Download 1 Biology 4361 Developmental Biology Fertilization October 19

Biology 4361
Developmental Biology
Fertilization
October 19, 2006
Fertilization - fusion of sperm and egg - accomplishes:
A) sex (combining genes derived from two genomes)
B) reproduction
- transmits genes from parents to offspring
- initiates reactions in the egg cytoplasm that allows development to proceed
Generally, four major events occur:
1) Contact and recognition between sperm and egg.
- also ensures that egg and sperm are of the same species
2) Regulation of sperm entry into the egg
- only one sperm can fertilize
- usually, only one sperm is allowed to enter the egg; others are excluded
3) Fusion of the genetic material of the sperm and egg.
4) Activation of egg metabolism to start development.
Structure of the Gametes
Sperm
- rather obscure; took a bad rap for a long time, e.g.
- von Leeuwenhoek (co-discoverer of sperm) - considered sperm to be parasites in the
semen
- this “parasite” idea was popular through many generations of scientists
- note that sperm were discovered in 1676, but their role in fertilization was not fully
elucidated until 1876 (200 years! a long time to wait!; Oscar Hertwig, Herman Fol
(independently) demonstrated sperm entry into the egg and union of cells’ nuclei)
Sperm structure:
- head contains:
- streamlined haploid nucleus
- DNA in pseudo-crystalline form with protamines
- transcriptionally inactive
- acrosome (acrosomal vesicle)
- Golgi-derivative
- contains enzymes that digest proteins and complex sugars; used to lyse outer
coverings of the egg
- modified secretory vesicle
- many species contain region of globular actin proteins between sperm nucleus and
acrosomal vesicle
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- g-actin used to extend acrosomal process (filamentous actin) during early stages
of fertilization
- recognition between sperm and egg (i.e. species specificity) molecules located in
on the acrosomal process
- Flagellum (tail) contains:
- axoneme
- microtubules (tubulin) emanate form the basal centriole
- 9 + 2 construction
- 2 central microtubules
- 9 doublet microtubules
- one mt of doublet is complete (13 protofilaments)
- second mt of doublet is incomplete; C-shaped (11
protofilaments)
- NOTE - 9 +2 arrangement with dynein arms conserved in
axonemes throughout the eukaryotic kingdoms
- dynein attached to microtubules
- motor protein
- ATPase
- dynein activity causes outer mt to slide past inner, causing
flagellar bending
- midpiece
- mitochondria
- mammals - several in a spiral around axoneme
- sea urchins - one large mitochondrion in a ring around axoneme
Egg (ovum)
- all material necessary for the beginning of growth and development must be stored in the egg
- eggs actively accumulate material as they develop
- NOTE - volume of eggs v. sperm; sea urchin egg: 2 x 10-4 mm3 (200 picoliters); > 200X
volume of the sea urchin sperm
- proteins
- yolk (made in other organs; mostly liver)
- ribosomes and tRNA
- burst of protein synthesis after fertilization
- mRNA
- encodes proteins for use in the early stages of development
- messages remain repressed until after fertilization
- morphogenic factors
- direct the differentiation of cells into certain types
- transcription factors, paracrine factors
- localized regionally; segregated into different cells during cleavage
- protective chemicals
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- DNA repair
- UV filters
- antibodies
- alkaloids (defensive)
- in many (most) species, the final stages of egg meiosis take place while the sperm’s nuclear
material (male pronucleus) is traveling toward what will become the female pronucleus
Recognition of egg and sperm
The interaction of egg and sperm generally proceeds according to five basic steps
1. The chemoattraction of the sperm to the egg by soluble molecules secreted by the egg.
2. The exocytosis of the acrosomal vesicle to release its enzymes.
3. The binding of the sperm to the extracellular envelope (vitelline layer or zona pellucida)
of the egg.
