Class 5, Part II: Sperm-Zona Class 5, Part II: Sperm-Zona Interactions & Acrosomal Exocytosis Interactions & Acrosomal Exocytosis

(AE; Formerly Known as AR)(AE; Formerly Known as AR)

Fertilization is a complex series of Fertilization is a complex series of reactions that, if once completed, reactions that, if once completed,

excludes all possibilities of excludes all possibilities of repetitionrepetition

CR Moore (1916) On the Superposition of Fertilization in CR Moore (1916) On the Superposition of Fertilization in Parthenogenesis. Parthenogenesis. Biological BulletinBiological Bulletin 31:137 31:137

Fertilization: A Series of Irreversible, Fertilization: A Series of Irreversible, Precisely Orchestrated EventsPrecisely Orchestrated Events

Sperm-egg recognitionSperm-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

Unique Sperm “Accessory” Structures Unique Sperm “Accessory” Structures Are Generated During SpermiogenesisAre Generated During Spermiogenesis

Acrosome (AC)Acrosome (AC) Equatorial Segment (ES)Equatorial Segment (ES) Postacrosomal Sheath Postacrosomal Sheath

(PAS)(PAS) Perinuclear Theca (PT)Perinuclear Theca (PT) Reduced Sperm CentrioleReduced Sperm Centriole Mitochondrial SheathMitochondrial Sheath FlagellumFlagellum

During Fertilization, Sperm Accessory During Fertilization, Sperm Accessory Structures Fulfill Unique Roles, While Structures Fulfill Unique Roles, While Being Transformed or Lost IrreversiblyBeing Transformed or Lost Irreversibly

AC:AC: Facilitates sperm-ZP binding and penetration; lost in part to Facilitates sperm-ZP binding and penetration; lost in part to acrosomal exocytosis, remnants degraded by ooplasmacrosomal exocytosis, remnants degraded by ooplasm

ES:ES: Facilitates sperm-oolemma binding after ZP penetration, Facilitates sperm-oolemma binding after ZP penetration, membranes intermingle with oolemma, insoluble layers degraded in membranes intermingle with oolemma, insoluble layers degraded in ooplasmooplasm

PAS & PAS-PT:PAS & PAS-PT: Solubilize in ooplams while releasing the oocyte Solubilize in ooplams while releasing the oocyte activating factorsactivating factors

Reduced Sperm Centriole:Reduced Sperm Centriole: Actually duplicates to form zygotic Actually duplicates to form zygotic centrosome and sperm aster for PN apposition (exception: rodents)centrosome and sperm aster for PN apposition (exception: rodents)

Mitochondrial Sheath:Mitochondrial Sheath: Provides energy for motility/sperm transport; Provides energy for motility/sperm transport; degraded by ooplasm along with its contingent of mtDNAdegraded by ooplasm along with its contingent of mtDNA

Spermatozoa must undergo Spermatozoa must undergo acrosomal exocytosis to penetrate acrosomal exocytosis to penetrate

the zona pellucida and fuse with the the zona pellucida and fuse with the oolemmaoolemma

BUTBUT

Spermatozoa with exocytosed Spermatozoa with exocytosed acrosome can still bind to ZPacrosome can still bind to ZP

Zona Pellucida 1906Zona Pellucida 1906If the ovum should be impregnated, several important If the ovum should be impregnated, several important

