Chapter 6 - Fertilization (Beginning a New Organism)

BIO. SCI. 10 - Developmental Biology

Cristel Joy C. Mallari

Instructor I
Overview
A. Fertilization
B. Structure of Gametes
C. Internal Fertilization in Mammals
Fertilization
• gametes fuse to begin the
creation of a new individual
• genome from both parents
• to transmit genes from
parents to offspring
• to trigger the egg cytoplasm
- onset development
 Four (4) Major Events at Fertilization

• Contact and recognition between gametes

1

• Regulation of sperm entry into the egg

2

• Fusion of the genetic material of sperm and egg

3

• Activation of egg metabolism to start development

4
Structure of Gametes
The egg activates the sperm metabolism that is essential for fertilization, and the
sperm reciprocates by activating the egg metabolism needed for the onset of
development.
Sperm
• 135 years - role in fertilization
• Anton Van Leeuwenhoek - parasitic animals in semen
• spermatozoa = “seed animals”
• Preformationist - contained preformed embryo
• Sperms were seeds, female merely provided nutrient soil
Nicolas Hartsoeker
• co-discoverer
• Draw the picture of what he hoped to
find a miniscule human (homunculus)
within the sperm
Lazzaro Spallanzani
• importance of sperm in reproduction
• male toads to ejaculate and found toad semen so filtered
to be devoid of sperm; such semen did not fertilize eggs
• semen had to touch the eggs in order to be functional
• spermatic "animals" were parasites in the fluid
• egg contains the embryo
• activates by spermatic fluid
J. L. Prevost and J. B. Dumas (1824)
• sperm were not parasites
• active agents of fertilization
• existence in sexually mature males, absence in
immature and aged individuals
• sperm entered the egg and contributed materially to
the next generation
Albert von Kolliker
• described the formation of sperm
from cells in the adult testes
• denied there was any physical
contact between sperm and eggs
• sperm excites the egg – the same
way a magnet communicates its
presence to iron
Karl Ernst von Baer
• First description of fertilization
• Sea urchins and tunicates
• Fertilization envelope
• Migration of sperm nucleus to
the egg
• Early cell divisions of
development
Why sea urchins?
• (Paracentrotus lividus)
• common
• sexually mature
throughout most of the
year
• eggs were available in
large numbers
• transparent even at high
magnifications
Sperm Anatomy
Each sperm cell consists of a haploid nucleus, a propulsion system to move the
nucleus, and a sac of enzymes that enable the nucleus to enter the egg
Centriole - long flagellum at posterior end; Golgi apparatus - acrosomal vesicle at the future anterior
end; Mitochondria - collect around the flagellum, incorporated into the midpiece; Remaining
cytoplasm is eliminated; Nucleus condenses. *The size of the mature sperm has been enlarged relative
to the other stages.
Acrosome
• nucleus - DNA becomes tightly compressed
• derived from the Golgi apparatus
• modified secretory vesicle
• contains enzymes that digest proteins and complex
sugars
• digest outer layers of an egg cell
*acrosome and nucleus constitute the sperm head
Flagellum
• an individual sperm can travel by whipping
• Axoneme - flagellum’s major motor portion
• structure formed by microtubules emanating from the
centriole at the base of the sperm nucleus
• Dynein – provides force for sperm propulsion
• an ATPase, hydrolyzes ATP to release chemical energy
Axoneme Core
• consist of two central microtubules surrounded by a
row of a 9 doublet microtubules
• Only one microtubule of each doublet is complete (13
protofilaments)
• the other is C-shaped
• Has only 11 protofilaments
• made up of tubulin – dimeric protein
In mammals
• sperm released can move
• don’t yet have the capacity to bind and fertilize
• Capacitation - final stages of mammalian sperm
maturation
• don’t occur until inside the female reproductive tract
• increase in membrane fluidity, lateral movement of cholesterol
to the apical region, and cholesterol efflux from the sperm
plasma membrane to the extracellular environment
Egg (Ovum)
• Stored all the materials necessary for development
• Oocyte (developing egg) conserve its cytoplasm
• Oocyte either synthesizes or absorbs protein
• Yolk - act as food reservoir for the developing embryo
• Egg accumulates cytoplasmic storehouse during
maturation
Egg’s Cytoplasmic Trove Includes:
• Nutritive proteins - supply of energy and amino acids
• Ribosomes and tRNA - structural proteins and enzymes
• Messenger RNAs - encode proteins
• Morphogenetic factors - direct cell differentiation
• Protective chemicals - DNA repair enzymes, ultraviolet
filters
The Egg and its Environment
• Cell membrane
• capable of fusing and regulating the flow of certain ions
during fertilization
• Extracellular matrix – outside cell
• Forms fibrous mat around the egg
• sperm-egg recognition
• Most animals (invertebrates) - vitelline envelope
(glycoproteins)
Sea urchin egg cell surfaces. (A) Scanning electron micrograph of an egg before fertilization.
The cell membrane is exposed where the vitelline envelope has been torn. (B) Transmission
electron micrograph of an unfertilized egg, showing microvilli and cell membrane, which are
closely covered by the vitelline envelope. A cortical granule lies directly beneath the cell
membrane.(From Schroeder 1979, courtesy of T. E. Schroeder.)
Zona pellucida
• thick matrix that separates extracellular envelope
• glycoprotein layer surrounding the cell membrane of
mammalian oocytes
• Cumulus - layer of cells surrounding the mammalian egg
• ovarian follicular cells -nurturing the egg at the time of release
• Corona radiata – the innermost layer of cumulus cells,
