1. Fertilization-Part 3: Sperm-Oolemma Fusion & Oocyte Activation

2. 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.

3. Fertilization: A Series of Irreversible, Precisely Orchestrated Events Sperm-egg recognition √ Sperm binding to zona pellucida (ZP), acrosomal exocytosis and ZP- penetration√ 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

4. AE Prepares Sperm Head for Fusion With the Oolemma

5. 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, 1544-1556Theriogenology 62

6. Cascade of Sperm-Egg Interactions During Fertilization Cumulus ZP-surface ZP Interior Per. Space Egg Cortex Ooplasm

7. Sperm-Oolemma Binding: Actin-Rich Microvilli

8. 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 ???

9. Tetraspanin CD9 on the Oolemma Egg surface molecule with 4 membrane- spanning domains KO causes male infertility Mutations in its Ser-Phe-Gln (SFG) domain block membrane fusion Recombinant during IVF Recombinant Immunoglobulin superfamily ligand PSG17 blocks sperm-oolemma binding during IVF

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

11. ADAM* 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

12. Sperm-Oolemma Fusion is Followed by Oocyte Activation Release from CSF block () Release from CSF block (CSF=MPF+MAP+p90rsk) Completion of oocyte meiosis CG exocytosis Calcium oscillations Cytoplasmic reorganization PN development PN development Embryo cleavage and development

13. 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.

14. Calcium Signaling During Fertilization is Evolutionarily Conserved

15. Calcium Release at Fertilization Mouse Sea urchin

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

17. 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

18. 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-  (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

19. PLC  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

20. Anti-Polyspermy Defense Primary (fast): plasma membrane potential change (invertebrates) Secondary (slow): CG exocytosis & zona hardening

21. CG exocytosis ↓ Zona Hardening ↓ Secondary Block of Polyspermy

22. ZP Hardening: Proteolytic Cleavage of ZP2 Protein by CG Exudate Yi et al., 2007 Soc Reprod Fertil Suppl. 2007;63:385-408. Review Intact ZP2 Cleaved ZP2 M-II IVF MG 132

23. Factors Reducing Polyspermy Oviductal fluid glycoproteins Timing of sperm release from oviductal sperm reservoir Oocyte maturity/ZP integrity Oocyte age

24. Cytoplasmic relocation of tyrosine kinase c - Yes 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

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