Fertilization-Part 3:
Sperm-Oolemma Fusion
& Oocyte Activation
Ma il seme affinche esecriti la sua azione nei girini, deve
insinuarsi nei loro corpicelli, sembrando che non possa
animarli con la semplice impressione da esso fatta su la
loro cute.
L. SPALANZANI. “DISSERTAZIONI DI FISICA ANIMALE E VEGETABILE” VOL. II., MODENA, 1780
However, for the semen to exert its effect on
the eggs, it must enter their little bodies, as it
seems unlikely that it could animate them by
the mere contact with their skin.
Fertilization: A Series of Irreversible,
Precisely Orchestrated Events
Sperm-egg recognition √
Sperm binding to zona pellucida (ZP), acrosomal exocytosis and ZPpenetration√
Sperm-egg plasma membrane fusion and sperm perinuclear theca
solubilization
Egg activation and anti-polyspermy defense
Centrosome reconstitution and formation of sperm aster
Pronuclear development and apposition
Degradation of redundant sperm accessory structures
AE Prepares Sperm Head for
Fusion With the Oolemma
Sperm-Oolemma Binding & Fusion
•Mediated by sperm ES and oocyte microvilli
•Species specific
•Can acrosome-intact spermatozoa fuse with oolemma?
•Wu G-M et al., 2004. Birth of piglets by in vitro fertilization of zona-free porcine
oocytes. Theriogenology 62, 1544-1556
Cumulus ZP-surface ZP Interior Per. Space
Egg Cortex
Cascade of
Sperm-Egg
Interactions
During
Fertilization
Ooplasm
Sperm-Oolemma Binding: Actin-Rich
Microvilli
Ligands & Receptors:
Sperm Ligand
Oolemma Receptor
Izumo (Immunoglobulin superfamily)
CD9/Tetraspanin
PH30/Fertilin β (ADAM 2),
cyritestin (ADAM 3)
Integrin α6β1
GPI-anchored sperm proteins
???
Epid. proteins CRES, DE; cadherins
???
Tetraspanin CD9 on the Oolemma
Egg surface molecule with 4 membranespanning domains
KO causes male infertility
Mutations in its Ser-Phe-Gln (SFG) domain
block membrane fusion
Recombinant Immunoglobulin superfamily
ligand PSG17 blocks sperm-oolemma binding
during IVF
Izumo -/- Spermatozoa Can Reach &
Penetrate ZP, but Fail to Fuse with
Oolemma
ADAM* Family Disintegrins on Sperm PM
*A Disintegrin and A Metaloproteinase Domain Proteins
Found in snake venome
Contain RGD (Arg-Gly-Asp), TDE (Thr-Asp-Glu), QDE (Gln-Asp-Glu), or
KDQ (Lys-Asp-Gln) domains
Bind to integrins via the above domains
Integrins are plasma membrane receptors for ECM proteins
Cyritestin & fertilin KO and double KO did NOT cause
infertility
Sperm-Oolemma Fusion is
Followed by Oocyte Activation
Release from CSF block (CSF=MPF+MAP+p90rsk)
Completion of oocyte meiosis
CG exocytosis
Calcium oscillations
Cytoplasmic reorganization
PN development
Embryo cleavage and development
Contact & Fusion Hypothesis of
Oocyte Activation
Runft LL, Jaffe LA, Mehlmann LM. Egg Activation at fertilization: where it
all begins. Dev Biol. (2002) 245:237-54. Review.
Calcium Signaling During Fertilization is
Evolutionarily Conserved
Calcium Release at Fertilization
Mouse
Sea urchin
Model of Ooocyte Activation by The SpermBorne, Oocyte Activating Factor (SOAF)
Criteria: What is the SOAF?
Triggers CG exocytosis/PN development/embryo
cleavage
Induces Ca-oscillations
A male germ cell/testis specific gene product
Released by spermatozoon upon sperm-oolemma
fusion
Soluble, present in the freeze-thaw sperm extracts
A signaling molecule
Present in the acrosome-reacted sperm head and
absent from the sperm flagellum
Absent from round spermatids lacking SOAF activity
Absent from round headed spermatozoa lacking the
SOAF activity
Evolutionarily conserved/inter-specific
Candidate SOAF Molecules/Components*
Oscillin* (Parrington et al., Nature. 1996, 379:364-8 )
Truncated c-Kit tyrosine kinase (trKIT; Sette et al., J Cell Biol. 1998; 142:1063-74 )
PLC-z (Saunders et al., Development. 2002; 129:3533-44 )
Nitric Oxide (Thaler CD, Epel D, Curr Pharm Des. 2003;9(5):399-409)
RGD peptides (Fenichel P, Durand-Clement M., Hum Reprod. 1998 Dec;13 Suppl
4:31-46. )
PAWP (Wu et al. 2007, J Biol Chem 282(16):12164-75 )
Citrate synthase (Newt; Harada et al., 2007; Dev Biol 306: 797-808)
*Oscillin is NOT a signaling molecule, NOT the SOAF
*SOAF may be a multi-component factor
PLCz
Soluble PLC species
mRNA injection into oocyte cause calcium
oscillations somewhat similar to those
induced by the spermatozoon
After activation, the overexpressed PLCz
is sequestered in pronuclei
No KO available (still)
Localization in sperm unclear
Anti-Polyspermy Defense
Primary (fast): plasma membrane potential
change (invertebrates)
Secondary (slow): CG exocytosis & zona
hardening
CG exocytosis
↓
Zona Hardening
↓
Secondary Block of
Polyspermy
ZP Hardening: Proteolytic Cleavage of
ZP2 Protein by CG Exudate
Intact ZP2
Cleaved ZP2
M-II IVF MG
132
Yi et al., 2007 Soc Reprod Fertil Suppl. 2007;63:385-408. Review
Factors Reducing Polyspermy
Oviductal fluid
glycoproteins
Timing of sperm
release from
oviductal sperm
reservoir
Oocyte maturity/ZP
integrity
Oocyte age
Cytoplasmic Remodeling After
Oocyte Activation
•Organelle remodeling
(mitochodnria, Golgi, ER)
•Organele rearangement or
movement (mitochondria,
cytoskeleton)
•Maternal mRNA recruitment &
tarnslation
•Maternal RNA degradation
•Maternal protein degradation
Cytoplasmic relocation of tyrosine kinase c - Yes
Next : Are We (Males) Really So
Worthless?