Unformatted text preview: Biology 442 Biology
Developmental Biology Developmental
Lectures 5 & 6 – Fertilization Fertilization – Process by which 2 gametes fuse to create a new individual with genetic potentials derived from both parents. derived Major events in fertilization: Recognition and contact between egg and sperm Regulation of sperm entry into the egg Activation of the egg metabolism Fusion of the genetic material Rearrangement of the egg cytoplasm Recognition and contact between egg and sperm Recognition 5 basic steps common to all systems 1. 2. 3.
4. 5. Chemoattraction of sperm to egg by soluble Chemoattraction molecules secreted by egg. molecules Or 3. The exocytosis of the acrosomal vesicle to release its enzymes. to Or 2. Binding of the sperm to the extracellular envelope (vitelline layer or zona pellucida of the egg. egg. Passage of the sperm through the extracellular Passage envelope envelope Fusion of egg and sperm membranes. Focus on 2 systems – Sea Urchin where fertilization is external and Mammals were fertilization is internal 1) Chemoattraction of sperm to egg by soluble molecules secreted by egg.
Action at a distance – species where fertilization is external: Cnidarians, molluscs, echinoderms, -- sperm locate eggs by chemotaxis Eggs release chemoattractant when they are ready to be fertilized. Example – sperm added to eggs before 2ond meiotic division – no sperm attracted to egg. Sperm added to eggs after second meiotic division – sperm swim to eggs! In sea urchins the chemoattractant has been identified – a 14 amino acid peptide called resact. No Resact Sperm swim in circles. 20, 40 and 90 sec after Resact addition Sperm swim to site of Resact addition Resact attraction of sperm is Species Specific – binds to a receptor on sperm membrane Resact also activates sperm -- increases mitochondrial respiration and sperm motility Action at a distance – species where fertilization is internal: In mammals – sperm is ejaculated inside the reproductive tract, but only ~200 of 200-300 x10e6 sperm will get near the egg The female reproductive tract plays an active role in helping the sperm find the egg. 1.) transport of sperm into uterus – uterine wall makes muscular contractions that push the sperm in. 2.) Capacitation - in order to fertilize the egg the sperm must spend time in the reproductive tract otherwise it can not penetrate the cumulus cell layer around the egg. 3.) Hyperactivation – once in oviduct sperm swim even faster 4.) Chemotaxis – like resact –follicular fluid can attract sperm but only if it is from fertilizable eggs. Once in the vicinity of the egg, the sperm must get through Egg Jelly and Vitelline Envelope in Sea Urchin, or the Cumulus and then the Corona Radiata to reach the Zona Pleucida in mammals 1. Or 1. 3. The exocytosis of the acrosomal vesicle to release its enzymes. enzymes. 2. Or 2. Binding of the sperm to the extracellular envelope (vitelline layer or zona pellucida of the egg). (vitelline The Acrosome Reaction Acrosome
Two Parts 1. Fusion of the sperm cell membrane with the acrosomal vesicle – exocytosis 1. Extension of the acrosomal process The Acrosome Reaction The Acrosome
In Sea Urchin initiated by contact of the sperm with the egg jelly – causes exocytosis of acrosomal vesicle and release of proteolytic enzymes that digest a path through the jelly coat to the egg surface 3 components of egg jelly bind to receptors on sperm cell membrane directly above acrosome – binding opens calcium ion channels permitting Ca++ entry to sperm head. Exocytosis of acrosomal vesicle is caused by Ca++ mediated binding of acrosomal vesicle to sperm plasma membrane The egg jelly factors are species specific – will only bind to sperm membrane receptors of the same species – activation of the acrosomal reaction constitutes a barrier to interspecies fertilizations The second part of the reaction involves extension of the acrosomal process – results from polymerization of globular actin monomers into actin filaments. The influx of Ca++ is thought to activate RhoB (small GTP binding protein). Rho B then aids in Actin polymerization. Species specific sperm-egg recognition in Sea Urchins Species
