biol. Reprod. 66,1828–1837.

Goudeau, H., Depresle, Y., Rosa, A., Goudeau,M. (1994) Evidence by a voltage clamp study ofan electrically mediated block to polyspermy inthe egg of the ascidian Phallusia mammillata,Dev. Biol. 166, 489–501.

Grainger, J.L., Winkler, M.M., Shen, S.S.,Steinhardt, R.A. (1979) Intracellular pHcontrols protein synthesis rate in the seaurchin egg and early embryo, Dev. Biol. 68,396–406.

Guerrier, P., Leclerc-David, C., Moreau, M.(1993) Evidence for the involvement ofinternal calcium stores during serotonin-induced meiosis reinitiation in oocytes of thebivalve mollusc Ruditapes philippinarum, Dev.Biol. 159, 474–484.

Halet, G., Tunwell, R., Balla, T., Swann, K.,Carroll, J. (2002) The dynamics of plasmamembrane PtdIns(4,5)P(2) at fertilization ofmouse eggs, J. Cell. Sci. 115, 2139–2149.


Hamaguchi, Y., Hiramoto, Y. (1981) Activation ofsea urchin eggs by microinjection of calciumbuffers, Exp. Cell Res. 134, 171–179.

Han, J.K., Fukami, K., Nuccitelli, R. (1992)Reducing inositol lipid hydrolysis, Ins(1,4,5)P3receptor availability, or Ca2+ gradientslengthens the duration of the cell-cycle inXenopus laevis blastomeres, J. Cell Biol. 116,147–156.

Heinecke, J.W., Meier, K.E., Lorenzen, J.A.,Shapiro, B.M. (1990) A specific requirementfor protein kinase C in activation of therespiratory burst oxidase of fertilization, J. Biol.Chem. 265, 7717–7720.

Hesketh, T.R., Moore, J.P., Morris, J.D., Taylor,M.V., Rogers, J., Smith, G.A., Metcalfe, J.C.(1985) A common sequence of calcium andpH signals in the mitogenic stimulation ofeukaryotic cells, Nature 313, 481–484.

Hirose, K., Kadowaki, S., Tanabe, M., Takeshima,H., Lino, M. (1999) Spatiotemporal dynamicsof inositol 1,4,5-triphosphate that underliescomplex Ca2+ metabolism patterns, Science284, 1527–1530.

Ho, H.C., Suarez, S.S. (2001) Hyperactivationof mammalian spermatozoa: function andregulation, Reproduction (Cambridge, England)122, 519–526.

Homa, S.T., Carroll, J., Swann, K. (1993) The roleof calcium in mammalian oocyte maturationand egg activation, Hum. Reprod. (Oxford,England) 8, 1274–1281.

Hyslop, L.A., Carroll, M., Nixon, V.L., Mc-Dougall, A., Jones, K.T. (2001) Simultaneousmeasurement of intracellular nitric oxide andfree calcium levels in chordate eggs demon-strates that nitric oxide has no role at fertiliza-tion, Dev. Biol. 234, 216–230.

Inoue, S., Tilney, L.G. (1982) Acrosomal reactionof thyone sperm. I. Changes in the spermhead visualized by high resolution videomicroscopy, J. Cell Biol. 93, 812–819.

Jaffe, L.A. (1976) Fast block to polyspermy in seaurchin eggs is electrically mediated, Nature261, 68–71.

Jaffe, L.A., Sharp, A.P., Wolf, D.P. (1983) Absenceof an electrical polyspermy block in the mouse,Dev. Biol. 96, 317–323.

Jellerette, T., He, C.L., Wu, H., Parys, J.B.,Fissore, R.A. (2000) Down-regulation of theinositol 1,4,5-trisphosphate receptor in mouseeggs following fertilization or parthenogeneticactivation, Dev. Biol. 223, 238–250.

Johnson, J.D., Epel, D. (1976) IntracellularpH and activation of sea urchin eggs afterfertilisation, Nature 262, 661–664.

