BIOL 370 – Developmental Biology
Topic #4
Fertilization:
The Beginning of Development in
a New Organism
Lange
Fertilization: The process where the gametes fuse together to create
a new instance of an organism whose genome is derived from two
parents.
There are four major events in this process:
1. Contact and recognition between gametes (species recognition
is a key aspect of this process).
2. Regulation of the merging of gametes (typically regulation of the
sperm into the egg and the inhibition of additional sperm from
entering).
3. Fusion of the genetic material of the gametes.
4. Activation of metabolism in the fertilized product to start
development.
Sperm – the “male” gamete which typically is much smaller
than the ova.
First discovered by Anton van Leeuwenhoek in the 1670s, but
mistakenly suggested that sperm were actually parasites in the
semen.
The human infant preformed in the sperm, as depicted by Nicolas Hartsoeker (1694)
Hartsoeker proposed
that there would be
found within sperm a
homuncular body and
drew this image of
what he hoped would
be discovered with
improvements in
microscopy.
It was not until the 1876 that Oscar Hertwig and Herman Fol both
demonstrated that sperm were definitively entering the egg.
Oscar Hertwig
Herman Fol
Modification of a germ cell to form a mammalian sperm – SPERM ANATOMY
General overview of sperm formation in mammals.
Modification of a germ cell to form a mammalian sperm (Part 1)
•
Acrosomal vesicle (acrosome) – a structure derived from the Golgi
apparatus that associates with the highly condensed nucleus. The
acrosome produces enzymes and is a secretory vesicle. The use of these
enzymes is to digest through the outer coverings of the egg during
fertilization. This vesicle will form the acrosomal cap that is associated with
capacitation of sperm.
•
Notice how the flagellum develops from the centriole.
Modification of a germ cell to form a mammalian sperm (Part 2)
• Sperm head – the
acrosome and the
nucleus
• Flagellum – the tail-like
projection
Motile apparatus of the sperm (Part 1)
• Axoneme – the
flagellar structures
that are formed from
microtubules
originating at the
centriole within the
midpiece.
Motile apparatus of the sperm (Part 2)
• Tubulin – the major protein constituent of the microtubules
• Dynein – the protein that is going to hydrolyze ATP (it is actually
an ATPase) to release energy.
*
*
Modification of a germ cell to form a mammalian sperm (Part 3)
•
Capacitation – the final stages of sperm maturation. In most
mammals this occurs OUTSIDE of the testes and is typically within
the vaginal/uterine environment of the female. This occurs. The
uterus aids in the steps of capacitation by secreting variety of
chemicals that react with the outer aspects of the sperm.
•
Non-mammalian spermatozoa do not undergo capacitation step and
are ready to fertilize an oocyte immediately after release from the
male.
•
When capacitation occurs the sperm must still undergo the acrosome
reaction.
Structure of the sea urchin egg at fertilization
Be sure to notice
difference in sperm size
and egg size.
• Oocyte – a developing
egg that CANNOT yet
be fertilized
• Ovum (or egg) –
female gamete capable
of being fertilized
Eggs of different species
can be at different
developmental stages of
meiosis prior to
fertilization.
Other important aspects about the structure of the egg:
• Cytoplasm content in the egg is roughly 10,000 X
that of sperm in a typical sea urchin egg (similar
differences are seen in many other species).
• Cytoplasm of the egg contains nutritive proteins,
ribosomes, tRNA, mRNA, protective factors (such as
filters, repair enzymes, distasteful chemicals,
antibiotics), and morphogenetic factors (to regulate
development).
Stages of egg maturation at the time of sperm entry in different animal species
*
Figure 27.19
Sea urchin egg cell surface
Vitelline envelope – an extracellular matrix that forms a “fibrous” mat
around the egg. It is used typically in sperm-egg recognition
Egg jelly – seen in some species, resides outside the vitelline envelope,
but when present it serves to attract and activate (capacitate) sperm
(Egg) Cortex – a thin layer of cytoplasm along the interior of the cell
membrane of most eggs that is more dense than most other cytoplasm.
The cortex ** is especially rich in actin that helps in the formation of
microfilaments and microvilli.
Cortical granules – homologus structures to the acrosomal vesicles in
sperm.
Mammalian eggs immediately before fertilization
In mammals we see a zona pellucida & corona radiata – a
thick matrix (z.p.) and layer of cells (c.r.) that mammalian
sperm cells need to pass through prior to fertilization.
In this image, the
zona pellucida is the
thick, white band
and the corona
radiata (and its cells)
surrounds the zona
pellucida.
Summary of events leading to the fusion of egg and sperm cell membranes in the sea urchin and
the mouse
Sperm chemotaxis in the sea urchin Arbacia punctulata
Chemotaxis –
the phenomenon whereby
somatic cells, bacteria, and
other single-cell or
multicellular organisms
direct their movements
according to certain
chemicals in their
environment. In fertilization,
the chemicals are positively
chemotaxic.
Model for chemotactic peptides in sea urchin sperm
• Resact is a small
peptide chemical
that stimulates a
cell membrane
protein on the
head of the
sperm.
• The resulting
opening of
calcium channels
causes an influx
of calcium into
the sperm cell.
• This leads to
swimming
behavior in the
sperm.
Note that this image depicts
the SPERM CELL.
Species-specific induction of the acrosome reaction by sulfated polysaccharides characterizing the
egg jelly coats of three species of sea urchins
Acrosomal reaction the reaction that
occurs in the
acrosome of the
sperm as it
approaches the egg.
The acrosome is a
cap-like structure
over the anterior half
of the sperm's head.
