1. Chapter 4, Fertilization: Beginning a New Organism
Importance of The Fertilization
Combining of genes derived form two parents
Creation of a new organism
Processes of the fertilization
Contact
Sperm entry
Fusion of genetic materials
Activation of egg metabolism
Start development
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2. The modification of a germ cell to form a mammalian sperm (Part 2)
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3. The modification of a germ cell to form a mammalian sperm (Part 3)
1.acrosome; enzymes that digest protein and sugars
2. Flagellum; axoneme—motility, composed of microtubules emanating from centrioles
3. Mitochondria; E source
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Acrosome marked
by proacrosin-GFP
Mitochondria(Green)
MT (Red)

4. Figure 4.3 Motile apparatus of the sperm (Part 1)
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5. Figure 4.3 Motile apparatus of the sperm (Part 2)
9+2 structure;
Doublet microtubule; protofilaments
-dynein; ATP hydrolysis- Karagener triad disease
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The movements of cilia and flagella result from the sliding of outer microtubule doublets relative to one another, powered by the motor activity of the axonemal dyneins.
Dyneins provide driving force for microtubules and vend the flagella with their ATPase activity.

6. Movement of microtubules in cilia and flagella

7. Figure 4.5 Stages of egg maturation at the time of sperm entry in different animal species
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8. Structure of the sea urchin egg at fertilization
Mature egg (ovum) contains all materials necessary for the beginning of growth and development.
Developing egg (oocyte) actively accumulate these materials.
Proteins: ex. Yolk proteins as a nutrient source
rRNA and tRNA: preparing for the burst of protein synthesis after fertilization
mRNA: for the early onset of specific protein synthesis. Enable maternal effects in the early embryonic stage.
Morphogenic factors: transcription factors and paracrine factors.
Protective chemicals: UV filters and DNA repair enzymes
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9. The sea urchin egg cell surface (Part 1)
Glycoprotein-rich vitelline envelope locates at the outside of plasma membrane of egg. It involves in species-specific binding of sperm as well as sperm attraction.
Cortex area extends out to form microvilli, which eventually involve for sperm entry process.
Cortical granules are Golgi-derived structures containing proteolytic enzymes in similar to acrosomal vesicles in sperm, and additional materials involve in sperm entry or the prevention of polyspermy.
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10. Hamster eggs immediately before fertilization
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In mammal, vitelline envelope is a thick ECM called zona pellucida.
Cumulus is made up of the ovarian follicular cells.

11. Process of fertilization
The chemoattraction of sperm to the egg
The exocytosis of acrosomal vesicle to release enzymes
The binding of the sperm to the extracellular envelope
The passage of the sperm through the envelope
The membrane fusion between sperm and egg

12. Figure 4.8 Summary of events leading to the fusion of egg and sperm cell membranes in the sea urchin and the mouse
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13. Scanning electron micrographs of the entry of sperm into sea urchin eggs
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14. External Fertilization (Sea urchin)
Sperm diffuses to meet egg.
How can the sperm and egg meet in the diluted condition?
- by chemotaxis
- Resact: 14aa peptide chemoattractant expressed in sea urchin Arbacia punctulata
Speract: a chemoattractant in sea urchin Strongylocentrotus purpuratus
- Only A. punctulata has receptor for resact, which activates guanylyl cyclase activity and the consequent elevation of cGMP to induce calcium entry into sperm. Resact also activate mitochondrial respiration, which increase the motility.
Sperm chemotaxis in the sea urchin Arbacia punctulata

15. Figure 4.10 Model for chemotactic peptides in sea urchin sperm
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16. Acrosomal reaction
Initiated by the contact between sperm and egg jelly.
Receptor in sperm membrane recognize polysaccharides on the egg jelly.
The composition of the polysaccharides are different from species to the other species. Ex.) S. purpuratus acrosomal reaction is initiated by fucose sulfate.
Acrosomal reaction is mediated by
1) Initial Ca2+ entry into head
2) Na+/H+ exchanger pump in Na+ while pump out H+
3) PLC generates IP3, which induce Ca2+ release from the reservoirs to stimulate the fusion of acrosomal membrane to plasma membrane.
Ca2+ induces exocytosis of digestive enzymes to digest the path, though which acrosomal process adhere to egg envelope.
Ca2+ influx also associates with this process by activating RhoB in acrosomal region.

