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Fertilization
Chapter 7
I. Gametes
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Meiotic products
Two main types, sperm and egg
Sperm and Egg Comparison
Sperm
Egg
Stem Cell
Spermatogonia
Oogonia
Entering Meiosis I
(diploid)
Primary
Spermatocyte
Primary Oocyte
Completed Meiosis I
(haploid)
Secondary
Spermatocyte
Secondary Oocyte
Completed Meiosis II
(haploid)
Spermatid
Ootid
I A. Sperm
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First discovered by Leeuwenhoek in 1678
Thought sperm were parasites
Named spermatozoa (sperm animals)
First real recognition that sperm were important in fertilization not until 1824, 50 years
after sperm discovery
Oscar Hertwig and Herman Fol (1876) were first to watch fertilization (in sea urchins)
Formation includes loss of most cytoplasm
Meiotic reduction of nucleus to haploid state
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Head (blue)
Middle Piece (green)
Tail (red)
Head
• Haploid nucleus
• Acrosome, Golgi-derived, with digestive enzymes and complex sugars
• Actin in some species
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Midpiece
• Concentration of mitochondria for ATP production
• Centriole for base of flagellum
Tail
• Flagellum
• 9 + 2 microtubule array
Differentiation
• Meiosis completed before differentiation occurs
• Each spermatogonial cell yields 4 haploid sperm
Mammals
• Differentiation occurs in seminiferous tubules of testes
• Maturation of sperm (ability to move) gained in epididymus
• Capacitation (ability to fertilize egg) gained within female reproductive tract
I B. Egg
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Significantly larger than sperm
Sea urchin egg 10,000 times the volume of sperm
I B 1. Egg Cytoplasm
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Proteins
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Stored food and amino acids for embryos
May be derived from other organs in body and transported to ovary and egg
Ribosomes & tRNA
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For translation following fertilization
Messenger RNA
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Stored for translation after fertilization
25,000 to 50,000 different mRNA types in sea urchin eggs
Morphogenetic factors
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Cause determination and differentiation into specific cell types
Protective chemicals
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UV filtering
DNA repair enzymes
Distasteful to predators
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I B 2. Egg Nucleus
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Meiotic stage at which fertilization occurs extremely variable among species
Varies from NO meiosis initiated (Primary oocyte), Meiosis I begun, Meiosis I
completed, Meiosis II completed
Human Oogenesis
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Maximum follicle number at 6 mo development
Large die-off prior to birth
I B 3. Plasma Membrane/Cytoplasmic Cortex
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Just under plasma membrane is cortex
Cortex contains
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Actin (for microfilaments)
Cortical granules (15,000 in sea urchin)
Cortical granules
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Golgi-derived (like acrosome)
Digestive enzymes, mucopolysaccharides, adhesive glycoproteins, hyalin protein
I B 4. Outside Plasma Membrane
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Primary egg envelope
Derived from ovarian follicle cells
Vitelline envelope (sea urchin, frogs)
Primary egg envelope
Derived from ovarian follicle cells
Vitelline envelope (sea urchin, frogs)
Zona pellucida (mammals)
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Surrounded by cells of cumulus oophorus and corona radiata
Secondary egg envelope
Derived from oviduct/female reproductive tract
Jelly layers (sea urchin, frogs): glycoprotein
Egg white, shell membranes, shell (birds and reptiles)
Frog Egg with Jelly
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Chick Secondary Egg Envelopes
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Chalazae
Albumen
Inner Shell Membrane
Outer Shell Membrane
Calcareous Shell
II. Fertilization Events
Four major series of events make up the fertilization process
• Contact & recognition between sperm & egg
• Regulation of sperm entry into egg (polyspermy block)
• Fusion of sperm & egg genetic material (amphimixis)
• Activation of egg metabolism
II A. Contact & Recognition Between Sperm and Egg
Five basic steps (in somewhat varying order)
• Chemoattraction of sperm by egg
• Exocytosis of acrosome
• Binding of sperm to zona pellucida or vitelline envelope (2 & 3 reversed in
mammals)
• Passing of sperm through extracellular matrix
• Fusion of egg and sperm plasma membranes
II A 1. Chemoattraction of Sperm by Egg
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Eggs secrete chemicals that attract sperm
Demonstrated in animals as diverse as cnidarians and mammals
Eggs vary in timing of release of attractants
Secretions occur when eggs ready to be fertilized