changes take place which are as follows: The changes take place which are as follows: The zona zona pellucida,pellucida, or outer membrane of the egg, having thrown or outer membrane of the egg, having thrown off its outer cell covering, and the spermatozoa have no off its outer cell covering, and the spermatozoa have no difficulty in penetrating the soft albuminous membrane difficulty in penetrating the soft albuminous membrane that encloses the yelk. An additional change observed that encloses the yelk. An additional change observed taking place in the tubes is a deposit of albumen around taking place in the tubes is a deposit of albumen around zona pellucida, which takes place when the ovum is zona pellucida, which takes place when the ovum is passing the middle and lower third of the tube. These passing the middle and lower third of the tube. These occurrences are so uniform that occurrences are so uniform that the different offices for the different offices for different portions of the Fallopian tube may be readily different portions of the Fallopian tube may be readily determined.determined. The first or upper third is appropriated to The first or upper third is appropriated to the reception of the ovum, and for removing the the reception of the ovum, and for removing the adventitious covering of cells, while it also prepares the adventitious covering of cells, while it also prepares the ovum for the operation of the spermatozoa. In the middle ovum for the operation of the spermatozoa. In the middle third, the respiratory chamber is formed, and here the third, the respiratory chamber is formed, and here the rotation of the yelk commences. In the lower third the rotation of the yelk commences. In the lower third the cleverage takes place, as also the deposit of albumen. cleverage takes place, as also the deposit of albumen.

Frederick Wilson Pitcairn & Elizabeth J. Williard (1906) Frederick Wilson Pitcairn & Elizabeth J. Williard (1906) Woman’s Guide to Health, Beauty and Happiness. What Woman’s Guide to Health, Beauty and Happiness. What Every Woman Should Know.Every Woman Should Know. Horace C. Fray Publisher, Horace C. Fray Publisher, Washington DC., Pages 96-97.Washington DC., Pages 96-97.

Zona Pellucida 2008Zona Pellucida 2008General model (murine, bovine):General model (murine, bovine):

ZP3/ZP2 heterodimers cross-linked by ZP1 monomers.ZP3/ZP2 heterodimers cross-linked by ZP1 monomers. ZP3-sperm binding, induction of acrosomal exocytosis ZP3-sperm binding, induction of acrosomal exocytosis (O-(O-

linked, branched saccharide chains)linked, branched saccharide chains) ZP2-sperm anchoring to ZP, sustenance of sperm bindingZP2-sperm anchoring to ZP, sustenance of sperm binding ZP1-structural role (cross-linker)ZP1-structural role (cross-linker) Anti- polyspermy defense: After fertilization, ZP2 is cleaved, Anti- polyspermy defense: After fertilization, ZP2 is cleaved,

preventing sperm bindingpreventing sperm binding

Human & Rat ZP:Human & Rat ZP:

Four ZP proteins & genes: ZP1, ZP2, ZP3 & ZPB4Four ZP proteins & genes: ZP1, ZP2, ZP3 & ZPB4 Both ZP3 & ZP4 serve as sperm receptors in humansBoth ZP3 & ZP4 serve as sperm receptors in humans

Pig ZP:Pig ZP:

ssZPA=mmZP2ssZPA=mmZP2 ssZPB=mmZP1ssZPB=mmZP1 ssZPC=mmZP3ssZPC=mmZP3

(ZPB & ZPC responsible for sperm-ZP binding in pig)(ZPB & ZPC responsible for sperm-ZP binding in pig)

ZP K.O.ZP K.O.

ZP3 KO: no ZP, no fertilityZP3 KO: no ZP, no fertility

Rankin T, et al., (1996) Mice homozygous for an insertional Rankin T, et al., (1996) Mice homozygous for an insertional mutation in the Zp3 gene lack a zona pellucida and are mutation in the Zp3 gene lack a zona pellucida and are

infertile. infertile. Development Development 122:2903-10.122:2903-10.

ZP2 KO: abnormal ZP, no fertilityZP2 KO: abnormal ZP, no fertility

Rankin TL, et al. (2001) Defective zonae pellucidae in Zp2-Rankin TL, et al. (2001) Defective zonae pellucidae in Zp2-null mice disrupt folliculogenesis, fertility and development. null mice disrupt folliculogenesis, fertility and development. Development Development 128:1119-26. 128:1119-26.