Jones, K.T., Nixon, V.L. (2000) Sperm-inducedCa2+ oscillations in mouse oocytes andeggs can be mimicked by photolysis ofcaged inositol 1,4,5-trisphosphate: evidence tosupport a continuous low level productionof inositol 1, 4,5-trisphosphate duringmammalian fertilization, Dev. Biol. 225, 1–12.

Jones, K.T., Soeller, C., Cannell, M.B. (1998)The passage of Ca2+ and fluorescent markersbetween the sperm and egg after fusion inthe mouse, Development (Cambridge, England)125, 4627–4635.

Kaji, K., Oda, S., Shikano, T., Ohnuki, T.,Uematsu, Y., Sakagami, J., Tada, N., Miyazaki,S., Kudo, A. (2000) The gamete fusion processis defective in eggs of Cd9-deficient mice, Nat.Genet. 24, 279–282.

Kaltschmidt, J.A., Brand, A.H. (2002)Asymmetric cell division: microtubuledynamics and spindle asymmetry, J. Cell. Sci.115, 2257–2264.

Kawahara, H., Yokosawa, H. (1994) Intracellularcalcium mobilization regulates the activityof 26 S proteasome during the metaphase-anaphase transition in the ascidian meioticcell cycle, Dev. Biol. 166, 623–633.

Keizer, J., Li, Y.X., Stojilkovic, S., Rinzel, J.(1995) InsP3-induced Ca2+ excitability of theendoplasmic reticulum, Mol. Biol. Cell 6,945–951.

Kishimoto, T., Usui, N., Kanatani, H. (1984)Breakdown of starfish ovarian follicle inducedby maturation-promoting factor, Dev. Biol. 101,28–34.

Kline, D. (1988) Calcium-dependent events atfertilization of the frog egg: injection of acalcium buffer blocks ion channel opening,exocytosis, and formation of pronuclei, Dev.Biol. 126, 346–361.

Kono, T., Carroll, J., Swann, K., Whittingham,D.G. (1995) Nuclei from fertilized mouseembryos have calcium-releasing activity,Development (Cambridge, England) 121,1123–1128.

Kono, T., Jones, K.T., Bos-Mikich, A.,Whittingham, D.G., Carroll, J. (1996) A cellcycle-associated change in Ca2+ releasingactivity leads to the generation of Ca2+transients in mouse embryos during the firstmitotic division, J. Cell Biol. 132, 915–923.


Kranz, E., Lorz, H. (1994) In vitro fertilisation ofmaize by single egg and sperm cell protoplastfusion mediated by high calcium and high pH,Zygote (Cambridge, England) 2, 125–128.

Krylov, D.M., Nasmyth, K., Koonin, E.V. (2003)Evolution of eukaryotic cell cycle regulation:stepwise addition of regulatory kinases and lateadvent of the CDKs, Curr. Biol. 13, 173–177.

Kuo, R.C., Baxter, G.T., Thompson, S.H.,Stricker, S.A., Patton, C., Bonaventura, J., Epel,D. (2000) NO is necessary and sufficient for eggactivation at fertilization, Nature 406, 633–636.

Kuroda, R., Kontani, K., Kanda, Y., Katada, T.,Nakano, T., Satoh, Y., Suzuki, N., Kuroda, H.(2001) Increase of cGMP, cADP-ribose andinositol 1,4,5-trisphosphate preceding Ca2+transients in fertilization of sea urchin eggs,Development 128, 4405–4414.

Kyozuka, K., Deguchi, R., Mohri, T., Miyazaki,S. (1998) Injection of sperm extract mimicsspatiotemporal dynamics of Ca2+ responsesand progression of meiosis at fertilization ofascidian oocytes, Development 125, 4099–4105.

Labbe, J.C., Lee, M.G., Nurse, P., Picard,A., Doree, M. (1988) Activation at M-phaseof a protein kinase encoded by a starfishhomologue of the cell cycle control genecdc2+, Nature 335, 251–254.

Lambert, C., Goudeau, H., Franchet, C., Lambert,G., Goudeau, M. (1997) Ascidian eggs blockpolyspermy by two independent mechanisms:one at the egg plasma membrane, the otherinvolving the follicle cells, Mol. Reprod. Dev.48, 137–143.

