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 . • Formation and fusion of sperm and egg nuclei => new genome .dependent d d t upon sperm and d egg competence t / receptors / acrosome reaction / egg maturity /…….

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. ……: • Sperm activation factors (decondensation.decondense The oocyte contribution • Cellular mass (organelles. aster formation) • Mitotic spindle formation (filaments) .): capacitate and acrosome react bind – penetrate – fuse . growth factors.

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 .When and where?? 21.

Capacitation – A post-ejaculatory event. stimulated by oocyte secreted factors GDF-9/BMP-15) • Sperm secrete hyaluronidase. <1h – Ca2+ increase.and Ca2+  cAMP  PTK  capacitation – Cumulus cells secrete hyaluronic acid (hyaluronan) during oocyte maturation cumulus complex expansion (initiated by LH surge. 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. cAMP increase – Sperm plasma membrane increase fluidity y ((cholesterol loss)) – Can be seen as a hyperactivated movement pattern – Reversible – In the capacitated state. possibly support penetration through cumulus matrix Secretion of hyaluronidase Penetration of cumulus matrix Secretion of hyaluronic acid Expansion of cumulus cells .

ZP3 and ZP4. 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). the cell may undergo the acrosome reaction. p ( ). secreted by the oocyte – ZP1. – Only acrosomereacted sperm can fuse with the egg plasma membrane Plasma membrane Outer acrosomal membrane Inner acrosomal membrane Nuclear membrane Centrioles The acrosome reaction .Zona pellucida – The human zona pellucida is made up as an 3-D matrix. ZP2. by four major glycoproteins. ZP1.

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 .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.

competent to penetrate) 1. sperm recognition of zona pellucida (bind to ZP3.14:431-446 4. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. please email: journals. bind to ZP2. sperm penetration of expanded cumulus cells 3.Specific recognition and adhesion Binding Fusion Decondensation Diagram of six steps required for successful fertilization. 2nd PB) © The Author 2008. All rights reserved. 2nd meiosis completion. oocyte activation (polyspermy block. Fertility and development . 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.org Polyspermy block / 1. Pool T B Hum. For Permissions. Reprod. 2.permissions@oxfordjournals. Formation of pronuclei 5. AR. Sperm decondensation Swain J E . Update 2008. the sperm bind to the oolemma through interactions with microvilli and associated membrane proteins 6.

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. trypsin-like proteinases. 2nd meiosis PN formation . 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. The cortical reaction (z.Polyspermy block / 2.g.p. • Prevents polyspermy by inducing changes in the zona pellucida: • receptor inactivation (ZP3) • “zona hardening” (modification of ZP2) Liu. peroxidases….

Kashir et al 2010 . and cease around PN formation ICSI intact •Regulation by mitochondria /ATP production? ICSI manipulated * * release of sperm factors • Frequency.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. amplitude and duration influence subsequent fertilisation events and embryo development (altered gene expression? Epigenetics?) Tesarik and Testart 1994.

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 .

fuse • Oocyte – Similar to in vivo situation • vs. bind. penetrate.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. acrosome react. In vivo – Abundance of sperm – Environment .

Competence? – Communication with oocyte? • Oocyte – Invasive – Contamination? • vs. In vivo – Environment ICSI for non-male subfertility • Still only one randomised controlled trial (Bhattacharya. Davies et al 2012. ESHRE data . i l t ti rate implantation t or live li bi birth th rate t iin non-male l subfertility.ICSI • Sperm – Ability to decondense and activate oocyte – Selection. • ICSI is more invasive • Birth defects??? Van Rumste et al 2011. 2001) • No evidence that ICSI has a higher fertilisation rate.