FertilizationFertilization

SpermSpermEach sperm consists of a haploid nucleus, a propulsion system to Each sperm consists of a haploid nucleus, a propulsion system to move the nucleus, and a sac of enzymes that enable the nucleus to move the nucleus, and a sac of enzymes that enable the nucleus to enter the egg. Most of the sperm’s cytoplasm is eliminated during enter the egg. Most of the sperm’s cytoplasm is eliminated during maturation, leaving only certain organelles that are modified for maturation, leaving only certain organelles that are modified for spermatic function. spermatic function.

SpermSperm

During the course of sperm maturation, the haploid nucleus becomes very streamlined, and its DNA becomes tightly compressed. In front of this compressed haploid nucleus lies the acrosomal vesicle, or acrosome, which is derived from the Golgi apparatus and contains enzymes that digest proteins and complex sugars; thus, it can be considered a modified secretory vesicle. These stored enzymes are used to lyse the outer coverings of the egg. In many species, such as sea urchins, a region of globular actin molecules lies between the nucleus and the acrosomal vesicle. These proteins are used to extend a fingerlike acrosomal process from the sperm during the early stages of fertilization. In sea urchins and several other species, recognition between sperm and egg involves molecules on the acrosomal process. Together, the acrosome and nucleus constitute the head of the sperm.

SpermSperm

SpermSpermThe motile apparatus of the sperm. Cross section of the flagellum of a mammalian The motile apparatus of the sperm. Cross section of the flagellum of a mammalian spermatozoon, showing the central axoneme and the external fibers. Interpretive spermatozoon, showing the central axoneme and the external fibers. Interpretive diagram of the axoneme, showing the “9 + 2” arrangement of the microtubules and diagram of the axoneme, showing the “9 + 2” arrangement of the microtubules and other flagellar components. The schematic diagram shows the association of tubulin other flagellar components. The schematic diagram shows the association of tubulin protofilaments into a microtubule doublet. The first (“A”) portion of the doublet is a protofilaments into a microtubule doublet. The first (“A”) portion of the doublet is a normal microtubule comprising 13 protofilaments. The second (“B”) portion of the normal microtubule comprising 13 protofilaments. The second (“B”) portion of the doublet contains only 11 (occasionally 10) protofilaments. The dynein arms contain doublet contains only 11 (occasionally 10) protofilaments. The dynein arms contain the ATPases that provide the energy for flagellar movement. A three-dimensional the ATPases that provide the energy for flagellar movement. A three-dimensional model of an “A” microtubule. The α- and β-tubulin subunits are similar but not model of an “A” microtubule. The α- and β-tubulin subunits are similar but not identical, and the microtubule can change size by polymerizing or depolymerizing identical, and the microtubule can change size by polymerizing or depolymerizing tubulin subunits at either end. tubulin subunits at either end.

EggEggAll the material necessary for the beginning of growth and development All the material necessary for the beginning of growth and development must be stored in the mature egg (the must be stored in the mature egg (the ovumovum). Whereas the sperm has ). Whereas the sperm has eliminated most of its cytoplasm, the developing egg not only conserves its eliminated most of its cytoplasm, the developing egg not only conserves its material, but is actively involved in accumulating more. The meiotic material, but is actively involved in accumulating more. The meiotic divisions that form the oocyte conserve its cytoplasm and the oocyte either divisions that form the oocyte conserve its cytoplasm and the oocyte either synthesizes or absorbs proteins, such as yolk, that act as food reservoirs synthesizes or absorbs proteins, such as yolk, that act as food reservoirs for the developing embryo. Thus, birds’ eggs are enormous single cells, for the developing embryo. Thus, birds’ eggs are enormous single cells, swollen with their accumulated yolk. Even So, while sperm and egg have swollen with their accumulated yolk. Even So, while sperm and egg have equal haploid nuclear components, the egg also has a remarkable equal haploid nuclear components, the egg also has a remarkable cytoplasmic storehouse that it has accumulated during its maturation. This cytoplasmic storehouse that it has accumulated during its maturation. This cytoplasm includes the following:cytoplasm includes the following:

Proteins.Proteins. The early embryonic cells need a supply of energy and amino  The early embryonic cells need a supply of energy and amino acids.. Many of the yolk proteins are made in other organs (liver, fat body) acids.. Many of the yolk proteins are made in other organs (liver, fat body) and travel through the maternal blood to the egg.and travel through the maternal blood to the egg.Ribosomes and tRNA.Ribosomes and tRNA. The early embryo needs to make many of its own  The early embryo needs to make many of its own proteins, and in some species, there is a burst of protein synthesis soon proteins, and in some species, there is a burst of protein synthesis soon after fertilization. Protein synthesis is accomplished by ribosomes and after fertilization. Protein synthesis is accomplished by ribosomes and tRNA, which exist in the egg. tRNA, which exist in the egg. Messenger RNA.Messenger RNA. In most organisms, the instructions for proteins made  In most organisms, the instructions for proteins made during early development are already packaged in the oocyte. during early development are already packaged in the oocyte. Morphogenetic factors.Morphogenetic factors. They appear to be localized in different regions of  They appear to be localized in different regions of the egg and become segregated into different cells during cleavage.the egg and become segregated into different cells during cleavage.Protective chemicals.Protective chemicals. Many eggs contain ultraviolet filters and DNA repair  Many eggs contain ultraviolet filters and DNA repair enzymes that protect them from sunlight.enzymes that protect them from sunlight.

EggEggnucleus, cell nucleus, cell membrane, vitelline membrane, vitelline envelope (zona envelope (zona pellucida), cortex and pellucida), cortex and cortical granules, cortical granules, mitocondria, jellymitocondria, jelly

cortical granules cortical granules analogous to analogous to acrosome – contains acrosome – contains enzymes, enzymes, mucopolysaccharidesmucopolysaccharides, adhesive , adhesive glycopoteins, etcglycopoteins, etc

EggEggStages of egg maturation at the time of sperm entry in Stages of egg maturation at the time of sperm entry in different animal species. The germinal vesicle is the different animal species. The germinal vesicle is the name given to the large diploid nucleus of the primary name given to the large diploid nucleus of the primary oocyte. The polar bodies are seen as smaller cells. oocyte. The polar bodies are seen as smaller cells. 

Recognition of Egg and SpermRecognition of Egg and Sperm

The chemoattraction of the sperm to the egg by soluble molecules secreted by the eggThe chemoattraction of the sperm to the egg by soluble molecules secreted by the egg

The exocytosis of the acrosomal vesicle to release its enzymesThe exocytosis of the acrosomal vesicle to release its enzymes

The binding of the sperm to the extracellular envelope (vitelline layer or zona pellucida) of the eggThe binding of the sperm to the extracellular envelope (vitelline layer or zona pellucida) of the egg

The passing of the sperm through this extracellular envelopeThe passing of the sperm through this extracellular envelope

Fusion of egg and sperm cell plasma membranesFusion of egg and sperm cell plasma membranes

Chemo taxisChemo taxis

In many species, sperm are attracted toward eggs of their species In many species, sperm are attracted toward eggs of their species by by chemotaxischemotaxis, that is, by following a gradient of a chemical , that is, by following a gradient of a chemical secreted by the egg. The mechanisms of chemotaxis differamong secreted by the egg. The mechanisms of chemotaxis differamong species. One chemotactic molecule, a 14-amino acid peptide species. One chemotactic molecule, a 14-amino acid peptide called called resact,resact, has been isolated from the egg jelly of the sea  has been isolated from the egg jelly of the sea urchin urchin Arbacia punctulataArbacia punctulata. Resact diffuses readily in seawater and . Resact diffuses readily in seawater and has a profound effect at very low concentrations when added to a has a profound effect at very low concentrations when added to a suspension of suspension of ArbaciaArbacia sperm  sperm

Sperm – Egg InteractionSperm – Egg InteractionAcrosomal reactionAcrosomal reaction

components of egg jelly bind to receptors on sperm cell membranecomponents of egg jelly bind to receptors on sperm cell membranecalcium channels opened, calcium enters sperm head and induces calcium channels opened, calcium enters sperm head and induces fusion of acrosomal vesicle with membrane leading to exocytosis of fusion of acrosomal vesicle with membrane leading to exocytosis of enzymesenzymesacrosomal process forms from polymerization of actin also acrosomal process forms from polymerization of actin also facilitated by calciumfacilitated by calcium

Acrosomal reaction in hamster Acrosomal reaction in hamster sperm. sperm.

