FERTILIZATION TO IMPLANTATION

Complex sequence of coordinated molecular events that

begins with contact between a sperm & an oocyte
And
Ends with intermingling of maternal & paternal chromosomes
At metaphase of 1st mitotic division of zygote (a unicellular embryo)
 OOCYTE TRANSPORT

• Follicular fluid
• Fimbriae
• Cilia
• Peristalsis

Fertilized oocyte
reaches uterine lumen
in 3-4 days
 SPERM TRANSPORT

200-600 million sperms deposited in vaginal fornix

Only 1% (200) enter cervix—may survive for many hours
Most degenerate & reabsorbed
Move to uterine tubes by propulsion & fluid movements by uterine cilia (2-7 hours)
Sperm tail motility
Vesiculase (by seminal vesicles) semen coagulation—vaginal plug to prevent backflow
Cervical mucous—less viscid at ovulation

Prostaglandins (semen) stimulate ut motility

Fructose (semen) energy for sperms
Chemotaxis by ovum
Semen=2-6 ml (3.5 ml)
Motility varies=non-motile in epidid, more in uterus (alkaline pH)
Cervical mucus

Abundant mucus
- like “raw egg
Production
white”
of low Cervical
viscosity mucus
mucus
increases Thick, rubbery, high
Variable viscosity -
number of impenetrable to
“dry” days sperm.

0 4 8 12 16 20 24 28

Menstruation
OVULATION
 MATURATION OF SPERMS

1. Capacitation—epithelial interactions b/w sperm & tubal mucosa—

substances released from uterus & tubes; glycoprotein coat & seminal
pl pr removed from pl memb-- increases activity without morphological
change
2. Acrosome reaction– induced by Z.pr after binding to ZP3
 CAPACITATION
1. Epithelial interactions b/w sperm & tubal mucosa
2. Substances released from uterus and tubes
3. Glycoprotein coat & seminal pl pr removed from pl
memb
4. Sperm memb components altered
5. Increases sperm activity
6. No morphological change in sperms
7. In vitro fertilization—incubate sperms in a defined
medium for several hours

Completion of capacitation permits

acrosome reaction to occur
 ACROSOME REACTION
1. Capacitated sperms with intact acrosome
2. Contact C.radiata
3. Complex molecular changes (perforations in acrosome)
4. Intact acrosome binds to ZP3
5. Multiple point fusions of sperm pl memb & ext ac memb
6. Inner acrosomal memb dissolves—apertures formed
7. Acrosomal enzymes released—hyaluronidase, acrosin
8. Critical role by– sperm pl memb, Ca++, PG, Prog
9. Must be completed before sperm can fuse with oocyte

Enzymes facilitate fertilization

PHASES OF FERTILIZATION
 PHASES OF FERTILIZATION
Phase 1: Passage of sperm through corona radiata
– Hyaluronidase from acrosome
– Sperm tail
– Tubal mucosal enzymes
 PHASES OF FERTILIZATION
Phase 2: Penetration of zona pellucida (by acrosomal enzymes)
– Acrosin (proteolytic enzyme)
– Neuraminidase
– Esterases

Sperm penetrates Zona Pellucida

Zona reaction
Properties of ZP altered by
Release of Lysosomal enzymes of cortical granules
 PHASES OF FERTILIZATION
Phase 3: Fusion of oocyte and sperm pl
memb:
• Adhesion of sperm to oocyte--interaction of
integrins (oocyte) & disintegrins (sperms)
• Fusion of plasma membranes of oocyte & that
covers post region of sperm head
• Head & tail enter oocyte cytoplasm
 FERTILIZATION
1 Contact. The 2 Acrosomal reaction. Hydrolytic
sperm cell enzymes released from the
contacts the acrosome make a hole in the
egg’s jelly coat, jelly coat, while growing actin
triggering filaments form the acrosomal
exocytosis from the process. This structure protrudes
sperm’s acrosome. from the sperm head and
penetrates the jelly coat, binding
to receptors in the egg cell
membrane that extend through
the vitelline layer.
Sperm plasma
membrane
3 Contact and fusion of sperm
and egg membranes. A hole
is made in the vitelline layer,
allowing contact and fusion of
the gamete plasma membranes.
The membrane becomes
depolarized, resulting in the
fast block to polyspermy.
4 Entry of 5 Cortical reaction. Fusion of the
sperm nucleus. gamete membranes triggers an
increase of Ca2+ in the egg’s
cytosol, causing cortical granules
in the egg to fuse with the plasma
membrane and discharge their
contents. This leads to swelling of the
perivitelline space, hardening of the
vitelline layer, and clipping of
sperm-binding receptors. The resulting
fertilization envelope is the slow block
to polyspermy.

Sperm
nucleus
Acrosomal
process
Basal body
(centriole)
Sperm Fertilization
head envelope
Fused plasma
Cortical membranes
granule
Actin
Perivitelline
Hydrolytic enzymes
space
Acrosome Cortical granule
Jelly coat Vitelline layer membrane
Egg plasma
Sperm-binding EGG CYTOPLASM
membrane
receptors
 • Cortical & zona reactions
• Resumption of 2nd meiotic
division of oocyte
• Formation of female pronucleus
• Metabolic activation of egg
• Formation of male pronucleus
• Ootid
Haploid • Pronuclei lose their nuclear
envelopes
• Fusion of pronuclei to
Ootid
form
• Zygote
• Chromosomes on cleavage
spindle
 Results of Fertilization
❑ Stimulation to complete Meiosis-II
❑ Restoration of diploid chromosomes
❑ Biparental inheritance-- new combination of
chromosomes, different from the parents
❑ Variation of human species
❑ Crossing over of chromosomes in Meiosis--
shuffling of genes
❑ Determination of chromosomal sex of embryo
❑ Metabolic activation of ootid & initiation of
cleavage of zygote
 Early Pregnancy Factor (EPF)
• Immuno-supressant protein
• Secreted by trophoblastic cells
• Appears in maternal serum in 24-48 hrs
after fertilization
• Forms the basis for pregnancy test
during 1st 10 days
Dispermy

