Fertilization II

Gamete Fusion, Polyspermy Prevention, Activation of Egg Metabolism and Fusion of Genetic Material

Fusion of Membranes in Sea Urchins

• Following release of acrosomal enzymes is the fusion of plasma membranes of the sperm and egg

• Sperm-egg binding causes the extension of several microvilli to form the fertilization cone

– Extended by actin polymerization

• Egg and sperm membranes now join

• Sperm nucleus & tail pass through the resulting cytoplasmic bridge

– Similar in mammals

Entry of Sperm into Sea Urchin Eggs

Fusion of Membranes in Mammals

• The fertilin proteins in the sperm plasma membrane are essential for sperm membrane-egg membrane fusion

• Mouse fertilin localized to the posterior plasma membrane of the sperm head

• It first binds the sperm and egg plasma membranes and then also promotes their fusion

Entry of Sperm Into Golden Hamster Egg

Fusion of Sperm Acrosome & Plasma Membranes

Prevention of Polyspermy

• Normally - monospermy - one sperm enters & contributes its haploid nucleus - the centriole from the sperm divides - forms the spindle for cleavage

• Entrance of multiple sperm - polyspermy - typically disastrous

– May see multiple spindles

• Embryo dies or develops abnormally

Polyspermy & Aberrant Development

Prevention of Polyspermy in Sea Urchins

• Fast block to Polyspermy

– Achieved by changing electrical potential of the egg plasma membrane

– Normally more sodium ions outside than inside and more potassium ions inside than out

• This results in a resting membrane potential of about -70mv inside negative

• Within 1-3 seconds after binding of the first sperm - membrane potential shifts to a positive level - about +20mV (inside positive) - caused by small influx of sodium ions

• Only lasts about a minute

Membrane Potential & Fertilization

Slow block to Polyspermy in Sea Urchins

• After the membrane potential begins to return to normal other sperm bound to the vitelline membrane could enter

• Removal of bound sperm by the cortical granule reaction - slower, mechanical block to polyspermy - effective about one minute after first successful sperm attachment

• Below plasma membrane - 15,000 cortical granules - fuse with the plasma membrane upon sperm entry - release contents into space between plasma membrane & mat of vitelline envelope proteins

Slow block to Polyspermy in Sea Urchins

• Enzymes released - proteases - dissolve protein posts that hold vitelline envelope to the cell membrane

• Mucopolysaccharides released - produce an osmotic gradient - causes water to rush into the space between the plasma membrane and the vitelline membrane

• The envelope expands and becomes the fertilization envelope

• Peroxidase also released - hardens the fertilization envelope by crosslinking tyrosine residues on adjacent proteins

Slow block to Polyspermy in Sea Urchins

• A fourth cortical protein - hyalin forms a coat around the egg

• The egg extends elongated microvilli whose tips attach to the hyaline layer

• This layer provides support for the blastomeres during cleavage

Formation of Fertilization Envelope

Cortical Granule Excytosis in Sea Urchins

Cortical Granule Reaction in Mammals

• Released enzymes modify the zona pellucida sperm receptors - no longer bind sperm

• Called zona reaction - Both ZP3 and ZP2 molecules are modified

• Sperm can no longer bind to the zona

Calcium: Cortical Granule Reaction

• Rise of calcium that triggers granule release is not from an influx of calcium - from the egg itself

• A wave of calcium release occurs over the egg - takes about 30 seconds

• Calcium is stored in the endoplasmic reticulum - the ER surrounds the granules in the cortex region

Wave of Calcium release

Cortical Endoplasmic Reticulum

Activation of Egg Metabolism
- Early Responses

Activation of Egg Metabolism -
Late Responses

Fusion of Genetic Material in Sea Urchins

• Female pronucleus - egg haploid nucleus

• Male pronucleus - sperm haploid nucleus

• Sperm nucleus - nuclear envelope vesiculates into small packets - this exposes the sperm chromatin to the egg cytoplasm

• Proteins in sperm chromatin are exchanged for proteins derived from the egg cytoplasm - sperm chromatin can now decondense

Fusion of Genetic Material in Sea Urchins

• After the sperm enters the male pronucleus rotates 180° so the centriole is between the sperm & egg pronuclei

• The centriole - now acts as a microtubule organizing center - together with egg microtubules - from aster

• These microtubules extend to the egg pronucleus and the two pronuclei migrate toward each other

• Fusion of the two pronuclei - zygote nucleus

Nuclear events in Fertilization - Sea Urchin

Fusion of Genetic Material in Mammals

• In mammals the process of pronuclear migration takes about 12 hours compared to about 1 hour in the sea urchin

• Mammalian sperm enters almost tangentially to the surface

• Sperm nucleus breaks down as its chromatin decondenses - then it is reconstructed by coalescing vesicles

• DNA of sperm bound by basic proteins - protamines - tightly compacted by disulfide bonds

Fusion of Genetic Material in Mammals

• In the egg cytoplasm, glutathione - reduces disulfide bonds - allows chromatin to uncoil

• Male pronucleus enlarges as the oocyte nucleus completes 2nd meiotic division

• The centrosome - new centriole accompanying the male pronucleus produces asters

Fusion of Genetic Material in Mammals

• Then each pronucleus migrates toward each other - replicating its DNA as they travel

• Upon meeting the two nuclear envelopes break down - chromatin condenses

• Now the chromosomes orient themselves on a common mitotic spindle

• Thus a true diploid nucleus is not seen in the zygote but first in the two cell stage

Pronuclear Movements In Mammals During Fertilization