adjacent to the zona pellucida
Mammalian eggs immediately before fertilization. (A) The hamster egg, or ovum, is encased in
the zona pellucida, which in turn is surrounded by the cells of the cumulus. A polar body cell,
produced during meiosis, is visible within the zona pellucida. (B) At lower magnification, a mouse
oocyte is shown surrounded by the cumulus (R. Yanagimachi.)
Cortex
• thin layer of gel-like cytoplasm
• beneath the cell membrane
• high conc. globular actin molecules
• Polymerize -> microfilaments - cell division
• Extend the egg's surface into small projections - microvilli (aid
the sperm entry)
• Cortical granules - contain proteolytic enzymes
Cortical granules
• membrane-bound, Golgi-derived structures
• homologous to acrosomal vesicle of the sperm
• Mucopolysaccharides, adhesive glycoproteins, and
hyalin protein
• Enzymes and M - help prevent polyspermy
• AG & HP - support for the cleavage-stage blastomeres
Recognition of Egg and Sperm
5 Basic Steps of Gamete Interaction
1. Chemoattraction of sperm to egg by soluble molecules
secreted by the egg
2. Exocytosis of sperm acrosomal vesicle to release enzymes
3. Binding of the sperm to the ECM (VE/ZP) of the egg
4. Passage of the sperm through this ECM
5. Fusion of egg and sperm membranes
C. Internal Fertilization in
Mammals
1. Getting the gametes into the oviduct
• Ampulla – the region of the oviduct where fertilization
takes place
• In humans, approx. 300 million sperm are ejaculated
into the vagina, but only 1 in a million enters the
Fallopian tube
• Translocation of sperm from the vagina involves
several processes
Sperm motility
• Motility/flagellar action –
sperm through the cervical
mucus then to the uterus
• Promiscuous female – sperm
from the same male will form
“trains” or aggregates = faster
sperm
Uterine Muscle Contractions
• Sperm appeared to be
transported to the
oviduct by the muscular
activity of the uterus
Sperm Rheotaxis
• “Sperm also receive long-distance directional cues
from the flow of liquid from the oviduct to the
uterus.”
• Migrate against the direction flow using CatSper
calcium channels
• sense Ca influx and monitor the direction of the current
2. The acrosome reaction and recognition at the
Zona pellucida
• Bind and penetrate the
egg’s zona pellucida
• Mouse ZP, 3 glycoproteins
(ZP 1, 2, and 3)
• Human ZP, 4 glycoproteins
(addition of ZP4)
• ZP2 – critical for human
sperm-egg binding Gain-of-function experiment demonstrating that human
sperm bind to ZP2. Of the four human zona pellucida
proteins, only ZP4 is not found in mouse zona.
3. Gamete fusion and the prevention of
polyspermy
• Once the sperm penetrates ZP via secretion of
enzymatic contents of the acrosome – expose the
inner acrosomal membrane, equatorial region
• membrane fusion between sperm and egg begins
• In mammalian gamete fusion process, an
immunoglobulin-like protein called Izumo
• found in the acrosomal granule membrane
Izumo interacts with Juno
• Recruits the egg membrane protein CD9 to the area of
sperm-egg adhesion
• female mice with the CD9 gene knocked out are
infertile due to fusion defects
Izumo protein and membrane fusion in mouse
fertilization. (A) Localization of Izumo to the inner
and outer acrosomal membrane. Izumo is stained
red, acrosomal proteins are green. (B) Diagram of
sperm-egg cell membrane fusion. During the
acrosome reaction, Izumo localized on the
acrosomal becomes translocated to the sperm cell
membrane. There it meets the complex of Juno and
CD9 proteins on the egg microvilli, initiating
membrane fusion and the entry of the sperm into the
egg. (After Satouh et al. 2012; M. Okabe.)
Polyspermy
• the fertilization of an egg by more than one sperm
• a slow block to polyspermy occurs when enzymes
released by the cortical granules
• modify the zona pellucida sperm receptor proteins
such that they can no longer bind to sperm
• Ovastacin – protease found in the cortical granules
Polyspermy
• prevented by Juno protein
• “As the sperm and egg membranes fuse, Juno protein
appears to be released from the plasma membrane.
Thus, the docking site for sperm would be removed.”
4. Fusion of genetic material
• Mammalian pronuclear migration- about 12 hours
• single mammalian sperm that finally enters the egg
carries its genetic contribution in a haploid pronucleus
• DNA of the sperm pronucleus is bound by
protamines—basic proteins that are tightly
compacted through disulfide bonds. Glutathione in
the egg cytoplasm reduces these disulfide bonds and
allows the sperm chromatin to uncoil
4. Fusion of genetic material
• “Microtubules join the two pronuclei and enable
them to migrate toward one another. Upon meeting,
the two nuclear envelopes break down. However,
instead of producing a common zygote nucleus, the
chromatin condenses into chromosomes that orient
themselves on a common mitotic spindle.”
• Thus, in mammals a true diploid nucleus is seen for
the first time not in the zygote, but at the 2-cell stage.
5. Activation of the mammalian egg
• transient rise in cytoplasmic Ca2+ is necessary for egg
activation in mammals
• Fertilization triggers the production of the enzyme
phospholipase C (PLC) by the sperm head = activates
Ca2+ release
• Egg activation (resumption of meiosis, cortical granule
exocytosis) and formation of the pronuclei
Laboratory Activity 3 (Materials)
• Fertilized/fresh chicken eggs (min. 2 per group)
• Unfertilized chicken egg
• Egg tray (anything that keeps the egg in place)
• 1L beaker or glass container
• Rock Salt
• 1L distilled water
• Dissecting kit (spatula and scalpel)
• Microscope slides and cover slips
• Petri plate
• Stove and pan
• Pencil / coloring materials