ICSI with sperm derived from cultured testes (Nature 2011) Establishment of rabbit ips cells (J Biol Chem 2010)

Transcription

1

2 Efficient production of offspring in wild-derived strains of mice (Biol Reprod in press) (Presented by Keiji Mochida) Birth of offspring from ectopically transplanted PGCs (Biol Reprod 211) ICSI with sperm derived from cultured testes (Nature 211) Establishment of rabbit ips cells (J Biol Chem 21) Establishment of ES cells from immature oocytes (Stem Cells 211) Improvements of efficiency in somatic cell nuclear transfer by correcting aberrant Xist expression in cloned embryos (Science 21; PNAS 211)

3

4 Piezo-impact unit attached to an injection holder

5 Genotype +Ter/ The maturity of male germ cells Freezing treatment of male germ cells

6 Survival after injection Survival B6 Cleavage Cleavage Implantation Implantation ICSI ELSI ROSI ICSI Birth ELSI ROSI ICSI ELSI ROSI OverallOverall efficiency Birth ICSI ELSI ROSI ICSI ELSI ROSI ELSI: elongated spermatid injection; ROSI: round spermatid injection Ogonuki et al., PLoS One 21

7 Overall (Offspring/oocytes injected) (%) Sperm Elongated ELSI spermatid Round ROSI spermatid Ogonuki et al. PLoS One 21

8 Overall (Offspring/oocytes injected) (%) Sperm Elongated ELSI spermatid Round ROSI spermatid Ogonuki et al. PLoS One 21

9 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 ma 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

10 Sperm with a bent neck from a TG mouse B6;129S-Gtrosa26 Lgh DDXC-P SMXA-1 shl C3.KOR-Apoe CXDD-M STS(tremor) NOD.R29-LineT NOD-Spretus Congenic 18-1 C57BL/6J-TgN(b-actin-hBACE1)riken2 Floxed Sox2 mice mel18-/cnr33cp/v11ve1 KA FUBI Card15KO 129 x B6-G 129 x B6-G IVCS.129P(B6)-Akr1c18<tm1Mni> SMXA-45 SMXA-33 SMXA-34 NZB.Cg-Cd18<tm1Kmiy> B6;CB-Rab27a<Gt(pU-21T)58Imeg>/Rbrc FVB129P2(B6)-Mmp2<tm1Ito> B6C3-Tmem5b<Gt(venusTrap_fJAV17 B6.Cg-Tg(Scx-GFP)1Stzr C57BL/6-Tg(CAG-Nup85)3-3Sush

11 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 ma 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

12 o Epididymides and testes

13 Recipient medium Cells used n Sperm El. Spermatid GL-PBS NIM GL-PBS NIM No. pregnant No. transferred (Total) No. (%) implant (68.) (95.) (3.) (3.8) No.(%) offspring (22.) (65.) (1.) (17.9) Ogonuki et al., PNAS 13: , 26 in collaboration with Dr. Martin Fray

14 o (provided by Dr. Iwaki, Tokyo Jikei Medical University) Feb. 13, 26 Mar. 2, 26 Recipient females No. oocytes that survived inejction No. (%) oocytes cleaved BALB/c-nude (76.4) C3H/He (89.8) Strain of male Aug. 28, 26 No. embryos transferred No. (%) implant No. (%) offspring No. used No. pregnant (51.9) 17 (21.) (35.1) 12 (12.4) Ogonuki et al., PNAS 13: , 26

15 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 ma 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

16 First wave round spermatids in a 17 day-old testis Genomic DNA A pup born from a 17 day-old spermatid cdna M H19 B6JF1 Spermatid IVF Injection (B6 x JF1) H19, Igf2, Meg3, Igf2r Miki et al., JRD 24

17 22 Completion of congenesis Ogonuki et al., PLoS One 29

18 (79/86) (56/86) d 1 (/86) 2 RELMb (86/86) Markers homozygous for B6 (%) Markers homozygous for B6 (%) EdnraEGFP 43d 43d (86/86) 41d (85/86) d 9 (77/86) 85 97d (72/86) d (72/86) 5 1 (176/176) (154/176) (163/176) 43d 41d (123/176) 42d d (38/176) Kitlg 15 2 Markers homozygous for NOD (%) Markers homozygous for B6 (%) Gdf5 15 (7/72) (63/72) (6/72) 44d 45d 43d (38/72) d 2 (/72) 143d (/72) Ogonuki et al., PLoS One 29

19 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

20 12.5 dpc gonads Dissociation by enzyme Aggregation and transplantation

21 ROSI or ICSI IVM Matoba and Ogura, Biol. Reprod. 211

22 For the details of our lab: