PROF. DR.

ÇİĞDEM ELMAS
 5-45 minutes (2-7 hours)  sperm reaches fallopian tube from cervix.
Two events must occur before
fertilization:
 (1) sperm maturation in the
epididymis

 (2) sperm capacitation in the

female reproductive tract
 1) SPERM MATURATİON (2W; epididymis)

Sperm released from the testis and entering the

epididymal duct have circular motion. After a 2-week
maturation process, following epididymal transit and
storage in the tail or cauda of the epididymis, sperm
acquire forward motility necessary for fertilization
 2) CAPACİTATİON
 After ejaculation, sperm undergo a capacitation
process in the uterus and fertilization of the
ovum takes place in the oviduct.
 Capacitation can be induced in vitro, a
procedure that permits in vitro fertilization.
 Capacitated sperms show no morphologic
changes, but they are more active. Sperms are
usually capacitated in the uterus or uterine tubes
by substances secreted by these parts of the
female genital tract.
 Sperms must undergo a period of conditioning-capacitation-
lasting approximately 7 hours.
 During this period, a glycoprotein coat and seminal proteins are
removed from the surface of the sperm's acrosome.
 The membrane components of the sperms are extensively altered.
 Only capacitated sperm can bind to Zona pellucida.
 the sperm head
consists of three
components:
 (1) the condensed
nucleus,
 (2) the acrosomal sac,
 (3) the plasma
membrane.
The condensed nucleus consists of genomic DNA
coated by very basic protamines. Nucleosomes are
not present because somatic histones have been
replaced by protamines!
The acrosomal sac is formed by three constituents
(1) the outer acrosomal membrane,
(2) the inner acrosomal membrane,
(3) hydrolytic enzymes
(mainly hyaluronidase and acrosin,
the latter derived from the
precursor proacrosin).
The three main events during
fertilization are, sequentially

1.The acrosome reaction (for corona

radiata)

2. Sperm binding to ZP3, a

glycoprotein of the zona pellucida

3. Sperm-egg fusion
ACROSOME REACTİON
 In the presence of Ca2+, the sperm plasma membrane fuses with the outer
acrosomal membrane.
 This event is known as the acrosome reaction. Small openings created by
membrane fusion facilitate the release of hydrolytic enzymes.
 The equatorial region of the acrosome does not participate in the
membrane fusion process at this time
 1.Passage of a sperm through the corona radiata. Dispersal
of the follicular cells of the corona radiata surrounding the
oocyte and zona pellucida appears to result mainly from the
action of the enzyme hyaluronidase released from the
acrosome of the sperm, but the evidence of this is not
unequivocal. Tubal mucosal enzymes also appear to assist the
dispersal. Movements of the tail of the sperm are also important
in its penetration of the corona radiata
 2.Penetration of the zona pellucida. Passage of a
sperm through the zona pellucida is the important
phase in the initiation of fertilization. Formation of a
pathway also results from the action of enzymes
released from the acrosome. The enzymes esterases,
acrosin, and neuraminidase appear to cause lysis of the
zona pellucida, thereby forming a path for the sperm to
follow to the oocyte
 The zona pellucida is composed of only three glycoproteins ZP1, a
dimer of 200 kd; ZP2, 120 kd; and ZP3, 83 kd.
 ZP2 and ZP3 interact to form a long filament complex
interconnected by ZP1 dimers at regular intervals. During sperm
binding, O-oligosaccharides linked to ZP3 bind to sperm
receptors. Only acrosome-reacted and capacitated sperm can
interact with ZP3
 Dispersal of the follicular cells of the corona radiata
surrounding the oocyte and zona pellucida appears to result
mainly from the action of the enzyme hyaluronidase
released from the acrosome of the sperm
 The enzymes esterases, acrosin, and neuraminidase appear
to cause lysis of the zona pellucida, thereby forming a path
for the sperm to follow to the oocyte
 Tripsin for the lysis of the zona pellucida
 3.Fusion of plasma
membranes of the
oocyte and sperm.
The plasma or cell
membranes of the
oocyte and sperm
fuse. The head and
tail of the sperm
enter the cytoplasm
of the oocyte, but the
sperm's plasma
membrane remains
behind
 egg plasma membrane has α3β1, α6β1, and α5β1 integrins, which
associate with CD9, a protein member of the superfamily of
tetraspanins
 A lack of CD9 prevents sperm-egg fusion.
 Sperm; The fertilin α and β heterodimer
 Cortical and zona reactions
 Finishing meiosis II division
 metabolic activation of oocytes
 Oocyte cortical granules containing lysosomal enzymes
 oocyte membrane thickens
 Polyspermi X

Cortical granules and the vitelline envelope (fertilization envelope):

