Fertilization Notes
Fertilization: Beginning a New Organism
Fertilization is the fusion of two sex cells (gametes) to create a new individual with genetic potentials from both parents.
Fertilization serves two purposes:
Sex: Combining genes from both parents.
Reproduction: Creating new organisms.
Functions of fertilization:
Transmit genes from parent to offspring.
Initiate reactions in the egg cytoplasm to allow development.
Four major events in fertilization:
Contact and recognition between sperm and egg to ensure they are of the same species.
Regulation of sperm entry to allow only one sperm to fertilize the egg.
Fusion of the genetic material of sperm and egg.
Activation of egg metabolism to start development.
A complex dialogue exists between egg and sperm: the egg activates sperm metabolism, and the sperm activates egg metabolism.
Structure of the Gametes
Sperm's role in fertilization was discovered recently.
Anton van Leeuwenhoek (1678) co-discovered sperm and initially thought they were parasitic animals in semen.
Spermatozoa means "sperm animals."
He later thought each sperm contained a preformed embryo.
Leeuwenhoek (1685) believed sperm were seeds and the female provided nutrient soil, similar to Aristotle's idea.
Nicolas Hartsoeker, another co-discoverer, drew a preformed human ("homunculus") within the sperm.
Early Experiments on Sperm
Lazzaro Spallanzani in the late 1700s demonstrated that filtered toad semen without sperm would not fertilize eggs.
He concluded the viscous fluid, not sperm, was the agent of fertilization.
Spallanzani also considered spermatic "animals" as parasites.
In 1824, J. L. Prevost and J. B. Dumas claimed sperm were active agents of fertilization.
They noted sperm's universal presence in sexually mature males and absence in immature/aged individuals and sterile mules.
They proposed sperm entered the egg to contribute to the next generation.
In the 1840s, A. von Kolliker described sperm formation from cells in the testes.
He dismissed the idea of semen supporting numerous parasites.
Von Kolliker denied physical contact between sperm and egg, suggesting sperm excited the egg to develop, like a magnet.
In 1876, Oscar Hertwig and Herman Fol independently showed sperm entry into the egg and union of nuclei.
Hertwig used Mediterranean sea urchin, Toxopneustes lividus, for microscopic observations due to its transparent eggs available in large numbers.
Hertwig observed a sperm entering an egg and the two nuclei uniting.
He noted only one sperm entered each egg and all embryo nuclei derived from the fused nucleus.
Fol detailed sperm entry mechanism.
Fertilization was recognized as the union of sperm and egg.
Sperm Structure
Each sperm has a haploid nucleus, a propulsion system, and enzymes to enter the egg.
Most cytoplasm is eliminated during maturation, leaving modified organelles.
The haploid nucleus becomes streamlined, and DNA is tightly compressed.
(or acrosome) is derived from the Golgi apparatus and contains digestive enzymes.
It lyses the outer coverings of the egg.
In many species, globular actin molecules lie between the nucleus and the acrosomal vesicle forming the during early fertilization.
Sperm Propulsion
Sperm are propelled by amoeboid motion or flagella, depending on the species' environmental adaptations.
Flagella:
The major motor portion of the flagellum is called the .
Formed by microtubules emanating from the centriole at the base of the sperm nucleus.
Core: Two central microtubules surrounded by nine doublet microtubules.
One microtubule of each doublet is complete (13 protofilaments); the other is C-shaped (11 protofilaments).
Protofilaments are made of the dimeric protein tubulin.
Proteins critical for flagellar function:
: attached to microtubules, hydrolyzes ATP, converting chemical energy into mechanical energy for propulsion.
Active sliding of outer doublet microtubules causes flagellum to bend.
Individuals lacking dynein have Kartagener triad: sterile males (immotile sperm), bronchial infections (immotile respiratory cilia), and a 50% chance of reversed heart position.
Histone H1: Stabilizes flagellar microtubules to prevent disassembly.
The "9 + 2" microtubule arrangement with dynein arms is conserved for energy transmission.
for flagellar movement comes from mitochondria in the neck region of the sperm.