4. The passage of the sperm through the extracellular envelope.
5. Fusion of egg and sperm cell membranes.
NOTE - sometimes steps 2 and 3 are reversed (e.g. mammalian fertilization); sperm binds
to extracellular matrix of the egg before releasing acrosomal contends
- after these 5 steps, sperm and egg pronuclei meet and development is initiated
External Fertilization in Sea Urchins
Fertilization challenges:
- how to bring two very small cell together in a large place
- how to ensure that only sperm and eggs of the same species join
Sperm attraction: Action at a distance
Species-specific sperm attraction documented in numerous species, including
cnidarians, molluscs, echinoderms, urochordates
chemotaxis - sperm are attracted towards eggs of their species
- sperm follow chemoattractant gradient
- Orthopyxis caliculata (cnidarian) regulates taxis (movement) and timing of sperm
- prior to 2nd meiotic division = no chemotaxis
- after 2nd meiotic division = chemotaxis
chemotaxis mechanisms
- molecular agents can be different even in closely related species (need to be!)
- sea urchins - motility acquired shortly after exposure to seawater
- in testes, pH is kept low (pH ~7.2) by high CO2 content
- in seawater, pH elevated to ~7.6
- results in activation of dynein ATPase (flagellar motor protein)
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- chemotaxis: guided by egg-derived peptides, e.g. resact
- Resact - 14aa peptide isolated from egg jelly of Arbacia punctulata
- sperm in sea water swim in circles
- add resact - sperm congregate
- resact specific for A. punctulata; will not activate sperm from
Strongylocentrotus purpuratus
- S. purpuratus chemotactic peptide - speract
- A. punctulata sperm have resact receptors in membrane
- resact binding activates guanylyl cyclase activity in cytoplasmic side of receptor
- cGMP activates a calcium channel
- Ca2+ influx provides directional cue
NOTE - a single resact molecule can provide directional information for sperm;
swim up a concentration gradient until they reach the egg
- resact also acts as a sperm-activating peptide (resact = respiration activating)
- increased mitochondrial respiration and sperm motility
- increase in cGMP and Ca2+ activates mitochondrial ATP generating apparatus
and dynein ATPase that stimulates flagellar movement
Acrosome reaction
- in marine invertebrates AR has two components:
1. Fusion of the acrosomal vesicle with the sperm cell membrane (exocytosis
results in the release of acrosomal contents
2. Extension of the acrosomal process
- sea urchin AR initiated by contact of sperm with egg jelly
- specific complex sugar in the egg jelly
- polysaccharide bind to specific receptors located on the sperm cell membrane
directly above the acrosomal vesicle
- egg jelly polysaccharides often highly specific to each species
- components from one species fail to activate AR in closely related species
- in S. purpuratus AR initiated by repeating polymer of fucose sulfate
- sulfated carbohydrate binds to its sperm receptor
- receptor activates :
1) Ca2+ transport channel - allows Ca2+ entry into sperm head
2) Na+/H+ exchanger that pumps Na+ ions into sperm as it pumps
H+ out
3) Phospholipase enzyme that make IP3 (inositol trisphosphate)
second messenger
- IP3 release Ca2+ from inside sperm (probably AR)
2+
- elevated Ca level in relatively basic cytoplasm triggers fusion of
acrosomal membrane with the adjacent sperm cell membrane =
initiates AR
- contact causes exocytosis of sperm acrosomal vesicle
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- release of proteolytic enzymes
- enzymes digest a path through jelly coat to the egg surface
- at the egg surface, the acrosomal process adheres to the vitelline envelope and
tethers the sperm to the egg
- extension of the acrosomal process
- polymerization of globular actin molecules into actin filaments
- Ca2+ influx activates protein RhoB in acrosomal region and midpiece (sea urchin)
- GTP = binding protein helps organize the actin cytoskeleton
- thought to be active in polymerizing actin to make acrosomal process
Species-specific recognition
- 1st event - jelly-coat stimulated AR
- 2nd event - binding of sperm protein - bindin
- species specific bindin
- bindin is located specifically on the acrosomal process
- bindins from closely related sea urchin species are different
- implies species-specific bindin receptors on egg vitelline envelope
- also, evenly spaced binding of saturating numbers of sperm to egg
indicates spaced binding sites
- EBR1 - 350kDa glycoprotein - displays properties expected of a bindin
receptor
- bindin receptors aggregate into complexes on the vitelline envelope; hundreds
