Efficient production ofoffspring in wild-derivedstrains of mice (Biol Reprod inpress)

Birth of offspring from ectopicallytransplanted PGCs (Biol Reprod2011)

ICSI with sperm derived fromcultured testes (Nature 2011)

Establishment of rabbit iPS cells(J Biol Chem 2010)

Establishment of ES cells fromimmature oocytes (Stem Cells2011)

Improvements of efficiency insomatic cell nuclear transferby correcting aberrant Xistexpression in cloned embryos(Science 2010; PNAS 2011)

(Presented by Keiji Mochida)

Piezo-impact unit attachedto an injection holder

Genotype

+Ter/

The maturity ofmale germ cells

Freezing treatmentof male germ cells

Implantation

0

20

40

60

80

100

ICSI ELSI ROSI

Overall

0

5

10

15

20

25

ICSI ELSI ROSI

Survival

0

20

40

60

80

100

ICSI ELSI ROSI

Cleavage

0

20

40

60

80

100

ICSI ELSI ROSI

Birth

0

10

20

30

40

50

60

ICSI ELSI ROSI

Birth Overall efficiency

Survival after injection Cleavage Implantation

Ogonuki et al., PLoS One 2010

B6

ELSI: elongated spermatid injection; ROSI: round spermatid injection

・

・

・

Ogonuki et al. PLoS One 2010

・

・

・

Overall (Offspring/oocytes injected)

0

5

10

15

20

25

ELSI ROSI

(%)

Sperm Elongated Roundspermatid spermatid

・

・

・

Ogonuki et al. PLoS One 2010

・

・

・

Overall (Offspring/oocytes injected)

0

5

10

15

20

25

ELSI ROSI

(%)

Sperm Elongated Roundspermatid spermatid

Genetic conservation Poor reproductive performance by aging

Motility failure of frozen sperm due to cryoinjury

Simple cryopreservation of male gametes

Fertilization of cumulus-free cryopreserved oocytes

Gene function analysis Sperm-oocyte fusion protein KO mice (CD9, Izumo)

Cumulus-deficient KO mice

Spermatogonial stem cell transplantation Allogeneic spermatogonial transplantation

Confirmation of clonal origin of spermatogonial colonies and many…

Gene therapy Rescue of Sl/Sld mice by virus-mediated gene therapy

Production of interspecies F1 C57BL/6 x Spretus

Transgenesis Sperm-mediated transgenesis by ICSI

Speed congenic Use of first wave spermatogenic cells

Analysis of sperm factor Sperm-borne oocyte-activating factor

In vitro manipulation of immature germ cells ES cell-derived sperm

Spermatids derived from in vitro-meiosis

PGC ectopic transplantation

B6;129S-Gtrosa26LghDDXC-PSMXA-1

C3.KOR-Apoeshl

CXDD-MSTS(tremor)NOD.R209-LineTNOD-Spretus Congenic 18-1C57BL/6J-TgN(b-actin-hBACE1)riken2Floxed Sox2 mice

mel18-/-CNR303cp/v11ve1KAFUBICard15KO129 x B6-G129 x B6-GIVCS.129P(B6)-Akr1c18<tm1Mni>SMXA-45SMXA-33SMXA-34NZB.Cg-Cd180<tm1Kmiy>B6;CB-Rab27a<Gt(pU-21T)58Imeg>/RbrcFVB129P2(B6)-Mmp2<tm1Ito>B6C3-Tmem50b<Gt(venusTrap_fJAV0017-B)B6.Cg-Tg(Scx-GFP)1StzrC57BL/6-Tg(CAG-Nup85)3-3SushSperm with a bent neck

from a TG mouse

Genetic conservation Poor reproductive performance by aging

Motility failure of frozen sperm due to cryoinjury

Simple cryopreservation of male gametes

Fertilization of cumulus-free cryopreserved oocytes

Gene function analysis Sperm-oocyte fusion protein KO mice (CD9, Izumo)

Cumulus-deficient KO mice

Spermatogonial stem cell transplantation Allogeneic spermatogonial transplantation

Confirmation of clonal origin of spermatogonial colonies and many…

Gene therapy Rescue of Sl/Sld mice by virus-mediated gene therapy

Production of interspecies F1 C57BL/6 x Spretus

Transgenesis Sperm-mediated transgenesis by ICSI

Speed congenic Use of first wave spermatogenic cells

Analysis of sperm factor Sperm-borne oocyte-activating factor

In vitro manipulation of immature germ cells ES cell-derived sperm

Spermatids derived from in vitro-meiosis

PGC ectopic transplantation

Epididymides and testes

o

medium

nNo.