Is Postranslational Modification of ZP-Proteins Is Postranslational Modification of ZP-Proteins Responsible for Species Specificity of Responsible for Species Specificity of

Fertilization?Fertilization?Experiment 1 (Rankin et al., 1998, Experiment 1 (Rankin et al., 1998, DevelopmentDevelopment 125:2415-24): 125:2415-24): Mouse lines with human-mouse Mouse lines with human-mouse

chimeric zonae pellucidae have been established.chimeric zonae pellucidae have been established.

Results: Mouse, but not human, sperm bind to huZP2 and huZP2/huZP3 rescue eggs; Results: Mouse, but not human, sperm bind to huZP2 and huZP2/huZP3 rescue eggs; humanized ZPs did not undergo ZP2 cleavage.humanized ZPs did not undergo ZP2 cleavage.

Interpretation by authors: The cleavage status of ZP2 modulates the three-dimensional Interpretation by authors: The cleavage status of ZP2 modulates the three-dimensional structure of the zona pellucida and determines whether sperm bind (uncleaved) or do not structure of the zona pellucida and determines whether sperm bind (uncleaved) or do not (cleaved). (cleaved).

Alternative interpretation: Human ZPs were postarnslationally modified in a mouse-like Alternative interpretation: Human ZPs were postarnslationally modified in a mouse-like manner, thus promoting the binding of mouse sperm to “humanized” mouse ZPs.manner, thus promoting the binding of mouse sperm to “humanized” mouse ZPs.

Experiment 2 (Chakravarti et al., 2005, Experiment 2 (Chakravarti et al., 2005, Mol Hum RepMol Hum Rep 11:365-72): 11:365-72): Human ZPB & ZPC proteins Human ZPB & ZPC proteins were expressed in yeast and in baculovirus-transfected insect cells. Both types of were expressed in yeast and in baculovirus-transfected insect cells. Both types of recombinant ZPs were incubated with human spermatozoa and assessed for their ability to recombinant ZPs were incubated with human spermatozoa and assessed for their ability to induce AE.induce AE.

Result: Only the viral-transfection rZP3 induced AEResult: Only the viral-transfection rZP3 induced AE

Interpretation: Postranslational modification of ZP3 (i.e. glycosylation) rather than its AA Interpretation: Postranslational modification of ZP3 (i.e. glycosylation) rather than its AA sequence determines its ability to interact with sperm acrosome.sequence determines its ability to interact with sperm acrosome.

Sperm AcrosomeSperm Acrosome

Derived from GolgiDerived from Golgi

Undergoes changes during epididymal Undergoes changes during epididymal maturation and capacitationmaturation and capacitation

Components:Components:

1.Inner acrosomal membrane-IAM1.Inner acrosomal membrane-IAM2.Outer acrosomal membrane-OAM2.Outer acrosomal membrane-OAM3. Acrosomal matrix-AM3. Acrosomal matrix-AM4. Segments: Apical, principal4. Segments: Apical, principal equatorial (lip formed by OAM)equatorial (lip formed by OAM)

Lysosome and Secretory Granule?Lysosome and Secretory Granule?

LL

Lysosomal Hydrolases: Lysosomal Hydrolases:

Arylsulfatase A Arylsulfatase A

B-N-acetyl-glucosamidase B-N-acetyl-glucosamidase Phospholipase A Phospholipase A

Various proteasesVarious proteases

SGSG

Secretion coupled to Secretion coupled to extracellular stimulus (binding extracellular stimulus (binding to ZP)to ZP)

Secretory products are Secretory products are concentrated and condensedconcentrated and condensed

Granules are stored for long Granules are stored for long timetime

Biogenesis from Golgi is Biogenesis from Golgi is similar to SG biogenesis similar to SG biogenesis (preacrosomal vesicles, (preacrosomal vesicles, acrosomic granule)acrosomic granule)

Acrosomal Exocytosis/Acrosome Acrosomal Exocytosis/Acrosome ReactionReaction

Induced by sperm Induced by sperm binding to sperm binding to sperm receptor protein receptor protein on ZP (ZP3 &/ on ZP (ZP3 &/ ZP4 / ZPB+ZPC)ZP4 / ZPB+ZPC)

