Germ Cell Transplantation in Fish
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1 Larvi 2009 Germ Cell Transplantation in Fish Goro Yoshizaki (Tokyo University of Marine Science and Technology, SORST/JST)
2 Tuna Mackerel Body weight; 300 kg 300 g Body length; 3 m 30 cm Scombridae family
3 Aquaculture of tuna *conventional aquaculture is capture-base Maintain adult tuna in cages kg Market size: kg
4 Maintenance of adult tuna requires a lot of space, cost, and labor Mackerel can spawn in a small fish tank If we can obtain mackerel that produce tuna gametes
5 Aquaculture of tuna Maintain adult tuna in cage
6 Mackerel as surrogated broodstock Egg taking in small tanks *save a lot of space, labor, and cost. *environmental manipulation and hormonal treatment can be easily performed.
7 How can we obtain mackerel that t can produce tuna eggs and sperm??? 1. Isolate the progenitor cells of eggs and sperm from tuna. 2. Transplant them into recipient mackerel.
8 What are the progenitor cells of eggs and sperm? Primordial Germ Cell oogonia oocyte egg 20 μm Sex differentiation spermatogonia spermatocyte sperm
9 Primordial germ cells Male Female recipients Donor sperm Donor egg
10 How can we transplant primordial germ cells into recipients? 8M years Donor: rainbow trout Recipient: masu salmon
11 gonad immunorejection Gonads are too small
12 Migration of Primordial Germ Cells Migrating primordial germ cells ll Gonad Cross section of a hatchling h t hli Primordial g germ cells emerge g in extragonadal g areas and then migrate to the immature gonads
13 PGCs Transplantation into the Peritoneal Cavity of Hatched Embryos Xenogenic recipient (non-transgenic) vasa-gfp rainbow trout PGCs 10 PGCs /embryo Micropipette
14 Behavior of Transplanted PGCs in Recipient Fish 10 days after t.p. 30 days after t.p. Immature gonad Pseudopod Takeuchi et al., Biol Reprod, 2003
15 Male Female Transplanted primordial germ cells proliferated and started meiosis in gonads of xenogeneic recipients
16 Can the transplanted PGCs differentiate into functional gametes in the gonads of xenogenic recipients?
17 Donor (Trout) Recipient (Salmon) Mature Recipient Trout Eggs Mixture of trout and salmon sperm Trout Sperm Trout Eggs Salmon Sperm X Trout Lethal hybrid
18 34 days post-fertilization at 10 o C Trout embryos Trout/salmon hybrid
19 F1 embryos (34 dpf) derived from recipient salmon gonad of hatchlings Takeuchi et al., Nature, 2004
20 hatchlings un-hatched Takeuchi et al., Nature, 2004
21 Rainbow trout juveniles produced by surrogated masu salmon Donor-derived offspring from inter-species germ-cell transplantation were successfully produced Takeuchi et al., Nature, 2004
22 1)Number of PGCs is limited (20-30 from one embryo) 2)Difficult to capture newly hatched embryos of commercially important species
23 ?? Spermatogonia *Possessed by any age of male fish
24 Intraperitoneal Transplantation of Testicular Germ Cells Testes of vasa-gfp trout Recipient (non-tg) Testicular cell suspension Intraperitoneal transplantation
25 Incorporation of Spermatogonia in Allogenic Genital Ridges Bright field view Fluorescent view 18 days after transplantation
26 Proliferation of Donor-Derived Spermatogonia in a Recipient Testis Testis of 6-month-old recipient IHC with GFP-specific antibody : Endogenous germ cell Okutsu et al., PNAS, 2006
27 Can donor-derived d d spermatogonia mature and produce normal sperm in recipient gonads?
28 Wild-type recipient X vasa-gfp transgenic Albino If the recipients produce donor-derived derived sperm, F1 embryos with albino body color and green germ cells will be obtained. Wild-type eggs
29 Albino vasa-gfp Donor-derived d spermatogonia resumed spermatogenesis and produced functional sperm in the recipient testes Okutsu et al., PNAS, 2006
