FERTILIZATION

AND
EARLY EMBRYONIC
DEVELOPMENT
Dr. Ashok Kumar Rangra
M.V.Sc. Veterinary Anatomy
GADVASU, Ludhiana
 Fertilization:
• It can be defined as multiple step phenomenon initiated
by the interaction, binding, and subsequent fusion of
male and female gametes/ pronuclei. And result in
formation of single cell of new individual, called Zygote,
which has bi-parental nuclear heredity.
• Site: ampulla-isthmus junction
• Of the few hundred sperm which reach the egg, only one
will successfully fertilize it.
• This process ultimately leads to the formation of a
diploid cell called a zygote. The zygote begins to divide
and form a blastocyst and when it reaches the uterus, it
performs implantation in the endometrium. At this point
the female is said to be pregnant.
  Transport of spermatozoa
• Testicular spermatozoa of most of species are immotile and
movement in epididymis is entirely passive. sperms are
moved passively toward the epididymal tail. And ejaculated
sperms are motile.
• After sexual intercourse: sperm reach very rapidly in ampulla.
Due to combined effect of: sperm motility, uterine contactions
and reverse ciliiary movements of oviductal epithelium.
• During Mating or AI, the estrogen-sensitized uterus responds
with muscular contractions due to the reflexogenic release of
oxytocin in response to genital stimulation. After mating or
insemination, uterine contractions are far more important than
spermatozoan motility in spermatozoal transport. However,
spermatozoan motility does enhance the probability of a
sperm-oocyte collision.
  Transport of oocyte

• Ovulated oocytes normally enter the fimbria of the same side

oviduct, which is in close contact with the ovary during estrus.
• By ciliary movements , ovum is pushed to the lower end of ampulla

• The proper transport of fertilized eggs in the oviducts is essential

to assure their arrival in the uterus at the proper time. Premature or
delayed arrival results in embryonal death.

• Transport in the oviducts depends upon ciliary movements,

segmental and peristaltic contractions of the oviduct, and probably
the flow of oviductal secretiens.
• Oocytes are capable of fertilization and development, 12 to
24 hours following ovulation in most species.

• Fertility is highest in the ampulla, decreases significantly in

the isthmus, and is lost in the uterus.

• Ejaculated spermatozoa of several mammalian species

must be exposed to the female reproductive tract for a
variable period of time
 Stages of fertilization
 Hyperactivation

 Capacitation

 Transport of spermatozoa
 Zona binding
 Acrosome reaction
 Zona penetration
 Sperm-oocyte membrane fusion
 Formation of male pronucleus
1. Sperm
hyperactivity:
 Sperms acquire hypersensitivity.
 Linear and progressive motility is converted
to non-linear, frenzied, localized motility.
 Due to:
 Narrow isthmic lumen,
 Secretions of isthmus,
 Reduced local temp.,
 Ciliary beat of oviduct
 Purpose: to make contact between sperm
and ova.
 2. Capacitation:
 Ejaculated spermatozoa must be exposed to secretions of female
reproductive tract for a variable period of time before they attain
capacity to fertilize oocyte. This process of enabling spermatozoa
for in-vivo fertilization is called Capacitation.
 The secretions of the uterus and oviducts participate in the
capacitation process, and the follicular fluid released at ovulation
may also contribute to capacitation.
 It involves release or activation of enzymes, possibly associated
with loosening or detachment of the acrosome.
 Surface of spermatozoa in epididymis acquire certain molecules.
These surface molecules are masked by surface proteins. These
are removed in female reproductive tract. i.e. removal of
glycoprotein coat and seminal proteins
 During capacitation:
 Destablishment of sperm plasma membrane
 Removal of cholesterol and glycosamine glycons from the sperm
surface
 Removal of decapacitaion factors
 Activation of receptor sites present on sperm head. These sites
are:
zona binding region (ZBR) And
acrosome reaction promoting region (ARPR).
 Activation of acrosomal enzymes.
 Non-mammalian spermatozoa do not require this capacitation
step and are ready to fertilize an oocyte immediately after
release from the male.
 Site: isthmus
 Time req: sheep: 1-42 hrs,
rabbit: 4 min., cattle<4 min.
3. Transport of
spermatozoa
 Transport of spermatozoa to cumulus oocyte
complex and corona radiata.
 From the site of semen deposition in the female
genital tract to the ampullary region of the oviducts is
essential for successful fertilization.
 Enzymes hyaluronidase released from acrosome helps
in passage of spermatozoa through cumulus
oophorus.
 Corona penetrating enzyme is Aryl Sulfatase.
4. Sperm bind to Zona
Pellucida:
 ZP consists of 3 proteins: ZP1, ZP2, ZP3.
 ZP1 and ZP2 is structural proteins
 ZP3 is receptor site for sperm plasma membrane and
contains 2 sites:
 Zona Binding region (ZBR)
 Acrosome reaction promoting region(ARPR)
 Binding of ARPR with ZP3 initiates acrosomal reaction.
 ZP has species specific receptor, so spermatozoa of
one species can not penetrate oocyte of other
species.
 If ZP is removed, interspecies fertilization can be
done.
5. Acrosome reaction:
 It is release of acrosomal contents.
i.e. exyocytosis of acrosome contents (Ca mediated)
 It involves fusion of outer acrosomal membrane with sperm
plasma membrane resulting in formation of vesicles through
which acrosomal enzymes are released.
 Acrosomal reaction is indicator of complete capacitation
because, sperm do not undergo AR, unless they become
capacitated.
 During AR, there is massive influx of Ca2+ . Carbohydrate is
distinct component of acrosomal matrix.
 Glycoprotein layer covering inner surface of outer acrosmal
membrane holdes vesiculated plasma /outer acrosomal memb
together during AR.
 Enzymatically inactive proacrosin in sperm acrosome is
converted to active acrosin by glycosaminoglycancs in uterine
fluid.
 Acrosome Reaction

