FERTILIZATION

Fertilization is the process whereby the gametes-sperm and egg

fuse together to begin the creation of a new organism.
Fertilization accomplishes two separate ends:
(i)the combining of genes derived from two parents
(ii)the generation of a new organism
Thus, the first function of fertilization is to transmit genes from
parents to offspring, and the second is to initiate in the egg
cytoplasm those reactions that permit development to proceed.
Site of fertilization: mammalian fertilization occurs inside the
oviducts (ampulla) of the female.
MECHANISM OF FERTILIZATION:
Approach of spermatozoa to ovum (Translocation)
Capacitation and contact of sperm and ovum
Acrosomal reaction and entry of sperm
Activation of ovum
Amphimixis
Approach of spermatozoa to ovum (Translocation):
The meeting of sperm and egg must be facilitated by the female
reproductive tract.When the oocyte-cumulus complex enter the
oviduct a combination of ciliary beating and muscle contractions
transport it to the appropriate position for its fertilization in the
oviduct.The sperm must travel a longer path. In humans, about
200–300 million sperm are ejaculated into the vagina, but only
one in a million enters the oviducts (fallopian tube). And half the
sperm enter the “wrong” oviduct, the one that has no oocyte.
Only about 200 sperm probably reach the vicinity of the egg.
The translocation of sperm from the vagina to the oviduct
involves several processes that work at different times and
places:
Sperm motility: Motility (flagellar action) is probably important
in getting sperm through the cervical mucus and into the uterus.
Uterine muscle contractions: sperm appear 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.
Sperm display rheotaxis—that is, they migrate against the
direction of the flow—using CatSper calcium channels (like sea
urchin sperm) to sense calcium influx and monitor the direction
of the current.
Sperm attraction: In many animals, sperms are attracted
towards eggs of their species by “chemotaxis” i.e. following a
gradient of a chemical secreted by the egg.
Chemotaxis has been demonstrated in cnidarians, molluscs,
echinoderm and urochordates.
Similarly in the egg jelly of the sea urchin, chemotactic factors
resacet are present for sperm attraction.
Capacitation and contact of sperm and ovum:
Newly ejaculated sperm cannot fertilize an egg. They are
immature. The cells of the oviduct induce the sperm to mature,
and mammalian sperm complete their development in the
female oviducts. This maturation is called capacitation (the
gaining of capacity). The capacities the sperm gain are those for
(1) recognizing the cues that will guide them to the egg, (2)
undergoing the acrosome reaction, and (3) fusing with the egg
cell membrane.Sperm that are not capacitated are “held up” in
the cumulus matrix and are unable to reach the egg.Capacitation
is possibly initiated by an efflux of cholesterol from the sperm
cell membrane, an efflux caused by proteins in the female
reproductive tract.Capacitation is result of some chemical
reactions like loss of decapacitation factors and calcium influx.
The changes that follow unmask receptors on the sperm that
allow binding to the zona pellucida and prepare the acrosome
for the acrosome reaction.
There is a thermal gradient of 2 degrees celsius between the
isthmus of the oviduct and the warmer ampulla. Capacitated
mammalian sperm can sense this thermal difference
(thermotaxis).The capacitated sperm are also able to detect and
respond to the hormone progesterone, which is secreted by the
cumulus cells surrounding the egg . Thus, as the sperm enter the
ampulla, they are told where the egg can be found.
Fertilizin and Anti fertilizin interactions: Factors that mediate
sperm– egg interactions even before they make contact were
identified by F.R. Lillie (1912). He proposed the first theory of
physiology of fertilization called fertilization theory.
He observed that the egg water (seawater surrounding
unfertilized sea urchin eggs), agglutinated the sperm and
activated their motility.The reaction was species specific. This
factor called fertilizin came from the egg jelly coat. It slowly
dissolved as in sea water. Fertilizin was later shown to be the
constituent of both jelly coat and egg membrane such as
vitelline membrane and plasma membrane.Fertilizinis a
proteoglycan. Both the amino acids and monosaccharides of
fertilizin vary from one species to another so that each species
possesses its specific type of fertilizin. Each molecule of
fertilizin has more than one ‘active group’ so that one fertilizin
particle may attach to two or more sperms and bind them
together.The receptor sites for fertilizing are present on the
sperm plasma membrane called antifertilizin.These are acid
proteins. Adhesion of spermatozoa to the surface of the egg is
brought about by linking of fertilizin molecule with antifertilizin
molecules.The reaction between fertilizin and antifertilizin is
similar to antigen – antibody reaction. In both cases, a chemical
lock is formed between two complimentary substances. It has
been suggested that the main function of fertilizin – antifertilizin