IRREVERSIBLEIRREVERSIBLE

Involves fusion of Involves fusion of PM with OAM & PM with OAM & formation of formation of hybrid acrosomal hybrid acrosomal membrane membrane vesiclesvesicles

Acrosome Reaction: Two State/Binary ModelAcrosome Reaction: Two State/Binary Model

Acrosome is either intact Acrosome is either intact (ON/1) or reacted (OFF/2) and (ON/1) or reacted (OFF/2) and there are no intermediatesthere are no intermediates

Intermediate state is short-lived Intermediate state is short-lived and spontaneous AR is not and spontaneous AR is not physiologicalphysiological

Assumes that sperm-ZP Assumes that sperm-ZP interactions are governed by interactions are governed by acrosomal status (intact acrosomal status (intact acrosome needed for binding*)acrosome needed for binding*)

****Not true: AR sperm do bind and penetrate ZPNot true: AR sperm do bind and penetrate ZP

Acrosomal Exocytosis: Analog Model Acrosomal Exocytosis: Analog Model

•Assumes the existence of Assumes the existence of transitional intermediatestransitional intermediates of AE of AE•Capacitation promotes AE, sperm-ZP binding accelerates itCapacitation promotes AE, sperm-ZP binding accelerates it•Spontaneous AE is physiological event, slower yet Spontaneous AE is physiological event, slower yet mechanistically similar to ZP-induced AEmechanistically similar to ZP-induced AE•Acrosomal matrix is not dispersed at once, but layer after layerAcrosomal matrix is not dispersed at once, but layer after layer

Gerton G. (2002). Function of the sperm acrosome. In” Fertilization” (D.Hardy,Ed.), pp. 265-302, Academic Press, San Gerton G. (2002). Function of the sperm acrosome. In” Fertilization” (D.Hardy,Ed.), pp. 265-302, Academic Press, San Diego.Diego.

Acrosomal Matrix Domains in Acrosomal Matrix Domains in Guinea Pig* Guinea Pig*

*Differential Release Hypothesis (Hardy, 1991): Components of acrosomal matrix released at different times during ZP penetration.

Acrosomal ExocytosisAcrosomal Exocytosis1.1. Priming of OAM during capacitationPriming of OAM during capacitation

2.2. Binding of PM/OAM to ZPBinding of PM/OAM to ZP

3.3. Vesiculation of PM/OAMVesiculation of PM/OAM

4.4. Processing of proacrosin Processing of proacrosin → → acrosinacrosin

5.5. Dispersion of the outer layer of matrixDispersion of the outer layer of matrix

6.6. Shedding of acrosomal shroudShedding of acrosomal shroud

7.7. Dispersion of the inner layer of AMDispersion of the inner layer of AM

8.8. Binding of IAM to ZPBinding of IAM to ZP

9.9. Localized ZP degradation & penetrationLocalized ZP degradation & penetration

10.10. Sperm–oolemma bindingSperm–oolemma binding

Acrosomal ShroudAcrosomal Shroud

Signaling During AESignaling During AE Heterotrimeric G-proteins in plasma Heterotrimeric G-proteins in plasma

membranemembrane

Tyrosine phosphorylationTyrosine phosphorylation

Voltage-sensitive Ca2+ channels, other Voltage-sensitive Ca2+ channels, other ionsions

Phospholipase C activation and internal Phospholipase C activation and internal calcium release via IP3-receptorcalcium release via IP3-receptor

Adenyl cyclase/cAMPAdenyl cyclase/cAMP

SNARE hypothesis (membrane vesicle SNARE hypothesis (membrane vesicle fusion proteins: VAMP, syntaxin 1, fusion proteins: VAMP, syntaxin 1, synaptotagmin). synaptotagmin).

Progesterone (receptor on sperm PM; is it Progesterone (receptor on sperm PM; is it physiologically relevant?)physiologically relevant?)