1. Pores
2. Emerging of acrosomal contents A. Head
3. Inner acrosomal membrane B. Neck
4. Acrosomal content C. Mid piece
5. Outer acrosomal membrane
6. Cell membrane
Acrosomal reacted spermatozoon

7 Membrane residues dropping behind

 The acrosome reaction is completed. The residues of

the vesicle membrane are partly still held together
by a sticky molecular bonding.
Acrosomal reacted spermatozoon

 Post-acrosomal membrane region

 The forward part of the sperm cell head is now
covered only by the former inner membrane of the
acrosome. For binding to the oocyte the now active
post-acrosomal region is decisive.
 Functions of Acrosomal Enzymes:
 Hyaluronidase: released from acrosome reacting sperm
digest cumulus matrix
 Acrosin: carried on the surface of acrosome reacted sperm
helps in passage of sperm through zona pellucida
 Proacrosin: has a strong zona-binding ability

 Function of acrosome reaction :

 Eggs are surrounded by glycoproteins coats through which
sperms must pass before reaching egg plasma memb.
Acrosome reacted sperm dissolve coat locally to produce a
hole through which sperm swim.
 Equitorius segment of sperm head is exposed. This segment
fuses with vitelline memb.
 Make effective fertilization
 Rendering sperm capable of penetrating zona pellucida.
 Rendering sperm capable of fusing with egg plasma
membrane.
 6. Penetration
through
Zona Pellucida:
 Acrosomal enzymes (zonalysin/acrosin) are responsible for
sperm penetration.
 Penetration of Zona by sperm occurs with in 5-15 min after
sperm attachment. Penetration occurs in curved path leading
to penetration slit. Spermatozoa come in perivitteline space.
 The zona pellucida of most mammals allows a single
spermatozoon to reach the plasma membrane. The zona
pellucida is an effective barrier to prevent the fertilization of an
oocyte by spermatozoa from a different species.
 Enzymatic digestion of the zona pellucida allows for inter­
species fertilization in vitro.
 Primary block to polyspermy is a reaction in the zona pellucida.
The reaction is secondary to changes in the vitelline cortex and
the release of agents which alter the zona pellucida.
 7. Gamete Fusion:
 vitelline membrane may have less specificity than ZP in binding
foreign sperms.
 In perivitteline space, sperm settle in microvilii. Now real fusion
occurs. Sperm plasma membrane at equitorius region fuses with
microvilli of vitelline membrane. Sperm nucleus is engulphed in
vitellus.
 Fusion proteins are present on SPM. They are inactive till
acrosome rexn takes palace. They become active when
acrosomal contents are released. They are responsible for fusion
of sperm with vitelline membrane.
 With entry of sperm nucleus in ooplasm, meiosis-II (metaphase
II) is resumed, resulting in release of Polar body and oocyte.
 Sperm contributes - centriole which organizes mitotic spindle
 Oocyte contributes - mitochondria (maternally inherited)
8. Pronuclei
formation:

 After release of 2nd polar body, oocyte new nuclear envelop called
female pronuclei.
 Simultaneously, new nuclear envelop is formed around sperm
nucleus, called male pronuclei.
 Both pronuclei moves to center of oocyte and both nuclear envelop
fuses together resulting in formation of Zygote (2n). This process is
called syngamy.

