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 devbio8e-chptopener-07.jpg

The modification of a germ cell to form a mammalian sperm (Part 2) devbio8e-fig-07-02-2.jpg

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 devbio8e-fig-07-02-3.jpg Acrosome marked by proacrosin-GFP Mitochondria(Green) MT (Red)

Figure 4.3 Motile apparatus of the sperm (Part 1) DevBio9e-Fig-04-03-1R.jpg

Figure 4.3 Motile apparatus of the sperm (Part 2) 9+2 structure; Doublet microtubule; 13 + 11 protofilaments -dynein; ATP hydrolysis- Karagener triad disease DevBio9e-Fig-04-03-2R.jpg 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.

Movement of microtubules in cilia and flagella

Figure 4.5 Stages of egg maturation at the time of sperm entry in different animal species DevBio9e-Fig-04-05-0.jpg

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 devbio8e-fig-07-04-0.jpg

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. devbio8e-fig-07-06-1.jpg

Hamster eggs immediately before fertilization devbio8e-fig-07-07-0.jpg In mammal, vitelline envelope is a thick ECM called zona pellucida. Cumulus is made up of the ovarian follicular cells.

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

Figure 4.8 Summary of events leading to the fusion of egg and sperm cell membranes in the sea urchin and the mouse DevBio9e-Fig-04-08-0.jpg

Scanning electron micrographs of the entry of sperm into sea urchin eggs devbio8e-fig-07-15-0.jpg

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

Figure 4.10 Model for chemotactic peptides in sea urchin sperm DevBio9e-Fig-04-10-0.jpg

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.

Figure 4.12 Acrosome reaction in sea urchin sperm (Part 1) DevBio9e-Fig-04-12-1R.jpg

Figure 4.12 Acrosome reaction in sea urchin sperm (Part 2) DevBio9e-Fig-04-12-2R.jpg

Figure 4.13 Species-specific binding of acrosomal process to egg surface in sea urchins DevBio9e-Fig-04-13-0.jpg

Figure 4.15 Bindin receptors on the egg DevBio9e-Fig-04-15-0.jpg

Figure 4.14 Localization of bindin on the acrosomal process DevBio9e-Fig-04-14-0.jpg

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 devbio8e-fig-07-16-1.jpg

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

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 DevBio9e-Fig-04-18-1R.jpg

Membrane potential of sea urchin eggs before and after fertilization 490 mM Na+ 120 mM Na+ devbio8e-fig-07-17-3.jpg

The slow block to polyspermy; Figure 4.19 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 DevBio9e-Fig-04-19-0.jpg

Figure 4.20 Cortical granule exocytosis (Part 1) CGSP: cortical granule serine protease TG(transglutamunases), OVOP/Dbdx; perosidase DevBio9e-Fig-04-20-1R.jpg

Figure 4.21 Wave of Ca2+ release across a sea urchin egg during fertilization DevBio9e-Fig-04-21-0.jpg

Figure 4.23 Probable mechanisms of egg activation DevBio9e-Fig-04-23-0.jpg

Figure 4.24 Roles of inositol phosphates in releasing calcium from the endoplasmic reticulum and the initiation of development DevBio9e-Fig-04-24-0.jpg

Figure 4.25 G protein involvement in Ca2+ entry into sea urchin eggs Red: hyaline Green: Gaq DevBio9e-Fig-04-25-0.jpg

Figure 4.26 Postulated pathway of egg activation in the sea urchin DevBio9e-Fig-04-26-0.jpg

Figure 4.27 A burst of protein synthesis at fertilization uses mRNAs stored in the oocyte cytoplasm DevBio9e-Fig-04-27-0.jpg

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

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.

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 -- DevBio9e-Fig-04-28-0.jpg

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. devbio8e-fig-07-29-0.jpg

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

Figure 4.29 Hypothetical model for mammalian sperm capacitation DevBio9e-Fig-04-29-0.jpg

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.

Figure 4.31 Sperm-zona binding DevBio9e-Fig-04-31-0.jpg

Figure 4.33 Acrosome reaction in hamster sperm DevBio9e-Fig-04-33-0.jpg

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.

Figure 4.34 Entry of sperm into a golden hamster egg (Part 1) DevBio9e-Fig-04-34-1R.jpg

Figure 4.34 Entry of sperm into a golden hamster egg (Part 2) DevBio9e-Fig-04-34-2R.jpg

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

Figure 4.35 Pronuclear movements during human fertilization DevBio9e-Fig-04-35-0.jpg

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 DevBio9e-Table-04-02-0.jpg