Candidate Sperm Receptors for ZP:Candidate Sperm Receptors for ZP:

““The lack of general agreement on The lack of general agreement on the means of sperm adhesion to and the means of sperm adhesion to and penetration of the zona pellucida” penetration of the zona pellucida” underlines “the need for new underlines “the need for new approaches to this problem.” approaches to this problem.”

Olds-Clarke P. Unresolved Issues in Olds-Clarke P. Unresolved Issues in Mammalian Fertilization. Int. Rev. Cytoll. Mammalian Fertilization. Int. Rev. Cytoll. 2003; 232:129-184. 2003; 232:129-184.

How Does The Spermatozoon Pass Through ZP?

Models of Zona PenetrationModels of Zona Penetration

1. Biophysical/mechanical model: motile force of flagellum/oscillative thrust creates penetration slit

2. Noncatalytic, non-enzymatic disassembly of zona (hydrophobic abalone sperm lysin)

3. Proteolytic model:Proacrosin glycosidases (PH-20 hyaluronidase; ASA)TESP proteases (non-acrosin serine proteases)

26S proteasomes

Mechanical Model-ProsMechanical Model-Pros

Conventional protease inhibitors may delay but Conventional protease inhibitors may delay but not completely prevent sperm-ZP penetration. not completely prevent sperm-ZP penetration.

Sperm flagellar beating persists during ZP Sperm flagellar beating persists during ZP penetration and stops only at sperm-oolemma penetration and stops only at sperm-oolemma bindingbinding

Proacrosin KO mice are fertile, albeit with delayed AR; PA may be involved in matrix dispersal)

Mechanical Model-ConsMechanical Model-Cons

SSperm head of some perm head of some species is not shaped for species is not shaped for easy penetration easy penetration (marsupials, rodents)(marsupials, rodents)

FFertilization slit is much ertilization slit is much larger than sperm head larger than sperm head diameterdiameter

Measurements of sperm Measurements of sperm motile force do not support itmotile force do not support it

Enzymatic Model-ProsEnzymatic Model-Pros

The slit is digested irreversiblyThe slit is digested irreversibly

ZP may be too rigid for mechanical ZP may be too rigid for mechanical penetrationpenetration

Sperm flagellum may not generate sufficient Sperm flagellum may not generate sufficient thrust for push sperm head through ZPthrust for push sperm head through ZP

Enzymatic Model-ConsEnzymatic Model-Cons

Conventional protease inhibitors delay, but do Conventional protease inhibitors delay, but do not prevent sperm-ZP penetration completely. not prevent sperm-ZP penetration completely.

Acrosin KO mice are fertile* Acrosin KO mice are fertile*

SOLUTION: A stochiometric, non-hydrolytic SOLUTION: A stochiometric, non-hydrolytic disassembly mechanism (abalone), or a disassembly mechanism (abalone), or a protease other than conventional proteases protease other than conventional proteases already identified in the acrosomealready identified in the acrosome

*Baba et al., 1994; J Biol Chem. 69: 31845-9*Baba et al., 1994; J Biol Chem. 69: 31845-9

Inhibition of the 26S Proteasome Inhibition of the 26S Proteasome Prevents FertilizationPrevents Fertilization

ZP Digestion Visualized by Anti-ZP Digestion Visualized by Anti-Ubiquitin AntibodiesUbiquitin Antibodies

ZP-Penetration is Not the Only ZP-Penetration is Not the Only Purpose of Acrosomal ExocytosisPurpose of Acrosomal Exocytosis

Intact Sperm Head:Intact Sperm Head:

Plasma membranePlasma membrane

Acrosome:Acrosome:

- Anterior sac-like, filled with enzymes (AAS)- Anterior sac-like, filled with enzymes (AAS)- Posterior narrow pouch-like equatorial region - Posterior narrow pouch-like equatorial region ((ERER))

Perinuclear Theca:Perinuclear Theca:

- Subacrosomal LayerSubacrosomal Layer- Postacrosomal Layer- Postacrosomal Layer

Nucleus (N)Nucleus (N)