may be needed to tether the sperm to the egg
- closely related sea urchin eggs have different binding specificities; eggs will
adhere only to the bindin of their own species
- species-specific recognition of sea urchin gametes occurs at the levels of sperm
attraction, sperm activation, and sperm adhesion to the egg surface
Fusion of the egg and sperm cell membranes
- sperm-egg fusion causes polymerization of egg actin to form a fertilization cone
- actin from both gametes forms a connection that widens the cytoplasmic bridge
between the egg and sperm
- sperm nucleus and tail pass through this bridge
- in sea urchins all regions of egg membrane are capable of fusing with sperm
- however, other species have specialized regions for sperm recognition and fusion
- fusion often mediated by specific “fusogenic” proteins
- bindin may play a role as a fusogenic protein
Prevention of Polyspermy
- monospermy (entrance of one nucleus and one centriole) necessary to restore diploid
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condition
- polyspermy = disastrous consequences
- triploid nucleus
- sperm centrioles divide to form four poles; chromosomes may be divided into four cells
- chromosomes apportioned unequally
- cells die or develop abnormally
Fast block to polyspermy
- achieved by changing the electric potential of the egg cell membrane
- membrane provides selective ion barrier between the egg cytoplasm and outside
environment
- seawater has high Na+ concentration; e.g. cytoplasm contains relatively little Na+
- seawater has low K+; cytoplasm has relatively high K+
- ionic imbalance maintained by the cell membrane; inhibits entry of Na+ and
prevents K+ leakage
- this imbalance creates an electrical potential across the membrane
- resting membrane potential ~-70 mV (expressed as negative because
inside of the cell is negatively charges with respect to the exterior)
- within 1-3 sec after binding of the first sperm, membrane potential shifts positively;
~ +20 mV
- change caused by a small influx of Na+ ions into the egg
- sperm can fuse at -70 mV, but cannot fuse at positive resting potentials
- polyspermy can be induced if sea urchin eggs are artificially supplied with an electric
current theat keeps their membrane potential negative
- conversely, fertilization can be prevented entirely by artificially keeping the membrane
potential of eggs positive
- fast block can be circumvented by lowering Na+ concentration in surrounding water
- if Na+ concentration not sufficient to cause positive shift in membrane potential,
polyspermy occurs
- fast block is transitory; membrane potential remains positive for only about a minute
- not sufficient to permanently prevent polyspermy
NOTE - electric block to polyspermy occurs in frogs, but probably not in most mammals
Slow block to polyspermy
- cortical granule reaction (slow block to polyspermy)
- slower, mechanical block to polyspermy
- becomes active about 1 min after first successful sperm-egg fusion
- found in most animals; including mammals
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- cortical granules contained just beneath the sea urchin cell membrane
~ 15,000
~ 1 :m
- upon sperm entry, cortical granules fuse with the egg cell membrane and release their
contents into the space between the cell membrane and fibrosis mat of vitelline
envelope proteins
- cortical granule serine protease - trypsin-like protease
- dissolves protein posts that connect the vitelline envelope proteins to the
cell membrane
- clips off bindin receptors and any sperm connected to them
- components of cortical granules fuse with vitelline envelope to form a
fertilization envelope
- fertilization envelope elevated from the cell membrane by
mucopolysaccharides released by the cortical granules
- sticky compounds
- produce an osmotic gradient - forces water to rush in
- causes envelope to expand and radially move away from
egg
- peroxidase enzyme released from cortical granules hardens fertilization envelope
by crosslinking tyrosine residues on adjacent proteins
- fertilization envelope begins to form at the site of sperm entry; continues expansion
around egg
- process starts about 20 sec after sperm attachment; complete by the end of the
first minute of fertilization
- hyaline (cortical granule protein) forms a coating around the egg - hyaline layer
- egg extends elongated microvilli
- microvilli tips attach to the hyaline layer
- hyaline layer provides support for the blastomeres during cleavage
Calcium as the initiator of the cortical granule reaction
- mechanism of cortical granule reaction similar to acrosome reaction
- may involve many to the same molecules
