Signal Transduction Mechanism of Fertilization

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Signal Transduction Mechanism of
Fertilization Basics of Reproductive Science and
Biotechnology SOI Asia Course Feb. 8, 2006 Yasuo
Fukami Ph.D. Research Center for Environmental
Genomics Graduate School of Science and
Technology Kobe University
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Fukami Lab
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Mechanism of Reproduction and Development of Our
Body
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????????
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No! Genome DNA is the inheritance.
A small infant inside?
Nicolas Hartsoeker (1694)
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(Dr. Nagai)
(Dr. Miyano)
Sato et al. BBA 2004
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(Gerald Karp, Cell and Molecular Biology 2nd ed.)
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Xenopus laevis (adult) Albinos ?
?
1.21.3 mm
5 mm
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000
100
200
300
400
500
600
700
800
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Rafts
Sperm
Protease?
Sperm receptor?
Egg
plasma membrane
xSrc (Xyk)
H2O2? A23187?
Tyr-P
Src/PTK
Unfertilized egg
inhibitors
PLC
g
Tyr-P
PLC inhibitors
Calcium transient
2
Ca
chelator
Egg activation
10 min after insemination
Sato et al. Dev Biol 2000 Proteomics 2002
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Fontanilla et al. Biophys J. 2003
Fontanilla and Nuccitelli Biophys J. 1998
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Ca2 transient in mouse and frog
Mouse egg Mouse egg Xeno
pus egg Xenopus egg extract
Kurokawa et al. Biol Cell 2004
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What is Egg Activation?
Fertilization Envelope
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(Scott F. Gilbert, Developmental Biology 7th ed.)
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(Scott F. Gilbert, Developmental Biology 7th ed.)
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Phospholipid and its cleavage by phospholipases
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PLC
(Scott F. Gilbert, Developmental Biology 7th ed.)
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(Scott F. Gilbert, Developmental Biology 7th ed.)
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Phosphorylation of Tyrosine Residues
Protein-tyrosine Kinases
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SDS-PAGE Western blottingSDS Sodium dodecyl
sulfatePAGE Polyacrylamide gel
electrophoresis Immunoprecipitation
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Dephosphorylation of MAP kinase monitored by
Immunoblotting
PP1 Src inhibitorPP3 Inactive
analogueU-73122 PLC inhibitorU-73343 Inactive
analogue
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Involvement of Xenopus Src in Fertilization
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Anti-pepY antibody
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Effect of anti-pepY antibody on v-Src and c-Src
activities
v-Src
c-Src
Fukami et al. J. Biol. Chem., 268, 1132-1140, 1993
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Dose-dependent activation of c-Src by anti-pepY
IgG
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A Immature oocytesB Mature oocytes
(Unfertilized eggs)C Fertilized eggs
Anti-pepY IgG
Sato et al., J. Biol. Chem., 1996
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Purification of Xyk (Xenopus tyrosine kinase)
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Sato et al. Dev Biol 1999
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Activation and translocation of Xyk
IP ImmunoprecipitationIB Immunoblotting
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Paired inhibitors used

• Genistein Tyrosine kinase-specific inhibitor
• Daidzein Inactive analogue of Genistein
• Peptide A7 Src-specific inhibitor peptide
• Peptide A9 Inactive counterpart of peptide
  A7
• PP1 Src-specific inhibitor
• PP3 Inactive analogue of PP1
• U-73122 PLC inhibitor
• U-73343 Inactive analogue of U-73122