Lawrence, Y., Whitaker, M., Swann, K. (1997)Sperm-egg fusion is the prelude to theinitial Ca2+ increase at fertilization in themouse, Development (Cambridge, England)124, 233–241.

Lawrence, Y., Ozil, J.P., Swann, K. (1998) Theeffects of a Ca2+ chelator and heavy-metal-ionchelators upon Ca2+ oscillations and activationat fertilization in mouse eggs suggest a rolefor repetitive Ca2+ increases, Biochem. J. 335,335–342.

Leckie, M.P., Empson, R.M., Bechetti, A.,Thomas, J., Galione, A., Whitaker, M. (2003)The NO pathway acts late during thefertilization response in sea urchin eggs, J.Biol. Chem. 278, 12247–12254.

Lee, M.G., Nurse, P. (1987) Complementationused to clone a human homologue of thefission yeast cell cycle control gene cdc2,Nature 327, 31–35.

Levasseur, M., McDougall, A. (2000) Sperm-induced calcium oscillations at fertilisationin ascidians are controlled by cyclinB1-dependent kinase activity, Development(Cambridge, England) 127, 631–641.

Li, S.T., Huang, X.Y., Sun, F.Z. (2001) Floweringplant sperm contains a cytosolic solubleprotein factor which can trigger calciumoscillations in mouse eggs, Biochem. Biophys.Res. Commun. 287, 56–59.

Li, W., Llopis, J., Whitney, M., Zlokarnik,G., Tsien, R.Y. (1998) Cell-permeant cagedInsP3 ester shows that Ca2+ spike frequencycan optimize gene expression, Nature 392,936–941.

Longo, F.J., Lynn, J.W., McCulloh, D.H., Cham-bers, E.L. (1986) Correlative ultrastructuraland electrophysiological studies of sperm egginteractions of the sea-urchin, Lytechinus var-iegatus, Dev. Biol. 118, 155–166.

Lorca, T., Abrieu, A., Means, A., Doree, M. (1994)Ca2+ is involved through type II calmodulin-dependent protein kinase in cyclin degradationand exit from metaphase, Biochim. Biophys.Acta 1223, 325–332.

Lorca, T., Galas, S., Fesquet, D., Devault, A.,Cavadore, J.C., Doree, M. (1991) Degradationof the proto-oncogene product p39mos is notnecessary for cyclin proteolysis and exit frommeiotic metaphase: requirement for a Ca2+-calmodulin dependent event, EMBO J. 10,2087–2093.

Lorca, T., Cruzalegui, F.H., Fesquet, D.,Cavadore, J.C., Mery, J., Means, A., Doree, M.(1993) Calmodulin-dependent protein kinaseII mediates inactivation of MPF and CSFupon fertilization of Xenopus eggs, Nature366, 270–273.

Machaca, K., Haun, S. (2002) Induction ofmaturation-promoting factor during Xenopusoocyte maturation uncouples Ca(2+) storedepletion from store-operated Ca(2+) entry,J. Cell Biol. 156, 75–85.

Marangos, P., FitzHarris, G., Carroll, J.(2003) Ca2+ oscillations at fertilization inmammals are regulated by the formation ofpronuclei, Development (Cambridge, England)130, 1461–1472.

Markoulaki, S., Matson, S., Abbott Allison,L., Ducibella, T. (2003) Oscillatory CaMKIIactivity in mouse egg activation, Dev. Biol. 258,464–474.

McCulloh, D.H., Chambers, E.L. (1992) Fusionof membranes during fertilization - increases


of the sea-urchin eggs membrane capacitanceand membrane conductance at the site ofcontact with the sperm, J. Gen. Physiol. 99,137–175.

McDougall, A., Levasseur, M. (1998) Sperm-triggered calcium oscillations during meiosisin ascidian oocytes first pause, restart, thenstop: correlations with cell cycle kinaseactivity, Development (Cambridge, England)125, 4451–4459.

McDougall, A., Levasseur, M., O’Sullivan,A.J., Jones, K.T. (2000) Cell cycle-dependentrepetitive Ca2+ waves induced by a cytosolicsperm extract in mature ascidian eggs mimicthose observed at fertilization, J. Cell. Sci. 113,3453–3462.

