Cryopreservation of Primordial Germ Cells by Rapid Cooling of Whole Zebrafish (Danio rerio) Embryos

Abstract

Abstract. 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.

Cryopreservation of Embryos from Model Animals and Human

“…Of the cryoprotecting agents tested, ethylene glycol solution specifically led to the blastoderm being dissociated from the yolk (Hagedorn et al, 1997c). A similar phenomenon was observed on 14 to 20-somite stage embryos of Danio rerio (Higaki et al, 2010b). Treating the embryos with glycerol at a concentration of 2M for 30 minutes did not yield any viable cells in the embryos.…”

“…In comparison, methanol and DMSO are moderately toxic. Propylene glycol and 1,3-butylene glycol are mildly toxic, killing only 58-78% of cells even at a concentration of 5M (Higaki et al, 2010b).…”

“…Blastomeres may have advantages over primordial germ cells in generating germline transmitting chimera. Firstly, identification and isolation of primordial germ cells relies on a readily observable transgenic marker (Higaki et al, 2010b;Kobayashi et al, 2007). Breeding of a strain to a marker transgenic strain or freshly injecting DNA constructs is required before cryopreservation procedures, making the procedures more complicated.…”

“…For example, glycerol and dimethylformamide are very toxic to fish embryos and mammalian morulae, respectively (Higaki et al, 2010b;Kasai et al, 1981).…”

Technical note: Viability and motility of vitrified/thawed primordial germ cell isolated from common carp (Cyprinus carpio) somite embryos1

“…However, the toxicity of these vehicles is species-specific in teleosts (Cabrita et al, 2006). Higaki et al (2010b) compared EG, DMSO, glycerol, methanol, propylene glycol, and 1,3-butylene glycol for use in vitrified zebrafish somite embryos. It was apparent that EG was a better cryoprotectant than DMSO.…”