is to thin out the number of spermatozoa around the egg, so that
the chances of two or more spermatozoa fusing with the egg at
the same time are diminished.
Acrosomal reaction and entry of sperm:
The mammalian egg is surrounded by extracellular envelope
called zona pellucida. Around zona pellucida is a layer of
cumulus cells (corona radiate) embedded in a cementing
substance, hyaluronic acid. Hyaluronidase activity on the
surface of the sperm head helps it to penetrate this layer. Next,
sperm must bind to zona pellucida before they make contact
with the surface of egg itself. The zona pellucida in mammals
plays a role analogous to that of viteline envelope in lower
vertebrates and invertebrates. The zona pellucida is a
glycoprotein matrix synthesized and secreted by the growing
oocyte. It plays two important roles in fertilization .It binds the
sperm and initiates acrosome reaction. Binding of the
spermtozona triggers the acrosome reaction, which allows the
sperm to penetrate the zona pellucida of ovum in animal pole.
Acrosome reaction in mammals involves the fusion of the outer
membrane of the acrosome with the sperm plasma
membrane.After the fusion, the acrosomal membrane
vesiculates which results in the release of acrosomal contents.
Subsequently, the outer portion of the acrosomal membrane
disappears and only the inner portion adjacent to the nucleus
remains intact. When the acrosomal contents are exocytosed,
several enzymes are released. These enzymes allow the
spermtozoa to approach the egg plasma membrane.
Hyaluronidase: It hydrolyse hyaluronic acid matrix between
cumulus layer and corona radiata.
Corona penetrating enzyme: dissolves corona radiata
Acrosin: dissolves zona pellucida
During sperm penetration, only nucleus and middle piece enter
the ovum while tail is lost. The mammalian acrosome reaction
differs from that of sea urchin in that no acrosomal process is
formed.
Acrosomal reaction in Sea urchin:
Once the sperm makes contact with the egg, then it has to
penetrate surface coats that surround the egg. The penetration is
facilitated by the acrosome reaction in which the membrane
enclosing the acrosome is shed, releasing the contents of
acrosome. The acrosomal reaction involves two processes:
a) exocytosis of acrosomal vesicle and
b) extension of acrosomal process.
Exocytosis of acrosomal vesicle: Contact of the sperm with the
egg jelly coat component, a fucose containing polysaccharide
triggers the acrosome reaction and causes influx of calcium into
the sperm head.This initiates fusion of the outer acrosomal
membrane with sperm plasma membrane and ultimate
breakdown of acrosomal vesicle. Hydrolytic enzymes called
lysins present in the acrosomal vesicle, are released. Lysins
digest the egg envelope locally and clear the path for
spermatozoa to reach the egg surface (vitelline membrane). The
exocytosis of acrosomal vesicle is thus caused by calcium –
mediated fusion of acrosomal membrane with the adjacent
sperm plasma membrane. The egg jelly factors that stimulate the
acrosome reaction are highly species specific.
Extension of acrosomal process: The second part of the
acrosomal reaction involves the extension of the acrosomal
process / tube / filament. It is formed by polymerization of
globular active molecules into actin filaments.
Activation of ovum:
A) Fertilization cone formation: In sea urchin, all regions of
the egg plasma membrane are capable of fusing with sperm. The
cytoplasm of the egg bulges forward at the point of contact
producing a process of hyaline cytoplasm called the
fertilization cone. Fertilization cone engulf spermatozoa.
B) Cortical reaction: When sperm and egg cell membranes
fuse some changes occur in egg cortex called Cortical reaction
to prevent polyspermy. Upon sperm entry, cortical granules fuse
with the egg cell membrane and release their contents into the
space between the cell membrane and the fibrous mat of
vitelline envelope proteins. The components of the cortical
granules bind to the vitelline envelope to form a fertilization
envelope. The fertilization envelope starts to form at the site of
sperm entry and continues its expansion around the egg.The
fertilization envelope is elevated from the cell membrane by
glycosaminoglycans released by the cortical granules.These
viscous compounds absorb water to expand the space between
the cell membrane and the fertilization envelope, so that the
envelope moves radially away from the egg. In sea urchins and
mammals, the rise in Calcium ion concentration responsible for
the cortical granule reaction is not due to an influx of Calcium
ion into the egg, but comes from the endoplasmic reticulum
within the egg itself. In mammals, perivitelline space develop
between zona pellucida and egg plasma membrane.This entire
process is slow and permanent block to polyspermy but there are
fast block to polyspermy also in sea urchins where the resting
membrane potential of the egg changed from -70 mV to + 20
mV. Sperm entry into ovum stimulates MPF (M- phase
promoting factor) and APF (Anaphase promoting factor) which
complete metaphase-II.
 At 10 sec. At 25 sec.