Mehlmann, L.M., Terasaki, M., Jaffe, L.A., Kline,D. (1995) Reorganization of the endoplasmicreticulum during meiotic maturation of themouse oocyte, Dev. Biol. 170, 607–615.

Mehlmann, L.M., Carpenter, G., Rhee, S.G.,Jaffe, L.A. (1998) SH2 domain-mediatedactivation of phospholipase C gamma is notrequired to initiate Ca2+ release at fertilizationof mouse eggs, Dev. Biol. 203, 221–232.

Miller, B.J., Georges-Labouesse, E., Primakoff,P., Myles, D.G. (2000) Normal fertilizationoccurs with eggs lacking the integrinalpha6beta1 and is CD9-dependent, J. Cell Biol.149, 1289–1296.

Miyazaki, S., Igusa, Y. (1982) Ca-mediatedactivation of a K current at fertilization ofgolden hamster eggs, Proc. Natl. Acad. Sci.U.S.A. 79, 931–935.

Miyazaki, S., Yuzaki, M., Nakada, K., Shirakawa,H., Nakanishi, S., Nakade, S., Mikoshiba, K.(1992) Block of Ca2+ wave and Ca2+ oscillationby antibody to the inositol 1,4,5-trisphosphatereceptor in fertilized hamster eggs, Science 257,251–255.

Moolenaar, W.H., Tertoolen, L.G., de Laat, S.W.(1984) Phorbol ester and diacylglycerol mimicgrowth factors in raising cytoplasmic pH,Nature 312, 371–374.

Nakano, Y., Shirakawa, H., Mitsuhashi,N., Kuwabara, Y., Miyazaki, S. (1997)Spatiotemporal dynamics of intracellularcalcium in the mouse egg injected witha spermatozoon, Mol. Hum. Reprod. 3,1087–1093.

Nixon, V.L., Levasseur, M., McDougall, A., Jones,K.T. (2002) Ca2+ oscillations promote APC/C-dependent cyclin B1 degradation duringmetaphase arrest and completion of meiosis

in fertilizing mouse eggs, Curr. Biol.: CB 12,746–750.

Nuccitelli, R. (1980) The fertilization potential isnot necessary for the block to polyspermy orthe activation of development in the medakaegg, Dev. Biol. 76, 499–504.

Ozil, J.P., Swann, K. (1995) Stimulation ofrepetitive calcium transients in mouse eggs,J. Physiol. 483, 331–346.

Ozil, J.P., Huneau, D. (2001) Activation of rabbitoocytes: the impact of the Ca2+ signal regimeon development, Development (Cambridge,England) 128, 917–928.

Parrington, J., Brind, S., De Smedt, H.,Gangeswaran, R., Lai, F.A., Wojcikiewicz,R., Carroll, J. (1998) Expression of inositol1,4,5-trisphosphate receptors in mouse oocytesand early embryos: the type I isoform isupregulated in oocytes and downregulatedafter fertilization, Dev. Biol. 203, 451–461.

Patel, R., Holt, M., Philipova, R., Moss,S., Schulman, H., Hidaka, H., Whitaker,M. (1999) Calcium/calmodulin-dependentphosphorylation and activation of humanCdc25-C at the G2/M phase transition in HeLacells, J. Biol. Chem. 274, 7958–7968.

Poenie, M., Alderton, J., Tsien, R.Y., Steinhardt,R.A. (1985) Changes of free calcium levelswith stages of the cell division cycle, Nature315, 147–149.

Poenie, M., Alderton, J., Steinhardt, R., Tsien,R. (1986) Calcium rises abruptly and brieflythroughout the cell at the onset of anaphase,Science 233, 886–889.

Reimann, J.D.R., Jackson, P.K. (2002) Emi1is required for cytostatic factor arrest invertebrate eggs, Nature 416, 850–854.

Rice, A., Parrington, J., Jones, K.T., Swann,K. (2000) Mammalian sperm contain aCa2+-sensitive phospholipase C activity thatcan generate InsP3 from PIP2 associatedwith intracellular organelles, Dev. Biol. 228,125–135.