After Acrosomal Exocytosis:After Acrosomal Exocytosis:

•The plasma membrane and the The plasma membrane and the anterior, sac-like acrosome are lost anterior, sac-like acrosome are lost

•The The equatorial acrosomeequatorial acrosome is retained. is retained. Some of its content is released on the Some of its content is released on the surface of the plasma membrane of that surface of the plasma membrane of that regionregion

•The inner acrosomal membrane is The inner acrosomal membrane is exposed in the anterior region exposed in the anterior region

Acrosomal Exocytosis Exposes Antigens in IAM Acrosomal Exocytosis Exposes Antigens in IAM in Preparation for Sustained Sperm-ZP Bindingin Preparation for Sustained Sperm-ZP Binding

Case in Point: IAM 32/ZPBP1A&BCase in Point: IAM 32/ZPBP1A&B

Resides on IAMResides on IAM Antigenic site exposed only after AEAntigenic site exposed only after AE KO is infertileKO is infertile Co-immuno-precipitates with SED1 antigenCo-immuno-precipitates with SED1 antigen

(SED1 immuno-saturation prevents fertilization in (SED1 immuno-saturation prevents fertilization in vitro and KO reduced male fertilityvitro and KO reduced male fertility

The acrosome-exocytosed/reacted The acrosome-exocytosed/reacted spermatozoa show labeling on the spermatozoa show labeling on the exposed inner acrosomal exposed inner acrosomal membranemembrane

The acrosome The acrosome intact intact spermatozoa spermatozoa display display exclusive exclusive labeling on the labeling on the equatorial equatorial region.region.

IAM32

TEM-Gold of IAM 32:TEM-Gold of IAM 32: 1. The acrosome intact spermatozoa The acrosome intact spermatozoa display exclusive labeling on the equatorial regiondisplay exclusive labeling on the equatorial region

2. The acrosome-exocytosed spermatozoa show 2. The acrosome-exocytosed spermatozoa show labeling on the exposed inner acrosomal labeling on the exposed inner acrosomal membranemembrane

Acrosomal Exocytosis Exposes Antigens in ES in Acrosomal Exocytosis Exposes Antigens in ES in Preparation for Sperm-Oolemma Binding and Preparation for Sperm-Oolemma Binding and

Oocyte ActivationOocyte Activation..

Case in Point: Equatorin MN9Case in Point: Equatorin MN9

Antigen not detected in intact Antigen not detected in intact sperm headsperm head

Antigenic site on ES exposed only Antigenic site on ES exposed only after AEafter AE

MN9 immunosaturation prevents MN9 immunosaturation prevents sperm-oolemma binding during sperm-oolemma binding during fertilization fertilization in vitroin vitro

Source:Source: Manandhar & Toshimori, 2001, Manandhar & Toshimori, 2001, Biol. Reprod.Biol. Reprod. 65:1425-1436 65:1425-1436

Sperm Antigens in PAS are not Exposed by Sperm Antigens in PAS are not Exposed by Acrosomal Exocytosis, Preserving their Function Acrosomal Exocytosis, Preserving their Function

in Oocyte Activationin Oocyte Activation..

Case in Point: PAS antigen MN13Case in Point: PAS antigen MN13

Antigen not detected in intact Antigen not detected in intact sperm head or after AEsperm head or after AE

Antigenic site on ES exposed Antigenic site on ES exposed only after sperm incorporation only after sperm incorporation into ooplasminto ooplasm

MN13 immunosaturation MN13 immunosaturation prevents oocyte activation after prevents oocyte activation after ICSIICSI

Source: Manandhar & Toshimori, Source: Manandhar & Toshimori, 2003; Biol. Reprod. 68:655-6632003; Biol. Reprod. 68:655-663

Next Week: Fertilization-Part II-Next Week: Fertilization-Part II-Oocyte Activation & Zygotic Oocyte Activation & Zygotic

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