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

Class 5, Part II: Sperm-Zona Interactions & Acrosomal Exocytosis (AE; Formerly Known as AR) Fertilization is a complex series of reactions that, if once completed, excludes all possibilities of repetition CR Moore (1916) On the Superposition of Fertilization in Parthenogenesis. Biological Bulletin 31:137 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 Unique Sperm Accessory Structures Are Generated During Spermiogenesis

Acrosome (AC) Equatorial Segment (ES) Postacrosomal Sheath (PAS) Perinuclear Theca (PT) Reduced Sperm Centriole

Mitochondrial Sheath Flagellum During Fertilization, Sperm Accessory Structures Fulfill Unique Roles, While Being Transformed or Lost Irreversibly AC: Facilitates sperm-ZP binding and penetration; lost in part to acrosomal exocytosis, remnants degraded by ooplasm ES: Facilitates sperm-oolemma binding after ZP penetration, membranes intermingle with oolemma, insoluble layers degraded in ooplasm PAS & PAS-PT: Solubilize in ooplams while releasing the oocyte activating factors

Reduced Sperm Centriole: Actually duplicates to form zygotic centrosome and sperm aster for PN apposition (exception: rodents) Mitochondrial Sheath: Provides energy for motility/sperm transport; degraded by ooplasm along with its contingent of mtDNA Spermatozoa must undergo acrosomal exocytosis to penetrate the zona pellucida and fuse with the oolemma BUT Spermatozoa with exocytosed acrosome can still bind to ZP

Zona Pellucida 1906 If the ovum should be impregnated, several important changes take place which are as follows: The zona pellucida, or outer membrane of the egg, having thrown off its outer cell covering, and the spermatozoa have no difficulty in penetrating the soft albuminous membrane that encloses the yelk. An additional change observed taking place in the tubes is a deposit of albumen around zona pellucida, which takes place when the ovum is passing the middle and lower third of the tube. These occurrences are so uniform that the different offices for different portions of the Fallopian tube may be readily determined. The first or upper third is appropriated to the

reception of the ovum, and for removing the adventitious covering of cells, while it also prepares the ovum for the operation of the spermatozoa. In the middle third, the respiratory chamber is formed, and here the rotation of the yelk commences. In the lower third the cleverage takes place, as also the deposit of albumen. Frederick Wilson Pitcairn & Elizabeth J. Williard (1906) Womans Guide to Health, Beauty and Happiness. What Every Woman Should Know. Horace C. Fray Publisher, Washington DC., Pages 96-97. Zona Pellucida 2008 General model (murine, bovine):

ZP3/ZP2 heterodimers cross-linked by ZP1 monomers. ZP3-sperm binding, induction of acrosomal exocytosis (Olinked, branched saccharide chains) ZP2-sperm anchoring to ZP, sustenance of sperm binding ZP1-structural role (cross-linker) Anti- polyspermy defense: After fertilization, ZP2 is cleaved, preventing sperm binding

Human & Rat ZP: Four ZP proteins & genes: ZP1, ZP2, ZP3 & ZPB4 Both ZP3 & ZP4 serve as sperm receptors in humans Pig ZP: ssZPA=mmZP2

ssZPB=mmZP1 ssZPC=mmZP3 (ZPB & ZPC responsible for sperm-ZP binding in pig) ZP K.O. ZP3 KO: no ZP, no fertility Rankin T, et al., (1996) Mice homozygous for an insertional mutation in the Zp3 gene lack a zona pellucida and are infertile. Development 122:2903-10.

ZP2 KO: abnormal ZP, no fertility Rankin TL, et al. (2001) Defective zonae pellucidae in Zp2null mice disrupt folliculogenesis, fertility and development. Development 128:1119-26. Is Postranslational Modification of ZP-Proteins Responsible for Species Specificity of Fertilization? Experiment 1 (Rankin et al., 1998, Development 125:2415-24): Mouse lines with human-mouse chimeric zonae pellucidae have been established. Results: Mouse, but not human, sperm bind to huZP2 and huZP2/huZP3 rescue eggs; humanized ZPs did not undergo ZP2 cleavage.

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 (cleaved). Alternative interpretation: Human ZPs were postarnslationally modified in a mouse-like manner, thus promoting the binding of mouse sperm to humanized mouse ZPs. Experiment 2 (Chakravarti et al., 2005, Mol Hum Rep 11:365-72): Human ZPB & ZPC proteins

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 induce AE. Result: Only the viral-transfection rZP3 induced AE Interpretation: Postranslational modification of ZP3 (i.e. glycosylation) rather than its AA sequence determines its ability to interact with sperm acrosome. Sperm Acrosome

Derived from Golgi Undergoes changes during epididymal maturation and capacitation Components: 1.Inner acrosomal membrane-IAM 2.Outer acrosomal membrane-OAM 3. Acrosomal matrix-AM 4. Segments: Apical, principal equatorial (lip formed by OAM) Lysosome and Secretory Granule? L

SG Lysosomal Hydrolases: Secretion coupled to extracellular stimulus (binding to ZP) Arylsulfatase A B-N-acetyl-glucosamidase Phospholipase A Various proteases

Secretory products are concentrated and condensed Granules are stored for long time Biogenesis from Golgi is similar to SG biogenesis (preacrosomal vesicles, acrosomic granule) Acrosomal Exocytosis/Acrosome Reaction Induced by sperm binding to sperm

receptor protein on ZP (ZP3 &/ ZP4 / ZPB+ZPC) IRREVERSIBLE Involves fusion of PM with OAM & formation of hybrid acrosomal membrane vesicles Acrosome Reaction: Two State/Binary Model Acrosome is either intact