In this example,
different egg jelly
layer polysaccharides
initiate this reaction in
a species specific
fashion.
Acrosome reaction in sea urchin sperm
In sea urchins, the protein BINDIN is seen at the base of
globular cluster of acrosomal enzymes. During the
acrosomal reaction, the formation of the acrosomal process
occurs and the bindin is important in the recognition of the
differences in egg jelly.
(Look at Vacquier and Moy, 1977)
Species-specific binding of acrosomal process to egg surface in sea urchins
Scanning electron micrographs of the entry of sperm into sea urchin eggs
Following the acrosome
reaction, the sperm enters
the egg causing actin to
polymerize and the egg
forms a fertilization cone
(the homologous process for
the egg).
• Monospermy – the normal condition of fertilization that results in the
formation of a diploid cell.
• Polyspermy – the enterance of more than one sperm into the ovum. In
animals and most organisms this leads to abnormal development and
typically fatal consequences for the cell or early stage embryo.
Theodor Boveri – demonstrated through experimentation the
problems associated with polyspermy in 1902.
Ova combat the risks of polyspermy in ways that have
tremendous similarities to how neurons function:
“Fast Block” to polyspermy – “electrical” membrane potential of the egg
changes immediately after sperm entry. The this change is due to the closing
of sodium channels. The change can be similar to that of a neuron stimulated
and activating an action potential. The change in this fast block is from the
resting potential of -70 mV to a new potential of +20 mV.
Membrane potential of sea urchin eggs before and after fertilization
Notice how in the sea urchin,
we can see the “fast block”
occurs very quickly following
addition of sperm.
Formation of the fertilization envelope and removal of excess sperm
“Slow Block” to polyspermy – more
technically called the “CORTICAL
GRANULE REACTION”.
• In this process, cortical granules that
line the inner layer of the egg’s cell
membrane fuse with the membrane.
• The release of their contents via
exocytosis will put a layer of an
enzyme between the cell membrane
and the vitelline envelope.
• The enzyme is called cortical granule
serine protease, and it helps in
breaking protein bonds and is part of
the formation of the fertilization
envelope.
Cortical granule exocytosis
This “slow block”
mechanism
develops the
fertilization
envelope.
Aberrant development in a dispermic sea urchin egg
This diagram shows how
the rare occurrence of
polyspermy results in
developmental defects that
prevent in virtually all
cases, the survival of the
organism.
Wave of Ca2+ release across a sea urchin egg during fertilization
The cortical granule reaction is initiated by a wave of calcium ions that
begins where the single sperm has entered the egg. This calcium wave
flowing across the egg takes on average about 30 seconds.
Endoplasmic reticulum surrounding cortical granules in sea urchin eggs
In most (but not all) organisms, the calcium used in this process is
stored in the ER associated with the cortical granules.
Postulated pathway of egg activation in the sea urchin
Variations in the process of fertilization related specifically to internal fertilization
occurring in mammals:
•
•
•
Occurs in the oviducts, making it difficult to study
Heterogeneity of sperm development
The potential of MULTIPLE mechanisms of sperm acrosome reaction in
mammals
Fertilization in mammals typically occurs in the ampulla of the oviduct.
DES – Endocrine Disrupting Prescription Medication Used in the 1950s & 1960s
DES
Diethylstilbestrol (DES) is a synthetic nonsteroidal estrogen first synthesized in 1938.
• It is classified as an endocrine disruptor.
Human exposure to DES occurred through diverse sources:
• dietary ingestion from supplemented cattle feed
• medical treatment for certain conditions (breast and prostate cancers)
• from about 1950 to 1971, DES was given to pregnant women in the mistaken belief
it would reduce the risk of pregnancy complications and losses.
Sperm Transport in Mammals:
• Ejaculation by the male will deposit sperm into the vaginal orifice
• Timing from release by the male and reaching of the ampulla in
humans is roughly around 30 minutes in many instances of
fertilization. Sperm are not able to navigate that distance by
flagellar swimming in that short a time frame.
• Muscular activity of the uterus is critical for sperm movement from
vagina to ampulla.
****See Baylis & Baker, 1993
Capacitiation of sperm:
• Newly ejaculated sperm are not able to undergo the acrosome
reaction (and hence cannot fertilize the egg)
• Time must be spent in the female reproductive tract where
physiological changes occur called CAPACITATION.
• Sperm cell membrane is denuded of cholesterol
• This removal leads to changes in position of lipids in the
membrane surface (called “lipid rafts”) that surround specific
receptor proteins.
• These receptor proteins (on the “lipid rafts”) then migrate to the
anterior head of the sperm
• These proteins are associated with the acrosome reaction and
binding to the zona pellucida.
Hypothetical model for mammalian sperm capacitation
SEM (artificially colored) showing bull sperm as it adheres to the membranes of epithelial cells in
the oviduct of a cow prior to entering the ampulla
Hyperactivation – following capacitation, the swimming rate of sperm
increases dramatically
Chemotaxis “Part A” – capacitation and hyperactivation may permit the
sperm to sense a chemical gradient within the oviduct
Thermotaxis – capacitiation allows sperm to be more sensitive to
temperature gradients. Studies have suggested that capacitated sperm
can sense differences in temperature as small as 0.014 degrees Celcius.
Sperm have been shown to be positively thermotaxic (meaning they will
migrate toward warmer temperatures).
Chemotaxis “Part B” – in the ampulla, it appears that progesterone is a
chemical mediator that attracts sperm.
Acrosome reaction in hamster sperm
Entry of sperm into a golden hamster egg
End.