Vitrification of primordial germ cells using whole embryos for gene-banking in loach, Misgurnus anguillicaudatus

Authors

  • D. Inoue,

    1. Laboratory of Aquaculture Genetics and Genomics, Division of Marine Life Science, Faculty and Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido, Japan
    2. Laboratory of Soft & Wet Matter, Division of Biological Sciences, Graduate School of Life Science, Hokkaido University, Sapporo, Hokkaido, Japan
    Search for more papers by this author
  • T. Fujimoto,

    Corresponding author
    • Laboratory of Aquaculture Genetics and Genomics, Division of Marine Life Science, Faculty and Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido, Japan
    Search for more papers by this author
  • Y. Kawakami,

    1. Nanae Fresh-Water Laboratory, Field Science Center for Northern Biosphere, Hokkaido University, Nanae, Kameda, Hokkaido, Japan
    Search for more papers by this author
  • G. S. Yasui,

    1. Laboratory of Aquaculture Genetics and Genomics, Division of Marine Life Science, Faculty and Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido, Japan
    2. Laboratório de Reprodução Animal, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, São Paulo, Brazil
    Search for more papers by this author
  • E. Yamaha,

    1. Nanae Fresh-Water Laboratory, Field Science Center for Northern Biosphere, Hokkaido University, Nanae, Kameda, Hokkaido, Japan
    Search for more papers by this author
  • K. Arai

    1. Laboratory of Aquaculture Genetics and Genomics, Division of Marine Life Science, Faculty and Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido, Japan
    Search for more papers by this author

Author's address: Takafumi Fujimoto, Laboratory of Aquaculture Genetics and Genomics, Division of Marine Life Science, Faculty and Graduate School of Fisheries Sciences, Hokkaido University, 3-1-1 Minato-cho, Hakodate, Hokkaido, 041-8611, Japan.

E-mail: fujimoto@fish.hokudai.ac.jp

Summary

In gene-banking, primordial germ cells (PGCs), which are embryonic precursor cells of germ cells, are useful for cryopreservation because PGCs have a potential to differentiate into both eggs and sperm via germ-line chimera. Here, we have established vitrification methods for PGCs cryopreservation using 12- to 17-somite stage embryos in loach, Misgurnus anguillicaudatus, which were dechorionated, removed their yolk and injected with green fluorescent protein (GFP) -nos1 3′UTR mRNA to visualize their PGCs. In order to optimize cryopreservation medium for vitrification, the toxicity of cryoprotectants was analyzed. Different concentrations (2, 3, 4, 5 m) of dimethyl sulfoxide (DMSO), methanol (MeOH), ethylene glycol (EG) and propylene glycol (PG) as cryoprotectants were tested. Then, 5 m DMSO showed significantly-high toxicity. Based on this information, combinations called DMP (2 m (14.2% [v/v]) DMSO, 2 m (8.1% [v/v]) MeOH and 2 m (14.4% [v/v]) PG), DP (2 m (14.2% [v/v]) DMSO and 4 m (28.7% [v/v]) PG) and DE (2.1 m (15% [v/v]) DMSO and 2.7 m (15% [v/v]) EG) were evaluated for their toxicities and efficacy of PGCs cryopreservation using two types of equilibration step: direct immersion of cryopreservation media (one-step) and serial exposure to half and full concentration of cryopreservation media (two-step). Viable PGCs were obtained from post-thaw embryos which were cryopreserved by DP and DE with both 1- and 2-step equilibrations. Despite DP showing the highest toxicity, it gave the highest survival rate of embryonic cells after cryopreservation. When PGCs recovered from vitrified embryos were transplanted into host embryos at the blastula stage, the transplanted PGCs were able to migrate to a host genital ridge similarly as endogenous PGCs. It suggests that our methods could be useful to create a germ-line chimera for the production of gametes from PGCs of cryopreserved embryos.

Ancillary