Once the acrosomal process is extended the sperm acrosome contacts the egg surface and binds to the egg surface in a species specific manner. The sperm acrosomal protein responsible for species specific binding to the egg surface is Bindin Binding is species specific Bindin is located on the sperm acrosomal process Experiment Results There are a limited number of bindin receptors on the egg surface –Viteline Envelope ( note the space on the egg surface where sperm are not bound) sperm Polystyrerne beads coated with Bindin Receptor Protein A 350 Kd glycoprotein has been isolated from egg Viteline Envelope that has the properties of a Bindin Receptor Bindin In Sea Urchins Species Specific Recognition Between Gametes Occurs at 3 Points Between
Sperm attraction to the egg (Resact peptide mediates species specific attraction) mediates Sperm activation (acrosomal reaction – species Sperm specific proteins in egg jelly initiate reaction) specific Sperm adhesion to the egg surface – Viteline Sperm Envelope (Bindin –receptor interaction is species Envelope specific specific Gamete binding and recognition in Mammals Mammals
Zona Pellucida in mammals is analogous to the Viteline Envelope in invertebrates Viteline Binding of Sperm to the Zona Pellucida is Binding relatively but not absolutely species specific – species specificity is not as important in species where fertilization is internal ! where Acrosomal Reaction occurs AFTER sperm Acrosomal binding to the Zona Pellucida in mammals. binding Zona Pellucida is made up of 3 major glycoproteins – ZP1, ZP2 and ZP3 ZP1,
ZP3 is the sperm binding site
Binding competition experiment showing that ZP3 competes binding by sperm and carbohydrate portion is necessary There are 3 ZP3 binding proteins located in the sperm head Which one or if all three are necessary is still not known In addition to binding sperm the ZP3 glycoprotein In also initiates the acrosomal reaction also
The zona pellucida is a thick structure – by undergoing the acrosomal reaction after binding, the sperm can concentrate its proteolytic enzymes at the point of attachment and “drill a hole” through the zona. Sperm that have undergone the acrosomal reaction before binding can not penetrate the Zona The mouse sperm acrosomal reaction is induced when ZP3 crosslinks the receptors on the sperm cell membrane. One of the sperm proteins that is crosslinked is galactosyltansferase-1 a transmembrane enzyme galactosyltansferase-1 whose active site faces outward and binds to ZP3 This binding reaction initiates a G protein cascade that opens membrane Ca++ channels and causes Ca++ mediated exocytosis of the sperm acrosomal vesicle The anterior portion of the sperm membrane is shed during the acrosomal reaction Enzymes released from the acrosomal vesicle include proteases that digest a hole through the Zona Pellucida that the sperm can travel through toward the egg the As sperm penetrate the zona pellucida, proteins on the inner As acrosomal membrane bind to the egg ZP2 protein in a second binding reaction. binding ZP2
ZP3 binds binds Acrosome intact Acrosome sperm will NOT bind ZP2 but acrosomal reacted sperm do! reacted Once the sperm has undergone the acrosomal reaction and Once traveled to the egg the fusion of the sperm cell membrane and the egg cell membrane can begin. and Sperm-Egg Fusion Sperm-Egg
Causes polymerization of actin in the egg to from a Causes Fertilization Cone – a discrete site - exists for several days days Marks a point that will 1) direct the plane of first cleavage Marks 1) and 2) is the site around which cytoplasmic 2) rearrangements will occur rearrangements The actin polymerization widens a cytoplasmic bridge The between the egg and sperm and the sperm nucleus and tail pass through while the acrosomal matrix is shed tail Prevention of Polyspermy Prevention
Normal fertilizaton – sperm Normal contributes 1 haploid nucleus and 1 centriole and egg provides 1 haploid nucleus and you get a diploid zygote. diploid Polyspermy – fertilization by more than one sperm is disaster for the egg !!! Species have evolved ways of Species preventing polyspermy preventing
Fast block to polyspermy Slow block to polyspermy Slow
Fast block to polyspermy – fusion of the sperm with the Fast