II A 2. Exocytosis of Acrosome
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In sea urchins, reaction to egg jelly causes influx of Ca++ into sperm (intracellular
Ca++ levels increase)
Ca++ increase causes two effects:
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Exocytosis of acrosomal contents, release of proteolytic enzymes to digest jelly coat
Polymerization of actin to form acrosomal process
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Process covered with bindin from acrosome
Polymerization due to pH increase (Na+/H+ exchange)
Note: no acrosomal process in mammals)
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Acrosomal Reaction
II A 3. Binding of Sperm
i. Sea urchins
• Species-specific recognition between bindin and vitelline envelope receptors
Figure 7.14 Species-Specific Binding of Acrosomal Process to Egg Surface in Sea Urchins
ii. Mammals
• Sperm bind to ZP3 glycoprotein on zona pellucida
• Binding of ZP3 to sperm initiates acrosomal reaction
• Binding NOT as species-specific as in sea urchins
• (A) immunofluorescence of ZP3-binding protein in sperm plasma membrane
• (B) radioactively labeled ZP3 bound to sperm
II A 4. Passing of Sperm thru Matrix
Sea Urchins
• Acrosomal reaction
• Digest thru jelly
• Bind to vitelline envelope
• Digest thru vitelline envelope
Mammals
• Bind to zona
• Acrosomal reaction
• Digest thru zona pellucida
II A 5. Fusion of Egg & Sperm Plasma Membranes
Sea Urchin
• Egg microvilli fuse to form fertilization cone
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Actin polymerization in egg
Egg & sperm membranes fuse
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Sperm perpendicular to egg membrane
Mammals
• Egg & sperm membranes fuse
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Sperm parallel to egg membrane
CD9 receptor protein in egg membrane essential
Following fusion ALL of sperm enters egg
Nucleus, centriole, mitochondria, flagellum
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Mitochondria typically degraded
II B 1. Regulation of Sperm Entry (Polyspermy Block)
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Why is this step important?
Failure has at least two consequences
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Polyploid nucleus
Extra centrioles for cytokinesis
II B 1a. Polyploid Nucleus
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Two sperm nuclei (two 1N genomes) + egg nucleus (1N) 3N zygote)  3N zygote
Polyploidy typically lethal in many species (unbalanced/excessive gene doses)
II B 1b. Extra Sperm Centrioles
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Centrioles for cytokinesis typically derived from sperm
One sperm  1 centriole  divides to 2 centrioles  2 poles for cytokinesis
(normal)
Two sperm  2 centrioles  divides to 4 centrioles  4 poles for cytokinesis
Unbalanced segregation of chromosomes at anaphase
II B 2. Regulation of Sperm Entry: Polyspermy Blocks
Sea Urchin has TWO blocks to prevent polyspermy
• Fast Block
• Slow Block
II B 2a. Fast Polyspermy Block
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Plasma membrane depolarization
Membrane potential changes from -70 mvolts to +20 mvolts
Na+ influx starts at point of sperm entry and spreads over surface
Depolarization takes 1-3 seconds to complete
Depolarization lasts about 60 seconds
If prevent depolarization, increase frequency of polyspermy
(B) Depolarization
(C) No depolarization
As decrease quantity of Na+ in artificial seawater, increase frequency of polyspermy
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II B 2b. Slow Polyspermy Block
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Ca released from ER
Ca++ wave spreads over surface within 30 seconds
Ca++ release causes cortical granule exocytosis
Causes rising of vitelline envelope to form fertilization membrane
Cortical granule exocytosis releases
• Proteases
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Degrade bindin receptors
Degrade connections between vitelline envelope and plasma membrane
Mucopolysaccharides
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Osmotic influx water
Raise vitelline envelope off surface as fertilization membrane
Raising begins at +20 seconds, completed +60 seconds
Cortical granule exocytosis releases
• Peroxidase protein
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Harden fertilization membrane
Hyalin protein
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Forms coating around egg
Holds blastomeres together
Fertilization Membrane
II B 2. Polyspermy Block
Mammals
• Most species lack fast block (membrane depolarization)
• Do have slow block (cortical granule exocytosis)
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ZP3 and ZP2 enzymatically modified to prevent sperm binding
No fertilization membrane
II C 1. Amphimixis in Sea Urchins
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Takes about 1 hour
Sperm mitochondria and flagellum degenerate
Centriole divides to provide two pole microtubule assemblies for cleavage
Sperm nucleus “decondenses” as pronucleus
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Sperm-specific histones replaced with egg-derived histones