17. Figure 4.12 Acrosome reaction in sea urchin sperm (Part 1)
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18. Figure 4.12 Acrosome reaction in sea urchin sperm (Part 2)
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19. Figure 4.13 Species-specific binding of acrosomal process to egg surface in sea urchins
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20. Figure 4.15 Bindin receptors on the egg
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21. Figure 4.14 Localization of bindin on the acrosomal process
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22. Aberrant development in a dispermic sea urchin egg (Part 1)
Why can only one sperm enter into the egg?
To prevent improper numbers of chromosomes in daughter cells.
Conflict between centriole numbers and chromosome sets
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23. Prevention of Polyspermy
Mechanisms preventing polyspermy
1. Electric change in the egg cell membrane (fast)
Egg membrane potential shits to a positive level (+20mV) from -70mV resting membrane potential through Na+ influx after the first sperm binds
2. Exocytosis of the cortical granuels (slow) - Cortical granule reaction
To separate vitelline membrane from egg membrane.
Cortical granule serine protease dissolves the protein linking the vitelline membrane to egg membrane.
Mucopolysaccharides from cortical granules contribute to form a very sticky fertilization envelope pulling in the water and expand to move away from the egg
Peroxidase hardens the fertilization envelope

24. Figure 4.18 Membrane potential of sea urchin eggs before and after fertilization (Part 1)
By changing the electric potential of the egg memb;
Environmental; high Na, low K, but low Na and high K in cell cytoplasm
-resting mem poteintial; -70 mV; after binding to sperm; +20mV
-can not fuse the memb at 20 mV
Exp) low Na in lowering Na in outside; -polyspermy
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25. Membrane potential of sea urchin eggs before and after fertilization
490 mM Na+
120 mM Na+
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26. The slow block to polyspermy;
Figure Formation of the fertilization envelope and removal of excess sperm
The slow block to polyspermy;
Cortical granule fuse with egg cell memb;
-release contents;
To separate vitelline membrane from egg membrane.
Cortical granule serine protease dissolves the protein linking the vitelline membrane to egg membrane.
Mucopolysaccharides from cortical granules contribute to form a very sticky fertilization envelope pulling in the water and expand to move away from the egg
Peroxidase hardens the fertilization envelope
-hyalin; forms a coating around the egg; hylain layer
In mammals; the cortical granule reaction—
Enzymes; modify the ZP sperm receptor; no longer binding
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27. Figure 4.20 Cortical granule exocytosis (Part 1)
CGSP: cortical granule serine protease
TG(transglutamunases), OVOP/Dbdx; perosidase
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28. Figure 4.21 Wave of Ca2+ release across a sea urchin egg during fertilization
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29. Figure 4.23 Probable mechanisms of egg activation
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30. Figure Roles of inositol phosphates in releasing calcium from the endoplasmic reticulum and the initiation of development
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31. Figure 4.25 G protein involvement in Ca2+ entry into sea urchin eggs
Red: hyaline
Green: Gaq
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32. Figure 4.26 Postulated pathway of egg activation in the sea urchin
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33. Figure 4.27 A burst of protein synthesis at fertilization uses mRNAs stored in the oocyte cytoplasm
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34. Fusion of genetic material
Sperm’s nucleus and centriole separate from the mitochondria and flagellum.
The sperm nucleus undergoes dramatic decondensation to form pronucleus.
Nuclear envelope vesiculates into small packets, and the sperm’s proteins associated with condensed chromosomes are exchanged by egg’s to decondensate chromosomes.
Phosphorylation of lamin and histones by PKA after the binding to glycoprotein in the egg jelly.
Rotation of male pronucleus results in the localization of centriole between male and female nuclei

35. Mammalian Fertilization
Egg is release from ovary together with cumulus cells into oviduct, where sperm meets egg.
Sperm migration into oviduct is not only mediated by flagellar movement, but also supported by muscular contraction of the uterus.
Sperm become hyperactive and flagellar motility is increased when they’re getting closer to oocyte.
Sperms slow down before ampulla, where sperm and egg actually meet.
Sperm receive directional cues from temperature gradient along reproductive track and chemoattractants from oocyte or cumulus.
The sperm also mature during the trek from vagina to oviduct.