- at fertilization, egg cytoplasm Ca2+ concentration increases
- high Ca2+ causes cortical granule membranes to fuse with egg cell membrane
- in mammals and sea urchins, Ca2+ increase is from internal stores
- Ca2+ wave propagates across the egg
- release of Ca2+ starts at one end of the cell and proceeds actively to the other end
- entire release of Ca2+ is complete within ~ 30 sec
- free Ca2+ re-sequestered shortly after release
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- Ca2+ directly responsible for propagating cortical granule reaction
- in sea urchins and vertebrates (not snails and worms), Ca2+ stored in endoplasmic
reticulum of egg
- sea urchins and frogs- ER content is pronounced in the cortex and surrounds the
cortical granules
- cortical granules are tethered to the cell membrane by a series of integral
membrane proteins that facilitate calcium-mediated exocytosis
- as soon as Ca2+ released from ER, cortical granules fuse with the cell
membrane above them
- once initiated, release of Ca2+ is self-propagating
- free calcium is able to release sequestered Ca2+ from its storage sites
- causing a wave of Ca2+ release and cortical granule exocytosis
Activation of Egg Metabolism in Sea Urchins
Fertilization results in:
(1) merging of two haploid pronuclei, and
(2) initiating the processes that start development
- the latter events happen in the cytoplasm and occur without the involvement of parental
nuclei
- sperm fusion activates egg metabolism
- stimulates a preprogrammed set of metabolic events into action
- “early” responses - occur within seconds of cortical reaction
- “late” responses - minutes after fertilization
Early responses
- Ca2+ release at fertilization also responsible for re-entry of the egg into the cell cycle and
reactivation
- egg Ca2+ levels increase from 0.1 - 1.0 :M
- occurs as a wave or succession of waves that sweep across the egg beginning at
the site of sperm-egg fusion
- Ca2+ release activates series of metabolic reactions that initiate embryonic development
- activation of NAD+ kinase
- converts NAD+ to NADP+
- NADP+ (but not NAD+) can be uses as a coenzyme for lipid biosynthesis
- conversion may be important for construction of cell membranes
- Ca2+ release affects O2 consumption
- burst of O2 reduction (to hydrogen peroxide, H2O2) seen during fertilization
- much of respiratory rust is used to crosslink fertilization envelope
- enzymatic activity is also NADPH-dependent
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- NADPH helps regenerate glutathione and ovothiols (scavengers of free radicals)
- injecting EGTA (chelates Ca2+ - removes it from the system) = no cortical
granule reaction, no change in membrane potential, no re-initiation of cell
division upon fertilization
- increase in intracellular Ca2+ leads to artificial activation of egg
Late responses: Resumption of protein and DNA synthesis
- new burst of DNA and protein synthesis
- increase in intracellular pH
- due to production of diacylglycerol
- species-specific; e.g. mouse - no increase in pH, no increase in protein synthesis
- increased pH begins with second influx of Na+; causes 1:1 exchange between Na+ ions
from seawater and H+ ions form egg
- loss of H+ causes pH of egg to rise
- pH increase and Ca2+ elevation act together to stimulate new DNA and protein synthesis
- experimental pH elevation in unfertilized egg - DNA synthesis and nuclear
envelope breaks down ensue, just as if the egg were fertilized
- Ca2+ critical to new DNA synthesis
- Ca2+ inactivates MAP kinase, converting it from a phosphorylated (active) to an
unphosphorylated (inactive form; removes inhibition on DNA synthesis
- DNA and protein synthesis can then resume
- in sea urchins - burst of protein synthesis usually occurs within several minutes after
sperm entry
- protein synthesis does not depend on synthesis of new mRNA
- utilizes mRNAs already present in oocyte cytoplasm
- induce production of histones, tubulins, actins, morphogenetic factors
used during early development
- burst of protein synthesis can be induced artificially by raising pH of cytoplasm
- one mechanism for global rise in translation of stored messages: release of inhibitor from
mRNA
- e.g. maskin (inhibits translation in unfertilized amphibian oocyte)
- sea urchin, similar inhibitor binds translation initiation factor eIF4E at the 5' end
of several mRNAs; prevents translation
- upon fertilization, the inhibitor becomes phosphorylated and is degraded
Fusion of genetic material
- after sperm and egg cell membranes fuse, sperm nucleus and its centriole separate from
the mitochondria and flagellum
- flagellum and mitochondria disintegrate
