11/5/12! Lecture 16: Fertilization! Animal Science 434! John J. Parrish! Capacitation! Chang Experiment - 1951! 1951! Ovulation! Austin - rat! Chang - rabbit ! After! Before! Fertilization does not occur until several hours after sperm are placed in the uterus or oviduct! Percent Fertilization! Effect of Capacitation! Capacitation! Epididymal! Ejaculated! Capacitated! The changes within sperm that confer upon the sperm the ability to acrosome react in response to the appropriate Seminal! stimulus.! Plasma! Female! +! +! and/or ! Tract! Zona Pellucida! Epididymal! Secretions! Cumulus Cells! Follicular Fluid! 1! 11/5/12! DF (BSP in Bovine) Cholesterol BSP Heparin Acceptor Plasma Membrane! Plasma Membrane! Decapacitation Factor – Stabilizes plasma membrane and inhibits a premature acrosome reaction! BSP + - BSP Cholesterol H HCO3 Cholesterol Heparin Heparin Acceptor Acceptor Zp binding Zp binding Plasma Membrane! Ra: Migra=on clustering Ra: Migra=on clustering (+) sAC + H - HCO3 (+) cAMP (+) pH i PK-A (+) (-) PTK Ptyr-Ptase (+) (-) Protein Tyrosine Phosphorylaon + - BSP H HCO3 Cholesterol Heparin Extracellular Space! Acceptor Zp binding Ra: Migra=on clustering Diffusion! (+) sAC + H - + - HCO3 CO2 + H2O H2CO3 H + HCO3 (+) cAMP (+) pH i PK-A Carbonic Anhydrase! (+) (-) PTK Ptyr-Ptase (+) (-) Protein Tyrosine Phosphorylaon 2! 11/5/12! BSP 2+ Ca2+ Cholesterol Ca Heparin Acceptor Ra: Migra=on ATP ADP ATP ADP Ca2+ Ca2+ Ca2+ ATP ADP ATP ADP Ca2+ Ca2+ Acrosome Acrosome + - BSP H HCO3 Cholesterol 2+ Heparin Ca Acceptor Capacitation ! No change in sperm morphology! (+) sAC (+) + Motility change! H - HCO3 Ca2+ – hyperactivation! (+) cAMP (+) pH i PK-A (+) (-) Ca2+ Acrosome PTK Ptyr-Ptase cAMP! (+) (-) Power output! Protein Tyrosine Phosphorylaon The Acrosome Reaction! Plasma Membrane! Fused Plasma! Membrane and ! Outer Acrosomal! Membrane! Acrosin! Acrosome! Hyaluronidase! Nucleus! Modification! of the ! Equitorial! Region! 3! 11/5/12! The Zona Pellucida! Sperm Interaction with the Zona! Attachment! Z o n a Binding! P ZP3! e Acrosome Reaction! l Induction! l u Acrosome Reaction! c i d a Z Secondary Binding! Zona Pellucida! o ZP n 2! a ZP2! Zona Penetration! P ZP e 2! l l ZP2! u Motility! c i d a 6-12 hr! 18-24 hr! Sperm tract through zona pellucida! 4! 11/5/12! Maturation Promoting Factor" Zona Pellucida! LH Surge! (MPF)! Oocyte! Metaphase Promoting Factor! 2+ Primary Oocyte! GVBD! Ca ! Metaphase I! Ca2+! MPF! MPF! Ca2+! GV-Intact! Ca2+! PB-1! Ovulation! MPF! MPF! Perivitelline! Space! Metaphase II! Secondary Oocyte! Zona Pellucida! Zona Pellucida! Oocyte! Oocyte! Ca2+! Ca2+! Ca2+! Ca2+! Ca2+! Ca2+! Ca2+! Ca2+! Perivitelline! Perivitelline! Space! Space! Zona Pellucida! Zona Pellucida! Oocyte! Oocyte! Ca2+! Ca2+! Ca2+! Ca2+! Ca2+! Ca2+! Ca2+! Ca2+! Perivitelline! Perivitelline! Space! Space! 5! 11/5/12! Calcium Transients During Bovine Fertilization! Initiation of Calcium Pulse by 500 500 450 450 Soluble Sperm Component! 400 400 ) ) 350 350 M M 300 300 (n (n 250 250 um i 200 200 c l a 150 150 Calcium C 100 100 50 50 DAG! 0 0 0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90 Oocyte! PIP ! Time (m in) Time (min ) PLCzeta! 2 500 500 450 450 IP3! 400 400 ) 2+ 350 ) 350 Ca ! nM 300 nM 300 ( 250 ( 250 ium cium 200 200 l alc Ca 150 150 C 100 100 50 50 2+ 0 0 Ca ! 0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90 Time (min) Time (m in) Endoplasmic Reticulum! Zona Pellucida! Oocyte! Zona Pellucida! Oocyte! Decondensation! Pronuclei! Zygote! Pronuclei! Pronuclei! Ca2+! Ca2+! AII! AII! TII! MPFX" ! TII! MPFX" ! Perivitelline! MII! Perivitelline! MII! Space! PBII! Space! PBII! Zona Pellucida! Zona Pellucida! Oocyte! Oocyte! Ca2+! Ca2+! Zona Block to ! Zona Block to! Cortical! Cortical! Granules! Polyspermy! Granules! Polyspermy! Perivitelline! Perivitelline! Space! Space! 6! 11/5/12! Blocks to Polyspermy! Species! !1. !Mammals! 1! !3. !Invertebrates" Amphibians" Fish" 2! Rabbits! !4. !Amphibians" 3! Avians! 4! Gamete Aging! !Oocyte life span approx. 6 – 12 hours after ovulation! !Sperm life span approx. 24 – 48 hours after ejaculation! 7!.