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Vitrification of primordial germ cells using whole embryos for gene‐banking in loach, M isgurnus anguillicaudatus
Author(s) -
Inoue D.,
Fujimoto T.,
Kawakami Y.,
Yasui G. S.,
Yamaha E.,
Arai K.
Publication year - 2012
Publication title -
journal of applied ichthyology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.392
H-Index - 62
eISSN - 1439-0426
pISSN - 0175-8659
DOI - 10.1111/jai.12058
Subject(s) - cryoprotectant , cryopreservation , biology , dimethyl sulfoxide , vitrification , andrology , misgurnus , embryo , microbiology and biotechnology , chemistry , fish <actinopterygii> , fishery , medicine , organic chemistry
Summary In gene‐banking, primordial germ cells ( PGC s), which are embryonic precursor cells of germ cells, are useful for cryopreservation because PGC s have a potential to differentiate into both eggs and sperm via germ‐line chimera. Here, we have established vitrification methods for PGC s 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 m RNA to visualize their PGC s. 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 ( M e OH ), 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]) M e OH 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 PGC s 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 PGC s 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 PGC s recovered from vitrified embryos were transplanted into host embryos at the blastula stage, the transplanted PGC s were able to migrate to a host genital ridge similarly as endogenous PGC s. It suggests that our methods could be useful to create a germ‐line chimera for the production of gametes from PGC s of cryopreserved embryos.