Species-specific recognition in sea Species-specific recognition in sea urchinsurchins

Once the sea urchin sperm has Once the sea urchin sperm has penetrated the egg jelly, the penetrated the egg jelly, the acrosomal process of the sperm acrosomal process of the sperm contacts the surface of the egg . A contacts the surface of the egg . A major species-specific recognition major species-specific recognition step occurs at this point. The step occurs at this point. The acrosomal protein mediating this acrosomal protein mediating this recognition is called recognition is called bindinbindin. In . In 1977, Vacquier and co-workers 1977, Vacquier and co-workers isolated this nonsoluble 30,500-Da isolated this nonsoluble 30,500-Da protein from the acrosome protein from the acrosome of of Strongylocentrotus Strongylocentrotus purpuratuspurpuratus and found it to be  and found it to be capable of binding to dejellied capable of binding to dejellied eggs of the same species Further, eggs of the same species Further, its interaction with eggs is its interaction with eggs is relatively species-specific bindin relatively species-specific bindin isolated from the acrosomes of isolated from the acrosomes of S. S. purpuratuspurpuratus binds to its own  binds to its own dejellied eggs, but not to those dejellied eggs, but not to those of of Arbacia punctulata.Arbacia punctulata.  

Immunochemical technique used to localize bindin. Rabbit antibody Immunochemical technique used to localize bindin. Rabbit antibody was made to the bindin protein, and this antibody was incubated was made to the bindin protein, and this antibody was incubated with sperm that had undergone the acrosomal reaction. If bindin with sperm that had undergone the acrosomal reaction. If bindin was present, the rabbit antibody would remain bound to the sperm. was present, the rabbit antibody would remain bound to the sperm. After any unbound antibody was washed off, the sperm were treated After any unbound antibody was washed off, the sperm were treated with swine antibody that had been covalently linked to peroxidase with swine antibody that had been covalently linked to peroxidase enzymes. The swine antibody bound to the rabbit antibody, placing enzymes. The swine antibody bound to the rabbit antibody, placing peroxidase molecules wherever bindin was present. Peroxidase peroxidase molecules wherever bindin was present. Peroxidase catalyzes the formation of a dark precipitate from diaminobenzidine catalyzes the formation of a dark precipitate from diaminobenzidine (DAB) and hydrogen peroxide. Thus, this precipitate formed only (DAB) and hydrogen peroxide. Thus, this precipitate formed only where bindin was present.  where bindin was present. 

Gamete binding and recognition in Gamete binding and recognition in mammalsmammals

ZP3 glycoprotein of egg binds sperm and initiates acrosomal ZP3 glycoprotein of egg binds sperm and initiates acrosomal reaction by binding to receptors that activates G protein and opens reaction by binding to receptors that activates G protein and opens calcium channels leading to exocytosiscalcium channels leading to exocytosisenzymes digest opening in zona, as sperm crosses zona loses enzymes digest opening in zona, as sperm crosses zona loses ZP3 binding sites but ZP2 binding sites come into playZP3 binding sites but ZP2 binding sites come into play

ZP3-binding proteins on the mouse sperm are located in ZP3-binding proteins on the mouse sperm are located in the plasma membrane, overlying the acrosome. In this the plasma membrane, overlying the acrosome. In this confocal image, a ZP3-binding protein is stained red by confocal image, a ZP3-binding protein is stained red by antibody immunofluorescence.  antibody immunofluorescence. 

Induction of the mammalian acrosomal reaction by ZP3Induction of the mammalian acrosomal reaction by ZP3

Unlike the sea urchin acrosomal reaction, the acrosomal Unlike the sea urchin acrosomal reaction, the acrosomal reaction in mammals occurs only after the sperm has bound to reaction in mammals occurs only after the sperm has bound to the zona pellucida. The mouse sperm acrosomal reaction is the zona pellucida. The mouse sperm acrosomal reaction is induced by the crosslinking of ZP3 with the receptors for it on induced by the crosslinking of ZP3 with the receptors for it on the sperm membrane.This crosslinking opens calcium the sperm membrane.This crosslinking opens calcium channels to increase the concentration of calcium in the sperm. channels to increase the concentration of calcium in the sperm. The mechanism by which ZP3 induces the opening of the The mechanism by which ZP3 induces the opening of the calcium channels and the subsequent exocytosis of the calcium channels and the subsequent exocytosis of the acrosome remains controversial, but it may involve the acrosome remains controversial, but it may involve the receptor’s activating a cation channel (for sodium, potassium, receptor’s activating a cation channel (for sodium, potassium, or calcium), which would change the resting potential of the or calcium), which would change the resting potential of the sperm plasma membrane. The calcium channels in the sperm plasma membrane. The calcium channels in the membrane would be sensitive to this change in membrane membrane would be sensitive to this change in membrane potential, allowing calcium to enter the sperm.potential, allowing calcium to enter the sperm.

Secondary binding of sperm to the zona pellucidaSecondary binding of sperm to the zona pellucida

During the acrosomal reaction, the anterior portion of the sperm plasma During the acrosomal reaction, the anterior portion of the sperm plasma membrane is shed from the sperm. This region is where the ZP3-binding membrane is shed from the sperm. This region is where the ZP3-binding proteins are located, and yet the sperm must still remain bound to the zona proteins are located, and yet the sperm must still remain bound to the zona in order to lyse a path through it. Acrosome-intact sperm will not bind to in order to lyse a path through it. Acrosome-intact sperm will not bind to ZP2, acrosome-reacted sperm will. Moreover, antibodies against the ZP2 ZP2, acrosome-reacted sperm will. Moreover, antibodies against the ZP2 glycoprotein will not prevent the binding of acrosome-intact sperm to the glycoprotein will not prevent the binding of acrosome-intact sperm to the zona, but will inhibit the attachment of acrosome-reacted sperm. The zona, but will inhibit the attachment of acrosome-reacted sperm. The structure of the zona consists of repeating units of ZP3 and ZP2, structure of the zona consists of repeating units of ZP3 and ZP2, occasionally crosslinked by ZP1. It appears that the acrosome-reacted occasionally crosslinked by ZP1. It appears that the acrosome-reacted sperm transfer their binding from ZP3 to the adjacent ZP2 molecules. After sperm transfer their binding from ZP3 to the adjacent ZP2 molecules. After a mouse sperm has entered the egg, the egg cortical granules release their a mouse sperm has entered the egg, the egg cortical granules release their contents. One of the proteins released by these granules is a protease that contents. One of the proteins released by these granules is a protease that specifically alters ZP2. This inhibits other acrosome-reacted sperm from specifically alters ZP2. This inhibits other acrosome-reacted sperm from moving closer toward the egg.moving closer toward the egg.

Sperm – Egg InteractionSperm – Egg Interactionfusion of sperm and egg membranesfusion of sperm and egg membranespolymerization of egg actin leads to formation of fertilization cone in sea polymerization of egg actin leads to formation of fertilization cone in sea urchinurchinin some species parts of egg membrane specialized to fuse with sperm, in in some species parts of egg membrane specialized to fuse with sperm, in sea urchins all regions are capablesea urchins all regions are capablefusion mediated by certain proteins – bindin in sea urchins has secondary fusion mediated by certain proteins – bindin in sea urchins has secondary rolerole

Gamete Fusion and the Prevention of Gamete Fusion and the Prevention of PolyspermyPolyspermy

Sperm-egg binding appears to cause the Sperm-egg binding appears to cause the extension of several microvilli to form extension of several microvilli to form the the fertilization conefertilization cone. The sperm and . The sperm and egg plasma membranes then join egg plasma membranes then join together, and material from the sperm together, and material from the sperm membrane can later be found on the egg membrane can later be found on the egg membrane. The sperm nucleus and tail membrane. The sperm nucleus and tail pass through the resulting cytoplasmic pass through the resulting cytoplasmic bridge, which is widened by the actin bridge, which is widened by the actin polymerization.polymerization. A similar process occurs during the A similar process occurs during the fusion of mammalian gametesIn fusion of mammalian gametesIn mammals, the mammals, the fertilinfertilin proteins in the  proteins in the sperm plasma membrane are essential sperm plasma membrane are essential for sperm membrane-egg membrane for sperm membrane-egg membrane fusion Mouse fertilin is localized to the fusion Mouse fertilin is localized to the posterior plasma membrane of the posterior plasma membrane of the sperm head). It adheres the sperm to the sperm head). It adheres the sperm to the egg by binding to the α6β1 integrin egg by binding to the α6β1 integrin protein on the egg plasma membrane. protein on the egg plasma membrane. Moreover, like sea urchin bindin fertilin Moreover, like sea urchin bindin fertilin has a hydrophobic region that could has a hydrophobic region that could potentially mediate the union of the two potentially mediate the union of the two membranes. Thus, fertilin appears to membranes. Thus, fertilin appears to bind the sperm plasma membrane to the bind the sperm plasma membrane to the egg plasma membrane and then to fuse egg plasma membrane and then to fuse the two of them togetherthe two of them together. .