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- few, if any, sperm-derived mitochondria found in developing or adult organism
- mitochondrial genome contributed primarily by the maternal parent
- centriole - in almost all animals (exception - mouse) the centrosome needed to produce
the mitotic spindle of subsequent divisions is derived from the sperm centriole
- fertilization occurs after 2nd meiotic division; haploid female pronucleus in egg cytoplasm
when sperm enters
- sperm nucleus decondenses to form haploid male pronucleus
- nuclear envelope vesiculates into small packets
- protein holding sperm chromatin in condenses inactive state are exchanged for
other proteins derived from egg cytoplasm
- exchange permits decondensation of sperm chromatin
- decondensation permits transcription and replication
- sea urchins - sperm chromosome decondensation appears to be initiated by
phosphorylation of nuclear envelope lamin protein & phosphorylation of two
sperm-specific histones that bind tightly to DNA
- cAMP-dependent protein kinase
- facilitates replacement of sperm -specific histones with egg histones
- sea urchin sperm pronucleus rotates 180° so that the sperm centriole is between the
sperm pronucleus and egg pronucleus
- sperm centriole acts a microtubule organizing center
- extends microtubules and integrates them with egg microtubules to form an aster
- microtubules extend throughout the egg and contact the female pronucleus
- the two pronuclei migrate toward each other
- fusion forms diploid zygote nucleus
- DNA synthesis can begin either in the pronuclear stage (during migration)
or after formation of the zygote nucleus
- depends on the level of Ca2+ released earlier in fertilization
Mammalian Fertilization
- difficult to study because of internal fertilization
- sperm population ejaculated into the female is probably very heterogeneous
- sperm at many different maturation states
- out of ~ 280 x 106 sperm in typical human ejaculate, ~ 200 reach the egg
- difficult to assay those molecules necessary to enable sperm to reach the egg
Getting the gametes into the oviduct: Translocation and capacitation
- female reproductive tract - highly specialized; actively regulates transport and maturity of
both gametes
- both gametes use small-scale biochemical interactions and large-scale physical propulsion
to get to the ampulla - region of oviduct just past the ovary where fertilization
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takes place
- mammalian oocyte released form the ovary is surrounded by a matrix containing cumulus
cells
- cumulus cells - ovarian follicle cells to which developing ovum is attached
- if cumulus is removed or altered, oviduct fimbriae will not pick oup
oocyte-cumulus complex
- complex will not be able to adhere or enter oviduct
- once picked up, combination of ciliary beating and muscle contractions transport oocytecumulus complex to the appropriate position for its fertilization in the oviduct
- sperm translocation from vagina to oviduct - flagellar motility: minor contribution to late movements (however, in mouse
sperm, motility required for movement though the cervical mucus and for
sperm to encounter the egg once it is in the oviduct)
- in rabbits, dead sperm can be transported within minutes from the vagina to the
oviduct
- mice, hamsters, guinea pigs, cows, humans: sperm found within 30 min of vaginal
deposition
- too short for sperm to have traveled by motility
- transported by muscular activity of the uterus
Sperm motility generalizations:
1. Uterine muscle contractions are critical in getting sperm to oviduct
2. The oviduct region just before the ampulla may slow sperm down; release them over
time
3. Flagellar motility is important once sperm arrives within the oviduct; sperm become
hyperactive in the vicinity of the oocyte
4. sperm may receive directional cues from temperature gradients between the regions of
the oviduct form chemical cues derived from the oocyte or cumulus
5. Sperm maturation must be completed during transport from vagina to oviduct
Capacitation
- newly ejaculated mammalian sperm are unable to undergo the AR until they have resided
in the female reproductive tract for some time
- set of physiological changes necessary is called capacitation
- uncapacitated sperm are “held up” in the cumulus matrix; unable to reach the egg
- capacitation requirements vary by species
- capacitation can be accomplished in vitro by incubation in tissue culture medium
(contains, e.g. Ca2+, HCO3-, serum albumin) or oviduct fluid
- timing: nearly all human pregnancies result from sexual intercourse during a 6-day period
ending on the day of ovulation
