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

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

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)

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,

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

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

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

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

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?)

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 -

histones

Pronuclear formation

Transcriptional competence of the gametes restored

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

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