Advertisement

Marine Biotechnology

, Volume 16, Issue 3, pp 309–320 | Cite as

Intraperitoneal Germ Cell Transplantation in the Nile Tilapia Oreochromis niloticus

  • Rodolfo Farlora
  • Shoko Hattori-Ihara
  • Yukata Takeuchi
  • Makoto Hayashi
  • Anna Octavera
  • Alimuddin
  • Goro YoshizakiEmail author
Original Article

Abstract

Germ cell transplantation offers promising applications in finfish aquaculture and the preservation of endangered species. Here, we describe an intraperitoneal spermatogonia transplantation procedure in the Nile tilapia Oreochromis niloticus. Through histological analysis of early gonad development, we first determined the best suitable stage at which exogenous germ cells should be transplanted into the recipients. For the transplantation procedure, donor testes from a transgenic Nile tilapia strain carrying the medaka β-actin/enhanced green fluorescent protein (EGFP) gene were subjected to enzymatic dissociation. These testicular cells were then stained with PKH26 and microinjected into the peritoneal cavity of the recipient fish. To confirm colonization of the donor-derived germ cells, the recipient gonads were examined by fluorescent and confocal microscopy. PKH26-labeled cells exhibiting typical spermatogonial morphology were incorporated into the recipient gonads and were not rejected within 22 days posttransplantation. Long-term survival of transgenic donor-derived germ cells was then verified in the gonads of 5-month-old recipients and in the milt and vitelogenic oocytes of 1-year-old recipients, by means of PCR using EGFP-specific primers. EGFP-positive milt from adult male recipients was used to fertilize non-transgenic oocytes and produced transgenic offspring expressing the donor-derived phenotype. These results imply that long-term survival, proliferation, and differentiation of the donor-derived spermatogonia into vitelogenic oocytes and functional spermatozoa are all possible. Upon further improvements in the transplantation efficiency, this intraperitoneal transplantation system could become a valuable tool in the conservation of genetic resources for cichlid species.

Keywords

Germ cell transplantation Spermatogonia Oreochromis niloticus Endangered species Transgenic fish Green fluorescent protein (GFP) 

Notes

Acknowledgments

This research was partly supported by the Japan Science and Technology Agency/Japan International Cooperation Agency through their Science and Technology Research Partnership for Sustainable Development program to GY.

