Fertilization

Reading

• Chapter 12
• Additional diagrams or pictures not found in text

Capacitation

• Experiments (1951) by Austin in the rat and Chang in the rabbit
  • sperm must reside in the female tract before it gains the ability to fertilize oocytes
  • modified now to be the ability to undergo a stimulus induced acrosome reaction
    • zona pellucida
    • cumulus cells
    • follicular fluid
• Effects of Capacitation
  • addition of sperm membrane components in epididymis allows fertility (fertilization)
  • epididymis and/or accessory gland fluid adds decapacitation factors
  • in female tract there is removal of sperm surface components to allow sperm to interact and acrosome react near the surface of the oocytes
• Model of Capacitation in the Bovine
  • displacement or release of decapacitation factors
    • in bovine heparin or heparan sulfate binds
    • in other species may just be time dependent
  • removal of cholesterol into cholesterol-acceptors
  • influx of bicarbonate, eflux of protons, increase in intracellular pH
  • soluble adenylate cyclase activated, increase in cAMP, PKA
  • cross talk and increase in tryosine phosphorylation
  • influx of calcium due to inactivation of plasma membrane calcium ATPase, filling of acrosomal calcium store
  • cytoplasmic changes lead to a change in the activity of cytoplasmic enzymes
  • modifications of the inner plasma membrane, outer acrosome membrane and outer plasma membrane to allow acrosome reaction
• Physical changes during capacitation
  • no morphological changes
  • change in motility with expression of hyperactivation
    • due to increase in cAMP
    • results in an increase in the power output of the flagellum

Acrosome Reaction

• fusion of the plasma membrane and outer acrosomal membrane
• release of acrosomal enzymes
• modification of equitorial region to allow fusion with the oocyte
  

Sperm interaction with the zona pellucida

• zona is made of 3 proteins, zp1, zp2 and zp3
• initial loose attachment
• binding occurs to zp3
  • specific receptor is present on surface of sperm which is exposed during capacitation
• binding initiates the acrosome reaction
• inner acrosomal membrane binds to zp2
  • needed because outer acrosomal membrane is lost and tethers sperm to zona pellucida
• motility of sperm is needed to drive sperm forward as enzymes digest the zona
• entry is usually at an oblique angle

Location of fertilizing sperm

• first 6-12 hours in the cervix, uterus
• next 18-24 hours in the isthmic sperm reservoir
• at ovulation moves up to ampullar-isthmic reservoir

Events in the oocyte

• Maturation promoting factor (MPF, metaphase promoting factor) in the oocyte
• synthesized after the LH surge
• increases in the primary oocyte and leads to the first metaphase of meiosis
• first division of meiosis (extrusion of polar body 1) is associated with a decrease in MPF
• MPF then increases again in the secondary oocyte to trigger metaphase II of meiosis
• the oocyte is ovulated with high MPF

Further events of fertilization

• Sperm passes through zona into the perivitelline space, binds and then fuses with the vitelline membrane
• fusion triggers the release of calcium in the oocyte which is repeated at 5 to 60 minute intervals depending on the species
• calcium oscilations
  • release of soluble sperm factor into egg cytoplasm
  • cause the break down of MPF in oocyte
    • meiosis is resumed and oocyte extrudes second polar body
    • oocyte chromatin then condenses followed by expansion to form a pronuclei
  • sperm decondensation
    • sperm nucleus enters the egg cytoplasm and calcium oscillations help remodeling of sperm chromatin to first condense and then decondense and form pronuclei in sequence with the oocyte chromatin
    • during epididymal maturation, condensation of the nucleus is associated with the addition of disulfide bonds to protamines
      • As the sperm nucleus enters the cytoplasm of the oocyte, the disulfide bonds are broken down by glutathione. This allows the sperm nucleus to expand or decondense.
  • cortical granules move to the surface of the oocyte, fuse and release contents into the perivitelline space
    • modifies the zona pellucida to prevent it from inducing the acrosome reaction thereby preventing polyspermy
• Blocks to polyspermy
  • zona pellucida (slow)
    • mammals
  • perivitelline space (slow)
  • vitelline membrane (fast)
    • invertebrates
    • amphibians
    • fish
    • rabbit
  • oocyte cytoplasm (very slow)
    • amphibians

Gamete aging

• oocytes have a viable life span of approx. 6-12 hours after ovulation during which they must be fertilized in order for the resulting embryo to develop normally
• sperm have a viable life span of approx. 24-48 hours in the female tract

Review Questions