EMMA Workshop: May 8 2012. - INFRAFRONTIER · In vitro fertilization 3. Application of our...

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



Sperm preincubation


3. Application of our technologies



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Unpublished data Takeo and Nakagata, Lab Animal 2010.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


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


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


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



Sperm preincubation


3. Application of the technologies



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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