The prevention of polyspermyThe prevention of polyspermyThe entrance of multiple sperm—The entrance of multiple sperm—polyspermypolyspermy—leads to disastrous —leads to disastrous consequences in most organisms. In the consequences in most organisms. In the sea urchin, fertilization by two sperm sea urchin, fertilization by two sperm results in a triploid nucleus, in which results in a triploid nucleus, in which each chromosome is represented three each chromosome is represented three times rather than twice. Worse, since times rather than twice. Worse, since each sperm’s centriole divides to form each sperm’s centriole divides to form the two poles of a mitotic apparatus, the two poles of a mitotic apparatus, instead of a bipolar mitotic spindle instead of a bipolar mitotic spindle separating the chromosomes into two separating the chromosomes into two cells, the triploid chromosomes may be cells, the triploid chromosomes may be divided into as many as four cells. divided into as many as four cells. Because there is no mechanism to Because there is no mechanism to ensure that each of the four cells ensure that each of the four cells receives the proper number and type of receives the proper number and type of chromosomes, the chromosomes would chromosomes, the chromosomes would be apportioned unequally. Some cells be apportioned unequally. Some cells receive extra copies of certain receive extra copies of certain chromosomes and other cells lack them. chromosomes and other cells lack them.

The fast block to polyspermyThe fast block to polyspermyThe The fast block to poly-spermyfast block to poly-spermy is  is achieved by changing the electric achieved by changing the electric potential of the egg plasma membrane. potential of the egg plasma membrane. This membrane provides a selective This membrane provides a selective barrier between the egg cytoplasm and barrier between the egg cytoplasm and the outside environment, and the ionic the outside environment, and the ionic concentration of the egg differs greatly concentration of the egg differs greatly from that of its surroundings. This from that of its surroundings. This concentration difference is especially concentration difference is especially significant for sodium and potassium ions. significant for sodium and potassium ions. Seawater has a particularly high sodium Seawater has a particularly high sodium ion concentration, whereas the egg ion concentration, whereas the egg cytoplasm contains relatively little sodium. cytoplasm contains relatively little sodium. The reverse is the case with potassium The reverse is the case with potassium ions. This condition is maintained by the ions. This condition is maintained by the plasma membrane, which steadfastly plasma membrane, which steadfastly inhibits the entry of sodium ions into the inhibits the entry of sodium ions into the oocyte and prevents potassium ions from oocyte and prevents potassium ions from leaking out into the environment. If we leaking out into the environment. If we insert an electrode into an egg and place insert an electrode into an egg and place a second electrode outside it, we can a second electrode outside it, we can measure the constant difference in charge measure the constant difference in charge across the egg plasma membrane. across the egg plasma membrane. This This resting membrane potentialresting membrane potential is  is generally about 70 mV, usually expressed generally about 70 mV, usually expressed as –70 mV because the inside of the cell as –70 mV because the inside of the cell is negatively charged with respect to the is negatively charged with respect to the exterior.exterior.

The slow block to polyspermyThe slow block to polyspermy

cortical granule reactioncortical granule reaction, a slower, mechanical block to polyspermy that becomes active about a minute after the first , a slower, mechanical block to polyspermy that becomes active about a minute after the first successful sperm-egg attachment. Directly beneath the sea urchin egg plasma membrane are about 15,000 cortical granules, successful sperm-egg attachment. Directly beneath the sea urchin egg plasma membrane are about 15,000 cortical granules, each about 1 μm in diameter. Upon sperm entry, these cortical granules fuse with the egg plasma membrane and release their each about 1 μm in diameter. Upon sperm entry, these cortical granules fuse with the egg plasma membrane and release their contents into the space between the plasma membrane and the fibrous mat of vitelline envelope proteins. Several proteins are contents into the space between the plasma membrane and the fibrous mat of vitelline envelope proteins. Several proteins are released. The first are proteases. These enzymes dissolve the protein posts that connect the vitelline envelope proteins to the released. The first are proteases. These enzymes dissolve the protein posts that connect the vitelline envelope proteins to the cell membrane, and they clip off the bindin receptor and any sperm attached to it .Mucopolysaccharides released by the cell membrane, and they clip off the bindin receptor and any sperm attached to it .Mucopolysaccharides released by the cortical granules produce an osmotic gradient that causes water to rush into the space between the plasma membrane and the cortical granules produce an osmotic gradient that causes water to rush into the space between the plasma membrane and the vitelline envelope, causing the envelope to expand and become the vitelline envelope, causing the envelope to expand and become the fertilization envelopefertilization envelope. A third protein released by the . A third protein released by the cortical granules, a peroxidase enzyme, hardens the fertilization envelope by crosslinking tyrosine residues on adjacent cortical granules, a peroxidase enzyme, hardens the fertilization envelope by crosslinking tyrosine residues on adjacent proteins .The fertilization envelope starts to form at the site of sperm entry and continues its expansion around the egg. As it proteins .The fertilization envelope starts to form at the site of sperm entry and continues its expansion around the egg. As it forms, bound sperm are released from the envelope. This process starts about 20 seconds after sperm attachment and is forms, bound sperm are released from the envelope. This process starts about 20 seconds after sperm attachment and is complete by the end of the first minute of fertilization. Finally, a fourth cortical granule protein, hyalin, forms a coating complete by the end of the first minute of fertilization. Finally, a fourth cortical granule protein, hyalin, forms a coating around the egg. The egg extends elongated microvilli whose tips attach to this around the egg. The egg extends elongated microvilli whose tips attach to this hyaline layerhyaline layer. This layer provides support for . This layer provides support for the blastomeres during cleavage. In mammals, the cortical granule reaction does not create a fertilization envelope, but its the blastomeres during cleavage. In mammals, the cortical granule reaction does not create a fertilization envelope, but its ultimate effect is the same. Released enzymes modify the zona pellucida sperm receptors such that they can no longer bind ultimate effect is the same. Released enzymes modify the zona pellucida sperm receptors such that they can no longer bind sperm (Bl. During this process, called the sperm (Bl. During this process, called the zona reaction,zona reaction, both ZP3 and ZP2 are modified.  both ZP3 and ZP2 are modified. in mice N-acetylglucosaminidase in mice N-acetylglucosaminidase enzymes cleave it from ZP3enzymes cleave it from ZP3

Calcium as the initiator of the cortical granule reactionCalcium as the initiator of the cortical granule reaction

Upon fertilization, the intracellular calcium ion concentration of the egg Upon fertilization, the intracellular calcium ion concentration of the egg increases greatly. In this high-calcium environment, the cortical granule increases greatly. In this high-calcium environment, the cortical granule membranes fuse with the egg plasma membrane, releasing their contents. membranes fuse with the egg plasma membrane, releasing their contents. Once the fusion of the cortical granules begins near the point of sperm Once the fusion of the cortical granules begins near the point of sperm entry, a wave of cortical granule exocytosis propagates around the cortex to entry, a wave of cortical granule exocytosis propagates around the cortex to the opposite side of the egg.the opposite side of the egg.The calcium ions responsible for the cortical granule reaction are stored in The calcium ions responsible for the cortical granule reaction are stored in the endoplasmic reticulum of the egg .In sea urchins and frogs, this the endoplasmic reticulum of the egg .In sea urchins and frogs, this reticulum is pronounced in the cortex and surrounds the cortical reticulum is pronounced in the cortex and surrounds the cortical granules .In granules .In Xenopus,Xenopus, the cortical endoplasmic reticulum becomes ten times  the cortical endoplasmic reticulum becomes ten times more abundant during the maturation of the egg and disappears locally more abundant during the maturation of the egg and disappears locally within a minute after the wave of cortical granule exocytosis occurs in any within a minute after the wave of cortical granule exocytosis occurs in any region of the cortex. Once initiated, the release of calcium is self-region of the cortex. Once initiated, the release of calcium is self-propagating. Free calcium is able to release sequestered calcium from its propagating. Free calcium is able to release sequestered calcium from its storage sites, thus causing a wave of calcium ion release and cortical storage sites, thus causing a wave of calcium ion release and cortical granule exocytosis.granule exocytosis.