In mammals, a layer of dense fibers between the mitochondrial sheath and axoneme stiffens the sperm tail.
The thickness decreases toward the tip, preventing sudden whipping of the sperm head.
Sperm Maturation
Mammalian sperm differentiation is completed in the epididymis where they acquire the ability to move.
Motility is achieved through changes in the -generating system (possibly dynein modification) and plasma membrane fluidity.
Released sperm can move but cannot bind to and fertilize an egg until capacitation in the female reproductive tract.
Egg Structure
The nature egg (ovum) stores all material for growth and development.
The egg is larger than the sperm.
The developing egg (oocyte) conserves material and actively accumulates more.
Meiotic divisions conserve cytoplasm (rather than halving it).
The egg synthesizes or absorbs proteins, like yolk, acting as food reservoirs for the developing embryo.
The volume of a sea urchin egg is about .
Egg cytoplasmic components include:
Proteins: Energy and amino acid supply via yolk proteins.
Ribosomes and tRNA: For early protein synthesis.
Amphibian oocytes produce up to ribosomes during meiotic prophase.
Messenger RNA: Instructions for proteins made during early development, dormant until fertilization.
Sea urchin eggs contain types of mRNA.
Morphogenetic Factors: Direct differentiation of cells into certain cell types, localized in different regions of the egg.
Protective Chemicals: UV filters, DNA repair enzymes, distasteful molecules, and antibodies.
A large nucleus resides within the cytoplasm:
Can be haploid or diploid depending on the species.
Egg plasma membrane:
Regulates ion flow during fertilization and fuses with the sperm plasma membrane.
Vitelline envelope:
Outside the plasma membrane.
Fibrous mat around the egg.
Contains glycoproteins involved in sperm-egg recognition.
Vitelline posts adhere to the membrane.
In mammals, the vitelline envelope is a thick extracellular matrix called the zona pellucida.
A layer of cells called the cumulus surrounds the egg
Made up of ovarian follicular cells nurturing the egg.
Egg Cortex
Lying immediately beneath the plasma membrane of the egg is a thin region () of gel-like cytoplasm called the cortex.
High concentrations of globular actin molecules.
During fertilization, these actin molecules polymerize to form long cables of actin (microfilaments).
Microfilaments are necessary for cell division and are used to extend the egg surface into small projections - microvilli.
The cortex also contains cortical granules:
Membrane-bound structures homologous to the acrosomal vesicle of the sperm.
Golgi-derived organelles containing proteolytic enzymes.
The sea urchin egg contains approximately 15,000 cortical granules.
In addition to digestive enzymes, the cortical granules contain mucopolysaccharides, adhesive glycoproteins, and hyalin protein.
Many types of eggs also have an egg jelly outside the vitelline envelope:
Glycoprotein meshwork used to attract or activate sperm.
The egg is a cell specialized for receiving sperm and initiating development.
Recognition of Egg and Sperm
Proceeds in five basic steps:
Chemoattraction of sperm to the egg by soluble molecules secreted by the egg.
Exocytosis of the acrosomal vesicle to release its enzymes.
Binding of sperm to the extracellular envelope of the egg.
Passing of the sperm through this extracellular envelope.
Fusion of egg and sperm cell plasma membranes.
In mammals, steps 2 and 3 are reversed.
After these steps, the haploid sperm and egg nuclei can meet, and development can begin.
Marine organisms release gametes into the environment, facing two problems:
How sperm and eggs meet in a dilute concentration.
How to prevent sperm from fertilizing eggs of another species.
Two major mechanisms:
Species-specific attraction of sperm.
Species-specific sperm activation.
Sperm Attraction
Species-specific sperm attraction is documented in cnidarians, molluscs, echinoderms, and urochordates.
Sperm are attracted toward eggs by chemotaxis, following a chemical gradient secreted by the egg.
In 1978, Miller demonstrated that the eggs of the cnidarian Orthopyxis caliculata regulate the timing of the release of a chemotactic factor.
