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A overview of the F tili tiFertilisation
Kersti LundinReproductive 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 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
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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
d d t d t /- dependent upon sperm and egg competence / receptors
/ acrosome reaction / egg maturity /.
Formation and fusion of sperm and egg nuclei => new
genome
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Development of a Human Embryo From Fertilization to
Implantation
The sperm contribution
To provide:The paternal chromosomesThe centrosome
(centrioles)Oocyte activating factor(s) inducing Ca2+
oscillations
Has to be able to (e.g.):capacitate and acrosome reactbind
penetrate fuse - decondense
The oocyte contribution Cellular mass (organelles, growth
factors, :
Sperm activation factors (decondensation, aster formation)
Mitotic spindle formation (filaments)
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When and where??
21.1 The fate of 20 hypothetical human eggs in the United States
and western Europe
Meeting of the gametes
Cytoplasmic maturation 1st meiosis
Spermatozoon Oocyte 1st + 2nd meiosis Metamorphosis
Maturation
Binding to ZPAcrosome reaction
Penetration
- A post-ejaculatory event,
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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 i d
tispermatozoa and induce 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), the
Plasma Plasma membranemembrane
Outer acrosomal Outer acrosomal membranemembranep ( ),
cell may undergo the acrosome reaction.
Only acrosome-reacted sperm can fuse with the egg plasma
membrane
Inner Inner acrosomal acrosomal membranemembrane
Nuclear Nuclear membranemembrane
CentriolesCentrioles
The acrosome reaction
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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 thefuse with the plasma membrane of the
oocyte
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Specific recognitionand adhesion
Binding
FusionFusion
Decondensation
Diagram of six steps required for successful fertilization.
1. sperm penetration of expanded cumulus cells
2. sperm recognition of zona pellucida (bind to ZP3, AR, bind to
ZP2, competent to penetrate)
3. the sperm bind to the oolemma through interactions with
Swain J E , Pool T B Hum. Reprod. Update 2008;14:431-446 The
Author 2008. Published by Oxford University Press on behalf of the
European Society of
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interactions with microvilli and associated membrane
proteins
4. oocyte activation(polyspermy block, 2nd meiosis completion,
2nd PB)
5. Sperm decondensation
6. Formation of pronuclei
Polyspermy block / 1.Membrane block
Decreased receptiveness of the oolemma The oolemma gradually
becomes resistant to
fusion with additional spermatozoafusion 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
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Polyspermy block / 2.The cortical reaction (z.p. block)
Release of cortical granulesInduced 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
1st + 2nd meiosis Metamorphosis Plasma maturation
Cytoplasmic maturation 1st meiosis
Spermatozoon Oocyte
Meeting of the gametes / part 2
Acrosome reaction
Activation, 2nd meiosis Decondensation
Binding
PenetrationAcrosome reaction
PenetrationFusionFusion
PN formation
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SNDF( l l i ) SAOAF
Sperm - Egg crosstalk
(nucleoplasmin) SAOAF (oscillin, phospholipase Cz)
Dozortsev et al 1998
Oocyte activationModifications to allow development to
proceedTriggered 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
SUZISUZI
ICSIICSIintactintact
*
Initiated by PLCz => IP3 Begin a few minutes after sperm
entry, and cease around PN formationintactintact
ICSIICSImanipulatedmanipulated *
* release ofsperm factors
Tesarik and Testart 1994, Kashir et al 2010
around PN formationRegulation by mitochondria /ATP production?
Frequency, amplitude and duration influence subsequent
fertilisation events and embryo development (altered gene
expression? Epigenetics?)
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Metaphase II arrest
Cytostatic factor causes metaphase blockCSF 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
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histones
Pronuclear formation
Transcriptional competence of the gametes restored
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Diploid sperm / 2 spermatozoa
No sperm activation
Number of pronuclei
??
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
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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
t li bi th t i limplantation rate or live birth rate in non-male
subfertility.
ICSI is more invasive Birth defects???
Van Rumste et al 2011, Davies et al 2012, ESHRE data