Transcript Fertilization Part 2 - Division Of Animal Sciences

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) Woman’s 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):
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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:
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Four ZP proteins & genes: ZP1, ZP2, ZP3 & ZPB4
Both ZP3 & ZP4 serve as sperm receptors in humans
Pig ZP:
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ssZPA=mmZP2
ssZPB=mmZP1
ssZPC=mmZP3
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(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.
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Results: Mouse, but not human, sperm bind to huZP2 and huZP2/huZP3 rescue eggs;
humanized ZPs did not undergo ZP2 cleavage.
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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).
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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.
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Result: Only the viral-transfection rZP3 induced AE
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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. Sperm–oolemma binding
Acrosomal Shroud
Signaling During AE
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Heterotrimeric G-proteins in plasma
membrane
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Tyrosine phosphorylation
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Voltage-sensitive Ca2+ channels, other
ions
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Phospholipase C activation and internal
calcium release via IP3-receptor
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Adenyl cyclase/cAMP
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SNARE hypothesis (membrane vesicle
fusion proteins: VAMP, syntaxin 1,
synaptotagmin).
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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
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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