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FERTILIZATION

Fertilization
The process by which the sperm initiates and participates, with the egg, in the
development of the embryo
What are the factors that affect the sperm’s potential for
successfully fertilizing the egg?
1. Capacitation - occurs in the female’s vagina. Vaginal secretions cause a molecular
change in the sperm plasmalemma (removal of decapacitating factor - semen proteins,
results in increased membrane fluidity,). Takes 4-5 hr in humans, 1 hr in mice, 6 hr in
rabbits.

http://www.erin.utoronto.ca/~w3bio380/l
ecture/Lect07/L7.htm
Effects of Capacitation on
Galactosyltransferase is a protein in the Sperm
sperm plasmalemma that MAY act as the
receptor that binds to the zona pellucida • Increased rate of metabolism
and initiates the acrosome reaction.
• Flagellum beats more rapidly; Result:
Sperm are more motile
• Changes in sperm plasmalemma proteins
allow sperm-egg binding and occurrence of
the acrosome reaction
• Pro-Acrosin (inactive) is converted to
acrosin (active)
• Sperm become capable of chemotaxis

http://www.erin.utoronto.ca/~w3bio380/l
ecture/Lect07/L7.htm
Factors that affect the sperm’s potential for successfully
fertilizing the egg? (cont.)
2. Dilution
Marine invertebrates - Free spawning of sperm into surrounding ocean water.
Mammals - Dilution: 40 - 400 million spermatozoa in vagina, only a few hundred to a
thousand reach the upper oviduct.

3. Egg secretions - importance varies


among species - fertilizins - F.R. Lille
(1919)
activating factors, agglutination,
chemoattraction

http://www.erin.utoronto.ca/~w3bio3
80/lecture/Lect07/L7.htm
Factors that affect the sperm’s potential for successfully
fertilizing the egg? (cont.)
4. Structures surrounding the egg - barriers
• Follicle cells
• Zona pellucida (vitelline membrane in non-
mammals)
• Oolemma (plasmalemma of egg)

http://www.talbotcentral.ucr.edu/mammalianfert.htm
http://arbl.cvmbs.colostate.edu/hbooks/pathphys/reprod/fert/gxport.html
6
Acrosome Reaction
How does the sperm get through the barriers surrounding
the egg?
1. Used to be thought that the acrosome enzymes did it all.
2. More recent data

a. Sperm arrives at zona pellucida with the


acrosome still intact. So, how does it get
through the follicle cells that surround the
egg?
b. Has to break down the intercellular cement
that holds the follicle cells together -
hyaluronic acid
c. Lin and co-investigators (1994) demonstrated
that a protein found in the sperm
plasmalemma (PH-20) had hyaluronidase
activity.

http://arbl.cvmbs.colostate.edu/hbooks/pathphys/reprod/fert/gxport.html
Getting through the zona pellucida
What happens when the sperm gets to the zona pellucida?
1. Attachment - loose association
2. Binding - strong attachment
3. Acrosome reaction - release of enzymes
4. Penetration of the zona pellucida by the sperm

http://www.talbotcentral.ucr.edu/mammalianfert.htm
How is this accomplished?
Wassermann and co-workers (1980, 1985, 1987, 1988)
Found that zona pellucida is composed of 3 glycoproteins
ZP1, ZP2, ZP3
Repeating subunits of ZP2 and ZP3 form filaments that are bound
together by ZP1
1. Wassermann et al. found that when the sperm
binds to ZP3 it causes a change in Ca+2 and Na+
flux across the sperm plasmalemma that results
in the acrosome reaction. (ZP1 and ZP2 will not
cause this to happen).
2. The sperm actually binds to an o-linked
oligosaccharide that is part of ZP3 - (a
carbohydrate component of the glycoprotein)
3. The identity of the receptor in the sperm
plasmalemma that binds to this oligosaccharide
is, as yet, uncertain. It has been suggested that
a part of PH-20 is the receptor; however, more
recent work indicates this may not be the case.
When the acrosome reaction occurs, a number
of proteolytic enzymes are exposed or released.
One or more of these enzymes is responsible for
digesting the hole through the zona pellucida
through which the sperm enters the perivitelline
space.
Following fusion of the sperm with the egg,
http://www.molbiolcell.org/cgi/content/full/9/7/1609/F6
In mammals:
1. The male nucleus enters the egg cytoplasm
and becomes the male pronucleus.
2. As a result of the sperm fusing with the egg
plasmalemma, the oocyte nucleus, which is at
metaphase of the second meiotic division,
completes that division giving rise to another
polar body.
3. Following the second meiotic division, what is
now the nucleus of the ovum becomes the
female pronucleus. Male and female pronuclei in a
fertilized hampster egg
4. The haploid male and female pronuclei move
toward one and other, meet, and fuse to form
the diploid nucleus of the zygote.
5. The zygote will now proceed to undergo cleavage.

http://www.talbotcentral.ucr.edu/mammalianfert.htm Cleavage
http://arbl.cvmbs.colostate.edu/hbooks/pathphys/reprod/fert/cleavage.html
Blocks to polyspermy
1. Dilution
2. Fertilizin like secretions cause agglutination of sperm in some
invertebrate species (Does not occur in mammals)
3. “Hardening” of vitelline membrane or zona pellucida that results from
cortical granule rupture - - slow block to polyspermy, takes 1 - 5 min

http://www.molbiolcell.org/cgi/content/full/9/7/1609
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=dbio.figgrp.1397
Blocks to polyspermy
4. Depolarization of the oolemma - membrane potential changes from
negative to positive, sperm cannot fuse with positively charged oolemma -
fast block to polyspermy, takes 1 - 2 seconds. Caused by influx of sodium
ions.

Sea urchins Frogs


Before sperm fusion -70 mv -28 mv
After sperm fusion +10 mv + 8 mv

Depolarization lasts longer than 5 min, but


eventually the potential difference across the
oolemma returns to its original value.
The change in membrane potential happens in
many species, but does not occur in mammals
Blocks to polyspermy

5. Hydrogen peroxide release by the eggs of some


invertebrate species (e.g. sea urchins) - inactivates
sperm - Does not occur in mammalian eggs.
6. Unknown mechanisms that cause degeneration of
extra sperm nuclei after they have entered the egg
cytoplasm. Seen in salamanders. As a result, even
though more than one sperm fuses with the egg, only
one male pronucleus survives to fuse with the female
pronucleus. - Does not occur in mammalian eggs.

Thus, only
1) dilution of the sperm and
2) hardening of the zona pellucida due to the
contents of the cortical granules
act to prevent polyspermy in most mammals,
including humans.
Egg Activation
A series of morphological, physiological and molecular changes that
occur in the egg in response to fusion of the sperm with the egg.
What does egg activation accomplish?
1. Frees egg from constraints that kept it from developing prior to
fertilization.
2. Prepares the egg for development.

Why do I say “frees the egg” ? Why not “frees the


egg and sperm”?
1. Egg activation does not require the sperm
nucleus
2. In some species, just pricking the egg with a
needle, shocking it, or simply changing the pH of
the solution it’s in will cause activation.
3. In some species eggs that are activated this way
will start to develop. Thus, we have a mechanism
that could allow for selection of parthenogenesis.
The seven events that characterize egg activation following
fusion of the sperm with the egg:
1. Release of Ca++ (calcium) stored in the
egg endoplasmic reticulum - appears to
be the critical step in the process.
2. Cortical reaction - rupture of cortical
granules that occurs concurrently with
the Ca++ release. Contents of granules
are released into perivitelline space and
cause “hardening” of the vitelline
membrane or zona pellucida. Causes
vitelline/fertilization membrane to rise
away from surface of egg in some
species.

http://www.molbiolcell.org/cgi/content/full/9/7/1609
“The fertilization transient can be seen at the
very start of the animation. Later there is a
Sea Urchin rise in calcium starting at the point of sperm
entry which correlates with the start of the
movement of the female pronucleus to the
center of the egg and then a further general
rise in calcium which correlates with the
fusion of the two pronuclei.”

This movie shows


A. calcium transients that occur firstly at fertilization
B. then as the female pronucleus starts to move
toward the male pronucleus
C. and finally general rise at the fusion of the
pronuclei
The data shown is the ratio of a calcium sensitive dye
at the time of the event with the background calcium
dye signal.

http://petrus.ncl.ac.uk/urchins/fertilizationb.html
The seven events that characterize egg activation following
fusion of the sperm with the egg:

3. In many species, an influx of Na+ (sodium) into the egg cytoplasm that
causes a change in membrane potential - fast block to polyspermy.
4. In many species a reorganization of the egg cytoplasm.
5. In most cases, completion of meiosis by the egg.
6. An efflux of H+ (hydrogen) ions causing an increase in cytoplasmic pH -
this activates previously inhibited synthetic pathways.
7. Increase in metabolism - zygote gears up for development.

Events that occur soon after egg activation:


a. DNA replication as male and female pronuclei
approach each other
b. Male and female pronuclei merge
c. Preparation for first cleavage