1. Fertilization • Structure of Gametes • External vs Internal Fertilization • Sperm Attraction • Sperm Binding to Egg and subsequent fusion • Activation of Egg • Fusion of Nuclear material

2. What is the purpose of Fertilization? • Transmit genes from Parent to offspring • Initiate functions in egg cytoplasm that permits development to occur

3. Common Elements of Fertilization • Contact and Recognition between Sperm and Egg • Regulation of sperm entry • Fusion of Genetic Material • Activation of egg metabolism to start development

4. Sperm Cell • First observed by Leeuwenhoek (Sperma, Semen= seed) • Homunculus • Believed to contain all information for embryo • Work demonstrated that Semen required sperm cells for fertilization of egg • 1876, Hertwig and Fol demonstrate sperm entering egg and fusion of their genetic material. Studies in Sea Urchin

5. Spermatogenesis

6. Sperm Structure • Haploid Nucleus and Acrosome. • Flagellum- axoneme • “9+2” arrangement • Dynein-Force generator! • Mitochondria

7. Egg Cell- Oocyte • All material needed for beginning of growth and development. • Large cytoplasm • Synthesis and absorption of proteins (yolk) • Other cytoplasmic factors: • Nutritive proteins • Ribosomes and tRNAs • Accumulated mRNA • Morphogenic factors (transcription and paracrine factors) • Protective elements against UV, give egg bad taste, and yolk contains antibodies.

8. Oocyte Structure

9. Fertilization patterns • External • Internal

10. Overview of External: Sea Urchin • Many Marine animals use this method • Chemotaxis main targeting mechanism. • Species specific sperm attraction and species specific sperm activation.

11. Overview of Internal: Mammals • Difficult to study • Sperm Heterogeneous at first. • Both gametes use biochemical and mechanical means to reach site of Fertilization(Ampula). • Translocation • Capacitation

12. External Sperm Attraction to Egg • Species specific • Resact (14 a.a.) • Regulates Calcium influx • Increases ATP formation from Mitochondria: increase flagellar motive force.

13. Internal: Sperm attraction to Egg • Regionally specific molecules-motility and capacitation • During capacitation, flagellum hyperactive • Directional cues: Heat and Chemotaxis (progesterone).

14. Capacitation of Mammalian Sperm

15. External: Recognition/fusion • Initial binding to Egg Jelly, Bindin • Species specific • EBR-1 interacts with Bindin. • Acrosome reaction • Once membranes fuse (Bindin), polymerization of actin in Egg: Fertilization cone. • Actin from gametes form a connection, sperm nucleus and tail pass into egg cell. • All regions of Egg can interact with sperm.

16. Internal: Recognition/fusion • Sperm binds to glycoprotein matrix of ZonaPellucida (ZP). • Second interaction occurs with ZP via SED1 protein of sperm • Final interaction between proteins (ADAM3, ADAM2) interact with ZP3. Crosslinking of galactosyltransferasesCa influx and acrosome reaction

17. Internal Recognition/Fusion • Interaction slightly different • Acrosomal fusion: • proteolytic enzymes • Secondary contacts: Zonadhesion • Other inner acrosomal proteins bind to ZP2. • Fusion not clear. Possible interaction with CD9 of oocyte.

18. External: Block of Polyspermy • Effects of Polyspermy • Fast block • Membrane potential switches from negative to positive • Transient (1 min) • Slow block • Shortly after fast block • Cortical Granules • Increase in Calcium, from internal stores. • Fertilization envelope

19. Slow-block for Polyspermy

20. Internal: Block of Polyspermy • No observed fast block • Slow block- similar to Sea Urchin • N-acetylglucosaminidase cleave NAG from ZP3. • Other proteins cleave proteins from cell surface.

21. External and Internal: Activation of Egg • Calcium-major signal. • Releases inhibitors from stored mRNAs-Maskin protein • IP3 created by phospholipase C, DAG also created • G proteins and Src induce PLC activity • IP3 causes Ca release from ER • Ca required for Na/H pump that regulates Egg internal pH. • Mammals similar…multiple waves of Ca release.

22. External: Fusion of Genetic material. • Nucleus/centriole separate from mitochondria and tail. • Nuclear envelope vesiculates into small packets. Expose chromatin • Phosphorylation of nuclear lamin and histones-starts early • Pronucleus rotates 180o, placing centriole between sperm and egg pronuclei • Zygote nucleus formed.

23. Internal: Fusion of Genetic material. • Takes longer in mammals • Sperm DNA bound by Protamines (disulfide bonds hold complex together) • Glutathione reduces protamine-relax chromatin • Ca release in waves. • As pronuclei meet, DNA condenses into chromosomes and orient on common mitotic spindle. • True zygote nucleus: 2 cell stage.