The transient rise in intracellular Ca2+ initiates exocytosis of the cortical
granules, elevating the vitelline envelope to provide a “slow” block to
polyspermy (a “fast” or “electrical” block to polyspermy results from the
transient depolarization of the egg membrane).
 Formation of the male pronucleus. Within the cytoplasm of the
oocyte, the nucleus of the sperm enlarges to form the male
pronucleus and the tail of the sperm degenerates
 Morphologically, the male and female pronuclei are indistinguishable.
 As the pronuclei fuse into a
single diploid aggregation of
chromosomes, the oocyte
becomes a zygote. The
chromosomes in the zygote
become arranged on a cleavage
spindle in preparation for
cleavage of the zygote
 Early pregnancy factor, an immunosuppressant
protein, is secreted by the trophoblastic cells and
appears in the maternal serum within 24 to 48 hours
after fertilization. Early pregnancy factor forms
the basis of a pregnancy test during the first 10
days of development
 Stimulates the penetrated oocyte to complete the second meiotic
division.
 Restores the normal diploid number of chromosomes (46) in the
zygote.
 Results in variation of the human species with maternal and
paternal chromosomes.
 Determines chromosomal sex of the embryo.
 Causes metabolic activation of the oocyte and initiates cleavage
(cell division) of the zygote
 Cleavage consists of repeated mitotic divisions of the zygote, resulting in a
rapid increase in the number of cells. These embryonic cells-
blastomeres-become smaller with each successive cleavage division
 Subsequent cleavage divisions follow one another, forming
progressively smaller blastomeres .
 After the nine-cell stage, the blastomeres change their shape and tightly
align themselves against each other to form a compact ball of cells.
 This phenomenon, compaction, is probably mediated by cell surface
adhesion glycoproteins
 When there are 32 to 60 blastomeres, the developing human is called a
morula
 The morula appears in the fallopian cavity about 2
to 3 days after fertilization
 Shortly after the morula enters the uterus
(approximately 4 days after fertilization), a fluid-
filled space called the blastocystic cavity appears
inside the morula
 The fluid passes from the
uterine cavity to form this
space. As fluid increases in
the blastocystic cavity, it
separates the blastomeres
into two parts:
 A thin, outer cell layer, the
trophoblast which gives
rise to the embryonic part
of the placenta
 A group of centrally
located blastomeres, the
inner cell mass, which
gives rise to the embryo;
because it is the
primordium of the
embryo, the inner cell
mass is called the
embryoblast
 4-4.5 day blastocyst58-107 cells
 107 cell blastocyst:

99 trophoblast, 8 embryoblast
 After the free blastocyst has floated in the uterine secretions
for approximately 2 days, the zona pellucida gradually
degenerates and disappears (Hatching)
 Approximately 6 days after fertilization (day 20 of a 28-day
menstrual cycle), the blastocyst attaches to the endometrium.
 The implantation of the blastocyst into a uterus endometrium
involves the initial unstable adhesion of the blastocyst to the
endometrial surface, called apposition, followed by a stable
adhesion phase and the decidualization of the endometrial
stroma
 the inner cell mass, which
gives rise to the embryo;
because it is the primordium
of the embryo, the inner cell
mass is called the
embryoblast
 the trophoblast which
gives rise to the embryonic
part of the placenta
 Trophobast
secrates integrins,
extracellular matrix proteins,
fibronectin and laminin that
are effective at implantation.
 Laminin,
integrin.......penetrating
 Fibronectin……migratio
n
 1.Superfetation or superfoetation: During the
pregnancy, new ovulation and fertilization occur
 2. Superfecundation: at one ovarian cycle more than
one oocyte separated and these oocytes fertilized by
different men’s sperm.
 In vitro fertilization (IVF) of oocytes and transfer of the cleaving
zygotes into the uterus have provided an opportunity for many
women who are sterile to bear children.
 The first of these in vitro fertilization babies was born in 1978.
Since then millions of children have been born after an in vitro
fertilization procedure.
 Ovarian follicles are stimulated to grow and mature by the administration of clomiphene
citrate or gonadotropin (superovulation).
 Several mature oocytes are aspirated from mature ovarian follicles during laparoscopy.
Oocytes can also be removed by an ultrasonography-guided large-gauge needle inserted
through the vaginal wall into the ovarian follicles.

 One or two of
 The oocytes the resulting
are placed in a embryos (four- to
Petri dish eight-cell stage
containing a or early
blastocysts) are
special culture transferred by
medium and introducing a
capacitated catheter through
sperms. the vagina and
 Fertilization of cervical canal
the oocytes into the uterus.
Any remaining
and cleavage embryos are
of the zygotes stored in liquid
are monitored nitrogen for later
microscopicall use.
y for 3 to 5  The patient lies
days. supine (face
upward) for
several hours.
 Obviously, the chances of multiple pregnancies
are higher than when pregnancy results from
normal ovulation, fertilization, and passage of
the morula into the uterus via the uterine tube.
The incidence of spontaneous abortion of
transferred embryos is also higher than normal.
 Cryopreservation of Embryos :Early embryos resulting
from in vitro fertilization can be preserved for long
periods by freezing them with a cryoprotectant (e.g.,
glycerol). Successful transfer of four- to eight-cell
embryos and blastocysts to the uterus after thawing is
now a common practice.
 Intracytoplasmic Sperm Injection : (ICSI):A sperm can
be injected directly into the cytoplasm of a mature
oocyte. This technique has been successfully used for
the treatment of couples for whom in vitro fertilization
failed or in cases where there are too few sperms
available for in vitro insemination
 Assisted In Vivo Fertilization: A technique enabling fertilization in
the uterine tube is called gamete intrafallopian transfer. (GIFT) It
involves superovulation (similar to that used for in vitro
fertilization), oocyte retrieval, sperm collection, and laparoscopic
placement of several oocytes and sperms into the uterine tubes. N
this technique, fertilization occurs in the ampulla.
 ZIFT: Zygote intrafallopian transfer
 Surrogate Mothers: Some women produce mature oocytes but are
unable to become pregnant, for example, a woman who has had her
uterus excised (hysterectomy). In these cases, in vitro fertilization
may be performed and the embryos transferred to another woman's
uterus for development and delivery.
 Sperms can be obtained from ejaculate, testicular
tissue and epididymis
 From ductus epididymis=MESA (Microsurjical
Epididimal Sperm Aspiration)
 From testicular tissue:TESA
 Rounded sperm injection:ROSI
 Elongated sperm injection:ELSI