Rongish, B.J., Wu, W., Kinsey, W.H.(1999) Fertilization-induced activation ofphospholipase C in the sea urchin egg, Dev.Biol. 215, 147–154.

Runft, L.L., Jaffe, L.A. (2000) Sperm extractinjection into ascidian eggs signals Ca2+release by the same pathway as fertilization,Development (Cambridge, England) 127,3227–3236.

Runft, L.L., Watras, J., Jaffe, L.A. (1999) Calciumrelease at fertilization of Xenopus eggs


requires type I IP(3) receptors, but not SH2domain-mediated activation of PLCgamma orG(q)-mediated activation of PLCbeta, Dev. Biol.214, 399–411.

Sato, K., Tokmakov, A.A., Iwasaki, T., Fukami, Y.(2000) Tyrosine kinase-dependent activationof phospholipase Cgamma is required forcalcium transient in Xenopus egg fertilization,Dev. Biol. 224, 453–469.

Sato, Y., Miyazaki, S., Shikano, T., Mitsuhashi,N., Takeuchi, H., Mikoshiba, K., Kuwabara, Y.(1998) Adenophostin, a potent agonist of theinositol 1,4,5-trisphosphate receptor, is usefulfor fertilization of mouse oocytes injected withround spermatids leading to normal offspring,Biol. Reprod. 58, 867–873.

Saunders, C.M., Larman, M.G., Parrington,J., Cox, L.J., Royse, J., Blayney, L.M.,Swann, K., Lai, F.A. (2002) PLC zeta: asperm-specific trigger of Ca2+ oscillations ineggs and embryo development, Development(Cambridge, England) 129, 3533–3544.

Schmidt, T., Patton, C., Epel, D. (1982) Is there arole for the Ca2+ influx during fertilization ofthe sea urchin egg? Dev. Biol. 90, 284–290.

SeGall, G.K., Lennarz, W.J. (1981) Jelly coat andinduction of the acrosome reaction in echinoidsperm, Dev. Biol. 86, 87–93.

Shapiro, B.M. (1984) Molecular aspects of sperm-egg fusion, Ciba Found. Symp. 103, 86–99.

Shearer, J., De Nadai, C., Emily-Fenouil, F.,Gache, C., Whitaker, M., Ciapa, B. (1999)Role of phospholipase Cgamma at fertilizationand during mitosis in sea urchin eggs andembryos, Development (Cambridge, England)126, 2273–2284.

Shen, S.S., Steinhardt, R.A. (1979) IntracellularpH and the sodium requirement atfertilisation, Nature 282, 87–89.

Shilling, F.M., Carroll, D.J., Muslin, A.J.,Escobedo, J.A., Williams, L.T., Jaffe, L.A.(1994) Evidence for both tyrosine kinase andG-protein-coupled pathways leading to starfishegg activation, Dev. Biol. 162, 590–599.

Shiraishi, K., Okada, A., Shirakawa, H.,Nakanishi, S., Mikoshiba, K., Miyazaki,S. (1995) Developmental changes in thedistribution of the endoplasmic reticulumand inositol 1,4,5-trisphosphate receptors andthe spatial pattern of Ca2+ release duringmaturation of hamster oocytes, Dev. Biol. 170,594–606.

Shiwa, M., Murayama, T., Ogawa, Y. (2002)Molecular cloning and characterization of

ryanodine receptor from unfertilized seaurchin eggs, Am. J. Physiol. Regul. Integr. Comp.Physiol. 282, R727–R737.

Snow, P., Yim, D.L., Leibow, J.D., Saini, S.,Nuccitelli, R. (1996) Fertilization stimulates anincrease in inositol trisphosphate and inositollipid levels in Xenopus eggs, Dev. Biol. 180,108–118.

Speksnijder, J.E. (1992) The repetitive calciumwaves in the fertilized ascidian egg are initiatednear the vegetal pole by a cortical pacemaker,Dev. Biol. 153, 259–271.

Speksnijder, J.E., Sardet, C., Jaffe, L.F. (1990a)The activation wave of calcium in the ascidianegg and its role in ooplasmic segregation, J.Cell Biol. 110, 1589–1598.

