Fertilization-Part 3:Fertilization-Part 3:

Sperm-Oolemma Fusion Sperm-Oolemma Fusion & Oocyte Activation& 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, Fertilization: A Series of Irreversible, Precisely Orchestrated EventsPrecisely Orchestrated Events

Sperm-egg recognition Sperm-egg recognition √√

Sperm binding to zona pellucida (ZP), acrosomal exocytosis and ZP-Sperm binding to zona pellucida (ZP), acrosomal exocytosis and ZP-penetrationpenetration√√

Sperm-egg plasma membrane fusion and sperm perinuclear theca Sperm-egg plasma membrane fusion and sperm perinuclear theca solubilizationsolubilization

Egg activation and anti-polyspermy defenseEgg activation and anti-polyspermy defense

Centrosome reconstitution and formation of sperm asterCentrosome reconstitution and formation of sperm aster

Pronuclear development and appositionPronuclear development and apposition

Degradation of redundant sperm accessory structuresDegradation of redundant sperm accessory structures

AE Prepares Sperm Head for AE Prepares Sperm Head for Fusion With the OolemmaFusion With the Oolemma

Sperm-Oolemma Binding & FusionSperm-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

Cascade of Cascade of Sperm-Egg Sperm-Egg Interactions Interactions

During During FertilizationFertilization

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Sperm-Oolemma Binding: Actin-Rich Sperm-Oolemma Binding: Actin-Rich MicrovilliMicrovilli

Ligands & Receptors: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 OolemmaTetraspanin CD9 on the Oolemma

Egg surface molecule with 4 membrane-Egg surface molecule with 4 membrane-spanning domainsspanning domains

KO causes male infertilityKO causes male infertility

Mutations in its Ser-Phe-Gln (SFG) domain Mutations in its Ser-Phe-Gln (SFG) domain block membrane fusionblock membrane fusion

Recombinant Recombinant Immunoglobulin superfamily ligand PSG17 blocks sperm-oolemma binding during IVFduring IVF

Izumo -/- Spermatozoa Can Reach & Izumo -/- Spermatozoa Can Reach & Penetrate ZP, but Fail to Fuse with Penetrate ZP, but Fail to Fuse with

OolemmaOolemma

ADAMADAM* Family Disintegrins on Sperm PM* Family Disintegrins on Sperm PM

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

*A Disintegrin and A Metaloproteinase Domain Proteins

Sperm-Oolemma Fusion is Sperm-Oolemma Fusion is Followed by Oocyte Activation Followed by Oocyte Activation

Release from CSF block (Release from CSF block (CSF=MPF+MAP+p90rsk))Completion of oocyte meiosis Completion of oocyte meiosis CG exocytosisCG exocytosisCalcium oscillations Calcium oscillations Cytoplasmic reorganizationCytoplasmic reorganization PN development PN development Embryo cleavage and developmentEmbryo cleavage and development

Contact & Fusion Hypothesis of Contact & Fusion Hypothesis of Oocyte ActivationOocyte Activation

Runft LL, Jaffe LA, Mehlmann LM. Egg Activation at fertilization: where it Runft LL, Jaffe LA, Mehlmann LM. Egg Activation at fertilization: where it all begins. Dev Biol. (2002) 245:237-54. Review.all begins. Dev Biol. (2002) 245:237-54. Review.

Calcium Signaling During Fertilization is Calcium Signaling During Fertilization is Evolutionarily ConservedEvolutionarily Conserved

Calcium Release at FertilizationCalcium Release at Fertilization

Mouse Sea urchin

Model of Ooocyte Activation by The Sperm-Model of Ooocyte Activation by The Sperm-Borne, Oocyte Activating Factor (SOAF)Borne, Oocyte Activating Factor (SOAF)

Criteria: What is Criteria: What is thethe SOAF? SOAF?