1. Two sperms fertilize an oocyte

2. Triploid embryos
3. Aborted
4. IUGR
 SEQUENCE AFTER
FERTILIZATION?
• 1st cleavage after 30 Hours
• Morula 3 days after F
• Morula enters uterine cavity
• Blastocyst formation 4 days after F
• Blastocyst floats in Uterus another 2 days
• ZP ruptures 6th day
• Blastocyst starts implantation 6th day
CONTRACEPTIVE METHODS
• Barrier techniques
-for males: condoms (latex)
-for females: condoms (polyurethane),
diaphragm, cervical cap, contraceptive
sponge
Contraceptive pill Hypothalamus
•inhibit ovulation

GnRH (gonadotrophin
Pituitary releasing
hormone)
LH
FSH
Feedback + (“gonadotrophins”)
Ovaries

Steroids
(estrogen+
progestin)

Reproductive tract
MENSTRUATION
Other targets
Depo-Provera
• Progestin
• S/dermally implanted (5 years)
• I/M inj (23 months)
Male pill (synthetic androgens)
• Prevent FSH & LH secretion
• Stop or reduce sperm production
IUD
• Inhibit preimplantation stages
RU-486 (mifepristone)
• Antiprogesterone
• Causes abortion if administered within 8 weeks of previous
menses
Vasectomy & tubal ligation
 INFERTILITY (15-30% couples)
Male Infertility
• Sperm number, structure & motility
• <10 million /ml FERTILE
20-100 million /ml

or
Female infertility 50-400 million/ejaculate

• PID-Tubal occlusion
• Hostile cervical mucus
• Immunity to spermatozoa
• Anovulation
• Uterine factor (endometriosis)
 COMMON CAUSES OF
INFERTILITY
Causes of Infertility
Cause Couples Women
Male 35% N/A
Ovulatory 15% 40%
Tubal 35% 40%
Unexplained 10% 10%
Other 5% 10%
 Clomiphene citrate (Clomid)
• Increase FSH

• Given in early endometrial cycle

 ASSISTED REPRODUCTIVE
TECHNOLOGY (ART)

Refers to all techniques

involving direct retrieval of oocytes

➢IVF**
from the ovary

Complications
• Prematurity ➢ICSI
•
•
Low birth weight
Infant mortality ➢GIFT
• Malformations
• Multiple births
IVF

M-1 late stages

Patient remains in supine position for several hours

Intracytoplasmic sperm injection
(ICSI)
• For male infertility

• Zygote till eight cell stage

• Placed in uterus
Gamete Intrafallopian (intratubal)
Transfer - GIFT
• Superovulation

• Oocyte retrieval

• Sperm collection

• Laparoscopic placement of several oocytes &

sperms into uterine tubes

• Fertilization occurs in ampulla

 PRESELECTION OF
EMBRYO SEX
• Primary sex ratio (1.00)

• Secondary sex ratio (1.05)

 PRESELECTION OF EMBRYO
SEX
In vitro techniques to separate X & Y sperms
• Differential swimming abilities of X & Y sperms
• Different speeds of migration of sperms in electric
field
• Microscopic differences in their appearance
• DNA difference – 2.8% more in than
 CRYOPRESERVATION

IVF – early embryos

• Freezing in liquid nitrogen with cryoprotectant
(glycerol or dimethyl sulfoxide [DMSO])
• Thaw
• Transfer to uterus
SURROGATE MOTHERS ??

Surrogate mother

a woman who agrees, usually by contract and for a fee,

to bear a child for a couple who are childless because
wife is infertile or physically incapable of carrying
a developing fetus. Often surrogate mother is the
biological mother of the child
 SURROGATE MOTHERS ??
• If hysterectomy in a natural mother
• IVF performed
• Embryo transferred to another woman’s
uterus
• Delivers it to natural mother at birth
 Holoblastic Cleavage
• Rapid mitotic division begins
• Cells gets progressively smaller
• Zygote does not get larger (yet)
• Morula: solid mass of cells (~32 cells)

~30 hours 4 days

 Cleavage
Repeated mitotic divisions of
zygote resulting in rapid
increase in no. of embryonic
cells “blastomeres”
Within Zona pellucida
1st cleavage—30 hours after
F
2nd cleavage---40 hours after
F
3rd cleavage--- blastomeres
maximize their contact

compaction
 CLEAVAGE
•Compaction -- Glycopr/tight junction
Segregates inner cells (gap junctions)
from outer cells -trop
•Morula-16/ 12-32 blastomeres
Formed 3rd day after F
•Morula enters uterine cavity 4 days after F
Morula
 BLASTOCYST
• Blastocele forms
• Inner cells at one pole--embryo proper
• Outer flattened cells--trophoblast (placenta)

• Floats for about 2 days

• Zona pellucida disappears—4-5 days after F

• Rapidly increases in size
• Gets nourishment from uterine secretions

• Starts implantation at embryonic pole 6th day after F

(20th day of Mensrual cycle )
If inner cell mass of a single blastocyst divides
monozygotic (identical) twins
 Cleavage:
Zygote to Blastocyst
 Epiblast, Hypoblast & Axis
Formation
• FGFs
• Embryoblast differentiate into epiblast &
hypoblast cells
• Initially scattered
 Epiblast, Hypoblast & Axis
Formation
• Segregate at implantation
Dorsal-ventral polarity is established
Anterior visceral endoderm (AVE)

Cranial-
caudal
embryonic
axis
established
near
implantation