Speksnijder, J.E., Sardet, C., Jaffe, L.F. (1990b)Periodic calcium waves cross ascidian eggsafter fertilization, Dev. Biol. 142, 246–249.

Speksnijder, J.E., Corson, D.W., Sardet, C., Jaffe,L.F. (1989) Free calcium pulses followingfertilization in the ascidian egg, Dev. Biol. 135,182–190.

Steinhardt, R.A., Epel, D. (1974) Activation ofsea-urchin eggs by a calcium ionophore, Proc.Natl. Acad. Sci. U.S.A. 71, 1915–1919.

Steinhardt, R.A., Alderton, J. (1988) Intracellularfree calcium rise triggers nuclear envelopebreakdown in the sea urchin embryo, Nature332, 364–366.

Steinhardt, R.A., Lundin, L., Mazia, D. (1971)Bioelectric responses of the echinoderm eggto fertilization, Proc. Natl. Acad. Sci. U.S.A. 68,2426–2430.

Steinhardt, R., Zucker, R., Schatten, G. (1977)Intracellular calcium release at fertilization inthe sea urchin egg, Dev. Biol. 58, 185–196.

Steinhardt, R.A., Epel, D., Carroll, E.J.,Yanagimachi, R. (1974) Is calcium ionophorea universal activator for unfertilised eggs?Nature 252, 41–43.

Stephano, J.L., Gould, M.C. (1997) Theintracellular calcium increase at fertilizationin Urechis caupo oocytes: activation withoutwaves, Dev. Biol. 191, 53–68.

Stith, B.J., Espinoza, R., Roberts, D., Smart,T. (1994) Sperm increase inositol 1,4,5-trisphosphate mass in Xenopus laevis eggspreinjected with calcium buffers or heparin,Dev. Biol. 165, 206–215.

Stricker, S.A. (1996) Repetitive calcium wavesinduced by fertilization in the nemerteanworm Cerebratulus lacteus, Dev. Biol. 176,243–263.


Stricker, S.A., Smythe, T.L. (2003) Endoplasmicreticulum reorganizations and Ca2+ signalingin maturing and fertilized oocytes of marineprotostome worms: the roles of MAPKs andMPF, Development (Cambridge, England) 130,2867–2879.

Stricker, S.A., Silva, R., Smythe, T. (1998)Calcium and endoplasmic reticulum dynamicsduring oocyte maturation and fertilization inthe marine worm Cerebratulus lacteus, Dev.Biol. 203, 305–322.

Stricker, S.A., Swann, K., Jones, K.T., Fissore,R.A. (2000) Injections of porcine spermextracts trigger fertilization-like calciumoscillations in oocytes of a marine worm, Exp.Cell Res. 257, 341–347.

Swann, K. (1990) A cytosolic sperm factorstimulates repetitive calcium increases andmimics fertilization in hamster eggs,Development (Cambridge, England) 110,1295–1302.

Swann, K., Whitaker, M. (1985) Stimulation ofthe Na/H exchanger of sea urchin eggs byphorbol ester, Nature 314, 274–277.

Swann, K., Whitaker, M. (1986) The part playedby inositol trisphosphate and calcium in thepropagation of the fertilization wave in seaurchin eggs, J. Cell Biol. 103, 2333–2342.

Swann, K., McDougall, A., Whitaker, M. (1994)Calcium signalling at fertilization, J. Mar.Biolog. Assoc. U.K. 74, 3–16.

Swanson, W.J., Vacquier, V.D. (1998) Concertedevolution in an egg receptor for a rapidlyevolving abalone sperm protein, Science 281,710–712.

Tachibana, K., Tanaka, D., Isobe, T., Kishimoto,T. (2000) c-Mos forces the mitotic cell cycleto undergo meiosis II to produce haploidgametes, Proc. Natl. Acad. Sci. U.S.A. 97,14301–14306.

Tang, T.S., Dong, J.B., Huang, X.Y., Sun,F.Z. (2000) Ca2+ oscillations induced by acytosolic sperm protein factor are mediated bya maternal machinery that functions only oncein mammalian eggs, Development (Cambridge,England) 127, 1141–1150.

