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Sperm freezing‐‐‐‐‐MBCD and GSH approach‐‐‐‐‐
Toru Takeo
Center for Animal Resources and Development (CARD), Kumamoto University, Japan
EMMA Workshop: May 8 2012.
Topics
1. Introduction
2. Technology Development:
Sperm cryopreservation
Sperm preincubation
In vitro fertilization
3. Application of our technologies
Inbred and hybrid strains
Cryopreserved sperm prepared by various protocols
2008 2009 2010 2011 20120
20
40
60
80
100
Year
% F
ertil
izat
ion
rate
CARD Protocol:Sperm cryopreservation and IVF
N=574 strains (C57BL/6 background: 435)
Technology Development
Methyl‐‐Cyclodextrin (MBCD):TYH with MBCD
Modified R18S3 (mR18S3):R18S3+L‐glutamine
Prof. Nakagata
Reduced glutathione (GSH):mHTF + GSH
Nakagata Method
Mammal Genome 2000.Biol Reprod 2008.Lab Animal 2010.Biol Reprod 2011.
C57BL/6 Mouse
1. Major inbred strain2. Most common background for genetically engineered mouse3. Low fertility of frozen‐thawed sperm
C57BL/6J is a major inbred strain and its geneticbackground is well known. Furthermore, this strain isused not only for the production of transgenic mice,but also applied as a back‐cross for targeted mutantmice.
2010
C57BL/6J86.1％
C57BL/6N1.9％
CD-14.3%
DBA/20.9%
NOD0.3%
BALB/cA3.3%
BDF11.8%
CBA:0.6%C3H/HeN
0.8%
（Total 1424 lines）
Fresh spermFrozen‐thawed sperm
0
50
100
BALB/c C3H/He C57BL/6 DBA/2 ICR BDF1 B6C3F1
Fertilization
rate（%）
We have addressed the problem of low fertility of frozen‐thawed C57BL/6 mouse sperm.
Topics
1. Introduction
2. Technology Development:
Sperm cryopreservation
Sperm preincubation
In vitro fertilization
3. Application of our technologies
Inbred and hybrid strains
Cryopreserved sperm prepared by various protocols
Cryopreservation: Preincubation： Fertilization：
1. Technology Development
Sperm Cryopreservation
120 L Cryoprotectant:R18S3 with L‐glutamine
Liquid paraffin
Cauda epididymides
3 minutes
Sperm suspension
CARD online manual: http://card.medic.kumamoto‐u.ac.jp/card/english/sigen/index.html
Cryoprotectant solution:18 % raffinose pentahydrate + 3% skim milk solution (R18S3)
+ L‐glutamine
ScreeningSugars, cyclodextrins, amino acidsin R18S3
Amino acidsglycine, alanine, glutamine
mR18S3:R18S3 + L‐glutamine
Unpublished data Takeo and Nakagata, Lab Animal 2010.
0 20 40 60control
HP-a-CDHP-b-CDHP-g-CDM-a-CDM-b-CD
SBE7-b-CDG2-b-CDCaramelBenecoat
XylitolGlycineAlanine
ValineIsoleucine
LeucineProline
Aspertic acidAspertate
Glutamic acidGlutamine
CysteineSerine
ThreoninePhenylalanine
ArginineLycine
% Fertilized eggs
Add
itive
s
Sperm preincubation
90 L Preincubation medium:TYH + methyl‐‐cyclodextrin
(MBCD)
Sperm suspension
HTF 30 minutes
IVF
CARD online manual: http://card.medic.kumamoto‐u.ac.jp/card/english/sigen/index.html
Component mg/100 mL
NaCl 697.6
KCl 35.6
Glucose 100
Sodium pyruvate 5.5
CaCl2/2H2O 25.1
MgSO4/7H2O 29.3
KH2PO4 16.2
NaHCO3 210.6
Potasium penicillin G 7.5
Streptomysin sulfate 5.0
Polyvinylalcohol 100
Bovine serum albumin 400
Methyl‐ ‐cyclodextrin 98.3
Sperm preincubation medium:TYH with methyl‐‐cyclodextrin (MBCD)
Prof. Toyoda：Reproductive Biology
TYH:ToyodaYokoyamaHoshi
Prof. Irie:Cyclodextrin Science
for Physical Pharmaceutics
Toyoda Y et al. 1971: J. Anim. Reprod. 16: 147‐151.
O
OH
HO
O
O
OHOO
OH
OH OH
OH
OHOH
O
O
HO
OH
O OH
O OHOH
HO
O
O
HO
OH
OHOH
OH
H
H
HH
H
HH
H
HO
O
Hydrophilic exterior
Hydrophobic cavity
1 2
65
43
)
(
‐CD, n = 1 ‐CD, n = 2‐CD, n = 3 nOH
Cyclodextrins
Compound n R1 R2 R3 D.S. a)
a) Average degree of substitution, b) Molecular weight, c) In water at 25�, d) The concentration of CyDs to induce 50% hemolysis of rabbit erythrocytes, e) 2‐Hydroxypropyl‐CyDs, f) 2,6‐Di‐O‐methyl‐CyDs, g) Methyl‐‐CyD.
M.W. b) Solubility(g/dL) c)
Hemolytic activity(mM) d)
‐CyD 6 ‐H ‐H ‐H ‐ 972 14.5 14
‐CyD 7 ‐H ‐H ‐H ‐ 1135 1.85 5.7
HP‐‐CyD e) 6 4.1‐H or ‐CH2CH(CH3)OH 1210 > 50 > 50
DM‐‐CyD f) 6 ‐CH3 ‐H ‐CH3 12 1141 > 50 6.0
DM‐‐CyD f) 7 ‐CH3 ‐H ‐CH3 14 1331 57 1.3
M‐‐CyD g) 7 ‐H or ‐CH3 12.6 1311 > 50 2.8
HP‐‐CyD e) 7 4.8 1413 > 50 14.5‐H or ‐CH2CH(CH3)OH
0 20 40 60
control
alpha-CD
HP-alpha-CD
beta-CD
M-beta-CD
HP-beta-CD
G1-beta-CD
G2-beta-CD
SBE-beta-CD
gamma-CD
HP-gamma-CD
% Fertilized eggs
Add
itive
s
Nature ‐CD and ‐CD derivatives enhance fertility of frozen‐thawed sperm
ScreeningNature CDs, various CD derivativesin TYH
‐CDsBCD, MBCD, HPBCD, G1BCD, G2BCD
Sperm preincubation:TYH + MBCD
Unpublished data
MBCD activates fertility of frozen‐thawed C57BL/6 mouse sperm
0
25
50
75 * *
2
1%
Fer
tiliz
ed e
ggs
MBCDBSA + +
++-- -
- 0 30 60 90 1200
25
50
75 * *
* *
21
% F
ertil
ized
egg
sIncubation time (min)
controlBSAMBCD
Takeo et al, Biol Reprod 2008.
MBCD facilitates cholesterol efflux from sperm membrane
0 15 30 45 600
25
50
75
100 BSAMBCD
* **
Incubation Time(min)
Rel
ativ
e M
ean
Fluo
resc
ence
Int
ensi
ty
control MBCD treated
Membrane cholesterol
Takeo et al, Biol Reprod 2008.
Cholesterol reductionin sperm membrane
‐CD
cholesterol
‐CD‐cholesterolcomplex
CD‐cholesterolcomplex
In vitro fertilization
SpermPreincubation medium 10
L
Motile sperm
Non‐motile and aggregated sperm
Tip
90 L Fertilization medium:mHTF + reduced glutathione (GSH)
Oocyte collection
Oviduct ampullaCumulus‐oocytes
complexes
Needle
Liquid paraffin
Insemination
CARD online manual: http://card.medic.kumamoto‐u.ac.jp/card/english/sigen/index.html
Component mg/100 mL
NaCl 593.8
KCl 35.0
Glucose 50.0
CaCl2 57.0
MgSO4/7H2O 4.9
KH2PO4 5.4
NaHCO3 210.0
Sodium pyruvate 3.7
Sodium lactate (L) 340.0
Penicillin G potassium salt 7.5
Streptomycin sulfate 5.0
Bovine serum albumin 400.0
Reduced glutathione 30.7
Fertilization medium:mHTF with reduced glutathione (GSH)
Antioxidants improve the fertility of frozen‐thawed sperm
Bath M, PLoS One, 2010.Ostermeier, PLoS ONE, 2008.
Oxidative stress is related to the reduction in fertility
0
20
40
60
80
100
% F
ertil
ized
egg
s
GSH treatment
SpermOocyte
--
+-
-+
++
Fertilization is facilitated by the treatment of GSH to oocytes
control Sperm OocyteSperm
+Oocyte
GSH treatment
MediumSperm preincubation: TYH + MBCD or TYH + MBCD + GSH
Fertilization: mHTF or mHTF +GSH
Takeo and Nakagata, Biol Reprod 2011.
GSH dissects disulfide bonds in ZP proteins and promotes the expansion of ZP
GSH (-) GSH (+)
GSH
ZPExpansion
Visualization of ‐SH ZP expansion
Takeo and Nakagata, Biol Reprod 2011.
CARD protocol:Sperm cryopreservation and IVF
Cryopreservation:R18S3 + L‐glutamine
Preincubation：TYH + MBCD
Fertilization：mHTF + GSH
Topics
1. Introduction
2. Technology Development:
Sperm cryopreservation
Sperm preincubation
In vitro fertilization
3. Application of the technologies
Inbred and hybrid strains
Cryopreserved sperm prepared by various protocols
Our protocol overcomes the strain‐dependency of recovery rate in frozen‐thawed sperm
0
50
100
BALB/c C3H/He C57BL/6JC57BL/6N DBA/2N ICR BDF1 B6C3F1 129T2/SvEmsJ
％Fe
rtili
zed
eggs
Fresh sperm
Frozen-thawed sperm
Our IVF protocol is applicable to cryopreserved sperm produced by different protocols
0
20
40
60
80
100%
Fer
tiliz
ed e
ggs
CARD CARD CARD CARD
CARDJacksonLab.
Univ. Missouri
MRCHarwell
Sperm freezing
IVF:MBCD and GSH
We can use sperm cryopreservation for archiving and distributing the samples in mouse bank community.
AcknowledgementsKumamoto University
Div. of Reproductive Engineering, CARD
Prof. Nakagata
Shuuji Tsuchiyama
Kiyoko Fukumoto
Yukie Haruguchi
Tomoko Kondo
Yuko Nakamuta
Yumi Takeshita
Mayuko Urakawa
Mari Iwamoto
Fumi Takahashi
Eri Kohagura
Shota Kita
Aki Tsutsumi
Taichi Omaru
Kazuhito Sako
Satohiro Nakao
Hidetaka Yoshimoto
Yuki Sakai
Yuka Horikoshi
MRC HarwellDr. FrayDr. PickardDr. GuanDr. WeaverDr. Brown
Kumamoto University
Div. of Developmental Biology
CARD
Prof. Yamamura
Dr. Miike
Dr. Imanaka
Division of Clinical Chemistry and Informatics
Prof. Irie
Division of Physical Pharmaceutics
Prof. Arima
European Mouse Mutant Archive: EMMA
GraciasThank you
謝謝ありがとう
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