Normal Semen Characteristics

Volume 2-6 ml

Sperm concentration 20-250 million/ml

Total Sperm Count Over 80 million/ejaculate
Motility (0.5-2 hr) Over 50 % motile
Progression at 37C (1-4) 3 or 4
Vitality Over 75%
Morphology
Head defects Less than 35%
Midpiece defects Less than 20%
• SPERMATOZOA
– 50 – 120 MILLION PER MILLILITER
• FRUCTOSE
– PROVIDES ENERGY FOR SPERM MOTILITY
• SPERMINE
– BASE, NEUTRALIZES VAGINAL ACIDITY
• FIBRINOGEN
– SEMEN CLOT, ADHERE TO VAGINA & CERVIX
• CLOTTING ENZYMES
– FIBRINOGEN  FIBRIN
• FIBRINOLYSIN
– DISSOLVES FIBRIN 15 – 30 MIN AFTER EJACULATION
• PROSTAGLANDINS
– STIMULATES PERISTALTIC CONTRACTIONS OF UTERUS
– DRAWS SPERM INTO UTERUS
– DECREASES VISCOSITY OF CERVICAL MUCUS
 Sperm's Journey

Emission
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Ejaculation
SECONDARY OOCYTE
 VIABILITY OF GAMETES

OVUM:

• Oocyte = 12 – 24 hours after ovulation, In vitro studies indicate, the

oocyte cannot be fertilised after 24 hours

SPERM:

• Most do not survive after 48 hours in female genital tract, however,

motile sperms have been recovered from the cervix 7 days after
ejaculation

FROZEN SPERMS:

• After being frozen to low temperatures ( - 700 C ), semen may be kept for
many years. Children have been born to women who have been
artificially inseminated with semen that had been stored for several years
 The secondary oocyte is surrounded by

Plasma membrane…….Vitelline membrane

Perivitelline space containing polar bodies

Zona pellucida

Cumulus cells……Granulosa cells arranged in radial

form
Zona Pellucida
• Consists of three major glycoproteins; ZP1, ZP2
& ZP3
• ZP3 is sperm binding protein
– Evidence:
• Purified ZP3 competes with intact zona for sperm binding
• Zona reaction (prevention of polyspermy) occurs
when ZP2 and ZP3 are modified by cortical
granule enzymes
(Zona reaction follows cortical reaction)
Fertilization
• The first step in development of a new
individual is fertilization
– Is the fusion of the plasma membranes of
sperm and sec. Oocyte and subsequent
union of their pronuclei to form the diploid
zygote
– It begins when the two membranes fuse and
ends when their pronuclei unite

This Union marks The Beginning of the development of

a New Potential ‘ HUMAN BEING ’
Fertilization
Typical ejaculate contains 200 – 500
million spermatozoa
– sperm move toward ovulated egg in uterine tube by
uterine muscles/ciliary action in tubes
– only 10,000 sperm make it through the uterus
– About 100 sperm reach egg
– require multiple sperm acrosomal activity to chew
through the egg follicular layer
– infertile male:
less than 20 million sperm/ml
 FERTILISATION

» Fertilisation usually occurs in the ampullary

region of the uterine tubes, probably within
24 hours of ovulation (may occur in other
parts of the tube BUT does not occur in
the uterus)

» Chemicals released by the oocyte and / or

surrounding cumulus cells appear to guide
the capacitated sperm to the oocyte
(SPERM CHEMOTAXIS)
 FERTILIZATION

» The process of fertilization is a

complex sequence of coordinated
molecular events that begins with
contact between a sperm and an
oocyte and

» Ends with the intermingling of

maternal and paternal chromosomes at
the metaphase of the first mitotic
division of the zygote (unicellular
embryo)

» The process lasts about 24 hours

A. BEFORE CONTACT BETWEEN THE GAMETES
1 Capacitation of sperms
2. Journey of sperm through the female genital tract to reach the ovum
3. Passage of sperm through the corona radiata
4. Sperm – zona binding …… triggering acrosome reaction
Sperm taking sigh of relief in the perivitelline space
B. AFTER FUSION OF SPERM AND OVUM MEMBRANES
1. Sperm contacts the egg
2. Sperm or its nucleus enters the ovum ……cortical reaction
3. Ovum becomes activated and developmental changes begin