30 Can donor-derived derived testicular germ cells colonize in female recipient gonads?
31 Proliferation of Spermatogonial Stem Cells in Ovaries Ovarian lamellall 2 months after transplantation
32 Differentiation of Spermatogonial Stem Cells in Ovaries Peri-nucleolus oocytes 100 μm Ovary of 6-month-old recipient IHC with antibody specific to GFP Okutsu et al., PNAS, 2006
33 100 rporation ion (%) 75 of incor roliferati Rates & pr 00 Inc Pro Male : Incorporation rate :Proliferation rate Inc Pro Female There were no significant differences in incorporation and proliferation efficiency i of spermatogonia in male and female recipients
34 Wild-type recipient vasa-gfp transgenic Albino Wild-type sperm
35 F1 offspring developed d from spermatogonia-derived eggs Spermatogonia can produce functional eggs Okutsu et al., PNAS, 2006
36 Making salmon that produces only trout gametes
37 2N 3N 2N vasa-gfp trout 2N 3N 3N salmon
38 Secondary sexual character 3N salmon recipient
39 2N male trout (fertile) 3N control salmon 2N trout spermatogonia 3N salmon recipients 3N female salmon 3N male salmon Progeny test
40 8 Gametes obtained from masu salmon Male salmon Female salmon Trout Salmon Trout Salmon 200 sperm sperm eggs eggs N recipients Control Milt volume (ml) Sperm number (X10 9 ) 3N recipients Control Egg number
41 Trout germ cell (2N) Fluorescent view Trout germ cell (2N) 2N-salmon recipient i 3N-salmon recipient i (Donor-derived F1 (Donor-derived F1 : 0.4%) : 100%) Okutsu et al., Science, 2007
42 RFLP analyses of mtdna F1 cytb CO1 All F1 fish derived from salmon recipients possess rainbow trout-derived mitochondria Triploid salmon recipients produce only trout eggs
43 Random Amplified Polymorphic DNA (RAPD) analysis of 3N-recipient i salmon-derived d F1 F1 Salmon-derived DNA Trout-derived DNA Triploid salmon recipients received trout germ cells produced only trout offspring Okutsu et al., Science, 2007
44 Trout offspring from triploid salmon parents Okutsu et al., Science, 2007
45 World salmon crisis ss Over-fishing Habitat destruction -196 o C
46 Preservation of genetic information in fish Sperm Eggs or Embryos Due to their large size and high yolk content Maternally-inherited cytoplasmic compartments t (mitochondrial i DNA) can not be preserved!
47 Cryopreservation Endangered of spermatogonia Extinct Inter-Species Transplantation Regeneration Kobayashi et al., Mol Reprod Develop, 2007
48 Pa acific Ocean Columbia R. Washington N Project Area Redfish Lake Oregon Idaho km
49 Can spermatogonial transplantation be applicable to marine fish?
50 Application of germ cell transplants to marine fishes Nibe croaker (Nibea mitsukurii) ) 1mm Intra-sp. TP. Donor: HSC-GFP-transgenic (heterozygous) Recipient: triploid sterile non-transgenic
51 10 μm donor testicular cells labeled with PKH26 Croaker larva (TL=4mm) PKH26 Microinjection i into body cavity Survival: 30-50% (2 weeks after TP)
52 Fluorescent observation of gonads at 3 wks after TP Bright field PKH26 Control Gonad Recipient (Scale bars : 20 μm) Takeuchi et al., Biol Reprod, 2009
53 PKH-positive cells were germ cells? Confocal microscopic analysis DIC PKH26 DAPI Merge (Scale bars :10μm) *no PKH(+) somatic cells were observed PKH (+) cells found in the recipient gonad were surely donor-derived germ cells
54 Progeny test using recipient and wild-type 6monthafterTP Donor-derived sperm (hetelozygous GFP) Wild-type eggs F1 embryos Genomic DNA extraction & PCR for GFP gene g If the male recipient produces only donor-derived sperm, 50% of F1 embryos should be GFP-positive
55 N P GFP β-actin Triploid nibe croaker recipients only produced donor-derived functional sperm Germ cell transplantation is applicable to marine fish species
56 recipients Donor sperm Donor egg
57 in vitro cultured germ cells recipients Donor sperm Donor egg
58 Yutaka Takeuchi Tomoyuki Okutsu Naoki Kabeya Ryosuke Yazawa Kentaro Higuchi
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