Egg ActivationEgg Activationincrease in calcium levels trigger many reactions increase in calcium levels trigger many reactions most protein synthesis comes from stored mRNA by most protein synthesis comes from stored mRNA by removing an inhibitorremoving an inhibitor

EARLY RESPONSESEARLY RESPONSESSperm-egg binding-0 secondsSperm-egg binding-0 secondsFertilization potential rise (fast block to polyspermy)- 1 secFertilization potential rise (fast block to polyspermy)- 1 secSperm-egg membrane fusion-6 secSperm-egg membrane fusion-6 secCalcium increase first detected-6 secCalcium increase first detected-6 secCortical vesicle exocytosis (slow block to polyspermy)-15–60 secCortical vesicle exocytosis (slow block to polyspermy)-15–60 sec

LATE RESPONSESLATE RESPONSESActivation of NAD kinase starts at 1 minActivation of NAD kinase starts at 1 minIncrease in NADH and NADPH starts at 1 minIncrease in NADH and NADPH starts at 1 minIncrease in O2 consumption starts at 1 minIncrease in O2 consumption starts at 1 minSperm entry1–2 minSperm entry1–2 minAcid efflux1–5 minAcid efflux1–5 minIncrease in pH (remains high)1–5 minIncrease in pH (remains high)1–5 minSperm chromatin decondensation2–12 minSperm chromatin decondensation2–12 minSperm nucleus migration to egg center2–12 minSperm nucleus migration to egg center2–12 minEgg nucleus migration to sperm nucleus5–10 minEgg nucleus migration to sperm nucleus5–10 minActivation of protein synthesis starts at 5–10 minActivation of protein synthesis starts at 5–10 minActivation of amino acid transport starts at 5–10 minActivation of amino acid transport starts at 5–10 minInitiation of DNA synthesis20–40 minInitiation of DNA synthesis20–40 minMitosis60–80 minMitosis60–80 minFirst cleavage85–95 minFirst cleavage85–95 min

Possible mechanisms of Possible mechanisms of

egg activation.egg activation.

The bindin receptor in the The bindin receptor in the egg plasma membrane egg plasma membrane has tyrosine kinase has tyrosine kinase activity. The tyrosine activity. The tyrosine kinase activates PLC. (B) kinase activates PLC. (B) The bindin receptor The bindin receptor activates a cytoplasmic activates a cytoplasmic tyrosine kinase. (C) An tyrosine kinase. (C) An activated tyrosine kinase activated tyrosine kinase in the sperm plasma in the sperm plasma membrane activates the membrane activates the egg pathways. (D) Soluble egg pathways. (D) Soluble activator pathways activator pathways activate PLC or directly activate PLC or directly release calcium from the release calcium from the endoplasmic reticulum.endoplasmic reticulum.

Fusion of Genetic MaterialFusion of Genetic Materialsperm contribute DNA & centriole to form initial spindle sperm contribute DNA & centriole to form initial spindle for division to the developing embryo.for division to the developing embryo.In sea urchins sperm nucleus must decondense to form In sea urchins sperm nucleus must decondense to form pronucleus – involves phosphorylation of lamin protein in pronucleus – involves phosphorylation of lamin protein in envelop and two sperm histones – can fuse with egg envelop and two sperm histones – can fuse with egg pronucleuspronucleus

In mammals fusion is longer process – sperm DNA bound to protamines in In mammals fusion is longer process – sperm DNA bound to protamines in compacted form – glutathione breaks dissulfide bonds to de-compact DNA; when compacted form – glutathione breaks dissulfide bonds to de-compact DNA; when sperm enters egg hasn’t complete meiosis; DNA synthesis occurs separately in sperm enters egg hasn’t complete meiosis; DNA synthesis occurs separately in each pronucleus; true diploid nucleus doesn’t form untill 2-cell stage.each pronucleus; true diploid nucleus doesn’t form untill 2-cell stage.