egg plasma membrane causes a change in the electrical potential of the egg plasma membrane that prevents fusion of additional sperm. Na+ +-+- + + + - Egg cytoplasm is low in Na+, high in K+ Sea water is high in Na+ and low in K+ Egg membrane keeps ions on both sides by pumping Na+ out. The resting potential is –70 mV (inside is more negative) - - Egg -70 mVolts 1-3 sec after sperm binding there is a small influx of Na+ ions. This changes the membrane potential from -70 mVolts to +20 mVolts. to Sperm can bind at -70 mVolts but not at +20 mVolts Sperm membrane potential. membrane The fast block to polyspermy is very short – only lasts about 30 seconds --- so need a more effective long term solution !! 30 Slow Block to Polyspermy: Slow The Cortical Granule Reaction The Results in removal of sperm bound to the egg cell Results membrane. membrane. Initiated about 1 minute after first successful fusion Initiated of sperm to egg. of Directly beneath the egg cell membrane are ~ Directly 1500 cortical granules. They are ~1um diameter cortical They Upon sperm entry the cortical granules fuse with Upon the egg cell membrane and release their contents into the space between the cell membrane and the vitelline envelope proteins. Exocytosis Exocytosis Cortical granule content in Sea Urchins includes: Cortical 1) a trypsin like protein – cortical granule serine protease that 1) dissolves protein posts that connect the vitelline envelope to the cell membrane. It removes the bindin receptors and the attached sperm. 2) Mucopolysaccharides – their release causes an osmotic gradient that causes water to rush into the space between the cell membrane and the vitelline envelope. This causes the envelope to expand and become the fertilization envelope. fertilization 3) Peroxidase enzyme that hardens the fertilization envelope 3) by crosslinking tyrosine residues on adjacent proteins. by The cortical granule reaction and formation of the fertilization envelope starts at the point of sperm entry and continues around the egg. and A 4th set of cortical granule proteins includes Hyalin Hyalin which forms a coating around the egg. The layer provides support for the blastomeres during cleavage cleavage In Mammals – cortical granule reaction does not In generate a fertilization envelope. Instead the released enzymes modify the zona pellucida sperm receptors so they can not bind sperm. sperm
Cortical granules of mouse eggs have Nacetylglucosaminidase enzymes that cleave Nacetylglucosamine from ZP3 carbohydrate acetylglucosamine chains. chains. ZP2 is cleaved by another released enzyme This removes the 2 main sperm binding proteins This from the egg surface from Ca++ is the initiator of the cortical granule reaction. Ca++
Upon fertilization – the intracellular free Ca++ concentration in Upon the egg increases. The high intracellular Ca++ causes the cortical granule membranes to fuse with the egg cell membranes ( exocytose ). membranes The rise in free Ca++ in the egg is not due to transport of The Ca++ into the egg but is due to release of Ca++ from egg intracellular stores. intracellular Endoplasmic reticulum Endoplasmic rich in Ca++ channel proteins surrounds cortical granules cortical Ca++ is released as a wave across the egg starting at the point of sperm entry. starting Activation of the Egg Metabolism Activation
The Mature Oocyte is a very sluggish cell that is activated by The sperm entry ! Activation sets into motion a preprogrammed set of metabolic Activation events leading to DNA replication and the first mitotic division of the fertilized egg. of Late Responses Early Responses Early Responses: occur within seconds of sperm binding Late Responses: take place Late several minutes after fertilization fertilization Several waves of Several Ca++ release Ca++ Late Responses: Late
1) Activation of 1) NAD+ kinase to form NADP, a form
coenzyme in lipid biosynthesis needed for new membrane synthesis during cleavage cleavage Ca++ plus Ca++ in pH cause in 2) Activation of Protein 2) Synthesis – stored maternal Synthesis
mRNAs recruited onto ribosomes and translated translated Increase in the % ribosomes in polysomes ribosomes The initial increase in The protein synthesis occurs even when transcription is inhibited by actinomycin indicating that stored maternal mRNAs are being translated translated Stored Maternal mRNAs Stored 3) Stimulation of DNA replication and restoration of the mitotic cell cycle mitotic
1) Releases the egg from it’s meiotic block allowing formation of a haploid egg nucleus ( female pronucleus) block 2) Sperm nucleus must decondense (male pronucleus) 3) Egg and Sperm pronuclei must fuse to form the diploid zygote nuclei 4) DNA replication must start and the cell must enter the rapid mitotic cell cycles. Can start before, after or during the nuclei fusion process cell 5) Cytoplasmic rearrangement During Oogenesis the egg has been arrested in Meiosis. Fertilization breaks the Meiotic arrest. Meiosis.