(ON/1) or reacted (OFF/2) and there are no intermediates Intermediate state is short-lived and spontaneous AR is not physiological Assumes that sperm-ZP interactions are governed by acrosomal status (intact acrosome needed for binding*) **Not true: AR sperm do bind and penetrate ZP Acrosomal Exocytosis: Analog Model

Assumes the existence of transitional intermediates of AE Capacitation promotes AE, sperm-ZP binding accelerates it Spontaneous AE is physiological event, slower yet mechanistically similar to ZP-induced AE Acrosomal 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 Diego. Acrosomal Matrix Domains in Guinea Pig* *Differential Release Hypothesis (Hardy, 1991): Components of acrosomal matrix released at different times during ZP penetration.

Acrosomal Exocytosis 1. Priming of OAM during capacitation 2. Binding of PM/OAM to ZP 3. Vesiculation of PM/OAM 4. Processing of proacrosin acrosin 5. Dispersion of the outer layer of matrix 6. Shedding of acrosomal shroud 7. Dispersion of the inner layer of AM 8. Binding of IAM to ZP 9. Localized ZP degradation & penetration 10. Spermoolemma binding

Acrosomal Shroud Signaling During AE Heterotrimeric G-proteins in plasma membrane Tyrosine phosphorylation

Voltage-sensitive Ca2+ channels, other ions Phospholipase C activation and internal calcium release via IP3-receptor Adenyl cyclase/cAMP

SNARE hypothesis (membrane vesicle fusion proteins: VAMP, syntaxin 1, synaptotagmin). Progesterone (receptor on sperm PM; is it physiologically relevant?) Candidate Sperm Receptors for ZP:

How Does The Spermatozoon Pass Through ZP? The lack of general agreement on the means of sperm adhesion to and penetration of the zona pellucida underlines the need for new approaches to this problem. Olds-Clarke P. Unresolved Issues in Mammalian Fertilization. Int. Rev. Cytoll. 2003; 232:129-184. Models 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-Pros Conventional protease inhibitors may delay but not completely prevent sperm-ZP penetration. Sperm flagellar beating persists during ZP penetration and stops only at sperm-oolemma binding Proacrosin KO mice are fertile, albeit with delayed AR; PA may be involved in matrix dispersal) Mechanical Model-Cons Sperm head of some species is not shaped for

easy penetration (marsupials, rodents) Fertilization slit is much larger than sperm head diameter Measurements of sperm motile force do not support it Enzymatic Model-Pros The slit is digested irreversibly ZP may be too rigid for mechanical penetration Sperm flagellum may not generate sufficient

thrust for push sperm head through ZP Enzymatic Model-Cons Conventional protease inhibitors delay, but do not prevent sperm-ZP penetration completely. Acrosin KO mice are fertile* SOLUTION: A stochiometric, non-hydrolytic disassembly mechanism (abalone), or a protease other than conventional proteases already identified in the acrosome *Baba et al., 1994; J Biol Chem. 69: 31845-9 Inhibition of the 26S Proteasome

Prevents Fertilization ZP Digestion Visualized by AntiUbiquitin Antibodies ZP-Penetration is Not the Only Purpose of Acrosomal Exocytosis Intact Sperm Head: Plasma membrane Acrosome: - Anterior sac-like, filled with enzymes (AAS) - Posterior narrow pouch-like equatorial region (ER)

Perinuclear Theca: - Subacrosomal Layer - Postacrosomal Layer Nucleus (N) After Acrosomal Exocytosis: The plasma membrane and the anterior, sac-like acrosome are lost The equatorial acrosome is retained. Some of its content is released on the surface of the plasma membrane of that

region The inner acrosomal membrane is exposed in the anterior region Acrosomal Exocytosis Exposes Antigens in IAM in Preparation for Sustained Sperm-ZP Binding Case in Point: IAM 32/ZPBP1A&B Resides on IAM

Antigenic site exposed only after AE KO is infertile Co-immuno-precipitates with SED1 antigen (SED1 immuno-saturation prevents fertilization in vitro and KO reduced male fertility The acrosome-exocytosed/reacted spermatozoa show labeling on the exposed inner acrosomal membrane IAM32

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

2. The acrosome-exocytosed spermatozoa show labeling on the exposed inner acrosomal membrane Acrosomal Exocytosis Exposes Antigens in ES in Preparation for Sperm-Oolemma Binding and Oocyte Activation. Case in Point: Equatorin MN9 Antigen not detected in intact sperm head Antigenic site on ES exposed only after AE

MN9 immunosaturation prevents sperm-oolemma binding during fertilization in vitro Source: Manandhar & Toshimori, 2001, Biol. Reprod. 65:1425-1436 Sperm Antigens in PAS are not Exposed by Acrosomal Exocytosis, Preserving their Function in Oocyte Activation. Case in Point: PAS antigen MN13 Antigen not detected in intact sperm head or after AE Antigenic site on ES exposed

only after sperm incorporation into ooplasm MN13 immunosaturation prevents oocyte activation after ICSI Source: Manandhar & Toshimori, 2003; Biol. Reprod. 68:655-663 Next Week: Fertilization-Part IIOocyte Activation & Zygotic Development

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