Feasibility of cryopreservation of zebrafish (Danio rerio) primordial germ cells by whole embryo freezing

Shogo Higaki, Kentaro Mochizuki, Hiroko Baba, Yuichiro Akashi, Etsuro Yamaha, Seiji Katagiri, Yoshiyuki Takahashi

Research output: Contribution to journalArticlepeer-review

13 Citations (Scopus)

Abstract

We investigated the feasibility of cryopreservation of zebrafish (Danio rerio) blastomeres and primordial germ cells (PGCs) by rapid freezing of dechorionated whole embryos at the blastula, gastrula and segmentation stages. Initially we examined the glass-forming properties and embryo toxicities of 5 cryoprotectants: methanol (MeOH), ethylene glycol (EG), dimethyl sulfoxide (DMSO), propylene glycol (PG), and 1,3-butylene glycol (1,3-BG). Embryos at the blastula and gastrula stages had high sensitivities to cryoprotectant toxicities and were fragile against mechanical damage Thus the segmentation stage embryos, the PGCs of which were visualized by injecting green fluorescence protein-nosi 3uTR mRNA, were frozen using solutions containing each cryoprotectant at 6 M (first trial) and 2 types of cryoprotectants at 3 M each (second trial). In the first trial, live PGCs were recovered from most of the embryos frozen with EG (about 2 cells/embryo); however, a few embryos had live PGCs when embryos were frozen with other cryoprotectants. In the second trial, a mixture of EG + PG better preserved the viability of PGCs in frozen embryos. Live PGCs were recovered from all embryos frozen with EG + PG (about 3 cells/embryo), and the survival rate of PGCs was estimated to be about 25% based on the number of live PGCs in fresh embryos (about 12 cells/embryo). The present study indicates that we can utilize rapid freezing of dechorionated whole embryos at the segmentation stage for the cryopreservation of PGCs.

Original languageEnglish
Pages (from-to)119-128
Number of pages10
JournalJapanese Journal of Veterinary Research
Volume57
Issue number2
Publication statusPublished - 2009 Aug

Keywords

  • Blastomere
  • Cryopreservation
  • Embryo
  • Primordial germ cell
  • Zebrafish

ASJC Scopus subject areas

  • veterinary(all)

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