- fertilizing sperm may take as long as 6 days to reach the ampulla
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- capacitation is a transient event; sperm become uncapacitated after a period
- molecular events of capacitation
1. Sperm cell membrane altered by the removal of cholesterol
- albumins in female reproductive tract remove cholesterol
- may bring clusters of proteins together at anterior end
2. Proteins and carbohydrates on surface are lost
- may block recognition sites for zona-binding proteins
- unmasking may be an effect of cholesterol depletion
3. Membrane potential becomes more negative ]
- K+ ions leave cytoplasm
- may allow Ca2+ channels to be opened; permits Ca2+ entry
- Ca2+ and HCO3- activate cAMP production; facilitate membrane
fusion events of the AR
4. Protein phosphorylation occurs; two chaperone (heat-shock) proteins migrate to
the surface of the sperm head when phosphorylated
- role in forming receptor that binds to the zona pellucida
5. Outer acrosomal membrane changes; comes into contact with sperm cell
membrane
- prepares membranes for fusion
- it is uncertain whether these events are independent of one another and to what extent
each of them contributes to sperm capacitation
- connection between capacitation and sperm translocation
- prior to capacitation, sperm bind actively to membrane of oviduct cells in the
ovarian isthmus
- binding is temporary; breaks off when sperm become capacitated
- life-span of sperm is significantly lengthened by binding; capacitation slowed
binding may function as block to polyspermy
- may maximize the probability that sperm will still be available to meet the egg in
ampulla, even if ejaculation does not occur at the same time as ovulation
Hyperactivation, thermotaxis, chemotaxis
- sperm of certain mammals (e.g. hamsters, guinea igs, some mouse strains) become
hyperactivated as they pass from the uterus to the oviduct
- swim at higher velocities; generate greater force
- mediated by open of sperm-specific Ca2+ channel located in the tail
- asymmetric beating of flagellum is changed into a rapid synchronous beat
- movement well suited for linear movement in the viscous fluid of oviduct
- hyperactivation, along with hyaluronidase enzyme on the outside of the sperm cell
membrane, enable sperm to digest a path through the extracelluar matrix of the
cumulus cells (hyaluronic acid is a glycosaminoglycan: hyaluron - polymer of
disaccharides composed of D-glucuronic acid and D-N-acetylglucosamine; major
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component of ECM)
- thermotaxis - sperm may be able to sense thermal gradient
- e.g. ampulla of oviduct is 2°C warmer than the isthmus
- apparently, only capacitated sperm are able to respond thermotactically
- chemotaxis - oocyte and cumulus cells may secrete chemotactic agents
- follicular fluid showed some chemotactic ability
- only fertilizable follicles had chemotactic activity
- only capacitated sperm respond
Recognition at the zona pellucida
- mammalian sperm first binds to and penetrates egg’s zona pellucida before binding to
oocyte membrane
- ZP is analogous to vitelline envelope in invertebrates
- ZP is far thicker and more dense than vitelline envelope
- binding of sperm to zona is relatively, but not absolutely, species-specific (remember,
species-specificity is not a major problem with internal fertilization)
- several steps in zona binding
- mouse ZP constructed of three glycoproteins: ZP1, ZP2, ZP3, along with accessory
proteins that bind to the zona’s integral structure
- glycoprotein matrix is synthesized and secreted by the growing oocyte
- binds sperm
- once bound, initiates AR
- sequential interaction between several sperm proteins and components of the zona
1. relatively weak binding accomplished by the recognition of a sperm protein by a
peripheral protein that coats the ZP
2. somewhat stronger association between Zona and sperm SED1 protein
3. sperm protein (one or more) form strong links with ZP3
- ZP3 stimulates AR
Early stages of gamete adhesion
- initial tethering: 250 kDa sperm protein binds to a protein associated with (but not an
integral part of) ZP
- zona-associated protein can be washed off
- second protein (sperm cell surface adhesion protein SED1 - binds to the zona protein
complex
- SED1 found in discreet domain of sperm cell membrane, directly overlying the
acrosome
- binds to zona of unfertilized oocytes (not fertilized oocytes)
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Final Stage of Sperm -zona recognition: binding to ZP3
- ZP3, but not ZP1 or ZP2, binds to heads of mouse sperm with intact acrosomes
- several sperm proteins bind to ZP3