The stage of meiosis at which the egg is fertilized The differs for different organisms differs Focus on mammals initially 2ond metaphase arrest is caused by high levels of MPF which MPF depends on mos protein and cytostatic factor which inhibits the mos degradation of cyclin. cyclin Ca++ release activates calmodulin (Ca++ binding protein) Ca++ calmodulin which activates Calmodulin dependent kinase which causes Calmodulin degradation of CSF, which allows degradation of cyclin and loss of MPF activity. loss Ca++ release inhibits MAP kinase which allows DNA Ca++ replication to take place replication While egg nucleus is finishing its meiotic divisions and becoming haploid (female pronucleus) the sperm nucleus must becoming the “decondense” its DNA on it’s way to becoming the male pronucleus pronucleus In Sea Urchins: 1. The sperm nucleus and centriole separate from the mitochondria 1.
2. 3. 4. 5. 5. and flagella. The sperm mitochondria and flagella disintegrate. and The sperm nuclear envelope vesiculates into many small vesicles The surrounding the chromatin surrounding The proteins holding the sperm chromatin in its condensed state The are removed and exchanged for other proteins including histones from the egg cytoplasm (less tight histones). from This decondensation is initiated by phosphorylation of the nuclear This lamin protein as well as phosphorylation of 2 sperm histone proteins. Phosphorylation of the histones is on basic amino acids and thus Phosphorylation loosens the DNA-histone interactions allowing the sperm histones to be replaced by histones stored in the egg cytoplasm. to 1. The male pronucleus rotates 180 degrees so that the 1. The centriole is between the sperm and egg pronuclei. centriole 2. The sperm centriole than sends out its asters and The functions as a microtubule organizing center and sets up the mitotic spindle. the 3. The two pronuclei migrate towards each other and fuse to The form the zygote nucleus which is now diploid. zygote diploid 4. Zygote DNA replication begins either during migration of Zygote the pronuclei or after their fusion. the 5. The process takes 1 hour. The In Mammals: 1. Process takes about 12 hours 2. DNA of the sperm is bound by protamines (very basic DNA protamines proteins that bind DNA very tightly). These are also further tightly compacted by disulfide bonds. further 1. Glutathione in the egg cytoplasm reduces these disulfide 1. Glutathione bonds and allows uncoiling of the sperm chromatin. bonds 2. The centriole accompanying the male pronucleus The produces its asters using tubulin stored in the egg cytoplasm. cytoplasm. 3. The microtubules join the 2 pronuclei and enable them to The migrate towards each other. migrate Male pronucleus with asters (centrioles) Female pronucleus without asters 1. DNA synthesis occurs separately in each pronucleus. 1. DNA 2. Upon meeting the two pronuclear envelopes breakdown Upon but the two nuclei don’t fuse. Instead the chromosomes condense and orientate themselves on a mitotic spindle. The first true diploid nucleus in mammalian embryos is first not formed until the 2 cell stage. 3. The sperm mitochondria are either selectively targeted 3. The for destruction in the egg or lost by dilution during cleavage so that all mitochondria are inherited from the egg (mother). Egg and sperm pronuclei migrating toward each other on microtubule tracks. on Migration and fusion of mammalian egg and sperm pronuclei Migration Female pronucleus Male pronucleus with asters Sperm tail Sperm flagella Rearrangement of the Egg Cytoplasm Rearrangement