Now ready for replication & transcription
Sperm nucleus rotates 180o, so centriole now central instead of peripheral
Sperm pronucleus moves towards egg pronucleus and fuses
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DNA replication can be either in pronuclear stage or after pronuclei fusion
Sperm pronucleus at top with aster formation
Egg pronucleus at bottom
Migration towards each other
Fusion of pronuclei
II C 2. Amphimixis in Mammals
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Takes about 12 hours
Sperm DNA originally bound with protamines
Protamines replaced in egg
Egg completes second meiotic division
Sperm centriole divides to form two centrioles for division
Both sperm and egg pronuclei replicate before fusion
When both pronuclei meet, nuclear envelopes breakdown
Chromosomes line up at metaphase plate
Thus, no true diploid nucleus formed until after first cleavage
Overview of Fertilization
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Fusion of sperm/egg through first two cleavage events
II D. Activation of Egg Metabolism
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Egg “preprogrammed” to develop
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Suite of metabolic responses
Series of two reactions
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Early
Late
Both reactions tied to increase in Ca++ levels
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Ca++ typically from ER
II D 1. Early Activation Response
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NAD+ kinase activated
Catalyzes NAD+  NADP+
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Lipid biosynthesis coenzyme
Increase in lipid biosynthesis for cell membranes necessary for cleavage and cell growth
Increased O2 use to make H2O2
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Crosslink and harden fertilization membrane
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II D 2. Late Activation Response
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Na+/H+ exchange (Na+ in, H+ out)
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pH increases from 6.9 to 7.2
Increase in DNA synthesis
Increase in Protein synthesis
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Due primarily to translation of stored mRNA
II D 3. Rearrangement of Egg Cytoplasm
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Fertilization can radically rearrange egg cytoplasm
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Changes not obvious in sea urchins and mammals
Changes are obvious in tunicates, amphibians
Rearrangements change contacts, different exposures to morphogens
In frogs, cytoplasmic cortex rotates 30o towards point of sperm entry (microtubules)
Animal hemisphere pigmented
Point opposite sperm entry has reduced pigmentation after rotation  gray crescent
Gray crescent becomes site of gastrulation
First cleavage bisects gray crescent
III. Unusual Aspects of Fertilization
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Non-equivalence of mammalian pronuclei
Parthenogenesis
Gynogenesis
Androgenesis
Double fertilization
III A. Non-equivalence of Mammalian Pronuclei
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Genes derived from sperm and egg not equivalent
Both needed for normal development
Genomic imprinting (single allele for specific locus expressed)
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Ifg-2 from sperm expressed
Ifg-2 receptor from egg expressed
Hydatidiform moles (covered shortly)
Pronuclear transplantation experiments
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No offspring if both sperm-derived
No offspring if both egg-derived
Some offspring if both sperm & egg pronuclei included
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III B. Parthenogenesis
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Activation of egg without sperm
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Activation without amphimixis
Natural in some lizards (3N), rotifers, hymenoptera, thrips
III C. Gynogenesis
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Sperm activate egg, but no amphimixis
Ambystoma (3N), African molly
Ambystoma platineum (3n) a hybrid of
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A. jeffersonianum (2n)
A. laterale (1n)
All female species
III D. Androgenesis
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Sperm activates egg, only genome sperm-derived
In mammals, forms hydatidiform mole
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No fetus
Abnormal chorion growth, as large as 6 month fetus, expelled at 20 weeks gestation
46 XX, both genomes duplicates of sperm nucleus
1 in 1,000-1,200 pregnancies in US
III D. Hydatidiform Mole
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“Grape-like” villi
Complete mole
Note “grape-like” villi
III E. Double Fertilization
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Nucleus of ootid and 2nd polar body both fertilized
Both undergo cleavage, two embryos fuse to form one mosaic
If X and Y-bearing sperm used, can get hermaphrodite
Last updated 1 March 2004
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