- sperm surface galactosyltransferase; recognizes N-acetylglucosamine residues
- ZP3 carbohydrate moieties crucial for sperm attachment
Induction of mouse AR by ZP3
- mouse sperm that undergo the AR before reaching the ZP are unable to penetrate it
- AR induced when ZP3 crosslinks receptors on the sperm cell membrane
- one crosslinked protein is sperm cell surface galactosyltransferase; active site
faces outward; binds to carbohydrate residues of ZP3
- crosslinking activates specific G-proteins in sperm cell membrane,
- initiates cascade that opens membrane Ca channels
- causes Ca-mediated exocytosis of acrosomal vesicle
Traversing the zona pellucida
- AR release a variety of proteases that lyse the ZP
- create a hole through which the sperm travels
- however, the anterior portion of the sperm cell membrane (containing ZP3 binding sites)
is shed
- secondary binding to zona accomplished by proteins in the inner acrosomal membrane
- bind ZP2 glycoprotein (analogous to sea urchin bindin binding to vitelline
membrane)
- acrosome-intact sperm will not bind to ZP2, acrosome-reacted sperm will
Gamete fusion and prevention of polyspermy
- mammalian sperm contact egg on the side of the sperm head
- fusion takes place at the junction between inner acrosomal membrane and sperm cell
membrane: the equatorial region
- sperm is bound to regions of the egg where actin polymerizes to extend microvilli to
sperm
- fusion may depend on interaction between sperm protein and integrin-associated CD9
protein on the egg
- sperm protein Izumo implicated in fusion
- immunoglobulin-like protein
- several candidates for sperm fusion proteins
- may be several operating
polyspermy prevention:
- enzymes released by the egg modify the zona pellucida sperm receptors such that
they can no longer bind sperm
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- mouse egg cortical granules contain N-acetylglucosaminidase enzymes; capable
of cleaving N-acetylglucosamine form ZP3 carbohydrate chains
- N-acetylglucosamine is one of the carbohydrate groups to which sperm
can bind
- ZP2 is also clipped by another cortical granule protease
- loses ability to bind sperm
Fusion of genetic material
- mammals - pronuclear migration takes ~ 12 h (compare to sea urchin; ~ 1 h)
- mammalian sperm enters almost tangentially to surface of egg (sea urchin: perpendicular)
- sperm membrane fuses with numerous microvilli
- sperm DNA is bound by basic proteins called protamines; tightly compacted through
disulfide bonds
[NOTE - protamines are present throughout vertebrates]
- glutathione in egg cytoplasm reduces disulfide bonds
- allows uncoiling of sperm chromatin
- mammalian oocyte nucleus is arrested in metaphase of 2nd meiotic division when sperm
enters [see Fig. 7.5]
2+
- Ca oscillations that are initiated by sperm entry inactivate MAP kinase;
- inactivated MAP kinase allows DNA synthesis
2+
- Ca oscillations also lead to proteolysis of cyclin
- cyclin degradation allows the cell cycle to continue; results in haploid female
pronucleus
2+
- Ca oscillations also lead to proteolysis of securin - protein holding metaphase
chromosomes together
- mammals undergo several waves of Ca2+ release
- DNA synthesis occurs separately in male and female pronuclei
- the centrosome (containing the new centriole) accompanying the male pronucleus
produces its asters (largely from proteins stored in the oocyte)
- microtubules join the two pronuclei and enable them to migrate toward one
another
- upon meeting, nuclear envelopes break down
- however, unlike sea urchins, which produce a common zygote nucleus, male and female
mammalian pronuclear chromatin condenses into chromosomes that orient
themselves on a common mitotic spindle
- thus, a true diploid nucleus in mammals is first seen not in the zygote, but at the
two-cell stage
- sperm contributes nucleus, mitochondria, centriole, small amount of cytoplasm
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- mitochondria and mitochondrial DNA are degraded
- therefore, all mitochondria are derived from mother
- in most mammals, the sperm centriole serves as the organizing agent for making the new
mitotic spindle
- several proteins and mRNA formed by the transcription of the pronucleus in the
spermatocyte are also brought into the egg
- include mRNAs for certain transcription and paracrine factors
- also microRNA that may down-regulate an important receptor involved in cell
division in early embryos
- significance of small amounts of RNAs and proteins still being debated
- one important protein is PLC.
- soluble small protein surrounds sperm head; crucial for egg activation and Ca2+
release in mammals
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