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Genistein inhibits fertilization of Xenopus eggs
Sato et al., FEBS Lett., 1998
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Inhibition of sperm entry by a peptide inhibitor
of Src
Sperm head
Egg chromosomes at metaphase
Peptide A7
Peptide A7
Peptide A7 inhibitory peptide Peptide A9
Non-inhibitory control
peptide Sato et al. Dev Biol 1999
Peptide A9
100 mm
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PP1 and U-73122 inhibited Ca2 transient
Sato et al., Dev. Biol. 2000
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Inhibition of IP3 formation by PP1 and U-73122
PP1 Src inhibitorPP3 Inactive
analogueU-73122 PLC inhibitorU-73343 Inactive
analogue
Uf Fertilized
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Effect of U-73122 on PLCg activity
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Effect of PP1 and U-73122 on tyrosine-phosphoryla
tion of PLCg
IP ImmunoprecipitationIB Immunoblotting
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(No Transcript)
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Activated Xyk (xSrc) is associated with PLCg
32P
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Preparation of LD-DIM (Rafts)
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LD-DIM low density detergent-insoluble
membranes DRM detergent-resistant membranes or
Rafts
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Xenopus Src, cholesterol and GM1 ganglioside are
concentrated in the raft fractions of
unfertilized eggs
PLCg is not found in the raft!
Sato et al. Development 2002
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Activation of xSrc in the isolated egg raft
fraction
Sperm Control Sperm GTPgS cAMP RGDS CaCl2
Sperm Control Sperm GTPgS cAMP RGDS CaCl2
Src activity ( of control)
Src activity ( of control)
Sato et al. JBC 2003
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PLCg translocates to rafts after activation
Uf Unfertilized F Fertilized A A23187 H
H2O2
Sato et al., J. Biol. Chem., 2003
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Reconstitution of calcium signaling with egg raft
and CSF extract
(CSF extract)
Sato et al. Focus on Genom Res 2004 Curr
Proteomics 2004 Methods Mol Biol 2005
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Activation of PLCg in CSF extract by the
activated raft
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Rafts
G-protein?
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Localization of tyrosine-phosphorylated proteins
Sato et al. Development 2002
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Sperm and H2O2 stimulate protein-tyrosine
phosphorylation in LD-DIM
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Tyrosine phosphorylation of egg raft proteins
upon fertilization or H2O2 treatment
pp30?
Sakakibara et al. JBC 2005
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GRR inhibitor peptide
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Inhibition of egg fertilization by anti-xUPIII
antibody
Sakakibara et al. JBC 2005
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Dev Biol. 1999 Apr 1208(1)79-92.Activation of
Xenopus eggs by proteases possible involvement
of a sperm protease in fertilization.Mizote A,
Okamoto S, Iwao Y.Faculty of Science, Yamaguchi
University, Yamaguchi, 753-8512, Japan.

• Egg activation in cross-fertilization between
  Xenopus eggs and Cynops sperm may be caused by a
  protease activity against Boc-Gly-Arg-Arg-MCA in
  the sperm acrosome. To determine the role of the
  sperm protease in fertilization, the protease was
  purified from Cynops sperm using several
  chromatographic techniques. We found that
  purified sperm protease readily hydrolyzes
  Boc-Gly-Arg-Arg-MCA and Z-Arg-Arg-MCA, that
  protease activity was inhibited by the trypsin
  inhibitors aprotinin and leupeptin, and that not
  only the purified protease, but also cathepsin B,
  induces activation in Xenopus eggs. We
  inseminated unfertilized Xenopus eggs with
  homologous sperm in the presence of various
  peptidyl MCA substrates or protease inhibitors
  and demonstrated that trypsin inhibitors or MCA
  substrates containing Arg-Arg-MCA reversibly
  inhibited fertilization of both fully jellied and
  denuded eggs. Sperm motility was not affected by
  the reagents. An extract obtained from Xenopus
  sperm showed hydrolytic activity against
  Boc-Gly-Arg-Arg-MCA, Z-Arg-Arg-MCA, and Arg-MCA.
  These results suggest that the tryptic protease
  in Xenopus sperm is involved in fertilization,
  most likely by participating in egg activation..

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Cathepsin B induces phosphorylation of xUPIII and
PLCg
Hasan et al. Dev Biol 2005
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Inhibition of xUPIII degradation and egg
activation by GRR peptide
Hasan et al. Dev Biol 2005
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Sperm extracts contain cathepsin B-like activity
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Hasan et al. Dev Biol 2005 Ou et al. Unpublished
results
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Rafts
Sperm
Protease?
Sperm receptor?
Egg
G-protein?
plasma membrane
H2O2? A23187?
xSrc (Xyk)
Tyr-P
Src/PTK
Unfertilized egg
inhibitors
PLC
g
Tyr-P
PLC inhibitors
Calcium transient
2
Ca
chelator
Egg activation
10 min after insemination
Sato et al. Dev Biol 2000 Proteomics 2002
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Different signaling molecules may be used in
different species
Uroplakins?
Sato et al., BBA, 2004
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Collaborators
Yasuhiro Iwao Ken-ichi Yoshino Kazuyoshi
Yonezawa Alexander A. Tokmakov Rafael A.
Fissore Manabu Korokawa Claudio Sette Maria
Paola Paronetto Kenji Miyado
Ken-ichi Sato Tetsushi Iwasaki Yasushi
Ueda Zhize Ou Kei-ichi Sakakibara AKM Mahbub
Hasan Teppei Fukuda Gunay Mammadova Natsumi
Yamamoto Yuta Koretomo Yukie Kono