4. Sperm and egg nuclei fuse

Sperm capacitation

• During ejaculation, sperm are mixed with

seminal plasma (combined secretions of the
accessory sex glands)
• Seminal plasma proteins coat the sperm to lengthen
the sperm’s fertile lifespan in the female
• Ejaculated sperm are not immediately fertile upon
deposition in the female
Rather, they must stay some hours in the female tract
before acquiring the ability to fertilize.
• During this period, seminal fluid proteins are removed
from the sperm membrane
Sperm capacitation Capacitation involves removal of seminal
Seminal fluid proteins block sperm fluid proteins, permitting interaction of
interaction with capacitating agents, sperm with ZP; sperm is now fertile, but
preventing pre-mature acrosome reaction lifespan is short.
Effects of Capacitation on Sperm
• Increased rate of metabolism
• Flagellum beats more rapidly;
– Result: Sperm are more motile (hyperactivated)
• Changes in sperm glycoprotein
– Allow sperm-egg binding
• Pro-Acrosin (inactive) is converted to acrosin (active)
– Able to digest zona pellucida proteins
Acrosomal Reaction and Sperm Penetration
 ACROSOME REACTION

» Once the sperms have

traversed the corona radiata,
they bind to specific
glycoprotein receptors on ZP

» Interaction of ZP3 with sperm

head induces ACROSOME
REACTION, which results in
release of a number of
acrosomal enzymes
 ACROSOME REACTION

» The essence of acrosomal

reaction is fusion of parts of the
outer acrosomal membrane and
the over lying plasma
membrane of the sperm
» Further, the fused membranes
form vesicles which pinch off
from the head resulting in
liberation of multitude of
enzymes that are stored in the
acrosome
 ACROSOME REACTION

» After acrosome reaction, the inner

acrosomal membrane forms the
outer surface covering of most of
the sperm head. Toward the base
of sperm head ( in the equatorial
region ), the inner acrosomal
membrane fuses with the
remaining POSTACROSOMAL
PLASMA MEMBRANE to maintain
membrane continuity around the
sperm head
 PENETRATION OF ZONA PELLUCIDA

» Only after completing acrosomal reaction can

sperm successfully begin to penetrate the ZP,
which is accomplished by a combination of:
• (1) Vigorous mechanical propulsion by
swimming movement of the sperm’s tail
and
• (2) Digestion of a path by the acrosomal
enzymes in the zona pellucida around the
sperm head
» After having traversed the zona, the sperm
reaches the PERIVITELLINE SPACE and can
make direct contact with the plasma
membrane of the oocyte
 Fertilization
• If timing is ideal,
sperm reach the
egg in the upper
third of the
uterine tube

• Sperm move by
flagellar action
but also receive
an assist from
uterine tube
peristalsis
Fertilization (continued)

• As fertilization
occurs, secondary
oocyte completes
2nd meiotic division.
Insert fig. 20.41
• Sperm enters ovum
cytoplasm.
• Ovum nuclear
membrane
disappears, zygote
formed.
• Centrosome of
zygote is derived
from sperm cell.
Process of Fertilization
Purpose of Fertilization
• Activate resumption of meiosis

• Activate embryo development

• Re-establish the diploid, chromosome complement

• Establish the unique chromosomal complement of a

new individual
Fertilization Activates the Egg

• Aerobic respiration increases

• Enzyme systems become activated
• A burst of protein synthesis begins
• In most animals, the nucleus undergoes the
second division of meiosis
Events Immediately Following Sperm Penetration
Acrosomal Reaction

An ovulated oocyte is encapsulated by:

The corona radiata and zona pellucida
Sperm binds to the zona pellucida and
undergoes the acrosomal reaction
Enzymes are released near the oocyte
Hundreds of acrosomes release their
enzymes to digest the zona pellucida
 Contact of sperm with egg’s zona pellucida causes:

1. Acrosome reaction: release of proteolytic enzymes to chew through

the egg’s zona pellucida coat
2. Acrosomal reaction activates the NOS (NO synthase) found in the
sperm’s cytoplasm
• Upon fusion with the egg, NOS and NO is released into the egg
• NO produced activates calcium release within the egg
• Calcium wave results in rising of the fertilization membrane
http://www.sciam.com/media/inline/0004D627-53B7-
1C75-9B81809EC588EF21_arch2.gif
Entry of Sperm
Acrosome reaction
Required for release of acrosomal Inner acrosomal
membrane studded with
enzymes critical for sperm bound acrosin
penetration Hyaluronidase

Hyaluronidase: Acrosin: digests

Digest pathway for pathway for sperm
sperm through cumulus through ZP
cells
 Sperm Binding to ZP