The eggs controlled not only the type of sperm they attract, but also the time at which they attract them.
Chemotaxis Mechanisms
Chemotactic molecule: resact
A 14-amino acid peptide isolated from the egg jelly of the sea urchin Arbacia punctulata.
Diffuses readily in seawater and has a profound effect at very low concentrations.
The sperm migrate towards the area of injection and congregate there.
Specific for A. punctulata and does not attract sperm of other species.
A. punctulata sperm have receptors in their plasma membranes that bind resact.
Resact also acts as a sperm-activating peptide.
Receptor for resact is a transmembrane protein.
Binding resact activates the receptor's enzymatic activity.
Activates mitochondrial ATP-generating apparatus and dynein ATPase, stimulating flagellar movement.
Acrosomal Reaction in Sea Urchins
Initiated by contact of the sperm with the egg jelly.
Two components:
Fusion of the acrosomal vesicle with the sperm plasma membrane, releasing the acrosomal vesicle's contents (exocytosis).
Extension of the acrosomal process.
Releases proteolytic enzymes that digest a path through the jelly coat to the egg surface.
The acrosomal reaction is thought to be initiated by a fucose-containing polysaccharide in the egg jelly that binds to the sperm and allows calcium to enter the sperm head.
Exocytosis of the acrosomal vesicle is caused by the calcium-mediated fusion of the acrosomal membrane with the adjacent sperm plasma membrane.
Egg jelly factors are often highly specific to each species.
The extension of the acrosomal process arises through the polymerization of globular actin molecules into actin filaments.
Species-Specific Recognition in Sea Urchins
After penetrating the egg jelly, the acrosomal process contacts the surface of the egg.
A major species-specific recognition step occurs.
Bindin is the acrosomal protein mediating this recognition.
In 1977, Vacquier and co-workers isolated this nonsoluble 30,500-Da protein from the acrosome of Strongylocentrotus purpuratus and found it to be capable of binding to dejellied eggs of the same species.
Different bindins exist among species.
There exists species-specific bindin receptors on the egg, vitelline envelope, or plasma membrane.
Vacquier and Payne (1973) experiments showed sperm binding does not occur over the entire egg surface, implying a limiting number of sperm-binding sites.
The bindin receptor on the egg has recently been isolated as a 350-kDa protein.
May have several regions that interact with bindin.
Recognizes only the bindin of the same species.
Other sites recognize a general bindin structure.
Receptors are aggregated into complexes on the egg cell surface.
Species-specific recognition of sea urchin gametes occurs at the levels of sperm attraction, sperm activation, and sperm adhesion to the egg surface.
Gamete Binding and Recognition in Mammals
The zona pellucida in mammals plays a role analogous to that of the vitelline envelope.
The glycoprotein matrix, which is synthesized and secreted by the growing oocyte, plays two major roles during fertilization:
It binds the sperm.
It initiates the acrosomal reaction after the sperm is bound.
The binding of sperm to the zona is relatively species-specific.
Bleil and Wassarman (1980, 1986 1988) isolated an 83-kDa glycoprotein, ZP3, from the mouse zona that was the active competitor for binding in this inhibition assay.
The other two zona glycoproteins they found, ZP1 and ZP2, failed to compete for sperm binding.
ZP3 also initiates the acrosomal reaction after sperm have bound to it.
The mouse sperm can concentrate its proteolytic enzymes directly at the point of attachment at the zona pellucida.
The current hypothesis of mammalian gamete binding postulates a set of proteins on the sperm capable of recognizing specific carbohydrate regions of ZP3.
Induction of Mammalian Acrosomal Reaction by ZP3
The acrosomal reaction in mammals occurs only after the sperm has bound to the zona pellucida.
The mouse sperm acrosomal reaction is induced by the crosslinking of ZP3 with the receptors for it on the sperm membrane.
This crosslinking opens calcium channels to increase the concentration of calcium in the sperm.
The mechanism by which ZP3 induces the opening of the calcium channels and the subsequent exocytosis of the acrosome remains controversial.
