Cryopreservation of primordial germ cells by rapid cooling of whole zebrafish (Danio rerio) embryos.
The feasibility of cryopreservation of zebrafish (Danio rerio) primordial germ cells (PGCs) by rapid cooling (i.e., vitrification) of dechorionated whole embryos at the 14- to 20-somite stage was investigated. Initially, we examined the glass-forming properties and embryo toxicities of six cryoprotectants: methanol (MeOH), ethylene glycol (EG), glycerol (GC), dimethyl sulfoxide (DMSO), propylene glycol (PG) and 1,3-butylene glycol (1,3-BG). According to the results of glass-forming and embryo toxicity tests, pretreatment solution (PS) containing 2 or 3 M cryoprotectant and vitrification solution (VS) containing 5 M cryoprotectant and 0.5 M sucrose were prepared using each cryoprotectant. Dechorionated embryos, the PGCs of which were visualized by injection of green fluorescence protein-nos1 3'UTR mRNA, were cooled rapidly by plunging into liquid nitrogen after serial exposure to PS and VS. All embryos cooled with MeOH, PG and 1,3-BG showed ice formation during cooling, and few embryos had live PGCs after warming. Most embryos cooled with GC did not show ice formation; however, few embryos had live PGCs. All embryos cooled with EG and most embryos cooled with DMSO had live PGCs when the embryos did not show ice formation during cooling. Based on the number of live PGCs in fresh embryos, the maximum survival rates of PGCs recovered from embryos cooled with EG and DMSO were estimated to be about 40 and 20%, respectively. The present study indicates that rapid cooling of dechorionated whole embryos, especially using EG-based solutions, could be utilized as a simple and promising tool for cryopreservation of PGCs.
Higaki
Shogo
S
Laboratory of Theriogenology, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
Mochizuki
Kentaro
K
Akashi
Yuichiro
Y
Yamaha
Etsuro
E
Katagiri
Seiji
S
Takahashi
Yoshiyuki
Y
D000818:Animals; D002072:Butylene Glycols / Q000494:pharmacology / Q000633:toxicity; D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation / Q000379:methods* / Q000662:veterinary*; D003451:Cryoprotective Agents / Q000494:pharmacology* / Q000633:toxicity; D004121:Dimethyl Sulfoxide / Q000494:pharmacology / Q000633:toxicity; D019855:Ethylene Glycol / Q000494:pharmacology / Q000633:toxicity; D005240:Feasibility Studies; D005854:Germ Cells / Q000166:cytology* / Q000187:drug effects*; D005898:Glass; D005990:Glycerol / Q000494:pharmacology / Q000633:toxicity; D000432:Methanol / Q000494:pharmacology / Q000633:toxicity; D019946:Propylene Glycol / Q000494:pharmacology / Q000633:toxicity; D015027:Zebrafish / Q000196:embryology*
D002072:Butylene Glycols; D003451:Cryoprotective Agents; C028491:1,3-butylene glycol; D019946:Propylene Glycol; D019855:Ethylene Glycol; D005990:Glycerol; D000432:Methanol; D004121:Dimethyl Sulfoxide