Seminal fluid proteins

ZP
Non-capacitated

Binding is species-specific!
Capacitated

Ovulated oocyte,
Arrested in metaphase of meiosis II

Sperm receptor protein

 Acrosome reaction and ZP
penetration
2. Acrosome reaction
• Releases hyaluronidase
1. Acrosome binds to • Exposes “acrosin”
ZP
• Species-
specific 3. Acrosin digests path through ZP
• Only occurs Hyperactivated tail motility propels
if acrosome is sperm through ZP
intact

ZP
 Cortical Reaction – block to polyspermy

First fertilizing sperm induces exocytosis

of enzymes from CG, diffuses across PVS
& catalyzes ZP “hardening”, “blocking”
further sperm entry

ZP

Sperm receptors

PVS

Oocyte Cortical granules (CG)

Pronuclear Fusion
• Once the second polar body is ejected the
female pronucleus can fuse with the male
pronucleus
• This is the genetic beginning of a new
organism
• The haploid genetic complements of the two
pronuclei form a 2N nucleus, which prepares
the nucleus, and cell, for cleavage
Pronuclear fusion
 Metaphase 1st polar body
chromosomes of
Meiosis II

Resumption of Meiosis
II; extrusion of 2nd
polar body

Sperm
Male & female pronuclei
Chromatin Chromatin Zygote
protein protein
core core
Genetic sex determined with
introduction of X or Y
chromosome from sperm

Male pronucleus Chromosomes replicate. Syngamy first blending

chromatin proteins Two-cell
of maternal & paternal
“remodeled” to embryo
Sperm tail degenerates chromosomes.
“match” female
First mitosis
Fertilization

Secondary Sperm binding to Female and male

oocyte: oocyte triggers chromosomes decondense
Arrested in resumption of to form haploid
Meiosis II; 2nd PB “pronuclei”
Metaphase of
extruded;
Meiosis II
Sperm binding also
“activates” embryo
development
Blocks to Polyspermy
• Only one sperm is allowed to penetrate the
oocyte
• Two mechanisms ensure monospermy
– Fast block to polyspermy – membrane
depolarization prevents sperm from fusing with the
oocyte membrane
– Slow block to polyspermy – zonal inhibiting proteins
(ZIPs):
• Destroy sperm receptors
• Cause sperm already bound to receptors to detach
Blocks To Polyspermy
• Fast block to polyspermy involves egg plasma
membrane action potential, prevents additional sperm
from entering
• The slow block to polyspermy involves the
cortical reaction
– Calcium ion entry causes fusion of large cortical
granules with plasma membrane that release
digestive enzymes
• In mammals, the zona pellucida sperm receptors are
modified to prevent further entry of sperm
• Some organisms do not block polyspermy.
– Some amphibians degrade the supernumery sperm
 At fertilization, only one sperm fuses with the egg

20_13_sperm_binds.jpg
Ca++ ions
released in
the egg
cytoplasm,
harden the
zona
pellucida (the
egg coat)

Fertilization reconstitutes a complete genome

http://www-personal.umich.edu/~segelj/Fertilization_clip_image004.jpg
Fertilization. SEM X4700.
Human sperm and egg during
fertilization. SEM X14,000 - colorized
Fertilization events ( review)

• Sperm capacitation
• Sperm binding to zona pellucida
• Acrosome reaction
• Penetration of ZP
• Entry into perivitelline space
• Sperm binding to oocyte cell membrane
• Cortical Reaction – block to polyspermy
• Endocytosis of sperm into oocyte cytoplasm
Completion of Meiosis II and Fertilization

• Upon entry of sperm, the secondary oocyte:

– Completes meiosis II
– Casts out the second polar body
• The ovum nucleus swells, and the two nuclei
approach each other
• When fully swollen, the two nuclei are called
pronuclei
• Fertilization – when the pronuclei come together
 FERTILIZATION EVENTS/ PHASES
» Sperm capacitation
» Penetration of corona radiata
» Sperm binding to zona pellucida
» Acrosome reaction
» Penetration of ZP
» Entry into perivitelline space
» Sperm binding to oocyte cell membrane
» Endocytosis of sperm into the oocyte cytoplasm
» Cortical Reaction – block to polyspermy
» Sperm nucleus enlarges / decondensation forming “ Male Pronucleus ”
» Resumption of meiosis – release of 2nd polar body
» Decondensation of the maternal chromosomes “ Female Pronucleus ”
» Chromosome replication

» Intermingling of maternal and paternal chromosomes at metaphase of first mitotic division of the
zygote