Terasaki, M., Jaffe, L.A. (1991) Organization ofthe sea urchin egg endoplasmic reticulum andits reorganization at fertilization, J. Cell Biol.114, 929–940.

Thomas, T.W., Eckberg, W.R., Dube, F., Galione,A. (1998) Mechanisms of calcium releaseand sequestration in eggs of Chaetopteruspergamentaceus, Cell Calcium 24, 285–292.

Tilney, L.G., Jaffe, L.A. (1980) Actin, microvilliand the fertilization cone of sea urchin eggs, J.Cell Biol. 87, 771–782.

Tilney, L.G., Inoue, S. (1982) Acrosomal reactionof Thyone sperm. II. The kinetics and possiblemechanism of acrosomal process elongation,J. Cell Biol. 93, 820–827.

Tokmakov, A.A., Sato, K.I., Iwasaki, T., Fukami,Y. (2002) Src kinase induces calcium releasein Xenopus egg extracts via PLCgamma andIP(3)-dependent mechanism, Cell Calcium 32,11–20.

Tombes, R.M., Simerly, C., Borisy, G.G.,Schatten, G. (1992) Meiosis, egg activation,and nuclear envelope breakdown aredifferentially reliant on Ca2+, whereasgerminal vesicle breakdown is Ca2+independent in the mouse oocyte, J. Cell Biol.117, 799–811.

Torok, K., Wilding, M., Groigno, L., Patel,R., Whitaker, M. (1998) Imaging the spatialdynamics of calmodulin activation duringmitosis, Curr. Biol. 8, 692–699.

Tsien, R.Y. (1986) New tetracarboxylatechelators for fluorescence measurement andphotochemical manipulation of cytosolic freecalcium concentrations, Soc. Gen. Physiol. Ser.40, 327–345.

Tunquist, B.J., Schwab, M.S., Chen, L.G., Maller,J.L. (2002) The spindle checkpoint kinasebub1 and cyclin e/cdk2 both contribute to theestablishment of meiotic metaphase arrest bycytostatic factor, Curr. Biol.: CB 12, 1027–1033.

Turner, P.R., Sheetz, M.P., Jaffe, L.A. (1984)Fertilization increases the polyphosphoinosi-tide content of sea urchin eggs, Nature 310,414–415.

Twigg, J., Patel, R., Whitaker, M. (1988)Translational control of InsP3-inducedchromatin condensation during the early cellcycles of sea urchin embryos, Nature 332,366–369.

Vacquier, V.D. (1998) Evolution of gameterecognition proteins, Science 281, 1995–1998.

Verlhac, M.H., Kubiak, J.Z., Weber, M., Geraud,G., Colledge, W.H., Evans, M.J., Maro, B.(1996) Mos is required for MAP kinaseactivation and is involved in microtubuleorganization during meiotic maturation inthe mouse, Development (Cambridge, England)122, 815–822.

Viets, L.N., Campbell, K.D., White, K.L. (2001)Pathways involved in RGD-mediated calcium


transients in mature bovine oocytes, CloningStem Cells 3, 105–113.

Walker, D.S., Gower, N.J.D., Ly, S., Bradley,G.L., Baylis, H.A. (2002) Regulated disruptionof inositol 1,4,5-trisphosphate signalling inCaenorhabditis elegans reveals new functionsin feeding and embryogenesis, Mol. Biol. Cell13, 1329–1337.

Webb, D.J., Nuccitelli, R. (1985) Fertilizationpotential and electrical properties of theXenopus laevis egg, Dev. Biol. 107, 395–406.

Whitaker, M., Irvine, R.F. (1984) Inositol 1,4,5-trisphosphate microinjection activates seaurchin eggs, Nature 312, 636–639.

Whitaker, M., Aitchison, M. (1985) Calcium-dependent polyphosphoinositide hydrolysis isassociated with exocytosis in vitro, FEBS Lett.182, 119–124.

Wilding, M., Dale, B. (1998) Soluble extracts fromascidian spermatozoa trigger intracellularcalcium release independently of the activationof the ADP ribose channel, Zygote (Cambridge,England) 6, 149–154.