TriggersTriggers CG exocytosis/PN CG exocytosis/PN developmentdevelopment/embryo /embryo cleavagecleavageInduces Induces Ca-oscillationsCa-oscillationsA male A male germ cell/testis specificgerm cell/testis specific gene product gene productReleased by spermatozoonReleased by spermatozoon upon sperm-oolemma upon sperm-oolemma fusionfusionSolubleSoluble, present in the freeze-thaw sperm extracts, present in the freeze-thaw sperm extractsA A signalingsignaling molecule molecule Present in the acrosome-reacted sperm headPresent in the acrosome-reacted sperm head and and absent from the sperm flagellumabsent from the sperm flagellumAbsent from round spermatidsAbsent from round spermatids lacking SOAF activity lacking SOAF activityAbsent from round headed spermatozoaAbsent from round headed spermatozoa lacking the lacking the SOAF activitySOAF activityEvolutionarily Evolutionarily conservedconserved/inter-specific/inter-specific

Candidate SOAF Molecules/Components*Candidate SOAF Molecules/Components*

Oscillin* (Parrington et al., Nature. 1996, 379:364-8 )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 )Truncated c-Kit tyrosine kinase (trKIT; Sette et al., J Cell Biol. 1998; 142:1063-74 )

PLC-PLC- (Saunders et al., Development. 2002; 129:3533-44 ) (Saunders et al., Development. 2002; 129:3533-44 )

Nitric Oxide (Thaler CD, Epel D, Curr Pharm Des. 2003;9(5):399-409)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 RGD peptides (Fenichel P, Durand-Clement M., Hum Reprod. 1998 Dec;13 Suppl 4:31-46. ) 4:31-46. )

PAWP (Wu et al. 2007, J Biol Chem 282(16):12164-75PAWP (Wu et al. 2007, J Biol Chem 282(16):12164-75 ))

Citrate synthase (Newt; Harada et al., 2007; Dev Biol 306: 797-808) Citrate synthase (Newt; Harada et al., 2007; Dev Biol 306: 797-808)

*Oscillin is NOT a signaling molecule, NOT the SOAF*Oscillin is NOT a signaling molecule, NOT the SOAF*SOAF may be a multi-component factor*SOAF may be a multi-component factor

PLCPLC

Soluble PLC speciesSoluble PLC species

mRNA injection into oocyte cause calcium mRNA injection into oocyte cause calcium oscillations somewhat similar to those oscillations somewhat similar to those induced by the spermatozooninduced by the spermatozoon

After activation, the overexpressed PLCz After activation, the overexpressed PLCz is sequestered in pronucleiis sequestered in pronuclei

No KO available (still)No KO available (still)

Localization in sperm unclearLocalization in sperm unclear

Anti-Polyspermy Defense Anti-Polyspermy Defense

Primary (fast): plasma membrane potential change (invertebrates)

Secondary (slow): CG exocytosis & zona hardening

CG exocytosisCG exocytosis

↓

Zona Hardening

↓

Secondary Block of Polyspermy

ZP Hardening: Proteolytic Cleavage of ZP Hardening: Proteolytic Cleavage of ZP2 Protein by CG ExudateZP2 Protein by CG Exudate

Yi et al., 2007 Soc Reprod Fertil Suppl. 2007;63:385-408. Review Yi et al., 2007 Soc Reprod Fertil Suppl. 2007;63:385-408. Review

Intact ZP2

Cleaved ZP2

M-II IVF MG 132

Factors Reducing PolyspermyFactors Reducing Polyspermy

Oviductal fluid Oviductal fluid glycoproteinsglycoproteinsTiming of sperm Timing of sperm release from release from oviductal sperm oviductal sperm reservoirreservoirOocyte maturity/ZP Oocyte maturity/ZP integrityintegrityOocyte ageOocyte age

Cytoplasmic relocation of tyrosine kinase c - YesCytoplasmic relocation of tyrosine kinase c - Yes

Cytoplasmic Remodeling After Cytoplasmic Remodeling After Oocyte ActivationOocyte Activation

•Organelle remodeling (mitochodnria, Golgi, ER)

•Organele rearangement or movement (mitochondria, cytoskeleton)

•Maternal mRNA recruitment & tarnslation

•Maternal RNA degradation

•Maternal protein degradation

Next : Are We (Males) Really So Next : Are We (Males) Really So Worthless?Worthless?