It may involve the receptor's activating a cation channel, which would change the resting potential of the sperm plasma membrane.
The difference between the acrosomal reaction in sea urchins and mammals may be due to the thickness of the extracellular envelopes surrounding the egg.
In mammals, the zona pellucida is a very thick matrix, so the sperm is far removed from the egg.
Sperm that undergo the acrosomal reaction before they reach the zona pellucida are unable to penetrate it.
Secondary Binding
During the acrosomal reaction, the anterior portion of the sperm plasma membrane is shed from the sperm.
In mice, secondary binding to the zona is accomplished by proteins in the inner acrosomal membrane that bind specifically to ZP2.
While acrosome-intact sperm will not bind to ZP2, acrosome-reacted sperm will.
After 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 specifically alters ZP2.
The structure of the zona consists of repeating units of ZP3 and ZP2, occasionally crosslinked by ZP1.
In guinea pigs, secondary binding to the zona is thought to be mediated by the protein PH-20.
Experiments show that the principle of immunological contraception is well founded.
Action at a Distance: Mammalian Gametes
It is very difficult to study the interactions that might be occurring between mammalian gametes prior to sperm-egg contact.
Fertilization occurs inside the oviducts of the female.
The sperm population ejaculated into the female is heterogeneous, containing spermatozoa at different stages of maturation.
There is a great deal of controversy concerning the mechanisms underlying the translocation of mammalian sperm to the oviduct.
Translocation and Capacitation
The reproductive tract of female mammals plays a very active role in the mammalian fertilization process.
Sperm motility is required for mouse sperm to encounter the egg once it is in the oviduct, it is probably a minor factor in getting the sperm into the oviduct.
Sperm appear to be transported to the oviduct by the muscular activity of the uterus.
Newly ejaculated mammalian sperm are unable to undergo the acrosomal reaction without residing for some time in the female reproductive tract.
The set of physiological changes that allow the sperm to be competent to fertilize the egg is called capacitation.
Sperm that are not capacitated are "held up" in the cumulus and so do not reach the egg.
The fertilizing sperm could have taken as long as 6 days to make the journey.
Capacitation is a transient event, and sperm are given a relatively brief window of competence in which they can successfully fertilize the egg.
Molecular Changes During Capacitation
Fluidity of the sperm plasma membrane is altered by the removal of cholesterol by albumin proteins found in the female reproductive tract.
Particular proteins or carbohydrates on the sperm surface are lost during capacitation.
The membrane potential of the sperm becomes more negative as potassium ions leave the sperm.
Protein phosphorylation occurs.
Sperm Translocation and Capacitation Connection
Uncapacitated sperm bind actively to the membranes of the oviduct cells in the narrow passage preceding it.
This binding is temporary and is broken when the sperm becomes capacitated.
The life span of the sperm is significantly lengthened by this binding, and its capacitation is slowed down.
This restriction of sperm entry into the ampulla, the slowing down of capacitation, and the expansion of sperm life span may have very important consequences.
Hyperactivation and Chemotaxis
When sperm pass from the uterus into the oviducts, they become hyperactivated, swimming at higher velocities and generating greater force than before.
While this behavior is not conducive to traveling in low-viscosity fluids, it is extremely well suited for linear sperm movement in viscous fluid.
Soluble factors in the oviduct may provide the directional component of sperm movement.
The ovum may secrete chemotactic substances that attract the sperm toward the egg during the last stages of sperm migration.
Human egg may secrete a chemotactic factor only when it is capable of being fertilized.
Gamete Fusion and the Prevention of Polyspermy
Recognition of sperm by the vitelline envelope or zona pellucida is followed by the lysis of that portion of the envelope or zona in the region of the sperm head by the acrosomal enzymes.
This lysis is followed by the fusion of the sperm plasma membrane with the plasma membrane of the egg.
Sperm-egg binding appears to cause the extension of several microvilli to form the fertilization cone.
The sperm and egg plasma membranes then join together, and material from the sperm membrane can later be found on the egg membrane.
