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 Zona Pellucida (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 180 o, 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.