Wilding, M., Torok, K., Whitaker, M.(1995) Activation-dependent and activation-independent localisation of calmodulin tothe mitotic apparatus during the first cellcycle of the Lytechinus pictus embryo, Zygote(Cambridge, England) 3, 219–224.

Wilding, M., Russo, G.L., Galione, A., Marino,M., Dale, B. (1998) ADP-ribose gates thefertilization channel in ascidian oocytes, Am.J. Physiol. 275, C1277–C1283.

Williams, C.J., Mehlmann, L.M., Jaffe, L.A., Kopf,G.S., Schultz, R.M. (1998) Evidence that Gqfamily G proteins do not function in mouseegg activation at fertilization, Dev. Biol. 198,116–127.

Winkler, M.M., Steinhardt, R.A., Grainger, J.L.,Minning, L. (1980) Dual ionic controls for theactivation of protein synthesis at fertilization,Nature 287, 558–560.

Witchel, H.J., Steinhardt, R.A. (1990) 1-Methyladenine can consistently induce a fura-detectable transient calcium increase which isneither necessary nor sufficient for maturationin oocytes of the starfish Asterina miniata, Dev.Biol. 141, 393–398.

Wood, C.D., Nisihigaki, T., Furuta, T., Baba,S.A., Darszon, A. (2005) Real-time analysis ofthe role of Ca2+ in flagellar movement and

motility in single sea urchin sperm, J. Cell.Biol. 169, 725–731.

Wu, G.J., Simerly, C., Zoran, S.S., Funte,L.R., Schatten, G. (1996) Microtubule andchromatin dynamics during fertilization andearly development in rhesus monkeys, andregulation by intracellular calcium ions, Biol.Reprod. 55, 260–270.

Wu, H., Smyth, J., Luzzi, V., Fukami, K.,Takenawa, T., Black, S.L., Allbritton, N.L.,Fissore, R.A. (2001) Sperm factor inducesintracellular free calcium oscillations bystimulating the phosphoinositide pathway,Biol. Reprod. 64, 1338–1349.

Wyrick, R.E., Nishihara, T., Hedrick, J.L. (1974)Agglutination of jelly coat and cortical granulecomponents and the block to polyspermy inthe amphibian Xenopus laevis, Proc. Natl. Acad.Sci. U.S.A. 71, 2067–2071.

Xu, X.Z.S., Sternberg Paul, W. (2003) A C.elegans sperm TRP protein required forsperm-egg interactions during fertilization,Cell 114, 285–297.

Yamamoto, S., Kubota, H.Y., Yoshimoto, Y.,Iwao, Y. (2001) Injection of a sperm extracttriggers egg activation in the newt Cynopspyrrhogaster, Dev. Biol. 230, 89–99.

Yoshida, M., Murata, M., Inaba, K., Morisawa,M. (2002) A chemoattractant for ascidianspermatozoa is a sulfated steroid, Proc. Natl.Acad. Sci. U.S.A. 99, 14831–14836.

Yoshida, M., Sensui, N., Inoue, T., Morisawa,M., Mikoshiba, K. (1998) Role of two seriesof Ca2+ oscillations in activation of ascidianeggs, Dev. Biol. 203, 122–133.

Yoshida, M., Ishikawa, M., Izumi, H., DeSantis, R., Morisawa, M. (2003) Store-operatedcalcium channel regulates the chemotacticbehavior of ascidian sperm, Proc. Natl. Acad.Sci. U.S.A. 100, 149–154.

Zhu, C.C., Furuichi, T., Mikoshiba, K.,Wojcikiewicz, R.J. (1999) Inositol 1,4,5-trisphosphate receptor downregulation isactivated directly by inositol 1.4.5-trisphosphate binding, J. Biol. Chem. 274,3476–3484.

Zucker, R.S., Steinhardt, R.A. (1978) Preventionof the cortical reaction in fertilized seaurchin eggs by injection of calcium-chelating ligands, Biochim. Biophys. Acta 541,459–466.

syngamy and cell cycle control - wiley-vchwiley-vch.de/books/sample/352730651x_c01.pdf · 2005. 10....

Documents