References

  1. Biswas AK, Morita T, Yoshizaki G, Maita M, Takeuchi T (2005) Control of reproduction in Nile tilapia Oreochromis niloticus (L.) by photoperiod manipulation. Aquaculture 243:229–239CrossRefGoogle Scholar
  2. Farlora R, Kobayashi S, França LR, Batlouni SR, Lacerda SMSN, Yoshizaki G (2009) Expression of GFP in transgenic tilapia under the control of the medaka β-actin promoter: establishment of a model system for germ cell transplantation. Anim Reprod 6:450–459Google Scholar
  3. Hamada K, Tamaki K, Sasado T, Watai Y, Kani S, Wakamatsu Y, Ozato K, Kinoshita M, Kohno R, Takagi S, Kimura M (1998) Usefulness of the medaka β-actin promoter investigated using a mutant GFP reporter gene in transgenic medaka (Oryzias latipes). Mol Mar Biol Biotechnol 7:173–180PubMedGoogle Scholar
  4. Higaki S, Eto Y, Kawakami Y, Yamaha E, Kagawa N, Kuwayama M, Nagano M, Katagiri S, Takahashi Y (2010) Production of fertile zebrafish (Danio rerio) possessing germ cells (gametes) originated from primordial germ cells recovered from vitrified embryos. Reproduction 139:733–740CrossRefPubMedGoogle Scholar
  5. Higuchi K, Takeuchi Y, Miwa M, Yamamoto Y, Tsunemoto K, Yoshizaki G (2011) Colonization, proliferation, and survival of intraperitoneally transplanted yellowtail Seriola quinqueradiata spermatogonia in nibe croaker Nibea mitsukurii recipient. Fish Sci 77:69–77CrossRefGoogle Scholar
  6. IUCN (2012) IUCN red list of threatened species. Version 2012.2. <http://www.iucnredlist.org>. Accessed 10 Apr 2013
  7. Kawakami Y, Goto-Kazeto R, Saito T, Fujimoto T, Higaki S, Takahashi Y, Arai K, Yamaha E (2010) Generation of germ-line chimera zebrafish using primordial germ cells isolated from cultured blastomeres and cryopreserved embryoids. Int J Dev Biol 54:1493–1501CrossRefPubMedGoogle Scholar
  8. Kise K, Yoshikawa H, Sato M, Tashiro M, Yazawa R, Nagasaka Y, Takeuchi Y, Yoshizaki G (2012) Flow-cytometric isolation and enrichment of teleost type A spermatogonia based on light-scattering properties. Biol Reprod 86(4):1–12CrossRefGoogle Scholar
  9. Kobayashi T, Takeuchi Y, Takeuchi T, Yoshizaki G (2006) Generation of viable fish from cryopreserved primordial germ cells. Mol Reprod Dev 74:207–213CrossRefGoogle Scholar
  10. Kobayashi S, Alimuddin, Morita T, Miwa M, Lu J, Endo M, Takeuchi T, Yoshizaki G (2007) Transgenic Nile tilapia (Oreochromis niloticus) over-expressing growth hormone show reduced ammonia excretion. Aquaculture 270:427–435CrossRefGoogle Scholar
  11. Lacerda SMSN, Batlouni SR, Silva SBG, Homem CSP, França LR (2006) Germ cells transplantation in fish: the Nile-tilapia model. Anim Reprod 3:146–159Google Scholar
  12. Lacerda SM, Batlouni SR, Costa GM, Segatelli TM, Quirino BR, Queiroz BM, Kalapothakis E, França LR (2010) A new and fast technique to generate offspring after germ cells transplantation in adult fish: the Nile tilapia (Oreochromis niloticus) model. PLoS One 5:e10740. doi: 10.1371/journal.pone.0010740 PubMedCentralCrossRefPubMedGoogle Scholar
  13. Lee S, Iwasaki Y, Shikina S, Yoshizaki G (2013) Generation of functional eggs and sperm from cryopreserved whole testes. Proc Natl Acad Sci U S A 110(5):1640–1645PubMedCentralCrossRefPubMedGoogle Scholar
  14. Mair GC, Little DC (1991) Population control in farmed tilapias. Naga 14:8–13Google Scholar
  15. Macaranas JM, Taniguchi N, Pante MJR, Capili JB, Pullin RSV (1986) Electrophoretic evidence for extensive hybrid gene introgression into commercial Oreochromis niloticus in the Philippines. Aquac Fish Manag 17:249–258Google Scholar
  16. Majhi SK, Hattori RS, Yokota M, Watanabe S, Strüssmann CA (2009) Germ cell transplantation using sexually competent fish: an approach for rapid propagation of endangered and valuable germlines. PLoS One 4:e6132. doi: 10.1371/journal.pone.0006132 PubMedCentralCrossRefPubMedGoogle Scholar
  17. Morita T, Kumakura N, Morishima K, Mitsuboshi T, Ishida M, Hara T, Kudo S, Miwa M, Ihara S, Higuchi K, Takeuchi Y, Yoshizaki G (2012) Production of donor-derived offspring by allogeneic transplantation of spermatogonia in the yellowtail (Seriola quinqueradiata). Biol Reprod 86:1–11CrossRefGoogle Scholar
  18. Nagasawa K, Shikina S, Takeuchi Y, Yoshizaki G (2010) Lymphocyte antigen 75 (Ly75/CD205) is a surface marker on mitotic germ cells in rainbow trout. Biol Reprod 83:597–606CrossRefPubMedGoogle Scholar