36. Figure 4.28 Nuclear events in the fertilization of the sea urchin
The sperm nucleus, centriole
Mito and flagellar disintegrate inside egg;
Mitochondrial DNA from egg , not from sperm
Sperm nucleus undergo dramatic changes;
--nuclear lamin breakage--pronucleus
--cAMP-dependent protein kinase phosphorylate the sperm specific Histone
--decondensation- sperm specific Histone are replaced by histone from egg --
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37. Capacitation: become competent to fertilize the egg.
Scanning electron micrograph (artificially colored) showing bull sperm as it adheres to the membranes of epithelial cells in the oviduct of a cow prior to entering the ampulla
Capacitation: become competent to fertilize the egg.
The capacitated sperm can’t survive long, so it should fuse to the egg promptly.
Binding of sperms to reproductive track delay the capacitation, but increase their survival
Capacitated sperms lose contact with reproductive track
It also decrease the chance of polyspermy.
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38. Molecular events of capacitation
Factors affecting to capacitation
Removal of sperm membrane cholesterol –> change the pattern of lipid rafts & membrane protein distribution
Changes in membrane proteins or carbohydrates –> unmasking of the binding site to zona pellucida
Membrane potential transition –> become more negative through the efflux of K+ & influx of HCO3- -> induce Ca2+ entry
Protein phosphorylation – ex. Heat shock proteins migrate to the surface upon phosphoylation

39. Figure 4.29 Hypothetical model for mammalian sperm capacitation
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40. Getting closer to the oocyte
Sperm become hyperactive when it move from the uterus into oviduct – Ca2+ entry at the tail speeds up the moving.
Themotaxis: capacitated sperm can sense thermal gradient between isthmus of oviduct and ampulla (about 2oC).
Chemotaxis: secreted molecules from cumulus cells and oocyte.
Sequential interaction between sperm proteins and zona pellucia components.
Zona pellucida contains glycoprotein ZP1, ZP2, and ZP3 induce acrosomal reaction when sperm binds to here.
ZP3 binding site containing outer membrane is shed away after acrosomal reaction.
ZP2 (or ZP1) involves the secondary binding after the acrosomal reaction.

41. Figure 4.31 Sperm-zona binding
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42. Figure 4.33 Acrosome reaction in hamster sperm
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43. Gamete fusion and prevention of polyspermy
Equatorial region: junction between inner acrosomal membrane and egg membrane
CD9, a binding partner for integrin at the sperm, at the egg membrane is important for the fusion.
Izumo(“Japanese shrine dedicated to marriage”), an Ig-like protein at the sperm, is essential for the fusion but not for the binding to the egg.
Polyspermy is prevented by cortical granule reaction.
Enzymes modify the sperm receptors in zona pellucida, rather than generating fertilization envelope by digesting away the component.
N-acetylglucosaminidase remove N-acetylglucosamine from ZP3, while a protease clipped ZP2 away.

44. Figure 4.34 Entry of sperm into a golden hamster egg (Part 1)
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45. Figure 4.34 Entry of sperm into a golden hamster egg (Part 2)
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46. Fusion of genetic materials
Sperm pronucleus migrate tangentially to the surface of the egg
Glutathione in the egg cytoplasm reduces disulfide bonds among protamines to uncoil sperm chromosome.
Proteolysis of cyclin B and securin, with the subsequent degradation of cohesin holding metaphase chromosomes, is induced by Ca2+.
The rest of sperm components in fertilized egg cytoplasm, such as mitochondria and other trace cytoplasmic content, except centromere is degraded

47. Figure 4.35 Pronuclear movements during human fertilization
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48. The Nonequivalnce of mammalian pronuclei;
Ex) Hydatidiform mole: a haploid sperm fertilize an egg in which the female pronucleus is absent; sperm
DNA can be duplicated>>> the egg become a mass of placental-like tissues
Parthenogenesis (“virgin birth”)>>the diploid egg divide to form embryos with spinal cord and organs(beating heart),
but these embryos do not further develop
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