pregnant

Sperm GL-PBS 4 4 50 34 (68.0) 11 (22.0)

NIM 2 2 20 19 (95.0) 13 (65.0)

El. Spermatid GL-PBS 1 1 10 3 (30.0) 1 (10.0)

NIM 4 3 39 12 (30.8) 7 (17.9)

Cells used

RecipientNo. transferred

(Total)

No. (%)

implant

No.(%)

offspring

Ogonuki et al., PNAS 103: 13098-13103, 2006 in collaboration with Dr. Martin Fray

o

(provided by Dr. Iwaki, Tokyo Jikei Medical University)

Feb. 13, 2006

Aug. 28, 2006

Ogonuki et al., PNAS 103: 13098-13103, 2006

No. used No. pregnant

BALB/c-nude 106 81 (76.4) 81 6 4 42 (51.9) 17 (21.0)

C3H/He 108 97 (89.8) 97 7 4 34 (35.1) 12 (12.4)

Strain of maleNo. (%)

offspring

Recipient femalesNo. embryos

transferredNo. (%) implant

No. oocytes

that survived

inejction

No. (%) oocytes

cleaved

Mar. 2, 2006

Genetic conservation Poor reproductive performance by aging

Motility failure of frozen sperm due to cryoinjury

Simple cryopreservation of male gametes

Fertilization of cumulus-free cryopreserved oocytes

Gene function analysis Sperm-oocyte fusion protein KO mice (CD9, Izumo)

Cumulus-deficient KO mice

Spermatogonial stem cell transplantation Allogeneic spermatogonial transplantation

Confirmation of clonal origin of spermatogonial colonies and many…

Gene therapy Rescue of Sl/Sld mice by virus-mediated gene therapy

Production of interspecies F1 C57BL/6 x Spretus

Transgenesis Sperm-mediated transgenesis by ICSI

Speed congenic Use of first wave spermatogenic cells

Analysis of sperm factor Sperm-borne oocyte-activating factor

In vitro manipulation of immature germ cells ES cell-derived sperm

Spermatids derived from in vitro-meiosis

PGC ectopic transplantation

First wave round spermatidsin a 17 day-old testis

Miki et al., JRD 2004

A pup born from a 17 day-oldspermatid

Genomic DNA cDNA

1 2 3 4 5 6 7 8M 9 10

SpermatidInjection

H19

JF1B6

H19, Igf2, Meg3, Igf2rIVF

(B6 x JF1)

22

Completion of congenesis

Ogonuki et al., PLoS One 2009

01020

3040506070

8090

100

0 20 40 60 80 100 120

Markers

homozygousforB6(%)

10

20

30

40

50

60

70

80

90

100

0 50 100 150 200

Markers

homozygousforB6(%)

80

85

90

95

100

0 50 100 150 200 250 300 350

Markers

homozygousforB6(%)

Gdf5

EdnraEGFP

Kitlg

RELMb

0

20

40

60

80

100

0 50 100 150 200 250 300

Markers

homozygousforNOD(%) (70/72)

(63/72)(60/72)

(38/72)

(0/72)(0/72)143d

20d

45d 43d44d

(72/86) (72/86)

(85/86)

(77/86)

(86/86)41d

43d

162d97d20d

43d

43d

(0/86)

(56/86)

(79/86) (86/86)

(38/176)

(123/176)

(154/176) (163/176)(176/176)

44d

42d41d

43d

Ogonuki et al., PLoS One 2009

Genetic conservation Poor reproductive performance by aging

Motility failure of frozen sperm due to cryoinjury

Simple cryopreservation of male gametes

Fertilization of cumulus-free cryopreserved oocytes

Gene function analysis Sperm-oocyte fusion protein KO mice (CD9, Izumo)

Cumulus-deficient KO mice

Spermatogonial stem cell transplantation Allogeneic spermatogonial transplantation

Confirmation of clonal origin of spermatogonial colonies

Gene therapy Rescue of Sl/Sld mice by virus-mediated gene therapy

Production of interspecies F1 C57BL/6 x Spretus

Transgenesis Sperm-mediated transgenesis by ICSI

Speed congenic Use of first wave spermatogenic cells

Analysis of sperm factor Sperm-borne oocyte-activating factor

In vitro manipulation of immature germ cells ES cell-derived sperm

Spermatids derived from in vitro-meiosis

Ectopic PGC transplantation

Dissociationby enzyme

Aggregationand

transplantation

12.5 dpcgonads

IVM

ROSIor

ICSI

→

Matoba and Ogura, Biol. Reprod. 2011

For the details of our lab: http://www.brc.riken.go.jp/lab/kougaku/indexE.html