  19. Nagasawa K, Miwa M, Yazawa R, Morita T, Takeuchi Y, Yoshizaki G (2012) Characterization of lymphocyte antigen 75 (Ly75/CD205) as a potential cell-surface marker on spermatogonia in Pacific bluefin tuna Thunnus orientalis. Fish Sci 78(4):791–800CrossRefGoogle Scholar
  20. Okutsu T, Suzuki K, Takeuchi Y, Takeuchi T, Yoshizaki G (2006) Testicular germ cells can colonize sexually undifferentiated embryonic gonad and produce functional eggs in fish. Proc Natl Acad Sci U S A 103(8):2725–2729PubMedCentralCrossRefPubMedGoogle Scholar
  21. Okutsu T, Shikina S, Kanno M, Takeuchi Y, Yoshizaki G (2007) Production of trout offspring from triploid salmon parents. Science 317:1517CrossRefPubMedGoogle Scholar
  22. Saito T, Goto-Kazeto R, Arai K, Yamaha E (2008) Xenogenesis in teleost fish through generation of germ-line chimeras by single primordial germ cell transplantation. Biol Reprod 78:159–166CrossRefPubMedGoogle Scholar
  23. Seehausen O, van Alpen JM, Witte F (1997) Cichlid fish diversity threatened by eutrophication that curbs sexual selection. Science 277:1808–1811CrossRefGoogle Scholar
  24. Shirak A, Seroussi E, Cnaani A, Howe AE, Domokhovsky R, Zilberman N, Kocher TD, Hulata G, Ron M (2006) Amh and Dmrta2 genes map to tilapia (Oreochromis spp.) linkage group 23 within quantitative trait locus regions for sex determination. Genetics 174:1573–1581PubMedCentralCrossRefPubMedGoogle Scholar
  25. Takeuchi Y, Yoshizaki G, Takeuchi T (2003) Generation of live fry from intraperitoneally transplanted primordial germ cells in rainbow trout. Biol Reprod 69:1142–1149CrossRefPubMedGoogle Scholar
  26. Takeuchi Y, Yoshizaki G, Takeuchi T (2004) Surrogate broodstock produces salmonids. Nature 430:629–630CrossRefPubMedGoogle Scholar
  27. Takeuchi Y, Higuchi K, Yatabe T, Miwa M, Yoshizaki G (2009) Development of spermatogonial cell transplantation in Nibe croaker, Nibea mitsukurii (Perciformes, Sciaenidae). Biol Reprod 81:1055–1063CrossRefPubMedGoogle Scholar
  28. Taniguchi N, Macaranas JM, Pullin RSV (1985) Introgressive hybridization in culture tilapia stocks in the Philippines. Bull Jpn Soc Fish 51:1219–1224CrossRefGoogle Scholar
  29. Witte F, Goldschmidt T, Wanink J, van Oijen M, Goudswaard K, Witte-Maas E, Bouton N (1992) The destruction of an endemic species flock: quantitative data on the decline of the haplochromine cichlids of Lake Victoria. Environ Biol Fish 29:1–28CrossRefGoogle Scholar
  30. Wong TT, Saito T, Crodian J, Collodi P (2011) Zebrafish germline chimeras produced by transplantation of ovarian germ cells into sterile host larvae. Biol Reprod 84:1190–1197PubMedCentralCrossRefPubMedGoogle Scholar
  31. Yazawa R, Takeuchi Y, Higuchi K, Yatabe T, Kabeya N, Yoshizaki G (2010) Chub mackerel gonads support colonization, survival, and proliferation of intraperitoneally transplanted xenogenic germ cells. Biol Reprod 82:896–904CrossRefPubMedGoogle Scholar
  32. Yoshizaki G, Ichikawa M, Hayashi M, Iwasaki Y, Miwa M, Shikina S, Okutsu T (2010) Sexual plasticity of ovarian germ cells in rainbow trout. Development 137:1227–1230CrossRefPubMedGoogle Scholar
  33. Yoshizaki G, Fujinuma K, Iwasaki Y, Okutsu T, Shikina S, Yazawa R, Takeuchi Y (2011) Spermatogonial transplantation in fish: a novel method for the preservation of genetic resources. Comp Biochem Physiol Part D Genomics Proteomics 6:55–61CrossRefPubMedGoogle Scholar
  34. Zhang T, Rawson DM, Pekarsky I, Blais I, Lubzens E (2007) Low-temperature preservation of fish gonad cells and oocytes. In: Babin PJ, Cerda J, Lubzens E (eds) The fish oocyte. Springer, Dordrecht, pp. 411–436Google Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Rodolfo Farlora
    • 1
  • Shoko Hattori-Ihara
    • 2
  • Yukata Takeuchi
    • 3
  • Makoto Hayashi
    • 2
  • Anna Octavera
    • 4
  • Alimuddin
    • 4
  • Goro Yoshizaki
    • 2
    • 5
    Email author
  1. 1.Laboratory of Biotechnology and Aquatic Genomics, Interdisciplinary Center for Aquaculture Research (INCAR)University of ConcepcionConcepciónChile
  2. 2.Department of Marine BiosciencesTokyo University of Marine Science and TechnologyTokyoJapan
  3. 3.Research Center for Advanced Science and TechnologyTokyo University of Marine Science and TechnologyChibaJapan
  4. 4.Department of Aquaculture, Faculty of Fisheries and Marine SciencesBogor Agriculture UniversityBogorIndonesia
  5. 5.Japan Science and Technology Agency/Japan International Cooperation Agency (JST/JICA) through their Science and Technology Research Partnership for Sustainable Development (SATREPS)ChibaJapan

Personalised recommendations