 As a consequence of fertilization, the diploid number of

chromosomes is restored, the sex of the individual is determined
and bio­logical variation results from the integration of paternal and
maternal hereditary characteristics.
 Errors in fertilization:
 Supplementary spermatozoa: When zona rexn is weak, slow or
absent, more spermatozoa enters perivascular space.
 Supernumerary spermatozoa: When vitelline block is weak, more
then one spermatozoa found in vitellus of egg. Such sperms are called
supernumerary spermatozoa. This leads to polyspermic fertilization and
polyploidy. In case of pigs it is common.
 Gynogenesis: During oogenesis, if egg fails to release polar body and
these polar bodies are fertilized, it is called gynogenesis.
 Androgenesis: Here 2 or more male pronuclei conjugate with female
pronuclei leading to triploidy. It is due to polyspermic fertilization.
 Immediate cleavage: when unfertilized ovum divides and 2 daughter
cells are fertilized by 2 different spermatozoa. Condition is called
mosaicism and leads to mosaics.
 Barriers to polyspermy
 Polyspermy may leads to the death of the zygote.
 Natural anatomical barriers of the female reproductive tract, the cervix and
the utero-tubal junction, prevent the mass movement of spermatozoa to the
site of fertilization.
 At the cellular level, the ovum has its own defence against polyspermy which
normally prevents the entry of more than one spermatozoon. This defence,
which is biphasic, operates at the zona pellucida and the cell (vitelline)
membrane of the ovum. In most mammals, both the zona pellucida and
vitelline membranes undergo alteration after entry of the first spermatozoon,
a change which makes these structures impenetrable to additional
spermatozoa.
 Secondary oocytes contain small, membrane-bound organelles termed
cortical granules beneath the vitelline membrane. These granules contain an
array of enzymes which are released when the head of the spermatozoon
comes in contact with the oocyte surface. Following the release of these
enzymes, the zona pellucida becomes altered with loss of species-specific
receptors for spermatozoa. This change, referred to as the zona reaction,
prevents spermatozoan adhesion and penetration of the zona by additional
spermatozoa.
 Comparable changes in the vitelline membrane of the oocyte which prevent
entry of spermatozoa are referred to as the vitelline block.
Cleavage:
 Is the process of cellular division without growth.
 The series of cell division that transforms the single
cell zygote into a multicellular embryonic stage- the
blastula, is called cleavage or segmentation.
 The mitotic divisions during this phase are called
cleavage divisions and resulting daughter cells are
called blastomere.
 Process is characterized by negative growth because
with each division there is decrease in cell size.
 It is a first step to increase cell number by continues
mitotic divisions. Cleavage always starts at animal
pole.
Characteristics of
cleavage:
 All divisions are mitotic
 During cleavage, there is negative growth
 General shape of embryo doe not change except for
the formation of cavity c/a blastocoel
 Great increase in synthesis of DNA takes place
 During cleavage, O2 consumption is increased
 Cleavage ends when blastocoels is formed
 Cleavage occurs by vertical division through main axis
starting from animal pole to vegetal pole.
 Animal pole is place where polar bodies are released
and vegetal pole is place where yolk reserve is present.
 Cleavage furrow is often goes through main axis where
pronuclei resides before syngamy. Resulting cell of
cleavage is K/a Blastomere.
 2nd division is also vertical and goes through main axis
but is perpendicular to 1st division. Resulting in
formation of 4 blastomeres.
 3rd division is perpendicular to 2nd division resulting in
formation of 8 blastomere.
 Upto 8 celled stage, blastomeres are totipotent and
after 8-celled stage, cleavage become asynchronous
(divide at different time) and blastomeres divide
independently and loose their totipotency.
 After 8-celled stage, 16-celled stage is called early
morula/ uncompact morula.
 32-celled stage is called compact morula.
 Name is because of embryo looks like mulberry fruit.
 It is stage before the blastocoel is formed.
 32-celled embryo is most important and is k/a stage of 5th
generation mitosis. This stage is used for embryo transfer.

 The central cell mass of morula gives rise to embryo

proper and peripheral cells form protective and nutritive
covering of embryo.
 Due to spheroidal shape of bastomere are separated by
small intercellular space. After morula stage, fluid
accumulation occurs in these spaces. And blastomeres are
pushed to periphery from the center.

 After this, blastomeres start secreting fluid forming cavity

k/a blastocoel and embryo is called blastocyst.

 This blastocyst consist of 2 cell masses – outer (tropho-

ectoderm or trophoblast) and inner cell mass
(embryoblast). Trophoblast forms chorion( fetal
membranes) and embryoblast forms embryo proper.

 Trophobast cell provide nourishment to embryo, secrete

enzymes for inducing attachment of blastocyst to
endometrium, and protect the inner cell mass.
 Zona Hatching: Realese of blastocyst from protective
zona pelucida is called zona hatching. It is enzymatic
process.
 In rabbit, enzymes called blastolemmase secreted by
trophoblast is responsible for weakening of zona
pelucida.
 In mouse, rhythmic contraction and expansion of
blastocyst initiated by zonalysin leads to zona pellucida
rupture.
 Zonalysin is released by uterine endometerium.

 After zona hatching, blastocyst looses its spherical

shape and becomes filamentous. It is k/a blastocyst
elongation. In cows, elongation occurs by 17th day. by
18th day elongated blastocyst enters contra lateral
horns. Growth of blastocyst depends on hyperplasia of
trophoblast.
 After blastocyst stage, Conversion of bilaminal
blastocyst to trilaminar structure is called gastrulation
and structure is called gastrula.
 Gastrulation is the process in which three germ layers-
ectoderm, mesoderm and endoderm are formed that
will give rise to the different organs and extra
embryonic membranes. In domestic animals it occurs
in uterus when blastocyst is still floating freely in
uterine fluid.
 In domestic animals, cleavage is indeterminate type i.e.
until a late stage in development; it is not possible to
show which cell is going to form which organ. And all
cells retain potency for overall development.
 In lower vertebrates, it is determinate type

i.e. particular cell going to form particular organ.

Thanx..