A overview of the

F tili ti
Fertilisation
Kersti Lundin
Reproductive Medicine
Sahlgrenska University Hospital

No conflicts of interest to declare

Learning objectives

To see the fertilisation process as an interaction

between the sperm and the oocyte
To understand the contribution of each gamete
To follow the time, timing and sequence of the
different parts in the fertilisation process
To be able to translate theory to clinical practice

Overview

Concepts of fertilisation
The sperm/oocyte contribution
The first ~20 hours

Sexual reproduction
Combination of genes from two
individuals (maternal / paternal)

A new unique organism (individual) is

formed
(Haploid) gametes are requiered !

Some prerequisites for (standard)

fertilisation

Contact and recognition (species-specific)

Sperm entry of one single sperm into egg
- dependent
d
d t upon sperm and
d egg competence
t
/
receptors / acrosome reaction / egg maturity /.

Formation and fusion of sperm and egg nuclei

=> new genome

Development of a Human Embryo From

Fertilization to Implantation

The sperm contribution

To provide:
The paternal chromosomes
The centrosome (centrioles)
Oocyte activating factor(s) inducing Ca2+
oscillations
Has to be able to (e.g.):
capacitate and acrosome react
bind penetrate fuse - decondense

The oocyte contribution

Cellular mass (organelles, growth factors, :

Sperm activation factors (decondensation, aster

formation)
Mitotic spindle formation (filaments)

When and where??

21.1 The fate of 20 hypothetical human eggs in

the United States and western Europe

Meeting of the gametes

Spermatozoon
1st + 2nd meiosis
Metamorphosis
Maturation

Oocyte
Cytoplasmic maturation
1st meiosis

Binding to ZP
Acrosome reaction
Penetration

Capacitation
A post-ejaculatory event, <1h
Ca2+ increase, cAMP increase
Sperm plasma membrane increase
fluidity
y ((cholesterol loss))
Can be seen as a hyperactivated
movement pattern
Reversible
In the capacitated state, the sperm cell
can undergo the acrosome reaction in
response to appropriate stimulus (not
reversible)

Hypothetical Model for Mammalian Sperm

Capacitation

K+

hyperpolarisation

+
Cholesterol
HCO3- and Ca2+
cAMP PTK
capacitation

Cumulus cells secrete hyaluronic acid (hyaluronan)

during oocyte maturation
cumulus complex
expansion (initiated by LH surge, stimulated by
oocyte secreted factors GDF-9/BMP-15)
Sperm secrete hyaluronidase, possibly support
penetration through cumulus matrix

Secretion of hyaluronidase
Penetration of cumulus matrix
Secretion of hyaluronic acid
Expansion of cumulus cells

Zona pellucida
The human zona pellucida is made up as an 3-D
matrix, by four major glycoproteins; ZP1, ZP2, ZP3
and ZP4, secreted by the oocyte
ZP1, ZP3 and ZP4 can bind capacitated human
t
d induce
i d
ti
spermatozoa
and
an acrosome reaction
Human ZP2 only binds to acrosome-reacted
spermatozoa and thus acts as a secondary sperm
receptor.

Gupta et al Cell Tissue Research 2012

The acrosome reaction

As a consequence of
the binding of
spermatozoa to the
ZP receptor(s),
p ( ), the
cell may undergo the
acrosome reaction.
Only acrosomereacted sperm can
fuse with the egg
plasma membrane

Plasma
membrane
Outer acrosomal
membrane
Inner
acrosomal
membrane
Nuclear
membrane
Centrioles

The acrosome reaction

The acrosome reaction in human sperm

Fusion of the plasma membrane and the acrosomal

membrane
Release of the soluble components acrosin and
hyaluronidase
Exposion of sperm fusogens on the inner acrosomal
membrane
Patrat et al 2000, Gupta & Bhandari 2011

Events Leading to the Fusion of Egg and

Sperm Plasma Membranes

Acrosomal enzymes
+ vigorous
movements of the
sperm tail drives the
sperm trough the
zona pellucida into
the perivitelline
space

Sperm-oocyte fusion
During the acrosome reaction, the
equatorial segment of the sperm head
acquires the capacity to recognise and
fuse with the plasma membrane of the
oocyte

Specific recognition
and adhesion
Binding

Fusion

Decondensation

Diagram of six steps required for successful fertilization.