In mammals, the fertilin proteins in the sperm plasma membrane are essential for sperm membrane-egg membrane fusion.
When the membranes are fused, the sperm nucleus, mitochondria, centriole, and flagellum can enter the egg.
Prevention of Polyspermy
After one sperm has entered the egg, the fusibility of the egg membrane becomes a liability.
In normal monospermy, a haploid sperm nucleus and a haploid egg nucleus combine to form the diploid nucleus of the fertilized egg (zygote).
Polyspermy: the entrance of multiple sperm leads to disastrous consequences in most organisms. Results in triploid nucleus.
Theodor Boveri demonstrated in 1902 that such cells either die or develop abnormally
Blocks to Polyspermy
Species have evolved ways to prevent the union of more than two haploid nuclei.
The most common way is to prevent the entry of more than one sperm into the egg.
The sea urchin egg has two mechanisms to avoid polyspermy: a fast reaction (electrical), and a slower reaction: cortical granule reaction (mechanical).
Fast Block to Polyspermy
Achieved by changing the electric potential of the egg plasma membrane.
The resting membrane potential is generally about because the inside of the cell is negatively charged with respect to the exterior.
Within 1-3 seconds after the binding of the first sperm, the membrane potential shifts to a positive level, about .
This change is caused by a small influx of sodium ions into the egg.
Sperm can fuse with membranes having a resting potential of , they cannot fuse with membranes having a positive resting potential, so no more sperm can fuse to the egg.
Importance of Sodium Ions
Polyspermy can be induced if sea urchin eggs are artificially supplied with an electric current that keeps their membrane potential negative.
Fertilization can be prevented entirely by artificially keeping the membrane potential of eggs positive.
The fast block to polyspermy can also be circumvented by lowering the concentration of sodium ions in the water.
Slow Block to Polyspermy
The eggs of sea urchins have a second mechanism to ensure that multiple sperm do not enter the egg cytoplasm.
The fast block to polyspermy is transient.
The membrane potential of the sea urchin egg remains positive for only about a minute.
This brief potential shift is not sufficient to prevent polyspermy.
The cortical granule reaction is a slower mechanical block that becomes active about a minute after the first successful sperm-egg attachment.
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.
Cortical Granule Exocytosis Proteins
Proteases 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 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 fertilization envelope.
A peroxidase enzyme hardens the fertilization envelope by crosslinking tyrosine residues on adjacent proteins.
Hyalin forms a coating around the egg.
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 sperm.
Called the zona reaction.
The cortical granules of mouse eggs contain enzymes capable of cleaving N-acetylglucosamine from ZP3 carbohydrate chains.
When the N-acetylglucosamine residues are removed at fertilization, ZP3 will no longer serve as a substrate for the binding of other sperm.
ZP2 is clipped by the cortical granule proteases and loses its ability to bind sperm as well.
Calcium as the Initiator of Cortical Granule Reaction
Similar to that of the acrosomal reaction.
Upon fertilization, the intracellular calcium ion concentration of the egg increases greatly.
In this high-calcium environment, the cortical granule membranes fuse with the egg plasma membrane, releasing their contents.
Starts near the point of sperm entry, a wave of cortical granule exocytosis propagates around the cortex to the opposite side of the egg.
Origin of Calcium
In sea urchins and mammals, the rise in calcium concentration responsible for the cortical granule reaction is not due to an influx of calcium into the egg, but rather comes from within the egg itself.
A drug, A23187, is a calcium ionophore
Transporting free calcium ions across lipid membranes.
The calcium ions responsible for the cortical granule reaction are stored in the endoplasmic reticulum of the egg.
Cortical endoplasmic reticulum becomes ten times more abundant during the maturation of the egg and disappears locally within a minute after the wave of cortical granule exocytosis occurs.
Free calcium is able to release sequestered calcium from its storage sites, thus causing a wave of calcium ion release and cortical granule exocytosis.
Activation of Egg Metabolism
Begins development.
The mature sea urchin egg is a metabolically sluggish cell that is activated by the sperm.
Responses of the egg to the sperm are