fertilization envelope is complete,

At 35 sec. and excess sperm have
been removed
 Cortical granule exocytosis and formation of the sea
urchin fertilization envelope

C) Metabolic activation:Increased rate of respiration due to

utilization of glycogen and other food stuffs for getting energy
ATP molecules.
Ionic changes: certain intracellular changes occur in the
concentration of cations such as sodium,potassium and calcium.
Activation of protein and there is increase in the rate of protein
synthesis by utilizing mRNA already present in the oocyte
cytoplasm.
Resumption of meiosis: in most animals meiosis is arrested at a
particular stage and resumes only after fertilization.
Initiation of mitosis: the initiation of mitosis occurs because (a)
the rate of DNA synthesis increases after fertilization and (b) by
the contribution of centriole by sperm to the egg, which is
needed for proper mitosis.
Amphimixis:
The fusion of male and female chromosomal sets is called
amphimixis. After the sperm and egg cell membranes fuse, the
sperm nucleus and its centriole separate from the mitochondria
and flagellum. The mitochondria and the flagellum disintegrate
inside the egg. The mitochondrial genome is transmitted
primarily by the maternal parent. The centrosome needed to
produce the mitotic spindle of the subsequent divisions is
derived from the sperm centriole.
But how do the sperm and egg pronuclei find each other?
Once the sea urchin sperm has entered the egg cytoplasm and its
nucleus has separated from the tail, the sperm nucleus rotates
180º so that the sperm centriole is between the developing male
pronucleus and the egg pronucleus. The sperm centriole then
acts as a microtubule organizing center, extending its own
microtubules and integrating them with egg microtubules to
form an aster. Microtubules extend throughout the egg and
contact the female pronucleus, at which point the two pronuclei
migrate along the microtubules toward each other. This leads to
fusion of both pronuclei and ulimately diploid zygote is formed.
In mammals the entry of sperm is tangential to the eggs surface.
Once inside the egg, the sperm nucleus becomes male
pronucleus. The sperm centrosome produces asters and contacts
the female pronucleus. Male & female pronuclei migrate
towards each other, become apposed but do not fuse. They
remain adjacent to each other; their nuclear envelopes break
down but instead of forming a zygote nucleus the chromatin
condenses into chromosomes orienting them on a common
mitotic spindle. Thus, only after completion of the first division
of fertilized egg, the paternal and maternal chromosomes
become enclosed by a common nuclear membrane to form the
nuclei of two blastomeres.