Fertilization can initiate a major reordering of the egg’s Fertilization cytoplasmic materials. cytoplasmic Rearrangement is more obvious in some species than others. Rearrangement Not obvious in sea urchins and mammals but very apparent in tunicates (colored cytoplasm) and amphibians that have a darkly pigmented animal pole and a lightly pigmented vegetal pole. Frogs: Initially egg is radially symmetrical around the animal vegetal axis . After sperm entry cortical (outer) cytoplasm shifts 30 degrees toward the point of sperm entry (fertilizaton cone) relative to the inner cytoplasm. to The underlying cytoplasm near the equator and directly opposite the point of sperm entry is “uncovered” by the cortical rotation. This is referred to as the Grey Crescent and is the point where gastrulation will initiate. gastrulation The motor for the rearrangement is a parallel array of The microtubule tracks that assemble between the outer cortical cytoplasm and the inner cytoplasm. They assemble just before rotation and disassemble right after rotation. rotation Microtubules initiate from the sperm centriole and microtubule Microtubules inhibitors block rotation (colchicine or UV). inhibitors Cortical rotation initiates a cascade of events that will form the Cortical dorsal ventral axis of the embryo dorsal Cytoplasmic Rearrangement in the Fertilized Egg of Styela partita, a tunicate with colored cytoplasm Styela
1. Before fertilization yellow cortical cytoplasm surrounds grey yoky Before inner cytoplasm. inner 2. After sperm entry in vegetal hemisphere yellow cortical cytoplasm After and clear cytoplasm derived from breakdown of egg nucleus stream toward sperm. toward 3. Sperm nucleus migrates toward animal end and yellow and clear Sperm cytoplasm move with it. cytoplasm 4. Cells arising from parts of the egg containing yellow cytoplasm will Cells become tail muscle Non-Equivalence of Male and Female Pronuclei Non-Equivalence
Males and females carry genetically equal haploid genomes Males but Genomic Imprinting makes some sperm or egg derived genes functionally non-equivalent. functionally Early evidence for Non-equivalence of male and female Early pronuclei comes from 2 sources. pronuclei 1) Hydatidform mole: haploid sperm fertilize an egg lacking 1) a pronucleus. Sperm chromosomes duplicate and restore the diploid state giving an egg with all of the chromosomal content from the sperm. content The cells survive and divide but there is no embryo – The just a mass of placenta like tissue. just Genomic imprinting caused by DNA methylation. by
Methylation can silence genes. In primordial germ cells all genes In are demethylated. are During gametogenesis some genes During are methylated and silenced in males and others in females. males Diploid zygote with a male and Diploid female contribution gets an active copy of each gene copy 2) Parthenogenic Activation of the egg: The ability of an embryo to develop without a sperm contribution. Many invertebrates and some vertebrates are capable of normal development parthenogenically. parthenogenically But not mammals!! Can artificially activate a mouse egg and suppress the last meiotic division so that the nucleus is 2N with all chromosomes from the female. Forms embryo with spinal chords, beating hearts, skeletons, organs but dies at 10 days. Pronuclear transplantation experiments confirm nonconfirm equivalence of equivalence male and female pronuclei in mammals. Specific Examples: Insulin-like growth factor gene is active only on sperm derived chromosomes . derived Insulin-like growth factor receptor gene is active only on egg derived chromosomes. Mouse pup that inherits a deletion of the receptor gene from its father is normal but if it inherits a deletion of the gene from its mother the fetus dies late in gestation. its Inheritance of a deletion of Inheritance chromosome 15 has different phenotypes depending on whether the deletion is from the male or female chromosome chromosome Just because it is a fertilized egg --- it is not necessarily a viable fetus !!! not ...
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