2. sperm recognition of
zona pellucida (bind to
ZP3, AR, bind to ZP2,
competent to penetrate)

1. sperm
penetration
of expanded
cumulus
cells

3. the sperm bind

to the oolemma
through
interactions with
microvilli and
associated
membrane
proteins

6. Formation of
pronuclei

5. Sperm
decondensation

Swain J E , Pool T B Hum. Reprod. Update 2008;14:431-446

4. oocyte activation
(polyspermy block,
2nd meiosis
completion, 2nd PB)

The Author 2008. Published by Oxford University Press on behalf of the European Society of
Human Reproduction and Embryology. All rights reserved. For Permissions, please email:
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Polyspermy block / 1.
Membrane block
Decreased receptiveness of the oolemma
The oolemma gradually becomes resistant to
fusion with additional spermatozoa

Does not seem to happen during ICSI

Reduced block in old eggs
At least partly calcium-induced

Gardner and Evans 2006 Reproduction, Fertility and development

Polyspermy block / 2.
The cortical reaction (z.p. block)
Release of cortical granules
Induced by the first calcium waves
Secretory vesicles derived from the Golgi complex
Translocated by microfilaments to the cortex during
oocyte maturation
Shown to contain e.g. trypsin-like proteinases,
peroxidases.
Prevents polyspermy by inducing changes in
the zona pellucida:
receptor inactivation (ZP3)
zona hardening (modification of ZP2)
Liu, 2011

Release of cortical granules

Meeting of the gametes / part 2

Oocyte

Spermatozoon
1st + 2nd meiosis
Metamorphosis
Plasma maturation

Cytoplasmic maturation
1st meiosis

Binding
Acrosome reaction
Penetration
Fusion
Decondensation

Activation, 2nd meiosis

PN formation

Sperm - Egg crosstalk

SNDF
((nucleoplasmin)
l
l
i )

SAOAF
(oscillin, phospholipase Cz)

Dozortsev et al 1998

Oocyte activation
Modifications to allow
development to proceed
Triggered by sperm entry
Release of cortical granules
Resumption of meiosis
Formation of male and female pronuclei
Starts with:
Increase in internal free calcium from the
endoplasmic reticulum (ER)
Generation of a series of calcium waves

Ca2+ oscillations
SUZI

*
Initiated by PLCz => IP3
Begin a few minutes after
sperm entry, and cease
around PN formation

ICSI
intact

Regulation by mitochondria
/ATP production?

ICSI
manipulated

*
* release of
sperm factors

Frequency, amplitude and

duration influence subsequent
fertilisation events and
embryo development (altered
gene expression?
Epigenetics?)
Tesarik and Testart 1994, Kashir et al 2010

Metaphase II arrest

Cytostatic factor causes metaphase block

CSF inactivated at fertilisation (Ca-flux
calmodulin CSF breakdown) MII

Sperm nucleus decondensation oocyte

maturation dependent
The sperm nucleus decondenses and enlarges:
Breakdown of the nuclear envelope
Replacement of protamines by maternally derived histones

Pronuclear formation

Transcriptional
competence of the
gametes restored

Number of pronuclei
No sperm activation

Diploid sperm / 2 spermatozoa

2nd polar body

?

Insemination / fertilisation technique

Standard IVF or ICSI ??

Routine IVF
Sperm
Ability to swim, bind, acrosome react,
penetrate, fuse

Oocyte
Similar to in vivo situation

vs. In vivo
Abundance of sperm
Environment

ICSI
Sperm
Ability to decondense and activate oocyte
Selection, Competence?
Communication with oocyte?

Oocyte
Invasive
Contamination?

vs. In vivo
Environment

ICSI for non-male subfertility

Still only one randomised controlled trial
(Bhattacharya, 2001)
No evidence that ICSI has a higher fertilisation rate,
i l t ti rate
implantation
t or live
li bi
birth
th rate
t iin non-male
l
subfertility.
ICSI is more invasive
Birth defects???

Van Rumste et al 2011, Davies et al 2012, ESHRE data