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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

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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 Migraon clustering Ra Migraon 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 Migraon clustering Diffusion (+) sAC + H - + - HCO3 CO2 + H2O H2CO3 H + HCO3 (+) cAMP (+) pH i PK-A Carbonic Anhydrase (+) (-)

PTK Ptyr-Ptase

(+) (-)

Protein Tyrosine Phosphorylaon

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BSP 2+ Ca2+ Cholesterol Ca Heparin Acceptor

Ra Migraon

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

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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

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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

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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

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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

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